UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS DE MONTES

INFLUENCIA DE LA LUZ Y LA SEQUÍA ESTIVAL EN LA

RESPUESTA FUNCIONAL DE BRINZALES DE Quercus petraea

(MATT.) LIEBL. Y Quercus pyrenaica WILLD.: IMPLICACIONES

PARA LA REGENERACIÓN

TESIS DOCTORAL

JESÚS RODRÍGUEZ CALCERRADA

Licenciado en Biología

2007

DEPARTAMENTO DE SILVOPASCICULTURA

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS DE MONTES

UNIVERSIDAD POLITÉCNICA DE MADRID

INFLUENCIA DE LA LUZ Y LA SEQUÍA ESTIVAL EN LA

RESPUESTA FUNCIONAL DE BRINZALES DE Quercus petraea

(MATT.) LIEBL. Y Quercus pyrenaica WILLD.: IMPLICACIONES

PARA LA REGENERACIÓN

JESÚS RODRÍGUEZ CALCERRADA

Licenciado en Biología

DIRECTORES:

JOSÉ ALBERTO PARDOS CARRIÓN ISMAEL ARANDA GARCÍA

Dr. Ingeniero de Montes Dr. en Ciencias Biológicas

2007

UNIVERSIDAD POLITÉCNICA DE MADRID

Tribunal nombrado por el Magfco. y Excmo. Sr. Rector de la Universidad

Politécnica de Madrid, el día .........de ..................de 200...

Presidente: .................................................................................................

Vocal: .................................................................................................

Vocal: .................................................................................................

Vocal: .................................................................................................

Secretario: .................................................................................................

Suplente: .................................................................................................

Suplente: .................................................................................................

Realizado el acto de defensa y lectura de la Tesis el día .........de ..................de

200... en la E.T.S.I. Montes

EL PRESIDENTE LOS VOCALES

EL SECRETARIO

Agradecimientos

Han sido muchas las personas que me han ayudado a lo largo de estos años. En primer

lugar quiero expresar mi agradecimiento a José Alberto Pardos, sobre todo por su

indestructible confianza en mi trabajo en todo momento. A Ismael Aranda, que

igualmente confió en mí desde el principio, y además, me ha enseñado a manejar tantos

equipos y me ha ayudado con el trabajo experimental. Y a Luis Gil, por sus comentarios

de ánimo y el inestimable sustento logístico.

Quiero agradecer la ayuda de mis “asesores matemáticos y estadísticos”: Sven

Mutke, Rosa Ana López y Nikos Nanos; y la ayuda todoterreno de Javier Cano y Jesús

Alonso, dos buenos compañeros de fatigas. Peter B. Reich, Rebecca A. Montgomery,

Lee Freelich, Eva Rosenqvist y Carl-Otto Ottossen me acogieron con una tremenda

amabilidad en sus departamentos. Ruben Milla y Melody Machala me hicieron sentir

como en casa cuando estuve en Minneapolis.

También han contribuido a sacar esto adelante: María del Carmen del Rey, Pablo

Fuentes, Antonio López, Matt Robson, Margarita Burón, Álvaro Soto, Martin Venturas,

Unai López de Heredia, María Valbuena, María Dolores Jiménez, Zaida Lorenzo, Javier

Navarro, José Climent, Jaime Puértolas, Miguel Pérez, Fernando Pardo, María del

Carmen García, Pilar Pita, Antonio Gascó, Francisco Masedo, y muchos de los guías del

Hayedo de Montejo.

Gracias a toda mi familia. En especial a mi padre y a mi madre por su obstinación

en que nunca me faltase nada y su apoyo en todas las cosas que he hecho; y a mi mujer,

que ha guiado esta tesis con su cariño en los momentos de mayor desilusión. Gracias.

ÍNDICE

Principales abreviaturas v

RESUMEN vii

ABSTRACT viii

1. INTRODUCCIÓN 1

1.1. Distribución del roble albar y el melojo 1

1.2. Ecología del roble albar y el melojo. Sus nichos de regeneración

en la Península Ibérica 4

1.3. La ecofisiología de brinzales como herramienta para evaluar las

posibilidades de regeneración de las especies 5

1.4. El comportamiento de las especies frente a la disponibilidad

de recursos: adaptación, aclimatación y plasticidad 6

1.5. Compromisos en los patrones de aclimatación a ambientes

sombreados y secos 9

1.6. La interacción entre la luz y el agua en los procesos de regeneración 11

1.7. El papel de los pinares en la expansión de los robles en el entorno

del Hayedo de Montejo de la Sierra 11

2. OBJETIVOS 15

3. MATERIAL Y MÉTODOS 17

3.1. Experimento en invernadero (anexos I y II) 17

3.1.1. Material vegetal y diseño experimental 17

3.1.2. Intercambio gaseoso foliar 18

3.1.3. Fluorescencia de la clorofila a 20

3.1.4. Parámetros bioquímicos de la hoja 22

3.1.5. Parámetros anatómicos y morfológicos de la hoja 24

3.1.6. Arquitectura de la planta 24

3.1.7. Crecimiento y distribución de la biomasa 24

3.1.8. Análisis estadístico 25

3.2. Experimentos en campo 26

3.2.1. Plantaciones en La Maleza 27

- Primera plantación (anexo III) 28

- Segunda plantación (anexos IV a VI) 29

- Tercera plantación (anexo V) 36

3.2.2. Plantación en Sierra Escalva (anexo VI) 37

4. RESULTADOS Y DISCUSIÓN 39

4.1. Experimento en invernadero: comparación interespecífica de las

respuestas de aclimatación a la luz en ausencia de estrés hídrico 39

4.2. Experimentos en el campo 45

4.2.1. Comparación de las respuestas de aclimatación a la luz con las

observadas en invernadero 45

4.2.2. Variación del “microclima” según la densidad del arbolado 47

4.2.3. Variación de la resistencia a la sequía estival según la

densidad del arbolado 49

4.2.4. Importancia del lugar de plantación en la supervivencia inicial 56

5. RECAPITULACIÓN 59

6. CONCLUSIONES 63

7. BIBLIOGRAFÍA 65

Anexos I. Leaf physiological versus morphological acclimation after high light exposure at

different foliage developmental stages.

II. Light response in seedlings of a temperate (Quercus petraea) and a sub-

Mediterranean species (Quercus pyrenaica): contrasting ecological strategies as

potential keys to regeneration performance in mixed marginal populations.

III. Summer field performance of Quercus petraea (Matt.) Liebl. and Quercus

pyrenaica Willd. seedlings planted in three sites with contrasting canopy cover.

IV. Acclimation to light in seedlings of Quercus petraea (Mattuschka) Liebl. and

Quercus pyrenaica Willd. planted along a forest-edge gradient.

V. Ability to avoid water stress in seedlings of two oak species is lower in a dense

forest understory than in a medium canopy gap.

VI. Effects of Scots pine overstory density on pressure-volume curves and survival of

two underplanted oak species in a Mediterranean mountain.

PRINCIPALES ABREVIATURAS Tratamientos HL Alta irradiancia (70 % de plena luz)

SH Baja irradiancia (5,3 % de plena luz)T1 Plantas transferidas antes de brotarT2 Plantas transferidas después de brotar

VariablesIntercambio gaseoso foliar Asat Tasa de fotosíntesis neta a saturación por luz

gsat Tasa de conductancia estomática a saturación por luzAa

max Tasa de fotosíntesis neta a saturación por luz y CO2

Vacmax Tasa máxima de carboxilación

Jamax Tasa máxima de transporte electrónico

Ammax Tasa de fotosíntesis neta a saturación por luz y CO2, por unidad de masa

Vmcmax Tasa máxima de carboxilación, por unidad de masa

Jmmax Tasa máxima de transporte electrónico, por unidad de masa

Relaciones hídricas Ψpd Potencial hídrico foliar al amanecerΨmd Potencial hídrico foliar a mediodía

Fluorescencia de la clorofila ETRmax Tasa máxima de transporte electrónico, estimada con un fluorómetroΦPSII Eficiencia fotoquímica efectivaNPQ Disipación no fotoquímica Fv/Fm Eficiencia fotoquímica potencial

Bioquímica de la hoja Na Concentración de nitrógeno por unidad de área foliarNm Concentración de nitrógeno por unidad de masa foliarEUN Eficiencia fotosintética del uso de nitrógenoPr Proporción de nitrógeno foliar en RubiscoPb Proporción de nitrógeno foliar en proteinas de transporte electrónicoPl Proporción de nitrógeno foliar en proteinas asociadas a la clorofilaPs Proporción de nitrógeno foliar estructuralCa Concentración de clorofila por unidad de área foliarCm Concentración de clorofila por unidad de masa foliar

Morfología foliar SLA Superficie foliar específicaGE Grosor de la epidermis GPP Grosor del parénquima en empalizadaGPE Grosor del parénquima esponjosoTH Tamaño de la hoja

Morfología de la planta SFT Superficie foliar totalLMF Proporción de biomasa seca en hojasSMF Proporción de biomasa seca en tallosRMF Proporción de biomasa seca en raícesR/S Relación entre la biomasa de raíces y la de tallos y hojas

v

RESUMEN

El estudio de las respuestas funcionales de brinzales de roble albar (Q. petraea) y

melojo (Q. pyrenaica) a la disponibilidad de luz y agua constituye la base para un buen

conocimiento de su capacidad de regeneración. En el desarrollo de esta tesis se ha

evaluado la capacidad de aclimatación a la luz y la influencia en la respuesta a la sequía

estival, de brinzales de las dos especies. Para ello se ha realizado un experimento en

invernadero en ausencia de estrés hídrico en el que se ha estudiado la capacidad de

aclimatación a la luz a corto y a largo plazo, y se han realizado varias plantaciones en

sitios próximos al “Hayedo de Montejo” (NE de Madrid) con diferente cobertura

arbórea, examinando la evolución de diversos parámetros ambientales y funcionales a lo

largo del verano y la supervivencia durante los 3-4 primeros años.

En invernadero, las hojas de los brinzales de roble albar tuvieron mayor superficie

específica e individual, mayor concentración de clorofila, y mayor proporción de

nitrógeno invertido en componentes del aparato fotosintético que las de melojo; en

condiciones de elevada iluminación crecieron más rápidamente que los brinzales de

melojo, invirtiendo menos biomasa en raíces y mostrando un patrón arquitectural más

eficiente en la captación de luz. Sin embargo, en el campo la supervivencia de melojos

fue igual o mayor que la de robles albares en un amplio rango de ambientes lumínicos,

probablemente porque la variación de la luz disponible para las plantas estuvo asociada

a la variación de su estado hídrico. La humedad del suelo en verano fue mayor en zonas

aclaradas de pinares de silvestre que en claros, o zonas sin aclarar con una elevada

densidad de pinos, donde además, el desarrollo de las plantas fue escaso. Ambas

circunstancias contribuyeron a que el mayor grado de estrés hídrico de los brinzales se

produjese en las zonas no aclaradas, y a que la supervivencia fuese mayor en las

parcelas establecidas bajo los pinares aclarados. No se detectaron diferencias entre las

dos especies en el estado hídrico, pero los brinzales de melojo siempre mantuvieron

tasas de intercambio gaseoso más altas en todos los ambientes; sus hojas mostraron una

eficiencia en el uso del agua menor que las de roble albar al final del verano.

Los resultados demuestran que las dos especies tienen requerimientos ecológicos

diferentes en la etapa de plántula, y apuntan a una mayor capacidad del melojo para

regenerarse en ambientes submediterráneos.

vii

ABSTRACT

The ecophysiological responses of seedlings to light and water resources can be a useful

tool to approaching their regeneration performance. The present PhD Thesis addresses

the light acclimatory responses of seedlings of sessile oak (Q. petraea) and pyrenean

oak (Q. pyrenaica) to light in a glasshouse, and the role of light in modifying water-

stress responses and initial survival of seedlings planted in sites of contrasting canopy

cover in a sub-Mediterranean mid-mountain field site.

Results at both leaf and plant level under glasshouse conditions pointed to a more

competitive ability to light in seedlings of sessile oak. They had larger leaves with more

specific area and higher nitrogen proportion in photosynthetic components than

pyrenean oak’ leaves, which all support its higher growth in low and high light (5.3 vs

70 % of full sunlight). In high light, seedlings of sessile oak showed a more efficient

pattern of growth in terms of light capturing, with longer internodes and lesser number

of basal sprouts resulting in reduced self-shading compared to pyrenean oak. Survival

was though similar or lower in sessile oak than in pyrenean oak in several plantations

carried out in gaps, clearings and Scots pine stands close to a beech-oak forest in

Montejo de la Sierra (NE Madrid, Spain). This could be so because variation of

understory light availability generated by the distinct canopy-tree densities was related

to the variation of seedlings’ water status. Summer soil moisture was higher in thinned

areas of Scots pine forests than in un-thinned areas, gaps or clearings. Both lower soil

moisture and plant development likely translated into the highest degree of water stress

found in understory seedlings. Survival was highest in thinned areas in all plantations

compared to un-thinned areas, gaps or clearings. There was barely any difference

between the two species in leaf water potentials across plantations, either at dawn or

midday, supporting a similar water-avoidance capacity. However, seedlings of pyrenean

oak had higher foliar gas exchange rates along the summer dates, its leaves being less

efficient in using water at the end of summer, the period of maximum water stress.

The results demonstrate that seedlings’ ecological requirements differ between the

two oaks, pointing to a better ability of Q. pyrenaica to regenerate in sub-Mediterranean

environments.

viii

Introducción

1. INTRODUCCIÓN

1.1. Distribución del roble albar y el melojo

El roble albar (Quercus petraea [Matt.] Liebl.) está ampliamente distribuido por

Europa. Se extiende desde el sur de la Península Escandinava hasta la Cordillera del

Caucaso por el Este y hasta Córcega, Sicilia y la Península Ibérica por el Suroeste. En

España ocupa 45.802 ha. (Maldonado et al. 1998) distribuidas principalmente desde la

Cordillera Pirenaica a la Cantábrica (Figura 1.1); también forma pequeños bosques en

zonas de montaña del centro peninsular (Sierra de Ayllón en el Sistema Central y

Sierras de Valdemeca y la Demanda, y el macizo del Moncayo, en el Sistema Ibérico),

en áreas cuya precipitación media anual supera normalmente los 600 mm y el periodo

seco estival es corto (al menos 150 mm). En general los bosques Ibéricos puros de roble

albar son poco extensos. En las localidades más septentrionales, comparte su área de

distribución con carballos, hayas, robles pubescentes o acebos en el piso montano. Se

halla formando rodales dispersos entre melojos, quejigos, o pinos silvestres en las

localidades más meridionales, donde se encuentra en una situación marginal refugiado

de un macroclima adverso en enclaves localmente húmedos o con mayor profundidad

de suelo (Díaz-Fernández et al. 1995; Costa et al. 1997).

El melojo (Quercus pyrenaica Willd.) tiene una distribución europea más

restringida que el roble albar, desde el Suroeste de Francia al norte de Marruecos. Se

extiende por 948.351 ha. en España (Maldonado et al. 1998) distribuidas principalmente

por los sistemas montañosos que rodean la submeseta norte (Figura 1.1). Se encuentra

sobre todo en laderas entre los 400 y 1500 m de altitud, con una precipitación media

entre 650 y 1200 mm anuales, una sequía estival ausente o poco marcada (entre 50 y

más de 125 mm de precipitación estival), y sustratos descarbonatados (Díaz-Fernández

et al. 1995; Costa et al. 1997). En puntos del Sur y Este peninsular se encuentra en los

enclaves más frescos o con suelos más profundos junto con especies de temperamento

algo más xerófilo como encinas, quejigos y pinos negrales (Sierras de Segura y de la

Batalla y Sierra Morena). En la vertiente septentrional de la cornisa cantábrica forma

pequeñas comunidades de transición entre bosques atlánticos y mediterráneos a baja

altitud. Melojo y roble albar se mezclan sobre todo en las montañas de León y sur de

Cantabria.

1

Introducción

Quercus petraea (Matt.) Liebl.

Quercus pyrenaica Willd.

Figura 1.1. Mapas de distribución española del roble albar y el melojo en situación dominante (verde

oscuro) y no dominante (verde claro), elaborados a partir del 2º y 3º Inventario Forestal Nacional (varios

años) y del Mapa Forestal de Ruiz de la Torre (varios años). Las áreas rodeadas en rojo corresponden a

las regiones de procedencia.

Fuente: INIA-DGB (www.inia.es/gcont/redestem/centrosydep.jsp?idcentro=69&tema=relint).

2

Introducción

Pese a la escasa importancia forestal de las poblaciones aisladas del núcleo

principal de distribución de una y otra especie, éstas constituyen por lo general bosques

de un alto valor ecológico por la diversidad y rareza de especies arbóreas, herbáceas,

liquénicas y briofíticas que congregan. Constituyen además un importante reservorio

genético, pudiendo albergar genotipos mejor adaptados a las condiciones locales que los

que proceden del núcleo de distribución (Díaz-Fernández et al. 1995; Lesica &

Allendorf 1995). Un paso importante para la conservación de estas poblaciones es

estudiar la ecología de las principales especies que las integran e identificar las pautas

de regeneración, con el objeto de establecer prácticas de gestión apropiadas (Díaz-

Fernández et al. 1995; Ajbilou et al. 2006).

Una de las poblaciones de roble albar más meridionales de la Península Ibérica, y

de Europa, la encontramos en la Sierra de Ayllón. Aquí, el roble albar sólo forma

rodales de cierta importancia en el bosque del Hayedo de Montejo, en el NE de la

Comunidad de Madrid (Figura 1.2), donde coexiste con melojos, hayas, serbales de

cazadores, acebos y cerezos. Su presencia se debe al avance post-glacial desde el norte

peninsular y posiblemente al ascenso desde reductos en el Sistema Central a menor

altitud. Su importancia ha suscitado numerosos trabajos de investigación en las últimas

décadas sobre aspectos relacionados con su estructura y dinámica (Pardo 2000), su

diversidad y composición florística (Hernández Bermejo et al. 1983), los ciclos de

nutrientes (Pardo 2000), los procesos de regeneración (Cano 2007), la estructura

genética (Valbuena 2006) y la ecofisiología de las principales especies arbóreas (Aranda

1996).

Figura 1.2. El Hayedo de Montejo se

localiza en el municipio de Montejo de

la Sierra (41º7’ Norte, 3º30’ Oeste),

integrado desde 2005 en la Reserva de l

Biosfera de la “Sierra del Rincón” de la

UNESCO.

Altitude (m)583 - 767767 - 11511151- 15351535 - 19201920 - 2204

MADRID

Montejo de la Sierra

a

3

Introducción

1.2. Ecología del roble albar y el melojo. Sus nichos de regeneración en

la Península Ibérica

El melojo tiene una buena capacidad de regeneración tanto por rebrote como por semilla

en masas de edad avanzada. Se regenera abundantemente bajo los doseles de pinares y

frondosas en muchas localidades de su área de distribución (Barrio et al. 2003; Novo et

al. 2003; Costa et al. 1997), pero también se extiende por zonas desarboladas, gracias

en parte a la posesión de un hábito radicular rizomatoso que favorece su persistencia en

zonas que han sufrido perturbaciones por talas, roturaciones o incendios, o en general el

impacto de procesos erosivos (Muller 1951; Ruiz de la Torre 1971; Calvo et al. 2003).

En las etapas juveniles ya muestra una elevada proporción de biomasa en raíces

(Antúnez et al. 2001). Da paso a encinares, quejigares, o pinares de pino negral o laricio

a medida que la humedad edáfica o ambiental disminuye, mientras que es en general

desplazado por otras especies planocaducifolias cuando la humedad aumenta. Es un

árbol de media luz, aunque los brinzales parecen necesitar bastante luz para su

desarrollo (Ruiz de la Torre 1971). Debido al retraso en la brotación de sus hojas, tiene

un periodo vegetativo corto, que le hace soportar bien la continentalidad; las hojas secas

suelen permanecer en las ramas en los individuos jóvenes.

El roble albar también posee un sistema radicular potente, y una importante

capacidad para rebrotar, aunque menor que la del melojo. De temperamento robusto y

requerimientos intermedios de luz; es algo más exigente en humedad que el melojo,

aunque como en general todas las especies de roble, resiste bien la escasez temporal de

precipitaciones, mejor que otros caducifolios atlánticos como hayas y carballos. Sus

hojas brotan antes que las del melojo.

Hay pocos trabajos que hayan estudiado la regeneración de las dos especies en los

lugares en los que coexisten. Barrio et al. (2003) en poblaciones de Galicia y Cano

(2007) en la población de Montejo de la Sierra, han observado que el regenerado de

roble albar es en general más escaso y está más restringido a entornos algo cubiertos, lo

que apuntaría a una mayor amplitud de nichos de regeneración del melojo, posiblemente

relacionada con un carácter más frugal. Los melojares, y en menor medida los

robledales albares Ibéricos, manifiestan un carácter de transición entre el clima atlántico

y el mediterráneo (Ruiz de la Torre 1971; Costa et al. 1997). No obstante, la

distribución más reducida y meridional del melojo hace pensar que los requerimientos

4

Introducción

ecológicos de estas especies difieran en alguna de sus etapas de desarrollo,

probablemente, en la capacidad para tolerar la sequía estival en las fases juveniles. Sin

embargo, hay pocos estudios que hayan examinado sus estrategias funcionales ante las

limitaciones hídricas (Aranda et al. 1996 y 2004a). Tampoco se conoce el efecto que

tiene el ambiente de crecimiento en la resistencia a la sequía en la fase de plántula en

zonas de clima Mediterráneo, ni la capacidad para tolerar la sombra o el exceso de

radiación, aspectos sobre los que se centra la presente tesis, y que son de gran

importancia para esclarecer tanto sus requerimientos ecológicos como sus pautas de

regeneración.

1.3. La ecofisiología de brinzales como herramienta para evaluar las

posibilidades de regeneración de las especies

La regeneración puede considerarse como un proceso cíclico en equilibrio con multitud

de factores de regulación bióticos y abióticos que interactúan a lo largo de sus distintas

fases secuenciales (Jordano et al. 2002). En general, los problemas de regeneración de

las especies vegetales surgen cuando las limitaciones que afectan al aporte de semillas

(problemas de floración, polinización o fructificación) o al establecimiento de plántulas

(consumo de semillas, herbivoría o estrés ambiental), sobrepasan la capacidad de

resiliencia del ciclo demográfico. La distribución de las especies a distintas escalas

territoriales depende en parte de las características ecológicas, resultantes de la

selección de diversos atributos funcionales, morfológicos y reproductivos que tiene

lugar a lo largo de estas etapas.

La fase de plántula es un filtro importante en la regeneración porque los

individuos no han desarrollado aún algunos de los mecanismos de resistencia que tienen

los individuos adultos, o lo han hecho en menor medida, lo que les hace especialmente

susceptibles a ciertas situaciones ambientales. Por ello, aunque en ocasiones, las

respuestas de los individuos adultos difieren de las que muestran en los primeros años

(Donovan & Pappert 1998; Cavender-Bares & Bazzaz 2000; Mediavilla & Escudero

2003a), la comparación de los rasgos fenotípicos de brinzales de varias especies en

condiciones óptimas o en respuesta a factores de estrés, permite clarificar cuáles son sus

requerimientos ecológicos en las etapas iniciales de desarrollo e inferir sus posibilidades

de regeneración en determinados ambientes, incluso en el caso de especies forestales

5

Introducción

longevas (e.g. Ashton & Berlyn 1994). Siendo la disponibilidad de agua y luz dos de los

factores que más influyen en el desarrollo de las plántulas en condiciones

mediterráneas, sus respuestas de aclimatación determinarán en buena medida la

capacidad específica para establecerse (Gómez-Aparicio et al. 2006) y, por tanto, son

relevantes para la planificación de actuaciones de gestión forestal, como por ejemplo las

relacionadas con las estrategias de manejo del bosque, y las técnicas y los lugares de

reforestación.

1.4. El comportamiento de las especies frente a la disponibilidad de

recursos: adaptación, aclimatación y plasticidad

La respuesta a los recursos, entendida de modo genérico como la modificación

fenotípica derivada de procesos de aclimatación a largo plazo (plasticidad) o a corto

plazo, está inversamente relacionada con el grado de adaptación del genotipo al

ambiente. Por ejemplo, las especies que completan su ciclo reproductivo bajo el dosel

arbóreo, las que persisten durante años en sotobosques muy umbríos (Strauss-

Debenedetti & Bazzaz 1991; Valladares 2000a), o igualmente, aquellas que se

regeneran en sitios abiertos bien iluminados (Ernstsen et al. 1997), muestran una baja

capacidad para modular el aparato fotosintético y la arquitectura de la planta en

respuesta a la variación de la luz. De igual forma cabe también esperar que la capacidad

de respuesta sea relativamente baja en taxones mediterráneos, con un alto grado de

canalización fenotípica como consecuencia de su adaptación a la escasez de recursos,

principalmente agua y nutrientes (Valladares et al. 2000b). La plasticidad del fenotipo

ante la luz abarca desde los cambios que se producen en la morfología, fisiología y

filotaxis de las hojas, a los que afectan a la forma y tasa de crecimiento, y la distribución

de carbohidratos entre los órganos. Si la selección no actúa por igual sobre todos los

caracteres fenotípicos de la planta cabe esperar que la plasticidad se exprese de manera

distinta según los niveles de organización. Por ejemplo, la plasticidad fisiológica parece

estar más relacionada con la capacidad para resistir la luz de alta intensidad que la

plasticidad de los caracteres anatómicos o morfológicos (Valladares et al. 2002).

Asumiendo que existe un fuerte componente genético, las hojas formadas en

ambientes con alta intensidad lumínica son en general más gruesas y tienen una

capacidad fotosintética superior a la de las plantas sombreadas (Givnish 1988). En

6

Introducción

algunas plantas mediterráneas se observa que su disposición en el tallo, a diferencia de

las plantas que crecen en sombra, no favorece tanto la captación de luz cuanto la

minimización de la radiación de alta intensidad (Valladares & Pearcy 1998), de manera

que su forma y fisiología, varían incluso en un mismo individuo según el grado de

exposición. La sombra produce en cambio modificaciones en la morfología y fisiología

de las hojas (grosor, superficie específica, tasa de respiración, concentración de

pigmentos, etc.) y la arquitectura de las plantas (principalmente en el reparto de biomasa

en tejidos fotosintéticos y no fotosintéticos) encaminadas a optimizar la captación de luz

(Givnish 1988).

Además, las plantas responden rápidamente a las continuas variaciones de la

intensidad de luz que tienen lugar a diario (nubes, movimiento de hojas por el viento,

etc.) a través de la regulación de los procesos fotosintéticos (Ort 2001). También

reajustan sus características fisiológicas (posición de los cloroplastos, degradación de

proteínas, translocación de nitrógeno, etc.) y morfológicas (espesor del mesófilo, masa

por unidad de área foliar, redistribución de biomasa, producción de hojas nuevas, etc.)

ante un cambio permanente en el régimen de luz. Los mecanismos y la magnitud de los

cambios implicados varían según las estrategias adaptativas de las especies (Walters

2005).

Las clasificaciones selvícolas de tolerancia a la sombra de muchas especies

forestales están basadas en la observación in situ de su capacidad para establecerse (i.e.

sobrevivir y crecer moderadamente) bajo la cubierta del bosque. La modificación

experimental de los niveles de radiación ha ayudado a individualizar las respuestas a la

luz y a esclarecer los procesos que las gobiernan. Hoy sabemos que la tolerancia a la

sombra se relaciona tanto con los mecanismos que maximizan la captación de luz y la

acumulación de biomasa, como con aquellos que maximizan la resistencia a las

perturbaciones. Se puede afirmar que una especie que mantuviera una estrategia

conservadora en sombra, es decir, profusa acumulación de sustancias de reserva, bajas

tasas de crecimiento en altura y bajas tasas metabólicas, y que tuviera hojas grandes y

delgadas con una importante proporción de nitrógeno en pigmentos y compuestos

tóxicos para los herbívoros, sería muy tolerante (Givnish 1988). Sin embargo, en

algunos aspectos ambas estrategias son contrapuestas. Por ejemplo, la inversión de

biomasa en raíces, que favorece la aparición de nuevos brotes tras la destrucción de la

parte aérea, reduce la que puede destinarse a las hojas, y por tanto a captar luz; al igual

7

Introducción

que la acumulación de alcaloides en las hojas podría repercutir negativamente en la

síntesis de pigmentos fotosintéticos. A pesar de que se han realizado numerosos

estudios para esclarecer las cualidades que confieren mayor tolerancia a la sombra, no

existe aún una idea clara al respecto, posiblemente, porque no exista un síndrome único

de tolerancia, sino varios, dependientes del grado de adaptación a otros factores como la

sequía o la presión de herbívoros (Sánchez-Gómez et al. 2006a; Niinemets &

Valladares 2006), y el grado de desarrollo ontogénico de los individuos (Niinemets

2006).

De igual modo no existe una respuesta única a la sequía. En general se distinguen

dos estrategias de resistencia no necesariamente excluyentes: la evitación y la tolerancia

(Valladares et al. 2004). En el primer caso, las plantas ponen en funcionamiento

mecanismos destinados a minimizar la desecación de los tejidos. El caso extremo de

esta estrategia adaptativa lo encontramos en las plantas que completan su ciclo

biológico en ausencia de estrés hídrico. En el caso de las especies leñosas, aquellas con

potentes sistemas radicales, capaces de extraer agua de horizontes profundos y

húmedos, se ven menos afectadas por la reducción de las precipitaciones que las que

poseen sistemas radicales más someros. En este sentido, algunas especies deciduas

mediterráneas, como es el caso del melojo, completan el desarrollo foliar en pocos días

antes de la disminución de las lluvias (Mediavilla & Escudero 2003b). La sensibilidad

de los estomas al déficit de presión de vapor del aire o a la desecación del suelo también

influye en el mantenimiento del estado hídrico, de manera que el cierre de los estomas

evita, o al menos reduce, la desecación de los tejidos. Por otro lado, las especies

adaptadas a fenómenos recurrentes de sequía, pueden desarrollar mecanismos que les

permiten mantener su actividad funcional en circunstancias de estrés hídrico; su

estrategia no radica tanto en evitar la sequía, como en tolerarla. Por ejemplo, sus hojas

son capaces de mantener la turgencia celular, la apertura estomática, la asimilación de

CO2 o la estabilidad de las membranas fotoquímicas, incluso cuando el potencial hídrico

foliar es bajo (Valladares et al. 2004).

8

Introducción

1.5. Compromisos en los patrones de aclimatación a ambientes

sombreados y secos

La heterogeneidad del dosel vegetal genera diferencias importantes en las condiciones

microclimáticas del sotobosque a distintas escalas espaciales y temporales (Martínez

Alonso 2007). Las plantas que crecen en ambientes umbríos reciben luz de menor

intensidad y durante menos tiempo que las que crecen en claros o zonas abiertas, lo que

modifica muchas de las características estructurales y funcionales del regenerado

(Montgomery 2004). Sin embargo, el grado de cobertura conlleva la variación de

diversos factores ambientales además de la luz, como la temperatura y la humedad

relativa del aire, la temperatura y el contenido hídrico del suelo, o su estructura y

propiedades físico-químicas (Aussenac 2000).

Dejando a un lado las variaciones edáficas y climáticas a pequeña escala, en

condiciones naturales se generan gradientes espaciales de disponibilidad de luz y agua

que pueden variar estacionalmente. De acuerdo con la teoría del reparto de los nichos

ecológicos, especies coexistentes en un territorio se repartirán estos espacios según la

idoneidad para sus requerimientos ecológicos (Silvertown 2004). Simplificando, las

especies más competitivas ocuparían las zonas con más recursos, mientras que las

especies con mayor capacidad para tolerar la sombra o la sequía ocuparían las zonas

donde estos factores fuesen limitantes. Sin embargo, la tolerancia a la combinación de

factores de estrés es un fenómeno raro en la naturaleza. Es decir, existen pocas especies

cuya estrategia adaptativa les permita tolerar la sequía y la sombra intensa al mismo

tiempo (Niinemets & Valladares 2006), del mismo modo que la aclimatación del

fenotipo a un extremo ambiental parece limitar la posibilidad de aclimatación a otros

factores ambientales (Smith & Huston 1989; Kubiske et al. 1996). La interacción entre

la luz y la humedad del suelo en el sotobosque es uno de los aspectos que actualmente

suscita más interés en los estudios de regeneración. Aunque la luz es un factor clave en

la regulación de los procesos de sucesión de especies en bosques templados (Kobe &

Coates 1997) y mediterráneos (Gómez-Aparicio et al. 2006), el establecimiento de los

brinzales raramente depende de un único factor (Sack 2004). La tolerancia a la sombra

puede quedar en un segundo plano en ambientes donde haya una reducción periódica de

las precipitaciones, como sucede en el sotobosque de la mayoría de ecosistemas

forestales Mediterráneos.

9

Introducción

La supervivencia de los brinzales en la sombra se puede ver comprometida por el

hecho de tenerse que enfrentar a una situación de estrés hídrico, que reduce la

asimilación de CO2 y altera el balance de carbono (Aranda et al. 2004b; Sánchez-

Gómez et al. 2006b). En el sentido opuesto, existen también evidencias de que la

sombra intensa limita la resistencia a la sequía. El retraso en el desarrollo de los

individuos o la inversión preferencial de biomasa en follaje que genera la escasez de

luz, pueden mermar la captación de agua y agravar el estrés hídrico de las plantas con

respecto a zonas más iluminadas donde la evapotranspiración es mayor (Valladares &

Pearcy 2002). La luz juega también un papel importante en la evitación del estrés

hídrico al influir en la regulación estomática y en el flujo de agua. Por un lado, la mayor

sensibilidad de los estomas a las variaciones del déficit de presión de vapor o del

potencial hídrico del suelo que muestran las plantas que crecen en ambientes bien

iluminados permite un mayor control de las pérdidas de agua por transpiración que las

que crecen en situaciones umbrías (Mendes et al. 2001); por otro, se ha puesto de

manifiesto el aumento de la conductancia hidráulica con la luz en especies de diversa

ecología (Barigah et al. 2006). Además, ciertas características foliares relacionadas con

el mantenimiento de la turgencia celular en respuesta a la desecación de los tejidos,

como son la concentración de solutos o la elasticidad de las paredes celulares, varían

con la disponibilidad de luz (Aranda et al. 2001). Así, la disminución del potencial

osmótico foliar a través de la acumulación activa de solutos depende en parte de la

síntesis de azúcares solubles, (Epron & Dreyer 1996) por lo que los individuos que

crecen en ambientes sombreados presentan menor grado de ajuste osmótico en respuesta

a la sequía que aquellos que crecen sin limitaciones lumínicas para la producción de

carbohidratos (Morgan 1984).

Entender los procesos morfo-funcionales que median las respuestas de

aclimatación a la luz y la sequía resultará útil para favorecer los procesos de

regeneración natural y artificial de las especies vegetales en el futuro. Hoy en día es una

cuestión con muchas incógnitas.

10

Introducción

1.6. La interacción entre la luz y el agua en los procesos de

regeneración

Las actuaciones de regeneración por repoblación en España se han realizado

tradicionalmente en lugares desarbolados previamente desbrozados de matorral para

evitar la competencia por los nutrientes y el agua, siguiendo en gran medida pautas

basadas en la selvicultura europea. En la actualidad hay numerosas evidencias de que el

dosel que forman los matorrales favorece la supervivencia inicial del regenerado natural

y artificial de diversas especies leñosas mediterráneas debido a la atenuación del estrés

ambiental (Castro et al. 2004; Gómez-Aparicio et al. 2004 y 2005). Además de tener

efectos positivos sobre la estructura y la composición de nutrientes del suelo, al

reducirse la radiación, se atenúa la demanda evaporativa del aire y la

evapotranspiración, favoreciéndose la conservación de la humedad del suelo en los

meses estivales, y reduciéndose el estrés post-transplante en el caso de plantaciones.

El signo de la interacción entre plantas parece variar según las condiciones

climáticas y edáficas del medio, de manera que a medida que el estrés ambiental

disminuye, la competencia por los recursos adquiere mayor importancia (Holmgren et

al. 1997; ver sin embargo Maestre et al. 2005). El resultado de las interacciones entre

plantas depende también de las especies de que se trate. Así, el papel protector de la

vegetación establecida sobre el regenerado podría ser especialmente beneficioso para el

establecimiento de aquellas especies más susceptibles al estrés hídrico y a las altas

temperaturas que se registran durante el verano. En este sentido, especies extendidas

ampliamente por centroeuropa se regeneran en las proximidades de matas, arbustos o

bosques abiertos en las poblaciones Ibéricas que constituyen su límite de distribución,

incluso en el caso de especies heliófilas o con altos requerimientos de luz para

regenerarse en su núcleo de distribución, como se ha puesto de manifiesto para el pino

silvestre (Rojo et al. 1994; Castro et al. 2004), o el pino negro, una especie subalpina

(Camarero et al. 2005).

1.7. El papel de los pinares en la expansión de los robles en el entorno

del Hayedo de Montejo de la Sierra

La conservación de la población actual de roble albar en el Hayedo de Montejo requiere

su extensión por medio de siembras, plantaciones u otras actuaciones selvícolas que

11

Introducción

favorezcan su regeneración natural. Pese a la creciente diversificación de las especies

empleadas en las actuaciones de repoblación, promovida sobre todo por el impulso de la

forestación de antiguos terrenos agrícolas, existen pocas experiencias con robles

planocaducifolios en España que ayuden a adoptar un plan de actuación. Debido a su

temperamento exigente se han obtenido peores resultados en zonas desprovistas de

vegetación que en plantaciones al amparo de matorral (Gómez-Aparicio et al. 2004). La

utilización de masas forestales para la plantación de especies es igualmente una buena

opción de regeneración en muchos lugares (Dey & Parker 1996; Paquette et al. 2006),

pero apenas se ha valorado en zonas Mediterráneas (Álvarez Linarejos et al. 1996).

Una práctica frecuente es la utilización de masas forestales repobladas con

coníferas como dosel “nodriza”, con el objeto, además, de diversificar su composición y

acelerar la dinámica natural de sucesión del bosque. Durante el siglo pasado se llevaron

a cabo numerosos programas de reforestación en zonas degradadas en las que se

plantaban habitualmente especies de pino (Pausas et al. 2004; Pardo & Gil 2005), más

frugales y resistentes a la sequía estival que los robles, en un primer paso para restaurar

la cubierta arbórea destruida sobre todo por la intensa acción humana (Grove &

Rackham 2001). El propósito final era en algunos casos la transformación de estas

masas en bosques mixtos o bosques caducifolios (Ceballos 1939), lo que habitualmente

no ha podido completarse debido a la falta de tiempo o de medios materiales. Existen

aún millones de hectáreas de pinar que se encuentran en la primera rotación cuya

diversificación tendría repercusiones ecológicas y económicas positivas.

Los extensos pinares de Pinus sylvestris que existen en las inmediaciones del

Hayedo de Montejo (Fotografía 1.1) pueden ser lugares adecuados para la repoblación

de robles al generar unas condiciones microclimáticas más favorables para las plantas

que las que existen en zonas abiertas, y haber favorecido el mantenimiento y posterior

desarrollo del suelo con respecto a zonas que quedaron sin repoblar.

Bajo esta perspectiva se ha estudiado profusamente el efecto de la densidad del

dosel arbóreo en el desarrollo de los individuos, especialmente en bosques templados.

En una revisión reciente se ha puesto de manifiesto que la reducción de la densidad

inicial a través de claras es positiva para el establecimiento de los brinzales en un

amplio rango de biomas debido al aumento asociado en la disponibilidad de recursos

(luz, agua y nutrientes) (Paquete et al. 2006). Densidades altas generan ambientes

excesivamente umbríos, donde la mineralización de la materia orgánica puede hacerse

12

Introducción

más lenta; además, la intercepción de las precipitaciones por parte de las copas de los

árboles y la hojarasca, así como el “drenaje” de agua a la atmósfera por las raíces de los

árboles adultos, pueden reducir la disponibilidad de agua en el suelo durante el verano

con respecto a zonas que han sido aclaradas, lo que se traduce en un mayor grado de

estrés hídrico en los brinzales (Kozlowski et al. 1991).

a

bc

Fotografía 1.1. Vista panorámica del Hayedo con numerosas repoblaciones de pinos en sus proximidades

llevadas a cabo en los años 50 y 60. Autor: Antonio López Santalla. En amarillo se indican las zonas

donde se realizaron plantaciones con robles albares y melojos en distintos años (ver Material y métodos).

No obstante, la peculiaridad del clima Mediterráneo impone una selvicultura

propia. Las escasas experiencias en el ámbito nacional avalan la mejora de los

resultados en zonas aclaradas (Álvarez Linarejos et al. 1996), pero aún existen pocos

trabajos que hayan examinado el efecto de la densidad del dosel arbóreo en las

características ambientales del sotobosque o en las características funcionales del

regenerado (Aranda et al. 2001, 2002 y 2004b; Bellot et al. 2004). Debido a la

marginalidad de la población del roble albar en el Hayedo de Montejo, al probable

aumento de la aridez en los próximos años (INM 2007), y al notable abandono de

muchas masas forestales repobladas, es necesario fomentar la investigación sobre estas

cuestiones.

Dentro de este contexto, se han realizado varios experimentos en los que se ha

examinado la capacidad de aclimatación a la luz en condiciones controladas y en

gradientes naturales, y se ha comparado la capacidad para establecerse en distintos

ambientes a través del estudio de las respuestas fisiológicas a la sequía estival y de la

13

Introducción

supervivencia en diversas plantaciones experimentales. Esta información puede resultar

muy útil para precisar las posibilidades de regeneración del roble albar en poblaciones

marginales mediterráneas, y establecer planes de gestión encaminados a su

conservación.

A partir de los antecedentes expuestos se plantearon como hipótesis generales de

trabajo (i) que los brinzales de roble albar presentan rasgos morfológicos y funcionales

menos adecuados para regenerarse en ambientes mediterráneos que el melojo, y (ii), que

la reducción parcial del dosel genera un ambiente de media sombra más propicio para

los brinzales que los lugares con mayor densidad de arbolado o aquellos plenamente

expuestos a la luz.

14

Objetivos

2. OBJETIVOS

Los objetivos de la presente tesis son los siguientes:

Comparar las características que muestran los brinzales de las dos especies bajo

las mismas condiciones de crecimiento con el fin de valorar la idoneidad para

regenerarse en poblaciones donde coexistan.

Comparar las respuestas de aclimatación a la luz a largo y a corto plazo, al nivel

foliar y de la planta entera con el fin de estimar la capacidad competitiva y el

grado de tolerancia a la sombra.

Examinar el comportamiento de las dos especies frente a la sequía estival en

diferentes lugares de plantación, evaluando el papel de la disponibilidad de luz

asociada al grado de cobertura arbórea en la modificación de las respuestas de

aclimatación a la sequía, y la influencia en la supervivencia durante los primeros

años.

15

Material y métodos

3. MATERIAL Y MÉTODOS

Se realizaron varios experimentos con brinzales de roble albar y melojo procedentes de

bellotas recogidas en el Hayedo de Montejo. Por un lado, brinzales cultivados en

condiciones de riego y fertilización idóneas en un invernadero se sometieron a la

variación experimental de la intensidad de luz (sección 3.1.). Por otro, se realizaron

cuatro plantaciones a lo largo de varios años (2000 a 2005) en claros y pinares de pino

silvestre adultos sometidos a claras de distinto peso (0, 25, 33 y 50 % de la densidad

original), cercanos al Hayedo de Montejo (sección 3.2.).

3.1. Experimento en invernadero (anexos I y II)

3.1.1. Material vegetal y diseño experimental

En otoño de 2003 se recogieron bellotas de varios árboles de roble albar y melojo. Se

conservaron en cámaras frigoríficas a 3 ºC de temperatura y 55 % de humedad relativa

hasta la primavera del año siguiente. En este momento, bellotas de tamaño similar se

sembraron en envases Super-Leach de 400 cm3 y 35 cm de profundidad que contenían

una mezcla de turba y arena (3:1, v/v) enriquecida con abono de liberación lenta (5 g l-

1). Las plántulas se cultivaron en un invernadero bajo dos niveles de luz (5,3 % de plena

luz, debido a una doble capa de malla negra y 70 % de plena luz, debido a la opacidad

de los paneles de PVC del invernadero). Al final del primer año, se transplantaron 80

brinzales de tamaño similar a envases de 3000 cm3 y 40 cm de profundidad, añadiendo

de nuevo abono de liberación lenta. Veinte individuos de cada especie se mantuvieron

en sombra (tratamiento de baja irradiancia, 5,3 %, SH), distribuidos en dos bancadas del

invernadero bajo cuatro estructuras metálicas; 10 individuos de cada especie se

mantuvieron entre medias (tratamiento de alta irradiancia, 70 %, HL), repartidos en las

dos bancadas del invernadero y suficientemente alejadas de la malla de sombreo. Antes

de que se produjera la brotación de las hojas (primera semana de marzo en todas las

plantas), seis individuos por especie se sacaron fuera de las estructuras de sombreo

junto con las del tratamiento de 70 % de luz (tratamiento T1). Unos noventa días

después de la brotación se movieron otras seis plantas por especie de sombra junto con

las plantas HL y T1 (tratamiento T2); aunque no se examinó la evolución de la

17

Material y métodos

expansión foliar, cabe esperar que las hojas estuvieran completamente expandidas en el

momento de la transferencia.

Los valores medios de luz fotosintéticamente activa (PAR) en un día despejado a

mediodía fueron 80 ± 7 μmol m-2 s-1 para SH y 1050 ± 28 μmol m-2 s-1 para el resto de

tratamientos (LICOR Li-185B; sensor cuántico: Li-190SB). La temperatura a mediodía

fue algo menor bajo la malla de sombreo (31,6 ± 0,2 ºC vs 33,7 ± 0,3 ºC). Todas las

plantas se regaron adecuadamente durante el transcurso del experimento. La

temperatura y la humedad relativa del aire dentro del invernadero fluctuaron entre 15 y

39 ºC y 50 – 90 %, respectivamente.

