Efecto de retama monosperma sobre el endemismo amenazado Thymus carnosus: luz, agua y patrón de distribución espacial en un sistema costero dunar

Efecto de Retama monosperma sobre el endemismo amenazado Thymus carnosus: Luz, agua y patrón de distribución espacial en un sistema costero dunar M. Paz Esquivias Segura Tesis doctoral 2012 DEPARTAMENTO DE BIOLOGÍA VEGETAL Y ECOLOGÍA FACULTAD DE BIOLOGÍA UNIVERSIDAD DE SEVILLA Efecto de Retama monosperma sobre el endemismo amenazado Thymus carnosus: Luz, agua y patrón de distribución espacial en un sistema costero dunar Memoria presentada por la Licenciada M. Paz Esquivias Segura para optar al título de Doctora por la Universidad de Sevilla, bajo la dirección de Dra. María Zunzunegui González Dr. Juan B. Gallego Fernández Sevilla, 6 de octubre de 2012 RESUMEN Las interacciones planta-planta suponen una parte fundamental de la ecología de las especies vegetales. Conocer los mecanismos subyacentes a las interacciones y la respuesta de las distintas especies a la presencia de otras plantas supone completar una información muy valiosa de su biología, ya que raramente ocurren de manera aislada en la Naturaleza. En esta tesis doctoral se aborda el estudio de la interacción entre Retama monosperma y Thymus carnosus. Ambas son especies nativas mediterráneas características de sistemas costeros dunares. Sin embargo R. monosperma ha mostrado en las últimas décadas un marcado carácter invasivo en las costas de Andalucía occidental; en cambio T. carnosus está considerada como especie en regresión y riesgo de extinción. Este estudio se realizó en un área compartida por ambas, la Flecha de El Rompido (Huelva, suroeste español). La mayor parte del trabajo se centró en estudiar la respuesta de ambas especies al uso de la luz y del agua en condiciones naturales. Los resultados mostraron que el efecto de la sombra de R. monosperma junto con los períodos de sequía ambiental crean unas condiciones negativas para T. carnosus, cuya respuesta fisiológica es peor que en ausencia de la influencia de R. monosperma. Profundizando en los mecanismos de uso del agua, se ha puesto de manifiesto que la presencia de R. monosperma modifica las fuentes de agua utilizadas por T. carnosus, que cuando está aislada puede utilizar agua subterránea y sin embargo en interacción con R. monosperma utiliza otras fuentes de agua no permanentes. Para determinar si la competencia entre las raíces de ambas especies era directa se desarrolló un experimento en condiciones controladas. Los resultados muestran el mismo patrón de respuesta ante la sequía que en condiciones naturales, y confirman la interacción directa de las raíces de ambas especies. Así mismo se observaron claras diferencias tanto en el patrón como en la velocidad de crecimiento y ramificación de T. carnosus y R. monosperma. Por último este estudio aborda la interacción indirecta del efecto del ganado sobre la competencia entre ambas especies, mediante el análisis de los patrones de distribución espacial. Los resultados muestran que tanto una presión herbívora alta como la ausencia total de herbivoría producen un patrón espacial de repulsión en la interacción T. carnosus - R. monosperma, en cada caso por causas muy diferentes. Al mismo tiempo, una presión baja del pastoreo permite una distribución agregada y por tanto una asociación entre ambas especies. Índice  Capítulo 1. Introducción general…………………………………….1 Objetivos generales…………………………………….17  Capítulo 2. Zona y especies de estudio……………………………19  Capítulo 3. La interacción de sombra y sequía como causa del efecto competitivo de Retama monosperma…....31  Capítulo 4. Cambios en el patrón de uso del agua en Thymus carnosus inducidos por la competencia con R. monosperma……………………...…….…….…65  Capítulo 5. Competencia intra e interespecífica entre T. carnosus y R. monosperma: crecimiento y sequía en condiciones controladas……….….………93  Capítulo 6. Efecto del ganado doméstico sobre la distribución espacial y la competencia entre dos especies de matorral de dunas costeras…………..…….………...131  Discusión general…………………………………………………..153  Conclusiones………………………………………………………..163 1 Capítulo 1 Introducción general Objetivos de la tesis …it may be argued, therefore, that the essential qualities that determine the ecology of a species may only be detected by studying the reaction of its individuals to their neighbours. John Harper, 1964 Capítulo 1 8 Sin embargo, otros autores apuntan que puede darse un cambio de facilitación a competencia bajo condiciones de alto estrés abiótico cuando los niveles del recurso más limitante sean tan bajos que los beneficios ofrecidos por la especie hospedadora no puedan superar su propia captación de recursos (Maestre & Cortina 2004). Así, podría darse facilitación sólo si los beneficios de una mayor disponibilidad de nutrientes o sombra excedieran la competencia por agua (Holmgren et al. 1997), o si la especie benefactora fuera capaz de aumentar la disponibilidad de agua a la especie facilitada (Zou et al. 2005, Maestre et al. 2009, Armas et al. 2011). PERSPECTIVAS ANTE EL CAMBIO GLOBAL Actualmente estamos asistiendo a un proceso de cambio global en el que, a diferencia de épocas anteriores, los cambios biosféricos se están produciendo a una velocidad tal que dificulta la adaptación de los sistemas naturales (Ozcáriz et al. 2008, Aponte 2010). A pesar de que el clima terrestre siempre ha oscilado entre períodos cálidos y fríos, hay una “gran certeza” de que la actividad humana está influyendo en las condiciones climáticas globales hacia un calentamiento, como resultado de la liberación de gases de efecto invernadero a la atmósfera (IPCC 2007). Por ello hay autores que utilizan la expresión “cambio climático” para referirse al cambio climático antropogénico, en oposición a las fluctuaciones climáticas naturales (Brooker 2010). Los posibles impactos asociados al cambio climático no son sólo variaciones de temperatura, sino también en la precipitación, en los patrones de vientos y en la frecuencia de eventos climáticos extremos (IPCC 2007). Las consecuencias del cambio climático no son espacialmente uniformes. Los modelos climáticos predicen un aumento de la aridez y temperatura en extensas áreas intertropicales incluyendo el sur de Europa, y en el caso de los ecosistemas mediterráneos las previsiones para las próximas décadas son particularmente severas (Bates et al. 2008), lo cual aumenta su vulnerabilidad. Una mayor aridez en estos ecosistemas puede tener importantes repercusiones para la diversidad, ya que implica la migración o la pérdida de especies (Thuiller et al. 2005). Esto puede verse potenciado por varios factores. Por un lado el aumento de especies invasoras, Introducción y objetivos 9 fomentado a su vez por el cambio climático, e incluso uno de sus componentes principales según algunos autores (Vitousek et al. 1997, Vilà et al. 2006, Thuiller et al. 2007). Se ha comprobado que los cambios en la composición de las comunidades debido a la invasión de especies exóticas puede llegar a modificar el funcionamiento del ecosistema y generar un efecto de retroalimentación que dé lugar a cambios irreversibles en la comunidad (Gómez-Aparicio & Canham 2008, Aponte 2010). Por otro lado, al referirnos concretamente a ecosistemas mediterráneos costeros se añade otra consecuencia del cambio climático, como es la subida del nivel del mar (Greaver & Sternberg 2010). La tasa media actual es de 3 mm/año de aumento de nivel en muchas costas, y se prevé una aceleración con un cambio climático continuado (Bindoff et al. 2007). La subida del nivel del mar puede conducir a la salinización de los acuíferos costeros por intrusión marina. La conservación de las dunas costeras mediterráneos es importante ya que, como la mayoría de ecosistemas mediterráneos, albergan una gran diversidad de especies vegetales endémicas (Médail & Quézel 1999). Además, al estar localizados en la franja litoral, protegen las líneas de costa de la erosión al acumular y estabilizar suelos arenosos que mitigan el oleaje marino de la mareas diarias, episodios de tormenta y eventos tipo tsunami (Liu et al. 2005). Resulta crítico aclarar la relación interacción de especies-severidad del estrés ambiental, ya que podría ayudar a predecir futuros cambios en la biodiversidad, estructura, funcionamiento y organización de las comunidades afectadas por el cambio climático global (Callaway 2007, Armas et al. 2011). REFERENCIAS Aponte C (2010) Interacciones planta-suelo en un bosque mediterráneo. Tesis doctoral. Universidad de Sevilla. Armas C, Pugnaire FI (2005) Plant interactions govern population dynamics in a semiarid plant community. Journal of Ecology 93: 978–989. Capítulo 1 10 Armas C, Pugnaire FI (2009) Ontogenetic shifts in interactions of two dominant shrub species in a semi-arid coastal sand dune system. Journal of Vegetation Science 20: 535-546. 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Para ello se analizaron los patrones de uso de luz y agua, y los patrones de distribución espacial de ambas especies. Se establecieron los siguientes objetivos específicos: 1. Determinar mediante el uso de medidas morfo-fisiológicas la relación entre R. monosperma y T. carnosus. Para ello se estudió el efecto de la sombra de Retama sobre Thymus en diferentes condiciones de disponibilidad hídrica (estaciones del año) (Cap. III). 2. Establecer la interacción entre estas dos especies en respuesta a la disponibilidad de recursos hídricos. Para ello se analizó estacionalmente la respuesta fisiológica al estrés hídrico y se determinaron las fuentes de agua utilizadas por ambas especies a lo largo del año, así como la respuesta a la salinización del freático (Cap. IV). 3. Estudiar mediante un experimento de invernadero con plantas juveniles obtenidas desde semilla, la competencia entre ambas especies en respuesta a un ciclo de sequía eliminando el factor sombra (Cap. V). 4. Establecer mediante el análisis de patrones de distribución espaciales el efecto de la herbivoría como factor añadido a la interacción interespecífica (Cap. VI). Capítulo 3 24 principales factores que explican la expansión de Retama en el área de estudio, cuya superficie ha aumentado hasta un 673.2 % entre 1956 y 2001 (Gallego-Fernández et al. 2006), colonizando las dunas interiores desde las 15 ha originales de superficie plantada hasta ocupar unas 125 ha en 2004 (Muñoz Vallés et al. 2009). Esta especie fue plantada a principios del siglo XX a lo largo de la costa SO española con el objetivo de estabilizar las dunas (Kith y Tassara 1946). A pesar de ser nativa, se considera que Retama exhibe un comportamiento invasivo en algunas de las áreas donde está presente (Muñoz Vallés et al. 2011) debido a su alta producción y dispersión de semillas y a su crecimiento rápido. Además se ha descrito como especie exótica invasora en California, EEUU (Cal-IPC 2006, JM Randall 1997) y sur de Australia (RP Randall 2007), habiendo llegado posiblemente debido a su utilización como especie ornamental. Los cladodios fotosintéticos podrían contribuir a explicar la naturaleza invasiva de ciertos arbustos leguminosos (Bossard & Rejmanek 1992, Nilsen et al. 1993), debido a su eficiencia en la asimilación de carbono ya sea con sequía (Nilsen 1992), calor (Smith & Osmond 1987) o altas radiaciones (Valladares 1999, Valladares et al. 2003). De hecho, se consideran una adaptación que permite asimilaciones prolongadas durante el estrés ambiental (Nilsen et al. 1989, Haase et al. 1999). Estudios recientes han mostrado la capacidad de Retama para modificar el ambiente físico, reduciendo temperaturas extremas y aumentando la humedad y nutrientes del suelo. De esta forma la especie es capaz de crear islas de fertilidad en las dunas, produciendo bajo su copa un aumento de la riqueza de especies y la biomasa de la vegetación, sobre todo de especies anuales (Muñoz Vallés et al. 2011). Estos factores predominantemente positivos, junto con su comportamiento invasivo, indican un alto potencial de Retama para inducir cambios funcionales significativos en los ecosistemas dunares, tanto al alterar la abundancia y composición de especies vegetales como modificando en definitiva la estructura del ecosistema dunar (Muñoz Vallés et al. 2009, Zunzunegui et al. 2012). La clasificación de Retama como especie nativa-invasiva ha sido realizada en el contexto de la definición de Alpert et al. (2000), según el cual las especies nativas pueden considerarse invasivas cuando aumentan en abundancia o distribución tras cambios recientes, especialmente cambios originados por humanos, en sus hábitats Interacción de sombra y sequía 25 naturales. Más recientemente, Valéry et al. (2009) también establecieron que cualquier especie, ya sea nativa o exótica, que se dispersa en una región puede denominarse invasiva ya que en ambos casos puede funcionar el mismo mecanismo básico, la competencia interespecífica sensu lato (Valéry et al. 2008). Por esta razón, analizar los cambios derivados de la invasión de Retama sobre otras especies nativas puede ser crucial para establecer su efecto potencial sobre la dinámica de comunidades vegetales de dunas costeras. Thymus carnosus Boiss (Labiatae, en adelante Thymus). Es conocida comúnmente como tomillo carnoso, o tomillo de las playas en Portugal. Arbusto leñoso perenne que suele alcanzar los 0.5 m de altura. Endémico de las costas arenosas del SO de la Península Ibérica, el litoral de la provincia de Estremadura (Portugal) representa el extremo septentrional de su distribución (ICN 2006), y la costa de Huelva el extremo meridional (Parra et al. 2000). Presenta hojas pequeñas, lineares y carnosas con los márgenes revolutos. Sus flores blancas aparecen agrupadas en capítulos desde mayo hasta julio-agosto y presentan polinización entomófila. Produce frutos tipo núcula normalmente menores de 1 mm, que suelen aparecer entre julio y octubre. Las semillas no presentan ningún mecanismo de dispersión especializado. De acuerdo con Morales (2010) pueden desarrollar un sistema radical de más de 1 m de profundidad. No hay constancia de que Thymus sea consumido por ningún herbívoro mamífero. Hojas revolutas y flores de Thymus carnosus Capítulo 3 26 Debido a alteraciones en su hábitat, principalmente por presión urbanística y turismo, Thymus está incluido en el catálogo de especies de interés comunitario de la Directiva Hábitats de la Comunidad Europea (92/43/CEE). Además al haber quedado su distribución