Ecología de la especie invasora Ailanthus altissima (Mill.) Swingle. Bases para su control y erradicación en Espacios Naturales Protegidos. Soraya D. Constán Nava. Facultad de Ciencias Departamento de Ecología TESIS DOCTORAL Ecología de la especie invasora Ailanthus altissima (Mill.) Swingle. Bases para su control y erradicación en Espacios Naturales Protegidos. Soraya D. Constán Nava Director: Andreu Bonet Jornet 2012 Departamento de Ecología Ecología de la especie invasora Ailanthus altissima (Mill.) Swingle. Bases para su control y erradicación en Espacios Naturales Protegidos. Memoria presentada por: Soraya D. Constán Nava para optar al titulo de Doctora en Ciencias Biológicas Director: Andreu Bonet Jornet Alicante, 2012 Dr. Andreu Bonet Jornet, Profesor titular del Departamento de Ecología de la Facultad de Ciencias de la Universidad de Alicante HACE CONSTAR: Que el trabajo presentado en esta memoria, con el titulo: “Ecología de la especie invasora Ailanthus altissima (Mill.) Swingle. Bases para su control y erradicación en Espacios Naturales Protegidos” ha sido realizado bajo su dirección por Soraya D. Constán Nava en el Departamento de Ecología y reúne todos los requisitos para su aprobación como Tesis Doctoral. Alicante, Enero de 2012 D. Andreu Bonet Jornet A Santi, mi mundo A mi familia, los que están y los que se fueron ÍNDICE Resumen Introducción. Objetivos y estructura de la tesis. Metodología y área de 1 estudio. Resultados generales. Discusión general. Conclusiones. Bibliografía Capitulo 1 Distribution and performance of populations of the invasive species 49 Ailanthus altissima on Mediterranean Protected Areas Capitulo 2 Genetic variability modulates the effect of habitat−type and 69 environmental conditions on early invasion success of Ailanthus altissima in Mediterranean ecosystems Capitulo 3 Direct and indirect effects of invasion by the alien tree Ailanthus altissima on riparian plant communities and 99 ecosystem multifunctionality Capitulo 4 Long-term control of the invasive tree Ailanthus altissima: Insights 133 from Mediterranean protected forests Agradecimientos 153 Afiliación de los coautores 155 RESUMEN Introducción. Objetivos y estructura de la tesis. Metodología y área de estudio. Resultados generales. Discusión general. Conclusiones. Bibliografía 2 INTRODUCCIÓN DEFINICIÓN. ANTECEDENTES. FASES GENERALES Las especies exóticas invasoras (sensu Richardson et al. 2000) son aquellas especies capaces de sobrevivir, establecerse y reproducirse en un lugar biogeográficamente distinto al original, superando los limitantes a su llegada, establecimiento y reproducción, tanto bióticos como abióticos (Williamson 1996; Mack et al. 2000; Balaguer 2004). Las invasiones biológicas por especies vegetales obedecen a un fenómeno natural, pero la presencia del ser humano ha acelerado este proceso exponencialmente (Vitousek et al. 1997; Mack et al. 2000; Mooney y Hobbs 2000; Vilà 2000). Actualmente, se consideran como la tercera causa principal del cambio global, solamente por detrás de la destrucción de hábitats y la fragmentación del paisaje (Vitousek 1990; Vitousek 1994; Williamson 1996), causando un enorme impacto sobre la biodiversidad a escala global (IUCN 2000; Mack et al. 2000; Didham et al. 2005). La introducción de especies vegetales ha sido tanto voluntaria como involuntaria por parte del ser humano, quien ha actuado como elemento diseminador de primer orden (antropocoria) (Vitousek et al. 1997; Vilà 2000; Sanz et al. 2001; Hulme et al. 2008). Entre las vías de introducción se encuentran el turismo, las migraciones y el desplazamiento y los intercambios comerciales, que aunque fueron iniciados principalmente en la Edad Moderna a partir del descubrimiento de las rutas transoceánicas, actualmente son de carácter global (McNeely et al. 2001; Vilà 2000; Kowarik 2005). El proceso de invasión está considerado como un continuum invasiónnaturalización en el que las especies han de atravesar diferentes tipos de barreras: geográficas, ambientales, reproductivas, de dispersión, ambientales en hábitats perturbados y finalmente, ambientales en hábitats naturales (Richardson et al. 2000; Pyšek y Richardson 2010). Solo unas pocas de las especies introducidas (alrededor del 10%) sobreviven a las nuevas condiciones a las que se enfrentan y pocas son las que se naturalizan, estableciéndose y reproduciéndose en el nuevo lugar, de manera que el número de individuos aumenta y la población comienza a expandirse (Williamson 1996; Mack et al. 2000; Balaguer 2004). En el caso de las especies invasoras, esta explosión demográfica normalmente se caracteriza por una alta tasa de crecimiento poblacional, es decir, el tamaño poblacional y la superficie ocupada crecen rápidamente 3 (McNeely et al. 2001). La mayoría de las especies naturalizadas no causan alteración en su nueva área de distribución, pero un 1 % se vuelven invasoras (Williamson y Fitter 1996; Mack et al. 2000; Mooney y Hobbs 2000; Balaguer 2004), estableciendo interacciones ecológicas y evolutivas con la biocenosis de la comunidad invadida (Vilà 2000). La invasión no solo depende de la especie introducida, sino que es el resultado de la interacción compleja con diferentes factores, incluyendo el clima y el hábitat, así como las especies nativas y otras especies invasoras que pueden acelerar y amplificar la invasión y sus efectos en el lugar invadido a través de procesos de retroalimentación (proceso conocido como invasional meltdown, Simberloff y Von Holle 1999; Richardson et al. 2000; Pyšek y Richardson 2010). LA ECOLOGÍA DE LA INVASIÓN. MARCO CONCEPTUAL La ecología de la invasión estudia todos los componentes y procesos que interfieren en la invasión de especies exóticas (Mack et al. 2000; Richardson y Pyšek 2006; Pyšek y Richardson 2010). Desde el libro “The ecology of invasions by animals and plants” (Elton 1958) hasta la actualidad, se puede encontrar numerosa literatura científica sobre estos componentes (véase por ejemplo: Lodge 1993; Richardson et al. 2000; Pyšek et al. 2004; Blackburn et al. 2011). Foxcroft et al. (2011) han propuesto un marco conceptual que integra todos estos mecanismos y el contexto en el que interactúan (Fig. 1). Este marco general está compuesto por tres componentes principales: las características de las especies invasoras, el contexto donde se desarrolla la invasión y la susceptibilidad del hábitat receptor. 4 Figura 1 Marco conceptual de la ecología de la invasión. Adaptado de Foxcroft et al. 2011 Respecto al primer componente, las características de una especie vegetal invasora, se encuentran numerosos mecanismos que pueden potenciar el carácter invasor de una especie, tales como la producción de propágulos para el éxito de reproducción (numero y viabilidad de semillas, tasa de germinación, crecimiento de plántulas), competitividad (por ejemplo, presencia de sustancias alelopáticas), reproducción vegetativa para expandirse, genotipos con elevada plasticidad fenotípica ya que se propagan por diferentes medios, y una elevada tasa de crecimiento (Rejmánek 1996; Vilà 2000; Castro-Díez et al. 2004; Foxcroft et al. 2011). Pero, no todas las especies vegetales invasoras poseen todas estas características, ni tampoco una especie con esas características actúa como invasora (Vilà 2000; Balaguer 2004). Existen diversos estudios comparativos entre especies vegetales nativas vs invasoras (e.g. Godoy et al. 2009; van Kleunen et al. 2010; Godoy et al. 2011) donde se ha encontrado que existen diferencias entre características relacionadas con el desarrollo (tasa de crecimiento, tamaño) siendo mayor en las invasoras, pero no hay diferencias en plasticidad fenotípica (por ejemplo, como respuesta a gradientes de nutrientes y luz). En este sentido, Thompson y Davis (2011) han concluido que las plantas invasoras poseen 5 rasgos característicos de las especies más exitosas, independientemente si son nativas o exóticas. El segundo componente es el contexto del sistema, es decir, dentro del cual se desarrolla la invasión. Este componente conecta las características de la especie invasora con la susceptibilidad del hábitat. Entre los mecanismos específicos que se incluyen en este componente se encuentran: − la conectividad entre hábitats. Por ejemplo, las zonas verdes, vertederos, áreas residenciales y parques periurbanos, dado que están sujetas al estrés ambiental y a causa de su proximidad a sitios de introducción de especies exóticas y su función como borde de otros hábitats, son el principal hábitat para las especies invasoras, las cuales se extienden hacia hábitats menos urbanos (Kowarik 1983; Gulezian et al. 2010; Hitchmugh 2011). − la presencia de vías de expansión que actúan como vías de dispersión, por ejemplo, debido al movimiento de semillas (Spellerberg 1998; Vilà y Pujadas, 2001; Hansen y Clevenger 2005; Kowarik y von deer Lippe 2006) − la presión de propágulos, es decir, nuevos eventos de introducción de individuos y su densidad (Von Holle y Simberloff 2005; Lockwood et al. 2009). − el tiempo de residencia (es decir, cuánto tiempo lleva una especie invasora en un determinado lugar (Lockwood et al. 2009; Vilà y Ibáñez 2011) − los valores y percepciones humanos, importante de cara a la gestión de las especies invasoras (Vitousek et al. 1997; Simberloff, 2009; Foxcroft et al. 2011). El tercer componente es la susceptibilidad del hábitat, y los mecanismos específicos que entran dentro de este componente están relacionados con las condiciones bioclimáticas, la presencia de depredadores (generalmente baja, pues escapan de sus enemigos naturales), la disponibilidad de recursos y la heterogeneidad del paisaje, que incluye la presencia de espacios vacíos (áreas perturbadas de origen antrópico) (Hobbs y Huenneke 1992; Maron y Vilà 2001; Sanz et al. 2004). EFECTOS DE LAS ESPECIES VEGETALES EXÓTICAS INVASORAS La presencia de especies vegetales invasoras supone nuevas situaciones ambientales tanto para las especies invasoras como para el ecosistema receptor (Castro-Díez et al. 2004), de manera que cambian las reglas de la existencia para las especies (Vitousek et al. 1997). Los efectos que causan estas especies ocurren a diferentes niveles. 