Fotografía 3.1. Disposición de las plantas en el

invernadero antes de sacar la segunda tanda de

plantas de las estructuras de sombreo.

Se midieron diversos parámetros relacionados con la fisiología y la morfología de

las hojas, así como con el crecimiento y la distribución de biomasa. Las medidas se

realizaron en plantas de dos savias, en hojas de la primera brotación completamente

expandidas y poco sombreadas por el resto de hojas de la planta.

3.1.2. Intercambio gaseoso foliar

Las medidas se realizaron con un equipo portátil de análisis de gases por infrarrojos

(IRGA, LCPro Analytical Development Corporation, UK).

Se examinó la evolución de las tasas de fotosíntesis (Asat) y conductancia

estomática (gsat) a saturación por luz (1000 μmol m-2 s-1 en HL y 700 μmol m-2 s-1 en

SH y T2) y CO2 ambiental (370 ppm) durante las seis semanas siguientes a la

transferencia de plantas T2, repitiendo las medidas sobre la misma hoja cuando fue

posible. Antes de su medición, las plantas de sombra se expusieron a luz de alta

18

Material y métodos

intensidad durante 10 – 15 min para inducir la apertura de estomas. Se utilizó un diodo

emisor de luz roja – azul como fuente de iluminación de las hojas. La temperatura en el

interior de la cámara se mantuvo entre 23,5 y 26,6 ºC, lo que produjo que la temperatura

foliar oscilara en el momento de la medición entre 23,8 y 28,5 ºC. Las medidas se

prolongaron desde las 8:00 a.m. hasta las 11:00 a.m.

Por otro lado se realizaron curvas de respuesta de la fotosíntesis a la concentración

de CO2 en 4 – 5 plantas por especie y tratamiento (HL, SH, T1 y T2), aproximadamente

un mes después de la segunda transferencia. Las curvas se realizaron a luz constante

(1000 μmol m-2 s-1 en HL y 700 μmol m-2 s-1 en SH, T1 y T2) y temperatura constante

(25,8 ± 0,1 ºC). Después de exponer la hoja durante 30 min a 380 ppm, se redujo la

concentración de CO2 en el flujo de aire de entrada en cuatro pasos (250, 200, 100 y 50

ppm), y a continuación, se incrementó en otros siete hasta 1800 ppm (380, 450, 700,

950, 1200, 1500 y 1800 ppm). Se estimó la capacidad fotosintética de cada planta

(Aamax) como el valor medio de las tres medidas realizadas a 1800 ppm. Se estimaron

también la tasa máxima de carboxilación (Vacmax) y la tasa máxima de transporte

electrónico (Jamax) ajustando las ecuaciones de Harley et al. (1992) a las fracciones de la

curva donde la tasa de fotosíntesis está limitada por la cantidad y grado de activación de

la enzima Rubisco (concentración intercelular de CO2 < 220 - 230 ppm; Ac) y por la

regeneración de la Ribulosa 1,5-bis fosfato (Aj):

)K/O(KCC

VAoci

*i

maxcc +⋅+Γ−

=1

, ⋅τ+⋅

Γ−=

)/(4

*

OCC

JAi

ij .

Se estableció una concentración de oxígeno en el estroma (O) de 20 KPa. Los

coeficientes de afinidad de la Rubisco por el CO2 (Kc) y el oxigeno (Ko), ambos

dependientes de la temperatura, se calcularon para la temperatura de cada hoja de

acuerdo a las ecuaciones de Bernacchi et al. (2001); el valor del factor de especificidad

de la Rubisco (τ) se ajustó a la temperatura de cada hoja siguiendo la ecuación de

Harley et al. (1992). El punto de compensación de CO2 en condiciones de iluminación

(Γ*) se calculó como O/2τ. El potencial de transporte electrónico (J) se calculó como:

19

Material y métodos

2

1 ⎟⎠⎞

⎜⎝⎛ ⋅α

+

⋅α=

maxa

sat

sat

JPPFD

PPFDJ ,

utilizándose un valor constante de 0,24 mol e- (mol quanta-1) como valor de la eficiencia

de las hojas en la conversión de luz (α).

Los valores de Vacmax y Ja

max se ajustaron a una temperatura de referencia de 25 ºC

a partir de las ecuaciones de Dreyer et al. (2001). Las tasas máximas de carboxilación

(Vmcmax) y transporte electrónico (Jm

max) por unidad de masa foliar, y la capacidad

fotosintética por unidad de masa foliar (Ammax), se estimaron a partir de los valores de

superficie foliar específica (SLA).

Ci (Pa)

An

(μm

olm

-2s-1

)

0 30 60 90 120 1500

10

20

30

40

50

Ac

Aj

Figura 3.1. Ejemplo de una curva de respuesta de la

fotosíntesis (An) a la concentración intercelular de C

(C

O2

i) en una planta de melojo del tratamiento T2.

3.1.3. Fluorescencia de la clorofila a

Se midió con un fluorómetro portátil de pulso modulado (FMS 2, Hansatech

Instruments LTD., UK). Para la nomenclatura y el cálculo de la mayoría de parámetros

se siguió a Maxwell & Johnson (2000).

Se examinó la evolución de algunos parámetros relacionados con la emisión de

fluorescencia de la clorofila durante las seis semanas siguientes a la transferencia de

plantas del tratamiento T2. Las medidas se repitieron sobre la misma hoja siempre que

fue posible. A partir de la fluorescencia máxima (Fm) y la fluorescencia mínima (Fo) de

hojas previamente oscurecidas, se calculó la eficiencia fotoquímica potencial del

fotosistema II (PS II) que indica el rendimiento cuántico si todos los centros de reacción

del PS II estuvieran abiertos (oxidados):

20

Material y métodos

m

ommv F

FFFF

−=/ .

Fm se obtuvo tras someter la hoja a un pulso de 0,8 s de luz de alta intensidad (6600

μmol m-2 s-1). Las medidas se llevaron a cabo a primera hora de la mañana (antes de las

8:00 a.m.) en hojas a las que se les había colocado la tarde anterior una pinza que cubría

una parte de la hoja, por lo que se puede considerar que indican el máximo valor diario

de Fv/Fm. Al finalizar las medidas de intercambio gaseoso, se estimó la eficiencia

fotoquímica efectiva (ΦPSII), es decir, la eficiencia en hojas aclimatadas a la luz

ambiental:

,'

'

m

smPSII F

FF −=φ

donde Fs es la fluorescencia emitida por la hoja y Fm’ es la fluorescencia en el momento

de ser de nuevo sometida a un pulso de luz saturante. Dado que las mediciones en

condiciones de oscuridad e iluminación se hicieron en la misma zona de la hoja, se pudo

estimar la disipación no-fotoquímica de la luz absorbida (NPQ):

.'

'

m

mm

FFF

NPQ−

=

Por otra parte, se realizaron curvas de respuesta de la fluorescencia de la clorofila

a la luz en 4 – 5 plantas por especie y tratamiento (HL, SH, T1 y T2), aproximadamente

un mes después de la segunda transferencia (3 – 7 de julio). Se realizaron en una cámara

de control climático a 23,5 ºC de temperatura y 65 % de humedad relativa. La

concentración de CO2 no pudo mantenerse en los niveles programados (380 ppm)

debido al aumento proveniente de la respiración de la persona en el interior de la

cámara. Antes de comenzar la curva se midió el parámetro Fv/Fm de acuerdo al

protocolo mencionado anteriormente. La misma superficie foliar fue entonces sometida

a valores crecientes de luz, emitida por una lámpara halógena dentro del equipo (PPFD;

40, 100, 250, 410, 710, 1060, 1400 μmol m-2 s-1). Cuando la señal de fluorescencia de la

21

Material y métodos

hoja alcanzaba un valor relativamente estable (Fs), la hoja era sometida a un pulso de

luz saturante, obteniéndose el valor de Fm’. Inmediatamente después la hoja se

iluminaba con un pulso de luz del espectro en el rojo-lejano de 5 s de duración para

favorecer el paso de electrones del PSII al PSI, y obtener así la fluorescencia mínima en

condiciones de iluminación (Fo’); a continuación se incrementaba la luz al siguiente

nivel de intensidad. En cada punto se obtuvieron la eficiencia fotoquímica efectiva

(ΦPSII), la tasa de transporte electrónico (ETR):

,84,05,0 PSIIPPFDETR φ×××=

y la disipación fotoquímica (qL) (según Kramer et al. 2004):

s

o

om

sm

FF

FFFF

qL'

'''

×−−

= .

Además, se calculó qué proporción de la energía que no se disipa por vía fotoquímica lo

hace de forma regulada (ΦNPQ) y no regulada por la planta (ΦNO) (Kramer et al. 2004).

La tasa máxima de transporte electrónico (ETRmax) se obtuvo ajustando un modelo

exponencial simple a la evolución de ETR con la luz.

3.1.4. Parámetros bioquímicos de la hoja

Se determinaron las concentraciones de clorofila (Cm) y nitrógeno (Nm) en hojas de 4-5

plantas por especie y tratamiento (HL, SH, T1 y T2), en varios momentos desde la

segunda transferencia. La clorofila se extrajo de discos foliares inmersos en tubos con 5

ml de Dimetil Sulfóxido (DMSO) durante 5 h a 60 ºC en oscuridad. La absorbancia del

DMSO se midió entonces con un espectrofotómetro a 648,2 y 664,9 nm de longitud de

onda, estimando la concentración de clorofila a y b en las hojas. La concentración de

nitrógeno se midió siguiendo el protocolo Kjeldahl. Brevemente, las muestras (~ 0,15

mg de hoja sin pecíolo) se sometieron a una digestión ácida con H2SO4 al 96 % a 400 ºC

durante aproximadamente 1,5 h (1016 Digestion System 12, Tecator, Sweden).

Posteriormente se destilaron (1026 Distilling Unit, Tecator, Sweden) en un medio

22

Material y métodos

básico (NAOH al 40 %), y por último se procedió a valorar con HCL 0,05 N el

destilado recogido en una solución de H3BO3 con dos indicadores de pH.

Los contenidos de clorofila (Ca) y nitrógeno (Na) por unidad de área foliar se

estimaron a partir de Cm y Nm, y los valores de SLA. El cociente entre Ammax y Nm se

empleó como indicador de la eficiencia en el uso del nitrógeno (EUN).

Se estimaron también las fracciones de nitrógeno en Rubisco (Pr), proteínas de la

cadena de transporte electrónico (Pb) y pigmentos captadores de luz (Pl) de acuerdo a las

ecuaciones de Niinemets & Tenhunen (1997):

m

cr NSLA

VP

×××=

)/1(5,2025,6max ;

siendo, 6,25 g Rubisco (g N en Rubisco)-1 un factor de conversión del contenido de

nitrógeno a cantidad de proteína y 20,5 μmol CO2 (g Rubisco)-1 s-1 el factor de

especificidad de la Rubisco;

mb NSLA

JP

×××=

)/1(15606,8max ;

siendo, 8,06 μmol citocromo f (g N en componentes del transporte electrónico)-1 un

factor de conversión y 156 mol e- (mol citocromo f)-1 s-1 el factor de actividad de

transporte electrónico por unidad de citocromo f;

Bm

ml CN

CP

×= ,

donde CB es la media ponderada de la cantidad de clorofila por cantidad de nitrógeno

que hay en los fotosistemas (PS II y PS I) y las antenas del PSII (LHC II). La

concentración de cada complejo enzimático por unidad de área y la proporción de

clorofila en cada complejo enzimático con respecto a la concentración total, se

calcularon de acuerdo a Hikosaka & Terashima (1995).

23

Material y métodos

La proporción de nitrógeno estructural se obtuvo a partir de las fracciones en los

anteriores componentes (Ps = 1– Pr – Pb – Pl).

3.1.5. Parámetros anatómicos y morfológicos de la hoja

Un mes después de la segunda transferencia, se introdujeron fragmentos de la zona

media de la hoja de 5 plantas por especie y tratamiento (HL, SH, T1 y T2) en viales que

contenían 1 ml de formaldehído al 37 %, 1 ml de ácido acético y 9 ml de etanol (70º).

Se mantuvieron en esta solución durante 24 h y después se sustituyó por etanol (70º)

para la conservación del material hasta su análisis. Se obtuvieron secciones de la

porción de la hoja comprendida entre el margen y el nervio medio central, midiéndose

los grosores de la epidermis adaxial y abaxial, el parénquima en empalizada y el

parénquima esponjoso. La densidad estomática se midió en las mismas hojas a partir de

huellas de la epidermis abaxial obtenidas tras aplicar una capa de esmalte.

La superficie foliar específica (SLA) se calculó dividiendo el área de un disco

foliar por su peso seco (48 h a 70 ºC) en 5 plantas por especie y tratamiento (HL, SH,

T1 y T2). El tamaño medio de la hoja se obtuvo dividiendo la superficie foliar total por

el número de hojas dos meses después de la segunda transferencia, sólo en los

tratamientos HL, SH y T2.

3.1.6. Arquitectura de la planta

Se calculó la longitud de los entrenudos a partir del cociente entre la longitud del tallo

principal comprendido entre la hoja basal y la yema apical, y el número de nudos. El

cociente entre SFT y la altura de la planta se utilizó como un índice de autosombreo

(IA). Ambos parámetros se obtuvieron dos meses después de la segunda transferencia

en 5 plantas por especie de los tratamientos HL, SH y T2.

3.1.7. Crecimiento, y distribución de la biomasa entre los órganos de la

planta

En el momento de transplantar los brinzales al final del primer año, se cosecharon 5 – 6

brinzales por especie de los tratamientos HL y SH, y se midió su peso seco. Al final del

experimento, dos meses después de la segunda transferencia, se cosecharon otras seis

24

Material y métodos

plantas por especie de los tratamientos HL, SH y T2, evaluándose la tasa de crecimiento

relativo a partir de la ecuación:

( )12

12 )ln()ln(DD

MMRGR−−

= ,

donde M2 y M1 son los pesos en la cosecha final e inicial, respectivamente, y D2 y D1

son los días de la cosecha final e inicial, respectivamente.

Dado que las hojas, tallos y raíces se pesaron separadamente, se pudieron estimar

las fracciones de biomasa en hojas (LMF), tallos (SMF) y raíces (RMF), a partir del

peso de cada órgano con respecto al peso seco de la planta. Se calculó la relación entre

la biomasa correspondiente a las raíces y la correspondiente a la suma de hojas y tallos

(R/S). Se calculó también la razón de área foliar (LAR), que es el cociente entre la

superficie foliar total y el peso seco de la planta. SFT se obtuvo midiendo la superficie

de todas las hojas de la planta con un analizador de imágenes (Delta-T Devices LTD,

UK). Las hojas del tallo principal y de los brotes basales se planimetraron y pesaron por

separado, así como las hojas de la primera brotación y las pertenecientes a brotaciones

sucesivas. Por último, se calculó la tasa de asimilación neta (NAR) según la ecuación:

[ ]( )

( )( )12

12

12

12 *)ln()ln(DDMM

SFTSFTSFTSFTNAR

−−

−−

= .

La altura y el diámetro del tallo principal también se midieron al final del primer

año y en varias fechas del segundo hasta el final del experimento.

3.1.8. Análisis estadístico

La normalidad de los datos se comprobó a través de la representación gráfica de la

distribución de los datos. La homocedasticidad se examinó con gráficos de cajas y

bigotes y con los tests de Cochran y Bartlett, que prueban la hipótesis de que las

desviaciones estándar de los niveles de un factor son iguales. Se corrigió la ausencia de

normalidad y homocedasticidad de algunas variables transformando el apuntamiento y

25

Material y métodos

la kurtosis de los datos con operadores matemáticos (logaritmo, arcoseno o raíz

cuadrada).

Se realizaron análisis de la varianza de dos vías para examinar el efecto del

tratamiento lumínico y la especie sobre las variables; para el análisis de aquellas que

fueron medidas reiteradamente sobre la misma hoja, o la misma planta, se consideró el

día de medición como factor de repetición. Se examinaron las interacciones de segundo

orden entre los factores. Cuando el término de interacción fue significativo (P-valor <

0.05), se fijó uno de los factores y se realizaron análisis de una vía para cada uno de sus

niveles. Las diferencias entre los valores medios de cada tratamiento de luz se

compararon a posteriori con el test de Tukey, más conservativo que otros tests de

comparaciones múltiples. El impacto de la transferencia de las plantas T2 a las

condiciones de mayor iluminación en Asat, gsat, Fv/Fm, ΦPSII y NPQ se evaluó

comparando la máxima variación en cada planta con respecto al valor medio en SH.

La intensidad y significación de las relaciones entre variables se determinaron

usando coeficientes de correlación de Pearson, ajustándose después los modelos de

regresión lineal que recogían el mayor porcentaje de variabilidad. Los términos

independientes que describen la evolución de los parámetros de fluorescencia en

respuesta al aumento gradual de la intensidad lumínica (pendiente, PPFD saturante y

valor a saturación) y la evolución de la fotosíntesis en respuesta a la variación de Ci

(Vmcmax, Jm

max) se estimaron con el programa Statgraphics plus 4.1 (Statistical Graphics

Corp.), a través de procedimientos iterativos que proporcionan las estimaciones que

minimizan la suma de cuadrados residual del modelo respecto a la nube de puntos.

Los resultados de la mayoría de los análisis se presentan en los anexos I y II.

3.2. Experimentos en campo

Se llevaron a cabo plantaciones en dos pinares de pino silvestre próximos al Hayedo de

Montejo: La Maleza (≈ 1250 m de altitud; Fotografía 1.1 a) y Sierra Escalva (≈ 1600 m;

Fotografía 1.1 c). Los suelos de la zona proceden de sustratos ácidos gneísicos y

micáceos; son en general profundos, fértiles y con una elevada capacidad para retener

agua. El clima es submediterráneo, con inviernos fríos y un periodo corto de sequía en

el verano. La temperatura media anual es de 8,7 ºC y la precipitación media anual es de

1124 mm (Pardo 2000).

26

Material y métodos

La precipitación en la zona se registró con un pluviómetro (AN1, Delta-T Devices

Ltd., UK), y la temperatura y la humedad relativa del aire con un sensor RHA1 (Delta-T

Devices Ltd., UK), ambos ubicados en una plataforma situada por encima del dosel de

copas y conectados a un compilador de datos. La precipitación se registró en intervalos

de una hora, y la temperatura y la humedad relativa cada minuto, disponiendo de

promedios cada diez minutos.

3.2.1. Plantaciones en La Maleza

Se trata de un pinar plantado en 1958. Las parcelas de estudio se situaron en una ladera

de pendiente suave (~ 15 %) orientada al sureste, en tres zonas con distinta densidad de

arbolado: una zona de pinar con la densidad original (1067 pies ha-1, 65,8 m2 ha-1 de

área basimétrica, y 22,5 m de altura dominante), una zona de pinar aclarado en 1998

contigua y al mismo nivel que la anterior (800 pies ha-1, 53,6 m2 ha-1 de área

basimétrica, y 21,6 m de altura dominante), y un cortafuegos adyacente de 10 m de

ancho realizado en 1992 que separa el pinar del Hayedo, y que representa un claro de

tamaño medio (Fotografía 3.2). La vegetación es abundante en el cortafuegos (Genista

florida L., Adenocarpus hispanicus (Lam.), Rubus ulmifolius Schott. y Lonicera

peryclimenum L. entre los taxones más abundantes), pero es escasa en la zona de pinar,

sobre todo en el de mayor densidad donde sólo Pteridium aquilinum L. (Kuhn) forma

pequeños rodales.

Fotografía 3.2. Los tres sitios se encontraban en un transecto de ≈150 m con orientación SW-NE.

Se hicieron tres plantaciones en distintos años. En la primera de ellas (año 2000)

se incluyó una zona desarbolada dentro del Hayedo pero no se plantó en el cortafuegos.

En la tercera plantación (año 2004) los individuos se protegieron con protectores

individuales, para evitar daños por herbívoros.

27

Material y métodos

- Primera plantación (anexo III)

Entre febrero y marzo de 2000 se plantaron 500 brinzales de roble albar y 500 de

melojo, de una savia, previamente cultivados en vivero sin restricciones de agua o

nutrientes. Doscientos cincuenta individuos con cepellón de cada especie se

distribuyeron en el pinar entre la zona aclarada y la que mantiene la densidad original de

plantación. No fue necesaria la eliminación de la vegetación preexistente antes de

proceder al ahoyado manual. Otros 250 individuos de cada especie se plantaron en una

zona sin cubierta arbórea dentro del Hayedo conocido como “Rozallano” (Fotografía

1.1 b), en fajas de dos metros de ancho donde el matorral, principalmente de

Adenocarpus hispanicus, A. complicatus y Juniperus communis, fue desbrozado

previamente a la instalación de los plantones. No hubo ninguna otra preparación del

terreno. El suelo en esta zona apenas alcanza los 60 cm de profundidad, frente a los más

de 100 cm que tiene en muchos puntos del pinar.

Se examinó la supervivencia al cabo del primer y cuarto año (octubre de 2000 y

2003). La luz en cada sitio se evaluó a partir de fotografías hemisféricas (n = 30 en las

dos zonas de pinar; n = 10 en el claro). Las fotografías se tomaron en días nublados o al

atardecer, para evitar la exposición directa a la luz solar, con una cámara analógica

Nikon FM a la que se le había acoplado una lente de 8 mm de tipo “ojo de pez”. Las

fotografías fueron posteriormente digitalizadas (Olympus ES-10, Olympus Optical Co

Europe GMBH) y analizadas con un programa informático (Hemiview 2.1 Canopy

Análisis Software, Delta T Devices Ltd., UK). La humedad del suelo se midió por

medio de un sistema por reflectometría TDR (Time Domain Reflectometry; Trase

System I, Soil Moisture Equipment, USA), que permite determinar el contenido

volumétrico de agua en el suelo a partir del cálculo de la constante dieléctrica. Se midió

la humedad hasta 80 cm de profundidad en tres tubos de PVC de 1 m de longitud, por

sitio. No se pudieron instalar tubos en “Rozallano” por lo que se tomaron los valores del

cortafuegos como aproximación. Ambas variables se midieron a lo largo del verano de

2003.

También se midieron el potencial hídrico foliar (PMS Instrument Co 7000,

Corvallis Oregon, USA), y la eficiencia fotoquímica potencial del fotosistema II (Fv/Fm)

(FMS 2, Hansatech Instruments Ltd., UK) en cinco plantas por especie y sitio, al

amanecer y a mediodía. Las medidas se realizaron en las primeras semanas de julio y

28

Material y métodos

agosto de 2002 (tercer año en campo), y julio, agosto y septiembre de 2003, aunque en

este último año no se pudo medir en el claro.

La valoración del efecto del lugar de plantación, la fecha y la especie en las

variables se hizo con análisis de la varianza. Cuando la interacción entre factores resultó

significativa (interacción máxima de segundo orden), se analizó el efecto de uno de

ellos para cada uno de los niveles del otro. El potencial hídrico al amanecer se tomó

como covariable para comparar los datos de Fv/Fm. La normalidad de los datos se

comprobó a través de la representación gráfica de la distribución de los datos. La

homocedasticidad se examinó visualmente con gráficos de cajas y bigotes, y con la

aplicación de los tests de Cochran y Bartlett. Los datos se transformaron con operadores

matemáticos cuando no eran normales u homocedásticos.

- Segunda plantación (anexos IV a VI)

En marzo de 2003 se plantaron 90 brinzales por especie, repartidos bajo el pinar

aclarado y sin aclarar, y el cortafuegos, en tres bloques completos distribuidos a lo largo

de la pendiente, cada uno con 30 plantas por especie situadas entre las hileras de pinos

separadas aproximadamente 1,5 m (diseño Split-Plot). Las plantas, de una año de edad

en el momento de su plantación, fueron cultivadas previamente en vivero en envases de

15 cm de profundidad; se transplantaron con cepellón en hoyos de aproximadamente

40×40×40 cm excavados manualmente. Algunos individuos fueron comidos el primer

año, por lo que se protegieron con una malla de alambre rígido de 1 cm de luz en el

siguiente. Durante los dos años que siguieron a la plantación se examinó el microclima

de cada sitio, y se evaluó el desarrollo de los brinzales determinándose distintos

parámetros fisiológicos y morfológicos al comienzo (final de junio) y al final del verano

(final de agosto).

Caracterización microclimática

En cada sitio se examinó la humedad del suelo a distintas profundidades, la humedad y

la temperatura del aire, y el régimen lumínico (irradiación relativa y duración de los

periodos de luz directa).

Se evaluó el perfil de humedad en el suelo hasta 60 cm de profundidad por medio

de una sonda móvil introducida en los tubos de PVC enterrados en los tres sitios (n = 3,

29

Material y métodos

uno en cada bloque de plantas). No se pudo medir la humedad del suelo por debajo de

los 60 cm de profundidad debido a la entrada de agua en los tubos. La humedad y

temperatura del aire se registraron por medio de sensores conectados a un compilador

de datos (HOBO H8 Pro, Onset); se programaron para tomar un registro cada 10 min.

Se colocó un equipo de medida por sitio. Con los valores de humedad relativa y

temperatura se calculó el déficit de presión de vapor del aire. Para evaluar el régimen de

luz de cada sitio se realizaron fotografías hemisféricas sobre el ápice de las plantas

utilizadas en las mediciones (n = 10 – 12). El tratamiento de las imágenes con el

programa Hemiview 2.1 permitió calcular las proporciones de luz difusa, directa y total

del lugar donde se había tomado cada fotografía, así como el número y la duración de

los momentos en que había luz directa a lo largo del día (“sunflecks”).

Relaciones hídricas

El estado hídrico de los brinzales se evaluó a través de la medida del potencial hídrico

foliar. Se midió con una cámara de presión (PMS Instrument Co. 7000, Corvallis

Oregon, USA) en una hoja por planta, y cinco plantas por especie y sitio, en torno al

alba (Ψpd) y al mediodía (Ψmd).

Se realizaron además curvas Presión-Volumen en cinco plantas por especie y sitio.

En primer lugar las hojas se rehidrataron por inmersión del pecíolo en agua destilada

durante varias horas. Posteriormente se dejaban deshidratar en las bancadas del

laboratorio, midiendo su potencial hídrico a intervalos de tiempo, representando

finalmente la relación entre el contenido hídrico relativo (CHR) y el inverso del

potencial hídrico (Robichaux et al. 1984). Cada curva contenía entre 10 y 17 puntos. El

contenido hídrico relativo se calculó como el cociente de la diferencia entre el peso

fresco (PF) y el peso seco de la hoja (PS), y el peso turgente (PT) y el peso seco:

PSPTPSPFCHR

−−

= .

El peso turgente se estimó a partir de la relación lineal que existe entre el peso fresco y

el potencial hídrico en los primeros puntos de la curva. La hoja se metió en una estufa a

65 ºC durante varios días para obtener su peso seco. A partir de la relación entre el

30

Material y métodos

contenido hídrico relativo y el inverso del potencial hídrico se obtuvieron diversos

parámetros fisiológicos.

Los potenciales osmóticos a turgencia plena (Ψπ100) y nula (Ψπ0) se calcularon a

partir de la regresión lineal entre el contenido hídrico relativo y el inverso del potencial

hídrico una vez que las células entran en estado de plasmolisis (Ψw = Ψπ):

nCHRm +⋅=Ψ

−π

1 .

En el punto de turgencia plena, CHR = 100 %, así que:

)(1

100 nm +−

=Ψπ ,

mientras que para calcular Ψπ0 se considera el contenido hídrico relativo en el punto de

plasmolisis (CHRpt):

)(1

0 nCHRm pt +⋅−

=Ψπ .

El módulo de elasticidad de las paredes celulares se calculó en los puntos de

mayor turgencia (Emax) según Robichaux et al. (1984):

)(max ap CHRCHR

CHRE −⋅

ΔΨ= .

El módulo de elasticidad depende de la pendiente de la relación lineal entre el contenido

hídrico relativo y el potencial de turgencia celular (Ψp = Ψw − Ψπ), y de la diferencia

entre el valor medio del contenido hídrico relativo en los puntos considerados y el

contenido hídrico del apoplasto (CHRa), que a su vez resulta de la extrapolación de la

relación entre CHR y 1/Ψw, cuando 1/Ψw = 0:

31

Material y métodos

mnCHRa = .

El contenido de agua del simplasto a plena turgencia respecto al volumen total de agua

(CHRs) se obtuvo a partir de la diferencia con el contenido de agua en el apoplasto.

Parámetros bioquímicos

Se determinó la concentración media de clorofila (Cm) y nitrógeno (Nm) en cinco

plantas por especie y sitio por los procedimientos anteriormente expuestos (sección

3.1.4.). El contenido medio de clorofila y nitrógeno por unidad de área foliar (Ca y Na,

respectivamente) se estimó a partir de los valores de SLA. Sólo se realizaron análisis de

nitrógeno foliar en 2004.

Intercambio gaseoso

Se utilizaron dos equipos de análisis de gases por infrarrojos. En 2004 las medidas se

realizaron con un IRGA-LCA 4 (Analytical Development Corporation, UK) y en 2005

con un IRGA-LCPro (Analytical Development Corporation, UK).

El protocolo de medición también difirió entre los dos años. En 2004, las hojas se

sometieron a luz saturante durante cerca de 15 min para inducir la actividad

fotosintética (aproximadamente 1300 μmol m-2 s-1 para las plantas del cortafuegos y 900

μmol m-2 s-1 para las del pinar). Tras tomarse tres registros de la asimilación neta (Asat)

y la conductancia estomática (gsat) a concentración de CO2 ambiental (365 ppm), se

reducía la concentración en el aire de entrada a la cámara foliar en tres pasos (300, 200

y 100 ppm), tomándose en cada punto tres registros más de la tasa de asimilación neta.

Se estimó la eficiencia de la carboxilación como la pendiente de la variación de la tasa

de asimilación neta con respecto a la variación de la concentración intercelular de CO2

(Ci). La temperatura en la cámara foliar durante este proceso se mantuvo en torno a 25

ºC (temperatura foliar: 24 – 30 ºC). Las medidas se hicieron entre las 9:30 y las 11:30

a.m. para evitar oscilaciones excesivas en el déficit hídrico. Se midieron cuatro plantas

por especie y sitio en junio, y tres en agosto.

En 2005 se midió la tasa de asimilación neta a distintas intensidades lumínicas.

Las hojas se sometían inicialmente a 800 μmol m-2 s-1 durante aproximadamente 15 min

32

Material y métodos

para lograr la apertura de los estomas y la activación de los procesos fotosintéticos. A

continuación se subía la intensidad a 1150 μmol m-2 s-1, y a 1600 μmol m-2 s-1 en las

plantas del cortafuegos. La luz se disminuía a continuación a 800 μmol m-2 s-1 y por

último a 40 μmol m-2 s-1 en cinco pasos (500, 350, 175, 90 y 40), tomándose en cada

punto tres registros de la tasa de asimilación neta. La temperatura dentro de la cámara

de medición se programó a 23 ºC para conseguir que la temperatura foliar se mantuviera

alrededor de 25 ºC. La concentración de CO2 se mantuvo en torno a 365 ppm durante

toda la curva. Se ajustó un modelo cuadrático a los datos con el programa informático

Photosyn Assistant 1.1 (Dundee Scientific, UK):

( )R

KAKPPFDAPPFDAPPFD

A satsatsat −⋅

⋅⋅⋅φ⋅−+⋅φ−+⋅φ=

242

,

que permitió estimar la tasa de asimilación neta a saturación por luz (Asat), el

rendimiento cuántico aparente (φ) y la tasa de respiración (R).

PPFD (μmol m-2s-1)

An

(μm

olm

-2s-1

)

0 400 800 1200 1600 20000

2.5

5.0

7.5

10.0

12.5

Figura 3.2. Ejemplo de una curva de respuesta de

la fotosíntesis (An) a la luz (PPFD) en una planta

de melojo en el cortafuegos.

Las estimaciones de φ y R fueron en varios casos incoherentes, debido al “ruido” de la

señal del equipo de medida y al escaso número de puntos de la porción lineal inicial de

la curva por lo que no se compararon. El parámetro K describe la forma de la curva. Las

medidas se hicieron entre las 8:30 y las 11:30 a.m., en cinco plantas por sitio y especie.

Se realizó una tercera medición a finales del mes de julio, en la que se midió la tasa de

asimilación neta a niveles de irradiación saturantes (1150 y 1600 μmol m-2 s-1, en el

pinar y en el cortafuegos, respectivamente).

33

Material y métodos

A partir de los valores de transpiración a saturación por luz (Esat) y de potencial

hídrico se estimó la conductancia hidráulica foliar aparente a través del continuo suelo-

atmósfera (KL) (Saito et al. 2003):

mdpd

satL

EK

Ψ−Ψ= .

Eficiencia en el uso del agua

Los cocientes entre la fotosíntesis neta y la tasa de transpiración, o la conductancia

estomática, son indicadores de la eficiencia foliar instantánea en el uso del agua, al

reflejar el agua que se pierde por transpiración para la asimilación de un mol de CO2. La

composición isotópica del carbono de un tejido vegetal (δ13C) es un indicador más

integrado de la eficiencia de la asimilación respecto al coste en consumo de agua que

los anteriores, ya que refleja la relación entre ambos procesos a lo largo de un periodo

de tiempo más amplio. Brevemente, las plantas discriminan positivamente el isótopo 12C respecto al 13C en los procesos de fijación del CO2. Esta discriminación se reduce

cuando la concentración intercelular de CO2 disminuye –por ejemplo debido al cierre

temporal de los estomas ante situaciones de estrés hídrico o al aumento de la

concentración de nutrientes y la tasa de fotosíntesis en hojas bien fertilizadas –, lo que

causa el incremento de la abundancia relativa del isótopo 13C en los tejidos (Farquhar et

al. 1989). δ13C se calcula como la relación 13C/12C respecto a la de la bentonita usada

como patrón. Por ello, valores más altos de δ13C (menos negativos) indican una

eficiencia en el uso del agua mayor.

En el año 2005, las hojas empleadas para medir el intercambio gaseoso se secaron

a 65 ºC durante tres días para determinar su valor de δ13C con un espectrómetro de masa

del Servicio Interdepartamental de Investigación (SIDI) de la Universidad Autónoma de

Madrid. El método analítico tenía una precisión de ± 0,2 ‰.

Fluorescencia de la clorofila a

Se midió con un fluorómetro portátil de pulso modulado (FMS 2, Hansatech

Instruments LTD., UK).

34

Material y métodos

En 2004 se realizaron curvas rápidas de respuesta de la fluorescencia de la

clorofila a la intensidad de luz siguiendo un protocolo similar al expuesto en la sección

3.1.3. Las mediciones se hicieron in situ en hojas no escindidas previamente

aclimatadas a oscuridad durante aproximadamente 30 min. El equipo se programó para

obtener el parámetro Fv/Fm en oscuridad y aumentar a continuación la intensidad de luz

hasta 1500 μmol m-2 s-1 en diez niveles. Antes de pasar al siguiente nivel de intensidad,

las hojas se sometían a un pulso de luz saturante (6600 μmol m-2 s-1 durante 0,8 s) y a

continuación a un pulso de luz del espectro rojo-lejano, lo que permitió estimar en cada

nivel la eficiencia fotoquímica efectiva (ΦPSII), la tasa de transporte electrónico (ETR),

la disipación no-fotoquímica de la luz absorbida (NPQ), la disipación fotoquímica (qP)

y la eficiencia de los centros de reacción oxidados (Fv’/Fm’):

.'

'''/'

m

ommv F

FFFF

−=

El tiempo de exposición a cada intensidad disminuyó entre 4 min en el primer nivel y

30 s en el último, según el tiempo necesario para la estabilización de la señal de

fluorescencia observado en ensayos previos realizados en el laboratorio. No obstante se

puede afirmar que las curvas no se realizaron en condiciones de aclimatación foliar a la

luz debido al retardo de la inducción fotosintética tras la iluminación de las hojas, en

concreto de la apertura estomática, que reduce la disponibilidad de CO2 (Ernstsen et al.

1997), y hace que la señal de fluorescencia máxima (Fm) se infravalore en cada punto de

la curva (White & Critchley 1999).

Análisis estadístico

Se realizaron análisis de la varianza (procedimiento GLM) para examinar el efecto del

sitio de plantación, la especie y la fecha sobre las variables. Las posibles diferencias en

el grado de estrés hídrico entre especies y sitios se controlaron considerando el potencial

hídrico al amanecer (Ψpd) como covariable. Se examinaron las interacciones de segundo

orden entre factores. Cuando el término de interacción fue significativo (P-valor <

0,05), se fijó uno de los factores y se realizaron análisis de dos vías para cada uno de sus

niveles, o de una vía cuando fue posible agrupar los valores de los otros dos factores (P-

35

Material y métodos

valor de la interacción > 0,05). Las diferencias entre las medias de cada nivel se

compararon a posteriori con el test de Tukey cuando existió un efecto significativo del

factor. No se siguió un análisis de medidas repetidas ya que las mediciones de la

mayoría de variables no se pudieron repetir sobre las mismas plantas; sólo en el caso de

la humedad del suelo se empleó una aproximación de medidas repetidas. Se corrigió la

ausencia de normalidad y homocedasticidad transformando el apuntamiento y la

kurtosis de los datos con operadores matemáticos.

La intensidad y significación de las relaciones entre variables se determinaron

usando coeficientes de correlación de Pearson, ajustándose después los modelos de

regresión lineal que recogían mayor porcentaje de variabilidad. La aclimatación de los

parámetros foliares al rango de luz generado entre ambientes en cada especie se

comparó mediante modelos lineales generales con GSF como variable cuantitativa

(covariable), ajustándose modelos distintos para cada fecha si las interacciones con GSF

eran significativas. Se emplearon modelos de regresión múltiple para analizar la

relación de algunas variables (Ψπ100 y Emax) con la luz (GSF) y el estrés hídrico (Ψpd);

las variables explicativas se introdujeron en el modelo “hacia delante” (forward

selection method), según la mejora en la predicción de la respuesta con respecto a un

modelo de regresión lineal simple. La evolución de los parámetros de fluorescencia en

respuesta al aumento gradual de la intensidad se describió por medio de funciones no

lineales. Para ello se proporcionan estimaciones iniciales de los parámetros

independientes del modelo hasta que por un procedimiento de iteración se alcanzan los

valores que minimizan la suma de cuadrados residual con respecto a los datos medidos;

se usó el programa estadístico Statgraphics plus 4.1 (Statistical Graphics Corp.).

La mayor parte de los análisis se presentan anexos a la memoria (anexos IV a VI).

- Tercera plantación (anexo V)

En marzo de 2004, se plantaron 60 brinzales de una savia por especie en los mismos

ambientes, repartidos en dos bloques de 30 individuos situados entre los bloques de la

plantación anterior. Se plantaron con cepellón y se rodearon con un protector de

alambre rígido desde el comienzo para evitar daños por herbívoros.

A finales de 2004 y 2005 se evaluó el desarrollo de los brinzales, excavando el

sistema radical de seis plantas por especie en el cortafuegos y la zona de pinar no

36

Material y métodos

aclarada. Se realizó un hoyo de unos 80 cm de profundidad y 30 cm de radio alrededor

de la planta. Las plantas extraídas se metieron en bolsas y se llevaron al laboratorio,

donde se lavaron y posteriormente se metieron en estufas durante aproximadamente una

semana. Se midió la biomasa de raíces (RDM) y la biomasa total de la planta (TDM),

calculando la fracción en raíces (RMF) y la proporción de biomasa subterránea respecto

a la aérea (R/S). En 2005 se midió también el área foliar total (SFT).

Se realizaron análisis de la varianza en cada año (procedimiento GLM), para

evaluar la significación estadística del sitio de plantación y la especie. El modelo de

varianza incluyó un término para el efecto del bloque, y otro para la interacción de

segundo orden entre el sitio y la especie. TDM se incluyó como covariable en los

análisis (anexo V).

3.2.2. Plantación en Sierra Escalva (anexo VI)

Se realizó con el objetivo principal de evaluar el efecto de la densidad de pinar en la

supervivencia de los robles. Para ello, en la primavera de 2005 se plantaron cerca de

1000 brinzales de dos savias de roble albar y cerca de 500 brinzales de melojo en un

pinar originado por una repoblación llevada a cabo en 1963. La pendiente oscila entre el

10 - 20 % y la orientación varía del SE al NE. En el año anterior, una zona de unas 10

ha se dividió en parcelas de tamaño semejante (60 – 80 m de ancho), que se asignaron a

tres tratamientos experimentales: clara de un 50 % de la densidad original, clara de un

33 % de la densidad original y pinar sin aclarar (C), cada uno replicado cuatro veces,

alternando en un gradiente Norte-Sur (Fotografía 3.3). En la zona central de cada

parcela se situaron otras parcelas de unos 530 m2 donde se plantaron los brinzales entre

las alineaciones de pinos (Fotografía 3.4). No se realizó ninguna operación del terreno

previa. Los árboles cortados se apearon.

Se registró la supervivencia al final del primer año en campo (octubre 2005) y al

comienzo del periodo vegetativo del segundo y tercer año desde la plantación (junio

2006 y 2007). La disponibilidad de luz se estimó un año después de la realización de las

claras con fotografías hemisféricas realizadas en el ápice de cinco brinzales formando

los vértices y el centro de un cuadrado de unos 10 m de lado localizado en el medio de

cada parcela experimental. La humedad del suelo se midió también a lo largo del primer

37

Material y métodos

año en campo a diferentes profundidades gracias a la colocación de tubos de PVC de un

metro de longitud por los que se hace desplazar la sonda de medición.

33

C

50

33

C50

33

C

50

33

C

501

2

3

4

5

6

7

8

9

10

11

12

#d

#

c

# e

#b

#

a

N

Bloques

Claros (a, b, c ,d, e)

1 al 12: Parcelas de control (C) y aclaradas al 33 y 50%.

Ensayos

100 0 100 200 Metros

Fotografía 3.3. Disposición de las parcelas

experimentales y los tratamientos.

Fotografía 3.4. Cada parcela albergaba 84 individuos

de roble albar y 42 de melojo entre las alineaciones de

pinos plantados en hoyos manuales de 40x40x40,

excepto las tres situadas más al norte, en las que el

número de robles y melojos fue de 80 y 40,

respectivamente.

La supervivencia de cada especie se analizó por medio de modelos de regresión

logística (GLZ), considerando las cuatro réplicas (bloque) como variable categórica de

predicción y el área basimétrica resultante después de las claras, el porcentaje de luz

global disponible (GSF) o la humedad del suelo como covariables caracterizadoras de la

clara. Los resultados de los análisis se describen en el anexo VI.

38

Resultados y discusión

4. RESULTADOS Y DISCUSIÓN

4.1. Experimento en invernadero: comparación interespecífica de las

respuestas de aclimatación a la luz en ausencia de estrés hídrico

Los brinzales de roble albar y melojo mostraron características distintas bajo

condiciones de riego y fertilización idóneas. Los caracteres foliares que de forma más

consistente y evidente difirieron entre las dos especies fueron la proporción de nitrógeno

en pigmentos y en componentes no fotosintéticos, la velocidad máxima de

carboxilación y de transporte electrónico por unidad de masa, el área foliar específica y

el tamaño de las hojas. En ciertas variables, las diferencias dependieron del régimen de

luz. La tasa de fotosíntesis, la conductancia estomática, la concentración de clorofila, o

tomando como referencia toda la planta, la superficie foliar total, el crecimiento y el

patrón de inversión de biomasa, fueron significativamente distintos entre las dos

especies, aunque con una marcada influencia de la luz reflejada en las diferencias que

exhibieron en plasticidad (en los anexos I y II pueden verse los valores de todas las

variables estudiadas y el efecto de la luz sobre ellas).

La expresión diferencial de caracteres bajo las mismas condiciones experimentales

puede reflejar la existencia de presiones selectivas diferentes (Villar et al. 2004). No

obstante, debemos ser cautos a la hora de atribuir un significado adaptativo a las

diferencias expuestas, ya que en todos los experimentos se ha empleado material

genético de varios árboles procedentes de una única población en el límite de

distribución. Estas podrían constituir ecotipos mejor adaptados a las condiciones

climáticas locales, por lo que es posible, que la magnitud de algunas diferencias variase

si se consideraran otras poblaciones. En el mismo sentido, hay que considerar la

existencia de un componente ontogénico en la expresión de los caracteres; el

comportamiento de las plántulas no es siempre igual al de los individuos maduros

(Cavender-Bares & Bazzaz 2000). Asumiendo estas limitaciones, los resultados

sugieren que los requerimientos ecológicos de los individuos de roble albar y melojo en

esta etapa de desarrollo son distintos, e indican cierto grado de segregación en los

ambientes de regeneración. El significado ecológico derivado de las características

funcionales de los brinzales guarda a menudo relación con la distribución de las

39

Resultados y discusión

especies a distintas escalas espaciales (Gómez-Aparicio et al. 2006; Schrader et al.

2006).

Con relación al aprovechamiento de la luz, la mayor concentración de clorofila,

superficie foliar específica y tamaño de las hojas del roble albar con respecto a las del

melojo, se relaciona con una tendencia superior a captar luz, que podría traducirse en

una mayor capacidad para establecerse en ambientes sombreados (Niinemets & Kull

1994; King 2003; Gómez-Aparicio et al. 2006). No obstante, para otros autores la

tolerancia a la sombra podría estar más relacionada con la capacidad para resistir el

impacto de la destrucción de la parte aérea que con la maximización de la captación de

luz. Una mayor proporción de biomasa subterránea (R/S) reflejaría una acumulación

preferencial de carbohidratos no estructurales en las raíces, que favorecería la formación

de brotes nuevos tras perturbaciones de la parte aérea y la persistencia de la planta, con

respecto a individuos con mayor razón de área foliar (LAR) o mayor inversión de

biomasa en hojas (LMF) (Lusk & del Pozo 2002). Ninguno de estos parámetros difirió

en condiciones controladas entre el melojo y el roble albar en sombra.