restringida al litoral occidental de la provincia onubense, fue clasificada como especie en peligro de extinción (CR, IUCN) en 2003 (Ley de Flora y Fauna de Andalucía), y en 2005 a nivel nacional (Cabezudo et al. 2005). En Portugal, a pesar de ser relativamente abundante en comparación con su estado en España, también ha sido catalogada como especie en regresión (ICN 2006) debido al deterioro de los hábitats costeros. Recientemente fue incluida en el Listado Andaluz de Especies Silvestres en Régimen de Protección (BOJA núm. 60, Decreto 23/2012). La Flecha de El Rompido constituye la principal área de distribución de la especie dentro de la costa de Huelva (Alés et al. 2003). A pesar de que la mayor parte de las poblaciones de Thymus han quedado fuera del espacio protegido, su presencia en el área fue un factor relevante para la creación del Paraje Natural, así como para la aceptación de la Flecha como Lugar de Importancia Comunitaria (LIC) (2006/613/EC). La expansión de Retama se está produciendo en las últimas comunidades dunares ocupadas por Thymus en la Flecha de El Rompido. Además, el hecho de que ambas especies cohabiten durante la casi totalidad del rango de distribución de Thymus hace extremadamente importante el estudio de la interacción entre ambas especies, con el objetivo de disponer de más información que permita la conservación de Thymus carnosus. REFERENCIAS Alés EE, Sánchez Gullón E, Peña J (2003) Consideraciones sobre la categoría de amenaza para Thymus carnosus en el suroeste de España. Conservación Vegetal. Boletín de la comisión de flora del comité español de la U.I.C.N 8: 9- 10. Interacción de sombra y sequía 27 Alpert P, Bone E, Holzapfel C (2000) Invasiveness, invasibility and the role of environmental stress in the spread of non-native plants. Perspectives in Plant Ecology, Evolution and Systematics 3: 52-66. Cabezudo B, Talavera S, Blanca G, Salazar C, Cueto M, Valdés B, Hernández Bermejo JE, Herrera CM, Rodríguez Hiraldo C, Navas D (2005) Lista Roja de la Flora Vascular de Andalucía. Consejería de Medio Ambiente, Junta de Andalucía, Sevilla. Cal-IPC (2006) California Invasive Plant Inventory. Cal-IPC Publication 2006-02. California Invasive Plant Council, Berkeley, CA, USA. Disponible: www.calipc.org/ip/inventory/weedlist.php (Último acceso: 24 Abril 2012). Bossard CC, Rejmanek M (1992) Why have green stems? Functional Ecology 6: 197- 205. Dabrio González CJ (1982) Historia y dinámica de nuestra costa. En: Cartaya 82. PF Martín (Ed). Ayto de Cartaya, Huelva, España, pp. 36-39. Dellafiore CM, Muñoz Vallés S, Gallego Fernández JB (2006) Rabbits (Oryctolagus cuniculus) as dispersers of Retama monosperma (L.) Boiss. seeds in a coastal dune system. Ecoscience 13: 5-10. Gallego-Fernández JB, Muñoz Vallés S, Dellafiore C (2006) Flora y Vegetación de la Flecha de Nueva Umbría (Lepe, Huelva). Ayto. de Lepe, Huelva, España. Haase P, Pugnaire FI, Clark SC, Incoll LD (1999) Diurnal and seasonal changes in cladode photosynthetic rate in relation to canopy age structure in the leguminous shrub Retama sphaerocarpa. Functional Ecology 13: 640-649. ICN (2006) Plano Sectorial da Rede Natura 2000. Instituto da Conservação da Natureza, Portugal. Disponible: http://www.icn.pt/psrn2000/caracterizacao_valores_naturais/flora/Thymus%20c arnosus.pdf (Último acceso: 15 Sept. 2012). Kith y Tassara M (1946) El problema de las dunas del SO de España. Revista de Montes 11: 414-419. Morales R (2010) Thymus L. En: Flora Ibérica, vol XII. S Talavera et al. (Eds), CSIC, Madrid, pp. 349-409. Capítulo 3 28 Muñoz Vallés S, Gallego Fernández JB, Dellafiore C (2009) Estudio florístico de la Flecha Litoral de El Rompido (Lepe, Huelva). Análisis y catálogo de la flora vascular de los sistemas de duna y marisma. Lagascalia 29: 43-88. Muñoz Vallés S, Gallego Fernández JB, Dellafiore C, Cambrollé J (2011) Effects on soil, microclimate and vegetation of the native-invasive Retama monosperma (L.) in coastal dunes. Plant Ecology 212: 169-179. Muñoz Vallés S, Gallego Fernández JB, Cambrollé J (2012) The biological flora of coastal dunes and wetlands: Retama monosperma (L.) Boiss. Journal of Coastal Research. DOI: 10.2112/JCOASTRES-D-12-00013. Nilsen ET, Meinzer FC, Rundel PW (1989) Stem photosynthesis in Psorothamnus spinosus (smoke tree) in the Sonoran Desert of California. Oecologia 79: 193- 197. Nilsen ET (1992) Partitioning growth and photosynthesis between leaves and stems during nitrogen limitation in Spartium junceum. American Journal of Botany 79: 1217-1223. Nilsen ET, Karpa D, Mooney HA, Field C (1993) Patterns of stem photosynthesis in two invasive legumes (Spartium junceum, Cytisus scoparius) of the California Coastal Region. American Journal of Botany 80: 1126-1136. Parra R, Valdés B, Ocaña ME, Díaz Lifante Z (2000) Thymus carnosus Boiss. En: Libro rojo de la flora silvestre amenazada de Andalucía. Vol. 2. G Blanca et al. (Eds). Consejería de Medio Ambiente, Junta de Andalucía, Sevilla, pp. 355-357. Randall JM (1997) Weed Alert! New invasive weeds in California. En: Symposium proceedings of the California Exotic Pest Plant Council, California, USA, pp. 1-6. Randall RP (2007) The introduced flora of Australia and its weed status. CRC for Australian Weed Management, Adelaide, Australia. Smith SD, Osmond CB (1987) Stem photosynthesis in a desert ephemeral, Eriogonum inflatum. Morphology, stomatal conductance and water use efficiency in field conditions. Oecologia 72: 533-541. Talavera S (1999) Retama Raf. En: Flora Ibérica, vol VII. S Talavera et al. (Eds). CSIC, Madrid, pp. 137-141. Valéry L, Fritz H, Lefeuvre JC, Simberloff D (2008) In search of a real definition of the biological invasion phenomenon itself. Biological Invasions 10: 1345-1351. Interacción de sombra y sequía 29 Valéry L, Fritz H, Lefeuvre JC, Simberloff D (2009) Ecosystem-level consequences of invasions by native species as a way to investigate relationships between evenness and ecosystem function. Biological Invasions 11: 609-617. Valladares F, Pugnaire FI (1999) Tradeoffs between irradiance capture and avoidance in semiarid environments simulated with a crown architecture model. Annals of Botany 83: 459-470. Valladares F, Hernández LG, Dobarro I, García-Pérez C, Sanz R, Pugnaire FI (2003) The ratio of leaf to total photosynthetic area influences shade survival and plastic response to Light of green-stemmed leguminous shrub seedlings. Annals of Botany 91: 577-584. Zunzunegui M, Esquivias MP, Oppo F, Gallego-Fernández JB (2012) Interspecific competition and livestock disturbance control the spatial patterns of two coastal dune shrubs. Plant and Soil 354: 299-309. 30 Interacción de sombra y sequía 31 Capítulo 3 La interacción de sombra y sequía como causa del efecto competitivo de Retama monosperma Este capítulo está basado en el siguiente manuscrito: Esquivias MP, Zunzunegui M, Díaz Barradas MC, Álvarez-Cansino L. Shade and drought interaction underlie competitive effects of the native-invasive Retama monosperma in a Mediterranean semi-arid sand dune system. Enviado a Journal of Vegetation Science (Octubre 2012). Capítulo 3 32 Interacción de sombra y sequía 33 Capítulo 3: La interacción de sombra y sequía como causa del efecto competitivo de Retama monosperma RESUMEN En los ecosistemas áridos y semi-áridos las interacciones entre plantas están sujetas normalmente a estrés tanto hídrico como lumínico. Numerosas investigaciones han demostrado que las condiciones creadas por la sombra de las especies de Retama pueden aminorar el estrés ambiental, posibilitando así el establecimiento de otras especies. En el presente trabajo se estudió el efecto de la sombra de Retama monosperma sobre Thymus carnosus en diferentes estaciones del año con distintas disponibilidades de agua y luz, con la intención de determinar si el efecto neto de ambos factores era positivo o negativo para Thymus. Se midieron estacionalmente la morfología y fisiología de plantas de Thymus creciendo al sol y a la sombra de Retama. Los resultados del índice de área foliar, peso específico foliar, contenido en pigmentos y la eficiencia fotoquímica real mostraron respuestas características de plantas de sol y de sombra. Sin embargo, los individuos de sol mostraron potenciales hídricos menos negativos y una mayor eficiencia de uso del agua, tanto instantánea como integrada, especialmente en primavera y verano. Estos resultados muestran una tolerancia baja de Thymus a la sombra de Retama. Cinco años después de estas medidas, la superficie de la copa en las plantas de sombra había disminuido y su mortalidad había sido mayor que en el caso de las plantas de sol, lo cual confirmaría el efecto competitivo de Retama. Retama aparece así como una especie potencialmente competidora sobre las comunidades de matorral de dunas, lo cual refuerza la idea de que posee un comportamiento invasivo, planteando además un serio riesgo sobre el amenazado Thymus. Este estudio demuestra también que los efectos eventualmente positivos de la sombra pueden verse superados por la competencia bajo la copa de plantas vecinas, especialmente en el caso de especies muy bien adaptadas al estrés hídrico y lumínico. Capítulo 3 40 LED at a 600 Hz frequency to assess F0, and then a saturating light pulse to determine Fm. Effective photochemical efficiency of PSII (ΦPSII) in a light-adapted state was determined on light-adapted leaves by means of a distance clip, as ΦPSII = (F’m-F)/F’m, where F’m= maximal and F= steady-state fluorescence under actinic irradiance (Genty et al. 1989). Three measurements per individual were carried out at complete darkness before dawn for Fv/Fm, and at midday for Fv/Fm and ΦPSI, and the average mean values per individual and light condition were calculated. Chronic and dynamic photoinhibition (PIchr and PIdyn respectively) were calculated following Werner et al. (2002). PIchr was calculated as the percentage reduction in pre-dawn Fv/Fm relative to the annual maximum Fv/Fm of both light conditions [(Fv/Fm)max] (Eqn 1), and PIdyn was calculated from midday Fv/Fm as the additional decrease relative to pre-dawn Fv/Fm (Eqn 2). Total photoinhibition (PItot) was calculated as the addition of PIchr and PIdyn (Eqn 3). PIchr = [(Fv/Fm)max – (Fv/Fm)pd]/( Fv/Fm)max x 100 (1) PIdyn = [(Fv/Fm)pd - (Fv/Fm)md]/ (Fv/Fm)max x 100 (2) PItot = (PIchr + PIdyn) (3) Where (Fv/Fm)pd and (Fv/Fm)md are pre-dawn and midday Fv/Fm values, respectively. (Fv/Fm)max is Fv/Fm annual maximum, calculated as the average of the maximum recorded values from the two months with highest Fv/Fm. Photoinhibition values were expressed as a percentage of reduction in (Fv/Fm)max values. Any photoinhibitory reduction in Fv/Fm at any time of the day may be composed of both chronic photoinhibition, such as in the case of the depressed predawn Fv/Fm values, and dynamic photoinhibition resulting in a further (fully reversible) decrease during the day (Werner et al. 2002). Interacción de sombra y sequía 41 Gas exchange and Leaf Mass Area Net photosynthetic (A) and transpiration (E) rates were measured on mature leaves of three terminal shoots per plant (several leaves per shoot) during clear sunny days, with an open system compact infra-red gas exchange analyzer (LCi-portable photosynthesis, ADC, UK). Instantaneous water use efficiency (WUEi) was calculated as the ratio of CO2 uptake per H2O transpired (A/E). The WUE of plants is a key aspect of their water economy and of their performance and survival in arid sites (Damesin et al. 1997, Valladares et al. 2008). Results were expressed on a projected leaf area basis, calculated using an image area analyser (Midebmp, R. Ordiales, Almería, Spain, 2000) from scanned images of the leaves from each measured shoot, collected following gas exchange measurements. After drying for 48 h at 70ºC, the leaf mass area (LMA) was calculated as the ratio of dry mass to leaf area. The LMA can be understood as the leaf-level cost of light interception (Gutschick & Wiegel 1988, Poorter et al. 2009). Due to technical problems, gas exchange data could not be carried out in winter. Leaf pigments content Two samples of leaves (about 1 g per sample) from each marked Thymus plant were collected; one sample was immediately stored in liquid nitrogen and kept in the laboratory at -24ºC until pigments extraction. The other sample was kept refrigerated in individual hermetic plastic bags, and fresh weight (fw) was registered in the lab within 3 h. Leaf samples were dried at 70ºC for 48h and dry weight (dw) was measured in order to obtain fw/dw relationship. Chlorophyll a and b and carotenoid pigments were extracted with 100% acetone, determined spectrophotometrically, and calculated on a dry mass basis following Lichtenthaler (1987). Leaf carbon and nitrogen content and stable isotopes composition In winter and spring, leaves of sampled Thymus (n = 5-10 individuals per light condition) were collected for isotopic (δ13C and δ15N) and elemental (carbon and nitrogen concentrations) analyses. Leaves were oven dried at 70ºC until constant weight and ball-milled to a fine powder (Retsch MM 400, Haan, Germany). Samples Capítulo 3 42 were transferred into tin capsules and injected into an elemental analyzer coupled in continuous flow to an isotope ratio mass spectrometer (EA-CF-IRMS, Thermo Delta V Advantage, Bremen, Germany) with a precision of 0.1-0.2 ‰, at the Universidad Autónoma de Madrid Stable Isotopes Laboratory (Spain). The isotopic abundance was expressed in delta notation (δ) in parts per thousand (‰) as δsample(‰) = (Rsample / Rstandard – 1) x 1000 (4) where Rsample and Rstandard are the molar ratios of heavy to light isotope of the sample and the international standard (NBS18 for δ13C , atmospheric N for δ15N). δ13C has been used in ecological research as a long-term integrator of ecophysiological processes, such as potential WUE integrated over the time of plant organ growth (Dawson et al. 2002). Stem water potential Stem water potential was measured before dawn (Ψpd) and at midday (Ψmd) in terminal shoots of every marked plant (1 x 10 plants x 2 light conditions). Measurements were performed immediately after the shoot was cut. Water potential was determined using a Scholander pressure chamber (Scholander et al. 1965; Manofrigido, Portugal). Statistical analyses One-way ANOVAs were carried out to test for differences between light conditions (open and understorey sites), in each season and time of the day (predawn or