Departamento de Biología Vegetal y Ecología
135
Transcript of Departamento de Biología Vegetal y Ecología
Departamento de Biología Vegetal y Ecología
Facultad de Ciencias Experimentales Universidad de Almería
Factores limitantes y estrategias de establecimiento de plantas leñosas en ambientes semiáridos.
Implicaciones para la restauración
Memoria presentada por el Licenciado Francisco Manuel Padilla Ruiz para optar al título
de Doctor en Ciencias Ambientales por la Universidad de Almería, dirigida por el Dr.
Francisco I. Pugnaire de Iraola.
Almería, septiembre de 2007
El Doctorando V.º B.º del Director
Francisco M. Padilla Ruiz Francisco I. Pugnaire de Iraola
A mi familia
Al Paquín†
Agradecimientos
En primer lugar, me gustaría agradecer a mi director de tesis, Paco Pugnaire, la confianza
depositada en mí durante todo este tiempo. Su apoyo y optimismo han sido decisivos para seguir
adelante y llegar a buen puerto. Gracias también por cederme tu foto de las avenas para la portada.
Mi madre, padre y hermanos siempre estuvieron conmigo en los mejores momentos, pero
también en los más difíciles, y de una manera u otra también han sentido las alegrías y los
desencantos de la investigación. Hasta diría que han soportado estoicamente todas las veces que les
he dado la lata con la misma cantilena. Sin duda, si no hubiera sido por su ánimo y cariño ahora
mismo no estaría aquí.
Llegado este momento no puedo dejar de recordar con emoción al Paquín. Él fue una
persona excepcional, terriblemente bueno con su gente y siempre disponible para lo que hiciera
falta. Su alegría, coraje y ganas de vivir aún cuando las cosas empeoraban me han marcado, y me
han servido de estímulo en las peores rachas.
Durante estos años también ha habido tiempo para la diversión. Los mejores momentos los
pasé con el Paquín, mi hermano Juan, Mónica y Juan, Vince, Francis, Chema, Moncho, Fernando y
Julián, y en Las Hortichuelas con el Makina, Juan Carlos, Antonio Miguel, Ángel, Ramón, Eli,
Jonathan, José Javier, Antonio Manuel, Diego y el Tuyú, compañeros de infancia y de marcha ya
más creciditos.
Los compañeros “precarios” de la EEZA, Rafa, Carmen, Ashraf, Sergio, Magda, Rosi, Ana
Were, Lupe, Miguel Ángel, Sebas y Ana amenizaron e hicieron mi estancia por la casa más
divertida gracias a las fiestas y demás eventos “sociales”. Mención aparte merecen los otros
ocupantes de “El Peñón”. Reyes y Cris me introdujeron en este mundillo y orientaron en mis
comienzos más verdes. Cris me ayudó además con la estadística y el diseño experimental. Mi
compañero de despacho Juande ha sido la persona que más cerca ha estado de mi trabajo en todos
estos años, ¡gracias por todo tu apoyo, joven!, y gracias también por acompañarme al campo en los
innumerables muestreos, por echar una mano en el invernadero y las cosechas, por compartir
desilusiones, madrugones, tostadas, etc. Ya en este último año, las incorporaciones de Laura, Iván y
Bea revitalizarón el cuarto con aires y acentos nuevos, y con sus ganas de comenzar en este
mundillo. Iván y Bea revisaron algunas secciones de la última versión de este documento,
aportaron buenas sugerencias y detectaron algún que otro gazapo.
Gran parte de las cosas que aparecen en esta tesis no hubieran sido posibles sin la ayuda en
el campo especialmente de Alejandro Moreno, y también de Floren, Pilar, Mar Candel y Carlos
Escudero. María José cuidó con esmero mis plantitas en el invernadero, incluso en los meses del
tórrido verano, y su inestimable ayuda con las muestras en el laboratorio fue todo un alivio cuando
la redacción me tenía liado. ¡Gracias por soportar tareas tan aburridas! Sebastián Vidal, Ramón,
Enrique y Miguel Ángel Domene solucionaron sin rechistar todos los problemas informáticos, de
sensores y de electrónica. El conocimiento y ayuda de Alfredo fueron valiosísimos para construir
artilugios estables que se mantuvieran en pie. Andrés, Manolo, Mari Carmen, Olga y Juan Leiva
me facilitaron todos los papeleos y trámites de la beca, los viajes, las estancias, etc. Paqui y
Ángeles me echaron más de una vez un cable con los envíos y faxes, e Isabel aguantó estoicamente
mis ráfagas de bombardeo con peticiones bibliográficas. Gracias también al resto de compañeros de
la EEZA por su hospitalidad y buen trato durante todos estos años.
Estoy en deuda con Jordi Moya por las innumerables consultas estadísticas que le he
venido haciendo en todos estos años, y por el rendimiento que le he sacado a las regresiones
logísticas. Su buen humor y el de Roberto, Zaza y Guliko hicieron estos años mucho más
divertidos.
Le doy las gracias también a José Miguel, a mi tutor Manuel Ortega y demás compañeros
de la Universidad de Almería por estos años, y en especial a Miguel Cueto por responder con
celeridad a todas mis dudas, mantenerme al corriente y facilitarme todos los trámites relacionados
con el DEA y esta tesis.
Aunque ellos no se dieron cuenta, los compañeros del pelotón ciclista Los Filósofos
(Ángel, Jaime, Joaquín, Paco, Antonio, Fede, Pepe, Luis, Carlos) contribuyeron a rebajar el nivel
de tensión acumulado durante la semana con nuestras rutas, demarrajes, ascensiones, sprints y
ritmo infernal a plato. Compartir horas encima de la bici da para mucho más que sólo un buen rato
de ciclismo.
Mis primeras salidas al extranjero las hice gracias a las estancias breves. Éstas me
sirvieron, además de para mejorar mi inglés y ver sitios que nunca hubiera pensado que visitaría,
para aprender cantidad de cosas y ganar tablas en este mundillo. Gracias a Silvia, Nina Buchmann,
Xin y Wengchao, Heather Reynolds, Dave, Becky, Mike, Barb y Brett por todas las facilidades y
por hacer la morriña en el extranjero más llevadera. Más cerquita, la experiencia y colaboración de
Eulogio Bedmar, Hamdi Zahran (Estación Experimental del Zaidín) y María Pérez Fernández
(Universidad Pablo de Olavide) fue decisiva para llevar a cabo el “experimento del Rhizobium”,
¡lástima que tuviéramos problemas con la inoculación!
A Joaquín Sánchez, Rafael Ortega y Manuel Hervás de Serfosur S.L. les agradezco todo el
apoyo personal y técnico que han ofrecido en todos estos años de colaboración. Sin su implicación,
muchos de los experimentos no se hubieran podido realizar. De vital importancia para los
experimentos de invernadero también resultó la donación de semillas y plantas por parte de
Serfosur, del vivero de plantas autóctonas de Rodalquilar (Consejería de Medio Ambiente, Junta de
Andalucía) y de Viveros Retamar S.L., así como la colaboración de Pepe del Cortijo La Sierra en
algunos de los experimentos de campo.
En el ámbito económico, todo este trabajo no hubiera sido posible sin el sustento de una
beca predoctoral I3P (CSIC, 2004-2007), y sin la financiación de los proyectos de investigación
REN2001-1544/GLO, AGL2000-0159-P4-02 y CGL2004-00090/CLI, del Ministerio de Educación
y Ciencia. Asimismo, gracias a la European Science Foundation y a su programa SIBAE por la
financiación de mi estancia en el ETH Zurich (Suiza) y al CSIC por las estancias en Indiana
University (EE.UU.).
Para terminar, me gustaría agradecer su apoyo y ayuda a todas las personas que, sabiéndose
merecedoras de estar en esta lista, no aparecen en ella por ningún otro motivo que no sea mi mala
cabeza y descuido. Mil disculpas y mi más sincero agradecimiento.
Factores limitantes y estrategias de establecimiento de plantas leñosas en ambientes semiáridos.
Implicaciones para la restauración
Índice
Introducción
Introducción general …………………………………………………………………………….. 19
Objetivos …………………………………………………………………………........................ 24
Referencias …………………………………………………………………………………………… 25
Síntesis
Síntesis de resultados …………………………………………………………………………….. 33
Referencias …………………………………………………………………………………………… 34
Capítulo I
LA PROFUNDIDAD DE ENRAIZAMIENTO Y LA HUMEDAD DEL SUELO CONTROLAN LA
SUPERVIVENCIA DE PLÁNTULAS DE ESPECIES LEÑOSAS DURANTE LA SEQUÍA ..………...... 35
Summary ……………………………………………………………………………………………... 37
Introduction ………………………………………………………………………..................... 37
Materials and Methods ……………………………………………………………................. 38
Species ……………………………………………………………………………………… 38
Field site and experimental design ………………………………………........... 39
Growth analysis and statistics ……………………………………………………… 40
Results ……………………………………………………………………………......................... 41
Discussion ……………………………………………………………………………………………. 44
Acknowledgements ……………………………………………………………….................... 47
References ……………………………………………………………………………………………. 47
Capítulo II
PLASTICIDAD EN EL DESARROLLO DE RAÍCES EN PLÁNTULAS DE TRES ESPECIES
LEÑOSAS MEDITERRÁNEAS ...………………….…………………………………………………………... 53
Summary ……………………………………………………………………………………………... 55
Introduction ………………………………………………………………………………………... 55
Materials and Methods ………………………………………………………………………….. 57
Species ……………………………………………………………………………………… 57
Experimental design …………………………………………………………………… 58
Measurements …………………………………………………………………………… 59
Growth analysis …………………………………………………………………………. 60
Statistics ……………………………………………………………………………………. 61
Results ……………………………………………………………………………......................... 62
Discussion ……………………………………………………………………………………………. 64
Acknowledgements ……………………………………………………………….................... 68
References ……………………………………………………………………………………………. 68
Capítulo III
RESPUESTA FISIOLÓGICA DE SIETE ESPECIES ARBUSTIVAS MEDITERRÁNEAS A PULSOS
DE AGUA …………………………………………………………………………………………………………. 73
Summary …………………………………………………………………………........................ 75
Introduction .……………………………………………………………………….................... 77
Materials and Methods ……………………………………………………………................. 77
Species ……………………………………………………………………………………… 77
Experimental design …………………………………………………………………… 78
Measurements and plant harvest …………………………………………………. 79
Statistics …………………………………………………………………...................... 80
Results ……………………………………………………………………………......................... 81
Discussion ……………………………………………………………………………………………. 85
Acknowledgements ……………………………………………………………….................... 87
References ……………………………………………………………………………………………. 87
Capítulo IV
EL PAPEL DE LAS PLANTAS NODRIZA EN LA RESTAURACIÓN DE AMBIENTES
DEGRADADOS ………………………………..………………………………………………………………… 93
Summary …………………………………………………………………………........................ 95
Introduction ………………………………………………………………………..................... 95
Competition and facilitation ………………………………………………………............. 96
The nurse effect ……………………………………………………………………………………. 96
Advantages of growing close to nurse plants ……………………………………………. 97
The role of nurse plants in restoration …………………………………………….......... 98
Considerations for management …………………………………………………………….. 101
Ecological conditions ………………………………………………………………………. 101
Rainfall variability …………………………………………………………….................. 101
Nurse species ………………………………………………………………….................... 102
Target species ………………………………………………………………………………….. 103
Positive and negative effects of nurses ………………………………………........... 103
Conclusions …………………………………………………………………………...................... 103
Acknowledgements ………………………………………………………………….................... 104
References ………………………………………………………………………………………………. 104
Capítulo V
LAS CONDICIONES AMBIENTALES Y EL USO DE PLANTAS NODRIZA EN RESTAURACIÓN … 109
Summary …………………………………………………………………………........................ 111
Introduction ………………………………………………………………………..................... 112
Materials and Methods ……………………………………………………………................. 113
Experimental site …………………………………………………………................. 113
Species and experimental design …………………………………………………. 114
Abiotic environment ………………………………………………………………….. 115
Survival, growth and physiological status …………………….……………….. 115
Statistics …………………………………………………………………...................... 116
Results ……………………………………………………………………………......................... 116
Abiotic environment ………………………………………………………………….. 117
Survival …………………………………………………………………………………….. 117
Growth and physiological status …………………………………………........... 118
Discussion ……………………………………………………………………………………………. 121
Acknowledgements ……………………………………………………………….................... 124
References ……………………………………………………………………………………………. 124
Conclusiones ……………………………………………………………………………………………….. 131
Introducción
Introducción
19
FACTORES LIMITANTES Y ESTRATEGIAS DE ESTABLECIMIENTO DE
PLANTAS LEÑOSAS EN AMBIENTES SEMIÁRIDOS. IMPLICACIONES PARA
LA RESTAURACIÓN
Introducción general
Los procesos de germinación y de
reclutamiento son aspectos importantes en el
modelado de las comunidades vegetales,
siendo los responsables últimos de la
estructura y composición de las mismas
(Grubb 1977). Tras la germinación, las
plántulas son muy vulnerables y están
expuestas a diversas amenazas tanto bióticas
como abióticas que limitan su
establecimiento. Esto provoca que la fase de
plántula sea considerada una de las etapas más
críticas en el ciclo de vida de una planta.
Como consecuencia, sólo una pequeña
fracción de los individuos germinados
conseguirá llegar a la fase adulta. Entre los
factores abióticos que limitan el
establecimiento destacan la sequía y la
desecación del suelo (Moles y Westoby
2004), si bien la elevada radiación o la
escasez de luz y las temperaturas extremas
también influyen en gran medida. Entre los
bióticos destacan la herbivoría, la
competencia por los recursos con la
vegetación existente (Rey-Benayas et al.
2002) o los efectos de las sustancias químicas
liberadas por plantas vecinas (i.e., alelopatía,
Fenner y Kitajima 1999). Si bien tanto
factores bióticos como abióticos inciden en el
éxito de establecimiento de las plántulas,
parece que los factores abióticos adquieren
especial protagonismo en ambientes
especialmente limitantes, mientras que en
hábitats más benignos son los bióticos los
principales causantes de la mortalidad en
juveniles (Fenner 1987). Esta observación
toma especial relevancia en sistemas áridos,
mediterráneos o alpinos, donde se ha visto que
las interacciones entre plantas de signo
positivo, como la facilitación, predominan
sobre las de carácter negativo, como la
competencia o la interferencia (Callaway
1995).
Tradicionalmente la competencia por los
recursos entre dos plantas que crecen
próximas entre sí ha sido el tipo de interacción
más estudiada, lo que provocó que los
modelos ecológicos se basaran durante mucho
tiempo en sus efectos. Sin embargo, en los
últimos años numerosos trabajos han puesto
de manifiesto que una planta se puede
beneficiar cuando vive cerca de otra, esto es,
es facilitada en términos de supervivencia,
crecimiento o éxito biológico (Callaway 1995,
Tirado y Pugnaire 2003). Actualmente se
acepta que entre dos plantas que crecen en
proximidad, ambas pueden ejercer sobre sus
vecinos tanto efectos positivos como
Establecimiento de plántulas
20
negativos, de manera que el balance entre
éstos determina el signo final de la
interacción. Así, encontramos competencia si
los efectos negativos prevalecen, y facilitación
si predominan los positivos (Pugnaire y Luque
2001). Aunque la facilitación se ha observado
en prácticamente todos los biomas del mundo,
desde desiertos hasta dunas costeras,
matorrales mediterráneos, sabanas tropicales,
salinas, tundra ártica y bosques y pastizales
templados, es más aparente en ambientes
severos (Callaway 1995, Holmgren et al.
1997), como zonas áridas (Pugnaire et al.
1996, Flores y Jurado 2003) y de alta montaña
(Callaway et al. 2002, Kikvidze 2002). En
estos ambientes, caracterizados por clima
extremo, suelos infértiles y/o altas tasas de
herbivoría, algunas especies suavizan las
condiciones extremas, mejoran la
disponibilidad de los recursos y/o protegen de
los herbívoros (ver revisión en Callaway 1995
y Callaway y Pugnaire1999), proporcionando
un hábitat más adecuado para el reclutamiento
y desarrollo. De hecho, ya antiguamente se
observó que las plántulas de determinadas
especies se beneficiaban de vivir próximas a
estas especies, y como resultado el
establecimiento en sus proximidades era
mayor que en zonas alejadas de ellas (Niering
et al. 1963). Esta asociación espacial entre
plántulas y plantas adultas se denominó
“efecto nodriza” (“nurse plant syndrome”), y
constituye un claro ejemplo en el que el
reclutamiento se ve facilitado por la presencia
de vegetación.
Sin embargo, las plantas no seleccionan de
una manera activa el hábitat donde crecen,
sino que les es impuesto por factores como la
dispersión, y factores bióticos y abióticos que
inciden sobre la supervivencia de las semillas
y la germinación de éstas (Schupp 1995). De
esta manera, si la semilla no es depositada en
un sitio favorable donde las condiciones sean
óptimas para la supervivencia de las plántulas,
el éxito de reclutamiento dependerá de la
habilidad de las plantas para adaptarse y/o
resistir los factores limitantes. En ecosistemas
mediterráneos, la supervivencia de las
plántulas se ve limitada principalmente por la
disponibilidad de agua en el suelo durante
varios meses (Noy-Meir 1985, Herrera 1992).
En estos ambientes, las plántulas han de hacer
frente, en general, a precipitaciones muy
bajas, y tras la geminación en invierno o
primavera, han de superar un largo periodo de
sequía estival que constituye un cuello de
botella para el establecimiento (García-Fayos
y Verdú 1998, Figura 1).
Figura 1. Temperatura media y precipitación mensual en Tabernas (Almería, 37º08’ N, 2º22’ W, 490 msnm). La temperatura media anual es 17.8 ºC y la precipitación anual 235 mm (periodo 1967-1997).
Introducción
21
Las distintas especies han desarrollado
diversas y variadas adaptaciones para tolerar
estos periodos de ausencia de lluvias, como
por ejemplo una alta tolerancia a la extrema
deshidratación de los tejidos (Balaguer et al.
2002), la presencia de hábitos deciduos
durante los periodos más secos (Haase et al.
2000) o una alta resistencia del xilema al
embolismo (Jacobsen et al. 2007). En cambio,
la estrategia de otras especies no reside en la
resistencia al estrés hídrico, sino en escapar de
las limitaciones hídricas a través de raíces
profundas que llegan a fuentes de agua
estables durante todo el año (Nepstad et al.
1994). Estos diferentes comportamientos
están relacionados con la tasa de
supervivencia de las plántulas al final del
periodo seco. De esta manera, las plántulas de
especies que toleran la sequía presentan tasas
de supervivencia mayores tras la sequía que
aquellas cuya estrategia es evitarla (Davis
1989, Ackerly 2004). Sin embargo,
independientemente del tipo de estrategia que
exhiba cada especie, todo apunta a que
determinados rasgos del sistema radical de las
plántulas relacionados con la captación de la
humedad del suelo, juegan un papel
primordial para superar este periodo de estrés
hídrico, particularmente una elevada
asignación de biomasa a las raíces con
respecto a la parte aérea (Lloret et al. 1999) y
la capacidad de desarrollar raíces profundas
que llegan a capas de suelo más húmedas que
las superficiales (Canadell y Zedler 1995).
Las plántulas son mucho más sensibles a la
deshidratación que las semillas o los
individuos juveniles, motivo por el cual los
procesos de reclutamiento y crecimiento se
ven afectados por la variabilidad climática, en
especial por la pluviometría (Figura 2). Años
especialmente húmedos constituyen una
ventana de oportunidad para el
establecimiento de las plántulas (Lázaro et al.
2001, Pugnaire et al. 2006), mientras que en
periodos secos la supervivencia es casi
inexistente (Kitzberger et al. 2000).
Estos procesos pueden agravarse aún más
en futuros escenarios climáticos (Lloret et al.
2005), que en particular predicen para el oeste
de la Cuenca Mediterránea una tendencia
hacia el aumento de la aridez y de los
periodos de sequía, principalmente debido a
cambios en la cantidad, frecuencia e
intensidad de las precipitaciones (IPCC 2001,
Figura 2. Variabilidad interanual de la precipitación en Tabernas. La línea de puntos muestra la precipitación media para el periodo 1940-2000.
Establecimiento de plántulas
22
Sánchez-Rodrigo 2002). De este modo,
eventos de lluvia de mayor volumen pero de
menor frecuencia, intercalados con largos
periodos de sequía, pueden afectar de manera
negativa especialmente a los ecosistemas en
los que la baja disponibilidad de agua limita la
actividad biológica (Knapp et al. 2002), y
pueden alterar el nicho de regeneración de las
especies (sensu Grubb 1977). Por tanto,
comprender la respuesta de las plántulas
durante las primeras etapas de desarrollo ante
cambios en la disponibilidad de agua del suelo
es importante en el marco de las condiciones
climáticas de la Cuenca Mediterránea y de los
escenarios de cambio climático, pues el éxito
de establecimiento dependerá en gran medida
de su habilidad para hacer frente a la sequía.