6 Genéticamente pueden afectar mediante la hibridación de especies exóticas con nativas (Huxel 1999; Ellstrand y Schierenbeck 2000; Vilà 2000; Lee 2002). En el ámbito paisajístico afectan a través de la homogeneización de los hábitats por la dominancia de especies exóticas invasoras (Vilà 2000; Hastings et al. 2005; Vuilleumier et al. 2011). Ecológicamente se encuentran la alteración de la estructura y funcionamiento del ecosistema invadido, mediante la alteración de ciclos biogeoquímicos, modificación de procesos de erosión y sedimentación, alteración de la fertilidad de los suelos, reducción o agotamiento de los niveles de agua, alteración de los patrones de drenaje, y/o modificación de los regímenes de incendios (Mack et al. 2000; Corbin y D´Antonio 2004; Didham et al. 2005; Castro-Díez et al. 2009). Asimismo, pueden competir con especies nativas, por ejemplo, mediante mecanismos competitivos que no están presentes en las comunidades donde invaden como es a través de sustancias alelopáticas (Callaway y Aschehoug 2000; Ens et al. 2009), o por competencia por polinizadores (Jakobsson et al. 2009; Morales y Traveset 2009). Esta competencia puede llegar a reemplazar a las especies nativas (Vilà 2000; Simberloff 2001). Por otro lado, pueden favorecer la invasión de otras especies exóticas y causar un impacto sobre la regeneración y dinámica natural de muchos ecosistemas terrestres (Groves 1986; Mooney y Drake 1986; Simberloff 2001; Valladares et al. 2004; Murphy et al. 2006). En el ámbito socioeconómico implican elevados costes debido a las inversiones realizadas en medidas de prevención (análisis de riesgos, educación ambiental), detección temprana (localización de especies invasoras, evaluación de impactos), control y erradicación, así como en medidas de restauración de ecosistemas invadidos (Dukes y Mooney 1999; Mack et al. 2000; Vilà 2000; Castro-Díez et al. 2004; Moore 2005). Por ejemplo, en Europa se ha estimado que el coste económico en relación a las especie invasoras es de 9-12 billones de euros anuales, siendo realmente mayor, pues en muchos países están comenzando a analizar los efectos de este problema (European Commission 2008). En el ámbito cultural implican una alteración de la percepción del ser humano, ya que en muchos casos las especies invasoras son consideradas como nativas (Balaguer 2004; Davis et al. 2011), por ejemplo, debido al uso común de especies exóticas en jardinería. 7 MARCO LEGISLATIVO SOBRE ESPECIES EXÓTICAS INVASORAS En la actualidad existe un gran interés en el desarrollo de normativas adecuadas, manifestada en diferentes mandatos internacionales de carácter ambiental, que regulen específicamente las especies exóticas invasoras. Hasta el momento se puede encontrar leyes que instan al control, prevención y protección frente a las especies invasoras. En el ámbito europeo, el Convenio de Berna, uno de los primeros antecedentes legales relativo a la conservación de la vida silvestre y el medio natural en Europa (1979), obliga a las partes contratantes a realizar un control estricto de la introducción de especies no nativas (Art. 11, 2b). Posteriormente, el Reglamento (CE) n.º 338/97 del Consejo, de 9 de diciembre de 1996, relativo a la protección de especies de la fauna y flora silvestres mediante el control de su comercio, incluye especies que son una amenaza ecológica para las especies silvestres autóctonas dentro de la Unión Europea. Posteriormente, dichas disposiciones son recogidas y ampliadas en la Directiva 92/43/CEE, del Consejo, de 21 de mayo de 1992, relativa a la conservación de los hábitats naturales y de la fauna y la flora silvestres, incluyendo la regulación de la introducción de especies exóticas. Asimismo, Comisión Europea (2008) adopta la Comunicación «Hacia una Estrategia de la Unión Europea sobre especies invasoras» (COM(2008) 789 final).La estrategia internacional queda recogida en el Convenio de Naciones Unidas sobre la Diversidad Biológica (1993), la cual manda a las partes firmantes a prevenir la introducción, control o erradicación de las especies no nativas. En el ámbito nacional, la Ley 4/1989 de 27 de marzo, de Conservación de los Espacios Naturales y de la Flora y Fauna Silvestre dicta normas en relación a la introducción o liberación no autorizada de especies alóctonas perjudiciales, considerándose como delito contra el medio ambiente en la Ley orgánica 10/1995, de 23 de noviembre, del Código Penal. Asimismo, la Ley 43/2002, de 20 de noviembre de Sanidad Vegetal incluye restricciones y prohibiciones relacionadas con especies vegetales alóctonas. La Ley 26/2007, de 23 de octubre, de responsabilidad medioambiental, a través del Real Decreto 2090/2008, de 22 de diciembre, de desarrollo parcial de dicha Ley, incluye a las especies vegetales invasoras como agente causante de daño biológico. La Ley 42/2007, de 13 de diciembre, de Patrimonio Natural y de la Biodiversidad, contempla la prohibición de la introducción de especies alóctonas por parte de las administraciones públicas competentes en el caso de que puedan afectar a las especies nativas (art. 52.5) y crea el Catálogo Español de Especies Exóticas 8 Invasoras (art. 61.1) de ámbito estatal. Finalmente, el Real Decreto 1628/2011, de 14 de noviembre, regula el listado y Catálogo Español de Especies Exóticas Invasoras. En el ámbito autonómico, podemos encontrar algunos ejemplos, como el Decreto 213/2009 sobre control de especies exóticas invasoras de la Comunidad Valenciana, el Plan Andaluz para el Control de las Especies Exóticas Invasoras, que aborda el problema de las invasiones biológicas, y la Estrategia Canaria de la Biodiversidad. PROBLEMÁTICA INVESTIGACIÓN EN LOS ESPACIOS NATURALES PROTEGIDOS. EL PAPEL DE LA EN LA GESTIÓN DE LAS ESPECIES INVASORAS Los espacios naturales protegidos generalmente representan las últimas áreas alteradas (Vitousek et al. 1997), pero no dejan de ser susceptibles a la presencia de especies invasoras, ya que muchas especies vegetales exóticas han invadido con éxito hábitats con un alto grado de conservación (Drake 1988; Luken 1988; Broncano et al. 2005). Este fenómeno, directa o indirectamente relacionado con la actividad humana, se está generalizando como un problema de manejo prioritario (Luken y Thieret 1997). Cada vez son más las áreas protegidas que se encuentran amenazadas por las invasiones biológicas (Luken y Thieret 1997; MacDougall y Turkington 2004; Traveset et al. 2008). Ante este problema, los planes de gestión de las áreas protegidas incluyen o deben incorporar el control de estas especies (Europarc-España 2002; Balaguer 2004; Worboys et al. 2005; Andreu et al. 2009; Davis et al. 2011). La investigación juega un papel de vital importancia en el ámbito de la gestión mediante el aporte de información sobre las características de las especies invasoras, la evaluación de sus impactos, la detección, el análisis de riesgos de expansión, las medidas de control más eficaces, etc. (Pyšek y Richardson 2010). Todo esto mejora el éxito en la gestión de las especies invasoras y puede implicar una reducción en los costes económicos. AILANTHUS ALTISSIMA, ESPECIE OBJETO DE ESTA TESIS DOCTORAL Ailanthus altissima (Mill) Swingle 1916 (ailanto, árbol del cielo) pertenece a la familia Simaroubaceae, y es una de las cinco especies del género Ailanthus Desf. Este árbol, nativo de China y del Norte de Vietnam, fue introducido en Francia en los años 1740 por el misionero Pierre d´Incarville en forma de semillas, y posteriormente (1751), al ser confundida con la especie Rhus verniciflua, fue transportada a Londres y a otras 9 partes de Europa y América (Hu 1979). Actualmente, esta especie está catalogada como especie invasora en todos los continentes salvo en la Antártida (Fig. 2; Kowarik y Säumel 2007). Está considerada como una de las plantas invasoras más extendidas en Europa y ha sido incluida en la lista Top 20 de especies de prioridad en esta zona (Sheppard et al. 2006; Pyšek et al. 2009). Asimismo, se encuentra en las Islas Mediterráneas (Moragues y Rita 2005; Bacchetta et al. 2009; Podda et al. 2010), donde está considerada como una de las 15 especies de mayor impacto dentro del proyecto Exotic Plant Invasions: Deleterious Effects on Mediterranean Island Ecosystems (EPIDEMIE) (Balaguer 2004; Moragues 2005). En España está incluida en la lista negra preliminar de especies exóticas invasoras (Capdevila et al. 2006), en el Decreto 213/2009 sobre control de especies exóticas invasoras de la Comunidad Valenciana, así como en el Anexo I del Real Decreto 1628/2011, de 14 de noviembre, por el que se regula el listado y catálogo español de especies exóticas invasoras, estando prohibida su introducción en el medio natural, posesión, transporte, tráfico y comercio. Figura 2 Área de expansión de Ailanthus altissima con una diferenciación del área nativa en China y Norte de Vietnam (rallado) y su distribución secundaria a lo largo del Mundo (negro) a partir de su introducción en Europa en los años 1740 (Kowarik y Saümel 2007) A. altissima es una especie pionera y de crecimiento rápido (1 a 1,5 m/año) (Zasada y Little 2002). El rango de tamaño que se puede encontrar es de 27−30 m en zonas templadas y de 18−20 m en zonas meridionales (Hunter 2000; Arnaboldi et al. 2003). Tiene una gran capacidad de rebrotado, formando densos rodales (Kowarik 1995). Es un árbol dioico; algunos autores señalan que las flores pueden ser bisexuales o los árboles monoicos, aunque esto aún no está demostrado (Kowarik y Säumel 2007). 