La tolerancia a los extremos lumínicos, además, depende de la capacidad para

modular el fenotipo según el ambiente lumínico, sobre todo en especies de media

sombra. No todos los caracteres de la planta presentan la misma plasticidad, ya que la

idoneidad del fenotipo en un ambiente determinado radica en la capacidad de cambio de

algunos caracteres y la homeostasis de otros. Especies más tolerantes a la sombra,

propias de etapas avanzadas en la sucesión natural del bosque, muestran mayor

estabilidad de los procesos fotosintéticos ante variaciones en la disponibilidad de luz

que otras menos tolerantes (Valladares et al. 2000a), pero mayor plasticidad en los

parámetros que conducen a una captación más eficiente de la luz, como la concentración

de nitrógeno, la concentración de clorofila, la superficie foliar específica, la arquitectura

de la copa y la inversión de biomasa (Valladares et al. 2002). Estas observaciones

coinciden parcialmente con nuestros resultados. Para el conjunto de variables la

plasticidad media fue prácticamente la misma para las dos especies, aunque al

considerar el valor medio de cada grupo de variables, se observó que la plasticidad de

las variables fisiológicas y anatómicas foliares era en general mayor en el melojo, y que

la plasticidad de las variables relacionadas con el crecimiento y la inversión de biomasa

era menor (Tabla 4.1).

40

Resultados y discusión

Tabla 4.1. Valor de plasticidad de algunas de las variables estudiadas según el índice de Valladares et al.

(2000a). Aparecen resaltados en negrita los valores superiores 0,10.

Tipo de variable Variable Q.pyrenaica Q.petraeaIntercambio gaseoso foliar A sat 0,47 0,33

g sat 0,49 0,30A a

max 0,43 0,29V a

cmax 0,38 0,38J a

max 0,51 0,35A m

max 0,05 0,13V m

cmax 0,05 0,02J m

max 0,17 0,03Fluorescencia de la clorofila ETR max 0,62 0,33

Φ PSII 0,30 0,30NPQ 0,76 0,79F v /F m 0,00 0,00

Bioquímica de la hoja N a 0,36 0,20N m 0,10 0,23EUN 0,11 0,13P r 0,04 0,25P b 0,23 0,20P l 0,27 0,34P s 0,08 0,04C a 0,04 0,13C m 0,39 0,48

Morfología foliar SLA 0,41 0,41GE 0,15 0,05GPP 0,65 0,48GPE 0,19 0,07TH 0,25 0,17

Crecimiento IA 0,79 0,56Altura 0,04 0,70Diametro 0,45 0,58RGR 0,74 0,75

Inversión de biomasa SFT 0,86 0,86LMF 0,12 0,25SMF 0,09 0,20RMF 0,00 0,19R/S 0,02 0,36

41

Resultados y discusión

Las diferencias expuestas entre los brinzales podrían tener un sentido ecológico

más amplio que el relacionado con la capacidad para tolerar la sombra. Algunos

resultados indican, más bien, un carácter más competitivo de los brinzales de roble

albar, como cabría esperar al comparar un taxón de distribución templada con otro de

distribución submediterránea. Las especies tolerantes a la sombra mantienen un

adecuado ritmo de crecimiento en sombra pero crecen menos al sol que las intolerantes

(Kobe & Coates 1997). Sin embargo, el crecimiento de ambas especies fue semejante en

el tratamiento de baja irradiancia, y en el tratamiento de alta irradiancia, la biomasa del

roble albar casi duplicó la del melojo (64,2 ± 4,9 vs 39,9 ± 3,1 g). Los brinzales de roble

albar crecieron más rápidamente a causa de un patrón morfo-funcional más eficiente en

la captación de la luz, derivado de un elevado desarrollo del tallo principal y la posesión

de entrenudos más largos (Fotografía 4.1), lo que redujo el autosombreo en los

individuos de roble albar pese a tener hojas más grandes y mayor superficie foliar total;

ello se tradujo en una tasa superior de asimilación neta al nivel de planta con respecto al

melojo (Falster & Westoby 2003). La relación entre la biomasa caulinar y radicular fue

dos veces más elevada en el roble albar (0,67 ± 0,05 vs 0,34 ± 0,02 g g-1).

Fotografía 4.1. Aspecto de algunas plantas de roble albar y melojo al final del experimento,

correspondientes a los tratamientos HL (foto izquierda) y T1 (foto derecha). La altura de las plantas fue

dos veces superior en el roble albar (75,9 ± 4,6 vs 32,6 ± 1,3 cm; tratamiento HL).

42

Resultados y discusión

Otro punto que corroboraría este aspecto sería el impacto que tuvo la transferencia

de las plantas de sombra al ambiente más iluminado, tanto en la evolución de la tasa de

fotosíntesis –que al menos en condiciones de riego y fertilización no limitantes no

difirió entre el melojo y el roble albar (Figura 4.1) –como en el patrón de crecimiento

(Fotografía 4.1).

El impacto de un aumento en los niveles de luz causa una inhibición de la

fotosíntesis mayor en las especies más tolerantes a la sombra (Gómez-Aparicio et al.

2006) y un retraso en la velocidad de recuperación, debido en parte al elevado tamaño

de las antenas colectoras de luz del fotosistema II con relación a los procesos de

disipación de la energía capturada (Osmond 1994). Llama la atención la flexibilidad de

muchos parámetros relacionados con la capacidad fotosintética en los brinzales de las

dos especies ante el cambio en las condiciones de iluminación. De hecho, la tasa de

fotosíntesis a saturación por luz y la capacidad fotosintética en las plantas cultivadas a la

sombra, aumentaron tras ser expuestas a un incremento de la luz durante

aproximadamente un mes hasta alcanzar valores similares a los de la plantas

continuamente sometidas a los mismos niveles de iluminación durante toda su vida,

hecho que ya se ha constatado en especies forestales templadas (Naidu & De Lucía

1998).

Asa

t(%

HL

)

20406080

100

a

Φ P

S II

(% H

L)

Tiempo (días)

NPQ

(% H

L)

20406080

100120140

050

100150200250300

0 10 20 30 400 10 20 30 40

c

b

Figura 4.1. Variación temporal desde la segunda

transferencia de plantas (T2-día 0) de algunos

parámetros fotosintéticos expresados en valores

porcentuales respecto a los correspondientes valores

medios de las plantas del tratamiento de alta irradiancia

(HL). Símbolos blancos: roble albar, símbolos grises:

melojo; las líneas delgadas continua y punteada indican

los valores medios porcentuales de las plantas SH de

melojo y roble albar, respectivamente.

43

Resultados y discusión

Esta rápida capacidad para reajustar muchos de sus caracteres funcionales (Figura

4.1) pudo derivar de la retranslocación de nitrógeno entre los componentes

fotosintéticos foliares, principalmente asociada a la degradación de clorofila y los

complejos proteínicos a los que se une (Figura 4.2), como han propuesto otros autores

(Yang et al. 1998); y sugiere, que los costes implicados en poner en marcha

mecanismos para evitar daños por fotoinhibición son menores, en términos ecológicos,

que los derivados de la reparación de los daños (ver en cambio Einhorn et al. 2004).

5

10

15

20

25

0 37 61Tiempo (días)

0 37 61Tiempo (días)

Cm

(mg

g-1)

a b

Figura 4.2. Evolución de la concentración de clorofila en los brinzales de melojo (a) y roble albar (b) de

los tratamientos de alta irradiancia (HL, rombos), baja irradiancia (SH, cuadrados) y segunda

transferencia (T2, triángulos).

Pese a la respuesta similar en los parámetros foliares, la aclimatación al nivel de la

planta, en especial en lo que respecta al crecimiento en altura y diámetro del tallo

principal, fue más evidente en los brinzales de roble albar (Figura 4.3). Es decir, más

que al potencial de aclimatación de las hojas previamente sombreadas, las diferencias

entre ambas especies parecieron deberse al desigual patrón de movilización de los

carbohidratos acumulados a la sombra, o de los recién sintetizados en las hojas

transferidas. El incremento de los niveles de luz supuso el escape de la situación de

estrés por falta de luz y el consiguiente aumento de la proporción de biomasa en hojas y

tallos (anexo II). El mayor crecimiento en altura, diámetro y biomasa que alcanzaron los

brinzales de roble albar tras ser expuestos a un incremento en la intensidad de luz,

confirmaría su carácter más competitivo en esta etapa de desarrollo, bajo la idea clásica

de que un crecimiento más rápido al aumentar la luz disponible permite sobresalir y

evitar la sombra de la vegetación circundante y escapar de los herbívoros (Küppers

1989).

44

Resultados y discusión

0

20

40

60

80

marzo mayo julioTiempo (mes)

Altu

ra (c

m)

marzo mayo julioTiempo (mes)

02468

101214

Diá

met

ro (m

m)

a b

c d

Figura 4.3. Evolución del diámetro del tallo principal y la altura de las plantas de melojo (a, c) y roble

albar (b, d) en el segundo año. Rombos: tratamientos de alta irradiancia (HL), cuadrados: baja irradiancia

(SH), círculos: primera transferencia (T1, 27 febrero) y triángulos: segunda transferencia (T2, 6 junio).

4.2. Experimentos en el campo

4.2.1. Comparación de las respuestas de aclimatación a la luz con las

observadas en invernadero

Existen motivos que dificultan la extrapolación de los resultados obtenidos en

condiciones controladas a situaciones naturales, como la restricción del desarrollo

radicular y, sobre todo, la ausencia de interacción entre factores ambientales (Parelle et

al. 2006). Por ello, es importante combinar estudios en invernadero o cámara climática,

que permiten asignar claramente las respuestas observadas a un único factor, con

estudios en el campo, que ofrecen una interpretación más realista y práctica.

Algunas de las variables foliares mostraron un patrón consistente entre los

experimentos de campo e invernadero. La masa por unidad de área foliar (1/SLA), la

concentración de clorofila, y las tasas de fotosíntesis y conductancia estomática a

saturación por luz fueron superiores en el melojo, tanto en situaciones de estrés hídrico

leve, al comienzo del verano, como moderado. Tomando como referencia la planta, la

superficie foliar y la proporción de raíces respecto a la biomasa aérea o total de las

45

Resultados y discusión

plantas cosechadas en el campo, fueron también más elevadas en el melojo (anexos III a

V).

Para otras variables, las diferencias entre especies no fueron consistentes entre

experimentos, sobre todo en sombra. Las tasas de fotosíntesis y conductancia

estomática, que no difirieron entre especies en el tratamiento de baja irradiancia del

invernadero, fueron mayores en las hojas de melojo en el campo. Incluso la tasa de

fotosíntesis por unidad de masa a saturación por luz fue mayor en el melojo que en el

roble albar y viceversa en el invernadero. Igualmente, la razón de área foliar no difirió

entre las dos especies en el invernadero en el tratamiento de baja irradiancia (35,6 ± 2,1

vs 36,8 ± 3,8 cm2 g-1, para los brinzales de melojo y roble albar, respectivamente), pero

sí fue diferente bajo el dosel del pinar no aclarado (25,4 ± 2,0 vs 39 ± 1,6 cm2 g-1, para

los brinzales de melojo y roble albar, respectivamente). Aunque no se pueden descartar

circunstancias asociadas a las condiciones de experimentación (disponibilidad de

nutrientes, grado de sombreo, estado hídrico, edad de los brinzales, inducción de la

apertura estomática, etc.), posiblemente las condiciones ambientales de crecimiento en

el campo modificaron el patrón de fraccionamiento de nutrientes entre los distintos

componentes foliares o la distribución de carbohidratos entre los órganos de la planta

que tuvo lugar en el invernadero.

El hecho de que el tratamiento de mayor irradiancia en el invernadero no superase

el 70 % no permite valorar la tolerancia a la luz de alta intensidad. Los resultados en el

campo corroboraron, sin embargo, el carácter de media sombra de los brinzales de las

dos especies, al mostrar síntomas de fotoinhibición crónica en ambientes próximos al

100 % de irradiancia (anexo III). La capacidad para usar la luz de alta intensidad en el

campo también pareció diferir respecto a la observada en el invernadero. Los brinzales

de roble albar exhibieron una eficiencia de los centros de reacción oxidados del PS II a

niveles saturantes de luz (Fv’/Fm’) menor que los de melojo (anexo IV), lo que

explicaría la mayor atenuación no-fotoquímica de la energía absorbida en forma de

calor (NPQ). Este es uno de los mecanismos fotoprotectores que operan en el interior de

los cloroplastos cuando la actividad fotosintética está temporalmente limitada, lo que

sugiere un mayor desequilibrio entre la luz absorbida y la utilización del NADPH y el

ATP en la asimilación de CO2 en el roble albar en condiciones naturales (Ort 2001).

46

Resultados y discusión

4.2.2. Variación del “microclima” según la densidad del arbolado

Como se indicaba anteriormente, la dificultad para extrapolar los resultados de los

experimentos en invernadero a situaciones naturales se deben en parte a la interacción

de múltiples factores ambientales en el campo. Así, la diferente densidad de pinos en los

lugares de plantación alteró la luz disponible para las plantas en el sotobosque, pero

también la humedad del suelo y el déficit de la presión de vapor de aire máximo diario.

La luz disponible para las plantas, estimada como la proporción de la suma de la

luz directa y difusa (GSF), fue mayor en los claros que bajo el pinar (anexos III y VI).

El régimen de iluminación también varió. En el caso de los tratamientos de La Maleza

el promedio para las cuatro fechas de muestreo del número de veces a lo largo del día

que las plantas recibían luz directa en forma de destellos (“sunflecks”) fue de 16 en el

cortafuegos, con una duración media de 23 min y máxima de 3,6 h en torno al mediodía,

32 en el pinar aclarado, con una duración media de 9 min y máxima de 39 min, y

finalmente 35 veces en la zona no aclarada, con 8 y 35 min de duración media y

máxima, respectivamente. En Sierra Escalva la disponibilidad de luz fue menor en las

parcelas que no se aclararon y no hubo apenas diferencias entre las que se aclararon un

33 y un 50 % (anexo VI).

Tanto la temperatura media como el déficit de presión de vapor del aire medio

fueron prácticamente iguales entre los tres lugares de plantación en La Maleza, pero los

valores máximos diarios fueron mayores en el cortafuegos que en el pinar (2,5 ºC y 0,30

KPa en 2004, y 3 ºC y 0,46 KPa en 2005 durante los meses estivales), con

independencia de la densidad de pies (Figura 4.4).

La humedad del suelo también varió entre sitios, siendo mayor en el pinar

aclarado a lo largo de todo el periodo vegetativo, y parecida en los otros lugares (Figura

4.5). Disminuyó en ambos años desde primavera a comienzos de otoño, con pequeñas

oscilaciones debidas a los episodios de precipitación, sobre todo en los horizontes

superficiales del cortafuegos. Los horizontes más profundos no fueron

significativamente más húmedos, al menos hasta los 60 cm de profundidad (Fprofundidad =

0,1n.s.), como ya ha sido observado en algunos bosques peninsulares (Bellot et al. 2004).

En Sierra Escalva, sin embargo, la humedad del suelo varió significativamente con la

profundidad, aumentando hasta los 40-60 cm, y disminuyendo a partir de ese punto

(Figura 4.6); la humedad fue menor en las parcelas no aclaradas.

47

Resultados y discusión

DPV

max

(KPa

)T

max

(ºC

)

0

1

2

3

4

5/04 6/04 7/04 8/04 9/0410/04 5/05 6/05 7/05 8/05 9/0510/05 Tiempo (mes) Tiempo (mes)

0

10

20

30

40a b

c d

Figura 4.4. Variación estacional de los valores máximos diarios de la temperatura (a, b) y del déficit de

presión de vapor (c, d), en el cortafuegos (línea discontinua), y bajo el pinar aclarado y no aclarado (líneas

continuas negra y gris, respectivamente) durante los periodos vegetativos de 2004 (a, c) y 2005 (b, d).

Es razonable pensar que al igual que sucede con la temperatura, la humedad

relativa del aire o la luz, las diferencias en humedad del suelo entre los sitios de

plantación se deban también a las variaciones de la densidad del arbolado, más que a

una posible variación de la textura del suelo. La intercepción de las precipitaciones en

las copas de los árboles y la hojarasca tiene un efecto negativo en la humedad del suelo

(Bellot et al. 2004). Y también la absorción de agua por parte de las raíces de los

árboles adultos pueden causar la disminución del contenido hídrico del suelo (Bellot et

al. 2004).

El grado de cobertura arbórea, o en general de la vegetación que forma el dosel,

tiene un papel determinante en la disponibilidad de luz y agua para las plantas que

crecen en su interior, de modo que coberturas medias podrían favorecer el

mantenimiento de la humedad sin reducir excesivamente la luz disponible para las

plantas (Aussenac 2000; Castro et al. 2004). La relación inversa de la humedad

superficial del suelo y la luz en el sotobosque con el área basimétrica apoyaría estas

observaciones (anexo VI).

48

Resultados y discusión

0

10

20

30 f (40 cm)

5/04 6/04 7/04 8/04 9/04 10/04

e

Hum

edad

del

suel

o (%

)

0

10

20

30 c

0

10

20

30

40a b (10 cm)

d (20 cm)

5/05 6/05 7/05 8/05 9/05 10/05 Fecha (mes) Fecha (mes)

0

10

20

30 g h (60 cm)

Figura 4.5. Variación estacional de la humedad del suelo a distintas profundidades en el cortafuegos

(cuadrados; línea discontinua), el pinar aclarado (círculos; línea punteada), y el pinar no aclarado

(triángulos; línea continua) durante los periodos vegetativos de 2004 (a, c, e, g) y 2005 (b, d, f, h). Se

indica el contenido volumétrico de agua por debajo del cual el potencial hídrico cae marcadamente (≈

12,5 %) según datos de Aranda et al. (2002) para el mismo pinar de estudio. N = 2-3 a 60 cm.

4.2.3. Variación de la resistencia a la sequía estival según la densidad del

arbolado

La variación de la luz disponible para las plantas generada por la cobertura del dosel de

los diferentes lugares de plantación tuvo un papel determinante en la modulación del

impacto que tuvo la sequía sobre los brinzales, como se ha manifestado en experiencias

previas (Aranda et al. 2001). No obstante, según los resultados anteriores, es difícil

49

Resultados y discusión

precisar hasta que punto la variación de algunas de las respuestas entre estos lugares se

vio influida por la variación del patrón de radiación, el déficit de presión de vapor del

aire, la humedad del suelo, o la de otros factores no evaluados, como la calidad espectral

de la luz (empobrecida en el componente rojo bajo el dosel de pinos) y la disponibilidad

de nutrientes (Niinemets & Valladares 2004).

Figura 4.6. Variación de la humedad del suelo

promediada a lo largo del verano de 2005 con

la profundidad en Sierra Escalva. Símbolos

blancos: zonas aclaradas un 50 %, símbolos

grises: zonas aclaradas un 33 % y símbolos

negros: zonas no aclaradas.

0

5

10

15

20

25

0 20 40 60 80Profundidad (cm)

Hum

edad

del

suel

o (%

)

Fprofundidad: 13***; Fbloque: 5,1**Ftratamiento: 8,5***; Fprofund. x trat.: 1,1n.s

El grado de estrés hídrico de las plantas a lo largo del verano fue mayor en el

ambiente más sombreado. En ambas especies, los individuos plantados en la zona de

pinar que no había sido aclarada tuvieron un potencial hídrico al amanecer menor que

en la zona aclarada o el cortafuegos (anexo V). Las diferencias en el contenido hídrico

del suelo entre los lugares de plantación explicarían, al menos en parte, estas

diferencias. Pero además, el hecho de que las plantas acumulasen al final del

experimento aproximadamente el triple de biomasa (16 ± 0,4 vs 5,1 ± 0,2 g, datos para

ambas especies) y de biomasa de raíces (9,3 ± 0.2 vs 3,3 ± 0,1 g, datos para ambas

especies) en el cortafuegos que en la zona no aclarada, refleja un mayor desarrollo del

sistema radicular, que probablemente favoreció el acceso de las plantas del cortafuegos

a zonas de suelo más húmedas (Fotografía 4.2). La sombra excesiva, al retrasar el

crecimiento de los brinzales, contribuiría así a aumentar la susceptibilidad a la sequía

estival. Otros trabajos han demostrado que el potencial hídrico de los brinzales es menor

bajo el dosel que forma la vegetación que en zonas menos cubiertas, más expuestas a la

luz y a un déficit mayor de la presión de vapor del aire (Crunkilton et al. 1992;

Stoneman et al. 1994; Poorter & Hayashida-Oliver 2000; Valladares & Pearcy 2002).

Estas diferencias en el estado hídrico de los brinzales limitan en cierta medida la

comparación de las respuestas de tolerancia al estrés hídrico entre ambientes, evaluadas

50

Resultados y discusión

a partir del comportamiento de los procesos funcionales (intercambio gaseoso y

relaciones hídricas) a lo largo del verano. Por ello se utilizó como covariable el

potencial hídrico de la rizosfera de cada planta, estimado a partir del potencial hídrico

de las hojas antes de amanecer, para comparar el valor medio de las variables entre

ambientes. En 2005, el lugar de plantación no tuvo entonces un efecto significativo en

las tasas de intercambio gaseoso, pero sí en los potenciales osmóticos a turgencia plena

(Fsitio: 16,04***) y en el punto de marchitez (Fsitio: 19,22***). Ello es debido a una

correlación más fuerte del potencial de base con las variables de intercambio gaseoso

que con los potenciales osmóticos (anexos V y VI). Además del estrés hídrico, la

variación de la luz disponible entre los tres ambientes influyó en la acumulación de

solutos.

Fotografía 4.2. Las plantas enraizaron mejor en el

cortafuegos (izquierda y centro) que en el pinar, donde

incluso no se observaron raíces fuera del cepellón de

algunas plantas, dos años tras ser transplantadas (derecha).

La influencia del lugar de plantación sobre los caracteres estudiados fue en general

parecida en las dos especies, como indica la ausencia de interacciones significativas

entre el sitio y la especie para la mayoría de variables. Sólo la capacidad de ajuste

osmótico disminuyó de forma más evidente en el pinar denso con respecto al

cortafuegos en el roble albar. Estudios previos han puesto de manifiesto que las dos

especies son capaces de reducir el potencial osmótico a través del acopio de solutos en

respuesta al estrés hídrico (Aranda et al. 1996 y 2004a), pero no se había comparado

aún la influencia de la luz sobre este proceso. Los resultados mostraron que al contrario

que las plantas de melojo, las plantas de roble albar apenas eran capaces de ajustar

osmóticamente al final del verano en el sotobosque del pinar sin aclarar, pese al alto

51

Resultados y discusión

grado de estrés hídrico al que se vieron expuestas; la reducción del potencial osmótico a

plena turgencia al final del verano en los dos años de estudio fue de 0,41 MPa (2004) y

0,53 MPa (2005) en las hojas de melojo y 0,10 MPa (2004 y 2005) en las de roble albar.

Dado que el nivel de ajuste osmótico entre especies fue parecido en el pinar aclarado y

en el cortafuegos, es razonable pensar que la reducción de la luz en la zona no aclarada

afectase en mayor medida a los brinzales de roble albar, indicando una menor capacidad

para acumular solutos en condiciones de sombra y sequía intensas (Morgan 1984; Kwon

& Pallardy 1989; Aranda et al. 2001). La acumulación activa de solutos en las células

del mesófilo es un mecanismo implicado en el mantenimiento de la presión de

turgencia. Sin embargo, el valor del potencial osmótico en el punto de marchitez (Ψπ0)

fue parecido en las dos especies, de modo que el potencial de presión, calculado a partir

de la diferencia entre el potencial hídrico y Ψπ0, no difirió significativamente ni al

amanecer ni a mediodía (Tabla 4.2). Una posible explicación radica en la variación

estacional de la elasticidad de las paredes celulares (anexo VI). Así, mientras el módulo

de elasticidad mostraba una tendencia a aumentar en el melojo de junio a agosto, la

tendencia se invertía en el roble albar, es decir, la elasticidad de las paredes celulares de

las hojas del roble albar era mayor al final del verano en el pinar no aclarado, lo que

atenuó la caída de la turgencia celular (Joly & Zaerr 1987).

Tabla 4.2. Valores medios del potencial de presión calculados al amanecer y a mediodía. Letras distintas

separan medias significativamente distintas en cada fecha de medición. No hubo diferencias significativas

entre tratamientos a mediodía.

Junio Agosto Junio AgostoQ. pyr. 2,17±0,11a 2,12±0,11a 2,21±0,07a 1,85±0,16aQ. pet. 2,13±0,09ab 1,99±0,11ab 2,13±0,04ab 1,72±0,26aQ. pyr. 1,84±0,04abc 1,82±0,11ab 1,67±0,04abc 1,28±0,15abQ. pet. 1,73±0,12bc 1,79±0,10ab 1,55±0,07c 1,12±0,25abQ. pyr. 1,59±0,09c 1,38±0,26b 1,60±0,10bc 0,77±0,18bQ. pet. 1,62±0,09c 1,39±0,21b 1,23±0,25c 0,50±0,26b

Q. pyr. 0,34±0,08 0,31±0,16 0,03±0,11 0,26±0,09Q. pet. 0,21±0,10 -0,01±0,10 -0,17±0,08 0,19±0,13Q. pyr. 0,58±0,06 0,43±0,28 0,36±0,27 0,27±0,19Q. pet. 0,63± 0,12 0,43±0,09 0,20±0,30 -0,07±0,17Q. pyr. 0,65±0,19 0,23±0,12 0,25±0,08 -0,17±0,11Q. pet. 0,56±0,16 0,32±0,16 0,01±0,03 -0,34±0,21

2004 2005

P pd

(MPa

) Cortafuegos

Clara 25 %

Sin aclarar

P md

(MPa

) Cortafuegos

Clara 25 %

Sin aclarar

52

Resultados y discusión

A pesar de que la turgencia celular fue parecida entre las dos especies, las tasas

instantáneas de conductancia estomática, transpiración y fotosíntesis máximas fueron

siempre superiores en las hojas de melojo en los tres lugares de plantación (Figura 4.7;

anexo V).

0

5

10

15

20

-3-2-10

cc

0

200

400

600

8000

2

4

6

8

Ψpd (MPa)

Asa

t(μ

mol

m-2

s-1)

g sat

(mm

olm

-2s-1

)E

sat(m

mol

m-2

s-1)

a

b

c

Figura 4.7. Variación de las tasas de transpiración (a),

conductancia estomática (b) y fotosíntesis (c) a

saturación por luz, con el potencial hídrico del suelo

entorno a las plantas de melojo (símbolos grises) y roble

albar (símbolos blancos). Datos de varias fechas de 2004

y 2005 en los tres lugares de plantación.

Se ha resaltado el hecho de que ciertos atributos foliares, como la masa por unidad

de área foliar, la cantidad de nitrógeno o la tasa de conductancia estomática, se

corresponden con la posesión de tasas elevadas de fotosíntesis. La acumulación de

tejido fotosintético en la hoja es responsable de la relación positiva entre la masa por

unidad de área y la asimilación neta de CO2 descrita en muchos trabajos, aunque esta

relación pueda invertirse o desaparecer debido a la mayor limitación a la difusión de la

luz y el CO2 en hojas más gruesas o densas (Niinemets 1999; Reich et al. 1999). Tanto

la masa por unidad de área como la conductancia de los estomas al CO2 fueron

superiores en las hojas de melojo, lo que puede explicar su elevado potencial de

asimilación por unidad de área.

53

Resultados y discusión

Es difícil aludir, no obstante, a un único motivo para explicar la mayor

conductancia y transpiración de las hojas del melojo, sobre todo en condiciones de

estrés hídrico, ya que la apertura de los estomas está sometida a un complejo sistema de

regulación hidráulico y hormonal. La densidad de estomas puede influir en la tasa de

conductancia al CO2 y, por tanto, en la tasa de fotosíntesis (Woodward et al. 2002). La

comparación de ambas especies en el invernadero puso de manifiesto que la densidad

estomática fue superior en las hojas del melojo (434 ± 20 mm-2 para el melojo y 371 ±

28 mm-2 para el roble albar, en el tratamiento de baja irradiancia y 606 ± 74 mm-2 para

el melojo y 469 ± 52 mm-2 para el roble albar, en el tratamiento alta irradiancia; Fespecie:

4,84*), lo que contribuye a explicar que en condiciones de adecuado estado hídrico la

conductancia estomática fuese también superior en el melojo; se observó una

correlación positiva entre ambas variables (Tabla 4.3). Igualmente existió una relación

inversa entre la superficie foliar total y la tasa de conductancia instantánea (Tabla 4.3),

probablemente debida a la modificación de aspectos hidráulicos de la planta (Kramer &

Boyer 1995). La menor superficie foliar del melojo podría influir en que sus hojas

mantuviesen tasas de conductancia mayores.

Tratamiento Variables R2

70 % de luz densidad 0,67*

SFT 0,62*

Na 0,10n.s.

SLA 0,08n.s.

5,3 % de luz densidad 0,25n.s.

SFT 0,76*

Na 0,38n.s.

SLA 0,25n.s.

Tabla 4.3. Coeficientes de regresión y significación

estadística para las relaciones de la densidad estomática, la

superficie foliar de la planta (SFT), el nitrógeno foliar (Na) y

la superficie foliar específica (SLA) con la conductancia

estomática. Datos del experimento en el invernadero.

El mantenimiento de la asimilación de CO2 a potenciales hídricos bajos se

relaciona con una mayor resistencia a la sequía basada en la tolerancia a la desecación

de los tejidos (Fotelli et al. 2000; Ngugi et al. 2004), aunque muchas de las especies que

evidencian esta respuesta, presentan también tasas de intercambio gaseoso elevadas en

condiciones de ausencia de estrés.

Por el contrario, el control de las pérdidas de agua por transpiración a través del

cierre de los estomas repercute positivamente en la eficiencia con que se asimila el CO2

(DeLucia & Schlesinger 1991; Ares & Fownes 1999). Así, la elevada conductancia

54

Resultados y discusión

estomática de las hojas de melojo pudo contribuir a que el flujo de entrada de CO2

superase la velocidad de asimilación (Figura 4.8); ello implicaría un cierto derroche de

agua, como indica la menor eficiencia intrínseca en el uso del agua bajo luz saturante en

las plantas de melojo al final del verano, estimada a través del cociente entre las tasas de

fotosíntesis y conductancia (Asat/gsat: 0,070 ± 0,003 μmol mmol para el melojo vs 0,067

± 0,002 μmol mmol para el roble albar, bajo el pinar sin aclarar y 0,060 ± 0,003 μmol

mmol para el melojo vs 0,099 ± 0,012 μmol mmol para el roble albar, en el cortafuegos;

Fespecie: 7,71*). Del mismo modo, la discriminación isotópica del isótopo 13C, que sirvió

como estimador de la eficiencia en el uso del agua cuando las condiciones de

intercambio gaseoso no fueron manipuladas experimentalmente, fue mayor en las hojas

del melojo al final del verano (δ13C: -29,1 ± 0,3 ‰ para el melojo vs -27,5 ± 0,3 ‰ para

el roble albar, bajo el pinar sin aclarar y -28,2 ± 0,2 ‰ para el melojo vs -27,5 ± 0,5 ‰

para el roble albar, en el cortafuegos; Fespecie: 13,06**). Bajo las condiciones del

invernadero, la eficiencia intrínseca en el uso del agua fue de nuevo menor en las hojas

de melojo (Asat/gsat: 0,045 ± 0,001 μmol mmol para el melojo vs 0,051 ± 0,002 μmol

mmol para el roble albar, en el tratamiento de baja irradiancia y 0,040 ± 0,002 μmol

mmol para el melojo vs 0,051 ± 0,002 μmol mmol para el roble albar, en el tratamiento

alta irradiancia; Fespecie: 5,13*). No obstante, el hecho de que no existiesen diferencias

significativas al comienzo del verano, cuando el estrés hídrico es menor y la

conductancia estomática al vapor de agua es máxima, apunta a la ocurrencia de otras

limitaciones de carácter no estomático en el campo. La evolución estacional de aspectos

relacionados con la capacidad de asimilación del carbono, como la resistencia a la

difusión de CO2 o del fraccionamiento de nitrógeno en las hojas, podría diferir entre las

dos especies, con relación a una respuesta distinta al estrés estival o a un desarrollo

ontogénico diferente.

Aunque en muchos casos las especies o las poblaciones de una misma especie que

habitan regiones más secas muestran un uso más eficiente del agua que aquellas de

zonas más húmedas (Sandquist & Ehleringer 2003), existen evidencias en el sentido

contrario (Golluscio & Oesterheld 2007), hecho que se relaciona con la capacidad para

aprovechar el agua disponible en los momentos del periodo vegetativo de mayor

disponibilidad hídrica, y que puede favorecer la competitividad de las plantas en las

primeras etapas de desarrollo (Mediavilla & Escudero 2003a).

55

Resultados y discusión

gsat (mmol m-2 s-1)

Asa

t(μ

mol

m-2

s-1)

02468

1012

0 100 200 300

Figura 4.8. Relación entre la conductancia estomática al

vapor de agua y la tasa de fotosíntesis, ambas medidas a

saturación lumínica, en las plantas de melojo (símbolos

grises) y roble albar (símbolos blancos). Datos de julio y

agosto de 2005 en los tres lugares de plantación.

Pese a las mayores tasas de transpiración instantánea detectadas en el melojo

durante el verano, ambas especies mostraron un estado hídrico semejante en la mayoría

de fechas y lugares de plantación. Esto lleva a pensar en un sistema conductor de agua

más eficiente en el caso del melojo (KL en agosto de 2005: 2,32 ± 0,18 MPa para el

melojo vs 1,20 ± 0,20 MPa para el roble albar, bajo el pinar sin aclarar y 2,24 ± 0,24

MPa para el melojo vs 1,07 ± 0,15 MPa para el roble albar, en el cortafuegos; Fespecie:

26,6***). También que ciertas características arquitecturales, como la menor superficie

foliar y el mayor autosombreo de las plantas de melojo, se hubieran traducido en una

reducción de la transpiración total (Kramer & Boyer 1995), lo que explicaría el similar

grado de estrés hídrico en el momento del día de mayor demanda evaporativa (Ψmd en

agosto de 2005: -2,99 ± 0,11 MPa para el melojo vs -2,95 ± 0,17 MPa para el roble

albar, bajo el pinar sin aclarar y -2,83 ± 0,07 MPa para el melojo vs -2,79 ± 0,08 MPa

para el roble albar, en el cortafuegos); más aún, al comprobar que el desarrollo del

sistema radicular en el campo en términos de acumulación de biomasa fue parecido en

ambas especies, lo que supuso que el potencial hídrico del suelo en el entorno de los

brinzales fuese similar en todas las fechas de medición (Ψpd en agosto de 2005: -2,05 ±

0,15 MPa para el melojo vs -2,12 ± 0,2 MPa para el roble albar, bajo el pinar sin aclarar

y -1,24 ± 0,24 MPa para el melojo vs -1,26 ± 0,2 MPa para el roble albar en el

cortafuegos).

4.2.4. Importancia del lugar de plantación en la supervivencia inicial

Las repoblaciones con frondosas han tenido un carácter minoritario en la selvicultura

mediterránea. La escasa experiencia con robles caducifolios apunta a la necesidad de

una cubierta protectora que atenúe la dureza del clima mediterráneo estival (sequía,

56

Resultados y discusión

elevada radiación, granizo, etc.) para aumentar el éxito de la repoblación (Gómez-

Aparicio et al. 2004), una circunstancia que refleja su escasa capacidad colonizadora.

Los resultados de las repoblaciones experimentales que hemos llevado a cabo en las

inmediaciones del Hayedo de Montejo indican que la supervivencia inicial aumenta

cuando se realizan bajo pinares de Pinus sylvestris adultos previamente aclarados, y

disminuye drásticamente en áreas desprovistas de vegetación o en pinares densos sin

aclarar, lo que demuestra el carácter inicial de media luz en ambos robles. El efecto de

la reducción del dosel fue más beneficioso para el melojo en la plantación realizada en

2005 en Sierra Escalva, probablemente porque la sequía fuese demasiado intensa como

para ver algún efecto en la supervivencia del roble albar asociado a la densidad.

En la plantación con robles llevada a cabo en la zona de matorral previamente

desbrozada, la mortalidad se situó en torno al 80 % al final del primer año en campo

para las dos especies, no llegando al 10 % en el interior del pinar. Tres años más tarde,

la mortalidad alcanzó aproximadamente un 95 % en el claro, y se duplicó (en el caso del

roble albar; 57 vs 28 %) o triplicó (en el caso del melojo; 54 vs 16 %) en la zona que no

había sido aclarada con respecto a la que sí lo había sido (anexo III). La ausencia de

vegetación en el claro aumenta la radiación que reciben las plantas, que al no poseer un

sistema radicular que pueda compensar las pérdidas de agua por transpiración se secan

en los primeros meses tras el transplante. Bajo el pinar, el impacto post-transplante es

menor, aunque la mortalidad es sostenida a lo largo de los primeros años en el campo

(Paquette et al. 2006), como puede verse en el anexo VI. La escasez de luz y humedad

en el suelo supone una restricción de la asimilación de carbono, cuyo efecto puede

traducirse en una mortalidad elevada, incluso en el primer año. Ya se ha hecho hincapié

en el compromiso que supone para las plantas la presencia de un dosel denso en la

aclimatación a la sequía. También existen evidencias que apuntan a una limitación de la

capacidad para tolerar la sombra debida al estrés hídrico (Sánchez-Gómez et al. 2006b)

derivada de la restricción adicional que ejerce el cierre estomático en el balance de

carbono de la planta (Aranda et al. 2004b). Ambas circunstancias podrían haber

intervenido en la aparente incapacidad de los brinzales para aclimatarse a las parcelas de

mayor densidad, más sombreadas y secas. No obstante, un hecho que dificulta la

interpretación de los resultados, al menos en Sierra Escalva, fue la herbivoría, que llegó

a alcanzar casi a la totalidad de individuos (91 % de melojos y 96 % de robles albares).

57

Resultados y discusión

La destrucción repetida de los brotes probablemente influyó en la supervivencia de las

plantas, e incluso pudo afectar de manera diferente a una y otra especie (anexo VI).

Estos resultados demuestran que es necesario aclarar el dosel de pinos antes de

llevar a cabo plantaciones en su interior, al generarse ambientes más favorables para el

desarrollo de las plantas, que favorecen además su capacidad de resistencia al estrés.

58

Recapitulación

5. UNA APROXIMACIÓN A LA REGENERACIÓN DEL ROBLE

ALBAR Y EL MELOJO A TRAVÉS DE LA ECOFISIOLOGÍA DE

BRINZALES. LIMITACIONES Y PERSPECTIVAS

Las respuestas morfo-funcionales que han mostrado los brinzales de roble albar y

melojo en el proceso de aclimatación a la variación de la disponibilidad de luz y agua en

los diferentes lugares de plantación contribuyen a explicar el desigual éxito de las

plantaciones. Estas respuestas pueden también relacionarse con la capacidad para

regenerarse de manera natural en las poblaciones donde coexistan, admitiendo la

posibilidad de que ciertos rasgos cambien a lo largo del proceso de desarrollo de los

individuos, o incluso sean diferentes de los que muestren otras poblaciones más

septentrionales (en nuestros experimentos sólo se ha utilizado material genético

procedente del Hayedo de Montejo). La coincidencia de algunos de nuestros resultados

con los que ofrecen otros estudios, nos ha llevado a especular sobre su significado

adaptativo, y su relevancia en los procesos de regeneración natural.

Los individuos de melojo, tanto jóvenes como adultos, poseen una elevada

proporción de biomasa subterránea respecto a la invertida en los brotes y una elevada

tasa de intercambio gaseoso foliar, comparativamente mayores que los valores que

muestran otras especies forestales simpátricas (Gallego et al. 1994; Antúnez et al. 2001;

Mediavilla et al. 2001, 2003c; Gómez-Aparicio et al. 2006; Quero et al. 2006).

Nuestros datos confirman estas observaciones, y sugieren una cierta coordinación entre

un crecimiento conservador y la posesión de hojas poco eficientes en el uso del agua,

una circunstancia consistente con las presiones selectivas que operan en su hábitat

actual: las laderas de la media montaña submediterránea. Aquí, la coincidencia de

periodos secos estivales y heladas tardías obliga a concentrar la fijación de carbono en

un periodo corto mediante la posesión de hojas bien adaptadas a ambientes

relativamente áridos, capaces además de mantener los estomas abiertos a potenciales

hídricos bajos y proporcionar así una adecuada cantidad de foto-asimilados,

primordialmente a las raíces; la elevada inversión de biomasa subterránea con respecto a

la aérea le confiere cierta persistencia ante perturbaciones que destruyen los brotes,

como incendios, movimientos de tierras, o daños por herbívoros.

59

Recapitulación

Por el contrario, la mayor competitividad de los brinzales de roble albar en un

rango más amplio de ambientes lumínicos es coherente con que su distribución

geográfica sea más amplia que la del melojo, dado que la competencia interespecífica

por la luz en las etapas iniciales es un factor determinante del patrón de regeneración en

la mayoría de ecosistemas forestales (Kobe & Coates 1997; Flores et al. 2006; Gómez-

Aparicio et al. 2006). Los brinzales de roble albar demostraron, además, cierto grado de

resistencia a la sequía, al ser capaces de disminuir el potencial osmótico en el punto de

marchitez permanente en situaciones de estrés y exhibir un estado hídrico foliar

parecido al del melojo; en este sentido, la supervivencia del melojo sólo superó a la del

roble albar cuando las condiciones de estrés en el año de plantación fueron extremas

(2005), hechos que confirman el carácter relativamente resistente a la sequía del roble

albar a lo largo de su área de distribución (Backes & Leuschner 2000; Raftoyannnis &

Radoglou 2002), donde desplaza a otras caducifolias como el haya o el carballo en las

localidades más secas.

Sin embargo, ciertas características de los brinzales de roble albar podrían mermar

la capacidad de esta especie para regenerarse en ambientes Mediterráneos. El rápido

crecimiento del roble albar, su tendencia a maximizar la superficie foliar expuesta a la

luz –i.e. mayor superficie foliar específica, individual y total, y menor autosombreo

debido en parte al elevado desarrollo de los tallos respecto al de las raíces –, así como la

menor capacidad para asimilar CO2 en situaciones de estrés hídrico, implicarían, a

priori, una desventaja competitiva frente al melojo en aquellos ambientes donde la

resistencia al estrés (e.g. sequía estival) o a las perturbaciones ambientales de origen

biótico (e.g. herbivoría) o abiótico (e.g. erosión), fuese un factor más selectivo que la

competencia interespecífica por los recursos. Ello contribuiría a explicar la segregación

geográfica entre ambas especies a distintas escalas espaciales. Por un lado, la escasa

presencia del roble albar en las formaciones Ibéricas submediterráneas, cuya

composición se debe en buena parte a la capacidad de adaptación que han tenido las

especies que las integran a los factores mencionados anteriormente; y por otro, la

probable distribución del roble albar en zonas con mayor profundidad de suelo que el

melojo (Pardo et al. 2004, datos de la población del Hayedo de Montejo).

Aunque los resultados con plantaciones podrían diferir respecto a los que se

observan en los procesos de regeneración natural, en los que las plántulas no son

favorecidas durante su cultivo inicial, Cano (2007), tras estudiar el regenerado natural

60

Recapitulación

de ambas especies durante los primeros años en un rango de ambientes de sombra en el

Hayedo de Montejo, observó varios resultados coincidentes con los que se presentan en

este trabajo: hojas más delgadas en el roble albar, con mayor concentración de clorofila

y más eficientes en el uso del agua en el pico de estrés estival que las de melojo. En este

estudio, la mortalidad de ambas especies al cabo del segundo año fue total bajo el dosel

de hayas y robles (< 7 % de luz), de acuerdo con el patrón de regeneración de algunas

especies templadas en poblaciones meridionales que necesitan condiciones de media

sombra durante las etapas iniciales para regenerarse (Castro et al. 2004).

El cambio del clima se revela como un elemento determinante de la composición

específica del Hayedo de Montejo en el futuro. La temperatura del planeta ha

aumentado 0,6 ºC en las tres últimas décadas (Hansen et al. 2006). Los escenarios de

cambio para la zona de estudio señalan incrementos de la temperatura máxima media

anual de unos 2 ºC en las próximas décadas, y de hasta 7 ºC a final de siglo respecto al

periodo de 1960 a 1990 (INM 2007). El aumento de la temperatura podría favorecer la

extensión de las especies arbóreas más sensibles a las heladas, como el haya, el cerezo o

el roble albar, cuya brotación es más temprana que la del melojo. Sin embargo, los

modelos de predicción también apuntan a una reducción de las precipitaciones, que

podría oscilar entre el 5-10 % y el 10-30 % para ambos periodos, según el modelo

empleado (INM 2007). Por ello, a la luz de las diferencias funcionales que existen entre

el melojo y el roble albar, u otras especies como el haya y el acebo (Aranda et al. 1996,

2007), es razonable pensar que a medio plazo se produzca una reducción de la

proporción de robles albares, hayas o acebos en los rodales de contacto o mezcla con el

melojo, tanto por la falta de individuos que se establezcan con éxito y alcancen la

madurez, como incluso por la muerte de individuos adultos.

No obstante, la interacción del clima con otros factores bióticos (herbivoría,

distancias y vectores de dispersión, etc.) o abióticos (profundidad del suelo, topografía,

etc.) hace difícil predecir cual será la evolución de las especies del bosque a corto plazo.