El regimen de precipitciones en ambientes
áridos es muy variable tanto en cuantía como
en la distribución temporal, y la recarga de las
capas del suelo responde a eventos discretos
de lluvia intercalados con largos periodos de
sequía (i.e., pulsos, Noy-Meir 1985). La
vegetación en estos ambientes no sólo
responde a cambios en el volumen de las
precipitaciones, sino también a las variaciones
temporales, de manera que pequeños cambios
en la frecuencia de las lluvias tienen
importantes efectos en la germinación,
supervivencia y crecimiento de las plantas
(Sala y Lauenroth 1982, Lázaro et al. 2001,
Reynolds et al. 2004). Este hecho es
especialmente frecuente en los ecosistemas
semiáridos del sureste de la Península Ibérica,
donde la variabilidad de las precipitaciones
influye en los procesos de germinación y
establecimiento (Pugnaire y Lázaro 2000,
Lázaro 2004, Pugnaire et al. 2006). En los
últimos años ha renacido el interés por la
respuesta de las plantas a pulsos de agua
(Novoplansky and Goldberg 2001, Sher et al.
2004, Heisler and Weltzin 2006, Maestre y
Reynolds 2007), pero hasta la fecha ningún
estudio ha analizado en particular cuáles son
sus efectos en especies arbustivas
mediterráneas. Muy poco se sabe sobre cómo
la variabilidad temporal de la humedad del
suelo afecta al crecimiento de las plántulas, y
asimismo se desconoce la respuesta de las
distintas especies. El estudio de estos efectos
no sólo es importante para una mejor
comprensión de las respuestas de las especies
de matorral mediterráneo del SE de la
Península, sino que esta información también
es valiosa dada la fuerte variabilidad climática
y las predicciones de cambio climático.
Los procesos de colonización y sucesión
secundaria que experimentan las zonas
degradadas son muy lentos y a menudo no
culminan dentro del periodo de vida de un ser
humano (Pugnaire et al. 2006). En este
sentido, el carácter impredecible del clima
Mediterráneo conlleva una baja frecuencia de
eventos positivos para el establecimiento
(Lázaro et al. 2001), y el ritmo de
colonización además se puede ver alterado por
la escasez de propágulos (Foster et al. 2004) y
las limitaciones en la dispersión y
Introducción
23
germinación de semillas (García-Fayos y
Verdú 1998). La restauración de los
ecosistemas puede acelerar estos procesos de
sucesión. Al igual que los procesos de
reclutamiento, las plántulas plantadas en las
restauraciones realizadas en ambientes
semiáridos también están expuestas durante
los primeros años a condiciones limitantes de
herbivoría, radiación y humedad del suelo
durante periodos más o menos prolongados.
Especialmente la falta acusada de
precipitaciones (inferiores a 300 mm año-1 en
gran parte del SE peninsular, Figura 3) y su
extrema irregularidad tanto estacional como
interanual (Lázaro et al. 2001) propician que
los eventos favorables para el éxito de las
restauraciones sean escasos y muy espaciados
en el tiempo (F.M. Padilla, sin publicar),
poniendo en peligro estos proyectos.
Diversos procedimientos se han propuesto
para aumentar el éxito de establecimiento de
las plántulas Las estrategias se han orientado
bien a mejorar la calidad del sitio de
plantación (Querejeta et al. 2000) y la calidad
de la planta introducida (Villar 2003), o bien a
atenuar las condiciones adversas que limitan
la supervivencia (Rey-Benayas 1998). Entre
estos últimos, destaca por su potencial
ecológico la reciente aplicación de la
facilitación en restauración a través del uso de
plantas nodriza (Maestre et al. 2001, Gómez-
Aparicio et al. 2004).
Así, el área bajo la cubierta de ciertas
especies adultas se ha mostrado como un
lugar idóneo para plantar los brinzales a
restaurar, pues las plantas se pueden
beneficiar del suavizado de las condiciones
microclimáticas, de una mayor disponibilidad
de recursos en el suelo y de la protección
ofrecida por el ramaje frente a los herbívoros.
Esto se traduce en una mayor tasa de
supervivencia en comparación con zonas en
claro desprovistas de vegetación. La
facilitación puede tener un gran potencial para
la restauración de ecosistemas, sin embargo
son necesarios experimentos de larga duración
que contrasten su éxito frente a otras técnicas
de restauración.
Figura 3. Distribución geográfica de la precipitación anual en la provincia de Almería (modificado de Lázaro y Rey 1991). Exceptuando las zonas montañosas, las lluvias apenas alcanzan los 300 mm año-1 en gran parte del territorio.
Establecimiento de plántulas
24
Objetivos
Los objetivos generales de esta tesis son
contribuir a un mejor conocimiento de a)
cuáles son las respuestas de distintas especies
leñosas mediterráneas ante condiciones de
menor disponibilidad hídrica durante la fase
de plántula, y b) cómo la sequía y la
protección por plantas nodriza afectan al
establecimiento de las plántulas en medios
mediterráneos semiáridos. Para conseguir
estos objetivos se realizaron experimentos de
invernadero y de campo de corta y larga
duración con especies mediterráneas arbóreas
y arbustivas que aparecen en el extremo
semiárido del sureste de la Península Ibérica,
así como una extensa revisión bibliográfica.
El primer objetivo específico de esta tesis
fue estudiar si existía una relación entre el
éxito de establecimiento de plántulas y la
capacidad de desarrollar raíces profundas.
Este objetivo se aborda en el Capítulo I y para
su consecución se realizó un experimento de
campo en una parcela semi-natural en el que
se siguió la tasa de supervivencia, la humedad
del suelo a distintas profundidades y la
profundidad de enraizamiento de plántulas de
cinco especies leñosas (Ephedra fragilis, Olea
europaea var. sylvestris, Pinus halepensis,
Retama sphaerocarpa y Salsola oppositifolia)
mensualmente desde el comienzo de la
estación de crecimiento hasta el final del
periodo de sequía. La hipótesis de partida fue
que las plántulas capaces de desarrollar raíces
profundas de manera temprana alcanzarían
tasas de establecimiento superiores a aquellas
con raíces superficiales debido al acceso a
capas de suelo más húmedas,
independientemente de la resistencia a la
sequía de los adultos.
El segundo objetivo fue estudiar la
plasticidad del crecimiento de las raíces en
respuesta a una reducción de la cantidad de
agua disponible en el suelo, centrándonos en
las primeras etapas del desarrollo de las
plántulas. Éste se aborda en el Capítulo II, y
para ello se realizó un experimento de
invernadero con plántulas muy jóvenes de tres
especies arbustivas (Genista umbellata,
Lycium intricatum y Retama sphaerocarpa)
en urnas de cristal traslúcido que permitieron
la observación directa de las raíces. La
hipótesis de partida fue que una reducción de
la cantidad de agua suministrada induciría
cambios en el crecimiento de las raíces de las
tres especies, concretamente aceleraría la tasa
de elongación e induciría una mayor
aportación de biomasa a las raíces. Sin
embargo, la respuesta sería más fuerte en la
especie menos tolerante a la sequía (Retama),
como medio para superar las limitaciones
hídricas, y en aquellas con semillas grandes,
debido a que las mayores reservas en los
cotiledones permitirían a las plantas crecer
bajo condiciones más desfavorables.
En el Capítulo III se estudió el efecto que
la variación en la cantidad y frecuencia en el
Introducción
25
suministro de agua tenían sobre el crecimiento
y varios atributos funcionales de siete especies
arbustivas. Se redujo la cantidad y la
frecuencia de riego durante 14 meses,
esperando que una serie de pequeños eventos
de riego no fueran equivalentes a la misma
cantidad de agua aplicada en eventos de
mayor cuantía pero más espaciados en el
tiempo. El experimento se realizó en macetas
en invernadero, y con especies de matorral
semiárido (Anthyllis cytisoides, Atriplex
halimus, Ephedra fragilis, Genista umbellata,
Lycium intricatum, Retama sphaerocarpa y
Salsola oppositifolia), que se podían clasificar
atendiendo a tres grupos funcionales distintos
establecidos según el hábito foliar (deciduas
de verano, siempre-verdes y con tallos
fotosintéticos) y la tolerancia a la sequía
(tolerantes vs. evitadoras). Se evaluó la
respuesta en términos de crecimiento y ajuste
de rasgos foliares y radicales relacionados con
la captación de la luz y el agua, medidos como
subrogados de la respuesta fisiológica de las
plantas. Se esperó que las respuestas fueran
distintas entre los grupos funcionales, siendo
las especies deciduas de verano y las
evitadoras las más perjudicadas debido a los
costes fisiológicos asociados a la caída de las
hojas y la incapacidad de tolerar la sequía.
Los capítulos IV y V abordan la aplicación
práctica del efecto nodriza y la facilitación en
la restauración de ecosistemas degradados. En
el Capítulo IV se realizó una extensa revisión
bibliográfica de los experimentos en los
cuales se plantaron brinzales de especies de
interés forestal debajo de la cubierta de la
vegetación existente que actuaba como
plantas nodriza, y en zonas en claros
desprovistas de la influencia de vegetación
leñosa. Fruto de los resultados obtenidos en
los distintos experimentos y del conocimiento
ecológico actual sobre las interacciones entre
plantas, se resalta el papel de esta técnica
reciente y se aportan consideraciones para la
gestión que pueden influir en su éxito o
fracaso.
En el Capítulo V se evalúa el papel
facilitador del arbusto leguminoso Retama
sphaerocarpa como planta nodriza. Se realizó
una plantación experimental en dos parcelas
con distinta orientación (umbría y solana), y
durante tres años se siguió la tasa de
supervivencia de tres especies arbustivas
(Olea europaea var. sylvestris, Pistacia
lentiscus y Ziziphus lotus) plantadas bajo la
cubierta de Retama y en claros cubiertos con
ramas secas de matorral. De esta manera se
pretendió contrastar el éxito de las plantas
nodriza frente al empleo de estructuras
artificiales de sombra que imitaban la
protección proporcionada por éstas.
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Establecimiento de plántulas
30
En los ambientes semiáridos de la provincia de Almería la sequía a menudo impide el establecimiento de las plántulas. En esta tesis estudio qué estrategias muestran las plantas para superar el estrés hídrico durante la fase de plántula y cómo la sequía y la protección por plantas nodriza afectan a la supervivencia.
Síntesis
Síntesis
33
FACTORES LIMITANTES Y ESTRATEGIAS DE ESTABLECIMIENTO DE
PLANTAS LEÑOSAS EN AMBIENTES SEMIÁRIDOS. IMPLICACIONES PARA
LA RESTAURACIÓN
Síntesis de resultados
A continuación se recogen los pricipales resultados obtenidos en esta tesis, así como las
publicaciones que de ella se han derivado hasta la fecha.
La habilidad de las plántulas de desarrollar raíces profundas fue decisiva para sobrevivir la
sequía estival, independientemente de la tolerancia a la sequía mostrada por las especies. Las
plántulas tanto de un especie tolerante a la sequía, como Salsola oppositifolia, como de una más
sensible, como Retama sphaerocarpa, desarrollaron raíces profundas durante los primeros meses
de crecimiento en campo, tuvieron acceso a capas más húmedas de suelo, y mostraron tasas de
supervivencia tras el verano muy elevadas. En cambio, la capacidad de profundizar de especies
como Ephedra fragilis, una especie muy tolerante, y Pinus halepensis, considerada más sensible a
la sequía, fue mucho menor, y murieron conforme las capas del suelo superficiales se secaron al
avanzar el verano. Una mayor asignación de biomasa al sistema radical con respecto a la parte
aérea no estuvo correlacionada con un mayor éxito de establecimiento en condiciones extremas de
sequía (Padilla y Pugnaire 2007).
En las primeras semanas de desarrollo, las plántulas de Genista umbellata, Lycium intricatum y
Retama sphaerocarpa respondieron a una disminución de la humedad del suelo aumentando la tasa
de elongación de las raíces, mientras que no se produjeron cambios en la inversión de biomasa al
sistema radical en detrimento de la parte aérea (Padilla et al. 2007). A pesar de las diferencias en el
tamaño de semilla y la estrategia de resistencia a la sequía, las tres especies, tanto tolerantes con
semillas pequeñas (Genista y Lycium) como la evitadora de semilla grande (Retama), respondieron
de la misma manera, aunque Genista mostró la respuesta más plástica. Retama, en cambio, exhibió
una plasticidad menor debido presumiblemente a una mayor dependencia de las reservas de los
cotiledones.
Los cambios en el suministro de agua tanto en cantidad como en frecuencia redujeron la
humedad del suelo y alteraron la dinámica desecación. Las siete especies arbustivas estudiadas
(Anthyllis cytisoides, Atriplex halimus, Ephedra fragilis, Genista umbellata, Lycium intricatum,
Establecimiento de plántulas
34
Retama sphaerocarpa, Salsola oppositifolia) respondieron a estos cambios aumentando la
asignación de biomasa al sistema radical y alterando el diámetro de las raíces. Sin embargo, pulsos
de agua de distinta cantidad y temporalidad no afectaron a la tasa de crecimiento de ninguno de los
siete arbustos seleccionados, así como tampoco se detectaron ajustes en rasgos funcionales como el
área foliar y el área específica de hoja.
En ambientes severos las plantas nodrizas suavizan las condiciones y mejoran la disponibilidad
de recursos, suministrando hábitats más adecuados para las plántulas. Su utilidad en restauración ha
sido corroborada en áreas de alta montaña, estepas semiáridas, salinas costeras, bosques tropicales,
matorrales áridos y sabanas. Sin embargo, hay ciertos aspectos relacionados con las características
de las plantas nodriza, la autoecología de las especies que se quieren implantar y la severidad del
sitio a restaurar que influyen en el éxito de este procedimiento (Padilla y Pugnaire 2006).
La efectividad del arbusto Retama sphaerocarpa como planta nodriza difirió dependiendo de las
especies y de la disponibilidad de recursos. Tras tres estaciones de crecimiento, la supervivencia de
Olea europaea bajo Retama fue el doble que bajo la protección artificial creada por ramas secas.
Los mecanismos de facilitación subyacentes están asociados a la mejora de los recursos del suelo
(efectos del suelo) y de las condiciones microclimáticas (efectos de cubierta). En cambio, Ziziphus
Lotus se vio notablemente perjudicado al vivir cerca de Retama y apenas sobrevivió en este
microambiente debido a su débil capacidad competidora. La facilitación por Retama fue más
aparente bajo condiciones de sequía, esto es, en los años más secos y cuando las plántulas no
recibieron riegos en verano, aumentando la competencia y disminuyendo la facilitación conforme
el estrés hídrico fue atenuado.
Referencias
Padilla, F.M. y Pugnaire, F.I. 2006. The role of nurse plants in the restoration of degraded
environments. Frontiers in Ecology and the Environment 4, 196-202.
Padilla, F.M. y Pugnaire, F.I. 2007. Rooting depth and soil moisture control Mediterranean woody
seedling survival during drought. Functional Ecology 21, 489-495.
Padilla, F.M., Miranda, J. y Pugnaire, F.I. 2007. Early root growth plasticity in seedlings of three
Mediterranean woody species. Plant and Soil 296, 103-113.
Capítulo I
La profundidad de enraizamiento y la humedad del suelo controlan la supervivencia de plántulas de especies leñosas durante la sequía†
† Publicado como “Padilla F.M. and Pugnaire F.I. 2007. Rooting depth and soil moisture control Mediterranean woody seedling survival during drought. Functional Ecology 21: 489-495”
Capítulo I
37
Capítulo I
ROOTING DEPTH AND SOIL MOISTURE CONTROL MEDITERRANEAN
WOODY SEEDLING SURVIVAL DURING DROUGHT
Summary
Seedling survival is one of the most critical stages in a plant’s life history and is often lowered
by drought and soil desiccation. It has been hypothesized that root systems accessing moist soil
layers are critical for establishment but very little is known about seedling root growth and traits in
the field. We related seedling mortality to the presence of deep roots in a field experiment in which
we monitored soil moisture, root growth, and seedling survival in five Mediterranean woody
species from the beginning of the growing season until the end of the drought season. We found
strong positive relationships between survival and maximum rooting depth, as well as between
survival and soil moisture. Species with roots in moist soil layers withstood prolonged drought
better, whereas species with shallow roots died more frequently. In contrast, biomass allocation to
roots was not related to establishment success. Access to moist soil horizons accounted for species-
specific survival rates, whereas large root-to-shoot mass (R:S) ratio did not. The existence of soil
moisture thresholds that control establishment provides insights into plant population dynamics in
dry environments.
Introduction
Seedling recruitment is a critical stage of
plant life history because high mortality rates
are often associated with the seedling phase
(Fenner 1987). Seedlings of different species
die from a wide variety of causes (Fenner &
Kitajima 1999; Moles & Westoby 2004),
including many biotic and abiotic factors such
as pathogens, herbivory, high or low
temperatures and radiation, allelopathy and
competition. Drought and soil desiccation are
primary limits to establishment in many
environments (Moles & Westoby 2004). One
such environment is Mediterranean-type
ecosystems, where establishment after
germination is severely limited by long, dry
summer periods (Herrera 1992). In such areas,
seedlings are very drought-sensitive, and
recruitment processes are often restricted to
sporadic rainfall periods (Holmgren &
Scheffer 2001; Pugnaire et al. 2006b) or wet
microsites (Padilla & Pugnaire 2006).
Root depth and seedling survival
38
Deep roots may improve water uptake and
increase the probability of survival in
Mediterranean communities since they can
access stable water reserves (Donovan,
Mausberg & Ehleringer 1993; Canadell &
Zedler 1995; Lloret, Casanovas & Peñuelas
1999), allowing plant growth extended into
the dry season (Nepstad et al. 1994).
However, differences in seedling survival
during drought is often due to varying
tolerance to low soil moisture (Hasting,
Oechel & Sionit 1989; Ackerly 2004). For
instance, Davis (1989) found in the California
chaparral that seedlings of drought-tolerant
species, often shallow-rooted, survived water
shortage better than seedlings of drought-
sensitive species.
Although the role of deep roots in plant
establishment has long been acknowledged
(Davis 1989; Enright & Lamont 1992;
Canadell & Zedler 1995; Pugnaire, Chapin &
Hardig 2006a), establishment failure due to a
lack of deep roots has rarely been quantified.
Very little is known about root growth in the
field as related to soil water, and many of the
mechanisms controlling establishment success
remain poorly understood (Hanley et al. 2004,
but see Lloret, Casanovas & Peñuelas 1999).
This question is particularly significant in dry
environments and under global change
scenarios with longer drought spells (IPCC
2001). Drier conditions may alter the
regeneration niche of many species (sensu
Grubb 1977) and species richness may be
limited if seedlings are unable to deal with
lower water availability (Brown, Valone &
Curtin 1997, Schenk & Jackson 2002).
Here we address how drought affects the
establishment of five woody species and relate
establishment to rooting depth and access to
soil moisture in a field experiment. We
hypothesized that deep-rooted seedlings
would attain higher survival after summer
than shallow-rooted individuals by keeping
roots in moister soil horizons.
Materials and Methods
Species
We selected five native perennial woody
species occurring in Mediterranean shrublands
in semi-arid southeast Spain. Two of the
species were nearly leafless shrubs with
photosynthetic stems, including a
gymnosperm, Ephedra fragilis Desf., and a
legume, Retama sphaerocarpa (L.) Boiss. The
other species included a succulent C4, Salsola
oppositifolia Desf., a large C3 shrub, Olea
europaea var. sylvestris Brot., and a tree
species, Pinus halepensis Mill. Hereafter we
refer to these species by their generic names
only. Ephedra, Olea and Pinus are frequent in
late-successional communities, whereas
Retama and Salsola successfully colonize
disturbed areas (Peinado, Alcaraz &
MartínezParras 1992). Our selected species
Capítulo I
39
differ in drought tolerance strategy based on
minimum pre-dawn water potential (Ψpd).
Salsola tolerates low water potentials, (Ψpd ≈ -
5 MPa, Pugnaire, Armas & Valladares 2004),
as does Ephedra (Ψpd ≈ -5.2 MPa, F.I.
Pugnaire, unpublished data). Our other
species could be considered as non-drought-
tolerant based on less negative Ψpd; around -
1.5 MPa for the deep-rooted Retama (Haase et
al. 1999), -2.5 MPa for Pinus (Oliet et al.
2002), and -2.25 MPa for Olea (Faria et al.
1998). Germination of Olea, Pinus and
Retama under Mediterranean conditions
begins in winter (Rey & Alcántara 2000;
Nathan & Ne’eman 2004; Pugnaire et al.
2006b). There are no accurate data for
germination patterns of Ephedra and Salsola,
but specific traits suggest that they germinate
in winter too, because seeds disperse in late
autumn or early winter (Rodríguez-Pérez,
Riera & Traveset 2005) and seeds do not
show dormancy (Navarro & Gálvez 2001).
Field site and experimental design
We tested our hypothesis by conducting a
transplant experiment in a semi-natural field
site rather than by monitoring seedling
occurrence in nature. This minimized
environmental heterogeneity and root losses at
harvest, allowed roots to grow without soil
impediments, and also allowed comparisons
of potential root growth under the same set of
abiotic conditions. The experiment was set up
in flat and homogeneous 15 x 15 m terrace for
vegetable crops in the foothills of the Sierra
Alhamilla range (Almería, Spain, 37º99’N,
02º99’W, 600 m elevation). The silt soil had
been ploughed regularly for years, was free of
rocks, and reached ca. 2 m in depth over a
mica-schist bedrock. Fertility and water
holding capacity were very low (Pérez-Pujalte
1989). Neither pesticides nor fertilizers were
applied at the site for at least five years. The
climate is typically Mediterranean semi-arid
with a mean annual temperature of 17.3 ºC
and mean annual precipitation of 282 mm and
a marked drought period from May to
September. Temperatures are mild in winter
and high in late spring and summer.