10 Produce semillas en forma de sámaras y se dispersan de manera anemócora a partir de septiembre-octubre permaneciendo muchas de ellas en el árbol hasta mayo del año siguiente (Fig 3; Bory y Clair-Maczulajtys 1980). También se ha encontrado dispersión hidrocora a través de cursos de agua, la cual juega un importante papel para su expansión a larga distancia (Kowarik y Säumel 2007, 2008; Kaproth y McGraw 2008; Säumel y Kowarik 2010). La producción de sámaras es elevada, de hasta 325.000 sámaras por individuo (Little 1974; Bory y Clair-Maczulajtys 1980). Produce una raíz pivotante y numerosas raíces laterales de una longitud media de 114,4 cm, donde almacena la mayor parte de las reservas de carbohidratos y proteínas (Dubroca y Bory 1981). La corteza de la raíz, y de otras partes de la especie, así como hojas, y sámaras contienen componentes herbicidas y alelopáticos que son tóxicos para muchas especies (Heisey 1990, 1996; Lawrence et al. 1991). En otras palabras, esta especie posee todas las características necesarias para ser una especie invasora: especie pionera de rápido crecimiento juvenil, rebrotadora, con sistema radicular potente y producción sustancias alelopáticas y herbicidas. Figura 3 Ejemplar ornamental adulto femenino de A. altissima y detalle de panícula de semillas Está considerada como una especie oportunista, intolerante a la sombra (Grime 1965), y como una pionera colonizadora de espacios abiertos. En el caso de ecosistemas forestales, se describe como “gap-dependent”, dependiente de la presencia de huecos en el dosel del bosque para su desarrollo (Knapp et al. 2000). 11 Fuera de su rango nativo está sujeta a una baja presión herbívora, lo que se ha atribuido a los componentes tóxicos de sus tejidos (Ohmoto y Koike 1984). En Europa se ha encontrado dos especies de artrópodos que se alimentan de esta especie, Hyphantria cunea en Austria y Samia cynthia en Italia y se han identificado 46 artrópodos fitófagos en su rango nativo (Kowarik y Säumel 2007). Asimismo, se han descrito 65 especies de hongos asociados a la especie (Ding et al. 2006). La distribución de la especie fuera de su área originaria se debe a su empleo como especie forestal y ornamental, así como en restauración de taludes de carreteras (Ruiz de la Torre et al. 1990). Existe un elevado número de espacios naturales protegidos en el mundo que han sido invadidos por A. altissima, como por ejemplo los Parques Nacionales de Richmond y Petersburg en Estados Unidos (Akerson et al. 2001), el Parque Nacional del Danubio (Austria) (Drescher y Ließ 2006) o el Parque Nacional Aggtelek (Hungria) (Kowarik 2003) en Europa. Concretamente en España, se encuentra en el Parque Nacional de Sierra Nevada (García et al. 2003) y el Parc Natural de la Sierra de Collserola (Barcelona) (Meggaro y Vilà 2002), en el Parque Natural de la Sierra de Cardeña y Montoro, donde su erradicación está planteado grandes dificultades (Algarra et al. 2005), así como en los Parques Naturales del Carrascal de la Font Roja y de Sierra de Mariola (Alicante y Valencia) (Constán-Nava et al. 2007, 2010). Para la gestión de especies vegetales invasoras en espacios naturales protegidos es importante seguir varios pasos (ver arriba apartado Problemática en los espacios naturales protegidos. El papel de la investigación en la gestión de las especies invasoras). Como primer paso, es importante conocer la distribución de la especie invasora y el grado de invasión que presenta en los diferentes ecosistemas que existen en el área natural, donde lo prioritario es la conservación de las especies nativas y los ecosistemas que lo conforman (Vilà et al. 2007; Chytrý et al. 2009). Por un lado, es necesario conocer la superficie ocupada por la especie invasora. Por otro lado, se ha de determinar los ecosistemas que han sido invadidos para llevar a cabo actuaciones de control, priorizando los ecosistemas de mayor interés para la conservación teniendo en cuenta el paisaje (Reid et al. 2009; Pyšek y Richardson 2010; Vilà y Ibañez 2011). En relación a A. altissima, estudios previos muestran que ha invadido tanto ecosistemas antrópicos (áreas periurbanas, cultivos en activo o abandonados, taludes de carretera, Fig. 4), como ecosistemas naturales, por ejemplo, pinares o bosques de ribera (Kowarik 1983; Danin 12 2000; Constán-Nava et al. 2007; Kowarik y Säumel 2007; pero ver Affre et al. 2010), y es menor la frecuencia a mayores altitudes (Traveset et al. 2008), pero son escasos los estudios que tienen el cuenta el paisaje en el proceso de invasión, así como el desarrollo de la especie en los diferentes hábitats que lo conforman. Figura 4 Expansión de A. altissima junto a borde de carretera (izquierda) e invasión en campo de cultivo de olivos (derecha). En segundo lugar, es necesario el conocimiento de rasgos ecológicos de la especie invasora que puedan influir en su expansión (Lake y Leishman 2004; Finnoff y Tschirhart 2005; Andreu y Vilà 2010). Entre estos aspectos, se encuentran la germinación y el establecimiento. Se conoce que A. altissima germina en diversos tipos de suelo, y es mayor la germinación con mayor disponibilidad lumínica y a bajas altitudes (< 1000 m), aunque parece no depender del tipo de hábitat (Mihulka 1998; Kota et al. 2007; Vilà et al. 2008; Moore y Lacey 2009) o de la fuente de semillas (Kota et al. 2007; Delgado et al. 2009). Aunque es importante el conocimiento del efecto de los factores genéticos como ambientales sobre el desarrollo de la especie invasora, no existen estudios que contemplen los efectos de ambos factores así como sus interacciones. Por otro lado, es importante el conocimiento sobre los efectos tanto directos como indirectos que puede provocar las especies vegetales invasoras en los ecosistemas invadidos. Estudios previos indican que A. altissima altera el ciclo del nitrógeno (Castro-Díez et al. 2009), y puede enlentecer la descomposición de hojarasca, aumentar el N total, C orgánico y pH del suelo, así como la proporción C/N (Vilà et al. 2006; Godoy et al. 2010; pero ver Castro-Díez et al. 2011). Asimismo, tiene un efecto en el funcionamiento ecosistémico y en la dinámica y estructura de la vegetación, 13 disminuyendo la biodiversidad (Lawrence et al. 1991; Vilà et al. 2006). A pesar de esto, aún no se han desarrollado estudios que analicen los efectos directos e indirectos (mediados por su efecto en la biodiversidad) sobre el múltiples funciones ecosistémicas así como sobre la diversidad filogenética, en comunidades vegetales invadidas, todo ello simultáneamente. Finalmente, dado la problemática que supone la presencia de A. altissima en los espacios naturales, es imprescindible el desarrollo de actuaciones que contemplen el control de las especies invasoras y su posible eliminación. En esta línea, se han desarrollado diversos métodos (manual, mecánico, químico, quema) para combatir a A. altissima (Hoshovsky 1988; Hunter 2000). El método aplicado más habitual ha sido el de eliminación mecánica, pero su tendencia a rebrotar hace que sea poco efectiva (Hoshovsky 1988; Bory et al. 1991; Hunter 2000). En áreas de clima templado se ha encontrado que el empleo de tratamiento mecánico junto con químico (en concreto, el glifosato) es el más eficaz (Meloche y Murphy 2006). Sin embargo, bajo clima mediterráneo no se conoce una técnica a largo plazo que sea efectiva, a pesar de la necesidad de su control, la cual está incluida en los planes de gestión de numerosos espacios naturales (Andreu y Vilà 2007; Andreu et al. 2009). OBJETIVOS Y ESTRUCTURA DE LA TESIS El objetivo general de esta tesis es analizar diferentes procesos que intervienen en la invasión de Ailanthus altissima en hábitats mediterráneos para mejorar el éxito en su gestión en áreas protegidas. Los objetivos específicos que se plantean en esta tesis son los siguientes: Analizar la distribución actual y el grado de invasión de la especie invasora A. altissima en la LIC Serra de Mariola i Carrascal de la Font Roja, así como determinar las variables ambientales que afectan a su desarrollo (Capitulo 1) Identificar la importancia relativa tanto de los factores genéticos como ambientales (variabilidad genética, condiciones climáticas y tipo de hábitat) y sus interacciones sobre la emergencia y establecimiento temprano de la especie invasora A. altissima bajo condiciones mediterráneas (Capitulo 2) 14 Analizar los efectos tanto directos como indirectos (mediados o no por su efecto sobre biodiversidad) de A. altissima sobre la multifuncionalidad ecosistémica en hábitats riparios bajo clima Mediterráneo (Capitulo 3) Determinar la mejor estrategia para controlar la invasión de A. altissima a largo plazo en bosques mediterráneos (Capitulo 4) Figura 5 Diagrama sintético en el que se incluyen los capítulos que conforma la tesis doctoral. Modelo adaptado de Foxcroft et al. 2011 Los cuatro capítulos que conforman esta tesis están escritos en inglés y se presentan en formato de artículo para su publicación en revistas científicas de carácter internacional. Esto implica algunas redundancias en relación a la descripción de la especie y de algunas de las áreas de estudio que son comunes entre algunos de los capítulos. 