Por ejemplo, la estructuración del monte en bosquetes de unos 200-300 m de radio

(Arcas et al. 2007) limita la dispersión de una especie bajo el dosel de otra, lo cual, en el

caso de que dicha estructuración se correspondiera con la profundidad del suelo (Pardo

et al. 2004), condicionaría que los brinzales de melojo crecieran en lugares con menor

desarrollo edáfico, y fuesen más vulnerables al aumento de la aridez en los próximos

61

Recapitulación

años. La desaparición de individuos de unas especies determinará la disponibilidad de

sitios de regeneración y la velocidad de cambio de otras especies.

Al igual que durante las glaciaciones se produjeron movimientos migratorios de la

vegetación en latitud y altitud, es muy posible que el calentamiento global del planeta, y

el aumento de la aridez, produzca el desplazamiento de especies en el hemisferio norte

hacia latitudes y altitudes superiores, con el consiguiente riesgo de extinción o

reducción de muchos ecosistemas sin posibilidad de desplazamiento, como los de alta

montaña (Williams et al. 2007). La extinción local de especies puede ser especialmente

notoria en las zonas donde coexisten especies en su óptimo de distribución con otras en

situación marginal, mucho más sensibles a pequeñas variaciones del clima; dada la

velocidad de cambio, el impacto será mayor en aquellas especies con ciclos de

regeneración largos. Evitar la desaparición del roble albar en el Hayedo de Montejo, o

de otras poblaciones de otras especies en el límite meridional de su distribución, no es

sólo una cuestión ética, ya que aquellas podrían albergar ecotipos mejor adaptados a la

sequía y constituir la base de futuras repoblaciones. Su conservación pasa por favorecer

la regeneración in situ en orientaciones de umbría, fondos de valle o altitudes superiores

con el fin de compensar el aumento de temperatura y la reducción de la precipitación.

En este sentido, los pobres resultados obtenidos bajo la cubierta de pinos en los ensayos

de Sierra Escalva impulsan a probar lugares de plantación, tratamientos selvícolas

(mayores intensidades de clareo, apertura de claros con distintos tamaños, etc.), fechas

de plantación o métodos de cultivo en vivero (endurecimiento a la sequía, contenedores

forestales, etc.) alternativos. Además, sería interesante comparar la respuesta a la sequía

y la sombra en individuos de varias procedencias del rango de distribución del roble

albar, con el fin de detectar si las poblaciones aisladas más meridionales son más

tolerantes al estrés hídrico o a la sombra (Lesica & Allendorf 1995; Hampe & Petit

2005). Los esfuerzos de investigación también deben extenderse al nivel intraespecifico,

con el fin identificar los individuos más resistentes dentro de la propia masa natural.

62

Conclusiones

6. CONCLUSIONES

1. Los brinzales de roble albar y melojo muestran atributos diferentes a distintos

niveles de organización bajo las mismas condiciones de crecimiento.

2. Los brinzales de roble albar tienen menor proporción de biomasa del sistema

radicular respecto a la parte aérea que los de melojo, con independencia de las

condiciones de crecimiento.

3. Los brinzales de roble albar poseen rasgos que aumentan la eficacia de la captación

de luz con respecto a los de melojo, a través de la posesión de hojas con más

superficie por unidad de masa y mayor concentración de clorofila, y un patrón de

crecimiento en el que el sombreo mutuo entre las hojas es menor.

4. Las respuestas de aclimatación a la luz a corto plazo son semejantes en las dos

especies a nivel foliar, predominando los ajustes fisiológicos sobre los morfológicos

o anatómicos, pero difieren a nivel de la planta entera.

5. Los brinzales de roble albar parecen poseer un fenotipo más competitivo que los de

melojo en un rango de ambientes de media sombra, lo que no repercute en la

supervivencia inicial bajo pinares con distinta cobertura en una zona de clima

submediterráneo.

6. Los brinzales de melojo presentan rasgos más adecuados que los de roble albar para

soportar las condiciones ambientales de zonas submediterráneas, entre ellos, un

patrón de crecimiento más conservador (menor superficie foliar y mayor proporción

de biomasa en estructuras subterráneas que aéreas) y la posesión de hojas capaces de

mantener tasas más elevadas de intercambio gaseoso en situaciones de estrés

hídrico.

7. Las diferencias en la capacidad para tolerar la sequía estival parecen más

determinantes que las diferencias en la capacidad para captar luz en los procesos de

regeneración artificial en zonas submediterráneas.

8. La supervivencia inicial de robles plantados en el entorno del Hayedo de Montejo

aumenta en pinares de pino silvestre previamente aclarados con respecto a zonas de

pinar no aclaradas o claros de distinto tamaño.

63

Conclusiones

9. El aumento de la densidad de pies de pino silvestre está relacionado con una

reducción de la disponibilidad de luz y agua para las plantas instaladas bajo su

dosel, lo que influye negativamente en su estado hídrico.

10. El aumento de la luz disponible en el sotobosque favorece el desarrollo de algunos

mecanismos de tolerancia a la sequía, como es el caso del ajuste osmótico en los

brinzales de roble albar.

11. El retraso ontogénico que genera la ausencia de luz bajo el dosel de pinar parece

aumentar la susceptibilidad de los brinzales de ambas especies a la reducción de la

humedad del suelo en verano.

64

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ANEXO I

Leaf physiological versus morphological acclimation after high light

exposure at different foliage developmental stages

Manuscrito aceptado en la revista Tree Physiology

Autores: Rodríguez-Calcerrada J., Reich P.B., Rosenqvist E., Pardos J.A., Cano F.J.,

Aranda I.

Leaf physiological versus morphological acclimation after high light exposure at

different foliage developmental stages

J. RODRÍGUEZ-CALCERRADA1, P.B. REICH2, E. ROSENQVIST3, J.A. PARDOS1,

F.J. CANO1 and I. ARANDA4, *

1 Unidad de Anatomía, Fisiología y Genética Forestal. Escuela Técnica Superior de

Ingenieros de Montes, Universidad Politécnica de Madrid (UPM), Ciudad Universitaria

s/n, E-28040, Madrid, Spain. Unidad Mixta INIA-UPM.

2 Department of Forest Resources, University of Minnesota, St. Paul, 55108, USA.

3 Department of Agricultual Science, University of Copenhagen, Hojbakkegaard Allé

21, DK-2630 Taastrup, Denmark.

4 Centro Nacional de Investigación Forestal (CIFOR). Instituto Nacional de

Investigación Agraria y Alimentaria (INIA), Apdo. 8111, E-28080, Madrid, Spain.

Unidad Mixta INIA-UPM.

∗ Author for correspondence (e-mail: aranda@inia.es; phone: 0034913476857; fax:

0034913476767).

Running head: LEAF RESPONSES TO LIGHT IN TWO CO-OCCURRING WHITE

OAKS

Anexo I - 1

Summary

We investigated the mechanisms of leaf acclimation to light in seedlings of two co-

occurring oak species, the temperate Quercus petraea (Matt.) Liebl. and the sub-

Mediterranean Quercus pyrenaica Willd. Seedlings were raised in a glasshouse for one

year under two contrasting light levels (LL – 5.3 % of full sunlight vs HL – 70 % of full

sunlight) and the next year subsets of the LL seedlings were transferred to HL either

before leaf flushing (BF) or after the leaf lamina had completely expanded (AF). Gas

exchange, chlorophyll a fluorescence, nitrogen fractions in photosynthetic components

and leaf anatomy were examined in all four treatments five months after transfer.

Differences in acclimation to high light of leaves of shade-developed plants between the

two species were minor. Area-based photosynthetic capacity (Aamax) and related

parameters, maximum rate of carboxylation (Vacmax), maximum rate of electron

transport (Jamax) and the effective photochemical quantum yield of PSII (ΦPSII) increased

in transferred plants of both species on exposure to high light to values similar to HL

plants. A rapid change in the pattern of nitrogen distribution among photosynthetic

components was observed in AF plants, which showed the highest photosynthetic

nitrogen use efficiency (PNUE). Increases in mesophyll thickness and dry mass per unit

area (MA) governed leaf acclimation in BF plants, which showed a tendency to have

lower nitrogen in photosynthetic components and had lower assimilation potential per

unit leaf mass or leaf nitrogen than AF plants. It is suggested that the phenological state

of the seedlings modifies the acclimatory response of leaf attributes to a change from

low to high light. Morphological adjustments in the leaves of shade-developed plants

exposed to high light before flushing enhance photosynthetic capacity per unit area, but

not per unit dry mass, whereas the strong redistribution of nitrogen among

Anexo I - 2

photosynthetic components in shade-developed leaves exposed to high light after

flushing enhance both mass- and area-based photosynthetic capacity.

Keywords: photosynthetic acclimation, nitrogen partitioning, competitive ability,

Quercus pyrenaica, Quercus petraea.

Anexo I - 3

Introduction

Plants are adapted to regulate the photosynthetic processes in response to the fluctuating

light conditions that take place at different time scales during their life span (Külheim et

al. 2002; Schurr et al. 2006). For example, the onset of mechanisms of thermal

dissipation in the light-harvesting complexes, as well as the engagement of alternative

non-photosynthetic electron pathways can protect the photosynthetic apparatus from

oxidative damages after shaded plants are exposed to sudden increases in light intensity

(Ort 2001). However, the capacity of a plant to acclimate its foliage to a new light

environment depends in the long run on the capacity to progressively readjust its

organization from one of high light-use efficiency at low light to one of high

photosynthetic capacity at high light (Hikosaka and Terashima 1995). Acclimation in

this way will result in increasing carbon assimilation while at the same time will reduce

the possibility of photo-inhibition (Baker and Oxborough 2004).

Nitrogen availability, allocation, and remobilization (e.g. increasing uptake by

roots, or translocation among plant organs or photosynthetic components) all play a role

in this process (Naidu and De Lucia 1997a; Ramalho et al. 2000; Frak et al. 2001;

Walters 2005). Within leaves, the relative amounts of chlorophyll to nitrogen (e.g.,

Ellsworth and Reich 1992), or more specifically, to Rubisco, cytochrome f, or the

electron transport rate, decrease with an increase in light intensity (Yin and Johnson

2000; Walters 2005) suggesting a turnover of nitrogen from light-harvesting complexes

to energy-processing components that balances antenna size relative to photosystem

content. Studies addressing nitrogen fractioning within leaves (i.e. light harvesting

pigments, electron transport chain proteins, carbon fixation enzymes, and non-

photosynthetic components) show a relatively rapid modulation if the intercepted light

Anexo I - 4

environment changes (Frak et al. 2001). On the contrary, anatomical features of shade-

developed leaves do not change so readily (Eschrich et al. 1989; Sims and Pearcy

1992), limiting in some instances a complete acclimation of photosynthesis (Tognetti et

al. 1998; Oguchi et al. 2003, 2005).

The extent and velocity of the acclimation of photosynthesis to increasing light

differ between species of contrasting adaptive strategies. For example, drought-adaptive

traits may act as carry-over effects constraining acclimation of photosynthesis to

increasing light (see Valladares et al. 2000), whereas shade-adaptive traits may entail a

trade-off to balance light absorption and use in a higher light environment (Seemann et

al. 1987; Strauss-Debenedetti and Bazzaz 1991). Studies of this kind of sympatric and

closely related species are rare however. To address this gap, we study the temperate,

deciduous species Quercus petraea (Matt.) Liebl. and the sub-Mediterranean,

marcescent species Quercus pyrenaica Willd. The two oaks coexist in several scattered

stands throughout central and Midwestern Spain, where Q. petraea is at the southern

extreme of its distribution. Studying the response of these seedlings to light can also

help to predict their performance within such populations, as interspecific differences in

light requirements appears to be an important factor for the distinct recruitment pattern

of Mediterranean tree species (Gómez-Aparicio et al. 2006).

The objective of the present work was to explore the relative contribution of

functional and structural leaf attributes in short- to long-term acclimation to light by

comparing key photosynthetic traits of leaves of shade-developed plants exposed to

high light before and after leaf flushing, as well as of leaves of plants always maintained

under constant low- or high-light intensity. The timing of exposure to high light has not

received much attention in studies of photosynthetic acclimation. We firstly

hypothesized that physiological changes, particularly in processes reflecting nitrogen

Anexo I - 5

fractioning within leaves, would mediate acclimation to high light of leaves of shade-

developed plants, while morphological changes, mainly in the accumulation of

mesophyll tissue, would determine acclimation for longer-term periods; secondly we

thought that differences in the timing of exposure, as well as in the leaf traits of

ecologically distinct Q. pyrenaica and Q. petraea would affect the magnitude of, or the

mechanisms for acclimation.

Material and Methods

Experimental treatment and design

In Spring 2004, seeds of Q. petraea and Q. pyrenaica taken from a mixed forest at the

extreme range of the distribution of Q. petraea (41º 7’ N, 3º 30’ W) were sown in 400-

cm3 plastic pots of 35 cm depth filled with a mixture of fertilized peat and sand (3:1,

v/v) containing slow-release fertilizer (5 g dm-3), and placed in a glasshouse under two

light treatments: low light (LL; double layer of neutral shadecloth, 5.3 % of full

sunlight; midday PAR on a sunny day: 80 ± 7 μmoles m-2 s-1) and high light (HL; no

shadecloth, 70 % of full sunlight due to the opacity of fiberglass claddings; midday

PAR: 1050 ± 28 μmoles m-2 s-1). At the end of the growing season, seedlings were

transplanted individually into 3000-cm3 PVC tubes of 40 cm depth, and newly enriched

with slow-release fertilizer. LL seedlings were distributed among four adjustable

shading structures placed on two benches of the glasshouse.

The next year, six containerized plants per species were moved out from low light

to high light before bud burst (27th February, approximately one week before bud burst;

BF treatment) and after the leaf lamina had completely expanded (6th June, three months

Anexo I - 6

after leaf emergence; AF treatment). They were put together with HL plants in between

the shading structures, at a sufficient distance as to avoid shading. First-flush leaves

from LL, AF, BF and HL plants were measured, or harvested for upcoming

measurements, five months after first transfer took place (3rd – 7th July). Photosynthesis

was also measured the week before second transfer. There were no significant

differences between LL plants and those about to be moved (AF), either between dates

(June vs July) for LL and HL plants (data not shown). Night-day ranges for temperature

and relative humidity in the glasshouse were 15-39 ºC and 50-90 %, respectively. All

seedlings were well watered over the course of the experiment.

Gas exchange measurements

Net CO2 assimilation rate (An) was measured with an IRGA (LC pro Analytical

Development Corporation, UK) at constant light (1000 μmol m-2 s-1 for HL and 700

μmol m-2 s-1 for LL, BF and AF plants) and leaf temperature (25.8 ± 0.1 ºC) over a

broad range of intercellular CO2 (Ci) resulting from changing the CO2 supply in twelve

steps from 50 to 1800 ppm. After 30 min at saturating light and 380 ppm of CO2, the

supply of CO2 was reduced step-wise to minimum values; then increased to 380 ppm

again, and lastly increased to high values. Photosynthetic capacity on a leaf area basis

(Aamax) was estimated as the mean value of the three records taken at 1800 ppm and

saturating light. A non-linear least squares fitting procedure was performed to estimate

the maximum rate of carboxylation (Vacmax) and the maximum rate of electron transport

(Jamax) from An – Ci curves. Regression models were constructed according to equations

of Farquhar et al. (1980), in which An is modelled as the minimum value of Rubisco-

limited (Ac) and RuBP-limited (Aj) rate of photosynthesis:

Anexo I - 7

djcn ),min( RAAA −= (1)

)/O1(C oci

*i

cmaxa

c KKC

VA++Γ−

= (2)

)/(4 i

*i

j τ+Γ−

=OC

CJA (3)

Rd is the mitochondrial respiration, and was estimated from Ac. The concentration of

oxygen (O) was considered 20 KPa. Temperature-dependent parameters Kc (Michaelis-

Menten coefficient of Rubisco for CO2) and Ko (Michaelis-Menten coefficient of

Rubisco for O2) were calculated for the leaf temperature of each curve following the

equations derived by Bernacchi et al. (2001); temperature dependency of τ (CO2

specificity factor) was accounted for using the equation derived by Harley et al. (1992).

The CO2 compensation point in the absence of mitochondrial respiration in light (Γ*)

was calculated as O/2τ. The light dependence of the potential rate of electron transport

(J) was calculated as:

2

maxa1 ⎟

⎠⎞

⎜⎝⎛ α

+

α=

JQ

QJ (4)

where α is the efficiency of light utilization 0.24 mol e- (mol quanta-1) and Q is the light

intensity.

All these parameters were entered for modeling functions. Estimates of Vacmax and

Jamax at 25 ºC were then obtained by re-arranging temperature-dependent equations of

Vacmax and Ja

max given by Dreyer et al. (2001) for Q. petraea. Mass-based estimates

(Ammax, Vm

cmax and Jmmax) were obtained by dividing area-based values by the leaf dry

mass per unit area (MA).

Anexo I - 8

Nitrogen content and nitrogen fractioning among leaf proteins

Values of Vacmax and Ja

max at 25 ºC were used to calculate leaf nitrogen fractions in

Rubisco (Pr) and photosynthetic electron transport proteins (Pb), respectively, following

equations in Niinemets and Tenhunen (1997):

mAcr

maxca

r 25.6 NMVVP = (5)

mAmc

maxa

b 06.8 NMJJP = (6)

where the value 6.25 g Rubisco (g N in Rubisco-1) converts nitrogen content to Rubisco

protein content, and the value 8.06 μmol cytochrome f (g N in bioenergetics)-1 is a

conversion factor that results from assuming a constant 1:1:1.2 (cytochrome f:

ferredoxin NADP reductase: coupling factor) stoichiometry controlling the electron

transport. Vcr is the specific activity of Rubisco at 25 ºC (20.5 μmol CO2 (g Rubisco)-1 s-

1) and Jmc is the capacity of electron transport per unit of cytochrome f at 25 ºC (156

mol electrons (mol cytochrome f)-1 s-1). Leaf nitrogen content per unit dry mass (Nm)

was measured, petioles excluded, using the Kjeldahl procedure (Bradstreet 1965). MA

was used to quantify nitrogen content on an area basis (Na = Nm MA).

Values of leaf chlorophyll content per dry mass (Cm) (determined following

Barnes et al. 1992) were used to calculate the fractions of chlorophyll associated with

PSI, PSII and LHCII. Previously, we calculated the concentration of these protein

complexes on a leaf area basis according to equations in Hikosaka and Terashima

(1995) and Niinemets and Tenhunen (1997). Fraction of nitrogen in light-harvesting

components (Pl) was computed as:

Bm

ml CN

CP = (7)

Anexo I - 9

where CB is the weighted average of chlorophyll bindings of the three protein

complexes (see Hikosaka and Terashima 1995).

The proportion of structural nitrogen was calculated as Ps = 100 - Pl - Pr - Pb.

Photosynthetic nitrogen use efficiency (PNUE) was estimated as Ammax / Nm.

Chlorophyll fluorescence measurements

Light response curves of chlorophyll fluorescence parameters were measured with a

portable pulse-modulated fluorometer (FMS 2, Hansatech Instruments Ltd., Norfolk

UK) to examine PS II acclimation. Attached leaves of five plants per treatment of Q.

petraea and Q. pyrenaica were alternately measured during three days 11:00–14:00 h

local time. Measurements were carried out in a growth chamber where the leaf

temperature was kept around 25 °C by setting the air temperature to 23.5 °C and relative

air humidity to 65%. However, for the two highest light levels a fan was also used to

prevent the leaf temperature from rising to 28 °C. The leaves were darkened for 20

minutes prior to measurements to obtain minimum (Fo) and maximum (Fm) values of

fluorescence by applying a 0.8 s saturating pulse (PPFD = 6600 μmol m-2 s-1). When the

fluorescence signal approached steady-state (Fs) in actinic light, a similar flash was

applied to obtain a value of the maximum fluorescence in light (Fm'). The minimum

fluorescence in light (Fo') was measured at each light level by applying a 5 s far-red

light pulse in temporary darkness to drain electrons from electron acceptors of PS II.

The redox state of the primary electron acceptor QA of PSII (qL) was calculated

according to Kramer et al. (2004):

s

o

om

sm '''

'qL

FF

FFFF

−−

= (8)

Anexo I - 10

The yield of the three competing pathways of de-excitation of chlorophyll in PSII, i.e.

the yields of photochemistry of PSII (ΦPSII), downregulatory non-photochemical

processes (ΦNPQ) and other energy losses (ΦNO) were also calculated (Kramer et al.

2004):

'm

s'

mPSII F

FF −=Φ (9)

NOPSIINPQ 1 Φ−Φ−=Φ (10)

and

)1/(qL1NPQ(1

o'

mNO

−++=Φ

FF (11)

where NPQ is the non-photochemical quenching of absorbed energy at each light level:

'm

'mmNPQ

FFF −

= (12)

The rate of electron transport through PSII was calculated following Rosenqvist and van

Kooten (2003):

84.0PPFD5.0ETR PSIIΦ= (13)

Non-linear regression models were fitted to describe the variation of

photochemical and non-photochemical yields with PPFD for each seedling. The light

response of ETR was modeled using a single exponential function (Rascher et al. 2000)

to estimate the maximum electron transport rate (JETR). Steady-state was estimated from

the Fs value, not from Fm', which requires longer time to reach a true steady-state. The

curves thus resemble rapid light response curves where the apparent rate of electron

transport is slightly underestimated (White and Critchley 1999). Due to the human

presence in the growth chamber the CO2 concentration increased from 380 to 550 ppm.

Anexo I - 11

Hence we could not assess mesophyll conductance from combined gas exchange and

chlorophyll fluorescence measurements.

Anatomical variables

Sections of leaf blades held in fresh carrot pith were cut around the middle region and

immersed in formalin-acetic acid-ethanol (FAA; 5:5:90) for 24 hours. The FAA was

then changed for ethanol (70º) until analysis. Measurements were made with a light

microscope, in cuts halfway between the mid-rib and the edge. The cell content was

destroyed with sodium hypochlorite and further stained to better distinguish tissues. The

thickness of adaxial plus abaxial epidermis (TE), palisade parenchyma (TPP), spongy

parenchyma (TSP) and leaf lamina (TL) were measured at three locations on five leaves

per species and light treatment.

Statistical analysis

Most variables were transformed to meet assumptions of parametric analysis. We

performed two-way analyses of variance (ANOVA) to test for the significance of light

treatment and species factors on each variable. To test the hypothesis that acclimation is

different between species, we included the interaction term between light treatment and

species in the variance model, as it tests for the statistical significance of species

difference in acclimation of variables. We used the Tukey’s Honest Significant

Difference (HSD) test to explore which means were significantly different (at P < 0.05)

among the treatments.

Anexo I - 12

Results

Gas exchange parameters

Parameters expressed on an area basis were higher in HL than in LL. For both species,

Aamax increased following transfer in AF and BF plants to values slightly lower but non-

significantly different to HL plants, and significantly higher (50%) than LL plants.

Maximum rates of electron transport (Jamax) and carboxylation (Va

cmax) increased on

exposure to high light in BF and AF plants with respect to LL (Table 1). On a mass

basis, Ammax, Vm

cmax and Jmmax did not differ between LL and HL plants. Am

max was

greater in AF plants than in all other treatments, and it did not differ significantly

among LL, HL and BF seedlings. Vmcmax and Jm

max were higher in AF than in BF and

were generally at intermediate values in LL and HL (Table 1). There were clear

differences between Q. pyrenaica and Q. petraea irrespective of the light treatment, but

both species responded similarly to the four different treatments. Q. petraea had higher

Ammax, Vm

cmax and Jmmax than Q. pyrenaica, but differences disappeared when the

variables were expressed on an area basis (Table 1). No effect of species or light

treatment was noticed for Jmax/Vcmax. Mesophyll resistances to CO2 diffusion, not taken

into account in this study, would have lowered estimates of Vcmax from real values, but

barely affected Jmax, resulting in the large Jmax/Vcmax ratios observed across treatments

(Piel et al. 2002).

Anexo I - 13

Chlorophyll fluorescence parameters

As expected, JETR and qL were greater in HL than in LL in both species (Table 2). JETR

and qL at high light (1100 μmol m-2 s-1) were similar among AF and BF plants,

significantly greater than LL and lower, but not significantly than HL plants (Table 2).

The relative contribution of photochemical (ΦPSII) and downregulatory non-

photochemical (ΦNPQ) mechanisms of processing absorbed light was similar among BF,

AF and HL plants, and different from LL plants (Table 2; Figure 1). In LL plants, ΦPSII

was lower and ΦNPQ higher than in the other treatments. Accordingly, the light level at

which ΦNPQ > ΦPSII was lowest in LL plants (~ 225 μmol m-2 s-1) and higher (~ 700

μmol m-2 s-1) in BF, AF and HL plants of Q. pyrenaica, and similarly ranged from ~

350 μmol m-2 s-1 in LL to ~ 500-550 μmol m-2 s-1 in BF, AF and HL plants of Q.

petraea (Figure 1). The pattern of unregulated non-photochemical mechanisms (ΦNO)

did not vary between species or light treatments. Seedlings of Q. pyrenaica had greater

JETR and ΦPSII(1100) than Q. petraea. It is worth noting that ΦNPQ(1100), and the

curvature of the light-response curves of ΦPSII and ΦNPQ (parameter b of non-linear

regression models; Figure 1), barely changed in AF seedlings of Q. petraea with respect

to LL, but they did in Q. pyrenaica (Table 1; Pinteraction < 0.05 only considering LL and

AF treatments).

Biochemical parameters

Nm was lower in HL and BF than in LL, while it was intermediate in AF seedlings. On

the contrary, Na was similarly higher in HL and BF than in the other two treatments

(Table 3). LL plants had a higher proportion of leaf nitrogen in light-harvesting

Anexo I - 14

components (Pl) than HL and AF plants, with BF having intermediate values.

Proportion in photosynthetic electron transport proteins (Pb) was slightly higher in HL

than in LL, AF plants exhibiting the highest values, and BF values more similar to LL

than to HL. Light treatment had a weak effect on nitrogen fractioning to Rubisco (Pr) (P

= 0.048), being higher in AF than in BF and similar between LL and HL. There was not

a significant effect of light treatment on non-photosynthetic nitrogen (Ps); however,

there was a tendency of a higher proportion of non-photosynthetic nitrogen in BF.

When nitrogen fractions were calculated per unit of photosynthetic nitrogen, LL and BF

plants had lower nitrogen in Rubisco [Pr/(Pr+Pb+Pl)] than AF and HL plants, and

nitrogen in bioenergetics [Pb/(Pr+Pb+Pl)] was similarly higher in HL, BF and AF plants

than in LL plants (data not shown). PNUE was highest in AF, and intermediate in HL

plants and similar in both species. There were differences between Q. pyrenaica and Q.

petraea in the concentration of nitrogen and the pattern of fractioning within the leaf,

which did not change with the light treatment. Nm and Pl were significantly higher in Q.

petraea than in Q. pyrenaica, while the reverse was true for Na and Ps.

There was a clear tendency for seedlings having a greater fraction of nitrogen in

photosynthetic components to show higher photosynthetic capacity (Figure 2). Ammax

was positively correlated with both Pr and Pb in both oaks (Figure 2 a, b). As a result, a

negative relationship was found between Ammax and non-photosynthetic nitrogen (Figure

2 d). No significant relationship was observed between Ammax and Pl or Nm (Figure 2 c,

e). Similar results were obtained with Vmcmax and Jm

max, instead of Ammax (data not

shown). There was a positive correlation between MA and Ps (r2: 0.19, P < 0.1 for Q.

pyrenaica and r2: 0.26, P < 0.05 for Q. petraea).

Anexo I - 15

Morphological and anatomical parameters

As expected, MA was lower in LL than HL plants. For transferred plants, leaves that

developed in high light (BF) had similar MA as HL plants, with MA intermediate in AF

plants (Table 4). MA was higher in Q. pyrenaica than in Q. petraea across all light

treatments. Lamina thickness changed with light mainly owing to changes in the

palisade parenchyma (Table 4, Figure 3). Leaves of HL and BF plants were thicker and

had thicker palisade parenchyma (generally with two layers of palisade cells) than LL

and AF plants, with some differences between species in these patterns (Pinteraction <

0.05). There was a trend for seedlings of Q. pyrenaica to have thicker palisade

parenchyma in HL than Q. petraea, but thinner in LL. Shade-developed leaves (AF)

barely altered their anatomy on exposure to high light, however, two out of the five

samples of Q. pyrenaica (none of Q. petraea) had a double layer of palisade cells,

resulting in a slight increase in lamina thickness with respect to LL samples, which had

a single layer of palisade cells. There were not significant effects of light or species on

the thickness of epidermis and spongy parenchyma tissues. Therefore, the ratio of

palisade to spongy parenchyma thickness was similarly greater in BF (1.63) and HL

(1.46), than in LL (0.67) and AF (0.87) (Pspecies > 0.15; Pinteraction > 0.15).

Discussion

Plasticity to light of leaves in long-term acclimated plants (LL vs HL)

Both oaks acclimated to low and high light by displaying contrasting functional and

structural leaf features consistent with general expectations. HL plants had thicker

Anexo I - 16

leaves, with a thicker palisade parenchyma, and superior maximum carboxylation rate,

electron transport rate and photosynthetic capacity per unit area than LL ones (e.g.

Ellsworth and Reich 1992; Evans and Poorter 2001). Such adjustments make HL plants

better able to take advantage of high radiation than LL ones, as they enable a more

active regeneration of NADP and ADP that alleviates the over-reduction of PS II

centers at high PPFD and the risk of suffering photoinhibition with respect to LL plants

(Chow 1994; Baker and Oxborough 2004). This would partly explain the gentler decline

in ΦPSII with increasing PPFD in HL (Figure 1), although differences in the rate of

photosynthetic induction between LL and HL plants cannot be excluded.

Acclimation of chloroplast ultrastructure to high light extends in some instances to

the plastic fractioning of nitrogen pools among its components, such that photosynthetic

capacity increases but the efficiency in low light decreases (Hikosaka and Terashima

1995; Niinemets and Tenhunen 1997; Niinemets et al. 1998). Nitrogen in Rubisco per

leaf nitrogen content was similar between treatments, but there was a trend for nitrogen

in electron transport components to increase in HL plants at the expense of a lower

investment in light harvesting (Table 3). Both nitrogen fractions in Rubisco and

bioenergetics per unit of photosynthetic nitrogen were also higher in HL than in LL

plants. Light-induced increases in leaf dry mass per area (i.e. increased mesophyll

tissue) are sometimes more important to enhancing photosynthetic capacity than

variations in nitrogen allocation within leaves (Ellsworth and Reich 1992; Evans and

Poorter 2001; Parelle et al. 2006; Katahata et al. 2007), which is here further supported

by the constant amount of photosynthetic machinery per unit dry mass observed in LL

and HL seedlings of both species (i.e. similar values of Vmcmax, Jm

max and Ammax ; Table

1).

Anexo I - 17

Hence seedlings of both Q. petraea and Q. pyrenaica had the ability to acclimate

to the prevailing light level, and they did it in slightly different ways. The former had

greater plasticity on nitrogen concentration and partitioning to light-harvesting

pigments, and the latter had greater plasticity in mesophyll thickness, chlorophyll

fluorescence parameters and area-based gas exchange parameters. These results are

consistent with a slightly superior tolerance to shade of Q. petraea (Rodríguez-

Calcerrada et al. 2007a, b) as discussed elsewhere (Niinemets 1997; Cao 2000;

Niinemets and Valladares 2004).

Acclimation of leaves of shade-developed plants to high light before flushing (BF)

The leaf dry mass per unit area (MA) of BF leaves increased to HL values five months

after transfer, at least in part corresponding to an increase in mesophyll thickness,

namely in the palisade parenchyma (Table 4). Other authors have reported significant

increases of the lamina thickness in tree species of various successional positions and

shade-tolerances after light intensity increased before bud break (Goulet and Bellefleur

1986; Aranda et al. 2001), which suggest a considerable anatomical flexibility in shade-

induced primordia (see Eschrich et al. 1989). It is likely that cells of the palisade

parenchyma enlarged to a greater extent than LL leaves while developing in high light

and then divided into a new layer, increasing the proportion of chloroplasts in the

palisade, better able to take advantage of high light than chloroplasts in the spongy

parenchyma (Terashima and Inoue 1984).

Therefore, leaf acclimation to high light was governed by the accumulation of

photosynthetic tissue per unit area. Area-based photosynthetic parameters of BF leaves

increased as much as in AF plants, and were not significantly different from HL. On the

Anexo I - 18

contrary, mass-based estimates (Vmcmax, Jm

max and Ammax) were similar or slightly lower

than in LL and HL, but clearly lower than in AF plants (Table 1). Poor nitrogen

readjustments among photosynthetic components on exposure to high light would have

contributed to reduce photosynthetic efficiency per unit dry mass in BF plants. It is hard

to tell whether differences in nitrogen assignment and mass-based photosynthetic

parameters between BF and AF seedlings were due to the longer exposure to high light

in BF plants (five vs one month), which had eventually affected photosynthetic

performance, or rather to the moment of exposure (before vs after flushing). However,

since the HL leaves also were subjected to high light for the same time period as the BF

plants and in most other respects exhibited similar responses as the BF plants, it is likely

that the timing relative to leaf flushing of the transfer, rather than the length of time

transferred, was responsible for the mass-based and N-related physiology of the BF

plants. The completion of leaf development in a new, higher light environment in BF

seedlings could have shaped leaves not to maximize photosynthetic efficiency but rather

to resist other stress factors linked to high light (e.g. higher evaporative demand).

Increasing MA could be partly ascribed to increasing thickness of mesophyll cell-walls,

which would explain the tendency for higher structural nitrogen in these leaves and

could limit CO2 diffusion into the chloroplasts (Miyazawa and Terashima 2001), in both

cases reducing their nitrogen use efficiency (Table 3).

Acclimation of shade-developed leaves to high light after flushing (AF)

Both oaks showed a great ability to acclimate their leaves to high light after flushing in

low light. Other tree species, both evergreen and deciduous, have shown the ability to

fully acclimate the photosynthetic capacity of shade-developed foliage to increasing

Anexo I - 19

light intensity (Strauss-Debenedetti and Bazzaz 1991; Naidu and DeLucia 1997b and

1998). Although the risk of being photoinhibited for the remainder of the summer

season should pose a strong driving force for acclimation, even for leaves transferred to

HL half-way through their life-span, it was not obvious to us that fully expanded leaves

of that age would retain much plasticity. It has been observed that the modest change in

thickness of shade-developed mature leaves limits the extent to which photosynthesis

acclimates to increasing light (Oguchi et al. 2003). In this study, only four weeks after

exposure to high light, the photosynthetic capacity, the maximum carboxylation rate and

the maximum electron transport rate per unit area all increased to values non-

significantly different to leaves of plants constantly maintained in high light for two

years, despite little acclimation in leaf anatomy. Accordingly, estimates of key leaf traits

of photosynthesis on a mass basis (Vmcmax and Jm

max) were rapidly modulated by the

change in the light intensity, and showed a trend to be higher than in HL or LL plants;

photosynthetic capacity per unit dry mass and nitrogen use efficiency were also highest

in AF plants (Tables 1-3). We suggest that the rapid reorganization in the protein pool

largely accounted for this pattern (Yamashita et al. 2000; Frak et al. 2001; Han et al.

2006), and contributed to overcome carry-over effects from anatomical acclimation of

leaves to shade. On exposure to high light, nitrogen in light harvesting decreased in

parallel to increasing partitioning to electron transport components and Rubisco,

suggesting a rapid re-mobilization of nitrogen among photosynthetic components. The

degradation of chlorophyll-binding proteins in response to high light can facilitate the

synthesis of Rubisco and compounds involved in the electron transport (Yang et al.

1998; Walters 2005). These adjustments translate into a higher capacity for using high

levels of radiation in photosynthesis. As such, even though non-photochemical energy

quenching plays an important role in protecting the PS II from oxidative damages

Anexo I - 20

within the first days upon exposure to high irradiance (Naidu and DeLucia 1997b;

Müller et al. 2001; Rodríguez-Calcerrada et al. 2007a), acclimation of photochemistry

reduced the level of excess energy (Rosenqvist and van Kooten 2003; Baker and

Oxborough 2004) and thus leveled off thermal dissipation of AF plants to that of HL

and BF ones (Figure 1, Table 2). This was more evident for Q. pyrenaica, probably in

relation to a different acclimation in the rate of photosynthetic induction, as suggests the

similar acclimation of photosynthetic capacity in both species.

Other factors might have contributed to this large increase in photosynthetic

capacity. For instance, Oguchi et al. (2003 and 2005) found that the surface of

chloroplasts facing the intercellular spaces increased after transfer from low to high

light as a result of an enlargement in chloroplast volume. Besides, experimental

conditions could have influenced the response of seedlings. The use of fertilized, well-

watered plants could have favored their response to light (Ramalho et al. 2000; Parelle

et al. 2006), and the moderate light intensity to which seedlings were exposed when

moved out the shading structures (around 1000 μmol m-2 s-1 maximum PAR) may also

have enhanced acclimation potential of leaves (Naramoto et al. 2006), as proved by the

slight decrease in Fv/Fm immediately after transference (Rodríguez-Calcerrada et al.

2007a).

Interspecific differences in leaf acclimation of shade-developed plants

We hypothesized that interspecific differences in leaf traits would have an influence on

the response to high light. We saw clear differences in many leaf traits between the two

species, but these differences were in general not significantly altered by the light

regime. Whereas seedlings of Q. pyrenaica had a greater capacity for electron transport

Anexo I - 21

at high radiation (ΦPSII(1100) and JETR), those of Q. petraea had higher Ammax, Vm

cmax,

Jmmax, Pl, and lower MA and Ps than Q. pyrenaica (Tables 1-4). Poorer photosynthetic

performance per unit dry mass in seedlings of Q. pyrenaica could be partly attributed to

the higher proportion in non-photosynthetic nitrogen, but differences in photorespiration

and internal diffusion of CO2 should not be ruled out. Additionally, the lesser growth in

Q. pyrenaica in the month preceding photosynthetic measurements (66 % lower height

growth all treatments pooled; data not shown) could have caused an end-product

inhibition of photosynthesis with respect to Q. petraea (Paul and Foyer 2001). Overall,

these results point to a greater tolerance to shade, and a somewhat more “high-

metabolism” leaf physiological strategy in the seedlings of Q. petraea than Q.

pyrenaica (Reich et al. 1999), except for their similar photosynthetic nitrogen use

efficiency (Wright et al. 2001; Lusk et al. 2003). But despite these results, neither

difference in the mechanisms nor barely in the extent of acclimation was observed for a

plethora of leaf traits, perhaps because ecological differences between the two oaks are

small enough that distinct patterns of acclimation of photosynthesis in optimal nutrient

and water conditions should not be expected. The only differences in acclimation

response of the two species involved AF seedlings, in which Q. pyrenaica had a trend

for thicker leaves and palisade parenchyma than low-light plants, whereas Q. petraea

had not. Significant enlargement of mesophyll cells were observed in mature leaves of

deciduous species Acer rufinerve on exposure to high light (Oguchi et al. 2005).

However, it is likely that the second layer of palisade cells in some samples of Q.

pyrenaica was already formed before transfer.

In summary, leaves of both oaks strongly acclimated photosynthetic capacity to

high light via adjustments in physiological and morphological features. The nature of

Anexo I - 22

such adjustments varied with the time of exposure relative to the time of leaf expansion,

being quite similar in both species.

Acknowledgements

This work was supported by the Consejería de Medio Ambiente y Desarrollo General de

la Comunidad Autónoma de Madrid. J. Rodríguez-Calcerrada was supported by a

scholarship from the Consejería de Educación de la Comunidad de Madrid (C.M.) and

the Fondo Social Europeo (F.S.E.), whereas P. Reich’s participation was supported in

part by the National Science Foundation LTER program.

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Anexo I - 30

Table 1. Means (± S.E.) of gas exchange parameters (n = 4-5). Different letters indicate

significantly different treatment means at P < 0.05 (Tukey’s HSD test following

ANOVA), for both species combined when Pinteraction ≥ 0.05. Statistically significant

differences between species at P < 0.05 are denoted with an asterisk (ns: P ≥ 0.05), for

all treatments combined when Pinteraction ≥ 0.05. Abbreviations are LL: plants in low

light, BF: plants transferred to high light before leaf flushing, AF: plants transferred to

high light after leaf flushing, HL: plants in high light, Aamax (μmol m-2 s-1):

photosynthetic capacity per unit area, Vacmax (μmol m-2 s-1): maximum rate of

carboxylation per unit area, Jamax (μmol m-2 s-1): maximum rate of electron transport per

unit area, Ammax (μmol g-1 s-1): photosynthetic capacity per unit mass, Vm

cmax (μmol g-1 s-

1): maximum rate of carboxylation per unit mass, Jmmax (μmol g-1 s-1): maximum rate of

electron transport per unit mass, Jmax/Vcmax (μmol μmol-1): ratio of maximum electron

transport to maximum carboxylation.

Variable LL BF AF HLQ. pyr. 20.5 ± 1.5 33.9 ± 2.7 36.2 ± 2.2 41.9 ± 5.0Q. pet. 24.8 ± 1.5 34.1 ± 2.0 34.8 ± 2.3 35.4 ± 1.3Q. pyr. 54.0 ± 4.4 70.8 ± 5.8 72.9 ± 8.5 77.2 ± 4.8Q. pet. 57.9 ± 3.9 70.5 ± 1.2 76.9 ± 9.7 86.3 ± 10.6Q. pyr. 87 ± 9 144 ± 12 156 ± 14 183 ± 22Q. pet. 106 ± 5 149 ± 9 151 ± 13 158 ± 11Q. pyr. 0.50 ± 0.04 0.46 ± 0.04 0.73 ± 0.06 0.61 ± 0.11Q. pet. 0.68 ± 0.06 0.59 ± 0.04 0.85 ± 0.03 0.60 ± 0.04Q. pyr. 1.32 ± 0.08 0.97 ± 0.08 1.47 ± 0.20 1.12 ± 0.13Q. pet. 1.58 ± 0.12 1.22 ± 0.06 1.89 ± 0.23 1.48 ± 0.22Q. pyr. 2.14 ± 0.22 1.96 ± 0.14 3.15 ± 0.36 2.68 ± 0.49Q. pet. 2.89 ± 0.21 2.57 ± 0.18 3.70 ± 0.20 2.71 ± 0.26Q. pyr. 1.63 ± 0.15 2.04 ± 0.16 2.19 ± 0.14 2.37 ± 0.21Q. pet. 1.86 ± 0.16 2.11 ± 0.14 2.01 ± 0.14 1.87 ± 0.13

V mcmax

J mmax

J max/V cmax

A amax

V acmax

J amax

A mmax

Treatment

b b

b

b

a

a

*

*

ns

a

a

a

ns

ns

ns

*

a ab b

a

b

a

a

a

ab

Species

ab

ab

a

a

ab

bb

b

b

Anexo I - 31

Table 2. Means (± S.E.) of gas exchange parameters (n = 4-5). Different letters indicate

significantly different treatment means at P < 0.05 (Tukey’s HSD test following

ANOVA), for both species combined when Pinteraction ≥ 0.05. Statistically significant

differences between species at P < 0.05 are denoted with an asterisk (ns: P ≥ 0.05), for

all treatments combined when Pinteraction ≥ 0.05. Abbreviations are JETR (μmol m-2 s-1):

maximum electron transport rate from fluorescence, qL(1100): photochemical

quenching at 1100 μmol m-2 s-1 PPFD, ΦPSII(1100): effective photochemical quantum

yield of PSII at 1100 μmol m-2 s-1 PPFD, ΦNPQ(1100): yield of downregulatory non-

photochemical dissipation of energy at 1100 μmol m-2 s-1 PPFD, bΦPSII: curvature of

light response of ΦPSII, bΦNPQ: curvature of light response of ΦNPQ. See Table 1 for

abbreviations of treatments.

Variable LL BF AF HLQ. pyr. 53 ± 9 123 ± 12 123 ± 12 140 ± 16Q. pet. 61 ± 6 93 ± 16 93 ± 13 107 ± 20

Q. pyr. 0.13 ± 0.02 0.25 ± 0.03 0.23 ± 0.02 0.27 ± 0.03Q. pet. 0.12 ± 0.01 0.19 ± 0.03 0.23 ± 0.03 0.25 ± 0.06

Q. pyr. 0.12 ± 0.01 0.26 ± 0.02 0.26 ± 0.02 0.29 ± 0.03Q. pet. 0.13 ± 0.01 0.20 ± 0.03 0.20 ± 0.03 0.23 ± 0.04

Q. pyr. 0.63 ± 0.01 0.50 ± 0.04 0.50 ± 0.02 0.50 ± 0.04Q. pet. 0.59 ± 0.02 0.55 ± 0.03 0.58 ± 0.03 0.53 ± 0.02

Q. pyr. 4.30 ± 0.6 1.74 ± 0.2 1.47 ± 0.2 1.57 ± 0.2Q. pet. 2.89 ± 0.5 2.26 ± 0.5 2.41 ± 0.6 1.84 ± 0.6

Q. pyr. 4.11 ± 0.6 1.29 ± 0.2 1.12 ± 0.2 1.35 ± 0.2Q. pet. 2.69 ± 0.5 1.74 ± 0.3 2.08 ± 0.5 1.7 ± 0.6

Treatment

J ETR

qL(1100)

ΦPSII(1100)

ΦNPQ(1100)

b ΦPSII(10-3)

b ΦNPQ(10-3)

ns

ns

Species

*

ns

*

ns

a

a

a

b

b

b a

a

a

b

ab

a

a

b b

b

b

b b

b

a

a

a

b

Anexo I - 32

Table 3. Means (± S.E.) of gas exchange parameters (n = 4-5). Different letters indicate

significantly different treatment means at P < 0.05 (Tukey’s HSD test following

ANOVA), for both species combined when Pinteraction ≥ 0.05. Statistically significant

differences between species at P < 0.05 are denoted with an asterisk (ns: P ≥ 0.05), for

all treatments combined when Pinteraction ≥ 0.05. Abbreviations are Nm (mg g-1): nitrogen

content per unit leaf mass, Na (g m-2): nitrogen content per unit leaf area, Pl (%):

nitrogen fraction in light-harvesting components, Pb (%): nitrogen fraction in electron

transport proteins, Pr (%): nitrogen fraction in Rubisco, Ps (%): nitrogen fraction in

structural components, PNUE (μmol gN-1 s-1): photosynthetic nitrogen use efficiency. See

Table 1 for abbreviations of treatments.