In late winter 2004, eight 3 x 3 m plots
spaced 1.5 m apart were laid out on the terrace
in a 3 x 3 design. To homogenize soil and
facilitate root growth, the soil in each plot was
completely dug up to a depth of 0.5 m, using
an auger (BT 120 C, Stihl AG & Co. KG,
Waiblingen, Germany) to drill adjacent 30
cm-wide holes. Seedlings of 1-2 months were
transplanted in early April, after heavy spring
rainfalls. Seedlings were provided by local
nurseries and seeds had been collected in
areas with similar ecological conditions. Care
was taken to follow the natural recruitment
dynamics of all species, and transplanting was
done when seedlings of all species had
already emerged in the field.
In each plot, ten bare-root seedlings of
each species, similar in size and with intact
Root depth and seedling survival
40
root systems, were planted ca. 35 cm apart
from each other and from the plot borders.
The spatial arrangement of different species in
each plot was fully random. The terrace was
fenced to prevent herbivory, and each plot
was watered once with 5 L (≈ 0.1 L of
water/plant) immediately after transplanting.
One plot was harvested (H, hereafter) every
three weeks on average between April and
September, encompassing the spring growing
period and the summer drought. Initial data
(referred to as H0) consisted of ten randomly
harvested seedlings of every species before
transplanting. The remaining harvests (H1 to
H8) were done 13, 28, 48, 66, 81, 97, 121, and
153 days after transplanting. On each harvest
all living individuals in a randomly chosen
plot were dug out carefully, and maximum
root depth was recorded. Root recovery was
maximized by digging a 3-m-long trench
around the periphery of the plot. Initially the
trench was ca. 40 cm wide and 30 cm deep,
but the depth of the trench increased in
successive harvests until reaching ca. 100 cm
on the last sampling date. The front of the
trench was gently crumbled from side to side
with a hoe, which was then replaced by a
small punch when close to the base of the
plant. Roots were carefully brushed and then
manually extracted and stored in paper bags.
Soil containing roots that could not be
separated in the field were processed in the
laboratory. Roots could be matched with
individuals because all species had one or
several major tap roots, grew vertically, and
none spread horizontally. Fine roots attached
to major tap roots were collected as well. In
the laboratory roots and soil were repeatedly
submerged in water and finely sieved to retain
fine roots. Shoots and roots were dried at 71
ºC for at least 48 hours.
Survival on each harvest date was
calculated as the proportion of plants alive
after the first week, excluding this way
seedling deaths caused by transplant. Soil
moisture (ECH2O, Decagon Devices Inc.,
Pullman, WA, USA) and temperature (Onset
Computers, Pocasset, MA, USA) at depths of
5, 15, 30, 45, and 60 cm were continuously
monitored during the experiment in the last
plot harvested. Readings were taken every ten
minutes and averaged daily. Soil water
content at any given depth within an interval
was determined through interpolation between
neighboring readings, assuming that water
content in the interval changed linearly.
Similarly, we inferred soil depth
corresponding to a particular moisture content
by interpolating from readings of probes
immediately above and below that depth.
Growth analysis and statistics
Mean relative growth rate (RGR) for each
species during the monitoring period was
calculated from observed values between H8
and H0 (Hunt et al. 2002). Growth curves
were analyzed using ANCOVA on log-
transformed observed values with number of
Capítulo I
41
days after transplanting as a covariate.
Differences among species were considered
significant when the species x time interaction
resulted significant. Relative root extension
rate (RER) between two consecutive harvests
was obtained for each species from fitted
polynomial curves (HPcurves v.3.0, A. Pooley
et al.). Differences in biomass and maximum
rooting depth among species at H8 were tested
using one-way ANOVA followed by Scheffé
post-hoc comparison tests. Heteroscedastic
variables were log-transformed to meet
ANOVA assumptions. Differences in seedling
survival among species in September were
compared through simple binary logistic
regression where survival was the dependent
variable and species the predictor factor
(Agresti 2002). Regression analyses were
performed to test correlation strength between
variables, using adjusted R2 to correct for the
degrees of freedom. All analyses were
conducted with SPSS v13.0 (SPSS Inc.,
Chicago, IL, USA) and differences were
significant at P < 0.05. Sample size in all
analyses was 4-10 for each species, with the
exception of Pinus at H8, when only two
plants remained alive. Data are presented as
means ± one standard error.
Results
A rainy spring (205 vs. 101 mm average in
the 1967-1997 period, Confederación
Hidrográfica del Sur) was followed by a
summer without rainfall (Figure 1).
Survival in spring (April to June) was
100% for all species except Ephedra; in this
case seedling survival was 87.5% by mid-
June, and 60% in September. In contrast,
Retama and Salsola had complete survival
throughout the season. Olea survival at the
end of the drought period was 80% and Pinus
20% (Figure 2). Moisture decreased quickly
in top soil layers as the drought period
progressed, reaching values in September of
1.5% and 11.5% at 5 and 15 cm in depth
respectively; soil moisture remained at ~21%
from mid-June onwards at 45 and 60 cm.
Thus, soil moisture in September showed a
strong gradient, increasing with depth (Figure
2).
Species differed significantly in the
maximum depth reached by roots in
September (one-way ANOVA F4,23 = 11.7,
P<0.001, Table 1). The two early colonizers,
Salsola and Retama, rooted deepest and also
had the highest mean root extension rates
(RERmean, Table 1). In contrast, the shallowest
Figure. 1. Daily rainfall between April and September of 2004 in the experimental site.
Root depth and seedling survival
42
roots were found in Ephedra and Pinus, which
also had the lowest mean root extension rates.
In September, at least one tap root of Olea,
Retama and Salsola reached well below 35
cm, whereas roots of Ephedra and Pinus did
not surpass 25 cm (Figure 2). While the root:
shoot (R:S) ratio in all species was below 0.5,
it varied significantly among species (one-
way ANOVA F4,23 = 29.3, P<0.001). Pinus
allocated the most to roots, followed by Olea,
Retama and Ephedra. By contrast, allocation
to roots relative to shoots was rather small in
Salsola (R:S < 0.1). Root: shoot ratio did not
increase in response to increasing drought but,
on the contrary, decreased over the course of
the season in Olea, Pinus and Salsola, and
remained relatively constant in Ephedra and
Salsola (Figure 3).
Figure. 2. Maximum rooting depth (solid bars) and isoclines of soil moisture (shaded areas) on left Y-axis and seedling survival (white dots) on secondary right Y-axis. Soil layers with moisture above 20% are represented by dark grey; in grey area moisture ranged 14-20%; pale grey area indicate 8-14%, and white area below 8%. Values of rooting depth are means ± 1SE.
Table 1. Final plant mass, mean relative growth rate (RGR) and root extension rate (RER), maximum root depth and root: shoot (R:S) ratio of five woody species.
Species Ephedra Olea Pinus Retama Salsola F4,23
Total mass (g) 0.28±0.06ac 1.57±0.33b 0.15±0.02c 1.22±0.29ab 75.01±17.01d 67.96***
RGR (mg g-1 day-1) 17.1±3.4 13.7±0.5 4.4±1.9 25.5±7.8 48.0±5.1 -
RER (mm cm-1 day.1) 5.3±3.3 5±1.3 1.9±1.2 9.3±3.8 10.8±2.5 -
Rooting depth (cm) 20.6±3.1a 35.2±2.3ab 15.9±2.7a 47.3±6.9b 59.5±6.7b 11.65***
R:S ratio 0.22±0.03a 0.43±0.02a 0.49±0.07a 0.31±0.04a 0.08±0.01b 29.32***
Significant differences among species are indicated by F values: ***, P<0.001. Different letters in a row show differences at P<0.05 (one-way ANOVA, Scheffé’s test). Values are means ± 1SE.
Capítulo I
43
At the end of the drought season there
were significant differences in seedling
establishment among species (logistic
regression χ2 = 24.2, df = 4, P<0.001). There
were strong positive relationships between
seedling survival and maximum rooting depth
(logistic function, Radj2=0.99, P<0.01, Figure
4a), and between survival and the soil
moisture estimated at the maximum rooting
depth of the species at final harvest (logistic
function, Radj2=0.97, P<0.02, Figure 4b),
showing that the probability of establishment
success was strongly related to increasing
rooting depth and therefore soil moisture.
Species with roots accessing soil deeper than
45 cm had 100% survival (Salsola and
Retama), whereas much lower rates (20-40%)
were found in species that rooted in shallower,
drier soil layers (ca. 22.5 cm, Ephedra and
Pinus). Species rooting in layers with < 12%
soil moisture established poorly (Pinus,
Ephedra). In contrast, species with roots
reaching soil with moisture > 18% (Retama
and Salsola) had complete survival.
There was, however, no relationship
between survival at final harvest and R:S ratio
(linear regression, Radj2=0.12, P>0.38, Figure
4c). Initial plant size did not correlate with
maximum rooting depth at H8 or RGR (linear
regression, P>0.57 and P>0.3, respectively),
or survival at H8 (P>0.3 for all fitted
functions).
Figure 4. Relationships between survival and maximum rooting depth (a), moisture at the deepest soil layer reached by roots (b), and R:S ratios in September (c), after the summer drought. Values are means ± 1SE, with the exception of survival. Ef, Ephedra; Oe, Olea; Ph, Pinus; Rs, Retama; So, Salsola. n.s.= no significant correlation.
Figure 3. Root: shoot (R:S) ratio for each species. Values are means ± 1SE.
Root depth and seedling survival
44
Species also differed in root growth
patterns over spring and summer (ANCOVA
species x time F4,333 = 18.4, P<0.001, Figure
5). Roots of Salsola displayed a parabolic
growth curve characterized by rather low RER
values early in the season, followed by a
period of increasing growth rate until the
onset of the drought season in mid-June, at
which point RER started to decrease. In
contrast, Ephedra, Olea and Retama grew at a
constant rate from April to September.
Ephedra and Olea shared nearly identical
RER, whereas Retama exhibited larger values.
Growth rate of Pinus decreased from the
beginning, showing the highest value in the
first harvest and the lowest in the last one.
Discussion
The effect of summer drought on seedling
establishment has long been acknowledged in
Mediterranean environments (Herrera 1992),
but, to our knowledge, direct links between
rooting depth, soil moisture, and
establishment have never been quantified. The
ability to develop deep roots and access soil
moisture was decisive for seedlings to survive
summer drought, regardless of species-
specific drought tolerance. Deep-rooted
seedlings either from a drought-tolerant
species (based on minimum Ψpd reported)
such as Salsola or a drought-sensitive species
such as Retama had consistent access to moist
soil layers and showed the greatest survival
rates. In contrast, shallow-rooted seedlings of
Ephedra (a drought-tolerant species) and Olea
and Pinus (more drought-sensitive species)
relied on water from shallower soil layers and
died as summer drought progressed.
Climate change scenarios for western
Mediterranean predict reduced annual
precipitation, shifts in seasonal rainfall
patterns (decreasing in spring, summer and
autumn) and extended drought periods (IPCC
2001). Here, we looked at species ability to
Figure 5. (a) Log-transformed maximum rooting depth (mm) at each harvest (symbols) and fitted functions (lines). R2 shows regression coefficient at P < 0.05. (b) Root extension rate (RER, mm cm-1 day-1) for each harvest and species. Symbols and lines of Olea and Ephedra overlap. Symbols are means ± 1 SE. Initial and final RER values not shown for clarity due to widening of confidence limits.
Capítulo I
45
extend their roots fast enough to keep pace
with retreating soil moisture and showed that
deep-rooted seedlings were best able to
establish during a very dry growing season,
suggesting that these species may be favored
over species of shallow-rooted seedling
during extended droughts. Whether shallow-
rooted species would decrease in abundance
or be confined to more mesic patches or
microsites remains unknown (Schenk &
Jackson 2002); however, it is worth noting
that shifts in regional climates are currently
leading to changes in vegetation type
dominance, e.g., the encroachment of shrubs
into American grasslands (Brown, Valone &
Curtin 1997) most likely because new
conditions favor the establishment of deep-
rooted species (Schenk & Jackson 2002).
The relationship between soil moisture and
survival suggests the existence of a threshold
of soil moisture that controls plant
establishment (Figure 6). In our system, very
low establishment rates were achieved by
species that kept roots in shallow soil layers
with moisture around 12% (e.g., Ephedra and
Pinus), and according to our data, no
establishment would occur for plants rooting
in layers drier than ~8% (m0) . On the other
hand, higher establishment rates were found
for deep-rooted species reaching soil layers
wetter than 15% (Retama and Salsola), and
full establishment would be related to rooting
in soil layer moister than ~20% (m100). It is
likely that something similar occurs in natural
systems, where obviously the threshold will
vary depending on soil properties and the
species involved.
Rainfall-dependent recruitment dynamics
reported in dry environments can be
interpreted under such operating thresholds.
Kitzberger, Steinaker & Veblen (2000) and
Holmgren et al. (2006) showed that
recruitment in dry years is almost zero,
whereas rainy years constitute a window of
opportunity for establishment. We suggest
that plants in dry habitats may establish easily
in wet years without deep roots because soil
moisture remains above the critical threshold
along the soil profile (Sala & Lauenroth
1982). Conversely, when moisture in the soil
profile is below the threshold, it is not enough
to maintain seedlings alive. Soil moisture
Figure 6. Proposed control of soil moisture thresholds on seedling establishment. There is a point below which seedling establishment is impeded due to insufficient soil moisture (m0) and another above which full establishment is reached (m100). Conditional establishment occurs between m0-m100 depending on rooting depth and drought tolerance. Dots show survival of our species and solid line fitted logistic function.
Root depth and seedling survival
46
between both extremes would pivot around
the critical threshold, and rooting ability and
drought tolerance of the different species
would explain variation in establishment
patterns.
The lack of rain during our experiment
produced a vertical soil moisture gradient, but
summer rains could have replenished soils and
altered the gradient, most likely changing the
final outcome. Supplying water during
summer drought boosts establishment success
in Mediterranean environments (Castro et al.
2005) and summer irrigation considerably
increases survival in all our study species
(Sánchez et al. 2004). Small rainfalls (like
watering) keep soil moisture above certain
thresholds and improve survivorship (Sala &
Lauenroth 1982).
Plants may adjust to resource imbalance by
allocating biomass to organs that acquire the
limiting resource (Chapin et al. 1987), so that
higher root: shoot ratios are expected under
water stress. However, we found no
relationship between survival and R:S ratio,
and paradoxically the most successful
survivor, Salsola, allocated relatively the least
to roots, and the species with most failure,
Pinus, had the highest R:S ratio. Overall, we
did not find substantial changes in R:S during
the growth period, in contrast to reports that
found shifts in dry mass partitioning between
shoots and roots during plant growth (Klepper
1991). Biomass allocation to roots did not
increase in any species in response to drought,
suggesting that large R:S ratios may not be
enough to compensate for the deepening of
moisture along the soil profile in summer.
Rather, the ability to alter rates, timing and
placement of root proliferation may be more
important for plant success than changes in
biomass allocation between roots and shoots
(Reynolds & D’Antonio 1996). Lloret,
Casanovas & Peñuelas (1999), however,
reported a large positive correlation between
R:S and seedling survival in a Mediterranean
shrubland, but in their field site roots rarely
reached below 10 cm and the high summer
rainfall in the study years kept shallow soil
horizons moist. Under such circumstances
greater biomass allocation to roots may
increase water uptake.
Some species from dry environments have
dual root systems with shallow lateral roots
that exploit small rainfall events which hardly
penetrate into the soil, and deep roots that tap
deep water sources (Canadell & Zedler 1995).
However, dual systems often develop as the
plant matures, and the absence of lateral
branches is frequent in seedlings from xeric
habitats (Canadell et al. 1999; Nicotra,
Babicka & Westoby 2002). Our observations
agree with these patterns, since roots of the
five species grew vertically and none spread
horizontally. This suggests a primary
investment to develop root systems that
penetrate into deeper, more reliable water
sources rather than allocating biomass to
Capítulo I
47
develop both surface and deep roots, because
moisture in the soil surface is unreliable
(Ehleringer & Dawson 1992).
In conclusion, our work underlines the
importance of rooting depth for seedling
survival. In the absence of other constraints on
establishment like dispersal, seed germination
triggers, or herbivory, the ability to reach
deeper, moister soil horizons is critical to cope
with water stress at such early stage and
become established. Our data suggest that
species able to keep roots in moist soil layers
are better prepared to withstand drought. Also,
soil moisture thresholds seem to control plant
survival, so plant establishment may be
restricted if soil moisture is below certain
levels, which has direct implications for
population and community dynamics.
Acknowledgements
We are grateful to Alejandro Moreno, Juan
Padilla, Juan de Dios Miranda, María José
Jorquera, María Pilar Sánchez and Pepe del
Cortijo La Sierra, for helping with the field
work. Serfosur SL, Viveros Retamar and
Junta de Andalucía provided seedlings.
Comments by Ragan M. Callaway, Heather L.
Reynolds, Scott D. Wilson and two
anonymous reviewers greatly improved this
manuscript. Funds were provided by the
Spanish Ministry of Education and Science
(grant CGL2004-00090/CLI).
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Capítulo I
51
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Scanned roots of the five species studied in this experiment. From left to right, Salsola oppositifolia, Retama sphaerocarpa, Olea europaea var. sylvestris, Ephedra fragilis and Pinus halepensis.
Capítulo II
Plasticidad en el crecimiento de raíces en plántulas de tres especies leñosas mediterráneas†
† Publicado como “Padilla, F.M., Miranda, J. and Pugnaire, F.I. 2007. Early root growth plasticity in seedlings of three Mediterranean woody species. Plant and Soil 296: 103-113”
Capítulo II
55
Capítulo II
EARLY ROOT GROWTH PLASTICITY IN SEEDLINGS OF THREE
MEDITERRANEAN WOODY SPECIES
Summary
Since very young seedlings are sensitive to dehydration, soil desiccation is often responsible for
seedling death in water-stressed environments. Roots play a major role in overcoming water stress
and plant establishment, thus early root development in response to limited water availability
becomes a strategy that may ensure recruitment. We explored whether different water availabilities
altered growth patterns of very young seedlings, focussing on root elongation, and hypothesized
that seedling responses would depend on species-specific drought-tolerance and seed size. We
carried out a greenhouse experiment exposing two-week-old seedlings of three Mediterranean
shrubland species, the drought-tolerant and small-seeded Genista umbellata and Lycium intricatum,
and the drought-sensitive, large-seeded Retama sphaerocarpa to two watering quantities and
monitored plant and root growth weekly in glass cases. We found that at such early stages, reduced
water quantity enhanced root growth in all three species, regardless of drought tolerance and seed
size, although root plasticity was the highest in the small-seeded and drought-tolerant Genista. In
contrast, shoot elongation and mass allocation, root-to-shoot mass (R:S) ratio, were unaffected by
watering. Seedlings responded to lower water availability with faster root elongation and greater
absorptive root surface, which can account for the enhanced relative growth rate (RGR) of the
small-seeded Genista and Lycium under reduced watering. By contrast, a larger root absorptive
surface did not lead to higher RGR in the large-seeded Retama probably because of its greater
independence from external mineral resources. Our data evidence the importance of water
availability on the initial stages of these species regardless of seed size and drought tolerance. Root
growth can be interpreted as an adaptive strategy to deal with drying soils since larger roots enable
to exploit unexplored soil areas of soil, which may ensure recruitment success.
Introduction
Plant communities are shaped by
germination and recruitment processes
(Donovan et al. 1993), which ultimately affect
community composition and structure (Grubb
1977; Harper 1977). Plants do not actively
choose the habitat they grow in (Bazzaz
Drought and root growth
56
1991); rather, habitat choice is first imposed
on plants by seed dispersal, and then by
environmental factors which constrain seed
survival, germination, seedling establishment
and growth (Schupp 1995). After seed
dispersal, germination does give way to the
most critical phase in the regeneration
process, seedling establishment (Fenner and
Kitajima 1999). Very young seedlings are
susceptible to many hazards, such as extreme
temperatures and radiation, competition,
pathogens, herbivory or drought (Moles and
Westoby 2004a), and as a result high
mortality rates are often associated to this
stage (Fenner 1987). An important
determinant of successful seedling recruitment
is the microsite where the seed is placed, often
a safe site providing conditions and resources
required for germination and establishment
(i.e., the regeneration niche sensu Grubb
1977; Fenner 1987). However, seed-seedling
conflicts may arise when environmental
conditions promoting seed germination are
not favourable for seedling survival and
growth (Schupp 1995), e.g., conditions good
enough for triggering germination may not be
as good for seedling growth. Eventually,
seedling’s fate and recruitment success will
depend on seedling’s ability to cope with
limiting environmental conditions.
Because emerged seedlings are much more
sensitive to dehydration than seeds or juvenile
individuals (Evans and Etherington 1991),
drought is often the main cause of seedling
death in many environments (Moles and
Westoby 2004a). This is particularly true in
water-stressed Mediterranean ecosystems,
where a dry, long summer season jeopardizes
recruitment of seedlings emerged in winter
and spring (Herrera 1992). In addition,
seedlings in arid environments are exposed to
highly variable rainfall, both in duration and
amount, being characteristic the presence of
dry periods interspersed between rain events
(Lázaro et al. 2001). Establishment success in
such areas greatly depends on seedling ability
to overcome water shortage (Davis 1989), and
root systems play a major role. Large biomass
allocation to roots is often related to higher
survival rates through improved water and
nutrient uptake (Lloret et al. 1999, Pugnaire et
al. 2006) linked to reaching moister soil layers
and exploring larger soil volumes (Davis
1989; Donovan et al. 1993; Leishman and
Westoby 1994a). Consequently, deep-rooted
seedlings have a probability of surviving
summer drought higher than shallow-rooted
seedlings (Padilla and Pugnaire 2007).