midday Fv/Fm and Ψ). Seasonal differences were analysed by a repeated-measures analysis of variance (ANOVAR) with season (summer, autumn, winter, and spring) as within-subjects effect. Post-hoc differences were tested using Tukey´s test when necessary. Differences between years in morphology and LAI measurements for Thymus and Retama were tested by means of one-way ANOVAs. A two-way ANOVA was used considering season and site (light condition) for each of the measured variables. All values conformed normality and sphericity assumptions. Homogeneity of Interacción de sombra y sequía 43 A S O N D J F M A M J J Temperature (ºC), solar radiation (MJ m-2 day-1) & VPD (KPa) x10 0 5 10 15 20 25 30 35 Precipitation (mm) 0 50 100 150 200 250 Tmean Precip Mean precip VPD VPD s. days Solar radiation variances was checked using Levene´s test. When variables were heterocedastic we applied the alternative non-parametric test for one-way ANOVA and ANOVAR (Kuskal-Wallis and Friedman tests, respectively). Differences between years in Thymus canopy cover and mortality were tested by means of a chi-square test. Significance level was set at 0.05. Analyses were performed with the SPSS v.18.0.0 software (SPSS Inc., Chicago, IL). RESULTS Weather conditions Fig. 1. Monthly mean temperature (Tmean, dashed grey line), monthly precipitation (Precip, solid bars), mean monthly precipitation over 30 years (Mean precip, open bars), monthly solar radiation (open triangles), monthly mean vapour pressure deficit (VPD, open circles) and sampling day VPD (black circles) throughout the study period (August 2006-June 2007). Meteorological variables showed the characteristic Mediterranean pattern with a marked seasonality in temperatures and rainfall distribution. Nevertheless, precipitation was especially pronounced in November and February and scarce during December and January compared with the 30-year historical data (Fig. 1). VPD was maximum in August and minimum in February, although on sampling day 29 August Capítulo 3 44 T T+R T T+R T T+R T T+R Summer 1480 18.3 43.0 35.5 28.0 26.5 35.7 31.3 - - Autumn 1400 21.9 29.5 18.5 9.0 10.0 19.2 15.1 46.5 53.7 Winter 1580 13.6 32.5 25.0 15.5 18.0 22.5 22.2 56.0 58.7 Spring 1680 23.1 41.5 35.5 23.0 22.5 35.0 26.0 32.3 46.8 Tmean (ºC) RH (%) Max. midday PAR (mmol m-2s-1)% PAR shade-sun Tmax (ºC) Tmin (ºC) VPD was double than the mean of the month (2.6 KPa compared to 1.35 KPa). 29 August was also the hottest date of the whole study period, with air temperature reaching 38ºC during the day in the meteorological station. PAR in the understorey compared to open sun ranged between the minimum in winter (13.6 %) and the maximum in spring (23.1 %), being intermediate in summer (18.3 %) and autumn (21.9 %) (Table 1). According to microclimatic sensors, the reduction in midday maximum temperature in the understorey attained 7.5 ºC in summer and winter, 11 ºC in autumn and 6 ºC in spring. Mean temperature was also lower, whereas RH was maintained between 2.7-14.5 % higher in the understorey. Table 1. Maximum midday PAR in the open site, % PAR in the shade respecting sun site, maximum, minimum and mean temperatures, and relative humidity (RH) microclimatic measurements, taken in the open (T) and the understorey sites (T+R) during sampling days (11am-19pm). Morphology In 2006 all Thymus individuals were of similar size in terms of height and canopy cover in both habitats. However, LAI was significantly higher in the open site (P<0.001, Table 2), as expected for sun-exposed plants. Retama plants measured 2.77 ± 0.19 m high and 17.8 ± 2.71 m2 in cover, showing a LAI of 1.57 ± 0.24 (mean ± SE). Five years later, T individuals increased in height (mean variation = +0.038 m) and canopy cover (+0.271 m2), whereas T+R had decreased for both variables (-0.14 m and -0.319 m2 respectively, Table 2). Mean variations were significant comparing sites, and consequently open site plants were bigger than understorey plants in 2011 (P<0.05). LAI was maintained similar in the five-years period in both light conditions, also being significantly higher in T (P<0.05). Retama plants height and LAI were similar after five years, whereas canopy cover increased significantly (P<0.05). Interacción de sombra y sequía 45 2006 Canopy cover (m2) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 2011 Canopy cover (m2) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 T T+R Mean SE Mean SE Height (m) T 0.357 0.020 0.394 0.020 T+R 0.388 0.050 0.292 0.050 * R2.768 0.190 2.760 0.135 Cover (m 2 ) T0.247 0.040 0.495 0.097 † T+R 0.388 0.130 0.181 0.120 R17.844 2.710 23.816 3.405 ††† LAI T 3.030 0.170 2.823 0.275 T+R 1.953 0.180 *** 1.608 0.254 * R1.565 0.236 2.009 0.152 2006 2011 Table 2. Morphology measures (height and cover) and leaf area index (LAI) in open-site Thymus (T), understorey Thymus (T+R) and Retama plants (R) (mean ± SE; n=10 in 2006; n=6-10 in 2011). Significant differences between open-understorey sites per year are marked with asterisks (*P<0.05 and ***P<0.001). Significant differences between years per species and site are labeled with crosses (†P<0.05 and †††P<0.001). Fig. 2. 2006-2011 canopy cover comparison for every marked Thymus’ individual (T, open circles; T+R, black filled circles) (n=10 in 2006; n=9-10 in 2011). Points over the line indicate increase in canopy cover, points below the line decrease in canopy cover, and points on X-axis indicate dead plants. Capítulo 3 46 Summer Autumn Winter Spring LMA (g m-2) 0 50 100 150 200 T T+R a b a A B * ** ** * b CC Site < 0.001 Season < 0.001 Site * Season = 0.014 Respecting survival and canopy status in 2011 compared with 2006, the situation was as follows (Fig. 2): In the open site 1 out of 10 Thymus plants died, 1 decreased and 8 increased in canopy cover; whereas in the understorey 4 out of 9 plants died, 4 decreased and 1 increased in canopy cover (chi-square test, P<0.05 for site effect). One Retama individual dried up and, despite resprouting, the Thymus plant beneath its canopy was discarded for survival control purposes. LMA LMA was significantly higher in the open site plants in all seasons (Fig. 3). When comparing each site, the lowest values were found in autumn and spring for sun-exposed plants, and just in autumn for understorey Thymus. The highest LMA was found in summer for both sites. Two-way ANOVA showed significant differences for season, site and season x site (Fig. 3). Fig. 3. Leaf mass area (LMA) in open-site Thymus (T) and understorey Thymus (T+R). Values are mean ± SE (n=10). Asterisks indicate significant differences between sites in each season (*P<0.05; **P<0.01). Different letters indicate significant differences among seasons per site (ANOVAR post hoc Tukey test, P<0.05. Lower case letters for T, upper case letters for T+R). Two-way ANOVA significance results (P) are shown in the figure. Interacción de sombra y sequía 47 Pre-dawn Fv / Fm 0.60 0.65 0.70 0.75 0.80 0.85 0.90 T T+R a aab b Site = 0.001 Season < 0.001 Site * Season = 0.811 Summer Autumn Winter Spring PSII 0.0 0.2 0.4 0.6 0.8 *** *** *** *** c Site < 0.001 Season = 0.025 Site * Season = 0.544 Summer Autumn Winter Spring Midday Fv / Fm 0.60 0.65 0.70 0.75 0.80 0.85 0.90 b a bb a A BBAB ** Site = 0.002 Season < 0.001 Site * Season = 0.321 Fig. 4. (a) Predawn and (b) midday maximum photochemical efficiency of PSII (Fv/Fm), and (c) effective photochemical efficiency of PSII (ΦPSII) throughout the study periods in open-site Thymus (T, open circles) and understorey Thymus (T+R, black filled circles) (mean ± SE, n=10). Asterisks show significant differences between sites in each season (**P<0.01; ***P<0.001). Different letters show significant differences among seasons per site (ANOVAR post-hoc Tukey test, P<0.05; lower case letters for T, upper case letters for T+R). Two-way ANOVA significance results (P) are shown in the figure. Chlorophyll fluorescence PSII pre-dawn maximum photochemical efficiency (Fv/Fm)pd showed similar values independently of Retama presence (Fig. 4a). At midday, Fv/Fm was also similar between sites in all seasons excepting spring, when (Fv/Fm)md was significantly higher for understorey plants (P<0.01, Fig. 4b). Effective photochemical efficiency (ΦPSII) was also higher in the understorey for all seasons, as expected due to Retama shade effect (P<0.001, Fig. 4c). Seasonal differences per site were only found for open-site plants in (Fv/Fm)pd, and for both sites at midday following the expected seasonal pattern: lowest values in summer and spring, the periods with higher temperature and radiation input. The Capítulo 3 48 Summer Autumn Winter Spring T T+R T T+R T T+R T T+R Total (chronic and dynamic) photoinhibition 0 2 4 6 8 10 12 14 16 PIdyn PIchr * *** * A B B A A BB AB aa bb bc b ac b    theoretical optimum (0.7-0.8 for woody species, Maxwell & Johnson 2000) was reached in all Fv/Fm cases. No seasonal differences were found for ΦPSII in any site. Nonetheless, two-way ANOVA detected site and season effects for (Fv/Fm)pd, (Fv/Fm)md and ΦPSII (Fig. 4). Chronic and dynamic photoinhibition PItot (PIchr + PIdyn) was similar in both light conditions for all studied seasons except for spring, the growth period, when it was significantly higher in open-site Thymus (P<0.05, Fig. 5). No differences between sites were found for PIchr in any period, although there was a clear tendency toward higher values in understorey plants (P=0.06-0.143). PIdyn showed significant differences between light conditions in winter and spring (P<0.05 and P<0.001, respectively), open-site plants displaying higher values (Fig. 5). Two-way ANOVA revealed significant differences for site and season effects in PItot, PIchr, and PIdyn (Table 3). Fig. 5. Chronic (PIchr), dynamic (PIdyn) and total photoinhibition (PItot) throughout the study periods in opensite Thymus (T) and understorey Thymus (T+R) (mean ± SE). Asterisks show significant differences between sites in each season (*P<0.05; ***P<0.001; asterisks at bar level refer to PIdyn, and upper asterisk refer to PItot. No significant differences were found for PIchr). Letters show significant differences among seasons in each site (ANOVAR post-hoc Tukey test; upper case letters for PItot, lower case letters for PIdyn and symbols for PIchr; normal type letters for T and italics for T+R). Interacción de sombra y sequía 49 Effect F df P PI chro Site 11.771 1 0.001 Season 14.800 3 0.001 Site * Season 0.342 3 0.795 PI dyn Site 21.567 1 0.001 Season 24.547 3 0.001 Site * Season 2.270 3 0.088 PI tot Site 7.860 1 0.006 Season 14.721 3 0.001 Site * Season 1.532 3 0.213 PItot was low in general for Thymus, reaching values lower than 15% in all study periods. Nevertheless, in summer and spring PItot was significantly higher than in autumn and winter, the less stressful seasons (P<0.005). This tendency was not reflected in PIchr for any light condition, whose values were gradually increasing from summer (3.9 % in T and 5.0 % in T+R) to spring (6.2 % in T and 7.6 in T+R). On the contrary, it was reflected in PIdyn for open-site plants, with the lowest values also in autumn and winter, but not for understorey Thymus. In this site, PIdyn in spring (1.5 %) was maintained as low as in the two previous seasons (1.4 % in autumn and 0.1 % in winter, Fig. 5). Table 3. Results of the two-way ANOVA (F, df and P values) for chronic, dynamic and total photoinhibition (PIchr, PIdyn and PItot, respectively). Bolds indicate significant differences (P<0.05). Gas exchange Net photosynthetic rate (A) showed higher values in open-site Thymus, although it was just significant in autumn (P<0.001, Fig. 6a). Transpiration rate (E) was significantly different between sites in summer (Fig. 6b), with open-site plants displaying higher values. In autumn, despite the tendency to an opposite pattern respecting summer, differences were not significant (P>0.05, Kruskal-Wallis test). WUEi was significantly lower in understorey plants in autumn and spring, whereas it was almost the same in summer (Fig. 6c). Capítulo 3 56 In accordance to the assimilation pattern, transpiration (E) was lower in the understorey in summer. This result, partly explained by Retama shading, and together with the lower Ψpd in the same season, reinforces the higher drought stress statement under Retama canopies. Open-site Thymus showed a more conservative water-use pattern or WUEi, being able to fix more carbon with less transpirational water loss. Both integrated (δ13C) and instantaneous Thymus WUE were higher in the open site than in the shade, similarly again to results found in Arctostaphylos uva-ursi (Valladares et al. 2008). In addition, the greater WUEi in autumn than spring and summer for open-site plants is in line with other studies on Mediterranean species, which also reported a decrease in WUE as water deficit increased (Moriana et al. 2002, Llorens et al. 2003, Álvarez-Cansino et al. 2010). In Mediterranean climate autumn days, it is not surprising that shrub species display high WUE as photosynthetic rates can be higher due to the high radiation, with a concomitant low water loss due to rainfall recover and decreased VPD. Moreover, it has been suggested that efficient use of water is a conservative ecophysiological strategy that can be detrimental in a competitive water-limited environment (DeLucia & Heckathorn 1989, Read & Farquhar 1991, Llorens et al. 2003), as we observed both in the seasonal and site patterns (Fig. 6c). The higher leaf N content showed by understorey plants was not related to a change in δ 15N values, similar between sites in winter and spring (Table 4). Our results indicated a lack of relationship of understorey Thymus with fixed atmospheric N via direct transfer from Retama roots, in accordance with Rodríguez-Echevarría & Pérez-Fernández (2003) in a study with R. sphaerocarpa. The presence of multiple N- sources with distinct isotopic values, mycorrhizal associations, temporal and spatial variation in N availability, and changes in plant demand can all influence plant δ 15N (Dawson et al. 2002). In fact, the seasonal decreasing pattern in spring with respect to winter in both sites could be indicating N depletion in the ecosystem due to higher N use during the growing season (Högberg 1997). Despite not explained by δ 15N, higher N content in understorey plants can be explained by higher soil litter and N content under Retama canopy (Muñoz-Vallés et al. 2011). Greater N content