Variable LL BF AF HLQ. pyr. 29.9 ± 1.8 27.5 ± 1.4 28.8 ± 1.0 26.9 ± 0.9Q. pet. 35.1 ± 1.1 28.5 ± 1.1 32.2 ± 1.06 27.2 ± 1.2Q. pyr. 1.23 ± 0.10 1.99 ± 0.10 1.44 ± 0.07 1.93 ± 0.11Q. pet. 1.29 ± 0.05 1.61 ± 0.11 1.33 ± 0.15 1.61 ± 0.11Q. pyr. 24.9 ± 2.5 19.9 ± 2.8 18.5 ± 1.4 18.2 ± 1.7Q. pet. 30.7 ± 1.2 23.9 ± 2.3 25.2 ± 2.7 20.3 ± 1.8Q. pyr. 5.7 ± 0.4 5.8 ± 0.6 8.8 ± 1.1 7.7 ± 1.3Q. pet. 6.5 ± 0.3 7.1 ± 0.4 9.2 ± 0.4 8.0 ± 0.8Q. pyr. 34.4 ± 0.9 28.2 ± 3.5 39.9 ± 5.5 31.8 ± 3.8Q. pet. 35.2 ± 3.2 33.5 ± 2.6 45.7 ± 4.4 42.9 ± 6.5Q. pyr. 35.0 ± 2.9 46.0 ± 6.2 32.8 ± 7.4 42.4 ± 5.7Q. pet. 27.6 ± 4.4 35.4 ± 3.2 19.9 ± 5.6 28.8 ± 7.8Q. pyr. 16.9 ± 1.4 17.1 ± 1.3 25.3 ± 2.2 22.1 ± 3.5Q. pet. 19.2 ± 1.2 20.6 ± 1.4 26.8 ± 1.5 22.4 ± 1.7

Species

P b

P r

P s

P NUE

a

b

aN m

N a

P l

ab

a

ab

abb

b

a

b

a

ab

b

a

ns

*

ns

b

a

b

a

ab

a

a

*

*

*

ns

a

a

a

Treatment

ab

a

a

Anexo I - 33

Table 4. Means (± S.E.) of gas exchange parameters (n = 4-5). Different letters indicate

significantly different treatment means at P < 0.05 (Tukey’s HSD test following

ANOVA); differences specified for each species when Pinteraction < 0.05. Statistically

significant differences between species at P < 0.05 are denoted with an asterisk (ns: P ≥

0.05), for all treatments combined when Pinteraction ≥ 0.05. Abbreviations are MA (g m-2):

leaf dry mass per area, TL (μm): leaf thickness, TE (μm): epidermis thickness, TPP (μm):

palisade parenchyma thickness, TSP (μm): spongy parenchyma thickness. See Table 1

for abbreviations of treatments.

Variable LL BF AF HLQ. pyr. 40.9 ± 1.0 72.6 ± 1.5 50.3 ± 2.2 67.9 ± 3.7Q. pet. 37.4 ± 1.4 56.6 ± 2.2 40.9 ± 2.9 58.7 ± 2.5Q. pyr. 91 ± 3 a 132 ± 8 bc 112 ± 6 ab 154 ± 13 cQ. pet. 101 ± 4 a 134 ± 7 b 99 ± 3 a 128 ± 4 b

Q. pyr. 24.2 ± 1.3 28.3 ± 1.3 27.0 ± 0.9 28.5 ± 2.5Q. pet. 25.0 ± 0.9 27.4 ± 1.2 25.9 ± 1.5 26.4 ± 0.7Q. pyr. 26.3 ± 1.0 a 59.1 ± 6.2 bc 41.7 ± 5.7 ab 75.1 ± 7.0 cQ. pet. 30.5 ± 1.1 a 69.5 ± 5.6 b 30.9 ± 1.9 a 59.1 ± 2.9 b

Q. pyr. 40.3 ± 2.0 44.4 ± 3.3 43.2 ± 1.4 50.0 ± 4.3Q. pet. 45.3 ± 2.3 37.5 ± 3.6 41.7 ± 1.2 42.1 ± 2.3

a

ns

ns a

Treatment

a

c a

Species

*

ns

ns

a a

M A

T L

T E

T PP

T SP

a

b a

a

a

Anexo I - 34

Figure legends

Figure 1. Variation of photochemical and non-photochemical yields of absorbed energy

with light [ΦPSII = m+a exp(-b ppfd); ΦNPQ = m (1-exp(-b ppfd)); ΦNO = m-a (exp(-b

ppfd))] in seedlings of Q. pyrenaica (a, c, e, g) and Q. petraea (b, d, f, h). ΦPSII:

triangles, ΦNPQ: squares, ΦNO: circles. Vertical lines indicate PPFD at which ΦPSII =

ΦNPQ. Data points are means (n = 4-5) ± S.E.

Figure 2. Relationships between leaf nitrogen estimated in a) Rubisco (Pr), b)

photosynthetic electron transport (Pb), c) light harvesting (Pl) and d) structural

components (Ps), and e) nitrogen content (Nm), with mass-based photosynthesis in LL

(circles), HL (squares), BF (triangles) and AF (diamonds) seedlings of Q. petraea (open

symbols, dashed lines) and Q. pyrenaica (filled symbols, continuous lines). Regression

lines are a): y = 0.45 ln x - 0.73 for Q. pyrenaica and y = 0.28 ln x - 0.17 for Q. petraea,

b): y = 0.49 ln x - 0.25 for Q. pyrenaica and y = 0.57 ln x - 0.36 for Q. petraea, d): y = -

0.49 ln x + 2.58 for Q. pyrenaica and y = -0.36 ln x + 2.07 for Q. petraea; regression

lines indicated when P ≤ 0.1. Notice that x-axis scales are different.

Figure 3. Transverse sections of leaves of HL (a, e), BF (b, f), AF (c, g) and LL (d, h)

plants of Q. pyrenaica (a-d) and Q. petraea (e-h). Light micrograph magnification is

400X. Depth of sections is 20-30 μm.

Anexo I - 35

Figure 1.

0.0

0.2

0.4

0.6

0.8

1.0

PPFD (μmol m-2 s-1)

ΦPS

II; Φ

NPQ

; ΦN

O

0 250 500 750 100012501500 0 250 500 750 100012501500

g h (HL)

0.0

0.20.4

0.60.8

1.0e f (AF)

0.0

0.20.4

0.60.8

1.0c d (BF)

0.0

0.20.4

0.60.8

1.0a b (LL)

0.0

0.2

0.4

0.6

0.8

1.0

PPFD (μmol m-2 s-1)

ΦPS

II; Φ

NPQ

; ΦN

O

0 250 500 750 100012501500 0 250 500 750 100012501500 0 250 500 750 100012501500

g h (HL)

0.0

0.20.4

0.60.8

1.0e f (AF)

0.0

0.20.4

0.60.8

1.0c d (BF)

0.0

0.20.4

0.60.8

1.0a b (LL)

Anexo I - 36

Figure 2.

Pr (%) Pb (%)

Nm (mg N g-1)

Ps (%)Pl (%)20 30 40 50 60 70A

mm

ax(μ

mol

CO

2g-1

s-1)

R2 = 0.24; P = 0.04 R2 = 0.31; P = 0.02 dR2 = 0.24; P = 0.04 R2 = 0.31; P = 0.02R2 = 0.24; P = 0.04 R2 = 0.31; P = 0.02 d

10 20 30 400.00.20.40.60.81.01.2

R2 = 0.02; P > 0.10 R2 = 0.00; P > 0.10 cR2 = 0.02; P > 0.10 R2 = 0.00; P > 0.10R2 = 0.02; P > 0.10 R2 = 0.00; P > 0.10 c

10 20 30 40

R2 = 0.18; P = 0.08 R2 = 0.41; P = 0.006 a

10 20 30 40

R2 = 0.18; P = 0.08 R2 = 0.41; P = 0.006 aR2 = 0.18; P = 0.08 R2 = 0.41; P = 0.006R2 = 0.18; P = 0.08 R2 = 0.41; P = 0.006 a

0.00.20.40.60.81.01.2

2 4 6 8 10 12

R2 = 0.40; P = 0.006 R2 = 0.72; P = 0.000 b

2 4 6 8 10 12

R2 = 0.40; P = 0.006 R2 = 0.72; P = 0.000 bR2 = 0.40; P = 0.006 R2 = 0.72; P = 0.000R2 = 0.40; P = 0.006 R2 = 0.72; P = 0.000 b

20 25 30 35 40

R2 = 0.16; P > 0.10 R2 = 0.14; P > 0.10 e

20 25 30 35 40

R2 = 0.16; P > 0.10 R2 = 0.14; P > 0.10 eR2 = 0.16; P > 0.10 R2 = 0.14; P > 0.10R2 = 0.16; P > 0.10 R2 = 0.14; P > 0.10 e

0.00.20.40.60.81.01.2

Anexo I - 37

Figure 3.

(a)

(e)(e)

50 µm

(c)(c)

(g)(g)

(d)(d)

(h)

(b)(b)

(f)(f)

50 µm

Anexo I - 38

ANEXO II

Light response in seedlings of a temperate (Quercus petraea) and a sub-

Mediterranean species (Quercus pyrenaica): contrasting ecological

strategies as potential keys to regeneration performance in mixed

marginal populations

Manuscrito en prensa en la revista Plant Ecology, DOI: 10.1007/s11258-007-9329-2

Autores: Rodríguez-Calcerrada J., Pardos J.A., Gil L., Reich P.B., Aranda I.

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12

3 Light response in seedlings of a temperate (Quercus petraea)

4 and a sub-Mediterranean species (Quercus pyrenaica):

5 contrasting ecological strategies as potential keys to

6 regeneration performance in mixed marginal populations

7 Jesus Rodrıguez-Calcerrada Jose Alberto Pardos

8 Luis Gil Peter B. Reich Ismael Aranda

9 Received: 18 September 2006 / Accepted: 12 June 200710 � Springer Science+Business Media B.V. 2007

11

12 Abstract In order to understand better the ecol-

13 ogy of the temperate species Quercus petraea and

14 the sub-Mediterranean species Quercus pyrenaica,

15 two deciduous oaks, seedlings were raised in two

16 contrasting light environments (SH, 5.3% full

17 sunlight vs. HL, 70% full sunlight) for 2 years,

18 and a subset of the SH seedlings were transferred

19 to HL (SH–HL) in the summer of the second year.

20 We predicted that Q. pyrenaica would behave more

21 as a stress-tolerant species, with lower specific leaf

22 area (SLA), allocation to leaf mass, and growth rate

23 and less responsiveness to light in these metrics,

24 than Q. petraea, presumed to be more competitive

25 when resources, especially light and water, are

26abundant. Seedlings of Q. petraea had larger leaves

27with higher SLA, and exhibited a greater relative

28growth rate (RGR) in both SH and HL. They also

29displayed a higher proportion of biomass in stems

30(SMF), and a lower root to shoot ratio (R/S) in HL

31than those of Q. pyrenaica, which sprouted pro-

32fusely, and had higher rates of photosynthesis (An)

33and stomatal conductance (gwv), but lower whole-

34plant net assimilation rate (NAR). On exposure to a

35sudden increase in light, SH–HL seedlings of both

36species showed a short period of photoinhibition,

37but fully acclimated photosynthetic features within

3846 days after transference; height, main stem

39diameter, RGR and NAR all increased at the end

40of the experiment compared to SH seedlings, with

41these increases more pronounced in Q. petraea.

42Observed differences in traits and responses to light

43confirmed a contrasting ecology at the seedling

44stage in Q. petraea and Q. pyrenaica in consonance

45with differences in their overall distribution. We

46discuss how the characteristics of Q. petraea may

47limit the availability of suitable regeneration niches

48to microsites of high-resource availability in mar-

49ginal populations of Mediterranean climate, with

50potential negative consequences for its recruitment

51under predicted climatic changes.

52

53Keywords Acclimation to light � Ecological

54requirements � Competitive ability � Marginal

55populations

A1 J. Rodrıguez-Calcerrada � J. A. Pardos � L. GilA2 Unidad de Anatomıa, Fisiologıa y Genetica Forestal,

A3 Escuela Tecnica Superior de Ingenieros de Montes,

A4 Universidad Politecnica de Madrid, Ciudad Universitaria

A5 s/n, Madrid 28040, Spain

A6 J. Rodrıguez-Calcerrada � J. A. Pardos �A7 L. Gil � I. ArandaA8 Unidad Mixta INA-UPM, Madrid 28040, Spain

A9 P. B. Reich

A10 Department of Forest Resources, University of Minnesota,

A11 St. Paul, MN 55108, USA

A12 I. Aranda (&)

A13 Centro Nacional de Investigacion Forestal (CIFOR),

A14 Instituto Nacional de Investigacion Agraria y Alimentaria

A15 (INIA), Apdo. 8111, Madrid 28080, Spain

A16 e-mail: aranda@inia.es

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56 Introduction

57 The existence of a transitional area between the

58 Oceanic and Mediterranean regions in the Iberian

59 Peninsula favours the occurrence of mixed stands of

60 species with contrasting ecological requirements.

61 There are many woody temperate trees in Mediter-

62 ranean habitats in a relict situation (e.g. Pinus

63 sylvestris, Ilex aquifolium, Frangula alnus, Fagus

64 sylvatica, Quercus petraea) which is partly explained

65 by their being more sensitive to local environmental

66 stress conditions than co-occurring species that are at

67 an optimum of their distribution. Usually drought

68 arises as the principal limiting factor for these

69 populations, operating either at the formation and

70 dispersal of seeds (Hampe 2005) or the establishment

71 of seedlings (Aranda et al. 2000; Castro et al. 2004;

72 Valladares et al. 2005). The result is a patchy

73 distribution at a landscape scale where individuals

74 are constrained to relatively humid microsites. None-

75 theless, the identification of key ecological charac-

76 teristics in seedlings through the study of responses to

77 light (e.g. shade-tolerance, acclimation potential and

78 competitive ability) can provide valuable insights

79 about niche segregation and recruitment potential

80 even in drought-prone habitats. Comparative eco-

81 physiological studies of closely related species car-

82 ried out in controlled environments are useful for this

83 purpose (Ashton and Berlyn 1994; Valladares et al.

84 2002a; Miyazawa and Lechowicz 2004).

85 Interspecific differences under common-environment

86 conditions may indicate different selection pressures in

87 their original habitats. Plant functional traits differ

88 among species from a wide range of climate and

89 habitat types in relation to the variation of the factors

90 that determine the outcome of regeneration (Westoby

91 et al. 2002; Reich et al. 2003; Wright et al. 2005).

92 Species with high-specific leaf area, high-net assim-

93 ilation rate, high partitioning of biomass to shoots and

94 a rapid growth rate are likely to exhibit a high

95 competitive potential in sites where interspecific

96 competition for light is a key factor for regeneration

97 (Poorter and de Jong 1999). In contrast, species

98 adapted to arid- or semi-arid areas usually have

99 leaves with high mass per unit area, a great

100 partitioning of biomass to roots, a high root to shoot

101 ratio and a conservative growth strategy (Chapin

102 et al. 1993; Reich et al. 1999). This suite of traits

103 provides a greater resistance to water stress, but may

104result in a less competitive ability to intercept light

105than that of fast-growing trees.

106Responsiveness to light is also related with

107adaptation to the environmental conditions existing

108in the habitat. For instance, late successional species

109show lower phenotypic plasticity to light (Chazdon

110et al. 1996; Ribeiro et al. 2005) and lower acclima-

111tion potential to increasing light (Fetcher et al. 1983;

112Strauss-Debenedetti and Bazzaz 1991) than early

113successional species. Similarly, responsiveness to

114resource availability is relatively low in some

115Mediterranean woody species, as a result of an

116adaptation to limiting stressful environments

117(Valladares et al. 2002b; Chambel et al. 2005), so

118that one might expect a different response to light

119between seedlings of a sub-Mediterranean and a

120temperate species of similar successional status. The

121objective of this study was to identify ecological

122characteristics in seedlings of two co-occurring oaks

123differing markedly in their distribution, namely

124Q. petraea and Q. pyrenaica, through the leaf- to

125whole-plant examination of short- to long-term

126acclimatory responses to light. The possible impli-

127cations of such responses in relation to low-latitude

128marginal population of Q. petraea are discussed

129herein. Based on Grime’s classification of plant

130functional types (1977), we hypothesized that seed-

131lings of the temperate Q. petraea would show

132features of a more competitive species while those

133of the sub-Mediterranean Q. pyrenaica would reflect

134a more stress-tolerant character. Particularly we

135anticipated a more conservative growth pattern, and

136a lower responsiveness to light (both lower pheno-

137typic plasticity and acclimation potential to a sudden

138increase in light availability) in Q. pyrenaica than in

139Q. petraea.

140Materials and methods

141Study species and experimental design

142Q. petraea and Q. pyrenaica are two deciduous

143white oaks (section: Lepidobalanus) forming late-

144successional forests in Atlantic and sub-Mediterranean

145areas, respectively. Q. pyrenaica extends from the

146southwest of France to northern Morocco while

147Q. petraea is distributed from the southern Scandi-

148navian Peninsula to the northern Iberian Peninsula,

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149 where it coexists with Q. pyrenaica in a number of

150 scattered stands.

151 In the autumn of 2003 acorns of both species were

152 harvested in a small population of Q. petraea near the

153 southern edge of its distribution (‘‘El Hayedo de

154 Montejo’’ forest, 41870 N, 38300 W, 1,300 masl). The

155 seeds were kept at 38C and 55% relative humidity until

156 the next spring. Acorns of similar size from five

157 different trees were then sown in 400-cm3 plastic pots

158 of 35 cm depth, filled with a mixture of peat and sand

159 (3:1, v/v) containing slow-release fertilizer (5 g dm�3).

160 Plants were cultivated inside a glasshouse under two

161 light treatments: shading (SH; double layer of neutral

162 shadecloth, 5.3% of full sunlight) and high light

163 (HL; no shadecloth, 70% of full sunlight, due to the

164 opacity of fibreglass claddings). Photosynthetically

165 active radiation (PAR) was measured using a

166 LICOR LI-185B irradiance meter equipped with a

167 LI-190SB quantum sensor. Average values of midday

168 PAR were 80 ± 7 mmoles m�2 s�1 in SH and

169 1050 ± 28 mmoles m�2 s�1 in HL. Although midday

170 temperature in HL was slightly higher than in SH

171 (33.7 ± 0.38C vs. 31.6 ± 0.28C) it was assumed that

172 13-fold differences in relative irradiance were more

173 important in explaining seedling responses.

174 At the end of the first growing season 80 seedlings

175 of similar size were transplanted singly to 3,000-cm3

176 plastic cylinders of 40 cm depth. Each pot contained

177 1.9 kg of the same type of substrate used the first year

178 and was again enriched with slow-release fertilizer. A

179 total of 20 individuals per species were maintained in

180 shade, distributed under four adjustable metal-framed

181 shading structures placed on two benches of the

182 glasshouse. Plants of the HL treatment were distrib-

183 uted between the shading structures at a sufficient

184 distance as to avoid shading. About 90 days after leaf

185 emergence, six plants per species were moved out of

186 the shade frames (SH–HL seedlings) and mixed

187 together with HL seedlings. Plants of all treatments

188 were periodically moved on their benches and

189 regularly watered over the course of the experiment.

190 Bud burst was synchronous in both species (first

191 week of March 2005), which precluded any differ-

192 ence in the length of the growth period. Temperature

193 and relative humidity into the glasshouse were in the

194 range of 15–398C and 50–90%, respectively.

195Physiological variables—gas exchange and

196chlorophyll fluorescence

197Physiological acclimation was explored over the

19846 days that followed the transference of SH plants

199(from 6 June [day 0] to 21 July [day 46]). Gas

200exchange and chlorophyll a fluorescence measure-

201ments were periodically conducted (more thoroughly

202immediately after transference) on one leaf per plant

203and five individuals per species and treatment. Leaves

204were selected from the first flush of growth and

205maintained for further analysis, if possible. Gas

206exchange and chlorophyll fluorescence was also

207measured on second-flush leaves of HL and SH–HL

208seedlings on day 46.

209Photosynthesis (An) and stomatal conductance

210(gwv) were measured with an IRGA (LCpro Analyt-

211ical Development Corporation, UK), at saturating

212light (1,000 mmol m�2 s�1 for HL seedlings and

213700 mmol m�2 s�1 for SH and SH–HL) and 365 ppm

214CO2. A red/blue light emitting diode supplied mea-

215suring light. Temperature into the leaf chamber was

216set to leaf temperature being around 258C in all cases.

217In order to avoid excessive temperature inside the

218glasshouse, gas exchange measurements started at

21908:00 h and continued until 11:00 h local time. Prior

220to measurements, shade plants were allowed to

221acclimate to a level of artificial light similar to the

222measuring light for a period of 10–15 min.

223Chlorophyll fluorescence was measured with a

224pulse-modulated fluorometer (FMS 2, Hansatech

225Instruments Ltd., UK). Maximum photochemical

226efficiency of photosystem II (PS II) (Fv/

227Fm = [Fm � Fo]/Fm) was calculated after measuring

228maximal (Fm) and minimal fluorescence (Fo) at dawn.

229After measuring leaf gas exchange, effective photo-

230chemical efficiency of PS II (UPSII) was calculated as

231(Fm0 � Fs)/Fm

0, where Fm0 is the maximal fluores-

232cence in light and Fs is the steady-state light-adapted

233fluorescence. Dark- and light-adapted fluorescence

234was measured in the same area to estimate non-

235photochemical quenching (NPQ = [Fm � Fm0]/Fm

0).

236Since sunlight was used as the actinic light, mea-

237surements were made on cloudless days at roughly

238the same hour, avoiding either shading the sample

239area or modifying the leaf angle.

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240 Specific leaf area and chlorophyll content

241 Chlorophyll content was estimated on one first-flush

242 leaf from five individuals per species and treatment in

243 three dates (days 0, 37 and 61 from transference). For

244 chlorophyll analysis, one leaf disc was cut and

245 immersed in a tube containing 5 ml of dimethyl

246 sulfoxide. Tubes were bathed in darkness for 5 h at

247 608C and the absorbance of dimethyl sulfoxide was

248 further measured at 648.2 and 664.9 nm wavelength

249 with a spectrophotometer, determining chlorophyll

250 content per unit of leaf mass (Chlmass). Values of

251 specific leaf area (SLA) were used to calculate

252 chlorophyll content on a leaf area basis (Chlarea).

253 Leaf morphology and plant architecture

254 The SLA was determined on one fully exposed leaf

255 from the first flush of growth in five individuals per

256 species and treatment. Discs of known area were cut,

257 oven-dried at 708C for 48 h and then weighed to

258 estimate SLA as the ratio between the area and its dry

259 mass. At day 61, SLA of second-flush leaves was also

260 determined in HL and SH–HL seedlings. Internode

261 length (IL) was calculated by dividing the length

262 between the lowest leaf and the tip of the main stem

263 by the number of nodes. Mean leaf size (LA) was

264 calculated by dividing the total leaf area of the plant

265 (TLA) at the end of the experiment by the number of

266 leaves produced. In order to estimate the self-shading

267 in a seedling (SS) a rough index was calculated by

268 dividing TLA by the shoot height.

269 Growth and biomass distribution

270 Height and stem diameter at the root collar were

271 measured at the time of transplanting (end of the first

272 year), and in 3 days along the second growing season

273 (days 0, 38 and 67).

274 At the moment of transplanting, six seedlings per

275 species from the HL and SH treatments were

276 harvested. At the end of the experiment (day 67),

277 another six seedlings per species were harvested in

278 the HL, SH and SH–HL treatments. The leaves,

279 stems and roots were dried to a constant weight for

280 3–6 days, and further weighed separately. All of the

281 leaves on each plant were separated as pertaining to

282 the main stem versus the sprouts, as well as to the

283 first-flush of growth versus the following flushes.

284Leaf area ratio (LAR; total leaf area/total plant mass),

285leaf mass fraction (LMF; leaf mass/total plant mass),

286stem mass fraction (SMF; stem mass/total plant

287mass), root mass fraction (RMF; root mass/total plant

288mass) and root to shoot ratio (R/S; root mass/shoot

289mass) were calculated. The relative growth rate

290(RGR) for each species and light treatment combi-

291nation was determined using the Eq. 1:

RGR ¼lnðM2Þ � lnðM1Þ

D2 � D1ð Þ; ð1Þ

293293where M2 was the total plant-dry mass at the final

294harvest, M1 was the total plant dry mass at the time of

295transplanting, D2 was the day of the final harvest and

296D1 was the time of transplanting. The net assimilation

297rate (NAR) was calculated using the Eq. 2:

298

NAR ¼ln(TLA2)� ln(TLA1)� �

TLA2 � TLA1

� �

M2 �M1

� �

D2 � D1ð Þ; ð2Þ

300300where TLA2 was the total leaf area at the final

301harvest, obtained by measuring the area of all the

302leaves with an image analyser (Delta-T Devices LTD,

303UK), and TLA1 was the total leaf area at the time of

304transplanting, which was estimated as the product of

305LMF by SLA.

306Statistical analysis

307Effects of species and light were tested by analysis of

308variance (ANOVA). A repeated measures approach

309was followed for all variables except those measured

310only at the end of the experiment. Despite variability

311among dates of measurement, no seasonal trend was

312observed in any variable in HL or SH seedlings (date

313effect at least P > 0.1). A significant interaction

314between species and light factors was considered as

315indicative of interspecific differences in plasticity

316(Schlichting 1986). Acclimation of the various traits

317studied on each species was evaluated comparing the

318means of the light treatments (HL, SH and SH–HL)

319by Tukey’s HSD test. The impact of transference on

320each species was examined comparing the maximum

321variation of each SH–HL plant upon exposure to light

322with respect to the mean value for SH plants. Logistic

323or lineal models were fitted to individual plants to

324evaluate the velocity of acclimation of physiological

325variables; independent parameters of each curve

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326 (n = 5) were then compared using ANOVA. Variables

327 were arcsine or log-transformed to enhance homo-

328 scedasticity. Significance level was 5%.

329 Results

330 Gas exchange parameters

331 Both the rate of photosynthesis (An) and stomatal

332 conductance (gwv) were higher inQ. pyrenaica than in

333 Q. petraea in HL, but not in SH (Table 1; Fig. 1a, b).

334 Gas exchange of SH–HL plants declined below values

335 of SH plants for the week following transference

336 (Fig. 1c, d). The impact of the sudden increase in light

337 did not significantly differ among species. Photoinhi-

338 bition was temporary; An and gwv gradually increased

339 in shade-formed leaves so that they did not differ

340significantly from those of HL plants at the end of the

341measuring period. Leaves of SH–HL plants developed

342after transference had gas exchange rates not signifi-

343cantly different from second-flush HL leaves (16.6 in

344HL vs. 17.7 mmol m�2 s�1 in SH–HL for An [both

345species combined; P > 0.1] and 402 in HL vs.

346364 mmol m�2 s�1 in SH–HL for gwv [both species

347combined P > 0.1]).

348Chlorophyll fluorescence parameters

349Light regime had a strong effect on the non-photo-

350chemical quenching (NPQ) and the effective quantum

351yield (UPSII), but not on the maximum quantum yield

352of PS II at dawn (Fv/Fm) (Table 1; Fig. 2a–c),

353indicating a daily build-up of photoinhibition revers-

354ible overnight. There were no statistically significant

355differences between Q. petraea and Q. pyrenaica in

Table 1 Two-way analysis of variance aimed to test the effects of light, species and the interaction of both factors on leaf and plant

traits

Variables Factors

Species Light Species · light

Physiology A * *** *

gwv * *** **

FvFm n.s. n.s. n.s.

UPSII n.s. *** n.s.

NPQ n.s. *** n.s.

Chlorophyll content Chlmass *** *** **

Chlarea ** n.s. *

Morphology and architecture SLA * *** n.s.

LA ** * n.s.

TLA *** *** ***

IL * * n.s.

SS n.s. *** **

Growth and biomass distribution Height *** *** ***

Diameter *** *** ***

Total plant mass ** *** **

LAR n.s. n.s. n.s.

R/S *** ** **

RMF *** ** **

LMF n.s. * n.s.

SMF *** n.s. **

A repeated measures approach (ANOVAR) was followed for those variables repeatedly measured throughout the experiment

*** P < 0.001; ** P < 0.01; *P < 0.05; n.s. P � 0.05

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356 any chlorophyll fluorescence parameter. Transference

357 of shade-acclimated plants to HL affected the activity

358 of PS II (Fig. 2d–f). Immediately after transference,

359 Fv/Fm of SH–HL plants declined with respect to SH

360 plants, the reduction being slightly higher in Q. pyre-

361 naica (0.76 vs. 0.80 on day 2; P < 0.05). UPSII also

362 decreased with respect to SH plants (0.24, minimum

363 value inQ. pyrenaica on day 2, and 0.31 the minimum

364 value in Q. petraea on day 1; P < 0.05), while a nearly

365 3-fold increase over HL values occurred on NPQ in

366 both species. By the second to fifth day after

367 transference all chlorophyll fluorescence parameters

368 began to recover, so that 46 days later shade-

369 developed leaves reached values not significantly

370 different from leaves continuously subjected to high

371 light. The acclimation of chlorophyll fluorescence

372 parameters was slightly more rapid in Q. pyrenaica

373 (P < 0.05 for Fv/Fm and UPSII; P = 0.11 for NPQ)

374 (Fig. 2d–f). High-light formed leaves of SH–HL

375 plants had also values of UPSII and NPQ not signif-

376 icantly different from those of HL plants (0.54 in HL

377 vs. 0.57 in SH–HL for UPSII [both species combined;

378 P > 0.1] and 0.97 in HL vs. 0.79 in SH–HL for NPQ

379 [both species combined P > 0.1]).

380Chlorophyll content

381Chlorophyll content (either Chlmass as Chlarea) was

382higher in Q. petraea in both environments, but

383especially marked was the difference in SH (Tables 1

384and 2A). After transference, Chlmass declined in SH–

385HL plants to HL values (Table 2A), earlier in

386Q. pyrenaica (day 37, not shown). Chlarea was lowest

387in SH–HL leaves on day 61.

388Leaf morphology and plant architecture

389As expected, specific leaf area (SLA) was greatly

390increased by shading, and was slightly higher in

391Q. petraea in both light treatments (Tables 1, 2B).

392Interspecific difference in total leaf area (TLA) was

393only significant in HL, while the leaf size (LA) was

394significantly higher in Q. petraea than in Q. pyrena-

395ica. The ratio between TLA and shoot height, used as

396an index of self-shading (SS), was lower in Q. petraea

397in HL. Although this index may give a good

398estimation of within-plant self-shading in HL, given

399the huge differences in height between the two

400species, this may not be the case in SH, where leaf

HL SH

An

(µm

ole

s m

-2s-1

)

An

(% H

L)

Days after transference

HL SH

gw

v (m

mo

les

m-2s-1

)

gw

v (%

HL

)

Days after transference

0

5

10

15

20

0

100

200

300

400

500

20

40

60

80

100

0 20 30

20

40

60

80

100

120

b

a

d

c

4010

0 20 30 4010

Fig. 1 Plasticity to light (a, b) and acclimation of shade-grown

seedlings to high light (c, d) on Q. pyrenaica (grey symbols)

and Q. petraea (white symbols). (a, b) Mean values (+1 SE) of

photosynthesis (An) and stomatal conductance (gwv) of high

light (HL) and shade (SH) seedlings. (c, d) Mean values (±SE)

of An and gwv of transferred seedlings (SH–HL), expressed as a

percentage of HL mean value (thick line). The thin solid line

and the thin dotted line indicate mean percent values of

Q. petraea and Q. pyrenaica in SH, respectively

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401 arrangement along the stem should be considered. HL

402 seedlings of Q. petraea had twofold longer leaf

403 internodes than those of Q. pyrenaica (Tables 1, 2B).

404 SLA of shade-developed leaves decreased signifi-

405 cantly in both species, but by 61 days after transfer-

406 ence SLA did not reach HL values (Table 2B). Newly

407 developed leaves of SH–HL seedlings had similar

408 SLA as second-flush HL ones (125 in HL vs.

409 131 cm2 g�1 in SH–HL [both species combined

410 P > 0.1]). SH–HL seedlings had higher TLA, LA and

411 SS than SH seedlings; in the case of LA, the increase

412 in light had no effect in Q. pyrenaica (Table 2B).

413 Growth

414 All seedlings flushed two or three times in HL and

415 once in SH. Interspecific differences were more

416 evident in HL (Tables 1, 2C). Total plant-dry mass,

417 height and main-stem diameter, and RGR, were all

418 higher in Q. petraea. Differences in growth metrics

419between species were minimal in shade; hence

420plasticity in these variables was greater in Q. petraea.

421The pattern of shoot growth was also different in each

422species. In Q. pyrenaica new flushes did not emerge

423from apical or near-apical buds but mostly from basal

424sprouts near the root-collar, giving most seedlings a

425spherical-shaped aspect with older leaves shaded by

426the new ones. The net assimilation rate (NAR) was

427higher in Q. petraea, while no differences in LAR

428were apparent between species or light treatments.

429All SH–HL plants flushed at least once after trans-

430ference, showing an increase in growth with respect

431to SH plants, which was more evident in Q. petraea.

432Differences between species for SH–HL seedlings

433held similar to those in HL (Table 2C).

434Distribution of biomass

435Biomass distribution differed more between species

436than among light environments. In HL, the species

0

0.2

0.4

0.6

0.8

0

0.2

0.4

0.6

0.8

HL SH

HL SH

HL SH

Pre

da

wn

Fv

/F

m

Pd

. F

v/F

m (

% H

L)

Days after transference

ΦΦ P

S I

I(%

HL

)

ΦΦ P

SII

N

PQ

NP

Q (

% H

L)

0 10 20 30 40

Days after transference

0 10 20 30 40

Days after transference

0 10 20 30 40

80

85

90

95

100

20

40

60

80

100

120

140

0

0.2

0.4

0.6

0.8

1.0

0

50

100

150

200

250

300

a

b

c f

e

dFig. 2 Plasticity to light

(a–c) and acclimation of

shade-grown seedlings to

high light (d–f) on

Q. pyrenaica (grey symbols)

and Q. petraea (white

symbols). (a–c) Mean

values (+1 SE) of predawn

maximum photochemical

efficiency of PS II (Fv/Fm),

effective photochemical

efficiency of PS II (UPSII)

and non-photochemical

quenching (NPQ) of high

light (HL) and shade (SH)

seedlings. (d–f) Mean

values (±SE) of Fv/Fm, UPSII

and NPQ of transferred

seedlings (SH–HL),

expressed as a percentage of

HL mean value (thick line).

The thin solid line and the

thin dotted line indicate

mean percent values of

Q. petraea and

Q. pyrenaica in SH,

respectively

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Table 2 Mean values (±SE) of variables related with chlorophyll content (A), leaf morphology and plant architecture (B), and growth and biomass distribution (C) 2 months

after moving SH-grown plants to HL (SH–HL plants)

Variables SH SH–HL HL

Q. pyrenaica Q. petraea Q. pyrenaica Q. petraea Q. pyrenaica Q. petraea

(A) Chlmass(mg g�1) 17.4 (1.0)b** 24.2 (0.6)b 10.5 (0.4)a** 13.1 (0.5)a 10.6 (0.7)a* 12.6 (0.6)a

Chlarea (g m�2) 0.71 (0.10)ab** 0.90 (0.04)c 0.60 (0.05)a 0.67 (0.07)a 0.74 (0.09)b 0.80 (0.09)b

(B) SLA (cm2 g�1) 244 (9)c* 268 (8)c 175 (9)b 196 (7)b 143 (8)a 157 (1)a

LA (cm2) 15.8 (2.1)a* 22.8 (2.6)a 19.2 (2.0)a** 31.3 (3.8)b 21.1 (2.9)a* 27.6 (1.2)ab

TLA (m2) 0.02 (0.001)a 0.03 (0.001)a 0.06 (0.01)b 0.1 (0.02)b 0.14 (0.01)c** 0.21 (0.01)c

IL (mm) 4.8 (2.1) 6 (2.0) No data No data 5.8 (0.6)** 12.8 (1.6)

SS (cm2 cm�1) 8.7 (1.2)a 12.1 (1.6)a 18.3 (3.4)b 16.5 (1.9)b 42.9 (3.5)c** 27.6 (1.7)c

(C) Height (cm) 23.9 (3.2)a 22.7 (1.6)a 32.8 (4.4)a** 58.8 (11.3)b 32.6 (1.3)a** 75.9 (4.6)b

Diameter (mm) 4.9 (0.3)a 5.5 (0.3)a 5.7 (0.6)a** 9.2 (0.6)b 8.9 (0.6)b** 13.2 (0.4)c

Total plant mass (g) 5.7 (0.8)a 7.4 (0.7)a 14.9 (2.7)b* 25.0 (4.1)b 39.9 (3.1)c** 64.2 (4.9)c

LAR (cm2 g�1) 35.6 (2.1)a 36.8 (3.8)a 38.9 (3.1)a 37.7 (1.8)a 35.0 (1.7)a 32.7 (1.7)a

NAR (g m�2 day�1) 1.6 1.8 4.0 4.8 6.5 8.5

RGR (mg g�1 day�1) 5.2 6.4 14.3 17.9 20.2 25.4

Notice that SLA, Chlmass and Chlarea is of first-flush leaves, and that LA is calculated from all leaves (i.e. including those newly formed in after transference in SH–HL plants)

Different letters indicate significant differences among light treatments within each species. The symbol indicates a significant difference between Q. pyrenaica and Q. petraea

within each light treatment (* P < 0.1 and ** P < 0.05)

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437 had similar biomass fraction in leaves (LMF), but

438 the fraction in stems (SMF) was higher, and in

439 roots (RMF) was lower, in Q. petraea than in

440 Q. pyrenaica (Table 1; Fig. 3); accordingly, the

441 root to shoot ratio (R/S) was greater in Q. pyrenaica

442 (1.55 ± 0.05 vs. 0.94 ± 0.07 g g�1). Proportion of

443 biomass from sprouts was 28% of total aerial

444 biomass in Q. pyrenaica whereas it was negligible

445 in Q. petraea. Light barely modified distribution of

446 biomass in Q. pyrenaica but it did in Q. petraea,

447 SH seedlings having lower LMF and higher RMF.

448 Transference of shade-grown plants modified bio-

449 mass partitioning. Distribution of biomass in leaves

450 and stems increased at the expense of biomass

451 partitioned to roots, more markedly in Q. petraea

452 (Fig. 3). Number of flushes and leaves newly

453 produced in HL after transference was similar in

454 both species (not shown). However, larger leaves of

455 Q. petraea resulted in higher biomass proportion of

456 new to shade-grown leaves compared to Q. pyrena-

457 ica (83% vs. 68.4%; P < 0.01). While SH–HL

458 seedlings of Q. petraea flushed from apical buds,

459 most seedlings of Q. pyrenaica flushed from basal

460 buds (0% vs. 20% above-ground biomass in sprouts

461 in Q. petraea and Q. pyrenaica, respectively).

462Discussion

463Species differences in ecophysiological traits and

464in long-term responses to light

465The species differed in their response to light

466availability, especially regarding growth-related

467parameters. Seedlings of Q. petraea in HL had a

468faster growth rate likely associated with its higher

469NAR and SLA (Veneklaas and Poorter 1998). Lower

470light-saturated rates of net photosynthesis per unit

471area in Q. petraea was compensated for by morpho-

472logical and architectural traits—larger leaves, longer

473internodes and taller shoots—that likely increased

474both the plant’s efficiency for light capture and the

475overall carbon gain (Niinemets et al. 2002; Brites and

476Valladares 2005). On the other hand, both the

477sprouting habit and shorter internode length in

478Q. pyrenaica increased self-shading, which translated

479into a lower NAR despite its higher instantaneous leaf

480gas exchange rates in fully exposed leaves (Kama-

481luddin and Grace 1993; Valladares et al. 2002b).

482Lower growth rate of Q. pyrenaica could also be

483related to a higher storage of non-structural carbohy-

484drates in the roots, as is often the case with sprouting

485species (Kruger and Reich 1997; Pausas et al. 2004;

486Schwilk and Ackerly 2005), or a greater carbon use in

487root respiration (Lambers et al. 1998). Provided likely

488ontogenetic effects, the inherent conservative growth

489of Q. pyrenaica seedlings point to an adaptive stress-

490tolerance strategy in the species (Chapin et al. 1993;

491Reich et al. 2003; Valladares et al. 2005), which is

492consistent with the hypothesis of a high phenotypic

493inertia in genotypes specialized to stressful environ-

494ments (Valladares et al. 2000; Valladares et al.

4952002b; Chambel et al. 2005).

496Increases of chlorophyll concentration and specific

497leaf area in response to shade were consistent with an

498optimizing light-capture strategy reported in most

499species. At this respect, although an intermediate

500degree of shade-tolerance has been noted in seedlings

501of both species (Kelly 2002; Baraza et al. 2004), the

502higher SLA, Chlmass and LA exhibited by seedlings

503of Q. petraea in deep shade could reflect a greater

504capacity to tolerate shade (Niinemets and Kull 1994;

505King 2003). However, the results at the plant-level

506(mainly, rapid growth in high light, lower leaf mass

507fraction and similar leaf area ratio in shade compared

508to HL) were at odds with this conclusion (Walters

HLSH SH-HL

Bio

ma

ss d

istr

ibu

tio

n (

% )

0

20

40

60

80

100

aa aaba b

a

ab

a a

a b

aa a

ab a

*

*

*

*

Fig. 3 Percent fraction of biomass distributed to leaves (dotted

area), stems (hatched area) and roots (open area) at the end of

the experiment on seedlings of Q. pyrenaica (grey bars) and

Q. petraea (white bars). Different letters indicate significant

differences among light treatments within each species. An

asterisk indicates a significant difference between Q. pyrenaica

and Q. petraea within each light treatment (P < 0.05)

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509 and Reich 2000). Two years under deep shade could

510 severely limit the maintenance of a positive carbon

511 balance once carbohydrate supply from the acorns

512 was exhausted. The large root system generated in the

513 first year and the continuous lack of new surplus

514 photosynthate may explain the fewer and smaller

515 leaves produced in shade with respect to high light,

516 and both the higher fractional proportion of root

517 biomass and lower of leaf biomass. Values of RMF in

518 HL in this study were similar to the ones observed for

519 Q. pyrenaica and Q. robur (a closely-related species

520 to Q. petraea) grown under optimal conditions

521 (Antunez et al. 2001), supporting that RMF was

522 unusually high in our shade treatment. Further, we

523 must consider that individual acclimatory responses

524 to resources do not always match across-species

525 evolutionary trends (see Valladares and Sanchez-

526 Gomez 2006).

527 Acclimation to a sudden increase in irradiance

528 Our results indicated a rapid acclimation of shade-

529 developed foliage to a higher light environment, as

530 previously found in seedlings of other deciduous oaks

531 (Naidu and DeLucia 1998). Species’ response was

532 rather similar regarding leaf physiological traits, but

533 clearly distinct in growth-related metrics. On

534 exposure to high light, shade-developed leaves

535 experienced a much higher radiation load that they

536 could utilize in photosynthesis, resulting in a reduc-

537 tion in the maximum quantum yield of PS II at dawn

538 (Fv/Fm), indicative of photoinhibition (Maxwell and

539 Johnson 2000). The rapid onset of photoprotective

540 mechanisms (NPQ) contributed to dissipate part of

541 the excess of energy, preventing more severe dam-

542 ages through a down-regulation of PS II (as indicated

543 by a temporary reduction in UPSII). But photosyn-

544 thetic features rapidly acclimated to the new light

545 intensity. Within days, the effective photochemical

546 efficiency of PS II increased in parallel to a recovery

547 in Fv/Fm and the relaxation of non-photochemical

548 quenching, suggesting a rapid reorganization of PS II

549 (Strauss-Debenedetti and Bazzaz 1991; Yamashita

550 et al. 2000; Cai et al. 2005). Photosynthesis was also

551 gradually increasing in parallel to an increase in

552 stomatal conductance, whereas chlorophyll concen-

553 tration in shade-grown leaves decreased on exposure

554 to high light in both species as a result of chlorophyll

555 degradation. Acclimation of shade-developed leaves

556may be particularly important in Mediterranean

557environments, where summer water stress restricts

558the probability of forming new flushes. However,

559transferred plants produced a new flush whose leaves

560were functionally similar to leaves continuously

561submitted to high light, which likely contributed to

562explain overall plant acclimation (Naidu and DeLucia

5631998; Yamashita et al. 2000) and the greater accli-

564mation potential in seedlings of Q. petraea under

565these controlled conditions. The higher proportion of

566newly formed surface area in high light in Q. petraea

567than in Q. pyrenaica contributed to reach a higher net

568assimilation rate and growth after transference.

569Besides, there was a differential pattern of growth

570on exposure to high light between the two oaks. The

571less conservative response of the seedlings of Q. pet-

572raea, i.e. both the lower sprouting and root to shoot

573ratio, reveals a differential shift in carbohydrate

574allocation of enhanced partitioning to main-stem,

575which may give a competitive advantage in terms of

576suppression of neighbouring vegetation and improved

577light harvesting.

578Diverse ecological characteristics at the seedlings

579stage help to explain varying success in sub-

580Mediterranean habitats

581Seedlings of Q. petraea and Q. pyrenaica differed in

582ecophysiological traits and responses, which suggests

583they might have contrasting competitive ability, and a

584segregation of regeneration niches according to

585resources’ availability. Such differences could play

586a role in the differential regeneration success of the

587species in coexisting sub-Mediterranean populations.