Species-specific drought tolerance, however,
is a main factor for seedling survival in drying
soils (Ackerly 2004), and Davis (1989) and
Hasting et al. (1989) found in the California
chaparral that seedlings of drought-tolerant
species, usually shallow-rooted, survived
water shortage better than seedlings of
drought-avoider species, often deep-rooted,
because of the greater tolerance to low soil
water potentials of tolerant species. Seed size
has also been related to successful recruitment
Capítulo II
57
in dry habitats (Leishman and Westoby
1994a; Moles and Westoby 2004b). Large-
seeded species have storage reserves in
cotyledons that sustain growth during
unfavorable periods, and are more likely to
have large seedlings and longer roots than
small-seeded species (Buckley 1982; Jurado
and Westoby 1992; Fenner and Kitajima
1999), traits shown to be related to a higher
probability of survival by allowing access to
soil moisture at deeper levels (Donovan et al.
1993).
Given the typically unpredictable and
variable rainfall in arid environments and
Mediterranean ecosystems, and the fact that
climate change scenarios forecast for the
western Mediterranean Basin a mean annual
precipitation reduced by ~30% and shifts in
the frequency of rain events, i.e., greater, less
frequent events followed by longer drought
periods (IPCC 2001), understanding seedling
responses to changes in water availability is
important. Here, we explored whether
differences in watering altered growth
patterns of seedlings at the very early stages
of development, with cotyledons still
attached. We carried out an experiment in
mini-rhizotrons, subjecting very young
seedlings of three perennial woody species of
Mediterranean shrubs to reduced watering,
monitoring plant and root growth. We reduced
the amount of water supplied and its
frequency expecting that pulses of water of
different magnitude had different effects on
plants, even if the amount of water provided
was kept constant.
Research has shown that roots grow
towards resource patches (Reader et al. 1993;
Cahill and Casper 1999; Rajaniemi and
Reynolds 2004; Eapen et al. 2005), showing
an elongation response in low moisture
(Evans and Etherington 1991). Furthermore, it
is widely accepted that plants adjust to
resource imbalance by allocating biomass to
organs that acquire the limiting resource
(Chapin et al. 1987). Therefore, we expected
larger biomass allocation to roots relative to
shoots and larger root elongation rates in
response to drought as a means to overcome
water shortage. We hypothesized that 1)
seedling responses would depend on species’
water stress tolerance, so that drought-
sensitive species would show stronger
responses to drought than drought-tolerant
species as a means to overcome their lower
capacity of dealing with low water availability
and, following Leishman and Westoby
(1994a) 2) root growth would be positively
associated to seed size, so that large-seeded
species would show stronger responses to
drought than small-seeded species because
cotyledons allow plant to growth under
unfavorable conditions.
Materials and Methods
Species
Drought and root growth
58
Three perennial woody species occurring
in open Mediterranean semiarid shrublands of
southeast Spain were selected; Genista
umbellata (L’Hér.) Dum. Cours., Lycium
intricatum Boiss., and Retama sphaerocarpa
(L.) Boiss. Hereafter we refer to these species
by their generic names only. Two of the
species were nearly leafless legumes with
photosynthetic stems, the small shrub Genista
and the large shrub Retama, whereas Lycium
was a thorny shrub with drought-deciduous
succulent leaves. Our species differed in
drought-tolerance strategy based on rooting
depth and minimum pre-dawn water potential
(Ψpd) measured in the field during the water
shortage. Retama, a very deep-rooted species
accessing stable water sources through the
year (Haase et al. 1996), may be considered as
drought–avoider given the usually high Ψpd
reported (≈ -1.5 MPa, Haase et al. 1999). The
other two species can be classified more
properly as drought-tolerant. Lycium stands
very low water potentials (≈ -5 MPa, Tirado
2003) and its drought-deciduous habit
evidences shallow rooting depth. There are no
data available for Genista umbellata, a
shallow-rooted species (< 0.75 m, pers. obs.),
but a closely related species, G. hirsuta,
showed high tolerance to Mediterranean water
stress, reaching Ψpd under -6 MPa (Lansac et
al. 1994). Species also differed in seed mass.
Genista and Lycium are relatively small-
seeded species, whereas Retama is a larger-
seeded species with very heavy seed coat (up
to 35 mg, Table 1).
Table 1. Initial plant size (mg). Values are means ± 1SE. n=6 for each species, except seed mass (10).
Genista Lycium Retama
Seed mass 4.61±0.36 3.46±0.21 110.75±5.02
Shoot mass 3.17±0.75 4.40±0.78 24.43±1.85
Cotyledons mass 2.18±0.98 2.93±0.47 21.65±1.58
Root mass 1.53±0.35 1.58±0.27 4.32±1.09
R:S ratio 0.50±0.05 0.37±0.03 0.18±0.05
Experimental design
Freshly collected seeds of the three species
were sown separately in germination trays
containing type III vermiculite (Verlite®,
Vermiculita y Derivados SL, Gijón, Spain) in
laboratory at room temperature and light on
22 March 2005. Seeds were collected in the
field or provided by local nurseries. All seeds
germinated within two weeks, and very young
seedlings were carefully transferred to glass
cases on 13 April 2005, once that cotyledons
Capítulo II
59
had fully emerged from seed coats. Six
randomly selected seedlings of every species
were harvested before transplanting (Table 1).
Four transparent glass cases, 129 cm length,
43 cm depth, 3 cm width set at a 30º angle
from the vertical, were filled up with
vermiculite and placed in the greenhouse
(Figure 1). Because of the narrow design of
the cases, we selected vermiculite because of
its lower compaction and greater oxygenation
than other growing media. The case bottom
was perforated to allow for water drainage. At
transplant, individuals of each species were
placed completely at random 8 cm apart from
each other and near the lower side of each
case. Given the small seedling size, the lack of
lateral roots and the short monitoring period,
this distance seemed enough to prevent
competition. The lower side of the glass case
was covered by a black canvas so that roots
grew in darkness on this side and root growth
could be monitored through the glass. The
other side was left uncovered. Each individual
was watered with 40 ml every three days
during the first week following transplant.
After acclimation, on 19 April 2005, seedlings
were allocated to treatments following a
factorial design with two factors and two
levels each. Watering quantity included a
control (20 ml every time) and a watering of
30% less than the control (14 ml). A second
factor included frequency of watering, and
comprised a ‘normal’ level (two waterings per
week) and half the number of events (one per
week). Each of the four combinations
comprised five replicates per species. All
waterings were done with a syringe to prevent
flooding. Seedlings grew in a greenhouse
sheltered from direct radiation for five weeks
without fertilization and cases position was
rearranged weekly. The mean daily
temperature in the sheltered area was 18.9 ±
0.3 ºC, and the mean maximum and minimum
were 23.9 ± 0.4 ºC and 13.7 ± 0.3,
respectively.
Measurements
Shoot height and root length of each plant
were measured weekly during the
manipulation period. Shoot height was
measured with a calliper and new root
segments and trajectories were drawn on the
glass surface using different color markers. At
the end of the experiment, root length marks
on the glass were traced to acetate sheets and
digitalized with a portable scanner (Epson
GT7000, Seiko Epson Corp., Nagano, Japan)
at 300 dpi. Root length was measured from
digitalized traces using the macro
RootMeasure v.1.80 (Kimura and Yamasaki,
2003) implemented on the software Scion
Image Beta v. 4.02 (Scion Corp., Maryland,
USA). We calculated mean root and shoot
elongation rates for each plant between the
initial and final lengths. Growth curves were
obtained by plotting cumulative root length
data against time. Maximum rooting depth
was recorded before harvesting. At harvest, on
24 May 2005, shoots of each species were
Drought and root growth
60
clipped at surface level, stored in paper bags,
dried at 71ºC for at least 48 hours in a
ventilated oven and weighed. Glass cases
were then emptied out gently so as not to
damage root systems and vermiculite particles
attached to root hairs were removed by gently
washing and brushing them out. Roots were
then labelled, placed into wet paper towels
and kept cool in zip bags in a refrigerator until
they were scanned. Root length and root area
of each plant were digitalized and measured
following the procedure described above for
traced roots. Root biomass was obtained after
drying samples as with shoots, and root-to-
shoot mass (R:S) ratio for each plant was
calculated from these data. Specific root
length (SRL, cm g-1) on the entire root system
was computed from total root length and
mass.
Figure 1. Experimental glass cases and size (in cm). New root segments were traced on the glass weekly. Fifteen very young seedlings were placed at random in each case, but five plants have been drawn for clarity.
Growth analysis
Relative growth rate (RGR, mg g-1 day-1)
during the monitoring period was calculated
from data at harvest (W2) and transplant (W1)
following:
)(
)log(log
12
12
ttWWRGR
−−
= (1)
Capítulo II
61
where t2 - t1 was 41 days, using the Hunt et al.
(2002) spreadsheet tool. We calculated water-
use efficiency of productivity (WUE, mg L-1)
as the ratio between biomass gained and water
received during the experiment, taking into
account averaged initial biomass at transplant
(Kikvidze et al. 2006). From seedling root
length in reduced and control water levels at
harvest, we calculated for each species the
relative interaction index (RII, Armas et al.
2004) as an index of root plasticity to reduced
watering, expressed as:
where Rr and Rc were root length for reduced
and control plants, respectively. Although this
is not a specific plasticity index, its strong
mathematical and statistical properties make it
appropriate for comparisons between plants
growing in two treatment groups, in this case
control and reduced.
Statistics
Data were exploratory analyzed as a two-
factor design (watering quantity and
frequency); however, analyses showed no
differences in any variable between normal
watering and half the number of events in the
frequency factor. Likely, pulses of water of
different magnitude while keeping constant
the amount of water provided did not affect
soil moisture in our conditions. For this
reasons we excluded the frequency factor
from analyses to gain statistical power since
some plants died after transplant, and those
data were pooled either into corresponding
control or reduced quantity level since the
amount of water provided was kept constant
within the frequency factor, i.e., plants in the
control water quantity received 40 ml per
week in one (half events) or two events
(normal frequency), and similarly in the
reduced water quantity (28 ml distributed in a
single or two 14 ml events per week).
Data were then analyzed as a factorial
design with two factors, species and water
quantity. Differences in mean growth rate,
total root length, root area, maximum rooting
depth, biomass, SRL, R:S ratio and WUE
were tested using two-way analysis of
variance (ANOVA) for each variable
followed by Tukey HSD post-hoc comparison
tests. For total root length analysis we used
length of traced roots instead of length of
scanned roots since the former data were more
homoscedastic. Differences in root length
measurements between the two procedures
were not significant (paired t-test, P=0.47).
Because of the unequal sample size, we used
type III sum of squares. Heteroscedastic
variables were transformed to meet ANOVA
assumptions. When variables were still
heteroscedastic (as in WUE), we ran for each
species separately the non-parametric Mann-
Whitney U test (M-W U). Comparisons in
plasticity index (RII) among species were
)()(
cr
cr
RRRR
RII+−
= (2)
Drought and root growth
62
conducted from standard errors since all
replicates belonging to a treatment were
integrated in computation.
Since plotted data of cumulative root
length against time showed a linear trend,
growth curve analyses were conducted by
fitting individual data to a linear function Y =
mX + b, where Y was length (cm), X was time
(days), m was the slope and b the y-intercept.
Differences in growth curves between species
and water treatment were tested by comparing
regression slopes of each plant (m) through
ANOVA. We could not perform repeated-
measures and multivariate ANOVA to test
growth responses because our data violated
statistical assumptions (Von Ende 2001). Only
those individuals whose roots could be seen
through the glass case from the beginning of
the experiment were included into root growth
analysis. All tests were conducted with
Statistica v. 6.0 (Statsoft Inc, Tulsa, OK,
USA) and differences were considered
significant at P<0.05. Data are presented as
means ± one standard error.
Results
Cumulative root length over time was best
adjusted to a linear function. Growth curves
were statistically different between control
and reduced water quantity in all species
(P=0.014), with roots under drought growing
faster (Figure 2). This was reflected in root
elongation rate (ANOVAwater P=0.013); plants
subjected to lower watering elongated more
than control plants (8.58±0.74 vs. 6.74±0.65
mm day-1), regardless of species identity
(ANOVAspeciesxwater P=0.99, Figure 3a). We
found significant differences in mean root
elongation rate among species, with Lycium
having the highest rate (10.57±0.58 mm day-
1), followed by Retama (6.66±0.66) and
Genista (4.67±0.49, Table 2).
Figure 2. Root elongation curves. Cumulative root length over time in control (solid symbols) and reduced watering (white symbols), and fitted linear functions (lines) with r2 and P-values of regression. Growth curves of control and reduced treatments are statistically different (ANOVAwater F1,39=6.589, P=0.014), regardless of species (ANOVAspecies x water F2,39=0.062, P=0.940).
Capítulo II
63
Table 2. F-values of factorial ANOVA at harvest. RER, root elongation rate between harvest and the beginning of altering watering. SRL, specific root length. Superscripts show significance P-values.
F-values Effect (df)
RER Root mass
Shoot mass
Plant mass
R:S ratio
Root length
Root area SRL Rooting
depth
Species (2) 29.15<0.01 33.77<0.01 143.44<0.01 134.40<0.01 8.40<0.01 27.53<0.01 22.94<0.01 16.82<0.01 37.97<0.01
Water (1) 6.710.01 8.200.01 6.490.02 9.370.01 0.660.42 7.790.01 8.020.01 0.030.86 2.360.13
SpeciesxWater (2) 0.010.99 3.870.03 3.900.03 4.910.01 1.430.25 0.100.91 0.470.63 1.200.31 0.190.83
As for root plasticity, all species responded
to reduced watering by developing longer
roots (as reflected by positive values of RII),
though Genista showed the strongest response
(0.142±0.024), whereas in Lycium and
Retama it was lower (0.078±0.011 and
0.083±0.017, respectively).
Total root length and root area at harvest
differed among species, decreasing Lyicum >
Retama > Genista (Table 2, Figure 3b). There
were also significant differences in root length
and root area between water treatments
(ANOVAwater P<0.01), regardless of species
(ANOVAspeciesxwater P>0.6, Figure 2 and 3b).
When compared to control, plants supplied
with reduced water quantity showed longer
roots (28.65±2.26 vs. 35.75±2.57 cm) and
greater root area (2.69±0.26 vs. 4.02±0.47
cm2). On the contrary, we only detected a
tendency to root deeper in response to lower
water availability (25.55±2.03 for reduced vs.
22.45±1.71 cm for control plants, ANOVAwater
P=0.13, Tables 2 and 3). Roots of Lycium and
Genista had higher SRL than Retama,
although no significant adjustment in response
to altered watering quantity was detected in
any species (Pwater=0.86, Pspeciesxwater=0.31).
Root-to-shoot mass ratio was below 0.6 in all
species (Table 3), ranging from 0.47±0.05 in
Genista and 0.41±0.03 in Lycium to 0.28±0.02
in Retama. We did not detected significant
effects of water quantity on R:S ratio in any
species (ANOVAwater P=0.42).
We found differences among species in
plant, shoot and root mass at harvest
(ANOVAspecies P<0.001, Table 2), in contrast,
no differences were observed in mean shoot
elongation in any species in response to
drought (M-W Uwater, P>0.25). The effects of
watering quantity on biomass depended on
species, as revealed by the species x water
interaction (ANOVA P<0.03); plants supplied
with lower water quantity tended to exhibit
larger mass than those in control in Lycium
and Genista, whereas Retama performed
nearly the same both in control and reduced
levels. The same pattern was observed if plant
growth was considered with respect to initial
Drought and root growth
64
plant size (i.e., relative growth rate); RGR of
total plant, shoot and root masses were higher
under reduced water in Genista and Lycium,
whereas differences in Retama were less
patent (Table 3). This mirrored in water use
efficiency of productivity. Plants supplied
with lower water quantity produced
significantly more biomass per water received
than those in control in Lycium (M-W
Uwater=5, P<0.01), and marginally in Genista
(M-W Uwater=8, P=0.06). In Retama, however,
biomass gain was independent of water
provided (M-W Uwater=27, P=0.91, Figure 3c).
Discussion
A small reduction in water supply
enhanced root elongation in all our species at
very early stages of development, when
cotyledons were still attached. This could be
an analogous response to etiolation of shoots
under shaded conditions (Leishman and
Westoby 1994b). Despite the contrast in seed
mass and drought tolerance among Retama,
Lycium and Genista, all three species, either
drought-tolerant or sensitive, large or small-
seeded, responded equally to reduced
watering. These data evidence the importance
of water availability for seedling development
during such early stage. The increase in root
length and area in plants under reduced
watering can be interpreted as an adjustment
of absorptive surfaces to find water resources
(Hutchings and de Kroon 1994). By
increasing root length, plants exploit a larger
soil volume tapping otherwise unexplored
areas and increase their resource uptake
capacity, which paryl depends on root surface
area (Lambers et al. 1998b).
Figure 3. Plant growth at harvest. a) Mean root elongation rate (mm day-1) in control (solid bars) and reduced watering treatment (white bars), ANOVAwater P=0.013; b) root area (cm2) at harvest, ANOVAwater P=0.007; c) water use efficiency (mg L-1), M-W U test. A cross indicates marginal differences between water quantities (P<0.1) and asterisks significant differences (*, P<0.05; **, P<0.01). Values are means ± 1SE. n=6-9.
Capítulo II
65
Table 3. Plant growth and root traits. Plant, shoot and root mass (mg), root-to-shoot ratio, relative growth rate (RGR, mg g-1 wk-1) of total plant, shoot, and roots between transplant and harvest dates, maximum rooting depth (cm) and specific root length (SRL, cm mg-1) in control and reduced treatment. Different letters in a row show significant differences (P<0.05) after Tukey test. Values are means ± 1SE. n=6-9.
Genista Lycium Retama
Control Reduced Control Reduced Control Reduced
Plant mass 7.0±0.9a 10.5±0.7b 12.9±0.8bc 17.9±1.3c 38.4±3.0d 35.1±3.5d
Shoot mass 4.9±0.7a 7.2±0.5b 9.6±0.6bc 12.3±1.0c 30.4±2.8d 27.5±2.6d
Root mass 2.2±0.3a 3.3±0.4ab 3.3±0.4a 5.6±0.5bc 8.0±0.5c 7.6±1.1c
R:S ratio 0.48±0.08ac 0.46±0.05abc 0.35±0.04abc 0.47±0.04a 0.28±0.03bc 0.27±0.02c
Plant RGR 84.1±30.0 147.8±28.3 143.5±22.4 197.8±23.0 47.9±13.7 31.6±14.9
Shoot RGR 87.4±31.8 161.2±28.0 146.2±22.7 186.0±23.7 34.4±15.9 18.7±14.1
Root RGR 70.9±33.7 146.2±33.5 129.0±27.5 223.3±24.8 123.9±25.1 106.7±29.6
Rooting depth 13.1±1.6a 14.6±1.0ab 29.2±1.2cd 33±2.1c 23.2±2.5bc 25.4±2.9cd
SRL 9.1±1.0ac 8.1±1.4abc 13.8±2.2a 9.3±0.7ac 4.2±0.5bd 5.2±0.7cd
Our findings agree with reports showing
root elongation in response to low soil
moisture (Evans and Etherington 1991).
Reader et al. (1993) found that rooting depth
of seedlings of wild species increased in
response to drought due to higher elongation
rates, particularly in species that regenerate
mainly from seeds after disturbance (seeders),
suggesting that selective pressures favor
plasticity in root growth, affecting traits that
promote seedling survival. Although we do
not report significant differences in rooting
depth between control and reduced water
(P=0.13), most likely because of the short
time period considered, our data are consistent
with this explanation. Thus, early root growth
shows an adaptive strategy to deal with water
stress at the seedling stage (Fitter 1991). Root
elongation and deeper rooting depth in
response to water stress is presumably also an
adaptation that allows exploitation of
declining soil moisture (Lambers et al. 1998a)
and in fact, the ability to develop roots
accessing deep soil moisture has proved
decisive for survival of seedlings during
summer months in a Mediterranean semiarid
environment (Padilla and Pugnaire 2007). Our
hypothesis that root growth responses would
be stronger in the drought-sensitive and large-
seeded Retama because of its sensitivity to
dehydration and larger seed reserves could be
rejected since a drought-tolerant and small-
Drought and root growth
66
seeded species (Genista) showed a distinctly
plastic response. Developing seedlings of
large-seeded species acquire most resources
from seed reserves (Fenner and Kitajima
1999), and then they are relatively more
independent from external resources than
small-seeded species. However, the weak
response we found in Retama may not involve
a disadvantage in the field, since germination
timing and seedling size may offset low root
growth capacity. Interestingly, in agreement
with our findings, there are reports of greater
root elongation rate in drought-tolerant
turfgrass (Huang 1999) and phreatophyte
seedlings (Horton and Clark 2001), and in
seedlings of species restricted to dry sites
compared to humic environments (Evans and
Etherington 1991) when subjected to lower
water availability. It is clear that root
plasticity is under genetic control (Sydes and
Grime 1984; Sharp et al. 2004) and species do
not show the same ability to elongate;
however, whether root plasticity is linked to
species’ drought tolerance, and the underlying
mechanisms, still remains unclear and further
studies are necessary.