of litter from the leguminous shrub or changes in microbial activity may lead to the release of available N for the plants (Rodríguez-Echevarría & Pérez-Fernández 2003). Interacción de sombra y sequía 57 The decrease in leaf chlorophyll is common during the summer in Mediterranean Basin plants, and constitutes a reversible process that allows a decrease in light harvesting and reduces risks of over-excitation and photodamage (Balaguer et al. 2002). This reduction of the antenna size of up to 60% in Thymus allows a sustained functionality of PSII, as indicated by summer midday maximal Fv/Fm values no lower than 94% of the annual maximal values. When compared with other Mediterranean labiatae species such as Lavandula stoechas or Rosmarinus officinalis (Munné-Bosch et al. 1999) we found similar results, showing Thymus’ great level of adaptation to its environment. Even greater results are found comparing both Thymus sites: a significant reduction of 30% in August to 47% in May in total chlorophyll content for open versus understorey plants, led to less than 5% Fv/Fm reduction in any season, being only significant in spring. This, accompanied by the increase in the chlorophyll a/b and carotenoids/chlorophyll ratios, a common trait under high light conditions (Niinemets et al. 1998), partly explains the absence of damage to PSII in sun-exposed plants (Demmig-Adams & Adams III 1996). These results show how chlorophyll loss in open-site Thymus is an adaptation against photodamage, but not an irreversible event. Sun condition of open-site plants was reflected in higher PIdyn, but neither in an increase in PIchr nor in reduced carbon assimilation. Following Björkman & Powles (1984) and Valladares & Pearcy (1997), PI will be produced not only due to light excess, but also by water stress. In fact, understorey plants tended to have higher PIchr, coupled with lower A and Ψ. These results support the hypothesis of higher water stress in shaded Thymus. Overall, the capacity to withstand drought in Thymus plants is not only not enhanced in the shade, but decreased. In another study with a Mediterranean evergreen shrub, Heteromeles arbutifolia, Valladares & Pearcy (2002) also found that drought constrained photosynthetic rates to a much greater extent in the shade, and that sun plants exhibited a rather efficient photoprotection against strong irradiance conferred by both the architecture of the crown and the physiology of leaves, similarly to Thymus. Previous studies with plants from Mediterranean and arid ecosystems have also found that shade tolerance of each species influences the extent of Capítulo 3 58 facilitation (Prider & Facelli 2004, Valladares et al. 2008). In our case, competitive effect of Retama would be denoting low shade-tolerance of Thymus. The morphological measures in the five-year period also show a size decrease of understorey Thymus in height and cover, besides a higher mortality rate. Plant survival and growth are often observed to decline during drought more strongly in the understorey (Fisher et al. 1991, Valladares & Pearcy 2002, Sack 2004). While plant growth in arid systems is strongly limited by water availability, nutrients and light may also limit growth when water is available (Prider & Facelli 2004). Thus, despite Retama could enhance nutrients availability and its shade could be positive by lowering high temperatures, water scarcity in interaction with low light availability under its canopy possibly overrode facilitation by Retama over Thymus. As long as Retama is present all along Thymus range of distribution, the same mechanisms would be reproduced with the consequent displacement of Thymus. Thus, R. monosperma would arise as a new threatening factor for the endangered T. carnosus. In addition, and following Valéry et al (2009), Retama interspecific competition would be confirming the invasive behaviour of this native species. The present study also supports the idea that polytolerance to simultaneous but uncorrelated stresses such as drought and shade is difficult to achieve in Mediterranean environments. Very well drought adapted species, such as Thymus carnosus has been demonstrated to be, can face serious problems to deal with shade. Further studies should assess whether the relationship depends on the species bringing the shade, due to Retama effect over soil under its canopy, or on the contrary whether Thymus would be intolerant to any other species. Acknowledgements - This work was partly funded by the Spanish Ministry of Education and Science (CGL2005-02142). M.P.E. was supported by a FPDI-Junta de Andalucía fellowship. We thank Junta de Andalucía Environmental Agency for permission to work in the Natural Reserve and with the protected species. 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Capítulo 4 72 (18O) because of evaporation processes which imply isotopic fractionation with preferential loss of lighter isotopes (16O) (Feild & Dawson 1998). Leafless, lignified and mature stems fragments were sampled in the morning and immediately enclosed in screw-cap glass vials, sealed with Parafilm and kept refrigerated. Rain water was collected in pluviometers located at the sampling site, equipped with a 5-mm layer of liquid paraffin to prevent evaporation. Groundwater samples were extracted by means of a piezometer located in the study site. Atmospheric water vapour was collected following Helliker et al. (2002). All water samples were kept refrigerated in double cap polyethylene bottles sealed with Parafilm until analyses to prevent evaporation and isotopic fractionation. Plant samples were kept frozen in the lab until water was extracted using cryogenic vacuum distillation (Ehleringer & Osmond 1989) at the Department of Plant Biology and Ecology, University of Seville. The isotopic abundance was expressed in delta notation (δ) in parts per thousand (‰) as δ18O or δD sample(‰) = (Rsample / Rstandard – 1) x 1000 where Rsample and Rstandard are the molar ratios of heavy to light isotope of the sample and the international standard (Vienna standard mean ocean water, VSMOW). As δD in xylem water samples may fractionate during plant water uptake in species adapted to saline or xeric environments (Ellsworth & Williams 2007), only δ18O was used to determine water sources in plants. However, δD was also analysed in water table in order to compare with meteoric water line. Water isotope content was measured by CO2 equilibration using a Gas Bench coupled in continuous flow to a Delta V Advantage IRMS (Bremen, Germany) at the Universidad Autónoma de Madrid Stable Isotopes Laboratory (Spain). Statistical analyses One-way ANOVAs were carried out to test for differences in soil water content, stem water potential and proline content between sites (open and understorey), in each season and time (predawn or midday water potential). Xylem Cambios en el patrón de uso del agua 73 oxygen isotopic composition was analysed seasonally among study groups (open-site Thymus, understorey-site Thymus and Retama) also by one-way ANOVA. Seasonal differences in water potential and proline were analysed in Thymus and Retama by a repeated-measures analysis of variance (ANOVAR) with season (summer, autumn, winter and spring) as within-subjects effect. Post hoc differences were tested using Tukey´s test when necessary. Interaction effect between sites and seasons for Thymus was tested by means of a two-way ANOVA for each of the measured variables. All values conformed normality and sphericity assumptions, and significance level was set at 0.05. Linear regressions were conducted between groundwater electrical conductivity (EC) against rainfall amount between sampling dates, between δ18O against EC, and between δD against δ18O. Data analyses were performed with the SPSS v.18.0.0 software (SPSS Inc., Chicago, IL.). RESULTS Meteorological variables Meteorological variables showed a marked seasonal pattern, typical of the Mediterranean climate (Fig. 1a). Nevertheless, precipitation input was scarce during December and January for both study periods: 37 mm in 2006-07 and 47.6 mm in 2007-08, 78% and 72% lower than the 30 years historical rates for the same months (168mm), respectively. Rainfall was distributed mainly during spring and autumn, specially pronounced in November 2006. Minimum and maximum monthly mean temperatures were registered in January 2007 (6.1ºC) and July 2007 (31.2ºC) respectively (Fig. 1a). Soil water content and groundwater analysis Groundwater electrical conductivity (EC) varied seasonally, dropping from late summer 2007 (September) to spring and recovering in summer 2008 (Fig. 1a). EC was negatively related to rainfall between sampling dates (EC = -67.503rain+16791; r2=0.798; P=0.041). Water table level varied 0.39 m during the first sampling year, from 1.25 m (minimum) in autumn to 1.64 m (maximum) deep in summer and spring, Capítulo 4 74 ASONDJFMAMJJASONDJFMAMJJ Temperature (ºC) 0 10 20 30 40 50 Precipitation (mm) & Electrical Conductivity (mS cm-1) x10-2 0 50 100 150 200 250 Tmean Tmax Tmin 2006 2007 2008 ** * * Depht (m) 1.0 1.2 1.4 1.6 1.8 2.0 Water table level a b Precip Mean precip GW EC whereas during the second year the water table ranged 0.28 m, from 1.36 m (minimum) in autumn to 1.64 m (the same maximum than previous year) in summer 2008 (Fig. 1b). Fig. 1. (a) Monthly mean temperature (Tmean), monthly mean maximum (Tmax) and minimum (Tmin) temperatures; monthly precipitation (solid bars); mean monthly precipitation (open bars) over 30 years; first year physiological sampling dates (*); second year sampling dates and groundwater electrical conductivity (GW EC). (b) Water table depth below soil surface. Soil water content showed the highest value at 50 cm deep in spring for both sites, and in summer only in the understorey (Fig. 2). There were differences between sites just in spring at 25 and 50 cm deep, soil water content being lower in the understorey site in both depths. 2-way ANOVA showed significant differences for Site, Cambios en el patrón de uso del agua 75 Summer Autumn Winter Spring Water potential (MPa) -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 a * * a a A bBC bcbCbc aAB Summer Autumn Winter Spring T T+R R b aa A bb B bb B b a A * * Summer -1 0 1 2 3 4 5 6 A AB B Spring Soil gravimetric water content (%) 0 1 2 3 4 5 6 Depth (m) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 open understorey * *** a ab b A A B Depth (P<0.001) and Site x Depth interaction (P<0.05) in spring. Soil was dryer in summer than spring (P<0.001), with less than 1 % in summer water content (Fig. 2). Fig. 2. Soil gravimetric water content in spring and summer. Soil from open and understorey sites are compared (mean ± SD; n=3). Depth reference from soil surface. * indicate significant differences between sites in each depth (*P<0.05 and ***P<0.001); different letters indicate significant differences among depths within a site (ANOVA post hoc Tukey test, P<0.05. Lower case letters for open site, upper case letters for understorey site). Fig. 3. (a) predawn and (b) midday stem water potential in open-site Thymus (T, open bars), understorey Thymus (T+R, grey filled bars) and Retama over Thymus (R, black filled bars) plants from summer 2006 to spring 2007. Values are mean ± SD (n=10). Different letters indicate significant differences among seasons within each study group (ANOVA post hoc Tukey test, P<0.05. Lower case letters for T, lower case italic letters for T+R and upper case letters for R). * indicate significant differences between sites in each season (P<0.05). Capítulo 4 76 Water potential Differences in plant Ψpd between Thymus at different sites were significant in summer and spring, understorey Thymus displaying more negative values than opensite ones (Fig. 3a). Ψmd also showed differences between Thymus sites in winter and spring, but with opposite sign in both seasons: open-site Thymus had lower Ψmd in winter and higher values in summer. Retama Ψmd was more negative than Thymus in all studied seasons (Fig. 3b). The seasonal pattern was different depending on the Thymus site. Open-site plants reached the most negative Ψpd values in late summer, at the end of drought period, and recovered in autumn to display the highest in winter, whereas the situation in spring was intermediate between the preceding seasons (Fig. 3a). Ψmd showed the lowest value in summer, and remained constant from autumn to spring in the open site (Fig. 3b). Nevertheless, understorey Thymus displayed the minimum Ψpd and Ψmd values in both summer and spring, reaching maximum values during autumn and winter (Figs. 3a, 3b), similarly to Retama Ψmd seasonal pattern. Retama Ψpd reached the lowest values in summer, recovering in autumn to reach the highest values in winter, and in spring showed intermediate values between summer and autumn. Significant differences among Thymus sites were found across seasons, sites (excepting Ψmd) and for season x site (Table 1). Table 1. Results of the two-way ANOVA for the effect of season and Thymus sites on predawn water potential (Ψpd), midday water potential (Ψmd), free leaf proline content and xylem oxygen isotopic composition (δ18O). Numbers in bolds indicate significant differences. Free leaf proline Free leaf proline content was different between Thymus sites in summer and spring, higher in understorey Thymus in both cases (1.6 and 2 fold respectively) (Fig. 4). Seasonally, Thymus increased leaf proline content from summer to spring in both sites, although understorey Thymus did it in a more marked way in spring. Differently, Effect F df PF df PF df PF df P Season 22.406 3 <0.001 41.692 3 <0.001 44.355 3 <0.001 7.088 4 <0.001 Site 9.642 1 0.003 1.393 1 0.243 18.498 1 <0.001 0.064 1 0.802 Season x Site 4.005 3 0.011 6.234 3 0.001 9.903 3 <0.001 2.966 4 0.032 Ψpd Ψmd Proline δ18O Cambios en el patrón de uso del agua 77 Summer Autumn Winter Spring Proline (mmol g-1) 0 10 40 60 80 T T+R R * * aa bb ab c a A B B A Retama plants displayed the highest values in autumn and winter, with 31.9 and 38.6 μmol/g-1dw respectively. Two-way ANOVA showed significant differences for seasons, sites and season x site (Table 1). Fig. 4. Free leaf proline content in open-site Thymus (T, open bars), understorey Thymus (T+R, grey filled bars) and Retama over Thymus (R, black filled bars) plants from summer 2006 to spring 2007. Values are mean ± SD (n=10). Different letters indicate