588Both the rapid growth and acclimation potential

589showed by Q. petraea are likely to enhance its

590competitive ability in non-water-limited and produc-

591tive sites where among-species competition for

592resources is the driving force of regeneration (Keddy

593et al. 1997), as they enable overtopping of slower

594growing competitors (Kuppers 1989; Cornelissen

595et al. 1996). But in Mediterranean ecosystems

596recruitment relies to a greater extent on the capacity

597of seedlings to endure the combination of multiple

598stresses and disturbances, such as nutrient or water

599shortages, wildfires or herbivore damages. Several

600features exhibited by seedlings of Q. petraea are less

601suitable to such conditions (Villar et al. 2004). First,

602lower root to shoot ratio can be related with either a

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603 more limited access to belowground resources or a

604 lower ability to recover from above-ground distur-

605 bances. Second, high radiation may be a potential

606 source of stress, especially in periods of strong water

607 deficit and limited photosynthetic activity; even

608 though the leaf area to plant biomass ratio was

609 similar between species, higher self-shading in

610 Q. pyrenaica seedlings minimizes the leaf area

611 exposed to high-intensity radiation, thus maintaining

612 water balance by reducing transpiration at the plant

613 level and avoiding the probability of suffering severe

614 photoinhibition and overheating in open sites (Bragg

615 and Westoby 2002). Further, the sprouting habit of

616 Q. pyrenaica observed in this study at the seedling

617 stage is a typical trait of the species (Calvo et al.

618 2003), which constitutes an advantage to thrive in

619 fire-prone habitats and unstable hill slopes (Sakai

620 et al. 1995; Kruger and Reich 1997; Bond and

621 Midgley 2003).

622 These results are consistent with the modest

623 presence of Q. petraea in Mediterranean ecosystems,

624 where it only forms small stands in favourable areas

625 at high altitudes or northern exposures. Small-scale

626 habitat heterogeneity in less suitable, low-latitude

627 areas can thus play an important role in the recruit-

628 ment of this species. For instance, Pardo et al. (2004)

629 compared two nearby stands of Q. petraea and

630 Q. pyrenaica within a forest in a Mediterranean mid-

631 mountain site (the ‘‘Hayedo de Montejo’’ forest) and

632 found out that the former was at a site of deeper and

633 more humid soil. However, a recent inventory of the

634 whole forest addressing the evolution of vegetation

635 since year 1994 showed that Q. petraea was not able

636 to spread over open areas, and that under the forest

637 canopy the increase in measurable saplings was lower

638 compared to the increase of Q. pyrenaica (Nanos

639 et al. unpublished results). It is very likely that future

640 warming and severity of droughts will accelerate the

641 reduction of suitable regeneration niches for this and

642 likely many other temperate trees near the southern

643 edge of distribution, increasing the probability of

644 extinction of such populations (Willi et al. 2006).

645 Acknowledgements We thank Sven Mutke, Rebecca646 Montgomery, Alvaro Soto and Ruben Milla for valuable647 comments and suggestions. We are also grateful to Jesus648 Alonso for technical assistance and Maria del Carmen del Rey649 for help in plant measurements. This work was supported by650 the Consejerıa de Medio Ambiente y Desarrollo General de la651 Comunidad Autonoma de Madrid. J. Rodrıguez-Calcerrada

652was supported by a scholarship from the Consejerıa de653Educacion de la Comunidad de Madrid (C.M.) and the654Fondo Social Europeo (F.S.E.).

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830 Walters MB, Reich PB (2000) Seed size, nitrogen supply, and831 growth rate affect tree seedling survival in deep shade.832 Ecology 81:1887–1901833 Westoby M, Falster DS, Moles AT, Vesk PA, Wright IJ (2002)834 Plant ecological strategies: some leading dimensions of835 variation between species. AnnuRev Ecol Syst 33:125–159836 Willi Y, Van Buskirk J, Hoffmann AA (2006) Limits to the837 adaptive potential of small populations. Annu Rev Ecol838 Syst 37:433–458

839Wright IJ, Reich PB, Cornelissen JHC, Falster DS, Groom PK,840Hikosaka K, Lee W, Lusk CH, Niinemets U, Oleksyn J,841Osada N, Poorter H, Warton DI, Westoby M (2005)842Modulation of leaf economic traits and trait relationship843by climate. Global Ecol Biogeogr 14:411–421844Yamashita N, Ishida A, Kushima H, Tanaka N (2000) Accli-845mation to sudden increase in light favoring an invasive846over native trees in subtropical islands, Japan. Oecologia847125:412–419

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ANEXO III

Summer field performance of Quercus petraea (Matt.) Liebl and Quercus

pyrenaica Willd. seedlings planted in three sites with contrasting canopy

cover

Publicado en la revista New Forests 33: 67-80 (2007)

Autores: Rodríguez-Calcerrada J., Pardos J.A., Gil L., Aranda I.

Abstract In 2000, one-year-old seedlings of pyrenean oak (Quercus pyrenaicaWilld.) and sessile oak (Quercus petraea [Matt.] Liebl) were planted in a thinnedand an unthinned plot in a pinewood (Pinus sylvestris), and in a nearby clearing.In summer 2002 and 2003, water relations and gas exchange parameters weremeasured to address the impact of drought on the seedlings. Chlorophyll afluorescence was also measured to explore leaf photochemistry and a possiblenon-stomatal limitation to photosynthesis (A). Reduction in stomatal conduc-tance (g) in response to the decrease of predawn water potential (Ypd) resultedthe main cause affecting net carbon uptake. Water potential at midday (Ymd)was similar in both species but Quercus petraea was more sensitive to soil waterdeployment occurred along summer, showing slightly lower Ypd because worserecover of water potential during night. Rate of photosynthesis was higher inQ. pyrenaica probably in relation to its greater leaf mass per area (LMA) andnitrogen content per leaf area (Na). Mortality was highest in the clearing andlowest in the thinned pinewood. Throughout the summer, soil moisture washigher in the thinned area, possibly because of the reduction in tree transpiringsurface and interception of rainfall. Accordingly, Ypd of both species was higherin the thinned site.

Keywords Gas exchange Æ Water potential Æ Fv/Fm Æ Oaks Æ Thinning

J. Rodrıguez-Calcerrada Æ J. A. Pardos Æ L. GilUnidad de Anatomıa, Fisiologıa y Genetica Forestal. Escuela Tecnica Superior de Ingenierosde Montes, Universidad Politecnica de Madrid, Ciudad Universitaria s/n, E-28040 Madrid,Spain

I. Aranda (&)Centro de Mejora Genetica Forestal (CIFOR), Instituto Nacional de Investigaciones Agrarias(INIA), Apdo. 8111, E-28080 Madrid, Spaine-mail: aranda@inia.es

123

New Forests (2007) 33:67–80DOI 10.1007/s11056-006-9014-7

Summer field performance of Quercus petraea (Matt.)Liebl and Quercus pyrenaica Willd seedlings, plantedin three sites with contrasting canopy cover

J. Rodrıguez-Calcerrada Æ J. A. Pardos Æ L. Gil ÆI. Aranda

Received: 23 May 2006 / Accepted: 30 May 2006 / Published online: 20 October 2006� Springer Science+Business Media B.V. 2006

Introduction

Water stress caused by drought is the main constraint to natural and artificialregeneration in Mediterranean forest ecosystems. Irregular rainfall, high tempera-tures and vapour pressure deficit often faced by seedlings during summer reducetheir water potential, conditioning most of their physiological responses. Deep-rooting habit allows Quercus species to forage for water within the soil profile andsustain higher photosynthetic rates than other shallow-rooted species during thegrowing season, conferring a competitive advantage in dry-prone sites (Bahari et al.1985; Dickson and Tomlinson 1996; Wilson and Hanson 2003). At leaf level, osmoticadjustment (Abrams 1988; Aranda et al. 1996), strong stomatal control capacity(Bahari et al. 1985; Abrams 1990) and stability of photosynthetic metabolism up tosevere water stress (Epron et al. 1992; Damesin and Rambal 1995; Valladares et al.2005) are also typical drought-tolerant mechanisms in oaks. Regarding speciesstudied here, Quercus pyrenaica has been described as more tolerant to drought thanQuercus petraea in comparative studies with adult trees (Aranda et al. 1996) andwith seedlings under glasshouse conditions (Aranda et al. 2004b). However, despitethe large area covered by Q. pyrenaica in the Iberian Peninsula few works have beenundertaken aimed to discern the mechanisms underlying its resistance to drought atthe juvenile stage (Gallego et al. 1994; Mediavilla and Escudero 2003).

Stomatal limitation is the most frequent cause of assimilation impairment in dryperiods, in relation to stomata closure and decreasing inflow of CO2 to mesophyllcells. Non-stomatal limitations may also occur, especially under high water stress,favoured by stomata closure (Powles 1984; Faria et al. 1998). As a drop of photo-synthesis substrate occurs, accumulation of NADPH and ATP enhances an excess ofenergy that plants rapidly cope with by heat dissipation in the antennae of lightharvesting complexes of photosystem II (PS II). This response balances lightabsorption and utilization, avoiding injury of PS II and longer lasting occurrence ofphotoinhibition (Nogues and Alegre 2002; Franco 2004). Chlorophyll a fluorescenceof leaves permits to assess photoinhibitory responses to stresses affecting photosyn-thetic apparatus, such as water stress or light in excess (Maxwell and Johnson 2000).

Both the extent of drought and seedling susceptibility to drought varies acrossplanting sites. During summer, seedlings growing in open areas are exposed tohigher air and soil temperatures, higher vapour pressure deficit, and lower soil wateravailability than those in the understory (Aussenac 2000; Redding et al. 2003).Canopy protection is often necessary to ensure the success of reforestations carriedout in the Mediterranean basin (Gomez-Aparicio et al. 2004). Accordingly, anincreasing number of oak plantations have been conducted under secondary coniferforests, such as those formed by Pinus sylvestris. However, limits between facilitationand competition are not easily drawn. Excessive belowground competition andsummer rainfall interception in high-density stands may enhance soil waterdeployment, limiting seedling establishment (Dalton and Messina 1994; Valladaresand Pearcy 2002; Valladares et al. 2004). In addition, it has been pointed out thatcanopy effects over seedlings change with both site aridity and fertility, (Holmgrenet al. 1997; Machado et al. 2003), so that suitability of shelterwood has to be testedfor a particular region. In this regard, many studies have intended to ascertain mostconvenient stand densities for natural and artificial regeneration through morpho-logical and physiological responses, survival and growth of understory seedlings

68 New Forests (2007) 33:67–80

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(Collet et al. 1997, 2001; Gemmel et al. 1996; Agestam et al. 2003). The outcome ofnurse—target plants interaction is likely to be dependent of the ecology of thespecies. At this respect, although Q. petraea is said to be more shade-tolerant thanQ. pyrenaica we are not aware of any published work comparing functionality ofjuvenile seedlings of both species at different light environments.

In this study we aimed to address the influence of planting site on the survival ofQ. pyrenaica and Q. petraea seedlings, analysing the physiological mechanismsunderlying their responses to summer drought (water status, leaf gas exchange andphotoinhibition). Both oak species have flush type leaf development so that waterstress effects on leaf parameters was analysed in leaves that had developed undernon-stressed conditions in late spring. We hypothesized that survival is lower in openareas than in shelterwoods and, in turn, in dense shelterwoods than in light ones, andsecondly, that performance of Q. pyrenaica seedlings is better than that of Q. petraeain terms of water relations and survival.

Material and methods

The study site was located at Montejo de la Sierra (41�7¢ N, 3�30¢ W), in the ‘‘Sis-tema Central’’ of the Iberian Peninsula at 1,300 m above sea level. The experimentalsite was an even-aged Scots pine plantation carried out in 1958, southeastern ori-ented and 15% medium slope. It is close to the ‘‘Montejo de la Sierra’’ beech–oakforest, where Quercus petraea can be found in one of its southernmost populations ofEurope, co-occurring with Mediterranean mountain species such as Quercus pyre-naica (Gil et al. 1999). The soil is derived from micaceous gneiss to micacites sub-strate, generally well drained deep and fertile.

In 1998, a small area (2,000 m2) of the Scots pine stand, on level with an adjacentsite with the original tree density (unthinned area), was thinned to 75% of initialdensity (thinned area) (Table 1). In 2000, a total of 500 one-year-old seedlings ofQuercus petraea and Q. pyrenaica cultivated from seeds collected in the nearbybeech–oak forest were alternatively underplanted among rows of pines in two plotsin the unthinned and thinned sites. Understory vegetation was scarce in both plots;only bracken fern [Pteridium aquilinum (L.) Kuhn] was relatively abundant in thethinned site. Two hundred and fifty seedlings of the two species were also planted ina nearby clearing after brushing out 2 m wide stripes of low-size shrubs, consistingmainly of Adenocarpus hispanicus (Lam.) DC., Adenocarpus complicatus (L.) Gayand Juniperus communis L.

Table 1 Characteristics of the three experimental sites; n = 10–30 for GSF, n=5 for PPFDmax andVPDmax (±SE)

Clearing Shelterwood

Thinned Unthinned

Basal area (m2 ha–1) 0 53.6 65.8Density (trees ha–1) 0 800 1,067Dominant height (m) 0 21.6 22.5GSF (%) 95.2 ± 0.3 23.6 ± 1.0 19.3 ± 0.7PPFDmax (lmol m–2 s–1) 1332 ± 61 675 ± 110 365 ± 16VPDmax (KPa) 3.11 ± 0.04 2.58 ± 0.02 2.51 ± 0.02

New Forests (2007) 33:67–80 69

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Seedling survival was examined at the end of the first year and at the end of theexperiment (4th year). Leaf gas exchange, chlorophyll fluorescence and water statuswere measured in the first week of July and August 2002, and July, August andSeptember 2003. Five seedlings of each species were chosen avoiding the edge of theplots and one first-flush, completely expanded leaf from the upper third part of thecrown, selected for measurements. A portable infrared gas exchange analyser(IRGA LCA 4 Analytical Development Corporation, UK) was used to mea-sure photosynthesis (A, lmol CO2 m–2 s–1) and stomatal conductance (g, mmolH2O m–2 s–1) at early morning (10.00–11.00 h) and midday (12.00–13.00 h). Mea-surements were carried out under natural conditions, thus measurements in theforest understory were often made under non-saturating light conditions. Chloro-phyll a fluorescence was measured in the same leaf before dawn and at midday witha portable pulse-modulated fluorometer (FMS 2, Hansatech Instruments Ltd., UK)in order to obtain maximum photochemical efficiency of PS II (Fv/Fm, Fv = Fm – Fo,where Fm is the maximum fluorescence and Fo the minimum fluorescence emitted byleaves). At midday, leaves were darkened for a period of c 15 min before mea-surements. Predawn (Ypd, MPa) and midday water potential (Ymd, MPa) weremeasured with a pressure chamber (PMS Instrument Co. 7000, Corvallis Oregon). Ineach date, leaf mass per area (LMA, mg cm–2), chlorophyll and nitrogen concen-tration were analysed in sampled leaves. LMA was estimated by dividing the area ofa leaf disc by its dried weight. Chlorophyll content was examined spectrophoto-metrically after incubating leaf discs in 5 ml of dimethyl sulfoxide during 4 h at 65�C;values were expressed on an area (Chla) and mass (Chlm) basis. Nitrogen contentwas analysed in dried leaves, excluding petioles, following Kjeldahl procedure;values were expressed on an area (Na) and mass (Nm) basis. In the second samplingyear, it was not possible measuring plants in the clearing.

To characterize environment of each plot, light and soil moisture were quantifiedalong the last year of the study. Understory light availability was measured usinghemispherical photographs. Thirty photographs over the shoot tip of plants (10 inthe clearing) were taken around the plots centre in the evening to avoid directradiation exposition. A Nikon FM camera supplied with a sigma 8 mm fisheye wasused to photographs acquisition. Afterwards they were scanned (Olympus ES-10,Olympus Optical Co Europe GMBH) and further analysed with Hemiview 2.1Canopy Analysis Software (Delta-T), to determine global radiation (GSF; %)reaching the point where the photograph had been taken (Table 1); GSF is a sur-rogate of canopy opening ranging from 0% (no visible sky) to 100% (no plantobscures the sky) (Rich et al. 1993). Maximum daily water vapour deficit (VPDmax)and photosynthetic photon flux density (PPFDmax) were calculated as the average ofthe five highest values in the daytime. Measurements of PPFD were made with aquanta sensor mounted on the IRGA cuvette while air temperature and relativehumidity flowing inside the chamber were used to calculate VPD.

One-metre long PVC tubes were introduced into the soil to follow seasonalchanges in water content at various depths (10, 20, 40, 60 and 80 cm) with a TDR(Trase System I, Soil Moisture Equipment, USA). A total of six tubes were ran-domly introduced in the unthinned, and thinned plots, avoiding a one-metre widtharea either side of the rows of pines. It was not possible measuring soil moisture inthe clearing so that three additional tubes were buried in an adjacent firebreak tomake an estimate of soil moisture in the clearing.

70 New Forests (2007) 33:67–80

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Two-way analysis of variance (ANOVA), considering site and species as mainfactors, was run to examine leaf characteristics of seedlings within each date (i.e.,water potential, gas exchange, chlorophyll fluorescence, LMA and biochemicalcomposition). Ypd was controlled by ANCOVA when testing species and site effectson leaf gas exchange and Fv/Fm. Since leaf gas exchange was not obtained at satu-rating light, measuring PPFD was considered a covariate when testing seasonal andinterannual variability, and differences of A and g between the two species. Forwardstepwise multiple regression analysis was performed to assess the contribution of leafparameters on net assimilation variability. When necessary, data were square root orlog transformed to meet assumptions of normality and homoscedasticity. In all casesadopted significance level was 5%.

Results

Light availability to seedlings (GSF and PPFDmax) and evaporative demand(VPDmax) were highest in the clearing and lowest in the unthinned site (Table 1).Soil moisture in the thinned plot was higher than in the unthinned and firebreakplots, except at 40 cm depth, across all dates (Table 2). Soil moisture at 10 cm depthwas gradually decreasing from the first measurement in early July to 4th August2003, recovering after first rainfall events occurred (Fig. 1). In deeper layers, soilmoisture continued to decrease up to early October. Mortality at the end of the firstyear was much higher in the clearing than in the pine understory. By the end of thestudy period, mortality for both species was almost total in the clearing, lowest in thethinned site and intermediate in the unthinned site (Fig. 2).

Plants flushed only once along the vegetative period so that leaves expanded onlate May during non-limiting soil water conditions. Leaf water potential was rela-tively high at the beginning of summer, declined as summer progressed and recov-ered rapidly after late-season rains (Fig. 3), following seasonal evolution of topsoilmoisture (0–10 cm). Seedlings water potentials in 2002 were considerably lower asthis year was drier than 2003. In 2002, 37% of Ypd values were lower than –1.5 MPaunder the pinewood, while in the following year the lowest value was –1.2 MPa.Considering species, predawn water potential was higher in Quercus pyrenaica inalmost all dates (P-value <0.05 in August and September 2003) (Fig. 3a), but therewere no significant differences between species at midday (Fig. 3b). Ypd was lowestin the unthinned area and highest in the thinned area in both years. It is worth notingthat in 2002 Ypd in the unthinned area was even lower than in the clearing (Fig. 3a).

A, g, and Fv/Fm were affected differently by water stress along summer. Pooleddata for the 2 years showed that midday A and g values decreased steeply as Ypd

decreased, while Fv/Fm showed high stability up to –3 MPa (Fig. 4a, b, d); intrinsic

Table 2 Volumetric soilmoisture measured from 7 Julyto 12 September 2003 in thethinned and the unthinnedplots, and a nearby firebreak(±SE; n = 3)

Depth (cm) Soil moisture

Firebreak Thinned Unthinned

10 10.6 ± 1.1 14.9 ± 0.7 12.2 ± 0.720 10.7 ± 1.2 16.6 ± 0.5 14.4 ± 0.640 11.9 ± 1.1 11.8 ± 1.1 14.1 ± 0.660 12.2 ± 1.4 16.3 ± 1.1 8.7 ± 0.680 13.3 ± 2.0 18.8 ± 1.6 10.8 ± 0.9

New Forests (2007) 33:67–80 71

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water use efficiency (IWUE, A/g) increased as seedlings were subjected to increasingwater stress (Fig. 4c). Multiple regression analysis showed that g explained 58.3%and 68.7% of the variability observed in A for Q. pyrenaica and Q. petraea,respectively. When only values of Ypd < –1 MPa were selected for this analysis thecontribution of g was similar and the role of Fv/Fm was again non-significant. In 2002mean predawn Fv/Fm in August was higher than 0.8, except in the clearing

0

0

0

10

20

30

0

10

20

30

0

10

20

30

7/03 8/03 9/03 10/03 11/03 12/03

Vol

umet

ric

soil

moi

stur

e (%

)

Date

10 cm

20 cm

40 cm

60 cm

80 cm

10

20

30

40

10

20

30

Fig. 1 Seasonal variation ofsoil moisture (%) at variousdepths in the thinned(squares), unthinned(triangles) and firebreak plots(crosses) in 2003. Arrowsindicate dates in which waterpotential was measured

72 New Forests (2007) 33:67–80

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0

20

40

60

80

100

Year 1 Year 4Cl. Th. Un. Cl. Th. Un. Cl. Th. Un. Cl. Th. Un.

Mor

talit

y (%

)

Year 1 Year 4

a b

Fig. 2 Mortality of Q. petraea (a) and Q. pyrenaica (b) seedlings planted in a clearing (white bars), athinned pinewood (dashed bars) and an unthinned pinewood (black bars) at the end of the first yearand the fourth year after planting

–3

–2.5

–2

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–1

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u

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pd(M

Pa)

Ψm

d(M

Pa)

Ψ

Fig. 3 Seasonal evolution of predawn (a) and midday leaf water potential (b) in seedlings ofQuercus pyrenaica (white bars) and Quercus petraea (grey bars) planted in a thinned pinewood, anunthinned pinewood and a clearing. Species differences within a date were marked: *(P < 0.05).Different letters indicated site differences after Tukey’s test within a date

New Forests (2007) 33:67–80 73

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(0.75 ± 0.02 for the two species). Down-regulation of PS II at midday was evident inboth species, which brought about a declining in Fv/Fm, again to a higher extent inthe clearing than in the thinned or unthinned sites (0.65 ± 0.03, 0.76 ± 0.01 and0.77 ± 0.01, respectively; P-value < 0.0014). Fv/Fm values lower than 0.8 were nevermeasured in any date of 2003 at midday.

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Fig. 4 Midday response ofphotosynthesis (a), stomatalconductance (b), maximumphotochemical efficiency(c) and intrinsic water useefficiency (d) to decreasingpredawn leaf water potential inseedlings of Q. pyrenaica(white symbols) and Q. petraea(grey symbols) planted in aclearing (triangles), a thinnedpinewood (circles) and anunthinned pinewood (squares)

74 New Forests (2007) 33:67–80

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Insofar as measurements of gas exchange were not conducted at saturating light,it is difficult to compare photosynthetic performance between species. However,representing A against the spectrum of PPFD registered at the time of measure-ments made in 2003, it is clear that Quercus pyrenaica reached higher values of A atequal PPFD than Quercus petraea (Fig. 5). Quercus pyrenaica had also significantlyhigher LMA and Na. Superior Na despite similar Nm was the result of greater LMAin Quercus pyrenaica, which also explain that Chla were similar in both speciesdespite lower Chlm in this species (Table 3).

Discussion

Although stands tend to close after thinning, depending on how remaining trees andunderstory vegetation take advantage of the increase in resources availability(Kozlowski et al. 1991), in this study, we observed that the effects of a 4-year-oldthinning were still evident. Reduction in stand density brought about modificationsin environmental characteristics (i.e., light and soil moisture) that were facilitativefor the establishment of seedlings. Lower transpiring surface due to thinning of thepinewood might have been the major factor contributing to the higher wateravailability in this site (Aussenac 2000). However, other factors such as canopy andlitter rainfall interception should not be ruled out. Stogsdili et al. (1992) suggestedthat the effect of increased throughfall in thinned stands could be more importantthan the reduction in soil water usage. Litter accumulation in the unthinned plotcould also have enhanced surface soil layers desiccation because of precipitationinterception, particularly in summer months when only small rainfall events (lessthan 5 mm) were recorded (Kropfl et al. 2002).

Negative exponential relationship between soil moisture and seedling predawnwater potential causes great variation in water status following slight changes in soil

A;

mol

esC

O2

m–2

s–1

PPFD

0

2

4

6

8

10

0 500 1000 1500

nai Th.Q. pyre ca –Q. petraea – Th.Q. pyrenaica – Un.Q. petraea – Un.

Fig. 5 Relationship between photosynthetic photon flux density (PPFD) and photosynthesis (A) in2003, for seedlings of Q. pyrenaica (white symbols) and Q. petraea (grey symbols) planted in thethinned pinewood (circles) and the unthinned pinewood (squares). Equations were: A = 2.71 * Ln

(PPFD) – 10.1; r2: 0.84, P < 0.001 (Q. pyrenaica in the thinned pinewood); A = 1.19 * Ln (PPFD) –3.5; r2: 0.83, P < 0.001 (Q. pyrenaica in the unthinned pinewood); A = 1.59 * Ln (PPFD) – 5.2; r2:0.71, P < 0.001 (Q. petraea in the thinned pinewood); A = 1.05 * Ln (PPFD) – 3.0; r2: 0.80, P < 0.001(Q. petraea in the unthinned pinewood)

New Forests (2007) 33:67–80 75

123

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76 New Forests (2007) 33:67–80

123

moisture (Aranda et al. 1996; Kozlowski and Pallardy 1997), highlighting theimportance of minor differences in soil moisture between planting sites. Differencesin plant water status across sites may partly explain the results of mortality. Lowestwater potential in the unthinned understory seedlings across all dates may explainhigher mortality with respect to the thinned area. For instance, in 2002 Ypd inQ. petraea reached almost –2.5 MPa in the unthinned site, which is below waterpotential at turgor loss (–2.2 MPa) found in seedlings of Q. petraea and Q. pyrenaicagrowing under dense shelterwood (Rodrıguez-Calcerrada, unpublished data). Inaddition to low soil water content, low water potential in the unthinned understoryseedlings could be ascribed to a smaller root system, resulting from both a strongcarbon limitation beneath the forest canopy, where rarely saturating values wereattained during the exposition to sunflecks (Fig. 4), and a likely enhanced biomassallocation to shoots rather than roots compared to a higher light environment(Montgomery 2004).

Mortality was much higher in the clearing even though water potential in 2002was higher than in the unthinned area. This suggests that high mortality in theclearing probably occurred in the first year after planting, when plants lacking anextensive root system cannot meet high evaporative demand on open sites, sufferinga stronger transplant shock than under canopy protection (Johnson 1994). In addi-tion, mean predawn Fv/Fm in August was below the optimal range of c 0.84 in theclearing (Bjorkman and Demmig 1987) while midday Fv/Fm was lower than in thethinned or unthinned understories along the gradient of Ypd for both species. Pho-toinhibitory responses in the clearing reveal an inherent susceptibility to high irra-diance related with the late-successional character of both species (Kitao et al. 2000;Valladares et al. 2002, 2004).

Overall, these results highlight the necessity of shade to ensure the success ofplantations with Q. petraea and Q. pyrenaica in Mediterranean environments.Shelterwood would be particularly recommended to minimize drought-relatedmortality and photoinhibition during early phases of establishment (Evans 1984;Gomez-Aparicio et al. 2004). Nonetheless, thinning of highly dense stands prior toafforestations may ameliorate growth conditions as both light and seedlings waterstatus improve with respect to stands with the original density (Dalton and Messina1994; Aranda et al. 2004a).

Regarding species, it was surprising the slight difference between Q. pyrenaicaand Q. petraea in response to water stress. Decrease of seedling water potentialaffected negatively stomatal conductance and net assimilation. The relationshipbetween IWUE versus Ypd indicated that reduction in g caused by water stress washigher than reduction in A, indicating that stomatal closure was the main factorhampering net CO2 assimilation. Stability of Fv/Fm along the gradient of water stresssupports the high stability of photosynthetic apparatus up to severe water stress as ithas been already addressed in other works studying photosynthetic responses ofMediterranean species to drought (Flexas and Medrano 2002; Valladares et al.2005). Interspecific differences were found in Ypd, which was slightly lower inQ. petraea, probably in relation to a smaller or shallower root development thatrestrict its soil water acquisition (Castell et al. 1994; Asbjornsen et al. 2004). Dif-ferences were statistically significant just in 2003 in the thinned site, suggesting thatseedlings of Q. pyrenaica were more able to reflect the improvement in wateravailability associated to thinning when conditions are not so harsh as to equallystress both species, as occurred in 2002. Furthermore, Q. pyrenaica exhibited higher

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photosynthetic capacity in the relatively drought-free 2003 year. Higher LMA couldimply a greater accumulation of nitrogen and photosynthetic tissue per unit of leafarea, which increases area-based photosynthesis. Higher chlorophyll content in Q.petraea even when light is not a limited resource can be ascribed to a preferentialallocation of nitrogen to light harvesting, which reduces nitrogen availability tocarboxylation enzymes and may limit photosynthesis at higher PPFD (Seemannet al. 1987). Both lower LMA and higher chlorophyll concentration could be relatedto a greater tolerance to shade in agreement with a trade-off between drought versusshade-tolerance (Abrams et al. 1994; Aranda et al. 2005). Although Q. petraea isconsidered an intermediate shade-tolerant species in central Europe, light require-ments may differ with habitat such that its capacity to establish in shade may bemore pronounced in Mediterranean populations than in northern areas of its geo-graphic range (Kelly 2002).

Acknowledgements This research has been supported by the Consejerıa de Medio Ambiente yDesarrollo General de la Comunidad Autonoma de Madrid. J. Rodrıguez-Calcerrada was supportedby a scolarship from the Consejerıa de Educacion de la Comunidad de Madrid (C.A.M.) and theFondo Social Europeo (F.S.E.). We gratefully acknowledge Sven Mutke for reviewing this manu-script.

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ANEXO IV

Acclimation to light in seedlings of Quercus petraea (Mattuschka) Liebl.

and Quercus pyrenaica Willd. planted along a forest-edge gradient

Publicado en la revista Trees 21: 45-54 (2007)

Autores: Rodríguez-Calcerrada J., Pardos J.A., Gil L., Aranda I.

Trees (2007) 21:45–54DOI 10.1007/s00468-006-0095-x

ORIGINAL ARTICLE

Acclimation to light in seedlings of Quercus petraea (Mattuschka)Liebl. and Quercus pyrenaica Willd. planted along aforest-edge gradientJ. Rodrıguez-Calcerrada · J. A. Pardos · L. Gil ·I. Aranda

Received: 22 September 2005 / Revised: 8 August 2006 / Accepted: 31 August 2006 / Published online: 10 October 2006C© Springer-Verlag 2006

Abstract Photosynthetic acclimation of two co-occurringdeciduous oaks (Quercus petraea and Quercus pyrenaica)to a natural light gradient was studied during one grow-ing season. In the spring of 2003, 90 seedlings per specieswere planted along a transect resulting from a dense Pi-nus sylvestris stand, an adjacent thinned area and a 10-m-wide firebreak (16.5–60.9% Global Site Factor (GSF)). Intwo dates of the following summer, we measured leaf gasexchange, carboxylation efficiency (CE), chlorophyll andnitrogen content, light–response curves of chlorophyll a flu-orescence parameters, and leaf mass per area (LMA). Sum-mer was mild, as evidenced by leaf predawn water potential(�pd), which reduced the interactive effect of water stress onthe response of seedlings to light. Q. pyrenaica had higherLMA, CE, stomatal conductance (gs max) and photosynthesisper unit area (Aa

max) than Q. petraea at all growth irradiances.Aa

max, LMA, gs max and electron transport rate (ETR) all in-creased with light availability (GSF) in a similar fashion inboth species. Light had also a clear effect on the organiza-tion of Photosystem II (PS II), as deduced by chlorophyll afluorescence curves. Chlorophyll concentration (Chlm) de-

Communicated by U. Luttge

J. Rodrıguez-Calcerrada · J. A. Pardos · L. GilUnidad de Anatomıa, Fisiologıa y Genetica Forestal. EscuelaTecnica Superior de Ingenieros de Montes, UniversidadPolitecnica de Madrid,Ciudad Universitaria s/n,28040 Madrid, Spain

I. Aranda (�)Centro de Investigacion Forestal (CIFOR), Instituto Nacional deInvestigacion Agraria y Alimentaria (INIA),Carretera de La Coruna Km 7.5,28040 Madrid, Spaine-mail: aranda@inia.es

creased with increasing light availability in Q. pyrenaica butit did not in Q. petraea. Seedlings of Q. petraea acclimatedto higher irradiances showed a greater non-photochemicalquenching (NPQ) than those of Q. pyrenaica. This suggests ahigher susceptibility to high light in Q. petraea, which wouldbe consistent with a better adaptation to shade, inferred fromthe lower LMA or the lower rate of photosynthesis.

Keywords Quercus petraea . Quercus pyrenaica . Lightgradients . Photosynthetic acclimation . Chlorophyll afluorescence curves

Introduction

Acclimation to available light is achieved by an integral re-sponse at all plant levels. At leaf-level, a combination of sev-eral physiological and morphological changes are involvedin the photosynthetic acclimation to the prevailing light en-vironment. Changes in foliar nitrogen concentration and par-titioning of nitrogen between the components of the photo-synthetic apparatus play a role (Seemann et al. 1987; Evans1989; Niinemets 1997; Niinemets and Tenhunen 1997; LeRoux et al. 2001). The structural change that occurs in re-sponse to growth irradiance is also an important factor ina number of studies (Niinemets and Kull 1998; Evans andPoorter 2001; Muraoka et al. 2002; Robakowski et al. 2003;Niinemets and Valladares 2004); light increases mass perunit area (LMA), giving higher rates of area-based photo-synthesis in leaves with superior nitrogen content per unitarea (e.g. Reich et al. 1999). Yet the amount of photosyn-thetic machinery usually remains constant under differentgrowth irradiances, resulting in unaltered mass-based pho-tosynthesis (Evans and Poorter 2001).

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46 Trees (2007) 21:45–54

Light-induced photosynthetic adjustments vary withspecies’ light requirements (Ashton and Berlyn 1994; Naiduand DeLucia 1997; Valladares et al. 2002). Very shade-tolerant species show a limited photosynthetic plasticity(Valladares et al. 2000a; Mitchell 2001; Griffin et al. 2004)as well as a lower photosynthetic capacity in high light. Thisimplies that a smaller fraction of absorbed energy is dissi-pated in carbon fixation, so that plants lacking alternativemechanisms to cope with excess energy will be more likelyexposed to photoinhibitory damages on the photosyntheticapparatus (Ogren 1991). On the other hand, high photo-synthetic capacity of shade-intolerant species is positivelyrelated to the rate of respiration in low light and thus entailsa compromise for their long-term survival in the understory(Kaelke et al. 2001).

Non-photochemical quenching (NPQ) is involved in thethermal dissipation of excess energy. Xanthophyll cycle-dependent conformational changes in the light-harvestingcomplexes of PS II (LHC II) quench energy as heat in lieu ofdriving photochemistry (Walters and Horton 1993; Mulleret al. 2001), contributing to protect chloroplasts from ox-idative damage. A correlation of NPQ with growth irradi-ance has been observed in some species (Rosenqvist 2001;Einhorn et al. 2004), but not in others (Johnson et al. 1993;Anderson et al. 2001; Rosenqvist 2001), and the relation-ship of NPQ with species’ light requirements is yet far fromclear. Estimates of NPQ under ambient light conditions areaffected by fluctuating and often non-saturating irradiances.Chlorophyll a fluorescence curves in response to actinic lightprovide a reliable estimate of both the NPQ and the electrontransport rate that leaves display at saturating PPFD (Whiteand Critchley 1999).

Studies of acclimation to light carried out across naturallight gradients will be affected by the co-variation of air tem-perature, vapour pressure deficit and soil moisture (Ellsworthand Reich 1992; Muraoka et al. 1997; Valladares and Pearcy1997; Jifon and Syvertsen 2003; Redding et al. 2003), aswell as by changes in the spectral quality of light (Lei andLechowicz 1998). Even though a more realistic interpretationcan be deduced from such studies, one cannot easily separatethe interaction among all these factors. Microclimatic vari-ations linked to high-irradiance expositions are related withthe inability of some shade species to perform adequatelyout of tree canopy protection (Ellsworth and Reich 1992;Sipe and Bazzaz 1994), which is likely to be more dramaticin Mediterranean forest ecosystems (Faria et al. 1998).

The objectives of this study were (i) to examine the varia-tion of leaf photosynthetic characteristics along a forest-edgegradient in two contrasting oaks, namely Quercus petraeaand Quercus pyrenaica, (ii) to compare their acclimatoryresponses to the range of available light generated in thisgradient, and (iii) to discuss the ecological significance ofsuch responses. Q. petraea is a deciduous species and Q.

pyrenaica is a semi-deciduous marcescent species constitut-ing late-successional forests in temperate and Mediterraneanenvironments, respectively. A moderate degree of shade tol-erance is referred in both Q. pyrenaica (Baraza et al. 2004)and Q. petraea (Brzeziecki and Kienast 1994; von Lupke1998; Kelly 2002), while a higher sensitivity to water stresshas been pointed out in Q. petraea (Aranda et al. 1996, 2004).Traits conducive to a higher drought-resistance (e.g. highroot to shoot ratio, or high leaf mass per area) may restrictthe capacity of a plant to optimise light capture in low-lightenvironments, which is underlying the theory of a trade-offbetween drought and shade tolerance (Abrams 1990, Abramset al. 1994; Kubiske et al. 1996). Our hypotheses were(i) that leaf photosynthetic characteristics would change inrelation to light availability, (ii) that the degree of variationwould differ between both species, and (iii) that according toa trade-off between drought and shade tolerance, Q. petraeawould show both a higher capacity to tolerate shade and ahigher sensitivity to high light. To test these hypotheses, theacclimatory responses to a light-availability gradient werestudied in seedlings of Q. petraea and Q. pyrenaica duringone growing season.

Material and methods

The study was conducted in a mature Pinus sylvestris stand,at 1250 m a.s.l. This stand is located near the beech-oakforest of Montejo de la Sierra (41◦7′ N, 3◦30′ W), whereseveral temperate species at the southern boundary of theirgeographic range (e.g. Q. petraea, Fagus sylvatica) are co-occurring with other species at an optimum of their distri-bution (e.g. Q. pyrenaica). In early spring of 2003, one-year-old seedlings of Q. pyrenaica and Q. petraea wereplanted along a 150-m-long SW–NE aligned transect run-ning from a firebreak edge to the pinewood interior. Ninetyseedlings per species were alternately planted 1.5-m sepa-rated in the understory of a dense stand (1067 trees ha−1

density and 65.8 m2 ha−1 basal area), a contiguous thinnedarea (800 trees ha−1 density and 53.6 m2 ha−1 basal area)and in the centre of an adjacent 10-m-wide SE-oriented fire-break, forming the edge of the stand. Understory vegetationwas almost absent, except for the sparse presence of Pterid-ium aquilinum L. (Kuhn). Ferns were more abundant in thethinned stand adjoining the firebreak, which had been colo-nized by patches of pioneer woody shrubs (Genista floridaL., Adenocarpus hispanicus (Lam.) and Rubus ulmifoliusSchott.). The terrain was gently sloping (15%).

The following summer several leaf parameters were anal-ysed in order to evaluate the acclimation of the seedlings.Measurements were made in early summer (21–23 June)and late summer (26–28 August). Leaf gas exchange mea-surements were performed with a portable infrared gas

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Trees (2007) 21:45–54 47

analyser (IRGA LCA 4 Analytical Development Corpora-tion, UK), on attached leaves. For each species and site(dense pinewood, thinned pinewood and firebreak), fourmeasurements were made on each four different seedlings(three in August). Area-based rate of photosynthesis (Aa

max)and stomatal conductance (gs max) at saturating light (c1300 and 900 µmoles m−2 s−1 for edge and understoryseedlings, respectively) were initially measured at ambientCO2 (365 ppm) and down to 300, 200 and 100 ppm, throughreducing CO2 concentration entering the system with an ab-sorbing reactive (soda lime). Temperature inside the cuvettewas set to 25◦C. Carboxylation efficiency (CE) was esti-mated as the initial slope of the net assimilation rate againstthe intercellular concentration of CO2 (Ci). Due to the in-duction time for full photosynthetic activation, the leaf wassubmitted to near-saturating light for a period of c 15 minbefore starting the first measurements (three readings weretaken at each CO2 concentration). During this period, leaftemperature was measured with a copper–constantan ther-mocouple (24–30◦C). Measurements were made from 9.30to 11.30 local time, in order to avoid either excessive airtemperature (23.2◦C in June and 25◦C in August) or VPD(1.3 KPa in June and 1.7 KPa in August) that could lead todiurnal leaf water deficit.

Acclimation of PS II was examined by means of light–response curves of chlorophyll a fluorescence parametersin intact leaves. Curves were made with a portable pulse-modulated fluorometer (FMS 2, Hansatech Instruments Ltd.,UK). The unit was pre-programmed to gradually increaselight provided by a halogen lamp (Osram 64255, 25 W8 V). Prior to measurements, leaves were acclimated todark for 30 min with dark-adaptation leaf-clips. The fibre-optic cable linked to the light source was attached to theleaf-clip through an adapter that prevented variation of thedistance from the light source to the sample and expo-sition to ambient light. After measurement of minimumfluorescence in the dark-adapted leaf (F0) with a measur-ing light <0.05 µmol m−2 s−1, a 0.8 s saturating pulse(6600 µmol m−2 s−1) was applied, and maximum fluores-cence measured (Fm) to calculate potential quantum yieldof photosystem II (PSII) (Fv/Fm = (Fm–Fo)/Fm). Immedi-ately halogen lamp was turned on, and a new saturatingpulse applied (F′

m) after steady-state fluorescence (Fs) wasachieved. Time for reaching steady fluorescence varied fromapproximately 3 min at lower light intensities to 30 s atthe highest light level. Before changing to the next lightlevel, a 5 s far-red light pulse was applied to drain electronsfrom acceptors of PS II and measure light-adapted mini-mum fluorescence (F′

0). Light was increased in ten stepsfrom 26 to 1500 µmol m−2 s−1. At each actinic light wecalculated: the effective quantum yield of PSII (φPSII =(F′

m − Fs)/F′m), the efficiency of open reaction centres

(F′v/F′

m = (F′m − F′

0)/F′m), the photochemical quench-

ing (qP = (F′m − Fs)/(F′

m − F′0)), the non-photochemical

quenching (NPQ = (Fm − F′m)/F′

m), and the electron trans-port rate (ETR = øPSII × 0.5 × 0.84 × PPFD). Chlorophylla fluorescence curves were not realized under steady-stateconditions since Fm takes much longer to stabilize than Fs

(White and Critchley 1999). Measurements were performedon five seedlings per species and site. Two curves per sitewere alternately made to standardize environmental variationwithin a day.

The water status of the seedlings was estimated by mea-suring the leaf water potential at dawn (�pd) and at midday(�md). Measurements were made with a pressure chamber(PMS Instrument Co. 7000, Corvallis Oregon) in 23 June and28 August. Five plants per species and site were measured ateach date. The same leaves were used to estimate leaf massper area (LMA), and chlorophyll and nitrogen concentrationon an area (Chla, Na) and a mass basis (Chlm, Nm). Two discswere cut in the field. One was immersed in tubes containing5 ml of dimethyl sulfoxide (DMSO) and kept in dark. In thelaboratory tubes were bathed at 60◦C for 5 h. After that, ab-sorbance of the chlorophyll solved in DMSO was measuredwith a spectrophotometer at 648.2 and 664.9 nm, to estimatechlorophyll concentration. The other disc was oven-driedfor 2 days at 65◦C and weighed to calculate LMA. Nitrogencontent of leaves, petioles excluded, was determined usingthe Kjeldahl procedure. Nitrogen partitioning among photo-synthetic processes was estimated from the ratios of Chl/N,CE/N and ETRmax/N. The relative importance of photosyn-thetic processes was assessed by calculating CE/Chl andETRmax/Chl.

Hemispherical photographs were made to give an estima-tion of the proportion of light a plant was exposed to withrespect to an open place (Global Site Factor, GSF, %). Rain-fall was registered with a rain gauge (Delta-T) at the top ofa 16-m research tower located within a neighbouring stand.One air temperature sensor and one relative humidity sen-sor, both connected to a data-logger (HOBO H8 Pro, Onset)were placed in each site. Data acquisition was programmedto take readings at 10-min intervals. Additionally, volumetricsoil moisture was periodically recorded with a TDR (TraseSystem I, Soil Moisture Equipment, USA) at 10, 20, 40, 60and 80 cm depth in three probes per site.

The relationships between light availability (i.e. GSF) andleaf attributes, as well as among leaf attributes, were inves-tigated using correlation and regression analysis. Pearson’scorrelation analysis was used to explore the statistical signif-icance of the relationship and then a regression equation wasfitted to describe it. Analysis of covariance (ANCOVA) wasused to test the difference between Q. petraea and Q. pyre-naica in each trait, where GSF was the co-variable. Somevariables were log-transformed prior to run the analyses.When date was a significant factor (p<0.05), a different func-tion was adjusted for data of June and August. Non-linear

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functions were fitted to describe the evolution of chloro-phyll fluorescence parameters with actinic light (Rascheret al. 2000). Marquardt method was selected to estimate theindependent parameters of the functions. Species and sitedifferences in leaf water potential were tested by two-wayanalysis of variance (ANOVA).