Surprisingly, we found larger shoot mass
and higher RGR in Lycium and Genista
seedlings supplied with less water, whereas
differences were negligible in Retama. It is
improbable that this was due to greater root
biomass allocation or root length exploiting
potentially more soil volume of Retama, since
it allocated the least to roots (lowest R:S ratio)
and showed one of the shortest root lengths at
transplant. Rather, seed size and cotyledon
reserves can explain such response, since they
strongly affect seedling growth (Leishman
and Westoby 1994b; Cornelissen et al. 1996;
Bonfil 1998; Hanley et al. 2004; Hanley and
May 2006). Large-seeded species, indeed,
have storage cotyledons characterized by a
slow, prolonged mobilization of reserves
(Kidson and Westoby 2000), relying to a
greater extent on cotyledons than on soil
resources and light (Milberg and Lamont
1997), whereas small-seeded species are more
dependent on light and soil resources
(Leishman and Westoby 1994b; Fenner and
Kitajima 1999). In our experiment, all three
species retained green cotyledons until
harvest, but cotyledon reserves lasted longer
in Retama than in Lycium and Genista
because of its differences in seed size (up to
two orders of magnitude) and cotyledon mass.
All three species increased root absorptive
surface with lower water availability as a
strategy to maximize water uptake, allowing
secondarily greater nutrient uptake, but
Retama, however, did not show changes in
shoot growth due to its greater independency
from soil minerals (i.e., greater dependency
on cotyledon reserves). In this sense, Jurado
and Westoby (1992) found that seedlings from
large-seeded species thrived better under
nutrient stress than small-seeded species,
since their growth remained independent from
external resources, and similar results were
reported by Milberg and Lamont (1997).
Capítulo II
67
In conclusion, increased root absorptive
surface caused by low water availability was a
response of all three species to maximize
water uptake, which also allowed for greater
nutrient uptake. In fact, Wan et al. (2002) also
found that drought induced root production
and enabled droughted plants to produce
above-ground biomass similar to that of plants
receiving full watering. However, in our
experiment, growth depended on cotyledon
reserves. Shoot growth and RGR was higher
under reduced watering in Genista and
Lycium because of greater root exploitation
and resource uptake, while Retama growth
depended more on cotyledon reserves and
biomass was relatively unaffected by nutrient
uptake.
Having small-diameter roots (i.e., higher
SRL) favors greater rates of water and
nutrient uptake (Eissenstat 1992; Cornelissen
et al. 2003), therefore larger SRL under
reduced water availability could be expected
as a strategy to maximize absorptive surfaces
(Reich et al.1998; Wright and Westoby 1999).
All species showed increased root length
under reduced water availability, evidencing
changes in root architecture with water
quantity, but SRL did not differ between
watering treatments. This inconsistency can
be due to the fact that we used the whole root
systems to obtain this variable, and Nicotra et
al. (2002) showed that SRL of the entire root
systems can differ from that measured on the
main axis or secondary roots. Similarly, large
biomass allocation to roots relative to shoots
(i.e., higher R: S ratio) also favors water and
nutrient uptake (Chapin et al. 1987; Lambers
et al. 1998a), and therefore we expected larger
R: S ratios under reduced water availability.
However, plants did not respond to water
stress by shifting allocation patterns, and the
R:S ratio did not change. Although the
allocation model is widely accepted (see e.g.,
Chapin et al. 1987; Kozlowski and Pallardy
2002), other factors do impact upon R:S
partitioning. Evidence suggests that plasticity
in R:S ratio may be highly species-specific
(Joslin et al. 2000) and that in some species
R:S ratio is remarkably stable (Klepper 1991)
or subjected to developmental constraints
(Gedroc et al. 1996; McConnaughay and
Coleman 1999). Additionally, root
demography and the ability to alter rates and
place of root proliferation may have greater
importance for plants than changes in mass
allocation between roots and shoots (Reynolds
and D'Antonio 1996).
Overall, we showed that very young
seedlings responded to reduced water
availability by elongating roots, whereas no
significant changes in R:S ratio were detected.
Greater absorptive root surface likely allowed
seedlings to increase growth rate in the small-
seeded species, whereas growth of the large-
seeded species seemed independent from
external resources. Root growth may be
considered an important factor in early
seedling development, since rapid extension
Drought and root growth
68
of roots enables seedlings to tap water from
previously unexplored areas of soil (Schütz et
al. 2002). Regardless of seed size and drought
tolerance strategy, root elongation in our three
species is a common adaptive trait to cope
with soil dryness at early stages. However,
further research is needed to link root
plasticity to species-specific drought
tolerance.
Acknowledgements
We are grateful to Kazuhiko Kimura for
helping with the root macro, Florentino
Mostaza for root scanning and Consejería de
Medio Ambiente (Junta de Andalucía) for
seed donation. Tibor Kalapos and two
anonymous reviewers made valuable
comments on an earlier draft. The Spanish
Ministry of Education and Science funded this
work (grant CGL2004-00090/CLI).
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Capítulo III
Respuesta fisiológica de siete especies arbustivas mediterráneas a pulsos de agua
Capítulo III
75
Capítulo III
PHYSIOLOGICAL RESPONSES OF MEDITERRANEAN SHRUBS TO PULSED
WATER SUPPLY
Summary
Pulsed water supply has a strong effect on plant survival and seed germination in arid
environments, yet very little is known about the effects on growth and plant performance of
seedlings. Here we focused on the effects of both, amount and frequency of water inputs on
seedling growth and functional traits of seven Mediterranean shrub species occurring in semi-arid
SE Spain, and differing in leaf habit and drought tolerance, Anthyllis cytisoides, Atriplex halimus,
Ephedra fragilis, Genista umbellata, Lycium intricatum, Retama sphaerocarpa and Salsola
oppositifolia. In a 14-month greenhouse experiment we manipulated water supply expecting that
pulses of water of different magnitude would not have the same effects on plants, even if the
amount of water provided was the same. Different watering patterns altered soil drying dynamics
and water content at the end of watering cycles. We found that roots responded to such alterations
by changing biomass allocation patterns (root-to-shoot mass [R:S] ratio), and by altering root
architecture, measured in terms of specific root length (SRL). However, leaf traits such as leaf area
(LA) and specific leaf area (SLA), and biomass and relative growth rate (RGR) of all species were
insensitive to pulses of water and fluctuating soil moisture. Differences in RGR among species
were significantly linked to differences in SRL, presumably related to uptake capacity of roots, and
much less to differences in biomass allocation or leaf traits. These data show that roots are very
responsive to soil water heterogeneity at the juvenile stage, possibly a strategy to compensate for
lower water availability by maximizing uptake. We suggest that decreases in soil moisture over
long periods of time and extended drought spells are necessary to limit growth in these
Mediterranean shrubs because of their adaptation to severe drought and highly variable rainfall
patterns of semiarid environments.
Introduction
Understanding how plant species deal
with soil resource availability is a central
theme of plant ecological research (Chapin
1991, Lambers et al. 1998). Soil resources
required for plant growth are highly
heterogeneous at a wide variety of scales
both in time and space, so nutrients
(Schlesinger and Pilmanis 1998, Gallardo
Pulsed water supply and plant performance
76
2003) and water (Burgess et al. 1998, Cantón
et al. 2004) are not homogeneously
distributed in natural soils at a spatial scale,
and their temporal availability is not regular
(Austin et al. 2004, Reynolds et al. 2004).
This resource heterogeneity does impact
individuals in terms of survival, growth,
performance and biotic interactions
(Hutchings and Kroon 1994, Cahill and
Casper 1999, Poorter and Lager 2000, Hodge
2004, Padilla et al. 2007, Maestre and
Reynolds 2007), and therefore can alter
population dynamics.
Water availability is often the most
limiting factor for plant activity in arid
environments (Noy-Meir 1985). In such
environments, water availability is highly
pulsed, and discrete rainfall events
interspersed with drought periods are
important components of the annual water
supply. Vegetation not only responds to
rainfall quantity (Noy-Meir 1985; Reynolds
et al. 2004), but also to variations in time
(Sala and Lauenroth 1982, Turner & Randall
1989, Lázaro et al. 2001) in such a way that
relatively small changes in rainfall frequency
(i.e. pulsed inputs) can have strong effects on
survival and growth of individuals, yet
research has shown that there is some
variation in species responsiveness
(Novoplansky and Goldberg 2001, Sher et al.
2004). This is particularly true in
Mediterranean semi-arid ecosystems of SE
Spain. In this area, among the driest in
Europe with less than 250 mm year-1 (Capel-
Molina 2000), rainfall timing and amount
greatly influence germination and seedling
establishment (Pugnaire & Lázaro 2000,
Lázaro 2004, Pugnaire et al. 2006, Padilla
and Pugnaire 2007), but very little is known
about the effects on seedling growth after
germination.
Growing attention has been paid to the
relationship between pulsed water inputs,
species responses and arid ecosystems
dynamics in the last years (Reynolds et al.
2004, Schwinning et al. 2004, Heisler and
Weltzin 2006), with research conducted in
controlled environments in greenhouses
focusing on annual and grassland species
(Novoplansky and Goldberg 2001; Sher et al.
2004, Maestre and Reynolds 2007).
However, woody species should have
different behavior and growth responses,
although to our knowledge there is little
information on how shrub seedlings respond
to pulses of water. Addressing the effects of
pulsed water supply is not only important to
better understanding seedling responses, but
also to provide insights into how rainfall
variability can affect semiarid Mediterranean
ecosystems. Since seedlings and juveniles are
more sensitive to dehydration than seeds or
adults (Evans and Etherington 1991),
variations in quantity and frequency of water
supply (i.e., greater, less frequent events
followed by longer drought periods) is likely
to affect plant growth in different ways
Capítulo III
77
(Easterling et al. 2000, Weltzin et al. 2003,
Sher et al. 2004).
In this paper we focus on the effects of
variation in amount and temporal supply of
water on seven shrub species from semi-arid
SE Spain differing in leaf habit and drought
tolerance. In a greenhouse experiment we
modified water supply and analyzed its
effects on growth and plant functional traits,
expecting that pulses of water of different
magnitude did not have the same effects on
plants, even if the amount of water provided
was kept constant (Knapp et al. 2002;
Reynolds et al. 2004). In response to lower
soil moisture and modified drying dynamics
caused by altered watering supply, we
expected a decrease in seedling growth rate
and changes in leaf and root functional traits
related to light and water acquisition. We
hypothesized that species responsiveness
would depend on leaf habit and drought
tolerance, with deciduous and drought-
sensitive species showing the strongest
response because of the physiological costs
of leaf shedding and dehydration intolerance
(but see Reynolds et al. 1999).
Materials and Methods
Species
We selected seven native shrub species
occurring in Mediterranean semiarid
shrublands in the Tabernas basin (Almería,
SE Spain, 37º08' N, 2º22' W, 490 m
elevation). This area is characterized by mild
temperatures (17.8 ºC average annual
temperature), and low and variable rainfall
(235 mm annual rainfall, 1967-1997 period,
Confederación Hidrográfica del Sur), with a
markedly dry season from June to September
(Lázaro et al. 2001). Species can be classified
into three functional groups according to leaf
habit, a) nearly leafless shrubs with
photosynthetic stems, b) drought-deciduous
shrubs and c) evergreen species (Table 1).
Species also differed in drought tolerance
based on minimum xylem pre-dawn water
potential (Ψpd) recorded in the field. While
Anthyllis cytisoides L., a small, drought-
deciduous shrub (Haase et al. 2000), Lycium
intricatum Boiss., Atriplex halimus L.,
Salsola oppositifolia Desf. (the two latter C4
xero-halophyte shrubs; Pyankov et al. 2001;
Martínez et al. 2004), Ephedra fragilis Desf.,
and the shallow-rooted Genista umbellata
(L’Hér.) Dum. Cours., stand low water
potentials (Ψpd < -5 MPa; Lansac et al. 1994;
Pugnaire et al. 2004), Retama sphaerocarpa
(L.) Boiss., a deep-rooted species, shows a
more drought-sensitive behavior revealed by
less negative Ψpd (~ -1.5 MPa; Haase et al.
1999).
Pulsed water supply and plant performance
78
Table 1. Main plant traits.
Species Family Leaf habit Drought strategy Photosynthesis pathway
Ephedra fragilis Ephedraceae Leafless Tolerant C3, photosynthetic stems
Genista umbellata Leguminosae Leafless Tolerant C3, photosynthetic stems
Retama sphaerocarpa Leguminosae Leafless Avoider C3, photosynthetic stems
Anthyllis cytisoides Leguminosae Deciduous Tolerant C3, leaves
Lycium intricatum Solanaceae Deciduous Tolerant C3, succulent leaves
Atriplex halimus Chenopodiaceae Evergreen Tolerant C4, leaves
Salsola oppositifolia Chenopodiaceae Evergreen Tolerant C4, succulent leaves
Experimental design
Seeds of the seven species were separately
sown in germination trays containing type III
vermiculite (Verlite®, Vermiculita y
Derivados SL, Gijón, Spain) in laboratory at
room temperature and day light on 22 March
2005. Seeds from the Tabernas basin were
collected manually or provided by local
nurseries. All seeds germinated within three
weeks, and very young seedlings were
carefully transferred to pots on 14 April, once
cotyledons had fully emerged from seed
coats. Six randomly selected seedlings of
every species were harvested before
transplanting. At transplant, one seedling was
planted in each pot and tap water was
provided daily. Pots of 300 mL in volume
contained vermiculite and were 4.5 cm in
diameter and 18 cm deep (Forest Pot 300®).
We selected vermiculite because of its
relatively infertility, lower compaction and
greater oxygenation than other growing
media. A nutrient solution (2 mL/L water) of
a 4-5-6 NPK fertilizer (KB, Scott France,
Lyon, France) was added weekly for one
month, and seedlings that died during this
period were replaced.
Pots were arranged in a factorial design
with two factors (quantity of water and
watering frequency) on 16 May. Watering
treatments were established according to
climate change forecasts for the western
Mediterranean Basin, consisting in a
reduction of annual rainfall of ~30% with a
trend towards extended drought periods
(IPCC 2001; Sánchez-Rodrigo 2002).
Although potted experiments deviate from
natural conditions in the field, we would
rather to be consistent with these predictions
and not apply stronger yet arbitrary
reductions as the goal of the study was not to
decipher species responses to severe drought.
Water quantity included a ‘control’ and a
‘reduced’ level consisting of 30% less than
the control, and frequency comprised a
‘normal’ level (four watering events per
Capítulo III
79
week) and ‘half’ the number of events (two
per week). Since we focused on growth
rather than survival, we considered than two
watering events per weeks were necessary to
keep seedlings alive on the course of the
experiment. The quantity and frequency
factors were fully crossed in all species, and
seedlings subjected to ‘normal’ frequency
were watered four times a week, either with
20 mL (‘control’) or 14 mL (‘reduced’) each,
whereas those subjected to ‘half’ frequency
were watered twice a week, either with 40 or
28 mL.
Sample size of each combination at
transplant was 9 replicates, except for
legumes (18). Replicates of legumes doubled
those of other species because the initial
experimental design considered Rhizobium
inoculation of half the legume seedlings.
However, this could not be performed
because of the failure to isolate an
appropriate bacterial inoculum. Plants grew
in a greenhouse at the Estación Experimental
de Zonas Áridas (CSIC, Almería) under
natural irradiance and temperature without
further fertilization, and were kept for 14
months. Pot position was re-arranged at
random every two weeks.
Measurements and plant harvest
To estimate the effect of altered watering
on vermiculite moisture, we calculated the
gravimetric water content (%) corresponding
to each treatment during a two-week period.
Pot weight before and after watering was
recorded daily. At the end of the monitoring
period pots were dried at 105 ºC for 48 hours,
emptied out and weighed. Gravimetric water
content (GWC) was calculated following:
100)()(
(%) ×−
−=
potdry
drywet
WWWW
GWC (1)
where Wwet and Wdry was pot weight with
fresh and dry vermiculite respectively, and
Wpot was pot weight. Measurements were
done in five unplanted pots per treatment
because it is a destructive method.
Plant harvest was conducted in June 2006.
Before harvesting, plant physiological status
was assessed by measuring the
photochemical efficiency of photosystem II
(PSII, Fv/Fm), on 30-minute dark-adapted
leaves early in the morning with a portable
fluorimeter (PEA, Hansatech Instruments
Ltd., Kings Lynn, UK). Measurements were
carried out at the end of a watering cycle, at
the time of the strongest drought faced by
seedlings. To calculate leaf area (LA), 5 to 15
leaves from the same aspect of each plant, or
5 to 10 stem segments 5 cm long of leafless
shrubs, were excised, scanned with a portable
scanner (Epson GT7000, Seiko Epson Corp.,
Nagano, Japan) at 300 dpi, and the projected
area measured with appropriate software
(Midebmp v.4.2, R. Ordiales-Plaza, 2000).
Leaf area of cylindrical leaves and stems
Pulsed water supply and plant performance
80
were corrected by π/2. Due to the small leaf
size, leaves of each plant were scanned and
weighed together after drying at 72ºC for >
48 hours, and averaged. Specific leaf area
(SLA, m2 kg-1) was computed as the ratio
between leaf area and mass. Lycium leaves
were not measured because of their small
size.
At harvest, plants were clipped at ground
level and aboveground parts were
immediately labeled and stored in paper bags,
dried and weighed. Pots were emptied out
into water and vermiculite attached to roots
was removed by brushing gently. Roots were
then labeled, placed into wet paper towels
and kept cool in zip bags in a refrigerator
before processing. To calculate specific root
length (SRL, cm g-1), 5 to 10 root segments 5
cm long of each plant were excised and
digitalized. Segment length was measured
from digitalized traces using the macro
RootMeasure v.1.80 (Kimura and Yamasaki
2003) implemented on the software Scion
Image Beta v. 4.02 (Scion Corp., Maryland,
USA). Segment dry mass and root mass were
obtained as with leaves. Root-to-shoot mass
(R:S) ratio for each plant was calculated from
above and belowground masses. Relative
growth rate on plant mass (RGR, mg g-1 day-
1) during the monitoring period was
calculated from data at harvest (W2) and
transplant (W1) following:
)(
)log(log
12
12
ttWW
RGR−−
= (2)
where t2 - t1 was 425 days, using the Hunt et
al. (2002) spreadsheet tool.
Statistics
Vermiculite drying dynamics was
analyzed using ANCOVA on daily water
content with time as covariate. Differences
among treatments were considered
significant when the treatment x time
interaction resulted significant. We tested
differences in vermiculite water content at
the end of the monitoring period through
factorial analysis of variance (ANOVA)
followed by Tukey post hoc tests. This gives
an estimate of the lowest soil moisture plants
dealt with.
Plant data were analyzed as a non-
balanced nested factorial ANOVA with three
factors, species, watering quantity and
frequency. Since the ‘half’ level of the
frequency factor was lacking in Atriplex
because most replicates died by summer, we
nested this factor within species. We ran
independent ANOVA for each variable
followed by Tukey tests when significant
differences at P<0.05 were detected.
Heteroscedastic variables were transformed
to meet ANOVA assumptions. Since biomass
was unaffected by watering patterns,
differences in RGR among species were
Capítulo III
81
detected by one-way ANOVA using each
combination as a replicate (n = 4). Simple
linear regressions were performed to test
correlation strength between variables, using
adjusted R2 to correct for the degrees of
freedom. All tests were conducted with
Statistica v.6.0 (Statsoft Inc, Tulsa, OK,
USA) and data are presented as means ± one
standard error. Because of differing mortality
on the course of the experiment, the final
sample size of each combination ranged 6-14.
Results
Watering treatments led to differences in
vermiculite drying dynamics (ANCOVA
treatment x time F3,312 = 4.135, P<0.01, Figure 1).
Vermiculite moisture greatly fluctuated with
time, but in general it was lower in pots
supplied with reduced watering quantity
(ANOVA F1,16=80.580, P<0.001) and
normal frequency (ANOVA F1,16 = 52.869,
P<0.001). Considering the lowest vermiculite
moisture registered, our treatments created a
gradient that ranged from 32±2% in the
control quantity-half events combination, to
24±2% for the control-normal frequency, to
21±2% for the reduced quantity-half events,
and to 2±1% for the reduced quantity-normal
frequency, which entailed reductions of 25,
34 and 94%, respectively. It is worth noticing
that while vermiculite moisture remained in
control treatments always above 30%,
reaching peaks of 80%, in the driest
treatment moisture never surpassed 20%.
Figure 1. Mean gravimetric water content of vermiculite recorded in five unplanted pots for every combination during a 16-day watering cycle. Normal and half events refer to frequency of watering.
Altered patterns of water supply affected
root traits such as R:S ratio and SRL (Table
2). Plants subjected to reduced watering
allocated proportionally more biomass to
roots (i.e., higher R:S ratio, ANOVAquantity
F1,204 = 4.934, P<0.03, Figure 2) but no
consistent differences were found in species
responsiveness (species x water interaction,
Pulsed water supply and plant performance
82
F6,204 = 2.084, P>0.05). Frequency of water
supply had no effect on biomass allocation
patterns in any species (ANOVAfrequency F6,204
= 1.125, P>0.3), whereas it did affect SRL
(P<0.05), interacting with watering quantity
(amount x frequency, F6,204 = 2.363, P<0.04).
Neither amount nor frequency affected
consistently total, shoot or root mass at
harvest in any species (P>0.07). Leaf traits
such as LA and SLA did not differ among
watering treatments (P>0.1), and drought-
deciduous shrubs did not shed leaves
throughout the monitoring period. Similarly,
no effect on chlorophyll fluorescence was
detected (P>0.2) and all species showed
Fv/Fm values above 0.71 (Figure 2).