significant differences among seasons within each study group (ANOVA post hoc Tukey test, P<0.05. Lower case letters for T, lower case italic letters for T+R and upper case letters for R). * indicate significant differences between Thymus sites in each season (P<0.05). Isotope signature Groundwater δ18O signature varied from -2.98 ‰ in late summer 2007 to -4.1 ‰ in spring (Fig. 5), generally more enriched than precipitation excepting in summer 2008. There was a positive groundwater δ18O-EC linear regression across seasons (P=0.007, r2=0.94) (Fig. 6), as well as among groundwater δ18O-δD (P=0.001, r2=0.98) (Fig. 7). Precipitation δ18O signature varied among seasons between -6.2‰ (autumn) and -3.1‰ (summer 2008). These tendencies are consistent with precipitation values due to temperature and amount combined effects (Dansgaard 1964, Flanagan & Ehleringer 1991), as higher temperatures together with low precipitation quantities produce enriched isotopic signatures, whereas the contrary produces depleted values. Capítulo 4 78 Summer Autumn Winter Spring Summer d18O (‰) -6 -4 -2 0 2T T+R R GW Rain aba baa b ab a b a b ab Atmospheric water vapour apparently had no effect on the plants isotopic composition, ranging from -12.7 ‰ in winter to -11.63 ‰ in summer. Fig. 5. δ18O (‰) values of xylem water in open-site Thymus (T, open circles), understorey Thymus (T+R, grey filled circles) and Retama over Thymus (R, black filled triangles) plants, groundwater (grey lines) and rain water (black lines) from summer 2007 to summer 2008. Values are mean ± SD (n=3-6). Different letters indicate significant differences among study groups (T, T+R and R) in each season (ANOVA post hoc Tukey test, P<0.05). Atmospheric water vapour not shown. Retama plants showed δ18O xylem sap values similar to groundwater in any season, slightly mixed with rainfall in winter, spring and summer 2008 (Fig. 5). Opensite Thymus also showed intermediate values between groundwater and precipitation in autumn and summer 2008, and were similar to groundwater or more enriched in the rest of seasons. Understorey Thymus matched similar water sources than open-site plants from autumn to summer 2008, with the difference that Thymus beneath Retama showed enriched values in winter and summer, and was significantly different from Retama plants (Fig. 5). In summer 2007, a 37.8 mm rain event occurred two days before sampling, and only understorey Thymus reflected precipitation isotopic signature. δ18O xylem values in Thymus differed across seasons (P<0.001), and for season x site (P<0.032) (Table 1). Cambios en el patrón de uso del agua 79 EC (mS cm-1) 0 10000 20000 30000 40000 50000 d18O (‰) -5 -4 -3 -2 -1 0 1 r2=0.99 r2=0.94 d18O (‰) -6 -5 -4 -3 -2 -1 0 1 dD (‰) -40 -30 -20 -10 0 10 20 dD=5.12(d18O)-6.36 r2=0.98 , P=0.001 GMWL SMWL Fig. 6. Groundwater δ18O signature versus electrical conductivity (EC) for every season (black filled circles, dotted regression line; P=0.007, n=5). Regression line including sea water (black filled square) is also shown (P<0.001, n=6). Fig. 7. δD versus δ18O signatures of seasonal groundwater. Spanish meteoric water line (SMWL: δD = 8.49(δ18O) + 12.4) and global meteoric water line (GMWL: δD = 8.0(δ18O) + 10) are also shown. Capítulo 4 80 DISCUSSION The results show that Retama presence produced a physiological disruption on Thymus by lowering stem water potential and increasing leaf proline content in summer and spring, due to changes in water uptake patterns. Thus, our hypothesis was proved and water competition was demonstrated between Thymus and Retama, but the outcome for avoiding such water competition was adverse for the endangered Thymus. Consequently, Retama diminished Thymus drought tolerance in the understorey, whereas open-site Thymus was demonstrated to cope with drought periods by accessing the water table, in spite of being a brackish groundwater. Groundwater (GW) δ18O signature was higher (among -3 and -4‰) than usual values in Mediterranean coastal dune systems, normally around -5 or -6 ‰ (Alessio et al. 2004, Armas et al. 2010, Álvarez-Cansino et al. 2010), and similar to saline environments found in the Everglades, around -3 and -3.5 ‰ (Greaver & Sternberg 2006). Nonetheless, rainfall δ18O signatures were consistent with typical values found in the Atlantic coast of the Iberian Peninsula, among -4.1 and -6.1 ‰ (Araguas- Araguas & Diaz Teijeiro 2005). In addition, GW EC was high (4700 to 16700 μS cm-1, Fig. 1) compared to rainwater (430 μS cm-1 in the area), and also compared to the spit water table closest to the union with the mainland, which was around 530 μS cm-1 and constant throughout the year (unpublished data). The brackish groundwater found in our study site could be produced due to different mechanisms as dissolution and infiltration of NaCl compositions from sand dune deposits, or seawater intrusion (Yoon 1986, Kim et al. 2003). As a likely unconfined aquifer, typical of barrier and openocean islands, it would be a lens of low density fresh or brackish water that floats on underlying high-density saltwater (Gyben-Herzberg lens), recharged only by precipitation. This situation would explain that under decreasing precipitation in summer, groundwater recharge by freshwater diminished and lens would shrink, causing ocean water to intrude and salinize freshwater (Greaver & Sternberg 2010). The contrary would happen from autumn to spring with increasing rainfall. Groundwater level’s low variation (0.39 and 0.28 m in the first and second study years, respectively) could also be suggesting an equilibration with sea water. δD and δ18O are effective conservative tracers of mixing processes at the groundwater-seawater Cambios en el patrón de uso del agua 81 interface, because there is clear isotopic distinction between on-shore meteoric groundwater and seawater (Povinec et al. 2006). The study site groundwater δ18O and δD consistently deviated from both the global meteoric water line (GMWL) and the Spanish meteoric water line (SMWL), with a slope of 5.12 (Fig. 7). This is consistent with findings by Kim et al. (2003) in a Korean volcanic island, and Ewe et al. (2007) and Price & Swart (2006) in the Everglades, who found slopes of 5.79, 5.39 and 5.16, respectively. This lower than GMWL slope value indicates that either the waters experienced some extent of evaporation relatively to the local precipitation, or that they contain some isotopically enriched water probably due to mixing with marine sources (Kim et al. 2003, Ewe et al. 2007). The presence of groundwater at shallow depth suggests that it is potentially an important water source for vegetation capable of accessing it (Kite & Webster 1989, Zencich et al. 2002). Xylem water isotopic composition results show that Retama and open-site Thymus appear to use the brackish groundwater all over the year, and the shallow water table could confirm it (1.64 m the deepest). Nevertheless, understorey Thymus does not seem to reach groundwater, excepting in autumn when it is in the shallower level (1.36 m deep) due to water table recovery with high rainfall input. From winter to summer, understorey Thymus showed enriched isotopic composition respecting to rain, groundwater, and Retama plants signals. These results suggests upper soil layers water uptake for understorey plants, as water from deeper soil layers such as that found in the water table will present more depleted values of δ18O than water from the soil surface or from recent precipitation (Álvarez- Cansino et al. 2010). Máguas et al. (2011) also found more enriched signatures during summer than spring due to higher rates of evaporation from the soil, which was particularly evident for the shallow rooted Acacia longifolia, and a higher variability in summer such as that presented by understorey Thymus. These xylem water values could suggest a shortened root system, supported by the fact that understorey plants matched episodic rainfall in the shallower layers in summer 2007, unlike open-site Thymus that still matched groundwater (Fig. 5). The fact that precipitation isotopic signals appear generally more depleted than groundwater, instead of more enriched as usual, make it harder to elucidate the results. 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Plant Ecology 212: 127-142. 93 Capítulo 5 Competencia intra e interespecífica entre Thymus carnosus y Retama monosperma: crecimiento y sequía en condiciones controladas Este capítulo está basado en el siguiente manuscrito: Esquivias MP, Zunzunegui M, Díaz Barradas MC, Álvarez-Cansino L. Intra- and interspecific competition between Thymus carnosus and Retama monosperma. Growth and drought under controlled conditions (en preparación). Capítulo 5 94 Competencia intra e interespecífica 95 Capítulo 5: Competencia intra e interespecífica entre Thymus carnosus y Retama monosperma: crecimiento y sequía en condiciones controladas RESUMEN En este capítulo se describe un experimento de invernadero realizado para estudiar la competencia entre raíces de Thymus carnosus y Retama monosperma, al cual se le aplicó también un período de estrés hídrico. El experimento se desarrolló con juveniles de ambas especies de la misma edad, obtenidas a partir de semillas recolectadas en la zona de estudio de la Flecha de El Rompido. Se realizó un diseño factorial con combinaciones de las dos especies creciendo en interacción interespecífica, aisladas o en interacción intraespecífica. Se midió el crecimiento y la ramificación de los individuos durante varios meses, previo a la imposición del tratamiento de sequía. Durante este se midieron el potencial hídrico y la eficiencia fotoquímica de la clorofila a en luz y oscuridad. Al finalizar el experimento se calcularon diferentes parámetros de alocación de biomasa. Los resultados indican un efecto negativo de Retama sobre Thymus en el uso de los recursos subterráneos y como respuesta a la sequía. Además se ha descubierto un efecto facilitador intraespecífico entre las plantas de Thymus respecto al crecimiento y ante el estrés hídrico. A nivel de especies, Thymus presentó un menor desarrollo tanto en biomasa subterránea como aérea, y una mejor respuesta frente a la sequía que Retama. Abreviaturas - TER, tasa de elongación relativa; ΔR, incremento del número de ramificaciones; Ψ, potencial hídrico; ΦPSII, eficiencia fotoquímica real; Fv/Fm, eficiencia fotoquímica máxima; LMR, alocación de biomasa foliar; SMR, alocación de biomasa a tallos; RMR, alocación de biomasa a raíces; R:A, cociente biomasa raíces / biomasa aérea; δ13C, relación isotópica C13/C12; δ15N, relación isotópica N15/N14. Capítulo 5 96 INTRODUCCIÓN Los ambientes áridos y semiáridos se caracterizan por presentar pulsos de disponibilidad de agua, y los eventos de precipitación discretos intercalados con períodos de sequía son componentes importantes del aporte hídrico anual (Noy-Meir 1985, Padilla et al. 2009). La variabilidad e impredecibilidad de las precipitaciones imponen fuertes restricciones en las plantas que pueden ser muy importantes para la supervivencia de los individuos (Joffre et al. 1999). En los ecosistemas dunares mediterráneos, con una estacionalidad muy marcada por los veranos cálidos y secos, la vegetación depende en gran parte de los patrones de precipitación y disponibilidad hídrica (Van Der Maarel 1993). Las predicciones del clima apuntan a una disminución generalizada de la precipitación para la región mediterránea en el 2050, con menos días de lluvia más concentrados en invierno, y períodos de sequía más largos entre eventos de lluvia (Rind et al. 1989, Easterling et al. 2000, IPCC 2007). Por tanto, se considera que los ecosistemas mediterráneos pueden ser muy susceptibles a las fluctuaciones en el patrón de disponibilidad de agua debidas al cambio climático (Lavorel et al. 1998), lo cual posiblemente modificará la distribución de especies y las interacciones planta-planta (Domènech & Vilà 2008). La teoría del equilibrio funcional (Brouwer 1962,1963, Peperkorn et al. 2005) establece que las partes aéreas de la planta se favorecen cuando la luz o el CO2 son bajos, y que el crecimiento de la fracción subterránea se promueve si el agua o los nutrientes minerales escasean (Poorter & Nagel 2000, Arndt & Wanek 2002), en consonancia con la teoría de alocación de biomasa de Smith & Huston (1989). La competencia por los recursos subterráneos puede ser fuerte y se ha asociado con la segregación espacial de las raíces (Brisson & Reynolds 1997, Casper & Jackson 1997), ya que las plantas alteran su producción de raíces en presencia de otras plantas (Campbell & Grime 1989, de Kroon & Hutchings 1995, Gersani et al. 1998). Las raíces pueden bien evitar otras raíces (Mahall & Callaway 1991, Schenk 2006), o bien proliferar en su presencia (Gersani et al. 2001), interferiendo o inhibiendo el crecimiento de las vecinas (Bais et al. 2003, Bertin et al. 2007, Armas & Pugnaire 2011) mediante compuestos secundarios (Bertin et al. 2007). Por ello, conocer el funcionamiento de las especies a nivel subterráneo es muy importante a la hora de Competencia intra e interespecífica 97 determinar su capacidad competitiva frente a otras plantas. Además, la interferencia de raíces finas es un componente muy importante del éxito competitivo de una especie (Caldwell et al. 1996). El establecimiento de plántulas es una de las fases más cruciales que afectan a la presencia de especies aisladas en comunidades de plantas (Harper 1977, Grime & Hillier 1992). Además, tanto plántulas como juveniles son más sensibles a la deshidratación que semillas o adultos (Evans & Etherington 1991, Padilla et al. 2009). Por ello muchas especies se establecen a la sombra de otra especie hospedadora debido a una mayor humedad bajo su copa, fenómeno conocido como “efecto nodriza” (Niering et al. 1963). Por otro lado, diferencias en el tamaño y el estadío ontogenético de las plántulas pueden afectar al resultado de la competencia entre especies (Mingo 2009). El balance neto de las interacciones entre especies es el resultado de la suma de todas las influencias, directas e indirectas, positivas y negativas (Callaway & Pugnaire 1999, Pugnaire & Luque 2001). Por ello puede ser interesante estudiar de forma aislada los diferentes factores que pueden intervenir con el objetivo de conocer el efecto de la interacción a un nivel concreto, ya sea a nivel de raíces, de copa, por efecto del agua o de la sombra. En condiciones