15 METODOLOGÍA Y ÁREA DE ESTUDIO ÁREA DE ESTUDIO El área general de estudio es el Lugar de Interés Comunitario “Serra de Mariola i Carrascal de la Font Roja” (LIC, de aquí en adelante; Directive 92/43/EEC). Está situado al Sureste de España, entre el Norte de la provincia de Alicante y Sur de la provincia de Valencia, cuenta con una superficie de 19945.9 ha e incluye los Parques Naturales del Carrascal de la Font Roja y de la Sierra de Mariola (Fig. 6). Para el capítulo 1 se ha incluido el área general así como las carreteras que rodean el área natural, ya que se consideran los hábitats más invadidos por A. altissima, con una superficie total de muestreo de 22757 ha. Los capítulos 2 y 4 fueron desarrollados dentro del P.N. del Carrascal de la Font Roja. Para el capítulo 3, se seleccionaron los bosques de ribera presentes en la LIC. Figura 6 Mapa de localización del área general de estudio de la tesis doctoral Por un lado, el P. N. de la Sierra de Mariola está situado entre los municipios de Alcoy, Cocentaina, Muro de Alcoy, Agres, Alfafara, Onteniente, Bocairente, Bañeres de Mariola e Ibi y cuenta con una superficie de 16.926 ha, incluyendo el área de amortiguamiento (PORN, Fig. 6.; Decreto 76/2001). Por otro lado, el P. N. del 16 Carrascal de la Font Roja (situado entre los términos de Alcoy e Ibi, Alicante) se extiende en dirección este-oeste, e incluye la Sierra del Menejador, así como los valles y llanuras que hay a su alrededor. Comprende una superficie de 2298 ha, que junto a su área de influencia (Área PORN) ocupan una superficie de 6326 ha (Decreto 121/2004; Fig. 6). Geológicamente, el área general de estudio se engloba en el prebético y están formados mayoritariamente por rocas carbonatadas del Eoceno y Mioceno, y también con materiales del Triásico, facies de Keuper, de composición básicamente arcillosa (IGME 2010). El clima es Mediterráneo con influencia continental, con precipitación media anual y temperatura de 647 mm y 14,7 ºC, respectivamente (estación meteorológica de Bocairente, localizada en el área de estudio, a 641 m s.n.m., datos del periodo 19852006 para la temperatura y 1996-2006 para la precipitación; Rívas Martínez et al. 2007). El área de estudio incluye diversos hábitats. Por un lado se encuentran los bosques caducifolios (Acer granatense Boiss., Fraxinus ornus L., Quercus faginea Lam., Sorbus aria L., Polygonatum odoratum Mill.) y los encinares (Quercus ilex subsp. ballota (Desf.) Samp.). Asimismo aparecen encinares abiertos (Quercus coccifera L., Juniperus phoenicea L., Rhamnus lycioides L.), pinares (Pinus halepensis Mill.), con presencia de muchas especies aromáticas en el caso de la Sierra de Mariola (Serra 2007), y matorrales (Genista scorpius (L.) DC., Juniperus sp., Quercus coccifera L.). También aparecen bosques de ribera (Salix L. sp, Umus minor Mill., Populus alba L., Populus nigra L.), con especies de interés (Adiantum capillus-veneris L., Trachelium caeruleum L.), y que se encuentran incluidos como uno de los hábitats prioritarios (Directive 92/43/EEC; Serra 2007). En la Sierra de Mariola se encuentra un elevado número de endemismos iberolevantinos y setabenses (Serra 2007; Serra y Soler 2011). METODOLOGÍA A continuación se presenta un breve resumen de cada capitulo y la metodología utilizada en cada uno de ellos. 17 ANÁLISIS DE LA DISTRIBUCIÓN Y DESARROLLO DE POBLACIONES DE AILANTHUS ALTISSIMA EN ÁREAS PROTEGIDAS MEDITERRÁNEAS (CAPITULO 1) Este capítulo comprende el análisis de la distribución y grado de invasión de A. altissima en un área protegida a nivel nacional e internacional (LIC Serres de Mariola i el Carrascar de la Font Roja) y la influencia de variables ambientales en el desarrollo de las poblaciones de la especie. La hipótesis principal es que A. altissima invade numerosos hábitats, principalmente corredores de transporte, donde su desarrollo es mayor, y su crecimiento está influido por variables ambientales, como la altitud. Se localizaron todas las poblaciones presentes en el área natural, incluyendo hasta las carreteras que bordean el mismo (en total 22757 ha de área de estudio). Asimismo, se estimó el área de cada población y se indicó el tipo de uso del suelo en el que se desarrollaban. Se analizó el área invadida en los diferentes usos del suelo, así como respecto a las figuras de protección que abarca el área muestreada (parques naturales, LIC y área adyacente) mediante sistemas de información geográfica. Por otro lado, se seleccionaron aleatoriamente 99 poblaciones de A. altissima, se estimaron variables de crecimiento (área poblacional y densidad) y variables ambientales (altitud, pendiente, orientación, hábitat, presencia/ausencia de carreteras). LA VARIABILIDAD GENÉTICA MODULA EL EFECTO DEL TIPO DE HÁBITAT Y DE LAS CONDICIONES AMBIENTALES EN EL ESTABLECIMIENTO TEMPRANO DE A. ALTISSIMA (CAPITULO 2) Este capítulo se centra en el estudio de los factores ambientales y genéticos que limitan la expansión de A. altissima en las fases iniciales del proceso de invasión. La hipótesis principal de este capítulo es que los factores ambientales, no genéticos, afectan a la germinación de A. altissima. En este capítulo se analizó la germinación y el establecimiento temprano de semillas procedentes de diferentes árboles de A. altissima en experimentos de laboratorio y de campo. En cámara de germinación se analizó el efecto de la variabilidad genética, de la temperatura y sus interacciones en la germinación y viabilidad de las semillas, así como del tiempo de almacenamiento de las mismas. En campo, se consideraron seis hábitats mediterráneos (pinar solana, pinar umbría, encinar abierto, talud de carretera, borde de camino y cultivo abandonado) y se analizaron la germinación y establecimiento temprano de A. altissima, examinando las diferencias entre fuentes de semillas, hábitats, variables ambientales (relacionados o no con el tipo de hábitat) y condiciones climáticas durante el periodo de estudio. 18 EFECTOS DIRECTOS E INDIRECTOS DE LA INVASIÓN DE A. ALTISSIMA SOBRE COMUNIDADES RIPARIAS Y MULTIFUNCIONALIDAD ECOSISTÉMICA (CAPITULO 3) Este capítulo incluye el análisis de los efectos directos e indirectos (mediados por su efecto sobre biodiversidad) de A. altissima sobre el funcionamiento ecosistémico en comunidades vegetales de bosques de ribera mediterráneos. Las hipótesis principales son 1) A. altissima reduce además de la riqueza de especies, la diversidad filogenética, 2) los efectos de A. altissima sobre la diversidad vegetal reducen indirectamente la multifuncionalidad ecosistémica y 3) la reducción de la multifuncionalidad por A. altissima no es solo indirecta, mediada por su efecto sobre la diversidad vegetal, sino que también directamente por sus efectos conocidos sobre ciclos de nutrientes y propiedades del suelo. Para ello, se midieron variables de la vegetación (riqueza de especies y cobertura, diversidad filogenética), funciones del suelo (biomasa vegetal y actividades enzimáticas) y propiedades del suelo (pH, conductividad eléctrica, materia orgánica, P disponible) en diez parcelas de 100 m2 invadidas por A. altissima y en diez parcelas control (no invadidas). CONTROL DE A. ALTISSIMA A LARGO PLAZO EN ESPACIOS NATURALES MEDITERRÁNEOS (CAPITULO 4) Este capítulo se centra en determinar la mejor estrategia de control de la especie A. altissima en espacios naturales protegidos mediterráneos. Las hipótesis principales son 1) el tratamiento de desbroce anual incrementará el crecimiento de rebrotes y supervivencia durante los siguientes años, implicando cambios ecofisiológicos que le permitirán crecer y mantenerse, 2) los tratamientos de dos desbroces anuales y de desbroce junto a aplicación de herbicida serán los métodos más eficientes, porque afectarán negativamente a la especie invasora, reduciendo el desarrollo de la especie como resultado de una reducción de las reservas radiculares así como por sus efectos en la morfología causados por el herbicida, lo que reducirá la capacidad competitiva de la especie frente a las especies nativas. Se seleccionaron diferentes poblaciones de A. altissima y en cada una de ellas se aplicó un tratamiento: control, un desbroce anual, dos desbroces anuales y desbroce junto a aplicación de herbicida. Cada tratamiento fue aplicado sobre cada población a lo largo de 4 años consecutivos. Diferentes variables fueron medidas a lo largo de un periodo de cinco años de muestreo: biomasa, altura densidad de rebrotes, así como potencial hídrico, conductancia estomática e índice de área foliar (LAI). 