Table 2. P-values of nested factorial-ANOVA at harvest on chlorophyll fluorescence (Fv/Fm), plant, shoot and root mass, root-to-shoot mass (R:S) ratio, leaf area, specific leaf area (SLA), and specific root length (SRL). Frequency factor was nested within species. Significant effects are shown by bold at P<0.05.
Effect
Species (S) Quantity (Q) Frequency (F(S)) S x Q Q x F(S)
Fv/Fm <0.001 0.612 0.495 0.626 0.223
Plant mass <0.001 0.907 0.314 0.072 0.687
Shoot mass <0.001 0.633 0.470 0.091 0.787
Root mass <0.001 0.618 0.078 0.178 0.529
R:S ratio <0.001 0.027 0.349 0.057 0.627
Leaf area <0.001 0.895 0.434 0.329 0.914
SLA <0.001 0.793 0.103 0.193 0.482
SRL <0.001 0.589 0.048 0.929 0.031
Table 3. Relative growth rate (RGR, mg g-1 week-1) for each species x combination and average (± SE). Control and reduced refer to watering quantity, and normal and half to frequency. Significant differences among species are indicated at P<0.05 by differing lower-case letters (ANOVA after Tukey test).
Control Reduced
Species Normal Half Normal Half
Average
Anthyllis 100.9±14.9 97.0±15.4 101.8±14.8 98.2±13.3 99.5±14.6a
Atriplex 113.2±7.0 - 107.5±8.7 - 110.4±7.9b
Ephedra 76.8±6.7 73.1±13.0 73.7±9.6 77.9±7.5 75.4±9.2c
Genista 67.5±14.1 65.7±21.3 75.2±14.1 73.0±14.1 70.4±15.9c
Lycium 90.7±7.9 84.8±9.7 85.9±9.2 83.7±10.4 86.3±9.3d
Retama 57.6±7.6 61.3±6.5 56.8±9.4 57.5±7.4 58.3±7.7e
Salsola 32.2±4.8 31.3±4.8 35.8±7.3 33.2±5.4 33.1±5.6f
Capítulo III
83
Figure 2. Plant, root and shoot mass, R: S ratio, leaf area (LA), specific leaf area (SLA), specific root length (SRL), and chlorophyll fluorescence (Fv/Fm) for each species x combination at harvest (Ant, Anthyllis cytisoides; Atr, Atriplex halimus; Eph, Ephedra fragilis; Gen, Genista umbellata; Lyc, Lycium intricatum; Ret, Retama sphaerocarpa; Sal, Salsola oppositifolia). Control and reduced refer to watering quantity, and normal and half to frequency.
Pulsed water supply and plant performance
84
When comparing among species, we
found significant differences in biomass and
growth rate (ANOVARGR F6,19 = 268.02,
P<0.001). The highest RGR was achieved by
Atriplex, followed by Anthyllis and Lycium,
while Retama and Salsola showed distinctly
lower growth rates (Table 3). We also
detected differences in biomass allocation
(Figure 2), with the R:S ratio being especially
high in Anthyllis (2.59±0.16), and well above
1 in Retama (1.67±0.04), and Genista
(1.17±0.07). In contrast, Salsola allocated
proportionally the least to roots (0.81±0.04).
As for leaf traits, SLA showed considerable
contrast among species (P<0.001), with
Anthyllis and Atriplex having the highest
SLA, which differed from other species,
notably from the species with photosynthetic
stems Ephedra and Retama. As for root traits,
Salsola showed the lowest SRL (2210±157
cm g-1), and Lycium and Atriplex the largest
(~ 5100±330 cm g-1).
We found a positive relationship between
seedling growth rate (RGR) and specific root
length (R2=0.50, P<0.001). RGR was also
positively related to a lesser extend to leaf
area (R2=0.23, P<0.02), specific leaf area
(R2=0.29, P<0.01) and root-to-shoot mass
ratio (R2=0.12, P<0.05, Figure 3).
Figure 3. Relationships between relative growth rate (RGR) and, leaf area (LA), specific lead area (SLA), specific root length (SRL), and root:shoot (R:S) ratio. Each point represents mean value for each treatment.
Capítulo III
85
Discussion
By altering water supply we caused a
strong alteration of vermiculite drying
dynamics, as well as large decreases in the
water content at the end of the watering
cycles. However, our hypothesis that plant
responses would differ depending on the
species did not hold, as reduced quantity and
lower frequency of watering (i.e., pulsed
inputs) did not affect plant growth nor leaf
traits such as leaf area (LA) or specific leaf
area (SLA) of any species. We also
hypothesized that species would respond to
lower water availability by modulating root
traits responsible for resource acquisition;
and in this case our hypothesis held because
water supply led to changes in root traits such
as root-to-shoot mass (R:S) ratio and specific
root length (SRL), showing that seedlings
dealt with reduced water availability by
modifying root morphology, which supports
previous studies on root sensitivity and
plasticity of juveniles under soil water
heterogeneity (Padilla et al. 2007).
Chlorophyll fluorescence gives a potential
estimate of photosynthetic performance since
strong stress damages to PSII often manifest
in leaves (Maxwell and Johnson 2000). In
our experiment, however, Fv/Fm was
unaffected by water supply, and all species
showed values close to the optimum of 0.83
(ranging 0.72-0.78), evidencing that
seedlings were not subjected to severe water
stress. Nevertheless, it is worth noting that
we did not pursue to decipher physiological
responses to rather severe drought, but to
focus on the effects of heterogeneity of water
supply on growth and functional traits. As for
leaf traits, it is widely accepted that water
limitations select for smaller leaves and
lower SLA (Cornelissen et al. 2003, Wright
et al. 2006); however, we did not detect leaf
adjustments in response to lower soil
moisture and, because of the tight correlation
between SLA and growth rate (Cornelissen et
al. 1996; Wright and Westoby 1999), we did
not find differences in biomass or relative
growth rate (RGR). These data contrast with
reports of other experiments conducted under
controlled conditions. Fernández and
Reynolds (2000) found that biomass and
SLA of eight perennial C4 dessert grasses
were markedly reduced by severe drought. In
other Mediterranean perennial species, soil
water deficits also decreased SLA and
growth rate (Sack and Grubb 2002, Galmés
et al. 2005, Sánchez-Gómez et al. 2006).
Plant responses to soil water availability
depend on species and habitat occurrence, as
reflected by work reporting small responses
to pulses of water in species from very dry
habitats compared to species from more
mesic habitats (Novoplansky and Goldberg
2001; Sher et al. 2004). In this sense, our
species naturally occur in more limiting
Mediterranean environments than species
from the above reports, as it is one of the
Pulsed water supply and plant performance
86
driest in Europe, with large inter-annual (up
to 36%) and monthly rainfall variability
(ranging 55-207%, Lázaro et al. [2001]).
Given these conditions, selection pressures
could have led to plant adaptation to very
variable water inputs in all our species, and
therefore it is possible that stronger and more
prolonged droughts are needed to limit plant
growth. Valladares et al. (2005) have shown
that drought effects on seedlings of
Mediterranean species are noticeable at soil
moisture below 10%, which would suggest
that water content of our driest treatment was
most of the time low enough to constrain
plant activity. However, it is reasonable to
expect a soil moisture threshold lower than
10% in our species because of their
provenance.
It could be argued that the lack of growth
responses might be caused by the buffering
effect of vermiculite on water content and
drying dynamics. In our driest treatment,
water content reached as low as 2% (actually
lower since we calculated gravimetric water
content in unplanted pots), but plants faced
such low moisture for three days, remaining
within the range 2-20% in the remaining
days. In this sense it would be reasonable to
think that watering every three days
maximized water uptake at moisture peaks,
making irrelevant the following dry period.
In fact, authors have proposed that some
species of arid and semiarid environments
develop quick responses to water supply,
taking advantage of such pulses to increase
biological activity (Sala & Lauenroth 1982,
Reynolds et al. 2004, Schwinning and Sala
2004).
Importantly enough, unlike growth and
leaf traits, watering amount and frequency
affected seedling roots. Large biomass
allocation to roots relative to shoots (i.e.,
higher R:S ratio) and root diameter (i.e.,
SRL) are believed to alter rates of water and
nutrient uptake (Chapin et al. 1987;
Eissenstat 1992; Lambers et al. 1998,
Cornelissen et al. 2003). Thus, larger R:S
ratio in plants subjected to lower soil
moisture, and changes in root architecture,
can be interpreted as a strategy to maximize
absorptive surfaces to deal with water
limitations (Reich et al.1998; Wright and
Westoby 1999; Fernández and Reynolds
2000). This means that seedling roots
actually reflected lower water availability,
but we cannot state whether root responses
compensated for the reduction in soil
moisture and accounted for the lack of
growth differences between droughted and
control seedlings, as some authors suggest
(Ge et al. 2003).
Species wide, we detected no trend in R:S
patterns among functional groups. R:S ratio
of summer-deciduous species showed a great
variability, however, ranging from the largest
value in Anthyllis to one of the lowest in
Lycium. Nevertheless, the lack of clear
Capítulo III
87
patterns in leaf habit does not rule out the
existence of such links, which have been
revealed by Antúnez et al. (2001) in other
Mediterranean species, but may rather reflect
the small number of replicates within each
group. In agreement with published data
(Wright and Westoby 1999, Antúnez et al.
2001), we found that summer-deciduous
species, Anthyllis and Lycium, had greater
SLA and SRL and faster RGR than evergreen
species. Differences in growth rate among
species were linked to differences in traits
that maximize uptake capacity of roots and
leaves such as SRL and SLA (Garnier 1991,
Cornelissen et al. 1996, Reich et al. 1998,
Comas and Eissenstat 2004), rather than to
differences in biomass allocation to roots.
Overall, we showed that species
responded to altered patterns of water supply
by modulating biomass allocation patterns
and root diameter, whereas leaf functional
traits and growth of the seven species were
insensitive to water shortage. Regardless of
functional groups, roots were very sensitive
to soil water, presumably as a survival
strategy, and this plasticity might compensate
to some extend for lower soil moisture.
Stronger soil moisture decreases over longer
time periods seem to be needed to limit
seedling growth, partly due to species
adaptation to inherently variable rainfall
patterns of Mediterranean ecosystems.
Acknowledgements
We thank María José Jorquera for tending
the plants and Vivero de Rodalquilar
(Consejería de Medio Ambiente, Junta de
Andalucía) for seed donation. The Spanish
Ministry of Education and Science funded
this work (grant CGL2004-00090/CLI).
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General view of the experimental pots.
Capítulo IV
El papel de las plantas nodriza en la restauración de ambientes degradados†
† Publicado como “Padilla, F.M. and Pugnaire, F.I. 2006. The role of nurse plants in the restoration of degraded environments. Frontiers in Ecology and the Environment 4: 196-202”
Capítulo IV
95
Capítulo IV
THE ROLE OF NURSE PLANTS IN THE RESTORATION OF DEGRADED
ENVIRONMENTS
Summary
Traditional ecological models have focused mainly on competition between plants, but recent
research has shown that some plants benefit from closely associated neighbors, a phenomenon
known as facilitation. There is increasing experimental evidence suggesting that facilitation has a
place in mainstream ecological theory, but it also has a practical side when applied to the
restoration of degraded environments, particularly drylands, alpine, or other limiting habitats.
Where restoration fails because of harsh environmental conditions or intense herbivory, species
that minimize these effects could be used to improve performance in nearby target species.
Although there are few examples of the application of this “nursing” procedure worldwide,
experimental data are promising, and show enhanced plant survival and growth in areas close to
nurse plants. We discuss the potential for including nurse plants in restoration management
procedures to improve the success rate of such projects.
Introduction
Plant interactions strongly influence
community structure and dynamics, and are
responsible for the presence or absence of
particular species in a community.
Traditionally, competition has been the most
studied aspect of those interactions, so that
ecological models have focused for decades
on negative interactions, overlooking the
existence of positive effects between plants.
In the past 15 years, however, research has
highlighted the role of positive plant
interactions (facilitation) in almost all biomes
(Bertness and Callaway 1994; Bertness and
Hacker 1994; Callaway 1995; Brooker and
Callaghan 1998; Callaway et al. 2002; Bruno
et al. 2003; Lortie et al. 2004). Despite this
increasing recognition, the inclusion of
facilitation into mainstream ecological theory
has been slow (Bruno et al. 2003). Facilitation
appears to be essential process not only for
survival, growth, and fitness in some plants
(Callaway et al. 2002; Tirado and Pugnaire
2003; Cavieres et al. 2006), but also for
diversity and community dynamics in many
ecosystems (Pugnaire et al. 1996; Kikvidze et
al. 2005). Examples of facilitation are more
evident in harsh, limiting environments,
where some species are able to ameliorate the
The role of nurse plants in restoration
96
physical conditions in some way, or prevent
herbivory, thereby providing more suitable
habitats for other species. This interaction has
a practical side when applied to ecological
restoration. In degraded habitats with extreme
environmental conditions or large numbers of
herbivores, the area near or under the canopy
of certain species may be a safe site to place
the seeds or plants of the species being
restored (target species), and which otherwise
may fail to establish Here we review the
potential of this procedure for ecological
restoration.
Competition and facilitation
Plants growing close to each other
influence their neighbors in positive and
negative ways, resulting in a broad range of
detrimental or beneficial outcomes. If
negative effects prevail, the interaction results
in competition or interference, a consequence
of sharing limited resources (water, nutrients,
light, space), or of a release of chemicals that
will harm nearby plants (allelopathy).
Conversely, nearby plants may exert a
positive influence, termed facilitation, in
which at least one neighboring species
benefits from the interaction through
improved survival, growth, or fitness.
Both positive and negative effects can be
seen occurring at the same time, affect
different variables, and change with time and
in different areas (Armas and Pugnaire 2005).
The net balance between these effects
represents the magnitude and sign (either
positive or negative) of the interaction
(Callaway and Walker 1997; Holmgren et al.
1997; Figure 1). Several factors affect this
balance, including physiological and
developmental traits (Callaway and Walker
1997; Armas and Pugnaire 2005), but abiotic
conditions seem to be the overriding factor,
increasing the importance of positive effects
in harsher environments (Brooker and
Callaghan 1998; Pugnaire and Luque 2001;
Callaway et al. 2002; but see Maestre et al.
2005 and Lortie and Callaway 2006 for
discussion of the stress-gradient hypothesis).
The nurse effect
In some habitats, seedling establishment
may be enhanced in the vicinity of adult
plants that ameliorate extreme environmental
factors (eg Cavieres et al. 2006). The positive
influence of the adult plants on seedlings is
called “nurse plant syndrome” (Niering et al.
1963), and is one of the first recorded
examples of close spatial association between
plants being more advantageous than
detrimental. This effect is more common in
environments where abiotic factors or
herbivory limit plant performance, such as in
arid (Flores and Jurado 2003) or alpine
habitats (Cavieres et al. 2006).
Capítulo IV
97
Figure. 1. Facilitation and interference under nurse plants. The balance between positive and negative effects of closely placed species determines the net outcome of the interaction. (a) When positive effects outweigh negative ones, seedling survival or growth is enhanced as compared to survival of individuals in gaps; (b) opposite results are found when negative effects outweigh the positive ones.
The underlying mechanisms relate mainly
to the improvement of microclimatic
conditions, increased water and nutrient
availability, and protection against herbivory
(for more details see Callaway 1995;
Callaway and Pugnaire 1999).
The advantages of growing close to nurse
plants
Nurse plants may buffer non-optimal
environmental conditions. Shade reduces soil
water evaporation, lowers soil and air
temperature, and decreases the amount of
radiation reaching the plants, thus protecting
seedlings from the damaging effects of
extreme temperatures and low humidity in
arid environments. Canopy protection also
prevents salt enrichment in soil marshes and
wetlands, and may reduce frost injuries in
cold areas. Nurse plants also may improve the
availability of soil resources. Through the
process known as “hydraulic lift”, roots of
certain species lift water stored in deep soil
layers and released it near the soil surface.
Once in the surface layers, the water can be
used by understory plants, and improves their
water status and growth rate. Nutrients in the
understory are enhanced through litter and
sediment accumulation, higher mineralization
rates, and larger microorganism populations.
Positive root interactions between facilitator
and facilitated plants allow nitrogen transfer
between legumes and non-leguminous plants,
increase ectomycorrhizal infection, and make
The role of nurse plants in restoration
98
possible the exchange of nutrients and carbon
via mycorrhizal fungi. In heavily grazed areas,
plants growing beneath non-palatable or
thorny plants have an advantage, as compared
to unprotected plants. Finally, nurse plants
that are highly attractive to pollinators may
increase pollinator visits to the target plants.
Role of facilitation in restoration
Although some authors suggested that the
nurse effect could potentially play a role in
restoration (see Bradshaw and Chadwick
1980), by the mid-1990s only a few anecdotal
reports on this topic were available (Mitchley
et al. 1996). However, experimental evidence
addressing the role of nurse plants in
restoration has increased in the past few years
(Table 1). We reviewed restoration
experiments in which seeds or seedlings of
restored species were placed both near adult
plants that acted as nurses and in control gaps
(Figure 2), and provide suggestions for
management.
The first published research looking at the
use of natural nurse plants for restoration
purposes were carried out at the end of the
1990s, in southeast Spain (Castro et al. 2002;
Gasque and García-Fayos 2004). Since then
several experiments have been conducted in
alpine areas, semiarid steppes, arid
shrublands, coastal wetlands, and degraded
and burnt sites.
In the Sierra Nevada range (Spain), at an
elevation of 1800 m, Castro et al. (2002)
found that nurse shrubs decreased mortality in
two mountain pines without inhibiting their
growth. After two growing seasons, survival
of Scots pine (Pinus sylvestris) and European
Table 1. Experimental reports in which facilitation by nurse plants was used in restoration projects.
Environment Nurses Targets Reference Mediterranean mountain
Shrubs, legumes (Salvia, Genista)
Shrubs, trees (Pinus, Acer)
Castro et al. (2002); Gómez-Aparicio et al. (2004)
Semiarid steppes Perennial grass (Stipa)
Shrubs, trees (Quercus, Pinus)
Maestre et al. (2001, 2002); Gasque and García-Fayos (2004); Navarro-Cano et al. (pers comm)
Marshes Perennial grass (Spartina)
Deciduous shrub (Baccharis)
Egerova et al. (2003)
Tropical sub-humid forest
Trees (Acacia, Acalypha)
Tree (Brosimum)
Sánchez-Velásquez et al. (2004)
Arid shrubland Succulent shrubs (Drosanthemum)
Succulent shrubs (Drosanthemum)
Blignaut and Milton (2005)
Arid rangelands Shrub (Artemisia)
Grasses (Agropyron)
Huber-Sannwald and Pyke (2005)
This is not an exhaustive list of the species used
Capítulo IV
99
black pine (Pinus nigra) was markedly better
under sage (Salvia lavandulifolia) than in
control gaps (55 versus 22% and 82 versus
57%, respectively), and differences were still
present after four growing seasons (Castro et
al. 2004); survival was 1.8 to 2.6 times better
under sage than in gaps. When the nurse
plants were thorny shrubs such as Prunus
ramburii, establishment differed between the
north and south aspects of the plant; while
results in the north were similar to survival
levels seen under sage, in the south the results
were similar to those seen in open areas.
In the same Sierra Nevada range, but
including a wider altitudinal range (500–2000
m elevation), Gómez-Aparicio et al. (2004)
conducted a series of experiments to test the
effect of 16 native shrub species over 11 shrub
and tree species. One year after planting,
establishment success under shrubs was more
than double that seen in the gaps, reaching
fourfold higher numbers in some cases.
However, the outcome differed depending on
target species, type of nurse plant, and year.
The observed nurse effect of shrubs was
considerable for evergreen Mediterranean
species, such as Holm oak (Quercus ilex),
shrubs such as prickly juniper (Juniperus
oxycedrus), and deciduous species like maple
(Acer opalus), but was not significant for
pines (Scots and black pine). The most
successful nurse plant species were native
brooms (such as Genista spp), and small and
thorny shrubs. In contrast, a significant
negative influence was seen with rockroses
(Cistus spp), probably the result of
allelopathy. In fact, the harsher the ecological
conditions, the stronger the facilitative effect
of the nurse plants was.
A large number of experiments have been
carried out to test the potential of esparto
grass (Stipa tenacissima), a widespread
perennial tussock-forming grass, as a nurse
plant on degraded semiarid steppes in
southeast Spain. However, the results differed
depending on site, year, and target species
involved. Gasque and García-Fayos (2004)
found that the favorable conditions near
esparto grass tussocks increased germination
rate of Aleppo pine (Pinus halepensis; 43%
under Stipa versus 8% in control gaps) as well
as early establishment (19% versus 3% in
control gaps); after the summer drought,
however, all the plants died. Similar results
were obtained by Navarro-Cano et al. (pers
comm) with seedlings of Kermes oak
(Quercus coccifera) and Rhamnus lycioides,
and by Maestre et al. (2002) with Kermes oak.
Esparto grass increased germination and
survival before the drought period, but again
no plants survived beyond the summer. In
other experiments using seedlings of moon
trefoil (Medicago arborea), lentisc (Pistacia
lentiscus), and Kermes oak, Stipa did improve
survival after the drought period, and did not
affect plant growth (Maestre et al. 2001).
The role of nurse plants in restoration
100
Nurse plants have also helped in the
restoration of coastal marshes in Louisiana
(USA). Egerova et al. (2003) found higher
survival and growth rates in groundsel trees
(Baccharis halimifolia) growing inside clones
of the perennial smooth cordgrass (Spartina
alternifolia) than in gaps (45 versus 11%,
respectively), as a result of the more favorable
microclimate and soils.