naturales se ha observado que el efecto neto de la interacción de individuos adultos de Thymus carnosus con Retama monosperma es negativo para Thymus (Cap. 3). Pero aún se desconoce si es resultado de la alocación de biomasa hacia parte fotosintética en detrimento de la fracción de raíces debido al efecto de la sombra de la copa de Retama (de acuerdo con la teoría de Smith & Huston 1989), o si realmente existe una competencia directa entre las raíces de ambas especies por los recursos subterráneos. Por este motivo, se planteó un experimento de invernadero para evaluar la interacción entre raíces de Thymus y Retama. El objetivo fue determinar la competencia a nivel subterráneo en plantas sometidas a dos niveles de disponibilidad hídrica. Para eliminar el efecto de la sombra de Retama sobre Thymus, el experimento se realizó con juveniles de cinco meses de edad de ambas especies en interacción interespecífica, intraespecífica y aisladas. Tras varios meses creciendo juntas se las sometió a un período de estrés por sequía. Su respuesta a la Capítulo 5 104 0 50 100 150 200 250 ΔR total aa b b 0 2 4 6 8 10 12 14 16 TER total (cm día -1) T T+T T+R R+T R aa b a gl F Pgl F P Competencia (C) 2 6.830 0.001 1 4.678 0.036 Tiempo (T) 4 318.550 0.001 4 46.332 0.001 C * T 8 10.096 0.001 4 0.548 0.463 TER Thymus Retama ANOVAR, que analiza la evolución temporal, así como la TER total para todo el período de estudio (entre la primera y la última fecha de medida), mostraron que T+T presentaba las mayores tasas de elongación, siendo similares para T y T+R (Fig. 1 y 2a). Además de las diferencias entre combinaciones, se encontraron diferencias en el tiempo y en la interacción combinación X tiempo (Tabla 1). Fig. 2. (a) Tasa de elongación relativa (TER) total y (b) incremento en el número de ramificaciones (ΔR) total, para todo el período de estudio (media + SD). Con diferentes letras se muestran las diferencias significativas entre combinaciones por especie (ANOVA de una vía para TER; Kruskal-Wallis y U de Mann- Whitney para ΔR; P<0.05). Tabla 1. Resultados del ANOVA de medidas repetidas para los factores competencia y tiempo, en la tasa de elongación relativa (TER). Se muestran Thymus y Retama por separado. En negrita se indican los valores significativos (P<0.05). Competencia intra e interespecífica 105 Fig. 3. Incremento en el número de ramificaciones (ΔR) entre las sucesivas fechas de medida (media + SD). Sobre las columnas se muestran con diferentes letras las diferencias significativas entre combinaciones por especie (en minúscula Thymus y en minúscula cursiva Retama; Kruskal-Wallis y U de Mann-Whitney, P<0.05). La ramificación mostró un patrón semejante, con la misma tendencia en las primeras dos medidas hasta los 59 días, cuando T+R mostró menores incrementos de ramificación que T+T y T (Fig. 3). A los 88 días T+R igualó a T, mientras que a los 117 T+R y T+T mostraron similares incrementos de ramificación, a su vez significativamente superiores a T. Atendiendo al incremento total de ramificaciones (Fig. 2b), T+T resultó ser la combinación que más se ramificó durante todo el período de estudio, mientras que T y T+R fueron semejantes. En el caso de Retama, durante las primeras tres medidas tanto la tasa de elongación como el incremento en número de ramificaciones fueron significativamente superiores en los individuos aislados (R) respecto a aquellos creciendo con Thymus (R+T) (Figs. 1 y 3). Sin embargo en los días 88 y 117 los dos parámetros se igualaron en ambas combinaciones, llegando a ser incluso superior el incremento de ramificación para R+T en la última medida. El ANOVAR mostró una TER significativamente superior para R al analizar la evolución temporal (Fig. 1), 0 20 40 60 80 100 120 21 36 59 88 117 Días tras transplante ΔR T T+T T+R R+T R a b c b a a b b a b a b a b a b a b a Capítulo 5 106 0 50 100 150 200 250 300 0 500 1000 1500 2000 2500 3000 ΔE ΔR T T+T T+R R+T R0.745** 0.902*** 0.924*** 0.897*** 0.903*** aunque al analizar la TER total dichas diferencias desaparecieron (Fig. 2a). A su vez, el incremento de ramificación total tampoco mostró diferencias significativas entre ambas combinaciones de Retama (Fig. 2b). Comparando las especies entre sí, Retama mostró unas TER muy superiores a las de Thymus, llegando a casi 14 cm/día de media por planta, mientras que Thymus no superó los 6 cm/día de media (alcanzado por T+T) en todo el período de estudio (Fig. 1). Al contrario sucede con la ramificación, ya que Retama superó escasamente las 68 ramificaciones totales de media por planta, mientras que Thymus mostró entre 86 ramificaciones de mínima (T+R) y 142 ramificaciones totales de media máxima por planta (T+T). Estas diferencias entre ambas especies marcan dos patrones de crecimiento: el de Retama, que invierte más en crecer en longitud, y el de Thymus, que invierte más en ramificarse y menos en crecer longitudinalmente (Fig. 4), ambos parámetros altamente correlacionados (P<0.001). Fig. 4. Correlación entre los incrementos totales de elongación (ΔE) y de ramificación (ΔR) entre la primera y la última fecha de medida para cada combinación. Al lado de la leyenda se muestra la r de Spearman (** P<0.01 y *** P<0.001). Cada punto representa un individuo. Competencia intra e interespecífica 107 -100 -80 -60 -40 -20 0 20 Semana 0 Semana 1 Semana 2 Semana 3 Semana 4 Semana 5 Semana 6 % pérdida peso T Control T Sequía T+T Control T+T Sequía T+R Control T+R Sequía R Control R Sequía a b c c a a b ab Experimento sequía Tras la imposición del estrés hídrico las macetas sometidas a sequía (S) perdieron peso rápidamente, llegando en la última semana hasta un 38% menos de peso en T, 51% en T+T o 58% menos tanto en las macetas T+R (mismas macetas que R+T) como en las R (Fig. 5). Las diferencias fueron significativas entre todas las combinaciones, excepto T+R (R+T) con R. Por otro lado, es de destacar que las macetas T+R control (C) perdieran peso de forma más acusada que el resto hasta la segunda semana, cuando se volvieron a regar a capacidad de campo. Esta rápida pérdida de contenido hídrico al inicio produjo que al cabo de las 6 semanas de tratamiento fuesen significativamente diferentes a las otras macetas de Thymus (T y T+T), aunque no a las de R (Fig. 5). Fig. 5. Evolución del porcentaje de pérdida de peso de las macetas respecto a su capacidad de campo (C.C.) a lo largo del tratamiento de sequía (semana 0=previa al comienzo) (media ± SD). Al lado de la leyenda se muestran con diferentes letras las diferencias significativas entre combinaciones para todo el tratamiento (minúscula normal las macetas control, minúscula negrita las macetas de sequía; ANOVAR, P<0.05). La flecha indica el cambio de riego de las macetas control de nuevo a C.C. Capítulo 5 108 Fig. 6. Evolución del potencial hídrico (Ψ) de las combinaciones de Thymus (a) y Retama (b) a lo largo del tratamiento de sequía (semana 0=previa al comienzo; 7=recuperación). Se muestran al lado de la leyenda con diferentes letras las diferencias significativas entre combinaciones para todo el tratamiento (en minúscula los individuos control, minúscula negrita los de sequía, ANOVA de 2 vías; en mayúscula el global, ANOVA de 3 vías; P<0.05). Potencial hídrico Durante las seis semanas que duró el tratamiento no se observó una disminución significativa del Ψ ni en los Thymus aislados (T) ni en competencia intraespecífica (T+T), no mostrando síntomas de déficit hídrico (Figs. 6 y 7). El resto de combinaciones, con presencia de Retama, sí mostraron una disminución significativa del Ψ con la misma evolución temporal y en el mismo grado, llegando a sobrepasar ampliamente los -2 MPa en todos los casos. T+R comenzó a acusar el efecto del déficit hídrico en la semana 2 con valores de Ψ significativamente menores en los individuos de sequía que en los control (Fig. 7c), y fue disminuyendo paulatinamente y de forma paralela a las Retamas con las que interaccionaba, y ambas a su vez con las Retamas aisladas. Con el riego de recuperación todas volvieron a los valores control excepto las R+T, que seguían mostrando diferencias significativas (Fig. 7d). El ANOVA de 3 vías para Thymus reveló diferencias significativas para todos los factores (Tabla 2), y al comparar las distintas combinaciones entre sí T+R se separó de las otras dos con potenciales mucho más negativos (Fig. 6a). -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0023457 Semana de medida Ψ (MPa) T Control T Sequía T+T Control T+T Sequía T+R Control T+R Sequía a a a a a a b T T+T T+R A A B 0 2 3 4 5 7 Semana de medida R+T Control R+T Sequía R Control R Sequía b a b a a Competencia intra e interespecífica 109 Fig. 7. Evolución del potencial hídrico (Ψ) de las diferentes combinaciones (T, a), (T+T, b), (T+R, c), (R+T, d) y (R, e) a lo largo del tratamiento de sequía (semana 0=previa al comienzo y 7=recuperación) (Media - SD). Los asteriscos indican diferencias significativas entre tratamientos por semana (ANOVA; * P<0.05; ** P<0.005; *** P<0.001). La flecha indica el riego de recuperación. Al analizar los tratamientos por separado se puede observar que son los individuos de sequía los que repiten ese patrón, con T+R separándose significativamente de T+T y T ya desde la semana 3 y hasta la 5 (P<0.05 en todos los casos). Sin embargo no se obtuvieron diferencias al comparar T+R sequía con R+T y -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 00 2 3 4 5 7 Semana de medida Ψ (MPa) T Control T Sequía recuperación a R Control R Sequía ** * *** recuperación e -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 Ψ (MPa) R+T Control R+T Sequía recuperación *** * * d 0 2 3 4 5 7 Semana de medida T+R Control T+R Sequía ** * recuperación c -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 Ψ (MPa) T+T Control T+T Sequía * recuperación b Capítulo 5 110 Thymus gl F Pgl F Pgl F P Competencia (C) 2 15.830 0.001 2 26.994 0.001 2 15.486 0.001 Riego (R) 1 13.621 0.001 1 2.091 0.150 1 2.199 0.140 Tiempo (T) 5 13.089 0.001 4 0.954 0.434 4 20.989 0.001 C * R 2 8.641 0.001 2 3.156 0.045 2 0.883 0.415 C * T 10 2.805 0.003 8 2.630 0.009 8 0.454 0.887 R * T 5 5.607 0.001 4 5.110 0.001 4 5.057 0.001 C * R * T 10 2.437 0.009 8 2.424 0.016 8 0.229 0.985 Retama Competencia (C) 1 0.240 0.625 1 0.225 0.636 1 8.335 0.005 Riego (R) 1 52.657 0.001 1 0.000 0.983 1 0.001 0.977 Tiempo (T) 5 6.152 0.001 3 8.028 0.001 3 13.240 0.001 C * R 1 0.198 0.657 1 15.172 0.001 1 0.030 0.864 C * T 5 0.108 0.990 3 1.964 0.125 3 0.859 0.466 R * T 5 7.250 0.001 3 1.263 0.292 3 3.348 0.022 C * R * T 5 0.266 0.931 3 0.194 0.900 3 0.621 0.603 ΨΦPSII Fv/Fm R en ninguna de las semanas desde 0 a 5 (P>0.05), solamente en la semana 7 tras la recuperación (P<0.001). Retama sólo presentó diferencias en los factores riego, tiempo y riego X tiempo (Tabla 2), mostrando el mismo patrón frente a la sequía tanto en competencia con Thymus como aisladas (Fig. 6b). Sin embargo, los individuos control sí mostraron diferencias entre combinaciones, aunque al analizar cada semana de manera independiente dichas diferencias sólo se encontraron en la semana 2 (P=0.001). Tabla 2. Resultados del ANOVA de 3 vías para el efecto de los factores competencia, riego y tiempo durante el experimento de sequía sobre las variables potencial hídrico (Ψ), eficiencia fotoquímica real (ΦPSII) y eficiencia fotoquímica máxima (Fv/Fm). Se muestran Thymus y Retama por separado. En negrita se indican los valores significativos (P<0.05). Eficiencia fotoquímica Los resultados de eficiencia fotoquímica de la clorofila a no mostraron evidencias significativas del efecto de la sequía en luz ni en oscuridad, como puede comprobarse tanto en la evolución temporal (Fig. 8) como por el resultado no significativo para el factor riego del ANOVA de 3 vías en ambas especies y parámetros (Tabla 2). Competencia intra e interespecífica 111 Fig. 8. Eficiencia fotoquímica real (ΦPSII) de las combinaciones de Thymus (a) y Retama (b), y eficiencia fotoquímica máxima (Fv/Fm) de las combinaciones de Thymus (c) y Retama (d), a lo largo del tratamiento de sequía (semana 0=previa al comienzo). Se muestran al lado de la leyenda con diferentes letras las diferencias significativas entre combinaciones para todo el tratamiento (en minúscula los individuos control, en minúscula negrita los de sequía, ANOVA de 2 vías; en mayúscula el global, ANOVA de 3 vías; P<0.05). Sin embargo, centrándonos en la variable ΦPSII de Thymus sí se hallaron diferencias significativas para el factor competencia, así como en las tres interacciones dobles (competencia X riego, competencia X tiempo y riego X tiempo) más la triple (Tabla 2). Esto nos indica que la respuesta a la sequía de las plantas de Thymus era dependiente de la combinación en que crecía así como de la evolución temporal, aunque esta no mostró un patrón claro de disminución o aumento (Fig. 8a). Las diferencias entre combinaciones se tradujeron en valores más bajos para T+R, intermedios en T y los mayores en T+T si miramos los datos globales (individuos sequía y control conjuntamente). Si comparamos con los individuos de sequía, los 0.60 0.65 0.70 0.75 0.80 0.85 0.90 01235 Semana de medida Fv/Fm T Control T Sequía T+T Control T+T Sequía T+R Control T+R Sequía b ab a a a a b T T+T T+R A A B 0.00 0.10 0.20 0.30 0.40 0.50 0.60 ФPSII T Control T Sequía T+T Control T+T Sequía T+R Control T+R Sequía a a a b ab a b T T+T T+R A B C R+T Control R+T Sequía R Control R Sequía b a b a b 0 1 2 5 Semana de medida R+T Control R+T Sequía R Control R Sequía d a ba a R R+T A B Capítulo 5 112 T+R mostraron valores significativamente menores que los T+T. Incluso en el caso de los control, nuevamente T+R mostró valores inferiores a T y T+T (Fig. 8a). En el caso de las combinaciones de Retama, se encontraron diferencias para ΦPSII en el factor tiempo y en la interacción competencia X riego (Tabla 2). El resultado fue muy diferente según la combinación, ya que R+T control mostró valores más altos que R, mientras que para R+T sequía fueron más bajos (Fig. 8b). Al comparar los valores de ΦPSII entre las semanas 0 y 5 de tratamiento, se observa que son mínimos los casos en que hay diferencias entre los individuos control y sequía (Fig. 9a). Además sólo hubo diferencias significativas entre las semanas 0 y 5 de los individuos T+R y R+T de sequía, lo que indica que fueron esas macetas de competencia interespecífica las únicas afectadas por el déficit hídrico a nivel de ΦPSII. Tampoco se encontraron efectos evidentes