Results

Environmental parameters

The firebreak – forest interior transect generated a light gra-dient (GSF) that ranged from 60.9 to 31.9% in the firebreak,28.9 to 17.2% in the thinned site and 24.2 to 16.5% in thedense site. Daily maxima for air temperature and VPD wereon average 2.5◦C and 0.3 KPa higher in the edge than inthe dense and thinned sites, where almost identical valueswere observed (Fig. 1a, b). Soil moisture was highest in thethinned stand (Fig. 1c), probably because of lower evapo-ration than in the edge, and lower overall water uptake bytrees than in the dense site. The passage of several coldfronts produced 55.4 mm of rain between 5 and 25 August(Fig. 1d), which amounted to 67% of total summer rain-fall. This rendered temperatures and VPD lower than typi-cally at this time of the year, and lower than at the end ofJune.

Leaf water potential

Both �pd and �md decreased slight but significantly(p<0.001 and p<0.05, respectively) in August. In both dates,�pd was lowest in the dense stand whereas �md was lowest inthe edge (Table 1). There were no statistically significant dif-ferences between Q. petraea and Q. pyrenaica in both �pd

and �md. Relatively high values of �pd (minimum valuesaround − 1 MPa) evidenced a mild water stress.

Light–response curves of chlorophyll fluorescenceparameters

Chlorophyll fluorescence parameters followed a non-linearresponse to increasing actinic light. NPQ and ETR increased,

V P

Dm

ax(K

Pa)

Tm

ax (º

C)

So

il m

oist

ure

(%)

Rai

n f

all

(mm

)

Date

0.51.01.52.02.53.03.5

a

15

20

25

30b

5

10

15

20c

0

5

10

15

20

25

21-6 6-7 21-7 5-8 20-8 4-9 19-9

d

V P

Dm

ax(K

Pa)

Tm

ax (º

C)

0.51.01.52.02.53.03.5

a

0.51.01.52.02.53.03.5

a

15

20

25

30b

15

20

25

30b

5

10

15

20c

5

10

15

20c

0

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10

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20

25

21-6 6-7 21-7 5-8 20-8 4-9 19-

d

0

5

10

15

20

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21-6 6-7 21-7 5-8 20-8 4-9 19-

0

5

10

15

20

25

21-6 6-7 21-7 5-8 20-8 4-9 19-

d

Fig. 1 Daily maximum VPD a, daily maximum temperature b andmean values (n = 3) of soil moisture at 10 cm depth c, registered inthe dense stand (black line, triangles), the thinned stand (dashed line,squares) and the edge (dotted line, crosses) throughout the summer of2004; d precipitation in the study area

while qP, F′v/F′

m and φPSII all decreased as leaves weresubmitted to increasing light (Fig. 2). This pattern was similarin both dates, but in August NPQmax was lower (p<0.01), qPat a common PPFD was higher (p<0.01), and ETR saturatedboth at a higher value and a higher PPFD (p<0.05) than inJune (Fig. 2).

NPQmax and ETRmax increased with GSF (Fig. 3a, b).Q. petraea showed a more plastic response of NPQ to light

Table 1 Means ( ± SE) of predawn leaf water potential (�pd, − MPa) and midday leaf water potential (�md, − MPa)

Edge Thinned stand Dense stand p-valueQ. pyrenaica Q. petraea Q. pyrenaica Q. petraea Q. pyrenaica Q. petraea Species Site sp × site

June �pd 0.1 (0.03) 0.1 (0.01) 0.2 (0.04) 0.2 (0.1) 0.3 (0.02) 0.3 (0.05) 0.76 <0.001∗ 0.547�md 1.9 (0.1) 1.9 (0.1) 1.5 (0.1) 1.3 (0.1) 1.2 (0.1) 1.4 (0.1) 0.954 <0.001∗ 0.382

August �pd 0.5 (0.1) 0.4 (0.1) 0.5 (0.1) 0.4 (0.04) 1.0 (0.2) 0.6 (0.02) 0.089 0.016∗ 0.621�md 2.3 (0.2) 2.3 (0.3) 1.9 (0.3) 1.8 (0.1) 2.1 (0.1) 1.9 (0.1) 0.375 0.013∗ 0.826

ANOVA p-values (∗p<0.05) are indicated for species and site differences, and the interaction of both factors

Springer

Trees (2007) 21:45–54 49

Fv

/Fm

qP

0

20

40

60

80

0 400 800 1200

e

1600

j

400 800 1200

ΦΦ ΦΦP

SII

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Q

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a f

PPFD (µµµµmol m-2 s-1)

m+(1-m)*exp(-b*ppfd)

m*(1-exp(-b*ppfd))

d

m*(1-exp(-b*ppfd))

m+a*exp(-b*ppfd)

m+a*exp(-b*ppfd)

0

PPFD (µµµµmol m-2 s-1)

ET

R (

µµ µµm

ol

e-

m-2

s-1

)F

v/F

mqP

0

20

40

60

80

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20

40

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80

0 400 800 1200

e

1600

j

400 800 1200

ΦΦ ΦΦP

SII

0.2

0.4

0.6

0.8

0.4

0.6

0.8

c

i

h

1.0

2.0

3.0

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5.0

1.0

2.0

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5.0

NP

Q

b g

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0.8

1.0

a f

PPFD (µµµµmol m-2 s-1)

m+(1-m)*exp(-b*ppfd)

m*(1-exp(-b*ppfd))

d

m*(1-exp(-b*ppfd))

m+a*exp(-b*ppfd)

m+a*exp(-b*ppfd)

0

PPFD (µµµµmol m-2 s-1)

ET

R (

µµ µµm

ol

e-

m-2

s-1

)

Fig. 2 Light–response curves of photochemical quenching a, f, non-photochemical quenching b, g, efficiency of open reaction centres c, h,effective quantum yield d, i and electron transport rate e, j for seedlingsof Q. pyrenaica (circles) and Q. petraea (squares), growing in the edgeof the forest (open symbols), the thinned stand (grey symbols) and thedense stand (black symbols). Left panels are from 22 to 23 June a–eand right panels are from 27 to 28 August f–j. Error bars omitted forclarity (n = 3–5)

availability than Q. pyrenaica (species × GSF interaction,p<0.001) so that NPQmax was significantly higher in Q.petraea in the more exposed site. F′

v/F′m decreased with in-

creasing GSF, more pronouncedly in Q. petraea (species ×GSF interaction, p<0.001) (Fig. 3c). φPSII showed a low plas-ticity to light availability (Fig. 2). Since φPSII is the productof qP and F′

v/F′m, decreasing F′

v/F′m with light counter-

acted the higher capacity to maintain reaction centres oxi-dized (i.e. qP) for a given actinic light showed by more ex-posed seedlings (Fig. 3d). Fv/Fm did not correlate with light

Table 2 Regression analysis of the relationship between some leafattributes (light-saturated stomatal conductance [gs max], carboxyla-tion efficiency [CE], maximum electron transport rate [ETRmax], leafmass per area [LMA] and area-based nitrogen content [Na]) and light-saturated area-based photosynthesis [Aa

max] for seedlings of Q. petraeaand Q. pyrenaica

Species Variable × p-value R2

Q. pyrenaica gs max 0.001 0.46CE 0.012 0.37ln ETRmax 0.000 0.68LMA 0.001 0.45ln Na 0.003 0.4

Q. petraea gs max 0.002 0.42CE 0.000 0.66ln ETRmax 0.001 0.46LMA 0.107 0.13ln Na 0.483 0.03

availability and was similar in both species. All chlorophyllfluorescence parameters saturated at higher PPFD as theywere exposed to increasing GSF (data not shown). ETRmax

correlated positively with Aamax (Table 2).

Gas exchange

Aamax, gs max and CE were significantly higher in Q. pyre-

naica (all p<0.05) across all GSF (species × GSF inter-action, p>0.15) (Fig. 4). The variation of Aa

max and gs max

with light was similar in both species (Fig. 4a, b). A smallreduction in gs max occurred in both species on August(p<0.05), though Aa

max showed no decline (p>0.05). Ammax

was also higher in Q. pyrenaica (p<0.05), especially in thelower-light extreme of the gradient (species × GSF inter-action, p = 0.1). Am

max showed no relationship with GSFin Q. pyrenaica and a weak relationship in Q. petraea(Fig. 4c). CE increased significantly with light in Q. pyre-naica (Fig. 4d). CE and gs max correlated positively with Aa

max

(Table 2).

Morphological and biochemical variables

Mean LMA was higher in Q. pyrenaica across environments(p<0.05), but neither nitrogen nor chlorophyll content diddiffer significantly between Q. petraea and Q. pyrenaica(Fig. 5). LMA was positively correlated with GSF in bothspecies (Fig. 5a). Light had no effect on either Nm or Na

(Fig. 5b, c). Chlm decreased with increasing light availabilityin Q. pyrenaica, but not in Q. petraea (species × GSF inter-action, p<0.05) (Fig. 5d). When expressed on an area basis,chlorophyll content increased with light only in Q. petraea(species × GSF interaction, p<0.01) (Fig. 5e). Variationof light barely affected nitrogen partitioning to componentsof photosynthetic system. CE/N and ETRmax/N showed a

Springer

50 Trees (2007) 21:45–54

2.0

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Qm

ax

GSF (%) GSF (%)

ET

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ax

( µµ µµm

ol

e-

m-2

s-1

)

a

b

c

Fv

/F

m1

00

0

p < 0.05; R2: 0.37

p < 0.05; R2: 0.42

p < 0.001; R2: 0.64

p < 0.001; R2: 0.88

0.2

0.4

0.6d

qP

10

00

p < 0.001; R2: 0.82

p < 0.001; R2: 0.84

p < 0.01; R2: 0.64

p < 0.05; R2: 0.44

p < 0.001; R2: 0.74

p < 0.001; R2: 0.67

p < 0.01; R2: 0.60

p < 0.05; R2: 0.54

p < 0.001; R2: 0.75

p = 0.052; R2: 0.18

0

Fig. 3 Relationship ofmaximum non-photochemicalquenching a, maximum electrontransport rate b, efficiency ofopen reaction centres at 1000PPFD c and photochemicalquenching at 1000 PPFD d toglobal site factor (GSF) forseedlings of Q. petraea (greyline, filled symbols) and Q.pyrenaica (black line, opensymbols). A different regressionwas fitted for data of June(continuous line, circles) orAugust (dashed line, squares)when date was a significantfactor (p<0.05)

2

4

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p < 0.001; R2: 0.52

p < 0.001; R2: 0.53

p < 0.05; R2: 0.25

p < 0.01; R2: 0.88

p < 0.05; R2: 0.47

p < 0.01; R2: 0.81

p < 0.01; R2: 0.57

p < 0.05; R2: 0.58

p < 0.05; R2: 0.43

Aa

ma

x( µµ µµ

mol

CO

2m

-2s

-1)

gs

ma

x(m

mol

H20

m-2

s-1

)

Am

ma

x ( µµ µµm

ol

CO

2g

-1s-1)

CE

( µµ µµ

mol C

O2

m-2

s-1)

GSF (%) GSF (%)

a c

b d

Fig. 4 Relationship oflight-saturated rate ofphotosynthesis per unit area a,light-saturated rate of stomatalconductance b, light-saturatedrate of photosynthesis per unitdry mass c and carboxylationefficiency d to global site factor(GSF) for seedlings of Q.petraea (grey line, filledsymbols) and Q. pyrenaica(black line, open symbols). Adifferent regression was fittedfor data of June (continuousline, circles) or August (dashedline, squares) when date was asignificant factor (p<0.05)

trend to increase with light, but not statistically significant,whereas Chl/N decreased with light only in Q. pyrenaica(species × GSF interaction, p<0.05); Chl/N, CE/N andETRmax/N increased in August (data not shown). ETRmax ex-pressed per unit chlorophyll increased with light, the increase

being higher in Q. pyrenaica (species × GSF interaction,p<0.05) (Fig. 5f). LMA and Na were positively correlatedwith Aa

max in Q. pyrenaica (Table 2). No significant relation-ship was found when nitrogen was expressed on a dry massbasis (data not shown).

Springer

Trees (2007) 21:45–54 51

0

20

40

60

80

100

120

140

160

180

0 10 20 30 40 50 60 70 10 20 30 40 50 60 70

f

p < 0.05; R2: 0.29

p < 0.001; R2: 0.68

ET

Rm

ax /Chl

( µµ µµm

ol e-g C

hl -1s

-1)

GSF (%) GSF (%)

0.20.40.60.81.01.21.41.61.82.0

Na

(gm

-2)

79

1113151719212325

d

p < 0.001; R2: 0.35

Chlm

(mg g

-1)

79

1113151719212325

d

p < 0.001; R2: 0.35

Chlm

(mg g

-1)

5

10

15

20

25

30

35

40N

m(m

g g-1

)

b

5

10

15

20

25

30

35

40N

m(m

g g-1

)

b

c

0.10.20.30.40.50.60.70.80.91.0

Chla

(g m-2)

e

p < 0.05; R2: 0.42

p < 0.05; R2: 0.28

0

10

20

30

40

50

60

70a

p < 0.05; R2: 0.36

p < 0.05; R2: 0.27

LM

A (

g m

- 22 22)

Fig. 5 Relationship of leafmass per area a, nitrogencontent per unit dry mass b,nitrogen content per unit area c,chlorophyll content per unit drymass d, chlorophyll content perunit area e and maximumelectron transport rate per unitchlorophyll f to global sitefactor (GSF) for seedlings of Q.petraea (grey line, filledsymbols) and Q. pyrenaica(black line, open symbols). Adifferent regression was fittedfor data of June (continuousline, circles) or August (dashedline, squares) when date was asignificant factor (p<0.05)

Discussion

Variation of soil moisture, air temperature and VPD acrossthe forest-edge transect, confirm the uncertainties in fieldstudies of light acclimation in attributing the observed re-sponses to light. However, elevated precipitation recordedin the summer of 2004 reduced the effect of water stress,which is not common in Mediterranean environments. Pho-tosynthetic capacity increased non-linearly with light avail-ability in both species. This agrees with a number of stud-ies with oaks wherein maximum photosynthetic capacity(Hodges and Gardiner 1993; Ashton and Berlyn 1994) aswell as optimum growth (Gardiner and Hodges 1998) isachieved at intermediate irradiances. Light had no signifi-cant effect on foliar nitrogen concentration. The partition-ing of nitrogen among the components of the photosyn-thetic apparatus (estimated as Chl/N, CE/N and ETRmax/N)was also poorly conditioned by light. However, variation ofETRmax/Chl was consistent with an adjustment of photosyn-

thetic performance to the available light reported previously(e.g. Evans and Poorter 2001). Light-induced acclimationof the PS II brought about a greater capacity to deal withhigh PPFD. Together with higher electron transport rate,higher-light plants had also greater NPQ. This allowed alarger fraction of PS II reaction centres to remain open (i.ehigher qP than in the understory at a common PPFD), whichreduces energy in excess and thus the susceptibility to ox-idative damages (Demmig-Adams 1990; Ogren 1991). Lightacclimation of NPQ is consistent with the larger pool size ofxanthophyll cycle pigments observed in seedlings exposedto high irradiances (Barker and Adams 1997; Maxwell andJohnson 2000), as NPQ is related to the xanthophyll cy-cle activity. Besides, significant increases in Chl/N, CE/Nand ETRmax/N from June to August suggested an ontogeny-induced greater nitrogen investment in photosynthetic com-ponents (Niinemets and Tenhunen 1997; Miyazawa andTerashima 2001), which increased the capacity to use light inphotochemistry.

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52 Trees (2007) 21:45–54

It is difficult to assess what is limiting a larger increaseof photosynthetic capacity with light in these species. An in-ability to increase foliar nitrogen concentration in response tolight, as well as weak changes in the partitioning among thephotosynthetic components could have precluded a larger re-sponsiveness of photosynthesis. Furthermore, several workshave suggested shifts in temperature and VPD as factors lim-iting potential for photosynthetic acclimation in high-lightenvironments (Niinemets and Valladares 2004). Plants mayacclimate to high VPD and temperature by increasing boththe cuticle thickness and the epidermal cell wall thickness inorder to reduce water loss. This may imply an increase in theratio of non-photosynthetic to total leaf biomass (Strauss-Debenedetti and Berlyn 1994) that leads to an invariableAm

max across light environments (Montgomery 2004 ), as wasfound in the more drought-resistant Q. pyrenaica. Underthe hypothesis of Q. petraea being more tolerant to shadethan Q. pyrenaica, a less plastic response was expected inthe former (Strauss-Debenedetti and Bazzaz 1991; Ashtonand Berlyn 1994; Valladares et al. 2002), which was notconsistent with the similar light-related shifts observed inboth species. A limited plasticity to light has been observedin some Mediterranean species in relation to a conservativeresource-use strategy (Valladares et al. 2000b; Chambel et al.2005). This could help to explain the similar photosyntheticplasticity in these species.

However, some attributes in seedlings of Q. petraea sug-gested a higher susceptibility to high light and a betterperformance in shade than those of Q. pyrenaica. Non-photochemical quenching is a response to an excess of en-ergy. Such excess is partly related to the absorption of lightand the capacity of a plant to use it in photosynthesis. LowAmax together with a high chlorophyll concentration in themore exposed seedlings of Q. petraea is likely to increasesuch imbalance, triggering NPQ (Faria et al. 1998; Kyparissiset al. 2000; Martınez-Ferri et al. 2000; Williams et al. 2003).The rapid onset of NPQ suggested a higher sensitivity to ele-vated PPFD in Q. petraea. However, it contributed to keep ahigh oxidative state of electron acceptors (Rosenqvist 2001;Souza et al. 2004). As stated above, this may be useful inavoiding chronic photoinhibition, which is consistent withthe similar potential quantum yield of PS II (Fv/Fm) ob-served in Q. petraea and Q. pyrenaica. How severe droughtevents will affect the capacity for thermal regulation remainsunanswered, but it is likely that a greater imbalance willtake place if stomatal limitations of photosynthesis becomeimportant. Caution must be taken when interpreting rapidchlorophyll fluorescence curves, as the ETR may be under-estimated (White and Critchley 1999; Rosenqvist 2001).

The capability of a plant to establish in low light mayrely either on the capacity to maximise net carbon gain (i.e.low LMA and high Am

max) (e.g. Givnish 1988 ), or on theresistance to herbivore damages and the minimisation of

carbon losses (i.e. tough leaves with high LMA and lowAm

max) (e.g. Walters and Reich 1999 ). Having Q. petraeaand Q. pyrenaica leaves that live for less than 1 year, oneexpects interspecific differences in the maximisation of lightcapturing area and net carbon gain to be determinant of theircompetitive ability in shade. Lower LMA in seedlings of Q.petraea across the light-availability gradient is suggesting abetter suitability to shade than in those of Q. pyrenaica, asit entails an optimisation of carbon investment with respectto light acquisition (Niinemets and Kull 1994; King 2003;Kitao et al. 2006). Besides, leaves of Q. petraea had nogreater nitrogen concentration that could increase the sus-ceptibility to foliar feeders. Am

max in the lower-light extremeof the gradient was lower in Q. petraea than in Q. pyrenaica.The effect that low rates of photosynthesis may have on thenet carbon balance of shaded seedlings is not straightforward,as a positive correlation between mass-based photosynthe-sis and maintenance respiration rate (Reich et al 1998; Luskand del Pozo 2002) may reflect a low metabolic potentialand thus a better adaptation to low light in Q. petraea (Luskand Reich 2000).

The results of this study are restricted to the seedlingstage, since the responses showed at early ages may differfrom those of adult trees (Cavender-Bares and Bazzaz 2000).However, similar differences in LMA and Amax were foundbetween both species when comparing adult trees (Gil et al.1999). Studies of plant architecture and biomass allocationare needed to confirm the different capacity of both species totake advantage of light availability suggested here (Sipe andBazzaz 1994; Delagrange et al. 2004; Gratzer et al. 2004).

Acknowledgements We thank Dr. Sven Mutke for helpful com-ments on early versions of this manuscript. We acknowledge theadvices of two anonymous reviewers. This research has been sup-ported by the Consejerıa de Medio Ambiente y Desarrollo Generalde la Comunidad Autonoma de Madrid. J. Rodrıguez-Calcerrada wassupported by a scholarship from the Consejerıa de Educacion dela Comunidad de Madrid (C.A.M.) and the Fondo Social Europeo(F.S.E.).

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Springer

ANEXO V

Ability to avoid water stress in seedlings of two oak species is lower in a

dense forest understory than in a medium canopy gap

Manuscrito aceptado en la revista Forest Ecology and Management

Autores: Rodríguez-Calcerrada J., Pardos J.A., Gil L., Aranda I.

Ability to avoid water stress in seedlings of two oak species is lower in

a dense forest understory than in a medium canopy gap

J. Rodríguez-Calcerrada1, J.A. Pardos1, L. Gil1 and I. Aranda2, ∗

1 Unidad de Anatomía, Fisiología y Genética Forestal. Escuela Técnica Superior

de Ingenieros de Montes, Universidad Politécnica de Madrid, Ciudad Universitaria s/n,

E-28040, Madrid, Spain.

2 Centro de Investigación Forestal (CIFOR). Instituto Nacional de Investigación

Agraria y Alimentaria (INIA), Apdo. 8111, E-28080, Madrid, Spain.

Abstract

The ecophysiological response to summer drought in seedlings of two co-

occurring oak species (Quercus petraea and Quercus pyrenaica) outplanted below a

dense Pinus sylvestris stand and a medium canopy gap was examined in two

experiments carried out in central mountains of the Iberian Peninsula (Spain). Leaf

water relations and gas exchange were studied in the first experiment. For both species,

lower pre-dawn leaf water potential (Ψpd) evidenced a higher degree of water stress in

the understory seedlings, even though soil moisture was similar in both sites. Rates of

photosynthesis (Asat) and stomatal conductance (gsat) at saturating light were higher in

the gap seedlings in all measuring dates, but the earlier and more pronounced stress

imposed in the understory precluded the comparison of tolerance responses between

sites. Q. pyrenaica showed always higher Asat and gsat than Q. petraea, independently of

∗ Corresponding author. Tel.: +34-1-3476857; fax: +34-1-3572293. E-mail address: aranda@inia.es

Anexo V - 1

Ψpd, which did not differ significantly between species. At the end of summer, values

of Asat/gsat and leaf carbon isotopic composition reflected a less efficient water use in Q.

pyrenaica. In the second experiment, growth and root development were examined in a

different set of seedlings planted in the same sites. Q. petraea allocated less biomass to

roots, and attained 20 % higher total plant dry mass than Q. pyrenaica at the end of the

second growing season in the field. For both species total plant dry mass and coarse

plus fine root dry mass reached at this time were approximately 3-fold higher in the gap.

Poor root development could explain the more limited access to water of seedlings

outplanted in the understory. This study reveals that seedlings of Q. petraea and Q.

pyrenaica planted under a dense, mature pine stand are more susceptible to summer

drought and present a delay in growth with respect to a medium canopy gap.

Keywords: Underplanting; Mediterranean forests; Light availability; Drought; Quercus

1. Introduction

Forestations in Mediterranean ecosystems are severely limited by summer

drought, particularly when they are carried out in open sites and comprise late-

successional species. Lower survival of a wide range of species in open versus canopy-

protected areas (Castro et al., 2004; Gómez-Aparicio et al., 2004) is mainly owing to the

buffering effects of shrub or tree nurse canopies from high radiation and

evapotranspiration. However complex interactions between nurse and target species

govern the outcome of forestations. Existing vegetation may compete for resources

rather than facilitate seedling establishment depending on climatic, edaphic and species-

specific factors (Michalet, 2006). For example, either poor light availability (Holmgren

Anexo V - 2

et al., 1997; Zavala et al., 2000) or low soil moisture due to water use by nurse trees

(Buckley et al., 1998) can limit seedling establishment in the forest understory. In

addition, there are interactive effects regarding light and water resources affecting plant

development on multiple directions that further complicate anticipation of results.

Water stress may undermine the capacity to tolerate shade, as it imposes a

stomatal limitation in the assimilation of CO2 that precludes the maintenance of a

positive carbon balance (Aranda et al., 2004). Conversely, some changes in plant form

and function associated to shading make plants more susceptible to soil water depletion

(Smith and Huston, 1989; Kubiske et al., 1996). Numerous works have referred a shade-

induced decrease in the development of the root system or in the root to shoot ratio

(Climent et al., 2006; Wang and Bauerle, 2006); since seedling establishment is largely

dependent on the root growth after planting, both effects would translate into a lower

ability for water uptake and a greater unbalance with transpirational losses, such that

one might expect a more limited ability to avoid water stress in a lower-light

environment, if differences in light and evapotranspiration are moderate (Crunkilton et

al., 1992; Valladares and Pearcy, 2002; Sack, 2004). Light plays also a role in avoiding

water stress by regulating both water losses and transport capacity. On the one hand,

higher sensitivity of stomata to decreasing soil water potential in sun plants allows for a

better control of water loss than in shaded ones (Mendes et al., 2001), at the same time

that increasing investment in components of the photosynthetic apparatus is reflected in

a more efficient water-use (Aranda et al., 2005). On the other hand, under similar xylem

tensions, plants growing in high light environments maintain superior hydraulic

conductance than shaded ones as an acclimatory response to greater transpirational

losses (Cochard et al., 1999; Barigah et al., 2006). Further, mechanisms of drought-

resistance based on carbohydrate availability, such as osmotic adjustment, are affected

Anexo V - 3

by the reduction in the net assimilation rate with shade (Mendes et al., 2001; Aranda et

al. 2005).

Partial shade may thus provide the optimal conditions for drought-sensitive

seedlings as it might contribute to maintain soil moisture with respect to open sites

without imposing a severe low-light stress that restricts plant responses to other

potential sources of stress. Thinned stands, gaps or forest edges are especially

recommended in the case of oak plantations in droughty sites (Johnson et al., 2002). But

this issue has not been thoroughly addressed in Mediterranean environments where

afforestations have traditionally focused on pioneer Pinus species more suitable to

successfully establishing in open sites (Pausas et al., 2004). In this study, we explored

the physiological (Experiment 1) and morphological (Experiment 2) responses to soil

moisture reduction occurring in mountains of Central Spain during summer in seedlings

of the temperate Quercus petraea and the sub-Mediterranean Quercus pyrenaica

outplanted in a dense Scots pine understory and in a nearby medium gap. The results

were discussed in relation to the possible differential suitability to sub-Mediterranean

mountains between the two oaks, which co-occur in some sub-Mediterranean montane

forests in spite of their differential resistance to drought (Aranda et al., 1996). We

hypothesized that drought would have a different impact on each site by modifying

either the capacity to avoid or tolerate water stress; particularly, the hypothesis in the

first experiment was that both species would show higher stress and less tolerance in the

pinewood understory than in the gap. The second experiment was carried out to test the

hypothesis that overall growth and relative allocation to roots would be lower in the

understory.

Anexo V - 4

2. Methods

2.1. Experiment 1

2.1.1. Study area and experimental design

The study area was located in Central Spain (41º7’N 3º30’W) at 1300 m.a.s.l.

Climate is continental sub-Mediterranean, with cold winters and a period of drought in

summer. Mean annual temperature is 8.7 ºC and average annual rainfall is 1124 mm.

Soils are predominantly loamy with high water-holding capacity and fertility, derived

from mica gneiss to mica schist bedrocks (Pardo, 2000).

In March 2003, one-year-old seedlings of Q. petraea and Q. pyrenaica were pit-

planted in the understory of a 45-year-old Pinus sylvestris plantation (basal area: 65.8

m2 ha-1, tree density: 1067 trees ha-1) and in a nearby 10-m width gap-like site (ratio of

gap diameter to surrounding trees’ height: 0.46) resulting from cutting a stripe of pine

trees in 1992. Seedlings had been grown for the first year in the nursery in 15-cm-deep

plastic pots, at appropriate watering and fertilization. Care was taken not to bend the

root ball at the moment of transplanting. Three plots, approximately 5-m-separated

each, were distributed in each site along the slope (medium slope 15 %, orientation SE),

each one having ten seedlings per species ~1.5-m-spaced in five rows among lines of

Pine trees in the understory. The experimental layout was a split plot design with three

blocks along the slope, each one including the two sites (main plot factor) and the two

species (subplot factor). Vegetation was sparse in the understory, while shrubs

(Adenocarpus hispanicus [Lam.] DC., Adenocarpus complicatus [L.] Gay) and ferns

(Pteridium aquilinum L. [Kuhn]) were abundant in the gap.

Anexo V - 5

2.1.2. Environmental variables

Precipitation was measured throughout the study at 1-hour intervals by means of a

raingauge (AN1, Delta-T Devices Ltd., UK) installed in a 16-m-high research tower

close to the study area connected to a data logger; air temperature (RHA1, Delta-T

Devices Ltd., UK) was measured every minute and averaged in 10-min intervals. Air

temperature and relative humidity were also measured in the gap and the understory

placing one sensor in each site (HOBO H8 Pro, Onset). Records were taken

continuously at 10-min intervals, and were used to calculate the air vapour pressure

deficit (VPD). Volumetric soil moisture was periodically measured with a TDR at

different depths in three probes per site. The light environment was characterized using

hemispherical photography. In late summer of 2004 and 2005, photographs were made

over the tip of all seedlings sampled at least once over the course of the experiment,

with a Nikon FM camera supplied with a sigma 8-mm fisheye lens. Digitalized

photographs were analysed using Hemiview 2.1 canopy analysis software (Delta-T

Devices Ltd., UK). Global site factor (GSF) was employed as a surrogate of the

irradiance relative to an open place. We also estimated maximum duration of direct

sunlight reaching the seedlings, by reproducing the solar trajectory on a mid-summer

day (10 August) with Hemiview 2.1.

2.1.3. Physiological variables

Leaf water potential was measured before dawn (Ψpd) and at midday (Ψmd) on two

dates of 2004 (June 23 and August 28) and three dates of 2005 (June 23, July 27 and

Anexo V - 6

August 25) in five seedlings per species and site using a pressure chamber (PMS

Instrument Co. 7000, Corvallis Oregon, USA).

Gas exchange was also measured in five seedlings per species and site using a

portable infrared gas analyser (LC pro ADC BioScientific, Ltd. UK). Light response

curves were realized at the beginning (June 20-25) and end (August 21-26) of the

summer of 2005. All curves were made between 8:30 a.m. to 11:15 a.m. to avoid a

possible midday depression in photosynthesis. A red/blue light emitting diode provided

measuring light (Lc pro, ADC). Temperature inside the chamber was held at 23 ºC, so

that leaf temperature was at around 25 ºC during all curve acquisition. CO2

concentration in the supplied air was maintained at around 365 ppm. Measurements

began by submitting a leaf to 800 μmoles m-2 s-1 until steady readings of stomatal

conductance were observed (typically 10 - 20 min). At this point light was increased up

to 1600 μmoles m-2 s-1 in the gap plants in two steps, and up to 1150 μmoles m-2 s-1 in

the understory plants. Light was then returned to 800 μmoles m-2 s-1, and five additional

records were taken down to 40 μmoles m-2 s-1. Each curve typically involved 25 to 30

min. Three readings of photosynthesis (A), and stomatal conductance (gwv) were

recorded at each 7 (8) different light levels. A hyperbolic function was fitted to the

relationship of A and gwv with PPFD to estimate both parameters at saturating light (Asat

and gsat). In mid-summer 2005 (July 26-28), gas exchange was only measured at

saturating light (1150 and 1600 μmoles m-2 s-1 for understory and gap seedlings,

respectively), under the same conditions applied for the light response curves. Small

and deeply lobbed leaves of most Q. pyrenaica gap seedlings did not cover the entire

chamber window so that measurements were corrected by considering the actual area

enclosed.

Anexo V - 7

In 2005, the leaves used for gas exchange and water potential measurements were

further collected and carried to the laboratory. They were dried for 72 h at 60 ºC and

then grounded for determination of carbon isotopic composition (δ13C) using a

Micromass Isochrom mass spectrometer at the SIDI of the Autonomous University of

Madrid. The analytical method had a precision of ± 0.2 ‰. Hydraulic conductance (KL)

was also calculated for each seedling by dividing the maximum rate of transpiration

between the difference between daily maximum (~Ψpd) and minimum (~Ψmd) water

potential.

2.1.4. Statistical analysis

Differences between planting sites or species, and the interaction between these

factors, were examined on each sampling date with two-way analyses of variance

(ANOVA) using the General Linear Model procedure (GLM) on Statistica 6.1 (Statsoft,

Inc., Tulsa, USA). The variance model also included a term for effects of block, but not

for block interactions with site or species. Ψpd was typically treated as a covariate in

order to account for its influence on site and species comparison of leaf traits. We did

not use a repeated-measures approach because measurements could not be made on the

same individuals in all three dates. Only the effect of site on soil moisture was

examined on each year with a repeated measures analysis of variance, with sampling

date as a repeated measures factor. Before ANOVA, data were checked for normality

and homocedasticity, transforming data and excluding outside-range points when

necessary.

Anexo V - 8

2.2. Experiment 2

In March 2004, one-year-old seedlings of Q. petraea and Q. pyrenaica were pit-

planted in the same abovementioned sites. As in Experiment 1, seedlings had been

grown for the first year in 15-cm-deep plastic pots, at the same watering and

fertilization dosage. Two plots per site were located along the hill slope above and

below the upper plots of the Experiment 1, each one having ten plants per species

alternately planted ~1.5-m-spaced. The experimental design was a split plot design with

two blocks including the site, as the main plot factor, and the species, as the subplot

factor. Plant development was assessed at the end of 2004 and 2005. The root systems

of six plants per species and site were manually excavated. An approximately 80-cm-

deep hole was dug out within a 30-cm radius around the seedling stem. Rooting depth

could not be estimated because roots fractured at depths greater than 80 cm. Roots

spreading from the root ball were carefully separated from the soil, brushing out soil

rests from fine roots in the field. Plants were bagged with the root ball and taken to the

laboratory where they were carefully washed and further oven-dried at 75 ºC for five to

seven days. Since fine root losses were likely to be important, fine and coarse root

masses were put together for calculations. Total plant dry mass (TDM) was measured.

Root mass fraction (RMF) was calculated by dividing root dry mass (RDM) between

TDM. The root to shoot (leaves plus stems dry mass) ratio (R/S) was also calculated. In

2005, the area of all leaves was measured for each seedling with a digital area meter to

compute the total leaf area (TLA) and the leaf area ratio (LAR = TLA / TDM).

A GLM approach to ANOVA was performed in each year to test for the fixed

effects of site and species factors on the morphological variables at each year. The

variance model for dependent variables also included a term for the block, and a term

Anexo V - 9

for the second-order interaction between site and species factors. Total seedling dry

mass was considered as a covariate when testing differences on LAR, RMF and R/S.

3. Results

3.1. Environmental conditions and climatic data

Mean temperature in the summer months was 17.4 ºC in 2004 and 17.7 ºC in

2005, that is, 0.5 and 0.8 degrees higher than the 8-year average taken from the nearby

meteorological tower, respectively (data from 1995 to 2005; data from 1999, 2001 and

2002 were missing). Rainfall from June to September was 95.2 mm in 2004 and 40.4

mm in 2005, 33.6 and 71.8 % lower than average, respectively.

The average of daily mean vapour pressure deficits (VPDmean) over the summer

months was similar between plantation sites in both years, but the average of the daily

maximum values (VPDmax), which were registered around midday, was higher in the

gap than in the understory in 2004 and 2005 (Table 1). Soil moisture decreased along

the growing season in both sites; differences between sites were not statistically

significant at any depth across all sampling dates (one-way RM ANOVA: P-value >

0.15) (Fig. 1). Light availability, estimated by the Global Site Factor index (GSF), was

higher in the gap than in the understory (Table 1; Fsite: 157***). Also seedlings in the gap

were exposed to direct light in sunflecks of longer mean (Fsite: 19.8***) and maximum

(Fsite: 63.18***) duration than in the understory (Table 1). Neither of these variables

differed significantly between species (P-values > 0.15).

Anexo V - 10

3.2. Experiment 1

3.2.1. Water relations

Leaf water potential did not differ significantly between species throughout the

study period either before dawn or at midday. But there was a marked effect of site on

the pre-dawn leaf water potential (Ψpd), such that for both species, Ψpd was much lower

in the understory in 2004 (Fsite: 64.15*** and 9.43** at the end of June and August,

respectively; Fig. 2) and in 2005 (Table 2; Fig. 2). Furthermore water stress appeared

earlier in the understory seedlings. For instance, in June 2005, Ψpd was already as low

as –0.56 ± 0.08 MPa and –0.51 ± 0.07 MPa in Q. pyrenaica and Q. petraea,

respectively, whereas no symptom of water stress was found in the gap seedlings (Ψpd >

–0.1 MPa). Differences were not so pronounced in Ψmd. In 2004, Ψmd was lower in the

gap than under the forest in the two measuring dates (Fsite: 20.19*** and 5.95* at the end

of June and August, respectively; Fig. 2), while in 2005, Ψmd was lower in the gap in

June, but slightly higher in August (Fsite: 3.6, P-value: 0.079; Table 2).

3.2.2. Gas exchange

In all dates, the rates of photosynthesis and stomatal conductance at saturating

light (Asat and gsat) were higher in the gap in the two oaks (Fig. 3). However, when Ψpd

was accounted for by ANCOVA, differences in Asat and gsat between sites were non-

significant (Table 3). Asat and gsat decreased in response to decreasing Ψpd in both

species, apparently less steeply in the understory, although the few data at none-to-mild

water stress (Ψpd > –0.5 MPa) precluded a thorough comparison (Fig. 4). Q. pyrenaica

Anexo V - 11

exhibited higher Asat and gsat than Q. petraea across all sampling dates irrespective of

the growth environment; these differences were more significant as summer progressed

(Table 3). Mean hydraulic conductance (KL) did not differ between sites (Table 2; Fig. 3

d), because higher transpiration rates in the gap (not shown) were accompanied by a

higher daily water potential gradient. It did differ though between species in July and

particularly in August.

3.2.3. Water use efficiency

No relationship was found between instantaneous leaf water use efficiency

(Asat/gsat) and δ13C for the whole growing season, although in contrast to Asat/gsat, the

carbon isotopic composition reflects the efficiency under non-optimal ambient

conditions. There were no consistent differences in either Asat/gsat or δ13C between sites.

Observed difference between the gap and the understory in Asat/gsat in June 2005 was

again ascribed to the covariation of Ψpd; the negative relationship between Ψpd and

Asat/gsat in this date was not found in the following date (not shown). At the end of

summer, both Asat/gsat and δ13C were higher in Q. petraea (Table 2; Figs. 3 and 5).

3.3. Experiment 2

TDM was higher in the gap for both species (Table 3; Fig. 6). Accordingly, RDM

was also higher than in the understory, even though between-site differences were likely

underestimated because of greater root losses in the gap. No taproot had developed on

any seedling after planting; rather a large number of sinkers were produced at the

bottom of the container-formed taproot, which spread at least further than 80-cm deep in

Anexo V - 12

most individuals. Meaningfully, long lateral horizontal roots were observed in most gap

seedlings that were absent in the understory ones. But mass partitioning to roots was not

affected by planting site. When accounting for the effect of total plant dry mass,

differences in RMF and R/S were not significant in any species (Table 3), and for a

given value of root dry mass, shoot dry mass was not significantly different between

sites (ANCOVA: P-value = 0.97 for the comparison of site elevations; P-value = 0.38

for homogeneity of slopes). On the contrary, Q. petraea had superior shoot biomass

than Q. pyrenaica at any given value of root biomass (ANCOVA: P-value < 0.01 for the

comparison of species elevations; P-value = 0.49 for homogeneity of slopes; Fig. 7),

and both RMF and R/S were consistently higher in the two years (Table 3; Fig. 6). In

2005, TLA was significantly higher in Q. petraea vs Q. pyrenaica (211 vs 139 cm2 in

the understory, and 559 vs 449 cm2 in the gap), as well as in the gap vs the understory

(Fspecies: 4.34*; Fsite: 46.7***). LAR was significantly higher in Q. petraea, especially in

the understory (39.0 vs 25.4 cm2 g-1 in the understory, and 33.8 vs 29.5 cm2 g-1 in the

gap), but it did not differ between sites (Fspecies: 14.72**; Fsite < 0.01; F: 4.05 for the

interaction between species and site, P-value: 0.061).

4. Discussion

The results of two contrasting, dry years showed that seedlings outplanted in the

understory of a Scots pine stand suffered higher water stress than those outplanted in a

medium canopy gap. Provided TDR measurements were representative of soil water

content near the roots of the seedlings, such difference might have arisen from

differences in moisture below 40 cm caused by competition with overstory trees. High

root density often results in a strong below-ground competition for water in more-

Anexo V - 13

compared to less-dense sites, such as clear-cuts, gaps or thinned stands (Abrams and

Mostoller, 1995; Buckley et al., 1998; Ricard et al., 2003). Besides, as deduced by the

more profuse root growth observed in the Experiment 2, higher pre-dawn leaf water

potential in the gap seedlings might evidence a deeper rooting after planting, which

would allow for access to more humid soil horizons. In this case, observed greater

unbalances between transpiration and water absorption by midday in the gap in some

dates would be ascribed to the higher evaporative demand, and the larger total leaf area

of the seedlings in this site. Although several other traits related to root development,

such as root length, fine root mass or root architecture, could more precisely account for

a differential access to soil water, estimates of coarse plus fine root dry mass give an

idea of the different root growth rate between sites after outplanting. Light could have

played an important role in it. Evidence exists that plants growing in low-light

conditions allocate less biomass to roots and more to leaves compared to seedlings

growing in high-light conditions (e.g. McConnaughay and Coleman, 1999). Our data

suggest, though, that low root biomass in the understory did not occur as a result of a

greater partitioning to shoots (Table 3; Fig. 7), but rather that poor light within the

canopy did cause a marked delay in growth and root development after outplanting,

which further enhanced plant susceptibility to summer drought. If differences in light

availability were the main factor directing seedling performance, these results would

provide new evidence that intense shading reduces the ability of plants to facing

drought. However, this study does not address possible differences in soil characteristics

between the gap and the understory (nutrient content, texture…), which require us to be

cautious when discussing the extent of light influence on root growth. Another

limitation of the study is to cover only a study case in a humid area. The observed

canopy-related differences in water stress could possibly differ in more arid sites or

Anexo V - 14

when larger gaps are compared due to more intense evapotranspiration. In any case, our

observations are in agreement with the often-cited poor performance of seedlings

underplanted within heavy-dense Mediterranean pinewoods (Maestre et al., 2003) and

support other investigations revealing a negative effect of pine canopy on the functional

response to soil drying in understory woody species (Bellot et al., 2004). Leaf gas

exchange rates were also higher in the gap seedlings. The underlying reasoning is that

superior light availability increases the leaf mass per unit area, the stomatal density, plus

the investment in photosynthetic machinery (Kozlowski and Pallardy, 1997). These

features would increase the rate of photosynthesis in spring; however, as shown by

analysis of covariance, differences in gas exchange during summer were mostly due to

the different degree of water stress between sites across dates, precluding any

comparison of the response of gas exchange rates to water stress. Hence combined low

light and water stress led to reduced biomass accumulation observed in the Experiment

2.

The negative effect associated to the understory was similarly observed in

seedlings of the two oaks, which had comparable water status at dawn and midday,

despite differences shown in growth and leaf water relations. In spite of the large, early

reduction in gsat in Q. pyrenaica, this species maintained significantly higher rates of

photosynthesis and stomatal conductance than Q. petraea at all levels of water stress, in

consonance with its higher values of hydraulic conductance. This significant capacity to

display high leaf gas exchange at high and low soil water potentials has been previously

addressed in other drought-tolerant species (Ngugi et al., 2004), as well as in seedlings

(Quero et al., 2006) and adult trees (Gallego et al., 1994) of Q. pyrenaica. But this

pattern cannot be only seen as a strategy aimed to tolerate drought. Rather, we postulate

that it is to be attributed to its more conservative growth strategy (Experiment 2) that

Anexo V - 15

maximizes root mass partitioning in lieu of shoot growth and foliage area, and allows

for a high instantaneous stomatal conductance per unit leaf area (Kramer and Boyer,

1995; Mediavilla and Escudero, 2003). Higher stomatal conductance with respect to Q.

petraea could account for the lower Asat/gsat and δ13C at the end of summer (DeLucia

and Schlesinger, 1991). However, it is worth noting that poor leaf water-use efficiency

at this time did not translate into a worse water status, even though RDM was not

significantly different to Q. petraea. Again, a possible explanation resides in the lower

total leaf area and higher within-plant self-shading already exhibited by Q. pyrenaica in

other comparative works (Rodríguez-Calcerrada et al., submitted), which would reduce

both water using up and carbon gain at the plant-level. Although Q. petraea is a

relatively drought-resistant species compared with other sympatric broad-leaf trees of

Central Europe (Backes and Leuschner, 2000; Ponton et al., 2001; 2002), the suite of

features shown by seedlings of Q. pyrenaica is consistent with an expected, better

suitability to regenerate in Mediterranean mountains. On the one hand, superior

belowground biomass points to a greater ability to withstand stress and aboveground

perturbations (Kozlowski and Pallardy, 1997). On the other hand, adaptation to late

spring frosts through delayed leaf flushing (10–15 days later in Q. pyrenaica than in Q.

petraea; García-Esteban, pers. comm.) imposes a short leaf life span during which

individuals have to face low soil water content; maintenance of gas exchange may be

important, as water-stress avoidance via strong stomatal closure, a successful strategy

for some Mediterranean evergreen oaks (Abril and Hanano, 1998; Mediavilla and

Escudero, 2003), would reduce carbon assimilation and would negatively impact

competitive ability at the establishment phase. This proposed plant form-leaf function

coordination might partly explain failures in afforestations with Q. pyrenaica in

drought-prone, mountainous Mediterranean open sites (Rodríguez-Calcerrada et al.,

Anexo V - 16

2007), as they traditionally use short pots that restrict its inherently large root to shoot

ratio and exacerbate the effects of a non-conservative leaf water use.