In a secondary tropical dry forest,
Sánchez-Velásquez et al. (2004) looked at
four different types of nurse plants for
breadnut seedlings (Brosimum alicastrum).
Breadnut establishment after one year differed
depending on the type of species of nurse tree.
It was higher under Acalypha cincta and
guayabillo (Thouinia serrata; 55–40%) and
much lower (<5%) under thin acacia (Acacia
macilenta), trumpet tree (Tabebuia
chrysantha) and on open ground.
Blignaut and Milton (2005) looked at
survival of adult plants of three succulent
Karoo shrubs (Aridaria noctiflora,
Drosanthemum deciduum and Psilocaulon
dinteri) after transplanting. They moved all
three species either together or separately in
an arid shrubland in the Cape Province (South
Africa). Overall, survival of translocated
plants over the first 17 months was poorer for
clumped than for isolated plants.
The potential for seeding of native
bluebunch wheatgrass (Pseudoroegneria
spicata) and the introduced crested
wheatgrass (Agropyron desertorum), in the
vicinity of big sagebrush (Artemisia
tridentata) was examined by Huber-Sannwald
and Pyke (2005), as a means of thinning
woody shrubs in the Great Basin (USA)
rangelands. Sagebrush did not affect final
grass survival, but root interactions decreased
Figure 2. (a) A planted Aleppo pine thrives under the canopy of the drought-deciduous shrub Anthyllis cytisoides, which provides shelter against (b) high radiation levels in experiments on nurse plants conducted in dry mountains in Almería (SE Spain).
Capítulo IV
101
seedling biomass. Since light reduction (70–
90%) under sagebrush negatively affected
grass establishment, the authors recommended
seeding in gaps to minimize root interaction
with sagebrush as well as light interception.
Considerations for management
Successful tests in which seeds or
seedlings are placed near nurse plants
demonstrate the potential of this approach.
There are, however, several caveats regarding
species and site characteristics that could
influence the outcome and should be carefully
considered.
Ecological conditions
Using nurse plants is recommended for
restoring degraded sites where physical
conditions or grazing pressure seriously limit
establishment, since, where growing
conditions are optimal, spatial association
with such plants might not provide any
advantage. In such cases, the association
could have negative rather than positive
effects. Buckley (1984) found no positive
effects using nurse crops in fertile sites,
because their rapid growth depleted soil
resources, whereas in less fertile fields crops
grew less and the thinner cover improved the
survival of sycamore maple seedlings. In
research conducted by Marquez and Allen
(1996), at a site where soil resources and
climatic conditions did not constrain
establishment (reflected by 100% survival in
control plots) sagebrush seedlings growing
close to legumes were restricted rather than
favored by nurse plants.
The importance of facilitation increases
with increasing severity of the abiotic
conditions (Pugnaire and Luque 2001;
Callaway et al. 2002), and therefore the
possibility of benefiting from nurse plants
should also increase under such conditions.
Gómez-Aparicio et al. (2004), for example,
found that facilitation effects were stronger in
dry locations and on the south facing slopes of
a dry Mediterranean mountain.
Rainfall variability
In dry areas, changes in water availability
may make interactions among plants shift
from competition to facilitation and vice
versa, thereby increasing the importance of
facilitation during drought (Holmgren et al.
1997). This shift between positive and
negative effects may be relevant for nurse
plants success, since different results could be
obtained at the same site in different years,
depending on rainfall. Furthermore, in wet
years the nurse effect may not be as critical as
in dry years, because establishment may occur
without a nurse plant’s protection (see
Kitzberger et al. 2000). As described above,
Gómez-Aparicio et al. (2004) found that
shrubby nurse plants have considerable
influence on seedling survival in dry years,
but not in wet years. Similar results have been
The role of nurse plants in restoration
102
reported by Ibañez and Schupp (2001), in an
experiment conducted in Logan Canyon,
Utah, where they placed seedlings of curl-leaf
mountain mahogany (Cercocarpus ledifolius)
under big sagebrush; facilitation was apparent
in a dry year whereas negative effects were
seen during a wet year.
Nurse species
Selection of the best nurse species is an
important decision in restoration projects, as
this will determine the success or failure of
the project (Gómez-Aparicio et al. 2004;
Sánchez-Velásquez et al. 2004). In extreme
environments, the most suitable choices are
native species that are able to improve
environmental conditions for seedling
establishment. Although some exotic species,
such as black locust (Robinia pseudoacacia),
have been used successfully as nurse crops in
the south of England (Nimmo and Weatherell
1961), such options should be scrutinized
carefully because of the risk of biological
invasions. In heavily grazed sites, thorny,
non-palatable species are recommended,
although some herbivory and seed predation
may still occur, since the nurse plants may
actually provide refuge for small animals.
Species that release allelopathic compounds
should be avoided.
The nurse plant’s canopy structure may
also influence establishment success, in
particular in relation to shade intensity and
rainfall interception. The location of targets
under the canopy also affects seedling
survival (Castro et al. 2002), which is often
higher in the shadier positions. In a tropical,
sub-humid forest, the varying levels of
shading created by the nurse plants appeared
to be responsible for the variations in seedling
establishment reported by Sánchez-Velásquez
et al. (2004).
Many shrubs may limit water availability
in their understories by intercepting rainwater
during small precipitation events, making the
soil under shrubs dryer than in open areas
(Tielbörger and Kadmon 2000). Nonetheless,
during moderate to heavy rainfall, some
shrubs enhance water availability by directing
water intercepted by the canopy to the
understory through stemflow (García 2006).
Distance from the nurse plant is another
important factor; amelioration of negative
conditions and improved availability of
resources has been shown to decrease from
the canopy center outwards (Moro et al. 1997;
Dickie et al. 2005).
Factors such as competitive ability, use of
resources by the nurse plants themselves, and
the potential for root overlap between nurse
plants and target plants (Blignaut and Milton
2005; Huber-Sanwald and Pyke 2005) must
also be taken into account. Competition or
interference caused by species that occur
naturally under nurse plant canopies (eg
Capítulo IV
103
understory herbaceous species) may also
affect the outcome.
Target species
Interactions among plants depend upon
species characteristics, and thereby the
selection of target species (ie those being
restored) may influence the outcome of a
restoration project. Furthermore, the balance
of an interaction could be determined by the
ecological requirements of the species
involved and their ability to deal with
unfavorable abiotic conditions (see Bertness
and Hacker 1994, Liancourt et al. 2005;).
Walker et al. (2001), for example, reported
higher survival rates of Ambrosia dumosa in
the open than under shrubs in an arid
environment, because Ambrosia can
successfully cope with the conditions that
exist in open areas. Ambrosia was also
subjected to competition from the nurse shrub.
Gómez-Aparicio et al. (2004) reported that
shade-tolerant species and late-successional
shrubs showed a more positive effect in
response to nurse plants than did pioneer
shrubs and shade-intolerant pine trees (Castro
et al. 2002, 2004). In spite of this positive
influence, the nurse effect may be insufficient
to increase plant establishment if target
species have a low tolerance for the prevalent
abioitic conditions, or if these are particularly
severe. For example, Kitzberger et al. (2000)
and Maestre et al. (2002) found no seedling
establishment, either with or without nurse
plant protection, during especially dry years.
The age and size of target species must
also be considered, since several studies have
shown that the balance between facilitation
and competition varied with the life history of
plants. Nurse plants had strong positive
effects when the target species were relatively
young, but predominantly competitive
interactions were observed with older, larger
individuals (Callaway and Walker 1997;
Holmgren et al. 1997; Gasque and García-
Fayos 2004; Armas and Pugnaire 2005). The
use of plants of similar age and size, both as
nurse plants and target species, could have
exacerbated the negative effect of clumping
reported by Blignaut and Milton (2005).
Positive and negative effects of nurse plants
High recruitment rates close to nurse plants
do not preclude negative effects on target
species, but do ensure that the positive effects
outweigh the negatives ones. This may lead to
higher survival rates under nurse plants than
in gaps, but lower survival rates than those
seen when using other procedures, such as
artificial shading (Barchuk et al. 2005) or
watering (Sánchez et al. 2004).
Conclusions
The role of nurse plants in restoration
104
Published reports show that nurse plants
improve seedling establishment in some
systems, and that they may have potential for
use in restoration projects. Restoration
ecologists and land managers should take
facilitation effects into account, not only
because the role of facilitator species is key in
restoring the characteristics and functions of
the original system (Bruno et al. 2003), but
also because facilitation is believed to drive
succession in many habitats, particularly at
disturbed sites (Walker and del Moral 2003).
We see the need for additional
experiments, conducted under a variety of
environmental conditions and using different
nurse plant species, to identify the potential of
this process, and to encourage long-term
monitoring of target–nurse plant interactions.
Research aimed at determining the nurse
species’ zones of influence and their effects
on neighboring plants under differing
conditions of resource availability, will
provide us with a valuable technique for
improving the success of restoration projects.
Acknowledgements
This work was funded by the Spanish
Ministry of Science and Technology (grant
AGL2000-0159-P4-02). We thank Serfosur
SL for assistance during this project. James S
Gray made helpful comments on an earlier
draft of this manuscript.
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The role of nurse plants in restoration
108
Intense human pressure in the last centuries including agriculture but also overgrazing, burning, and logging for mining and shipyards, deforested most mountainous areas in SE Spain, like the Sierra Nevada (a) and Sierra Alhamilla (b) foothills. Woodland restoration in such sites is frequently impeded by drought spells and grazing; using nurse plants may improve the success of restoration projects.
Capítulo V
Las condiciones ambientales y el uso de plantas nodriza en restauración
Capítulo V
111
Capítulo V
ENVIRONMENTAL CONDITIONS AND THE USE OF NURSE PLANTS IN
RESTORATION PROJECTS
Summary
Seedling establishment in harsh environments is facilitated in the proximity of adults of some
species. This effect has successfully been applied to ecosystem restoration by placing species being
restored under the canopy of plants that act as nurses; however, there is a lack of long-term
monitoring of these processes, and comparisons with technical procedures that also provide
protection are scant. We tested the potential of the leguminous shrub Retama sphaerocarpa against
artificial protection that mimicked a nurse plant’s canopy. Retama shrubs form fertile islands where
environmental conditions are tempered and soil resource availability is improved compared to gaps
between shrubs, and we hypothesized that Retama fertile islands would enhance seedling survival
to a greater extent than artificial canopies. We planted seedlings of three shrubs (Olea europaea,
Pistacia lentiscus and Ziziphus lotus) either in Retama microsites or under artificial canopies
created with piled branches, and irrigated in summer half the seedlings to look at how water
availability affected the interaction between nurse plants and understorey seedlings. Retama islands
enhanced seedling survival over three growing seasons, but the outcome was species-specific and
depended on resource availability. Survival rate of Olea under Retama doubled that under artificial
canopies if not watered, whereas Pistacia resulted neither facilitated nor outcompeted. In contrast,
Retama had negative effects on Ziziphus, and most seedlings transplanted in this microsite did not
stand the first summer drought. Competitive abilities of the species likely accounted for such
discrepancy in nursing success since Ziziphus benefited from protection provided by artificial
canopies. Facilitation by Retama fertile islands was more apparent under dryer conditions –i.e., in a
dry year and without irrigation. According to the proposed stress-gradient hypothesis, competition
increased and facilitation decreased as water stress was lessened by rainfall or watering. Overall,
Retama fertile islands proved more beneficial for Olea survival than artificial protection in a
Mediterranean dry environment over a three-year period, and particularly in a very dry year.
Seedlings benefited from higher soil water availability and more fertile soils underneath Retama,
along with climatic amelioration. This shows that nursing has potential to become a relevant
technique in practice and contribute to more successful restorations; however, species identity
plays a major role, and seedling competitive-response ability determines the success of this
technique.
Seedling establishment in nurse plants
112
Introduction
Seedling establishment is the most critical
phase in the plant’s regeneration process
because young seedlings are very susceptible
to hazards like extreme temperatures and
radiation and soil desiccation (Franco and
Nobel 1989). High mortality rates are often
associated to the seedling stage, and the
ultimate determinant of recruitment success is
the microsite where seeds do germinate
(Schupp 1995). Competition with existing
vegetation has been pointed out as a factor
involved in recruitment failure (Tyler and
D’Antonio 1995, Ladd and Facelli 2005);
however seedling establishment in harsh
environments is often enhanced (i.e.,
facilitated) in the vicinity of adults of some
species that act as nurses, the so-called “nurse
effect” (Niering et al. 1963). Research over
the past years showed complex, and likely
synergistic mechanisms underlying the nurse
effect, coarsely related to climatic
amelioration (Franco and Nobel 1989,
Valiente-Banuet and Ezcurra 1991) and
protection from herbivory (Rousset and
Lepart 2000).
The establishment of seedlings under the
canopy of some species, however, does not
preclude negative effects of nurses on them.
Neighboring plants influence each other in
positive and negative ways, and the final
outcome is determined by the net balance
between these effects (Callaway 1995,
Callaway and Walker 1997, Holmgren et al.
1997, Pugnaire and Luque 2001). While
providing shelter, nurses can compete with
understorey seedlings for resources, and
survival may increase or decrease depending
on whether positive effects balance negative
ones. The importance of such effects changes
through time and space (Holzapfel and Mahall
1999, Tielbörger and Kadmon 2000, Armas
and Pugnaire 2005) and so does the net
balance in response to conditions like seedling
stress tolerance and competitive ability
(Bertness and Hacker 1994, Liancourt et al.
2005), life stage (juvenile vs. adults [Callaway
and Walker 1997, Armas and Pugnaire 2005,
Miriti 2006]), and abiotic harshness (Greenlee
and Callaway 1996, Brooker and Callaghan
1998, Pugnaire and Luque 2001, Callaway et
al. 2002, Cavieres et al. 2006, Sthultz et al.
2007). The importance of facilitation on
recruitment also depends on evolutionary
lineages. Valiente-Banuet et al. (2006) have
shown that the nurse effect is much more
important for recruitment of species evolved
in the Tertiary period (when species are
presumed to be less drought-tolerant, large-
seeded and fleshy-fruited) than for species
origined in the Quaternary; the former
recruiting preferably in mesic, cool
understories, and the later in open, harsher
environments.
The incorporation of facilitation in
ecosystem restoration has received growing
attention (Halpern et al. 2007), and especially
Capítulo V
113
the application of the nurse effect (see review
in Padilla and Pugnaire 2006). The area under
the canopy of certain species that act as nurses
is an appropriate site to plant seedlings of
perennial species being restored in a wide
range of environments, such as Mediterranean
mountains (Castro et al. 2002, Gómez-
Aparicio et al. 2004), semiarid steppes
(Maestre et al. 2001, Gasque and García-
Fayos 2004), coastal marshes (Egerova et al.
2003), tropical dry forests (Sánchez-
Velásquez et al. 2004), and dry Afromontane
savanna woodlands (Aerts et al. 2007).
However, most research was conducted by
comparing seedling performance under nurse
plants and in gaps (but see Gómez-Aparicio et
al. 2005), in contrast to common practices that
provide seedlings with some protection
against environmental conditions and
herbivory (Ludwig and Tongway 1996,
Pemán and Navarro 1998). Thus, the potential
of nurse plants in restoration might have been
overestimated, and therefore comparisons of
seedling survival under nurse plants versus
artificial protection, rather than gaps, may be
appropriate to fully acknowledge its potential
for practitioners in realistic terms. In addition,
research most often suffered from a lack of
long-term monitoring, focusing on seedling
performance restricted to a single growing
season (but see Castro et al. 2004). Thereby,
outcomes could have been determined by
particular climatic conditions and rainfall,
shown to determine the outcome of biotic
interactions (Kitzberger et al. 2000, Gómez-
Aparicio et al. 2004, Maestre and Cortina
2004, Sthultz et al. 2007).
In Mediterranean ecosystems, the
leguminous shrub Retama sphaerocarpa (L.)
Boiss. is well known because of its facilitative
effects. The Retama canopy buffers extreme
temperatures and radiation reaching the soil
surface, while its open structure allows light
to pass sufficiently. Moreover, Retama forms
fertile islands with higher organic matter,
nitrogen, water content, and improved clay
fraction and texture compared with between-
shrub spaces (Pugnaire et al. 1996, Moro et al.
1997a,b, Pugnaire et al. 2004, López-Pintor et
al. 2006), facilitating the establishment of
many small shrubs and herbaceous species
(Pugnaire et al. 1996, Rodríguez-Echeverría
and Pérez-Fernández 2003). Mediterranean
ecosystems experience a strong drought in
summer that threatens seedling establishment
(Padilla and Pugnaire 2007), and here we test
the potential of Retama as a nurse plant for
restoration, hypothesizing that Retama fertile
islands would help survive seedlings planted
under its canopy to a greater extent than
artificial protection provided by piled
branches due to soil effects (i.e., improved
fertility) in the Retama understorey.
Materials and Methods
Experimental site
Seedling establishment in nurse plants
114
We chose two environments with
contrasting abiotic conditions, selecting two
1-ha plots on opposite, moderate slopes in the
foothills of the Sierra Alhamilla range
(Almería, Spain, 37º99’N, 02º99’W, 650 m
elevation). Plant communities, soil, and slope
were very similar in both plots, differing only
in aspect; there was a relatively more humid,
east-facing slope and a relatively drier, west-
facing slope. The climate is Mediterranean
semi-arid with a mean annual temperature of
17.3 ºC with mild temperatures in winter and
high in late spring and summer, and 282 mm
of annual precipitation, with a marked drought
period from June to September. Plant
community is a degraded shrubland
dominated by the drought-deciduous shrub
Anthyllis cytisoides L. and scrubs such as
Artemisia barrelieri Bess. and Thymus
hyemalis Lange, interspersed with the large
shrub Retama sphaerocarpa, annual grasses
and herbs. Other shrub species belonging to
this community but almost absent are Olea
europaea L. var. sylvestris Brot., Pistacia
lentiscus L. and the thorny Ziziphus lotus (L.)
Lam. (Mota et al. 1997). Soils are loamy-
sandy, calcic regosols developed over a mica-
schist bedrock.
Species and experimental design
We tested the effect of Retama nurses on
Olea europaea var. sylvestris (Oleaceae),
Pistacia lentiscus (Anacardiaceae), and
Ziziphus lotus (Rhamnaceae). All three
species share reproductive traits such as fleshy
fruits and large seeds, and functional traits
such as sclerophylly, deep roots, and low
drought tolerance, pointing to a Tertiary
origin (Valiente-Banuet et al. 2006). In
January 2004, one-year-old seedlings of these
species, provided by local nurseries, were
transplanted in both slopes either under the
canopy of the shrub Retama sphaerocarpa
(Retama islands hereafter) or randomly in
gaps covered with piled branches of the shrub
Anthyllis cytisoides (artificial canopy)
imitating a nurse canopy (Figure 1). Selected
Retama shrubs were similar in age and height,
and lacked perennial species in their
understories. Seedlings were planted as close
to the trunk of Retama as possible since
amelioration of climatic extremes and
improved availability of resources decrease
from the canopy center outwards (Moro et al.
1997a), and on the upslope side of the shrub
to take advantage of the small soil mounds
formed by sediment accretion. At transplant,
we dug a 0.5-m-deep hole using an auger (BT
120 C, Stihl AG & Co. KG, Waiblingen,
Germany). Since summer drought is the major
constraint on seedling survival, and
competition for water is often more important
than competition for light or nutrients in dry
habitats (Casper and Jackson 1997), half the
seedlings were watered six times in the
summer of 2004 and 2005 every three weeks.
Around 2.5 L of water were supplied at the
root level through a pipe reaching 20 cm
depth in the soil (Sánchez et al. 2004).
Capítulo V
115
Figure. 1. Plantation of seedlings in gaps covered with piled branches of the shrub Anthyllis cytisoides (left) and under the canopy of the leguminous shrub Retama sphaerocarpa (right) in a degraded environment in the Sierra Alhamilla range (Almería, SE Spain).
Abiotic environment
We used sensors to record soil temperature
(Campbell Scientific Ltd, Leicestershire, UK)
and photosynthetically active radiation (PAR
quantum sensor SKP 215, Skye Instruments
Ltd, Powys, UK) at ground level under three
randomly selected Retama shrubs, under three
artificial canopies, and in two gaps. Data
collected for six days in March 2006 on a
sunny spell were recorded every minute and
averaged every ten minutes in a CR10X data
logger (Campbell Scientific Ltd,
Leicestershire, UK). Rainfall was collected
with a pluviometer (Davis Instruments Corp,
Hayward, CA, USA) and recorded daily
(Hobo, Onset Computers, Pocasset, MA,
USA) along the three years of
experimentation.
Survival, growth and physiological status
Since summer drought is the main threat to
seedlings in the Mediterranean (Padilla and
Pugnaire 2007), seedling survival was
recorded before and after the summer of 2004,
2005 and 2006. Survival rate was calculated
as a percentage of plants alive in spring 2004,
excluding this way seedling deaths caused by
transplant. In late spring 2006, plant growth
was assessed by measuring main shoot
extension with a digital caliper on 26 May and
30 June. Seedling physiological status was
also assessed in late June by measuring early
morning photochemical efficiency of
photosystem II (Fv/Fm) on 30-minute dark-
adapted leaves with a portable fluorimeter
(PEA, Hansatech Instruments Ltd., Kings
Lynn, UK). We also collected 5 to 15 leaves
similar in size and from the same aspect of
each plant to calculate specific leaf area (SLA,
Seedling establishment in nurse plants
116
cm2 g-1). Leaves were scanned with a portable
scanner at 300 dpi (Epson GT7000, Seiko
Epson Corp., Nagano, Japan), and the
projected leaf area was measured with the
software Midebmp v.4.2 (R. Ordiales-Plaza,
2000). Due to their small size, all leaves from
each plant were combined, scanned and
weighed after drying for at least 48 hours at
72ºC; SLA was computed as the ratio between
leaf area and mass.