de la sequía para la variable Fv/Fm. El ANOVA de 3 vías reveló diferencias significativas solamente en los factores competencia, tiempo y riego X tiempo para las dos especies (Tabla 2), y en ambos casos el efecto del tiempo conforme aumentó la sequía fue una subida de Fv/Fm (Fig. 8c y d). Además es de destacar que Fv/Fm no fue en ningún momento inferior a 0.74 de media. Las diferencias en Fv/Fm entre las combinaciones de Thymus resultaron en valores significativamente menores en el caso de T+R tanto a nivel global como de los individuos de sequía, y también inferiores a T+T para los control (Fig. 8c). En el caso de Retama también fue más baja la Fv/Fm de R+T tanto en los individuos control como en el global, aunque no en los individuos sometidos a sequía (Fig. 8d). Al comparar las semanas 0 y 5 del tratamiento no se hallaron diferencias en Fv/Fm debido al efecto de la sequía, y cuando las hubo fue por un aumento de Fv/Fm y en los individuos control, como en el caso de R+T y R (Fig. 9b). En definitiva, las medidas de fluorescencia mostraron el efecto de la competencia interespecífica Thymus-Retama tanto en control como en sequía, de manera más notable que el efecto de la sequía en sí. Competencia intra e interespecífica 113 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 PSII * * ab c aab ab abc abc abc bc abc abc abc abc ab cabc a abc abc bc 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 0505050505 Fv/Fm * abc ab c ab a bc bc a a a TT+T T+R R+T R a b Fig. 9. Eficiencias fotoquímicas real (ΦPSII, a) y máxima (Fv/Fm, b) de las combinaciones de Thymus (T, T+T, T+R) y Retama (R+T, R), comparando los individuos control (colores lisos) con los sometidos a sequía (colores rayados), en las semanas 0 (previa al tratamiento) y 5 del tratamiento de sequía. Los asteriscos indican diferencias significativas entre tratamientos por semana (ANOVA; * P<0.05). Diferentes letras muestran diferencias significativas entre combinaciones y semanas (en minúscula los individuos control, en minúscula negrita los de sequía; ANOVA de 2 vías; P<0.05). Biomasa final La alocación de la biomasa al final del experimento en las combinaciones de Thymus no mostró apenas diferencias entre tratamientos de sequía y control, a excepción de T+T que sí mostró una mayor proporción de raíces/aérea (R:A) en los individuos sometidos a sequía (Fig. 10a). Al comparar las tres combinaciones se encontró una mayor RMR y R:A en las plantas control de T+R respecto a T+T, siendo Capítulo 5 120 angustifolia (Peñuelas et al. 2004) o con otras labiadas como Rosmarinus officinalis o Lavandula stoechas (Munné-Bosch et al. 1999). El mantenimiento de valores constantes de Fv/Fm por encima de 0.75 tanto en Thymus como en Retama demuestra la falta de daños reales inducidos por la sequía en el fotosistema II (Genty et al. 1987). La disminución observada en ΦPSII en condiciones de luz fue completamente reversible tras unos minutos de oscuridad, lo cual indica que la fotoinhibición observada en ambas especies fue debida a un proceso fotoprotector y no a un daño fotoinhibitorio (Munné-Bosch & Alegre 2000). Estos datos concuerdan con los obtenidos en condiciones naturales en la Flecha de El Rompido, donde tampoco se registró un descenso de la eficiencia fotoquímica tras todo el período de sequía estival (Cap. 3). Al ser especies adaptadas a ambientes áridos, el aparato fotosintético no resultó dañado frente a condiciones de sequía como las establecidas en este experimento. Sin embargo, dentro de estos valores óptimos sí se encontró un efecto negativo de Retama sobre Thymus al mostrar T+R valores tanto de ΦPSII como de Fv/Fm más bajos que T o T+T, tanto en sequía como en condiciones control. Además también se puede apreciar un efecto facilitador de la interacción intraespecífica T+T, con valores de ΦPSII más elevados incluso que T. Respecto a Retama, parece haber también un efecto negativo por parte de Thymus, aunque sólo en los individuos de sequía, ya que R+T mostró valores más bajos de Fv/Fm y de ΦPSII que R (Fig. 8b). Igualmente, ambas combinaciones de competencia interespecífica fueron las únicas que mostraron una disminución significativa en el tiempo de ΦPSII entre el principio y el final del tratamiento de sequía (Fig. 9a), posiblemente relacionado con la bajada de potencial hídrico en estas mismas combinaciones. De manera general, la fluorescencia acusó menos el efecto de la sequía que el de la competencia, intraespecífica con carácter positivo e interespecífica con carácter negativo. Una adaptación característica del matorral perenne Mediterráneo a la sequía es aumentar la alocación de biomasa subterránea, disminuyendo la superficie de evaporación en relación a las raíces (Ludlow 1989). Así, ajustar la relación biomasa raíces/aérea (R:A) es un mecanismo efectivo para regular la pérdida de agua ante la sequía (Correia et al. 1992, Werner et al. 1999), ya que mayores R:A pueden interpretarse como una estrategia para maximizar la superficie de absorción de las Competencia intra e interespecífica 121 raíces (Reich et al. 1998, Fernández & Reynolds 2000, Padilla et al. 2009). Además se cree que la relación R:A está relacionada tanto con las tasas de captación de agua como con la de nutrientes (Chapin et al. 1987, Lambers et al. 1998, Cornelissen et al. 2003). El aumento del cociente R:A como respuesta a la sequía solamente se observó en nuestro experimento para T+T, con la misma tendencia para T, R+T y R, siendo T+R la única combinación que no mostró dicho patrón. Sin embargo, con la sequía las plantas de Retama de R+T invirtieron más en raíces que las de R, presumiblemente inducidas por la presencia de Thymus. Esto último pudo impedir el aumento de R:A en T+R, invirtiendo la tendencia que se observaba tanto en T como en T+T, ya que la competencia entre raíces puede provocar una bajada en la relación R:A (Pugnaire & Luque 2001). Así, el efecto conjunto de la competencia de Retama y la sequía habría modificado la respuesta debida únicamente a la sequía en T+R, limitando su desarrollo subterráneo y provocando la bajada del Ψ. Quedaría conocer si hay un efecto mediado por sustancias alelopáticas por parte de alguna de las especies. A pesar de que el contenido en N foliar final fue similar en todas las combinaciones, el δ15N fue menor tanto en T+R como en R+T. Una disminución en δ15N puede estar indicando escasez de N debido a un mayor uso durante el crecimiento (Högberg 1997). La aparición de nódulos de Rhizobium en las raíces de algunas plantas de Retama (observación personal) respaldaría este déficit de N, debido al agotamiento de los nutrientes del suelo abonado. Esta escasez de N del suelo en las macetas de competencia interespecífica habría provocado el aumento de la relación R:A en T+R control para favorecer su captación. De este modo, se podría decir que la competencia de Retama en T+R control no se expresó directamente mediante el R:A, ya que era mayor que para T+T, sino que fue indirecta a través del agotamiento de los nutrientes del suelo, que provocaron el aumento de R:A. Además, a pesar de estar regadas, las macetas de T+R perdían agua más rápidamente por el efecto de Retama, como se observó en los individuos control al principio del tratamiento de sequía (Fig. 5), lo cual también fomentaría un mayor desarrollo de sus raíces respecto de la biomasa aérea. Sin embargo, al añadir el factor sequía de manera acusada, las Retamas aumentaron su alocación a raíces para promover aún Capítulo 5 122 más la captación del agua, reduciendo la inversión subterránea de Thymus y consecuentemente su acceso a dicha agua, como demuestra su Ψ. La bajada de R:A puede reflejar competencia en relaciones tanto intra como interespecíficas (Peperkorn et al. 2005). Tanto en los individuos control como en el análisis global, parece que fue la combinación T+T la que más directamente reflejó esta respuesta por competencia intraespecífica. No obstante, esto no influyó en los parámetros fisiológicos como respuesta a la sequía, probablemente gracias al aumento en la alocación a raíces de T+T. Así, aunque normalmente la competencia intraespecífica se espera mayor que la interespecífica (Hodge et al. 2000, Olson & Blicker 2003, Peperkorn et al. 2005) debido a que individuos de la misma especie requieren condiciones ambientales semejantes, en nuestro caso esto sólo sería cierto atendiendo a la alocación de biomasa, pero no para el resto de parámetros medidos. En un estudio con otra especie de ambientes áridos, Stipa tenacissima, se comprobó una alta competencia por agua tanto a nivel intraespecífico (Ramírez et al. 2009) como interespecífico (Armas & Pugnaire 2011). En nuestro estudio se daría la situación intermedia en respuesta a la sequía, con competencia interespecífica y facilitación intraespecífica. El experimento se realizó con plantas de la misma edad exactamente, reproduciendo lo que ocurriría si crecen cerca dos plántulas de ambas especies. El mayor crecimiento de Retama (tanto en elongación como en biomasas finales) hace que la competencia por los recursos sea desigual, provocando una desventaja por parte de Thymus desde el primer año debido a que la primera explota mayores volúmenes de suelo (8 veces más de raíces de media). El tamaño del sistema radical está asociado positivamente con la habilidad competitiva bajo tierra (Rajaniemi 2002) lo cual le confiere a Retama ventaja competitiva ante un período de sequía. Probablemente esta respuesta frente al déficit hídrico sea más evidente para las combinaciones de Retama debido a sus mayores tasas de elongación, mientras que a la inversa para Thymus las 6 semanas de duración del experimento puede que fuesen poco tiempo como para mostrar efectos más claros y contrastados. En otro experimento de sequía con especies mediterráneas de ambientes áridos sí se encontraron respuestas a nivel de alocación de biomasa, pero fue tras 14 meses de sequía (Padilla et al. 2009). La magnitud de la respuesta frente al déficit hídrico Competencia intra e interespecífica 123 depende de la identidad de las especies y el hábitat, y otras investigaciones han mostrado que especies de hábitats secos tienden a exhibir menores respuestas que aquellas de ambientes menos áridos (Novoplansky & Goldberg 2001, Volis et al. 2002, Sher et al. 2004), presumiblemente debido a sus limitaciones fisiológicas, que les impiden responder a cambios puntuales en la disponibilidad de recursos (Chapin 1991, Sher et al. 2004, Padilla et al. 2009). En conclusión, las plantas jóvenes de Thymus favorecen a los individuos de su misma especie y no parecen afectar de manera importante a las de Retama, mientras que al contrario la influencia de Retama sobre Thymus frente a la sequía es negativa, existiendo competencia por el agua. Creciendo de forma aislada, los juveniles de Thymus muestran una respuesta menor o más lenta frente al estrés hídrico que los juveniles de Retama. Moles & Westoby (2004) apuntaron al défcit hídrico como la principal causa de mortalidad de plántulas en el medio natural, y la competencia por vecinos de mayor tamaño puede ser uno de los factores que hagan a la sequía más severa para las plántulas (Davis et al. 1998, Mingo 2009). En un futuro escenario de cambio climático, con sequías más largas y pronunciadas incluso fuera del período estival, podemos predecir a la vista de los resultados que creciendo en interacción y con la misma edad, las plantas de Thymus se verán afectadas negativamente por la presencia de plantas de Retama. Agradecimientos - Nos gustaría agradecerle a Celia Rodríguez y Alba Cuecas su inestimable ayuda durante las largas horas de invernadero. También a Jose y Jesús, encargados de las instalaciones, su voluntad y soluciones ante cualquier dificultad logística. REFERENCIAS Armas C, Pugnaire FI (2011) Belowground zone of influence in a tussock grass species. Acta Oecologica 37: 284-289. 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Werner C, Correia O, Beyschlag W (1999) Two different strategies of Mediterranean macchia plants to avoid photoinhibitory damage by excessive radiation levels during summer drought. Acta Oecologica 20: 15-23. Capítulo 6 136 INTRODUCTION Management of coastal sand dune plant communities may be difficult because of the instability and frequent perturbations typical of such ecosystems. The result of management in this ecosystem is often the exclusion of some less competitive species (Assmann 1970, Brokaw & Busing 2000). Traditional theory suggests that weaker competitors will be excluded when more than one species compete for a limiting resource, and therefore the survival of inferior competitors is an important issue for ecosystem management (Goreaud et al. 2002) particularly in the case of endangered species. Frequently, the natural dynamics of a species can facilitate the survival of inferior competitors when disturbances occur (Connel 1978). In this context, herbivory can play an important role in the survival of endangered species through modification of the competition conditions against the more competitive species. Although the effects of herbivory on vegetation have been investigated in many ecosystems throughout the world, most studies have focused on modifications to community composition, while few have concentrated on the consequences of browsing on vegetation spatial patterns (Adler et al. 2001, Seifan & Kadmon 2006). The spatial structure of a shrub community plays a key role in the dynamics of its own ecosystem (Menaut et al. 1990, Kubota & Hara 1995, Couteron & Kokou 1997) and may influence the interactions that take place within the plant community (Murrell et al. 1991). In a manner similar to morphological variables, the spatial distribution of vegetation canopies, as determined by the spatial arrangement of its elements, is an integrated result of selection that responds to a variety of environmental conditions (such as water, light or nutrient availability) and competitive interactions (either of the types plant-plant or animal-plant) (Sala et al. 1994). In this sense, herbivory may influence the spatial structure of vegetation mainly because the higher pressure of herbivores exerted upon the most palatable species will favour the establishment of the unpalatable species, but also indirectly through their natural activities such as trampling, trails, wallows or burrows. The impact of herbivory on vegetation dynamics depends largely on grazing pressure, and therefore management should take spatial aspects into account in order Efecto del ganado doméstico 137 to predict the effect of herbivory on spatial distribution (Adler et al. 2001). Two common methods for spatial point pattern analysis are the second-order statistics Ripley's K-function (Ripley 1976, 1977) and the pair-correlation g function, which can reveal non-random spatial structures in plant distribution on all interpoint distances. To investigate the role of domestic livestock herbivory pressure in dune shrub spatial patterns, we applied spatial point pattern analysis in the coastal dune system of El Rompido sand spit. The main objective of this paper was to examine the role of domestic livestock pressure in the spatial distribution patterns, relationships and cover expansion of Thymus and Retama. In order to determine the type of interaction that exists between these two species (competition or facilitation), and the effect of domestic livestock pressure on this interaction, we investigated Thymus abundance and distribution, relative to Retama, in three areas characterized by different livestock pressure intensities. Comparing different levels of herbivory, we expected that the plant cover of Retama would be reduced in the locations where herbivory was permitted, which would also feature a greater surface cover of Thymus than in protected areas, where Retama would be unimpeded by the activities of livestock. MATERIALS AND METHODS Study sites Three study sites were located along the 12 km of El Rompido spit: 1. High herbivory plots. Located in the western part of the spit, this site is easily accessible for livestock and is not included within the protected area. The vegetation is dominated by a shrub community composed mainly of Retama monosperma and Thymus carnosus. 