19 RESULTADOS GENERALES ANÁLISIS DE LA DISTRIBUCIÓN Y DESARROLLO DE POBLACIONES DE AILANTHUS ALTISSIMA EN ÁREAS PROTEGIDAS MEDITERRÁNEAS (CAPITULO 1) La presencia de A. altissima en el área de estudio estuvo principalmente asociada a carreteras, construcciones antrópicas y caminos (Fig. 7). A partir de estas zonas se encontró invadiendo numerosos tipos de uso del suelo, desde cultivos (en activo y abandonados) hasta ecosistemas semi-naturales y naturales, como matorrales, pinares, encinares y bosques de ribera. La pendiente, la orientación y el hábitat influyeron sobre el desarrollo de la especie. Figura 7 Localización de A. altissima (círculos rojos) en el área de estudio (línea gris: carreteras; línea verde: vía de tren abandonada). Arriba izquierda: borde de camino; arriba derecha: vía de tren abandonada; abajo izquierda: borde de carretera; abajo derecha: construcción antrópica 20 Tabla 1 Presencia de las poblaciones de A. altissima en los diferentes tipos de uso del suelo del área de estudio Área ocupada Área ocupada Área total Uso del suelo N % min (m2) máx.(m2) ocupada (m2) Construcción antrópica 46 16,4 0,8 795,8 2765,4 Desde construcción antrópica Invadiendo cultivos agrícolas 11 3,9 0,2 534,6 731,5 Invadiendo espacios verdes 2 0,7 6,5 79,7 86,2 Invadiendo bordes de camino 36 514,7 1927,5 12,9 0,7 Invadiendo encinares 2 0,7 72,1 506,8 578,9 Invadiendo pinares 6 2,1 1,1 226,2 474,5 Invadiendo matorrales 6 2,1 32,9 198,9 810,0 Bordes de carretera Desde bordes de carretera Invadiendo cultivos agrícolas Invadiendo cultivos abandonados Invadiendo encinares Invadiendo pinares Invadiendo bosques de ribera Invadiendo matorrales 48 17,1 0,2 235,4 1431,0 5 3 1 28 10 2 1,8 1,1 0,4 10,0 3,6 0,7 197,9 87,0 45,0 1505,6 4887,4 7436,9 454,2 107,6 45,0 8124,2 10325,1 13102,4 Bordes de camino Desde bordes de camino Invadiendo pinares Invadiendo bosques de ribera Invadiendo matorrales 32 11,4 0,2 883,5 2876,2 4 1 3 1,4 0,4 1,1 50,0 20,1 54,1 1906,8 20,1 135,3 2777,2 20,1 260,6 Vía de tren abandonada 16 5,7 0,1 1841,0 4321,6 Bosques Encinares Pinares Bosques de ribera 1 1 1 0,4 0,4 0,4 4,2 50,7 325,8 4,2 50,7 325,8 4,2 50,7 325,8 Canteras abandonadas Cultivos agrícolas Cultivos abandonados Total 2 12 1 280 0,7 4,3 0,4 100 626,0 0,2 557,0 737,5 28,7 557,0 1363,5 107,8 557,0 53628,4 LA 3,1 3,1 45,0 0,8 4,0 5665,5 VARIABILIDAD GENÉTICA MODULA EL EFECTO DEL TIPO DE HÁBITAT Y DE LAS CONDICIONES AMBIENTALES EN EL ESTABLECIMIENTO TEMPRANO DE A. ALTISSIMA (CAPITULO 2) La germinación y la supervivencia temprana de A. altissima estaban principalmente afectadas por factores externos, tales como las condiciones climáticas (pulsos de lluvia), el tipo del hábitat, el sitio y el suelo desnudo (Fig. 8). Sin embargo, la influencia de estos factores varió con la procedencia de las semillas (Fig. 9), sugiriendo que la 21 preferencia de hábitat y el óptimo ambiental varían dependiendo de la fuente de semillas. 180 A T min T max T media 140 30 120 20 100 80 10 60 40 0 Temperatura media (ºC) Precipitación (mm) 160 40 20 0 16 B Borde de carretera Borde de camino Pinar de umbría Pinar de solana Encinar abierto Cultivos abandonados Germinación (%) 14 12 10 = 0.07; P = 0.791 T: F 1,270 P: F = 3.8; P = 0.0541 1, 270 8 Ti: F = 14.9; P = 0.0001 12, 270 6 H: F = 1.2; P = 0.3707 S: F = 2.1; P = 0.0159 5, 270 12, 270 4 Ti × H: F = 1.6; P = 0.011 Ti × S: F = 1.4; P = 0.0162 70, 270 168, 270 2 0 J F M A M J J A S O N D J F M A M J 2010 2009 Mes y año Figura 8 Precipitación mensual (barras grises) y temperatura (puntos) (A) y curvas de germinación (media ± ES, n = 6) de A. altissima en seis tipos de hábitat incluidos (B) durante el periodo del estudio (T: temperatura, P: precipitación mes anterior, Ti: tiempo, H: hábitat, S: sitio) 22 30 Borde de camino Borde de carretera 1 2´ 3 4 6 7´ 8 9 10´ 11 12 20 10 Germinación (%) 0 30 Pinar de umbría H: F 20 10 Pinar de solana = 1.1; P = 0.3609 5,198 F: F = 4.2; P = 0.0001 10,198 S: F12, 198= 8.2; P = 0.0001 H × F: F = 1.4; P = 0.0781 50,198 F × S: F = 0.6; P = 0.9984 120,198 0 30 Cultivos abandonados Encinar abierto 20 10 0 J F M A M J J AS O N D J F M A M J 2009 J F M A M J J AS O N D J F M A M J 2010 2009 2010 Mes y año Figura 9 Curva de germinación (media ± ES) de semillas de A. altissima de 11 árboles en cada tipo de hábitat a lo largo del periodo de estudio (H: hábitat, F: fuente de semillas, S: sitio) EFECTOS DIRECTOS E INDIRECTOS DE LA INVASIÓN DE A. ALTISSIMA SOBRE COMUNIDADES RIPARIAS Y MULTIFUNCIONALIDAD ECOSISTÉMICA (CAPITULO 3) La riqueza de especies, la diversidad filogenética y la multifuncionalidad ecosistémica fueron reducidas en presencia de A. altissima (Tabla 2). El efecto de la especie invasora sobre la multifuncionalidad fue indirecto y estuvo mediado principalmente por su efecto sobre la diversidad filogenética y en menor medida sobre la riqueza de especies. La cobertura y composición de especies se vieron afectadas en las parcelas invadidas, pero no las propiedades del suelo. 23 Tabla 2 Resumen de los efectos de la invasión de A. altissima en los atributos del ecosistema medidos. Se incluyen el estadístico F y el P valor de los efectos analizados y la varianza (R2) explicada por el modelo. Aunque algunas de las variables utilizadas son derivadas de múltiples datos (p. ej. Propiedades del suelo y multifuncionalidad, ver capitulo 3), se ha añadido una interpretación de los resultados. Las etiquetas “efectos directos o indirectos” indican si se usaron los datos brutos o los residuales de regresiones lineales, respectivamente. En el último caso, la variable predictiva utilizada en la regresión lineal (con la multifuncionalidad como variable de respuesta) se muestra entre paréntesis. Atributos del suelo = primer eje del Análisis de Componentes Principales desarrollado con pH, conductancia eléctrica, materia orgánica, P disponible y cobertura de hojarasca. Diversidad filogenética = resultado del índices de similitud de especies filogenéticas. Multifuncionalidad = índice M construido mediante el promedio de Z-scores de tres variables funcionales utilizadas (actividades enzimáticas glucosidasa y fosfatasa, biomasa vegetal) F1,18 R2 P-valor Interpretación Atributos suelo 0,85 0,05 0,369 No efectos en pH, EC, OM o P disponible del suelo Riqueza especies 12,7 0,41 0,002 Diversidad filogenética (PSE) 29,4 0,62 <0,0001 11,3 0,39 0,004 3,23 0,15 0,089 1,36 0,07 0,26 EFECTOS DIRECTOS Atributos Ecosistema EFECTOS INDIRECTOS Multifuncionalidad Multifuncionalidad (riqueza especies) CONTROL Multifuncionalidad (PSE) DE A. A. altissima reduce la riqueza y diversidad filogenética A. altissima reduce la multifuncionalidad del ecosistema. Sin embargo, este efecto está mediado principalmente por su efecto en la diversidad filogenética y en menor medida, sobre la riqueza de especies ALTISSIMA A LARGO PLAZO EN ESPACIOS NATURALES MEDITERRÁNEOS (CAPITULO 4) El tratamiento más efectivo a largo plazo para controlar y reducir a la especie invasora A. altissima fue el de corte y aplicación del herbicida glifosato, por su reducción en biomasa, e índice de área foliar de los rebrotes (Tabla 3). Los tratamientos que solo incluyen métodos de corte no redujeron a la especie. 24 Tabla 3 Biomasa, diámetro basal (DB), altura y densidad de A. altissima (mean ± ES, n = 3) para cada tratamiento y año de estudio (ANOVA y test de Tukey, P < 0.05). Leyenda: Control; 1CT: tratamiento de un corte anual; 2CT: tratamiento de dos cortes anuales; CHT: tratamiento corte y aplicación de herbicida. Diferentes letras indican diferencias significativas entre tratamientos Tratamiento 2005 Biomasa (gr m-2 ) Control 1318 ±603 a 1CT 1879 ±1205 a 2CT 2262 ±1825 a CHT 828 ±246 a DB (mm) Control 11.7 ±2.2 a 1CT 14.8 ±4.0 a 2CT 14.5 ±3.6 a CHT 12.8 ±1.1 a Altura (m) Control 1.2 ±0.3 a 1CT 1.3 ±0.3 a 2CT 1.4 ±0.4 a CHT 1.0 ±0.2 a Densidad (num m-2) Control 8 ±2 a 1CT 4 ±0 a 2CT 8 ±3 a CHT 5 ±1 a 2006 2007 2008 2009 2422 ±1042 a 283 ±85 ab 341 ±144 ab 36 ±25 b 3330 ±1511 a 802 ±463 ab 414 ±163 ab 115 ±42 b 3129 ±1366 a 235 ±81 ab 534 ±55 a 45 ±30 b 3560 ±1656 a 160 ±69 a 206 ±101 a 15 ±15 b 14.3 ±3.2 a 7.4 ±1.3 ab 7.7 ±0.5 ab 4.4 ±0.9 b 15.5 ±3.4 a 7.6 ±1.8 ab 7.1 ±0.2 ab 4.5 ±0.3 b 15.7 ±3.1 a 5.7 ±1.6 bc 6.9 ±0.7 ab 2.4 ±0.7 c 17.3 ±4.3 a 6.2 ±2.1 a 6.0 ±0.3 a 1.4 ±0.5 b 1.3 ±0.4 a 0.6 ±0.1 ab 0.6 ±0.2 ab 0.2 ±0.0 b 1.5 ±0.3 a 0.7 ±0.2 ab 0.6 ±0.0 b 0.3 ±0.0 b 1.4 ±0.3 a 0.5 ±0.1 b 0.5 ±0.0 b 0.1 ±0.0 b 1.6 ±0.5 a 0.5 ±0.1 b 0.4 ±0.0 b 0.07 ±0.0 b 8 ±2 a 8 ±1 a 10 ±3 a 3 ±1 a 6 ±1 a 11 ±2 a 13 ±5 a 6 ±3 a 7 ±1 a 14 ±4 a 16 ±6 a 5 ±2 a 7 ±1 a 12 ±6 a 11 ±4 a 2 ±1 a 25 Figura 10 Diagrama de los resultados más significativos de la tesis doctoral (modelo adaptado de Foxcroft et al. 2011) DISCUSIÓN GENERAL Esta tesis doctoral abarca diferentes aspectos que intervienen en el proceso de invasión de la especie vegetal exótica Ailanthus altissima bajo clima Mediterráneo. Se han considerado sus características ecológicas, la susceptibilidad de los hábitats y el contexto del sistema, con la finalidad de incrementar nuestro conocimiento sobre la especie y sus efectos sobre ecosistemas nativos y sobre la mejora en la gestión de esta especie en espacios naturales protegidos. La invasión de A. altissima en ecosistemas mediterráneos está influida tanto por características genéticas como ambientales