Statistics
Differences among canopy treatments
concerning daily mean, maximum and
minimum air temperature and radiation were
tested through one-way ANOVA, followed by
Tukey post hoc comparison tests when
significant differences were found. When
variables were heteroscedastic, we ran the
Kruskal-Wallis non-parametric test, followed
by Mann-Whitney tests for paired
comparisons. For radiation analysis we only
considered the daylight time period, between
7:00-17:30 solar time, when PAR >100 μmol
m-2 s-1. For each species, seedling survival
analysis in early autumn was performed by
simple binary logistic regression where
survival was the dependent variable, and
aspect (east and west), watering (irrigated and
control), and canopy (Retama islands vs.
artificial canopies) were the predictor factors.
This is the appropriate method for analyzing
categorical variables where clearly one of
them is the response variable (Agresti 2002).
In Olea and Ziziphus, the statistical design
consisted of a three-factor factorial (Aspect x
Watering x Canopy); in Pistacia, however, the
design comprised two factors (Watering x
Canopy) because replication in the east plot
was very small. Logistic regression started
from the saturated model, and significance of
interactions and main factors were determined
through backwards elimination, first of
higher-order interaction terms and then of
main factors, and by comparing the goodness-
of-fit (G2) between the model with an
eliminated term and the preceding model
using the χ2 distribution as a significance
contrast (Tabachnick and Fidel 2001). Sample
size of each treatment ranged 15-28 plants
because of differing dieback caused by
transplant. Differences between treatments in
growth rate, SLA and Fv/Fm of irrigated Olea
and Pistacia seedlings were assessed by
independent two-way factorial analysis of
variance (Aspect x Canopy) followed by
Tukey tests. Ziziphus species and non-
irrigated seedlings were excluded from
analyses because very few seedlings remained
alive at the end of the experiment. Analyses
were conducted with the SPSS v14.0
statistical package (SPSS Inc., Chicago, IL,
USA) with significant differences set at P<
0.05. Data are presented as means ± 1
standard error.
Results
Capítulo V
117
Abiotic environment
Both Retama islands and artificial canopies
buffered air temperature and radiation
reaching the soil surface (Figure 2). Daily
mean temperature in both canopy treatments
was reduced by 3 ºC in comparison to open
areas (Table 1). Canopies also ameliorated
extreme temperatures, although the buffering
was greater under artificial canopies (up to 17
ºC lower than in gaps) than in Retama islands
(9 ºC). Radiation levels under both canopies at
noon were similar at around 500 μmol m-2 s-1,
which contrasts with radiation >1200 μmol m-
2 s-1 in gaps. Daily mean PAR under artificial
canopies and in Retama microsites was
reduced by 70 and 56 % when compared with
gaps, as well as maximum PAR (40%
reduction for both canopy treatments).
Table 1. Air temperature and photosynthetically active radiation (PAR) in spring 2006 under Retama shrubs, under artificial canopies, and in gaps; F-values of one-way ANOVA († H-value of Kruskal-Wallis test). Significant differences among canopy treatments are indicated at P<0.05 by differing lower-case letters after Tukey test (paired Mann-Whitney for PARmax). Daily temperature and PAR values are means (± 1 SE) of six days with three replicates for Retama islands and artifical canopies, and two for gaps. ***, P<0.001.
In gaps Artificial canopies Retama islands F
Temperature (ºC) Mean 16.1±0.6a 13.1±0.2b 13.3±0.3b 17.40***
Max 40.9±1.0a 23.8±0.5b 31.8±0.8c 128.89***
Min 2.1±0.4a 5.3±0.2b 3.1±0.3c 35.32***
PAR (μmol m-2 s-1) Mean 902.4±23.2a 275.1±20.6b 396.6±13.7c 263.36***
Max 1554.3±18.1a 900.9±91.4b 954.4±32.4b 26.55***†
Min 123.8±5.5a 44.3±1.5b 66.1±1.8c 217.08***
Survival
A rainy spring in 2004 was followed by a
summer with negligible rainfall (Table 2).
Watering during the first summer did enhance
survival rate in all three species (P<0.001,
Table 3), with irrigated seedlings of Olea and
Pistacia showing survival rates close to 100%
Figure. 2. Daily changes in soil temperature (above) and photosynthetically-active radiation (below) in the three canopy treatments. Values are means of three probes for Retama islands and artificial canopies, and two for gaps.
Seedling establishment in nurse plants
118
in autumn, and above 50% in Ziziphus under
artificial canopies (Figure 3). Regardless of
aspect and water supply, Retama plants had a
negative effect on Ziziphus (P<0.01), which
survived more under artificial canopies than in
Retama islands. In Olea and Pistacia,
however, survival rates were not affected by
Retama. Low rainfall characterized year 2005
(Table 2), which was also marked by a very
dry spring and summer. In this dry year,
summer irrigation also reduced mortality in all
three species (P<0.001). Retama shrubs
significantly enhanced the survival rate of
Olea, although the effect depended on water
supply (Watering x Canopy interaction,
P=0.02). Survival of non-irrigated seedlings
planted in Retama islands was greater than
under artificial shade (46±1 vs. 22±2 %), but
irrigated seedlings survived slightly less under
Retama (62±2 vs. 72±7 %, Figure 1). In
autumn 2006, an average year in terms of
annual and spring rainfall, seedlings that had
been irrigated in preceding summers (2004
and 2005) kept showing higher survival rates
than non-irrigated (P<0.01). Differences in
survival between microsites did increase in
Olea (P<0.01), being 48-56% higher for non-
irrigated seedlings planted in Retama islands
than for seedlings placed under artificial
canopies. Irrigated seedlings, in contrast,
survived more under artificial canopies.
Pistacia showed similar patterns but no
significant effects were detected.
Table 2. Seasonal and annual rainfall (in mm) in the years 2004, 2005, and 2006, and average of the 1950-2000 period (Confederación Hidrográfica del Sur) in the experimental site.
Season Year
Winter Spring Summer Autumn Annual
2004 90 205 2 18 315
2005 73 35 6 30 145
2006 113 90 26 52 281
Average 91 82 16 92 281
Growth and physiological status
Neither seedling growth nor specific leaf
area of Pistacia and Olea plants were affected
by nurses nor influenced by aspect (P>0.15,
Table 4). In the east-facing slope,
physiological status of Pistacia seedlings
planted under artificial canopies was slightly
better than in Retama islands, with higher
Fv/Fm values, whereas such differences were
non-significant in the western aspect (Aspect
x Canopy interaction, P=0.02). Fv/Fm values
of Olea seedlings were unaffected by Retama
shrubs.
Capítulo V
119
Table 3. Results of logistic regression performed with survival as the response variable and aspect (A), watering supply (W), and canopy treatment (C) as predictor variables in autumn 2004, 2005 and 2006 for Olea europaea var. sylvestris, Pistacia lentiscus and Ziziphus lotus. Bold letters show significant differences at P<0.05. Analyses could not be performed for Pistacia because of the low survival in the east aspect.
Aspect (A) Watering (W) Canopy (C) A x W A x C W x C A x W x C
Year Species χ2 P
χ2 P χ2 P χ2 P χ2 P χ2 P χ2 P
2004 Olea 0.114 0.736 49.434 <0.001 0.048 0.827 2.654 0.103 0.319 0.572 0.001 0.975 0 1
Pistacia - - 20.995 <0.001 2.653 0.103 - - - - 0.759 0.384 - -
Ziziphus 0.005 0.944 31.773 <0.001 8.718 0.003 0.709 0.340 1.299 0.254 0.003 0.956 3.588 0.058
2005 Olea 0.015 0.903 17.555 <0.001 0.983 0.321 0.241 0.623 0.251 0.616 5.281 0.022 0.552 0.458
Pistacia - - 16.123 <0.001 0.633 0.426 - - - - 1.239 0.266 - -
Ziziphus 0.064 0.800 28.441 <0.001 16.541 <0.001 1.521 0.217 1.492 0.222 0.191 0.662 0 1
2006 Olea 0.100 0.752 15.691 <0.001 0.052 0.820 3.808 0.051 0.014 0.906 6.879 0.009 0.195 0.659
Pistacia - - 8.463 0.004 0.109 0.741 - - - - 0.607 0.436 - -
Ziziphus 0.060 0.806 15.958 <0.001 28.972 <0.001 1.521 0.217 1.425 0.233 0.180 0.671 0 1
Seedling establishment in nurse plants
120
Figure 3. Seedling survival of Olea europaea var. sylvestris, Pistacia lentiscus and Ziziphus lotus in east and west-facing slopes, in Retama sphaerocarpa fertile islands and under artificial canopies, and with summer irrigation and control (n = 15-28). Some data for Pistacia in east aspect are not available because of low survival.
Capítulo V
121
Table 4. (a) Specific leaf area (SLA, cm2 g-1), shoot growth (mm wk-1), and chlorophyll fluorescence (Fv/Fm) in spring 2006 of irrigated seedlings of Olea europaea var. sylvestris and Pistacia lentiscus planted under Retama sphaerocarpa shrubs and under artificial canopies in east and west-facing slopes. Values are means ± 1 SE.
(a) East aspect West aspect
Species Variable Retama islands Artificial canopies Retama islands Artificial canopies
Olea SLA 56.34±6.47 52.49±1.80 58.93±3.23 56.53±3.77
Growth 10.93±3.78 4.94±1.27 6.48±1.87 5.46±1.94
Fv/Fm 0.72±0.02 0.72±0.02 0.76±0.03 0.70±0.04
Pistacia SLA 62.55±7.75 55.38±2.73 58.72±6.32 54.99±3.75
Growth 0.72±0.25 0.86±0.66 2.24±1.45 2.84±1.37
Fv/Fm 0.64±0.03 0.76±0.01 0.77±0.02 0.75±0.03
Table 4. (b) Results of two-way ANOVA for the effects of aspect and canopy protection (n = 5-10). Bold shows significant differences at P<0.05.
(b) Aspect (A) Canopy (C) A x C
Species Variable F P F P F P
Olea SLA 0.629 0.433 0.559 0.459 0.030 0.863
Growth 0.668 0.419 2.122 0.154 0.067 0.309
Fv/Fm 0.071 0.791 1.188 0.283 0.811 0.374
Pistacia SLA 0.103 0.751 0.687 0.414 0.069 0.795
Growth 1.175 0.287 0.156 0.696 0.563 0.459
Fv/Fm 4.453 0.043 2.688 0.112 5.668 0.024
Discussion
Our hypothesis that fertile islands formed
by the leguminous shrub Retama
sphaerocarpa would enhance survival of
seedlings planted under its canopy holds only
in part because growing in Retama microsites
resulted beneficial or detrimental depending
on seedling identity, which evidences that the
interaction with Retama is species-specific
(Callaway 1998). Thus, while most seedlings
of Ziziphus lotus planted under Retama died
the first summer, survival of Olea europaea
var. sylvestris in Retama microsites doubled
that of artificial canopies under natural rain
conditions (i.e., without watering supply).
This shows that seedling identity determines
the success of nurse plants, and highlights the
potential for planting species like Olea
europaea under the canopy of nurse shrubs in
Seedling establishment in nurse plants
122
the restoration of degraded, semi-arid
environments. This agrees with reports of
enhanced survival of the con-specific Olea
europaea var. cuspidate under the canopy of
Euclea racemosa in a dry Afromontane
savanna woodland (Aerts et al. 2007).
Moreover, growing evidence supports the
role of nurse plants in terrestrial restoration
(Padilla and Pugnaire 2006), but here, by
using a control treatment consisting of
artificial canopies rather than gaps, we truly
tested the potential of this procedure because
common restoration practices usually protect
seedlings against environmental harshness
through shelter tubes (Pemán and Navarro
1998) or brush (Ludwig and Tongway 1996).
In this sense, our data are relevant, and
consistent within the climatic variability of
our study site, because Retama islands proved
more beneficial for seedling survival than
artificial canopies in Olea over three years of
contrasting rainfall, and apparently neutral in
Pistacia.
Both protection provided by Retama shrubs
and artificial canopies did buffer maximum
and mean soil temperature and radiation
reaching the soil surface, but artificial
canopies were slightly more effective in
lowering extreme temperatures and PAR than
Retama canopies. However, despite this fact,
survival of Olea seedlings was greater in
Retama fertile islands. Our experiment design
does not allow to isolate soil versus canopy
effects underlying facilitation by Retama, but
our data evidence that seedlings benefited
from soil effects rather than from canopy
protection by itself. Canopy effects have often
been pointed as the main mechanism involved
in the nurse effect (Valiente-Banuet and
Ezcurra 1991, Callaway 1992, Maestre et al.
2001, Maestre, et al. 2003, but see García-
Moya and McKell 1970, Gutiérrez et al. 1993,
Pugnaire et al. 2004), and here we show that
the improvement of soil resources have a
synergistic effect when compared to solitary
canopy protection effects. In support of this
finding, Gómez-Aparicio et al. (2005) also
found that survival of tree species in a
Mediterranean mountain was generally
intermediate under artificial canopies (canopy
effects) and in sites where shrub canopies
were clipped (soil effects), but seedling
survived most under nurse plants, evidencing
that canopy and soil effects together were
responsible for enhanced survival in cases of
severe stress.
Shade provided by Retama shrubs decreased
radiation reaching the soil and lowered soil
and air temperature, and therefore also
reduced soil water evaporation; these
conditions have an important bearing on
photoinhibition and evapotranspiration, and in
fact can improve plant water status (Vetaas
1992, Valladares and Pearcy 1997, Armas and
Pugnaire 2005). Shade may have negative
effects if radiation under the canopy is
limiting; however, it is unlikely here because
Capítulo V
123
mid-day radiation levels under the sparse
canopy of Retama and piled branches was
>500 μmol m-2 s-1, usually a non-limiting level
for Mediterranean species (Valladares et al.
2005). The leguminous shrub Retama
sphaerocarpa is well known because of its
ability to modify the neighboring soil
environment. Soils under Retama shrubs are
more fertile than in open areas (Pugnaire et al.
1996, Moro et al. 1997b, Rodríguez-
Echeverría and Pérez-Fernández 2003,
Pugnaire et al. 2004, López-Pintor et al.
2006), and seedlings can have benefitted from
these improvements. Seedlings may also take
advantage of water released by Retama roots
through hydraulic lift (I. Prieto and Z.
Kikvidze, unpublished data), a process that
improves neighbors’ water status (Dawson
1993, Peñuelas and Filella 2003). Overall,
since we report enhanced seedling survival
but not facilitation for growth, the alleviation
of summer drought appears as the main
survival mechanism in this environment,
which is consistent with reports by Liancourt
et al. (2005).
We found that survival of Olea seedlings
was not affected by Retama islands the wet
year (2004), whereas in the very dry year
(2005) and average year (2006) Retama
shrubs did facilitate seedlings. In addition,
irrigated seedlings survived under artificial
canopies better than in Retama islands,
showing that higher water availability reduced
facilitation by Retama and led to competitive
interactions. Consistent with the stress-
gradient hypothesis (Bertness and Callaway
1994), facilitation decreased and competition
increased as water stress was alleviated by
watering or rainfall. The root system of
Retama consists of a dense layer of fine roots,
mainly in the top 20 cm of the soil, and
several deeply penetrating tap roots (Haase et
al. 1996). This dual system maximizes water
uptake from deep sources over the seasonal
drought, and from the upper soil horizons
when water is available after rain or watering
(Schwinning et al. 2002). Thus, irrigated
seedlings planted in nurse plants were
subjected to competition with Retama’s
shallow roots for water supplied whereas
seedlings in artificial canopies were not.
Contrary to our expectations, we found that
survival of Ziziphus seedlings planted under
artificial canopies was higher than in Retama
islands. Walker et al. (2001) in experimental
manipulations of fertile islands, and Gómez-
Aparicio et al. (2004) in a meta-analysis,
reported negative effects of nurse plants on
pioneer and shade-intolerant species, and
Liancourt et al. (2005) showed that facilitation
of stress-sensitive species was stronger than of
stress-tolerant species, because these species
can successfully cope with conditions
prevailing in open areas. Ziziphus, however,
had greater survival under irrigated artificial
canopies than in Retama islands, and this
evidences that mortality was not caused by
shade; incidentally, it can also show that the
Seedling establishment in nurse plants
124
regeneration niche of Ziziphus is not linked to
open areas. Ziziphus survival in the wet 2004
was lower than that of non-irrigated seedlings
of Olea planted outside Retama, which
suggests that Ziziphus has a more stress-
sensitive behavior than Olea, and therefore
rules out its stress tolerance as the cause of
failure in nurse plants. Rather, competitive
ability can account for differences in nursing
success between Olea and Ziziphus.
Facilitation is expected in species that tolerate
the negative effects of neighbors (i.e., have
strong competitive ability), minimizing costs
of negative interactions and maximizing
benefits (Brooker and Callaghan 1998,
Liancourt et al. 2005). Our survival data
reflect that Ziziphus is bound to be a weaker
competitor than Olea and it did not stand
competition from Retama.
Reports have shown that the herbaceous
community under Retama has negative effects
on seedling establishment (Espigares et al.
2004); however, this is unlikely in our system
because mortality in our species took place in
summer, when annual species are senesced.
As for Pistacia, we found that the
interaction with Retama was apparently
neutral, which contrasts with works reporting
a positive effect of the nurse plant Stipa
tenacissima on this species in semiarid
steppes (Maestre et al. 2001, Maestre et al.
2003). Nevertheless, it is worth noting that
Maestre’s control treatment consisted of
plantation in gaps, which entails a much more
stressful microsite than our artificial canopies.
Overall, our data show that fertile islands
have real potential in the restoration of
degraded, dry environments, as they enhance
seedling survival to a greater extent than
artificial protection. However, resource
availability and species competitive response
play a critical role in determining nursing
success, and should be considered carefully.
Acknowledgements
We are very grateful to Serfosur SL for
technical assistance in this project, and
especially to Rafael Ortega for suggestions on
the experiment. We also thank Alejandro
Moreno for field help, and Cristina Armas for
comments on an earlier version of this paper.
This project was founded by the Spanish
Ministry of Education and Science (grants
AGL2000-0159-P4-02 and CGL2004-
00090/CLI).
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Soil-digging in the experimental plots in January 2004 (Courtesy of Rafael Ortega).
Conclusiones
Conclusiones
133
FACTORES LIMITANTES Y ESTRATEGIAS DE ESTABLECIMIENTO DE
PLANTAS LEÑOSAS EN AMBIENTES SEMIÁRIDOS. IMPLICACIONES PARA
LA RESTAURACIÓN
Conclusiones
A continuación se enumeran las principales conclusiones obtenidas en la presente tesis doctoral:
1. La disponibilidad de agua en el suelo afecta a las primeras etapas del desarrollo de plántulas
de especies mediterráneas. La elongación de las raíces en respuesta a una disminución de la
humedad es independiente de la resistencia a la sequía y del tamaño de semilla y cotiledones,
y constituye una estrategia para captar los recursos hídricos necesarios.
2. La supervivencia de las plántulas en ambientes mediterráneos está controlada por la
presencia de umbrales de humedad del suelo. Existe un valor mínimo bajo el cual la
humedad no es suficiente para que las plantas sobrevivan, impidiendo el establecimiento. En
cambio, por encima de ese valor la supervivencia está asegurada.
3. La capacidad de las plántulas de desarrollar raíces profundas es decisiva para sobrevivir la
sequía estival mediterránea, independientemente de la mayor o menor resistencia a la sequía.
En cambio, una mayor asignación de biomasa a la parte radical con respecto a la parte aérea
no es suficiente para compensar el descenso de la humedad del suelo.
4. El comportamiento de las raíces ante cambios en la dinámica de desecación del suelo es muy
plástico, maximizando la toma de recursos a través de cambios en la asignación de biomasa y
la arquitectura radical.
5. Las especies arbustivas que aparecen en el extremo semiárido de la Península Ibérica están
bien adaptadas a una disponibilidad de agua del suelo variable, de manera que son necesarios
periodos de sequía prolongados para reducir sus tasas de crecimiento.
6. El empleo como plantas nodriza de la vegetación pre-existente tiene un gran potencial en
restauración. Sin embargo, el tipo de plantas nodriza, las características de las especies y las
condiciones del sitio a restaurar influyen en su éxito.
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7. Las plántulas colocadas bajo la cubierta de Retama sphaerocarpa se benefician de la
protección microclimática y de una mayor disponibilidad de recursos en el suelo, lo que
mejora su tasa de supervivencia. Sin embargo, este efecto es beneficioso sólo para aquellas
especies que toleran los efectos negativos de Retama, minimizando costes y maximizando
beneficios.
8. Los arbustos de Retama proporcionan a las plántulas hábitats más adecuados que la
protección artifical, por lo que su uso se debe implementar en restauración.
9. En condiciones de estrés hídrico atenuado, el balance de la interacción entre la planta nodriza
y la plántula se decanta hacia el lado de la competencia, mientras que los efectos positivos
son más aparentes bajo condiciones de sequía.