2. Low herbivory plots. This site is located 0.5 km to the east of the high herbivory site and 0.5 km from the western boundary of the protected area. It is not included within the protected area and is accessible to livestock, although their presence is less frequent. Vegetation is dominated by Retama monosperma, Thymus carnosus and Artemisia campestris. Capítulo 6 138 3. Non herbivory plots. Located 5.5 km to the east of the low herbivory plots. This site lies within the protected area and it is non-accessible for livestock. Consequently it can be considered as a control site, where competition or facilitation between the two study species is unaffected by livestock. In fact, physiological measurements from chapters 3 and 4 were developed in this site. The vegetation is composed mainly of a shrub community of Retama monosperma, Thymus carnosus, Helichrysum picardii, Artemisia campestris and Crucianella maritima (Gallego-Fernández et al. 2006). Methodology To select the three areas of different livestock pressure (high, low, none), the number of goat droppings, and Retama fruits below and alongside the Retama canopies, were counted in 20 plots of 0.5 x 0.5 m in each area (quantification was carried out at the end of the summer, when most of the fruits had already fallen). Two plots of 25 x 25 m were established in each study area. The coordinates of each individual plant were recorded in each plot, measuring angles and distances for each individual by means of a Laser range Impulse 200XL attached to a Mapstar Compass Module II (Laser Tech, USA). In addition, the size of each individual plant of both Retama and Thymus was calculated by measuring crown height and maximum and minimum horizontal orthogonal diameters. The percentage of total cover for each species was calculated as the sum of the crown cover of all individuals found within a plot (with shape approaching an ellipse), divided by the surface of the plot. Statistical analysis Data related to plant morphological characteristics, number of fruits and quantity of goat droppings were subjected to an analysis of variance (ANOVA) to detect significant differences among sites. A post-hoc Tukey test was used to compare pairwise differences between sites. All data reached normality assumptions of the ANOVA, except in the case of crown projection, which was Ln transformed. Analyses were performed using SPSS 17.0 for Windows software (SPSS Inc, Chicago, USA). To detect spatial relationships between the two species, the individual coordinates of the plants found in each plot were analyzed by the bivariate point– Efecto del ganado doméstico 139 process g(r), derived from the Ripley's K-function. Ripley’s K(r) function is the expected number of neighbors (points) within concentric circles of radius r centered on an arbitrary point and divided by the intensity λ of the pattern (Ripley 1976, 1977). Therefore, K(r) = λ-1 E (E: number of further events occurring within distance r), where λ is the average density of points, estimated as n/A in a data set of n points and where A is the area (m2) of the region containing all the points. The alternative pair correlation function g(r), which arises if the circles of Ripley`s K(r) function are replaced by rings (Stoyan & Penttinen 2000, Wiegand & Moloney 2004), gives the expected number of points at distance r from an arbitrary point and divided by the intensity λ of the pattern. g(r) = (1/2πr)dK(r)/dr (Wiegand et al. 2006) Ripley`s K(r) function and the pair correlation function g(r) describe the characteristics of the point pattern over a range of distance scales and allow the detection of mixed patterns, (e.g., dispersion at smaller distances and aggregation at larger distances) (Ripley 1981, Haase 1995, Wiegand & Moloney 2004). However, using rings instead of circles has the advantage that one specific distance class can be isolated, whereas the cumulative K-function confounds effects at larger distances with those at shorter distances and is consequently less informative and difficult to interpret (Law et al. 2009). To test H0 (random point pattern) versus H1 (attraction or repulsion point pattern), we assumed that the two point patterns were generated by independent processes: one process generating the locations of Retama, and the other generating the locations of Thymus. Consequently, we adopted the bivariate null model antecedent conditions (sensu Wiegand & Moloney 2004). This null model keeps the locations of individual plants of Retama, but distributes Thymus shrubs randomly over the area. Departure from independence indicates that the two processes display either attraction or repulsion. Ninety nine Monte Carlo simulations were used to generate a confidence interval of g(r) corresponding to 99% confidence limits. Observed values of g(r) > confidence intervals indicate an attraction between the two patterns to distance r, while values of g(r) < confidence intervals indicate Capítulo 6 140 repulsion between the two patterns to distance r. Values within the confidence interval indicate random distribution. The software Programita (http://www.oesa.ufz.de/towi/towi_programita.html), developed by Wiegand & Moloney (2004), was used to carry out the point pattern analyses. We used a minimum radius of 0.25 m up to a maximum radius of 12.5 m. This spatial resolution was sufficiently fine to examine the spatial distribution from a small scale to a larger one. Fig. 1. Goat droppings density and Retama fruits density outside and below Retama shrubs at three different intensities of livestock pressure: High herbivory, Low herbivory and Non herbivory (Mean ± SD, n=10 per treatment). Lower-case and capital letters indicate post hoc Tukey test results, outside and below the plants respectively. RESULTS As expected, the quantity of droppings was significantly different between sites, with no goat droppings found in the non herbivory plots. The highest dropping density was found below Retama plants in the high herbivory pressure plots, indicating that the goats spend more time below the canopies of these shrubs than outside (Fig. 1, Table 1). High livestock pressure depleted the Retama fruits almost completely, with Efecto del ganado doméstico 141 Factors Variables df F P Herbivory Droppings Outside 2 12.40 <0.001 intensity Below 2 7.79 <0.002 Fruits Outside 2 31.80 <0.001 Below 2 22.80 <0.001 Herbivory Thymus Height 2 70.56 <0.001 intensity Diameter 2 59.24 <0.001 Retama Height 2 57.33 <0.001 Diameter 2 12.73 <0.001 10 being the maximum number of fruits per m2 found in these plots, whereas densities of almost 2000 fruits m-2 were recorded in the protected plots (Fig. 1, Table 1). Fruit and dropping densities were always higher below Retama shrubs than alongside. Table 1. Summary of the one-way ANOVAs for the effect of herbivory intensity for droppings and fruits densities outside and below Retama canopies, and for height and diameter of Retama and Thymus plants. Individuals from the non herbivory plots of both shrub species were significantly larger and taller than those from plots with livestock (both of which exhibited comparable size and height, Fig. 2, Table 2). Individuals of Retama from non herbivory plots were 40% larger (16 m2) than those with herbivory presence (10 m2). A similar pattern was observed in Thymus plants where plants from non herbivory plots (0.23m2) were 48-26% larger than those from plots to which livestock had access (0.12-0.17 m2). Although the total number of Thymus plants present in high and low livestock pressure plots was similar, (1273 and 1260, respectively), the surface covered by this species was significantly higher in low herbivory plots (21%) than in high herbivory plots (12%, Table 2). On the other hand, the plots that were protected from herbivory, presented the lowest number of Thymus plants (284) and surface cover value (5.2%). The number of Retama plants, as well as the surface covered, decreased gradually with increasing intensity of herbivory: 65 individuals were registered in the nonherbivory plots compared to 41 and 35 in low and high herbivory plots, respectively, while surface cover was reduced from 82% in non herbivory to 23.6 % in the high herbivory plots (Table 2, Fig. 2). Capítulo 6 142 Factors Species Number of plants Total cover (%) Crown (m 2 ) High herbivory T 1,261 11.7 ± 0.3 0.12 ± 0.03 T+R 12 0.1 ± 0.02 0.11 ± 0.03 R 35 23.6 ± 6.7 10.1 ± 6.5 Low herbivory T 1,011 14.2 ± 5.2 0.17 ± 0.12 T+R 252 6.8 ± 1.9 0.34 ± 0.04 R 41 31.7 ± 0.8 9.8 ± 1.4 Non-herbivory T 221 3.6 ± 2.2 0.23 ± 0.08 T+R 63 1.6 ± 1.9 0.31 ± 0.01 R 65 81.8 ± 14.4 15.9 ± 1.0 Cover (%) 0 20 40 60 80 100 Diameter and height (m) 0 1 2 3 4 5 High herbivory Low herbivory Non herbivory Total cover Diameter T. carnosus R.monosperma T. carnosus R.monosperma Height T. carnosus R.monosperma abc A A B a a b A A B Fig. 2. Mean total cover, height and crown diameter ± SD of Thymus and Retama at three different intensities of livestock pressure: High herbivory, Low herbivory and Non herbivory. Lower-case and capital letters indicate post hoc Tukey test results for the two species (total cover was not included in ANOVA and Tukey analyses because n of this variable = 2). Table 2. Number of individual plants (sum of the two plots per site), mean total cover (%) and mean crown area of Retama (R), Thymus (T) and Thymus plants beneath Retama canopy (T+R) in the three levels of herbivory intensity. Efecto del ganado doméstico 143 Fig. 3. Spatial distribution maps of Retama and Thymus individuals at three different intensities of livestock pressure: High herbivory, Low herbivory and Non herbivory. Two plots of each herbivory pressure are included. Black filled circles represent Thymus plants, open circles: Thymus plants below Retama, +: represent the central location of Retama plants and grey areas represent canopies, to scale. Capítulo 6 144 Fig. 4. Results of bivariate pair correlation function g(r) of Retama and Thymus using the null model antecedent condition at three livestock pressure levels: A) High herbivory, B) Low herbivory and C) Non herbivory. Full lines represent values of g(r) function and dashed lines represent confidence intervals at 99% of complete spatial randomness. Values of g(r) falling within the confidence interval mean random distribution between the two patterns; those over the confidence interval imply a significant attraction and those under the interval mean significant repulsion between the two patterns. Efecto del ganado doméstico 145 The spatial distribution of both study species (Fig. 3) proves that Thymus can occur beneath Retama canopies. This occurrence varied non-linearly with livestock pressure. In the low herbivory pressure plots, we found 251 Thymus plants growing below Retama (Fig. 3 Low herbivory), while in the plots of high and no livestock pressure we found only 12 and 63 plants, respectively (Fig. 3 High and Non herbivory). Bivariate analysis under the null model of antecedent conditions testing for competition or facilitation between Retama and Thymus showed differences in spatial pattern between the three herbivory intensities (Fig. 4). In low herbivory plots, Thymus plants were randomly distributed in relation to Retama, however there was a positive association (statistically clumped) at scales of 0.75-2 m in plot 1 (Fig. 4 B1), and at scale of 2.25-2.5 m, 3.25 m in plot 2 (Fig. 4 B2). Conversely, in the high herbivory plots, Thymus plants showed a significant trend towards repulsion of Retama. There was a negative association between these species at scales of 0.25 to 3.5-4.25 m (Fig. 4 A1, A2), due to the no-neighbour case, with Thymus individuals being more frequent than expected around Retama plants. In high herbivory plot 2, which had a comparatively lower density of Thymus plants (542) than plot 1 (719), repulsion was markedly stronger. In contrast, the two study plots with no livestock did not show a common pattern: in plot 1 the bivariate analysis revealed a repulsion spatial relationship in the first 4 m (Fig. 4 C1), while in plot 2 the spatial distribution of the two shrubs did not differ significantly from random, with the exception of a weak repulsion between species at scales of 2.75 m and 3.75-4 m (Fig. 4 C2). DISCUSSION The main findings of this study indicate that under high and non livestock pressure, the less competitive Thymus is displaced by Retama. This brings about a reduction of the surface covered by the former smaller shrub and creates a repulsion point pattern between the two species. However, when disturbance imposed by livestock is low, our data show higher cover values of Thymus than was the case