las cuales afectan al desarrollo de la especie. A partir de su uso antrópico ha invadido numerosos ecosistemas, entre ellos los bosques de ribera, donde afecta negativamente sobre la riqueza de especies, la diversidad filogenética y múltiples funciones ecosistémicas. El método más efectivo para su control a largo plazo es el de desbroce y aplicación de glifosato. DISTRIBUCIÓN DE A. ALTISSIMA, LA IMPORTANCIA DE LA DISPERSIÓN SECUNDARIA Al igual que en estudios previos que fundamentan el carácter pionero de esta especie (Kowarik 1983; Hulme 2004; Kowarik y Säumel 2007), se ha observado que la aparición de A. altissima está principalmente asociada a perturbaciones de origen antrópico, principalmente las asociadas a las vías de transporte y las construcciones urbanas, periurbanas y rurales (Capitulo 1). El uso de la especie invasora en áreas mediterráneas como especie ornamental y la recomendación de su uso en restauración del paisaje hasta la actualidad (Ruiz de la Torre et al. 1990; Valladares et al. 2011), ha provocado que se extienda en estos lugares usando las vías de transporte y los cursos de agua como corredores, siendo el tráfico rodado y los cursos de agua mecanismos de dispersión secundaria a largas distancias bien conocidos para ésta y otras especies invasoras (p.ej. Timmins y Williams 1991; Tyser y Worley 1992; Kota 2005; Kowarik y von der Lippe 2006; 2011). La dispersión de las semillas, fase importante para el movimiento de las especies (Harper 1977) puede ser primaria (alrededor de la planta madre) y secundaria (mediada por agentes tras la dispersión primaria). La existencia de ambos tipos de dispersión contribuyen a la supervivencia de las especies vegetales (p.ej. Forget 1990; Moore 1997; Ruiz et al. 2010), especialmente en el caso de las especies invasoras, las cuales han visto aumentada su área de colonización debido en muchos 27 casos, a la intervención humana en la dispersión (Hodkinson y Thompson 1997; Kowarik y von der Lippe 2006; 2011; von der Lippe y Kowarik 2007). Concretamente, A. altissima, gracias a la dispersión secundaria, y su uso como especie ornamental, ha invadido hábitats naturales o semi-naturales rodeados por áreas antropizadas, como son comunidades ruderales, matorrales, pinares, cultivos abandonados, e incluso hábitats de interés para la conservación, como encinares y bosques de ribera (Kowarik 1983; Lepart y Debussche 1991). La presencia de espacios abiertos (gaps) en estos ecosistemas podría haber facilitado la entrada de la especie invasora (Davies 1944; Kowarik 1995; Knapp y Canham 2000; Kota 2005; Capítulo 2). La configuración del paisaje circundante (composición y estructura espacial) es también muy importante en relación a la presencia y establecimiento de especies invasoras (Pauchard y Alaback 2004; Vilà e Ibáñez 2011). Esta configuración de paisaje puede explicar la invasión de A. altissima en diferentes hábitats, por ejemplo, muchas de las poblaciones expandidas desde los bordes de carreteras o caminos a pinares cercanos, o aquellas poblaciones extendiéndose desde áreas construidas, principalmente rurales o periurbanas, a campos de cultivos circundantes. RASGOS ECOLÓGICOS DE LA ESPECIE VS CARACTERÍSTICAS DEL HÁBITAT Y CONDICIONES AMBIENTALES EN EL PROCESO DE INVASIÓN A pesar de que la presencia de las vías de comunicación (carreteras, pistas forestales) explican la distribución y colonización de A. altissima, estos factores no explican el desarrollo de tales poblaciones. El éxito de la invasión de especies exóticas puede estar influido por la disponibilidad de hábitats, de ahí la variación en el grado de invasión en los diferentes hábitats (Hansen y Clevenger 2005), y por el hábitat per se, ya que la dispersión de semillas está influida por el grado de perturbación de los mismos (Landerberg et al. 2007). En este sentido, en esta tesis doctoral se han observado diversas variables ambientales predictivas del desarrollo de las poblaciones de A. altissima. En primer lugar, el área poblacional ha sido mayor en umbría (Capitulo 1). Estudios previos son contradictorios. A pesar de que A. altissima ha sido considerada como especie intolerante a la sombra (Miller 1990; Facelli y Pickett 1991; Knapp y Canham 2000), Espenschied-Reilly y Runkle (2008) no encontraron un efecto significativo de la orientación en la presencia de A. altissima, y Kota et al. (2007) encontraron que A. altissima crecía más en umbría a partir del segundo año de crecimiento. El área poblacional ha sido también mayor en pendientes elevadas, lo que 28 puede ser debido a las condiciones del micrositio en áreas con gran pendiente, que favorecen la expansión de la especie invasora (Le Maitre et al. 1996; Kohama et al. 2006; Kowarik y Säumel 2007). La altitud no ha influido significativamente en el tamaño de la población, al contrario que lo encontrado en otros estudios (Traveset et al. 2008), excepto por su limite altitudinal, menor a 1050 m (Kowarik y Säumel 2007). En relación a la densidad de pies, se han encontrado mayores densidades en bosques/matorrales frente a los otros hábitats considerados. Esto podría deberse a un efecto secundario de las medidas de gestión forestal desarrolladas previamente al análisis. En estos hábitats, las medidas de control consistentes únicamente en el desbrozado de la especie han sido aplicadas con mayor frecuencia respecto a los otros hábitats (por ejemplo, en bosques de ribera aún no se han aplicado medidas de control) en base a los planes de gestión del área de estudio (Decreto 76/2001; Decreto 121/2004), provocando un incremento en los rebrotes y por tanto, de la densidad de pies (Hoshovsky 1988; Bory et al. 1991; Constán-Nava et al. 2010). Asimismo, Traveset et al. (2008) también encontraron diferencias entre hábitats, por lo que el tipo de hábitat puede influir de forma directa en el desarrollo de la especie. Otra condición ambiental importante que influye sobre A. altissima es la temperatura, la cual ha tenido un efecto significativo sobre la germinación, con mayores tasas de germinación a temperaturas más bajas (Capitulo 2). Estos resultados contrastan con estudios previos (Little 1974, Graves 1990) y con nuestros resultados en campo (Capitulo 2): los pulsos emergentes en el campo están relacionados con pulsos de lluvia, pero no con la temperatura. La explicación más posible para estos resultados contradictorios es que la temperatura en el campo fluctúa (tanto diaria como estacionalmente) y no es fija; la germinación, por lo tanto, no puede ser relacionada con un único valor de temperatura. Por otro lado, y en contraste con estudios previos usando la misma especie (Vilà et al. 2008) ha habido diferencias significativas en las curvas de germinación entre los diferentes tipos de hábitats considerados, los cuales están relacionados con las diferencias encontradas en relación a variables ambientales. El tipo de hábitat con menores tasas de germinación y supervivencia temprana es el talud de carretera, lo que puede estar relacionado con la baja disponibilidad de agua y la baja fertilidad en los suelos normalmente encontrados en estas áreas (Bochet and GarciaFayos 2004; García-Palacios et al. 2010). Los taludes de carretera son ecosistemas comúnmente invadidos por A. altissima bajo condiciones Mediterráneas (Kowarik 1983; Danin 2000; Constán-Nava et al. 2007; Traveset et al. 2008). El efecto del tipo de 29 hábitat en la tasa de germinación ha variado según las estaciones o las características microambientales de cada sitio dentro del hábitat. El experimento de campo ha revelado que el 61% de la varianza en la tasa de germinación de las semillas estaba explicado por el porcentaje de suelo desnudo en la parcela. Esto demuestra la importancia de las áreas con alto porcentaje de suelo desnudo para la germinación de A. altissima, particularmente en hábitats con relativamente poco estrés (por ejemplo encinares abiertos), como se ha indicado previamente para esta y otras especies invasoras (p.ej. Burke y Grime 1996; Bartuszevige et al. 2007; Kota et al. 2007). Esta dependencia con el suelo desnudo puede ser explicado por la baja competencia con otras plantas y una baja presencia de hojarasca en estas áreas, lo que puede tener efectos tanto efectos directos negativos (una menor disponibilidad de recursos e hidratación física de la emergencia de semilla) y efectos indirectos por el incremento de herbivoría por insectos (Facelli y Pickett 1991; Facelli 1994). El efecto del sitio en la germinación de A. altissima puede estar relacionado con factores microambientales no incluidos en el estudio de campo, tales como la humedad del suelo o la disponibilidad de luz (esta última fue medida indirectamente mediante la cobertura vegetal, pero no fue estadísticamente significativa), lo que ha sido encontrado relevante en el éxito de A. altissima (Kota et al. 2007; González-Muñoz et al. 2011). El hecho de que todas las plántulas germinadas en campo de A. altissima muriesen, ya fuese en invierno o verano, sugiere que las condiciones climáticas podrían ser el principal factor limitante para el éxito invasor de la especie. Sin embargo, esta conclusión debe ser considerada con cuidado, ya que los arboles de los que procedían las semillas utilizadas en los experimentos de laboratorio y campo se encontraban en un único tipo de ambiente. Además de los factores