5. Conclusion

This study shows that seedlings of two ecologically distinct oak species had worse

transplanting performance (i.e. higher water stress and lower growth) in the understory

of a mature Scots Pine stand than in a nearby medium canopy gap. Although these

results can hardly be extrapolated to other circumstances (i.e. different areas, gap

sizes…), they support previous findings that dense shelterwoods are not appropriate for

under-plantations. We suggest that poor growth in the understory is partly responsible

for the greater susceptibility of the seedlings to summer drought.

Acknowledgements

We thank Jesús Alonso and Javier Cano for their patient work on root

excavations. We gratefully acknowledge two anonymous reviewers for their enriching

comments, and Rosa Ana López, Sven Mutke and Nikos Nanos, who helped us with

statistical analyses. Thanks are also due to Maria del Rey for measurement assistance.

This work was supported by the Consejería de Medio Ambiente y Desarrollo General de

la Comunidad Autónoma de Madrid. J. Rodríguez-Calcerrada was supported by a

scholarship from the Consejería de Educación de la Comunidad de Madrid (C.M.) and

the Fondo Social Europeo (F.S.E.).

Anexo V - 17

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Anexo V - 22

TABLES

Table 1. Comparative statistics of daily mean (VPDmean) and maximum (VPDmax) values

of air vapour pressure deficit averaged over the summer months of 2004 and 2005 (June

to September, both included), light availability (GSF), and mean and maximum duration

of sunflecks.

Footnotes:

1: Data of both years are pooled since slight differences between years are due to

different points of sampling rather than to canopy closure.

2: Values for a typical mid-summer day (10 August).

Variable Year

Mean (± SD) Min-Max Mean (± SD) Min-Max VPDmean (KPa) 2004 0.77 (0.28) 0.09-1.35 0.78 (0.30) 0.11-1.47

2005 0.97 (0.37) 0.20-1.95 0.94 (0.37) 0.15-1.87

VPDmax (KPa) 2004 1.90 (0.60) 0.35-3.13 1.59 (0.52) 0.40-2.932005 2.29 (0.77) 0.61-4.34 1.83 (0.61) 0.45-3.42

GSF (%) both1 45.1 (11.2) 30.1-63.0 20.3 (2.6) 15.7-25.1

Sunfleckmean (h) both2 0.42 (0.27) 0.12-0.98 0.13 (0.02) 0.12-0.17

Sunfleckmax (h) both 3.68 (1.95) 0.72-5.77 0.50 (0.08) 0.33-0.63

SiteGap Understory

Anexo V - 23

Table 2. F-statistics of analyses of variance for factors effects on leaf traits in 2004 and 2005. Ψpd was considered a covariate for analyses of all

variables except Ψpd and Ψmd.

Date FactorsΨpd Ψmd Asat gsat Asat/gsat KL δ13C

June 05 Site 67.7*** 12.66** 1.21 2.98 1.11 0.12 2.37Species 0.16 1.48 7.6* 6.47* 0.62 2.39 0.02Site x Sp. 0.24 < 0.01 0.49 2.7 0.07 < 0.01 0.88

July 05 Site 68.21*** 0.63 < 0.01 0.08 0.02 < 0.01 0.18Species 0.03 1.93 15.84** 16.72** 3.46 6.77* 1.68Site x Sp. 0.02 < 0.01 0.01 0.38 0.83 0.9 2.14

Aug. 05 Site 40.92*** 3.6 0.11 1.84 0.04 0.64 0.28Species 0.1 0.12 18.32** 39.3*** 7.71* 26.59*** 13.06**

Site x Sp. 0.3 < 0.01 1.17 2.33 3.73 0.18 2.34

Dependent variables

Anexo V - 24

Table 3. F-statistics of analyses of variance for factors effects on plant traits in 2004 and

2005. TDM was considered a covariate for analyses of LAR, R/S and RMF.

Date FactorsTDM RDM R/S RMF

2004 Site 3.33 4.26* 0.76 0.7Species 1.17 2.69 12.68** 13.58**

Site x Sp. 0.18 0.35 0.03 0.1

2005 Site 51.12*** 49.12*** 1.41 2.7Species 1.72 0.11 7.69* 10.87**

Site x Sp. 0.9 0.01 0.44 1.22

Dependent Variables

Anexo V - 25

FIGURE LEGENDS

Fig. 1. Seasonal variation of precipitation, and soil moisture at 10 cm (a), 20 cm (b) and

40 cm depth (c) in the pinewood understory (triangles, solid line) and the gap (squares,

dashed line) during the growing seasons of 2004 and 2005.

Fig. 2. Pre-dawn (Ψpd; a, b) and midday (Ψmd; c, d) leaf water potentials of Q. pyrenaica

(grey bars) and Q. petraea (white bars) in the pinewood understory (u.) and the gap (g.)

along the summer of 2004 and 2005. N = 4-5.

Fig. 3. Mean values (+1SE) of photosynthesis (Asat; a), stomatal conductance (gsat; b),

instantaneous water use efficiency (Asat/gsat; c), and hydraulic conductance (KL; d) along

the summer of 2005, for seedlings of Q. pyrenaica (grey bars) and Q. petraea (white

bars) planted in the pinewood understory (u.) and the gap (g.). N=3-5.

Fig. 4. Responses of Asat (a) and gsat (b) to Ψpd in understory seedlings of Q. pyrenaica

(filled squares) and Q. petraea (filled circles), and gap seedlings of Q. pyrenaica (open

tsquares) and Q. petraea (open circles).

Fig. 5. Mean values (+1SE) of leaf carbon isotope composition (δ13C) along the summer

of 2005, for seedlings of Q. pyrenaica (grey bars) and Q. petraea (white bars) planted in

the pinewood understory (u.) and the gap (g.). N=4-5

Fig. 6. Mean values (+1SE) of total plant dry mass (TDM; a), root dry mass (RDM; b),

root to shoot ratio (R/S; c), and root mass fraction (RMF; d) at the end of 2004 and 2005

Anexo V - 26

for seedlings of Q. pyrenaica (grey bars) and Q. petraea (white bars) planted in the

pinewood understory (u.) and the gap (g.). N=5-6.

Fig. 7. Variation in root mass vs shoot mass, on a logarithmic scale, for seedlings of Q.

petraea (circles) and Q. pyrenaica (squares) growing in the pinewood understory (filled

symbols) and the gap (open symbols). Data points are individual plants harvested in

2004 and 2005. A linear model was fitted for data points of Q. pyrenaica (slope = 1.12,

R2 = 83.8***) and Q. petraea (slope = 1.03, R2 = 77.0***) pooled across sites.

Anexo V - 27

Rodríguez-Calcerrada et al. FORECO 3010

5101520253035

(a) 10 cm

51015202530

1020304050

Soil

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Figure 1

Anexo V - 28

Rodríguez-Calcerrada et al. FORECO 3010

g. u.

Ψm

d(-

MPa

)

3.53.02.52.01.51.00.50.0

2.5

2.0

1.5

1.0

0.5

0.0

June 05 July 05 August 05

u. g.

Ψpd

(-M

Pa)

u. g. u. g.

June 04 July 04 August 04

u. g. u. g. u. g.

2.5

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0

3.53.02.52.01.51.00.50.0

June 05 July 05 August 05

u. g. u. g. u. g.

June 04 July 04 August 04

u. g. u. g.

Q. pyrenaicaQ. petraea

Q. pyrenaicaQ. petraea

g. u.

Ψm

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3.53.02.52.01.51.00.50.0

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June 05 July 05 August 05

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June 04 July 04 August 04

u. g. u. g. u. g.

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2.0

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June 05 July 05 August 05

u. g. u. g. u. g.

June 04 July 04 August 04

u. g. u. g.

Q. pyrenaicaQ. petraea

Q. pyrenaicaQ. petraeaQ. pyrenaicaQ. petraea

Figure 2

Anexo V - 29

Rodríguez-Calcerrada et al. FORECO 3010

02468

1012141618

Asa

t(μm

olm

-2s-1

) a

0100200300400500600700

gsat (m

molm

-2s -1)

June 05 July 05 August 05

u. g. u. g. u. g.

Asa

t/gsa

t(μm

olm

mol

)

0

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June 05 July 05 August 05

u. g. u. g. u. g.

d

KL

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olm-2s -1 M

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Q. pyrenaicaQ. petraea

b

0

0.02

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02468

1012141618

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t(μm

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Asa

t/gsa

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mol

)

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June 05 July 05 August 05

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KL

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olm-2s -1 M

Pa-1)

Q. pyrenaicaQ. petraea

b Q. pyrenaicaQ. petraea

b

0

0.02

0.04

0.06

0.08

0.10 cc

Figure 3

Anexo V - 30

Rodríguez-Calcerrada et al. FORECO 3010

Asa

t(μ

mol

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g sat

(mm

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)

0

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8

12

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0100200300400500600700

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t(μ

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Ψpd (-MPa)

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(mm

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)

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0100200300400500600700

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Q. petraea – g.Q. petraea – u.

Q. pyrenaica – u.Q. pyrenaica – g.

Q. petraea – g.Q. petraea – u.

Q. pyrenaica – u.

Figure 4

Anexo V - 31

Rodríguez-Calcerrada et al. FORECO 3010

-30.0-29.5-29.0-28.5-28.0-27.5-27.0-26.5

δ13C

(‰)

June 05 July 05 August 05

u. g. u. g. u. g.

Q. pyrenaicaQ. petraea

-30.0-29.5-29.0-28.5-28.0-27.5-27.0-26.5

δ13C

(‰)

June 05 July 05 August 05

u. g. u. g. u. g.

Q. pyrenaicaQ. petraeaQ. pyrenaicaQ. petraea

Figure 5

Anexo V - 32

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Anexo V - 33

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Figure 7

Anexo V - 34

ANEXO VI

Effects of Scots pine overstory density on pressure-volume curves and

survival of two underplanted oak species in a Mediterranean mountain

Manuscrito en revisión en la revista Annals of Forest Science

Autores: Rodríguez-Calcerrada J., Mutke S., Pardos J.A., Alonso J., Gil L., Aranda I.

Effects of Scots pine overstory density on pressure-volume curves and survival of

two underplanted oak species in a Mediterranean mountain

J. Rodríguez-Calcerrada1, S. Mutke2, J.A. Pardos1, J. Alonso1, L. Gil1 and I. Aranda2,*

1 Unidad de Anatomía, Fisiología y Genética Forestal. Escuela Técnica Superior de

Ingenieros de Montes, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, E-

28040, Madrid, Spain.

2 Centro de Investigación Forestal (CIFOR). Instituto Nacional de Investigación Agraria

y Alimentaria (INIA). Carretera de La Coruña Km 7.5, 28040, Madrid, Spain.

* Corresponding author. Tel.: +34-1-3476857; fax: +34-1-3572293. E-mail address:

aranda@inia.es

Running title: Dense shelterwood hampers oak regeneration

Anexo VI - 1

Abstract

Pressure-volume curves and survival were investigated in seedlings of Quercus

petraea and Quercus pyrenaica planted in a dense Scots pine area, a 25 % thinned area

and a gap at a Mediterranean mid-mountain field-site. Survival was also measured in a

nearby stand, which had been assigned to three density treatments: uncut, 33 and 50 %

thinned. Osmotic adjustment at the end of summer in Q. petraea was 0.27 and 0.33 MPa

lower in the denser area compared to 25 % thinned and gap sites, respectively, and did

not vary among treatments in Q. pyrenaica. Basal values of osmotic potential at full

turgor were lowest for Q. pyrenaica overall. Survival was lower in both oaks in the

denser area. In the second stand, both thinning intensities had a positive effect on the

survival of Q. pyrenaica, while Q. petraea showed similarly higher mortality rates in all

three overstory treatments. These results stress the necessity to reduce stand density

before underplanting.

Keywords: osmotic adjustment / elastic adjustment / thinning / Quercus petraea /

Quercus pyrenaica

Anexo VI - 2

Résumé – Effets de la densité de l’étage dominant du pin sylvestre sur les courbes

de pression-volume et sur la survie de deux espèces de chênes de sous-étage en

montagne Méditerranéenne. Les courbes de pression-volume et la survie ont été

examinées chez des plantules de Quercus petraea et Quercus pyrenaica plantées dans

trois parcelles soumises à différents traitements : une aire dense de pin sylvestre, une

aire éclaircie à 25%, et un site ouvert dans une région Méditerranéene de moyenne

montagne. La survie a également été mesurée dans des parcelles voisines, qui ont été

soumises à trois traitements de densité de l’étage dominant : non coupé, 33 et 50%

d’éclaircie. Dans le premier dispositif, l’ajustement osmotique à la fin de l´été pour Q.

petraea était de 0.27 MPa entre les parcelles les plus denses et celles éclaircies à 25%,

et de 0.33 MPa entre les parcelles les plus denses et celles à site ouvert ; il n’a pas varié

entre les traitements chez Q. pyrenaica. Globalement, les valeurs de potentiel osmotique

à pleine turgescence étaient plus faibles pour Q. pyrenaica. La survie était plus basse

dans l’aire la plus dense pour les deux espèces de chênes. Dans le second dispositif, les

deux traitements d’éclaircie ont eu un effet positif sur la survie de Q. pyrenaica, alors

que Q. petraea a montré des taux de mortalité similairement élevés dans les trois

traitements de l’étage dominant. Ces résultats montrent la nécessité de réduire la densité

des stands avant de planter en sous-étage.

Mots clefs: ajustement osmotique / ajustement élastique / éclaircie / Quercus petraea /

Quercus pyrenaica

Anexo VI - 3

INTRODUCTION

Broadleaved woodlands of the mid-mountain Mediterranean regions are

potentially sensitive to a future deterioration of the climate [42, 48]. The problem may

be particularly critical for marginal forest populations of temperate species [10], given

their susceptibility to higher temperatures and more erratic rainfall characterized in

recent climate scenarios [25]. Conservation policies should promote the expansion of

such endangered populations by artificial means. Enrichment planting under mature

conifer forests is successfully used to improve recruitment of broadleaved tree species

with respect to open sites with higher evapotranspiration rates or higher occurrence of

frost damage. The method has alternative benefits in terms of transformation of pure,

even–aged coniferous stands into mixed stands.

However, while shelterwood regeneration method is one of the most common

permanent-cover techniques used in temperate forests, few experiences have dealt with

nurse-tree canopy effects on understory plantations in Mediterranean ecosystems [1, 2,

9, 35] and appropriate recommendations are uncertain. When light is the main limiting

factor, a closed canopy often hinders establishment of seedlings of different shade-

tolerances [34, 38], requiring its gradual opening by means of thinnings or the creation

of gaps. But owing to recurrent and severe episodes of drought during summer in

Mediterranean forests, the question remains whether initial thinning might result in

excessive evaporation and mortality or rather enhance soil moisture by diminishing rain

interception and transpiration of the overstory [6]. To a large extent, the outcome of the

canopy-understory relation seems to be mediated by the effect that shade has on the

conservation of soil moisture and the ability of seedlings to respond to water stress. But

facilitation is an issue of active debate [33, 37], as it is likely depending on the

Anexo VI - 4

ecological features of the target species (e.g. differing tolerance to late frosts in

temperate forests or to drought or excessive light in Mediterranean areas) and/or the

environmental characteristics of the site (e.g. fertility or aridity).

Because seedlings will increasingly have to cope with situations where both light

and water availabilities are low under tree canopies [1, 22, 56], the examination of how

mechanisms of drought-tolerance are affected by light will provide a valuable indication

of the limits to successful establishment in dry, shady Mediterranean forests. The

lowering of osmotic potential through an active accumulation of solutes is a positive

response to water stress [1, 16, 28], which may be hindered in light-limited plants given

the central role of the accumulation of hexoses during drought in this process [11, 17].

Light availability also modifies drought-induced responsiveness in cell-wall elasticity

[41]. Although successful establishment is rarely elicited by a single environmental

factor [49, 50, 56], but rather is affected by multiple above- and below-ground

interactions with mature trees [8] and with other less predictable factors, water relations

play a role in growth and survival of forest tree species [14, 19, 53, 57].

In this context, two experiments were carried out at a Mediterranean mid-

mountain field-site to address the effects of overstory retention on performance of

underplanted seedlings of two oaks, namely Quercus petraea and Quercus pyrenaica.

The first one aimed to assess the relative capacity to set up mechanisms of drought-

tolerance (i.e. pressure-volume curve parameters) under different canopy covers and

their importance in survival. We hypothesized that such mechanisms would be limited

under the denser canopy, and thus would affect survival. Additionally, oak seedlings’

survival was assessed in another Scots pine stand subjected to three overstory treatments

(uncut, 33 % thinned, and 50 % thinned of the original density), under the hypothesis

Anexo VI - 5

that mortality would be higher in the un-thinned plots because of a multiple reduction in

resources.

2. MATERIALS AND METHODS

2.1. Study area and species

Experiments were developed in Scots pine plantations located in the Spanish

Central System (3º30’W, 41º07’N; 1300 – 1600 m.a.s.l.). Pines were originally planted

in degraded lands during the twentieth century for soil and watershed protection [40],

and currently there are several thousands hectares of pine forests in the first rotation

whose transformation would report environmental as well as social benefits. In general,

soils in the area are loamy and fertile, and have a high water-holding capacity. Average

rainfall is 1,124 mm and annual mean temperature is 8.7 ºC [39]. They are included into

the “Sierra del Rincón” Biosphere Reserve of the UNESCO’s Man and the Biosphere

(MAB) Network and represent a potential expansion area for temperate hardwood

species in the region, such as Fagus sylvatica and Quercus petraea. Here they reach one

of their southernmost populations of Europe, co-occurring with other trees whose

ecological requirements are plainly met, such as Quercus pyrenaica. Q. petraea and Q.

pyrenaica are sufficiently close-related that intermixing and genetic introgressions

occurs [55], although their distinct leaf to whole-plant structural and functional features

confirm their segregation into the nemoral (Q. petraea) and the transitional nemoro-

Mediterranean phytoclimatic groups (Q. pyrenaica) [3, 12].

All plant material used in the experiments derived from seeds collected in the

study area. Current precipitation and temperature were recorded by means of a rain

gauge (1-hour interval) and a temperature sensor (1-min interval), respectively, both

Anexo VI - 6

connected to a data-logger. The sensors are placed at the top of a nearby 16-m height

tower, topping the canopy. It is operating since 1995 although data from 1999 and 2001-

2002 were missing.

2.2. Experiment 1

This experiment was conducted in an even-aged, 45-year-old Scots pine

plantation at 1300 m.a.s.l., SE facing, and moderate, uniformly sloping (15 %). Three

complete blocks were distributed in a ≈ 0.5 ha area along the slope in a split-plot design,

with overstory treatment (uncut, 25 % thinned, gap) as the main plot factor, and the

species (Q. petraea and Q. pyrenaica) as the subplot factor (table I). The thinning was

made in 1998 and the gap resulted from a 10-m-width firebreak made in 1992. The ratio

of gap width to surrounding trees’ height was 0.46. Here seedlings were growing under

a sparse shrub cover as no control of vegetation was made. Thirty one-year-old

seedlings per species and block were randomly distributed among treatments. Since

these treatments were contiguous to each other in a 150-m transect, seedlings were

planted far from the edges, in three rectangular experimental plots with 10 plants per

species 1.5-m-spaced. All seedlings had been cultivated in 300-cm3, 15-cm deep Forest

pots containing a mixture of peat and sand (3v/1v) plus slow release fertilizer (5 g l-1

[Osmocote Plus (8-9 months)]) for the first year; they were planted in early spring 2003.

Volumetric soil moisture was quantified on different dates of 2004 and 2005 (i.e.

seven to eight years after applying the thinning, and twelve to thirteen years after

making the firebreak) by Time Domain Reflectometry (TDR; Trase System I, Soil

Moisture Equipment Corp. USA). A sensor was moved inside buried PVC-tubes up to

40 cm depth; one tube per plot for a total of nine tubes. Light availability was assessed

by hemispherical photography in late summer 2004 and 2005. One photograph was

Anexo VI - 7

taken over each sampled seedling at the end of the day avoiding direct radiation

exposition (Nikon FM camera with a sigma 8-mm fisheye lens). Photographs were

scanned, and further analyzed with Hemiview 2.1 Canopy Analysis Software (Delta-T

Devices Ltd., UK) to estimate the percentage of global radiation received by the

seedling (Global Site Factor, GSF). Soil water potential was estimated by measuring the

leaf water potential before dawn (Ψpd), assuming there is little or no transpiration at

night. Completely expanded leaves from five plants per species and treatment were

measured with a pressure chamber (PMS Instrument Co. 7000, Corvallis Oregon).

Additionally, one fully expanded leaf per plant was excised from the same

seedling in which Ψpd was measured, its petiole immersed in distilled water, and

transported to the laboratory for the construction of P-V curves. Time for re-hydration

was no longer than 12 hours. We tested that no over-saturation took place by

determining the relationship between water potential and fresh weight during P-V

curves; no plateaus were observed among the first points of the curves [15, 30]. Curves

were only constructed where 10-or-more data points, typically 14, could be reliably

estimated. We followed the free transpiration method of Robichaux (1984). The osmotic

potentials at full turgor (Ψπ100) and zero turgor (Ψπ0) and the symplastic water volume

at full turgor (Vs) were derived from the P-V relationships [15]; Vs was related to the

total volume of leaf water within the leaf (Vt), as in Harayama et al. (2006). The

modulus of elasticity at maximum turgor (Emax) was also calculated following

Robichaux (1984). Curves were made in late June and late August of 2004 and 2005.

Survival was explored in late spring 2004 and 2007, i.e., after one and four years in the

field.

General Linear Models were run to test for the significance of block, date,

species and overstory effects on pressure-volume curve parameters. The variance model

Anexo VI - 8

also accounted for the second-order interactions between these factors, except those

with block. Pre-dawn water potential was treated as a covariate in the analyses of

variance. We further used the Tukey’s HSD test to separate means (at P < 0.05).

Bivariate relationships among variables were explored by Pearson’s correlation

coefficients, further comparing the slopes and intercepts of fitted regression lines

between treatments or species. Multiple regressions were used to analyze the response

of Ψπ100 and Emax to GSF and Ψpd as surrogates of water stress and light conditions. A

forward selection method was followed to enter predictor variables. Survival was

analyzed by a logistic regression model (Generalized Linear Model).

2.3. Experiment 2

This experiment was conducted in an even-aged, 42-year-old Scots pine

plantation, 1.5 Km apart from the previous stand, at 1600 m.a.s.l. It is E-NE facing and

moderately sloping (10-20 %). Four complete blocks were distributed in a 10 ha area

along an N-S gradient in a split-plot design, with overstory treatment (uncut, 33 %

thinned, 50 % thinned) as the main plot factor, and the species (Q. petraea and Q.

pyrenaica) as the subplot factor (table I). Thinnings were applied in twelve alternating

stripes in 2004, one year before planting (early spring 2005), and felled trees were

removed from the site. An experimental plot (≈ 0.053 ha) was established at the centre

of each replicate strip. Plots 1-3 in block I contained 80 individuals of Q. petraea and 40

individuals of Q. pyrenaica each, planted 1.5-m-separated in two rows among the pine

rows. Plots 4-12 (blocks II to IV) contained 84 individuals of Q. petraea and 42

individuals of Q. pyrenaica each, planted 1-m-separated in one row among the pine

rows. This different planting design was due to a different spacing of pine trees in Block

I (5 m between rows) vs the rest (3.5 m). No control of understory vegetation was made.

Anexo VI - 9

Seedlings were two-year-old when outplanted. For the first year, they were cultivated in

300-cm3 Forest pots containing a mixed peat/vermiculite substrate (3v/1v) enriched with

slow-release fertilizer at a dosage of 2.5 g l-1 [Osmocote Plus (8-9 months)] and in 3000-

cm3 Forest pots containing the same substrate type and fertilizer dosage for the second.

Plants were maintained in partial shade (30-40 % of full sunlight) in the second year.

Volumetric soil water content was measured in summer 2005 (i.e. one year after

applying the thinnings) with a TDR (Trase System I, Soil Moisture Equipment Corp.

USA) inside one PVC-tube buried in the center of each plot, for a total of 12 tubes.

Light availability was estimated with hemispherical photographs. Five digital

photographs (Nikon Coolpix 4500) were taken at seedling height in late summer 2005,

in the centre and corners of a c 10-m-side square located in the middle of each plot.

Photographs were analyzed with Hemiview 2.1 Canopy Analysis Software, quantifying

the Global Site Factor index. Survival was assessed at the end of the first growing

season in the field (October 2005), and in the beginning of the two following growing

seasons (June 2006 and June 2007).

The survival of each species under the three treatments was analyzed by a

logistic regression model, taking into account the four replicates (blocks) as categorical

predictor variable and the BA after thinning as covariable characterizing the thinning. In

an alternative approach, the correlated GSF was used in place of BA. The soil water

contents on different depths and dates were used as additional variables for exploratory

data analysis.

Anexo VI - 10

3. Results

Except the summer of 2003 in which seedlings of the first Experiment were

planted, summers 2004 to 2006 were drier and warmer than average (table II).

Especially dry was the summer of 2005, the year seedlings of Experiment 2 were

planted.

3.1. Experiment 1

Light availability (GSF) was highest in the gap, and slightly higher in the

thinned treatment than in the unthinned treatment (P < 0.001; table I). There were no

significant differences in GSF between species or years (not shown). There was a trend

for soil moisture within 40 cm depth to be higher in the thinned plots (P > 0.1; table I),

being more similar in the gap and the uncut plots. However, seedlings in the gap and

thinned plots exhibited higher values of Ψpd than those in the uncut plots (P < 0.001;

table III).

Seedlings of both species in the uncut and thinned plots had lower Ψπ0 and

Ψπ100 than in the gap (P < 0.001; table III). Both parameters decreased from June to

August in all treatments (P < 0.001; table III), more pronouncedly in the drier 2005

year. However, osmotic adjustment at the end of summer was lower in the seedlings of

Q. petraea in the uncut plots. Here they barely lowered Ψπ100 around 0.10 MPa in 2005

(table III), in contrast with large reductions in seedlings of Q. pyrenaica in the same

plots (0.53 MPa), or with seedlings of Q. petraea in the gap (0.43 MPa) and thinned

plots (0.37 MPa). Interspecific differences in Ψπ100 (P < 0.001) were most evident in

August (P date x species = 0.098; table III). At this time, Ψπ100 was lower in Q.

pyrenaica, more visibly under the pinewood canopy than in the gap (P site x species

Anexo VI - 11

excluding June values = 0.113; table III). Species effect on Ψπ0 was similar to the effect

on Ψπ100, but weaker (P < 0.05), such that turgor potential at dawn, calculated as the

difference between leaf water and osmotic potentials, did not differ significantly

between the species (not shown). Emax increased from June to August in Q. pyrenaica,

but remained unchanged in Q. petraea (P date x species < 0.05). Accordingly, Emax was

higher for Q. pyrenaica in August both years (P < 0.001), more visibly under the

pinewood cover than in the gap (P site x species excluding June values = 0.115; table

III).

Multiple regression analysis showed that GSF and Ψpd were significant

predictors of Ψπ100 for Q. pyrenaica (both coefficients P < 0.001; model R2 = 55.9 %)

and Q. petraea (both coefficients P < 0.001; model R2 = 40.3 %), while were marginally

significant predictors of Emax for Q. petraea (both coefficients P < 0.05; model R2 =

17.4 %). There was not a consistent relationship between Emax and Ψpd within

treatments, whereas Ψπ100 and Ψπ0 were positively correlated with Ψpd in both species

(figure 1), with differences among overstory treatments in the intercepts (all P < 0.001),

but not in the slopes (only P = 0.074 for Ψπ100 vs Ψpd in Q. petraea). In the uncut plots,

the slopes and intercepts of the Ψπ100 - Ψpd relationship differed significantly between

species (P < 0.01 and P < 0.05, respectively). Vs/Vt was positively correlated with Emax

in both species and all treatments, except in the gap in Q. pyrenaica (not shown).

Survival of both species was highest in the thinned plots, compared to the uncut

or gap plots, either at the end of the first or fourth year in the field (P < 0.05; figure 2).

Survival of Q. petraea and Q. pyrenaica was comparable in all treatments (P > 0.15).

Anexo VI - 12

3.2. Experiment 2

GSF differed significantly between the un-thinned and thinned plots, but hardly

at all between both thinning levels (table I). Soil moisture averaged over summer

months was lower in the uncut treatment than in the two thinning treatments (P < 0.01).

For all plots, GSF correlated negatively with the residual BA left after thinnings (figure

3). A negative correlation was also found between the residual BA and the soil moisture

at 10 cm depth (figure 3).

The survival of Q. petraea was poor in all plots (figure 2), though being

somewhat higher in the first block, with a wider between-row distance of the original

pine stand. At overall level, there was no significant effect of thinning, light

environment or soil-water content on the survival of Q. petraea. In Q. pyrenaica,

survival was significantly higher in the thinned plots (figure 2), though with strong

differences between blocks. Therefore, the logistic regression model for survival in all

three dates included as significant factors Block and Treatment, though since effects of

both thinning intensities did not differ significantly among each other, they can be

pooled into a common level of a new variable ‘thinned/unthinned’ that explains

practically the same proportion of deviation (D% 67.34 vs. 67.98% for the last date) with

a lower mean square error (MSE 0.167 vs. 0.186). The BA after thinning as

environmental proxy of each plot for fixed treatment effects matched plainly the

adjustment of the original model (D% 67.29%; MSE 0.152 in 2007) or even improved it

slightly (D% 69% vs. 67% in 2005, 63% vs. 61% in 2006) whereas the light

environment GSF performed worse (e.g. D% 54%; MSE 0,270 in 2007). As the ratios of

deviance, scaled deviance, Pearson Chi-Square and Scaled Pearson Chi-Square to the

respective degrees of freedom were in all cases close to 1.0, there was no evidence of

over-dispersion. The use of soil water content at any depth as covariable, either did not

Anexo VI - 13

improve the model or it did introduce correlations between estimations of model

coefficients that imply collinearity, therefore, they were discarded.

We observed symptoms of deer browsing on most seedlings already in the first

year (≈95 % of seedlings damaged); independently of the overstory density (P > 0.15),

but not of the species (91% in Q. pyrenaica vs 96% in Q. petraea; P < 0.001).

4. Discussion

4.1. Effects of the overstory on light and soil moisture

Our results confirmed that overstory density influences both light available in

the understory and soil moisture [6]. While light transmittance usually increases

exponentially with decreasing stand basal area [21, 32], the relationship of soil moisture

with basal area seems less straightforward, due to a stronger influence of climatic and

edaphic factors. Most experiences concerning tree or shrub effects on the

microenvironment in Mediterranean ecosystems lead us to hypothesize a non linear

relationship of moisture with density (figure 3), derived mainly from the changing

interplay among evaporation, water uptake by both canopy and understory vegetation,

and rainfall interception [10, 20, 24, 26, 36, 44, but see 9]. The negative, linear

correlation of moisture at 10-cm depth against the observed range of residual basal area

suggests that increases in relative irradiance did not cause too intense evapotranspiration

as to offset the reduction in living fine roots of Scots Pine in the upper soil layers and/or

the increased throughfall precipitation after thinning of the tree-canopy [29]; following

this reasoning, the 45 % available light in the gap could mark the beginning of a drop in

soil moisture, as it was similar to that in the more dense plots. Nonetheless, given the

trade-offs in plant acclimatory responses to light and soil moisture resources [see 50],

Anexo VI - 14

experiments should also aim to discern whether regeneration silvicultural practices

should target initial density levels at which soil moisture is maximized or rather at

which both light and soil moisture resources are optimized (figure 3).

4.2. Effects of the overstory on pressure-volume curves

The range of light created by the three sites played a role in modifying water

relations. Despite seedlings in the uncut plots showed consistently higher water stress

along dates when compared with the other two overstory treatments, low light in the

heavily dense uncut plots could preclude the ability of Q. petraea to osmotically adjust

in response to water stress, as previously observed in other species [1, 4, 5, 14], which

points to a species-specific threshold of light to maintain some drought-tolerance

mechanisms. Why Q. pyrenaica had superior ability to lower Ψπ100 at the end of

summer under the more dense pinewood is not clear. Given the importance of soluble

sugars as osmolytes [11, 17], interspecific differences in Ψπ100 under the pine cover

might be based on a differential ability to accumulate hexoses. Greater photosynthetic

capacity, and likely respiration and starch degradation, in the less shade tolerant Q.

pyrenaica [46] would help to produce a larger pool of hexoses, as shade-tolerance is

commonly associated with a more-conservative metabolic rate in shade.

However, more elastic cell walls of seedlings of Q. petraea contributed to

attenuate interspecific differences in Ψπ0 and maintain turgor at the end of summer in

the uncut plots despite their lower ability to decrease Ψπ100 in response to drought.

Increased cell-wall elasticity has previously been identified as a mechanism of

improved drought tolerance [51], which allows for the maintenance of turgor by

decreasing Ψπ0 rather than Ψπ100 [13, 27]. But the inconsistency of the relationship

between Emax and Ψpd suggests that the increase in Emax from June to August in Q.

Anexo VI - 15

pyrenaica responds not only to drought, but maybe to an ontogenetic process of leaf

maturation after full expansion. In any case, leaves of Q. petraea had less rigid cell-

walls at the end of summer (i.e. lower Emax) that could not accumulate as large amounts

of solutes as more sclerophyllous leaves of Q. pyreniaca [11]. Further, the positive

relationship between cell-wall elasticity and the symplastic water fraction may be

associated with water redistribution between the apoplast and the symplast [27, 43].

Combined increases in the concentration of solutes (i.e. lower Ψπ100) and cell-

wall rigidity in Q. pyrenaica enabled both cell turgor and volume at the time of

maximum drought to be maintained, as previously described in other drought-tolerant

trees [11, 52, 54], which should make it better able to withstand droughty conditions of

sub-Mediterranean understories.

4.3. Effects of the overstory on survival

Survival was only partially related with drought-tolerance features derived from

leaf pressure-volume curves, as seedling death is the complex interplay of multiple

factors upon the whole plant. Even though survival of Q. pyrenaica was higher than that

of Q. petraea in the Experiment 2, the exceptionality of meteorological conditions in the

year of planting plus the intense browsing complicate the interpretation of the results.

The clear light-related response to thinning in Q. pyrenaica in terms of survival

contrasted with the generalized outplanting failure of Q. petraea possibly owing to the

severe drought of 2005. This could also help to explain contrasting interspecific

differences in survival between experiments, as more similar survival rates between

species were found in milder years of planting (Experiment 1; [47]). Moreover, we

cannot rule out the influence of browsing on these aspects. Despite intensity was similar

among overstory treatments, light availability could have differentially influenced

Anexo VI - 16

resilience of seedlings of Q. pyrenaica to foliage removal [7]. Resprouting occurred in

all treatments after seedlings were browsed, however, higher light in the thinned plots

likely allowed for a higher leaf area re-development and carbon gain revenue with

respect to the uncut plots, which could help to rebuild-up non-structural carbohydrate

reserves in the root system and favour initial persistence, at least until reserves are

depleted in repeatedly producing new shoots to compensate for the continued lost of

foliage, if herbivory continues [7, 31]. In line with this reasoning, the greater root to

shoot ratio of seedlings of Q. pyrenaica, together with their higher resprouting ability

could have enhanced their survival over those of Q. petraea.

4.4. Conclusions and future directions

Despite the abovementioned uncertainties in explaining survival, the results of

both experiments conveyed a similar idea that leaving dense stands untouched before

underplanting is negative for seedling initial establishment. Overstory reduction by

means of thinnings and gaps might be advisable to foster artificial oak regeneration in

Mediterranean humid mountains, as seems to be a general pattern in other biomes [38],

given the observed increases in survival, light and soil moisture, and greater acclimation

potential to water stress, with respect to denser, uncut stands. If the severe conditions

encountered by seedlings over the study period are to be more common in the future

[25], regeneration of Q. petraea could be threatened in the study area, which urges to

develop further experiences to eventually draw clear statements about which density is

initially best, how this level varies across regions and conditions in the year of planting,

and how and when subsequent thinnings should be applied to enhance seedling

performance.

Anexo VI - 17

Acknowledgements: This work was supported by the Consejería de Medio

Ambiente y Desarrollo General de la Comunidad Autónoma de Madrid. J. Rodríguez-

Calcerrada was supported by a scholarship from the Consejería de Educación de la

Comunidad de Madrid (C. M.) and the European Social Fund. We thank Delphine

Grivet for the English-to-French translation and Matthew Robson for his review of the

manuscript and language.

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Anexo VI - 25

Figure legends

Figure 1. Osmotic potential at full turgor (Ψπ100) and at turgor loss point (Ψπ0), and

maximum modulus of elasticity (Emax) in relation to pre-dawn leaf water potential (Ψpd)

in seedlings of Q. pyrenaica and Q. petraea in the uncut (black symbols, continuous

lines), thinned (grey symbols, dashed lines) and gap (white symbols, dotted lines)

treatments.

Figure 2. Survival of Q. pyrenaica and Q. petraea. Notice that X-scales are different

between upper (experiment 1) and lower plots (experiment 2).

Figure 3. Variation of light (GSF; white squares, continuous line) and summer average

soil moisture at 10-cm depth (grey squares, dashed line) with the residual basal area in

plots of Experiment 2. Triangle symbols are mean data points from the thinned and

uncut plots of Experiment 1 (not included in the regression lines). The inset represents

the predicted evolution of light and soil moisture with basal area based on previous

research; the shady area indicates a possible range of basal area to be targeted (see text).

Anexo VI - 26

Tables

Table I. Mean values (± S.E.) of basal area (B.A), tree density, light availability (GSF)

and soil moisture (0-40 cm depth) averaged over the summer months of 2005 in

treatments of Experiments 1 and 2.

1: Data pooled for 2004 and 2005 years in Experiment 1.

Overstory n B.A. (m2 ha-1) Density (ha-1) n GSF1 n Soil MoistureUncut 1 65.8 1067 20 20.3 ± 0.7 3 10.7 ± 1.125 % thinned 1 53.6 800 16 22.5 ± 0.9 3 13.1 ± 0.6Gap 1 _ _ 15 45.1 ± 2.9 3 11.1 ± 1.6

Uncut 4 54.8 ± 1.4 947 ± 84 20 16.3 ± 0.6 4 14.0 ± 0.833 % thinned 4 40.1 ± 1.3 658 ± 16 20 25.8 ± 0.7 4 15.7 ± 1.450 % thinned 4 30.5 ± 0.5 446 ± 18 20 26.8 ± 0.9 4 17.0 ± 0.4

Exp.

1Ex

p.2

Anexo VI - 27

Table II. Precipitation and mean temperature in the study area over summer months

(June to September) from 2003 to 2006.

1: Averages from data of our meteorological tower between 1995 and 2006 (data of

1999, 2001 and 2002 were missing).

2: Averages estimated for the study area from precipitation- and temperature-altitude

regression lines constructed from data between 1955 and 1969 [18].

Year P (mm) T (ºC)2003 145.4 18.32004 95.2 17.42005 40.4 17.72006 112 17.3Average1 143.2 16.9Average2 168.2 16.6

Anexo VI - 28

Table III. Mean values (± S.E.) of pre-dawn leaf water potential (Ψpd; n = 4-5), osmotic

potential at full turgor (Ψπ100; n = 4-5) and at turgor loss point (Ψπ0; n = 4-5), and

modulus of elasticity at maximum turgor (Emax; n = 4-5) measured on four dates in each

treatment and species.

Treatment Species June August June AugustQ. pyr. -0.06±0.03 -0.52±0.06 -0.06±0.02 -1.24±0.24Q. pet. -0.07±0.01 -0.41±0.10 -0.07±0.02 -1.26±0.20Q. pyr. -0.24±0.04 -0.51±0.07 -0.25±0.04 -1.39±0.21Q. pet. -0.21±0.06 -0.42±0.04 -0.34±0.08 -1.47±0.17Q. pyr. -0.28±0.02 -0.95±0.25 -0.56±0.08 -2.05±0.15Q. pet. -0.33±0.05 -0.80±0.18 -0.51±0.07 -2.12±0.20

Q. pyr. -1.72±0.09 -2.07±0.04 -1.80±0.05 -2.18±0.05Q. pet. -1.73±0.07 -1.85±0.13 -1.72±0.04 -2.15±0.07Q. pyr. -1.61±0.06 -1.89±0.08 -1.46±0.07 -2.04±0.13Q. pet. -1.47± 0.08 -1.71±0.06 -1.47±0.08 -1.84±0.16Q. pyr. -1.50±0.07 -1.91±0.06 -1.64±0.11 -2.17±0.09Q. pet. -1.53±0.07 -1.63±0.04 -1.59±0.08 -1.69±0.06

Q. pyr. -2.23±0.10 -2.64±0.06 -2.27±0.06 -3.09±0.13Q. pet. -2.20±0.08 -2.42±0.14 -2.19±0.04 -2.98±0.11Q. pyr. -2.10±0.05 -2.33±0.13 -1.89±0.06 -2.67±0.10Q. pet. -1.94±0.08 -2.20±0.08 -1.89±0.07 -2.59±0.16Q. pyr. -1.87±0.09 -2.33±0.02 -2.16±0.08 -2.82±0.12Q. pet. -1.95±0.11 -2.19±0.06 -2.04±0.08 -2.62±0.18

Q. pyr. 10.4±0.9 12.9±1.0 11.3±1.3 10.0±1.0Q. pet. 10.6±0.9 10.1±1.9 10.1±1.2 9.5±0.7Q. pyr. 10.0 ± 1.2 12.4 ± 0.8 6.8 ± 1.3 12.2 ± 2.8Q. pet. 8.0 ± 1.4 8.8 ± 0.9 6.6 ± 1.0 7.1 ± 1.3Q. pyr. 8.9 ± 1.0 12.9 ± 1.6 7.3 ± 1.9 11.4 ± 1.2Q. pet. 10.5 ± 1.8 7.0 ± 0.9 7.9 ± 2.0 5.3 ± 0.8

E max

(MPa

) Gap

Thinned

Uncut

Ψπ 0

(MPa

) Gap

Thinned

Uncut

Ψπ 1

00 (M

Pa) Gap

Thinned

Uncut

2004 2005

Ψpd

(MPa

) Gap

Thinned

Uncut

Anexo VI - 29

Figures

-3.0

-2.5

-2.0

-1.5

-1.0

R2: 0.52; P < 0.001R2: 0.67; P < 0.001R2: 0.59; P < 0.001

-4.5-4.0-3.5-3.0-2.5-2.0-1.5-1.0

05

1015202530

-2.5-2.0-1.5-1.0-0.50.0 -2.5 -2.0-1.5-1.0-0.50.0

Ψpd (MPa) Ψpd (MPa)

Ψπ 1

00(M

Pa)

Ψπ 0

(MPa

)Ε m

ax(M

Pa)

R2: 0.18; P < 0.1R2: 0.20; P < 0.05R2: 0.53; P < 0.001

R2: 0.56; P < 0.001R2: 0.45; P < 0.01R2: 0.61; P < 0.001

R2: 0.66; P < 0.001R2: 0.72; P < 0.001R2: 0.77; P < 0.001

R2: 0.07; P > 0.15R2: 0.39; P < 0.01R2: 0.06; P > 0.15

R2: 0.16; P > 0.1R2: 0.09; P > 0.15R2: 0.04; P > 0.15

Q. pyrenaica Q. petraea

-3.0

-2.5

-2.0

-1.5

-1.0

R2: 0.52; P < 0.001R2: 0.67; P < 0.001R2: 0.59; P < 0.001

R2: 0.52; P < 0.001R2: 0.67; P < 0.001R2: 0.59; P < 0.001

-4.5-4.0-3.5-3.0-2.5-2.0-1.5-1.0

05

1015202530

-2.5-2.0-1.5-1.0-0.50.0 -2.5 -2.0-1.5-1.0-0.50.0

Ψpd (MPa) Ψpd (MPa)

Ψπ 1

00(M

Pa)

Ψπ 0

(MPa

)Ε m

ax(M

Pa)

R2: 0.18; P < 0.1R2: 0.20; P < 0.05R2: 0.53; P < 0.001

R2: 0.18; P < 0.1R2: 0.20; P < 0.05R2: 0.53; P < 0.001

R2: 0.56; P < 0.001R2: 0.45; P < 0.01R2: 0.61; P < 0.001

R2: 0.56; P < 0.001R2: 0.45; P < 0.01R2: 0.61; P < 0.001

R2: 0.66; P < 0.001R2: 0.72; P < 0.001R2: 0.77; P < 0.001

R2: 0.66; P < 0.001R2: 0.72; P < 0.001R2: 0.77; P < 0.001

R2: 0.07; P > 0.15R2: 0.39; P < 0.01R2: 0.06; P > 0.15

R2: 0.07; P > 0.15R2: 0.39; P < 0.01R2: 0.06; P > 0.15

R2: 0.16; P > 0.1R2: 0.09; P > 0.15R2: 0.04; P > 0.15

R2: 0.16; P > 0.1R2: 0.09; P > 0.15R2: 0.04; P > 0.15

Q. pyrenaica Q. petraea

Figure 1

Anexo VI - 30

0

20

40

60

80

100

Q. pyrenaica Q. petraea

Uncut25 % thinnedGap

0

20

40

60

80

100 Uncut33 % thinned50 % thinned

Months after planting

Surv

ival

(%)

Surv

ival

(%)

Exp. 1

Exp. 2

0 10 20 30 40 500 10 20 30 40 50

0 5 10 15 20 250 5 10 15 20 25Months after planting

Figure 2

Anexo VI - 31

R2: 0.75; P < 0.001

05

101520253035

0 20 40 60 800

4

8

12

16

20

Residual basal area (m-2 ha-1)

GSF

(%)

Soilmoisture

(%)R2: 0.47; P < 0.05

R2: 0.75; P < 0.001

05

101520253035

0 20 40 60 800

4

8

12

16

20

Residual basal area (m-2 ha-1)

GSF

(%)

Soilmoisture

(%)R2: 0.47; P < 0.05

Figure 3

Anexo VI - 32