ambientales, los rasgos ecológicos son importantes para el desarrollo de las especies. En el caso de las especies invasoras, los rasgos relacionados con el desarrollo, como las tasas de crecimiento, son mayores que en especies nativas (e.g. Godoy et al. 2009; van Kleunen et al. 2010; Godoy et al. 2011), y no se han encontrado diferencias en plasticidad fenotípica (por ejemplo, como respuesta a gradientes de nutrientes y luz). En A. altissima, los rasgos ecológicos influyen en fases tempranas de la invasión, presentando una elevada plasticidad a la sequia mediterránea por medio de la estrategia de ahorro del agua, estrategia que permite sobrevivir bajo condiciones de déficit hídrico al igual que muchas especies nativas (Levitt 1980; Vilagrosa et al. 2003; Vilagrosa et al. 2005). Esta estrategia le permite a la especie invasora crecer y desarrollarse durante periodos de sequía (Levitt 1980). El alto 30 potencial hídrico al alba encontrado en las plantas de A. altissima, principalmente durante la sequía estival, puede indicar una mejor rehidratación frente a especies nativas (Capitulo 4). Por ejemplo, Fraxinus ornus, un árbol nativo caducifolio, muestra potenciales más negativos (-2,56 ± 0,56 MPa) frente a A. altissima (-0,6 ± 0,04 MPa) bajo las mismas condiciones ambientales (Constán-Nava et al. 2009). Esta elevada rehidratación de A. altissima puede permitirle un mejor crecimiento y mejorar sus capacidades competitivas frente a las especies nativas bajo situaciones de sequía. Esta plasticidad a la sequia mediterránea demostrada por A. altissima se ve modificada al aplicar métodos de control sobre ella (Capitulo 4). En los rebrotes de los tres tratamientos aplicados (un desbroce, dos desbroces y desbroce junto a aplicación de herbicida) se detectaron cambios ecofisiologicos sustanciales. Los rebrotes tratados con un desbroce y dos desbroces mostraron altas tasas de conductancia estomática durante primavera, especialmente al mediodía, lo que podría ayudar a la especie invasora a recuperarse y crecer tras los tratamientos, lo que explicaría la falta de reducción de biomasa. Por otro lado, la alta conductancia estomática registrada en las hojas de los rebrotes de desbroce y aplicación de herbicida, principalmente durante la sequia estival, puede ser debida a los cambios producidos por el herbicida sobre la morfología foliar (S. Constán-Nava, observ. pers. Fig. 11), lo que podría perjudicar al estado hídrico de la especie y reducir su desarrollo, corroborando así, el resto de los resultados en este tratamiento. Figura 11 Ejemplar de A. altissima con efectos foliares como resultado de la aplicación de desbroce y herbicida en el tocón del que ha surgido Asimismo, en esta tesis se muestra que existe una variabilidad genética que influye en la capacidad germinativa y en el establecimiento temprano, la cual modula su capacidad de colonizar diferentes hábitats (Capitulo 2). Aunque se han encontrado 31 variaciones en la germinación de semillas entre fuentes maternales en otras especies (p.ej. Baskin y Baskin 1998), en el caso de A. altissima no se ha encontrado ningún efecto de este factor sobre la germinación (solo en el peso de las semillas; Kota et al. 2007; Delgado et al. 2009). Sin embargo, en esta tesis se demuestra que, tanto en condiciones de campo como de laboratorio, los factores genéticos afectan a la capacidad germinativa y establecimiento temprano de A. altissima. La tasa de supervivencia y de crecimiento de A. altissima difieren entre procedencias (Feret 1985), lo que sugiere la existencia de algún componente genético que afecta al desarrollo de la especie. Estos efectos genéticos vendrían determinados por el árbol maternal y su interacción con el ambiente, así como aquellos procedentes de las contribuciones parentales (Roach y Wulff 1987; Baskin y Baskin 1998; Bischoff et al. 2006). En este sentido, las condiciones ambientales que experimenta el árbol maternal durante la fructificación, como las diferencias en disponibilidad hídrica, nutrientes o luz, podrían explicar cambios en las tasas de germinación de las semillas procedentes del mismo árbol, pero recogidas en diferentes años. Esta variabilidad entre años distintos dentro del mismo árbol también puede estar relacionada con las contribuciones genéticas paternales, que pueden diferir de un año a otro. Independientemente de la variabilidad genética, se encontró un porcentaje de germinación en campo muy bajo (siempre menor de 35 %), algo que también se ha observado en otras zonas (Kota et al. 2007; Vilà et al. 2008). La baja viabilidad y germinación obtenidas para esta especie contrastan con el alto grado de invasión de A. altissima en muchos hábitats en todo el mundo (Kowarik y Säumel 2007). Posibles explicaciones para estos resultados contradictorios son 1) la alta fecundidad de la especie, con un único árbol capaz de producir un elevado número de semillas (Little 1974; Bory y Clair-Maczulajtys 1980), 2) el uso de esta especie en restauración de carreteras y el papel del tráfico rodado como dispersor secundario, 3) la capacidad rebrotadora de la especie (Kowarik y Säumel 2007), y 4) el sistema radicular potente que posee, con raíces laterales de casi 30 m de longitud (Kiermeier 1987). EFECTOS DE LA ESPECIE INVASORA SOBRE LOS ECOSISTEMAS NATIVOS Numerosos estudios han evaluado los efectos de las especies vegetales invasoras a diferentes niveles. En relación a las especies, las especies vegetales invasoras han causado, en general, efectos negativos sobre la biodiversidad vegetal y animal (Richardson et al. 1989; Maerz et al. 2005; Hejda et al. 2009; Vilà et al. 2011; Watling et al. 2011), con 32 algunas excepciones (ver Sax y Gaines 2003; Meffin et al. 2010). En el caso de A. altissima, se ha visto un efecto negativo sobre la biodiversidad (Vilà et al. 2006; Motard et al. 2011), causando una alteración y reducción de la composición y cobertura de las especies, de la riqueza especifica y de la diversidad filogenética (Capitulo 3). En relación a la composición de especies, se ha encontrado otras especies invasoras como Robinia pseudoacacia L. en las áreas invadidas por A. altissima. Se sabe que la presencia de especies invasoras acelera la invasión de otras especies exóticas y amplifica sus efectos en las comunidades nativas (invasional meltdown; ver Simberloff y Von Holle 1999; Richardson et al. 2000; Pyšek y Richardson 2010). Además, entre las especies presentes en las áreas no invadidas, se ha encontrado especies raras de alto interés para la conservación, como Cephalanthera damasonium (Mill.) Druce. Asimismo, la reducción de la diversidad filogenética bajo la presencia de A. altissima podría causar efectos negativos sobre los servicios y múltiples funciones ecosistémicos, ya que las comunidades formadas por especies funcionalmente más diversas (es decir, más filodiversas) son más probables que mantengan mayores niveles de funciones del ecosistema (Zavaleta et al. 2010). Por otro lado, las especies vegetales invasoras han causado efectos sobre las propiedades del suelo, ciclos de nutrientes o comunidades nativas microbianas así como sobre los procesos ecosistémicos asociados (p.ej. Vitousek and Walker 1989; Ehrenfeld 2003; van der Putten et al. 2007; Weidenhamer and Callaway 2010). Estudios previos sobre A. altissima indican efectos directos de la especie sobre varias funciones del ecosistema y atributos del suelo (descomposición de la hojarasca y pH, Godoy et al. 2010; ciclo del N, Castro-Díez et al. 2011); evidencias también encontradas en estudios observacionales (pH, C orgánico, proporción C/N, Vilà et al. 2006; Gómez-Aparicio y Canham 2008). El estudio de los efectos de las especies vegetales invasoras se ha centrado básicamente sobre la biodiversidad o sobre el funcionamiento de los ecosistemas por separado, pero no conjuntamente. En esta tesis se incluye uno de los pocos estudios que analizan el efecto de una especie invasora sobre diversas medidas de biodiversidad y la riqueza de especies, y sobre múltiples funciones del ecosistema, todo ello simultáneamente (Capitulo 3). Este es también el primer estudio que separa efectos directos e indirectos (mediados por su efecto sobre la biodiversidad) de plantas invasoras sobre el funcionamiento ecosistémico. Así, aunque A. altissima tiene un rápido crecimiento (Zasada y Little 2002), y puede incrementar la fertilidad del suelo y 33 la fijación de C (Vilà et al. 2006; Gómez-Aparicio y Canham 2008), los resultados encontrados en esta tesis muestran un efecto negativo neto de A. altissima en la productividad de herbáceas y arbustivas y sobre los ciclos de C y P. Estos resultados instan a futuras investigaciones que incluyan estudios manipulativos y la medida de múltiples funciones del ecosistema simultáneamente para concluir si la especie invasora altera directa o indirectamente la funcionalidad del ecosistema. La reducción de la multifuncionalidad en presencia de A. altissima encontrada en esta tesis está indirectamente mediada por la reducción en la diversidad filogenética y en menor medida, en la riqueza de especies. Entre estas funciones, y en línea con investigaciones previas (Strauss et al. 2006; Diez et al. 2008; Zavaleta et al. 2010) es posible que las comunidades más filodiversas (por lo tanto con mayores niveles de funciones en el ecosistema) puedan prevenir futuras invasiones. IMPLICACIONES PARA LA GESTIÓN Además de conocer la distribución, características que influyen el establecimiento y los efectos sobre los ecosistemas nativos, es necesario conocer cómo combatir a las especies invasoras. Existen numerosos métodos empleados sobre las especies vegetales invasoras, desde métodos mecánicos, biológicos, hasta químicos. Concretamente en A. altissima, el método usado más común ha sido el de desbroce de la parte aérea de la especie (Hoshovsky 1988; Hunter 2000). En áreas templadas se han aplicado diferentes herbicidas siendo el glifosato el más eficaz (Meloche y Murphy 2006), al igual que en bajo clima mediterráneo a largo plazo (Capitulo 4). En esta tesis se han probado 4 metodologías diferentes: control (ninguna actuación), un desbroce anual, dos desbroces anuales, y desbroce y aplicación de herbicida (glifosato). Ni el tratamiento de un desbroce ni el de dos desbroces han reducido significativamente la densidad de rebrotes, al igual que en otros estudios desarrollados en áreas templadas (Bory et al. 1991; Burch y Zedaker 2003; Meloche y Murphy 2006). El tratamiento combinado de desbroce y aplicación de glifosato sobre el tocón recién cortado es el más eficiente, principalmente debido a sus efectos negativos sobre el crecimiento de la parte aérea de los rebrotes de la especie (reducción de la biomasa, índice de área foliar, además de efectos sobre rasgos ecofisiológicos). Este tratamiento finalmente ha eliminado los rebrotes de más de la mitad de las parcelas tras cinco años de aplicación repetida, lo que sugiere que, si se prolonga por más tiempo, una aplicación más persistente del desbrozado junto a la aplicación de herbicida puede resultar en un control total. Además, este tratamiento 34 parece reducir su capacidad competitiva frente a las especies nativas como sugiere el hecho de que se ha observado recolonización natural tras cinco años de aplicación del tratamiento. Por ejemplo, existe una recuperación natural de especies nativas como Thymus vulgaris L. subsp. vulgaris, Brachipodium retusum (Pers.) P. Beauv., Cistus albidus L., o plántulas de Quercus ilex L., Viburnum tinus L.and Pinus halepensis Mill en parcelas con este tratamiento, pero no en el resto de las técnicas empleadas (S. Constán-Nava, observ. pers.). Esto puede indicar una reducción en la competición por luz y en la producción de componentes alelopáticos por una menor biomasa e índice de área foliar. Es importante reducir el impacto y crecimiento de A. altissima en áreas antropogénicas, y evitar la construcción de corredores de transporte junto a hábitats que son especialmente sensibles a la invasión. Asimismo, los modelos de distribución potencial de A. altissima (Albrigth et al. 2010) en espacios naturales protegidos pueden ser una herramienta muy útil para identificar las áreas potencialmente más propensas a la invasión, es decir, áreas disponibles para la especie invasora, de forma que se puedan determinar áreas de actuación prioritarias, por lo que deberían incluirse en los planes de gestión. En las áreas afectadas, medidas de restauración que incluyan la eliminación de A. altissima podrían favorecer la colonización de especies nativas y ayudar a preservar la riqueza (especialmente en el caso de especies amenazadas). Por tanto, en la aplicación de tratamientos de control y erradicación, habría que priorizar, aquellos hábitats de interés de conservación, por ejemplo encinares y bosques de ribera. Como se ha demostrado, A. altissima provoca efectos negativos en los ecosistemas. Mediante la eliminación de la especie invasora y la reintroducción de especies nativas, junto con otras actuaciones complementarias, se podría restablecer los servicios y funciones ecosistémicos importantes. En este aspecto, la reducción en la diversidad filogenética observada en parcelas invadidas por A. altissima, junto con su efecto indirecto sobre el funcionamiento ecosistémico, sugiere que el control directo sobre esta especie, sin otras medidas necesarias, podría ayudar a restaurar múltiples funciones y servicios ecosistémicos. Por otro lado, se sabe que la entrada de especies invasoras puede estar influida por la estructura filogenética de la comunidad receptora (Vacher et al. 2010), con especies exóticas más alejadas a la comunidad receptora con más probabilidades de acabar como invasoras en estas comunidades (Strauss et al. 2006; Diez et al. 2008). Aunque no ha sido estudiado antes, el conjunto de los resultados de ambas líneas de investigación sugiere que conservar conjuntos de especies 35 filogenéticamente más diversas podría incrementar la resistencia de las comunidades a futuras invasiones a la vez que acelera el restablecimiento de las funciones ecosistémicas perdidas. Comunidades formadas por especies funcionalmente más diversas (es decir, con mayor diversidad filogenética) son más probables que mantengan mayores niveles de funciones del ecosistema tales como la resistencia a la invasión de plantas (Zavaleta et al. 2010), y que incluyan entre esas especies algunos taxones cercanamente relacionados a posibles invasoras, por lo tanto les previene del establecimiento e invasión en la comunidad (Strauss et al. 2006; Diez et al. 2008). Como resultado de esta tesis doctoral se ha generado el manual para la gestión de Ailanthus altissima en espacios naturales protegidos de ámbito mediterráneo. PERSPECTIVAS DE FUTURO A partir de esta tesis doctoral, se han abierto diferentes líneas de interés para la investigación. Por un lado, se encuentra el desarrollo de modelos de distribución potencial de la especie invasora a partir de los datos obtenidos en campo, así como la comparación de la misma con respecto a modelos de distribución potencial de especies nativas, constituyendo una potente herramienta para la toma de decisiones. Por otro lado, es interesante conocer la diversidad genética y genotípica de A. altissima en el Mediterráneo (Dallas et al. 2005) para determinar los efectos genéticos en el desarrollo de la especie. Son necesarios más estudios relacionados con los efectos del tipo hábitat en la viabilidad de las semillas de A. altissima. En esta tesis, las semillas analizadas procedían de un tipo de hábitat, por lo que serían complementarios posteriores estudios que incluyan este factor. El uso de técnicas como las empleadas para determinar la multifuncionalidad ha mostrado ser muy útil para determinar conjuntamente efectos directos e indirectos de la especie invasora en ecosistemas invadidos como son los bosques de ribera. La aplicación de esta metodología en otros ecosistemas así como sobre otras especies puede ser de gran interés, así como el estudio a nivel filogenético. En relación al estudio sobre los tratamientos de control sobre A. altissima, sería interesante el seguimiento de las colonizaciones de especies nativas a largo plazo para determinar si existe una recuperación natural total de las áreas afectadas, o si finalmente, son necesarias medidas de restauración complementarias. 36 CONCLUSIONES 1. Las poblaciones de A. altissima están asociadas principalmente a perturbaciones de origen antrópico, como carreteras, construcciones y caminos. Los resultados sugieren que la especie invasora, a partir de estos usos del suelo, coloniza hábitats adyacentes como pinares, bosques de ribera o ecosistemas que si no fuera por la presencia de este tipo de perturbaciones no llegaría, como los encinares 2. Diferentes variables ambientales influyeron sobre el desarrollo de las poblaciones de la especie invasora, como la orientación, la pendiente y el hábitat 3. Es importante conocer la localización y el grado de invasión en áreas protegidas así como tener el cuenta la configuración del paisaje en el desarrollo de estrategias de control en áreas naturales 4. El componente genético no solo afecta al desarrollo de A. altissima, sino que también modula su respuesta a factores ambientales, tales como la lluvia, o el suelo desnudo, los cuales parecen ser los principales conductores de la germinación y el establecimiento temprano de la especie invasora 5. Tanto la preferencia del tipo de hábitat como el óptimo ambiental varía según la fuente de semillas 6. Existe una reducción de la riqueza, de la diversidad filogenética y de la multifuncionalidad ecosistémica en presencia de A. altissima en bosques de ribera mediterráneos 7. Es importante determinar los efectos directos e indirectos de las especies invasoras para mejorar las estrategias de gestión en espacios naturales. Esta tesis incluye una metodología fácil para analizar tales efectos directos e indirectos mediante datos observacionales 37 8. El método de control más efectivo para eliminar a A. altissima en ecosistemas mediterráneos a largo plazo es el de desbroce y aplicación de herbicida (glifosato), una metodología que debe ser incluida en los planes de manejo de las áreas protegidas 9. Aquellos métodos basados únicamente en la eliminación mecánica (aún repitiendo el tratamiento dos veces al año) no reducen a la especie. 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Biological Conservation 78: 163–170 Worboys G, DeLacy T, Lockwood M (2005) Protected Area Management: Principles and Practice (second ed). Cambridge University Press, Cambridge Zasada JC, Little S (2002) Ailanthus altissima (P. Mill.) Swingle. In: Bonner, Franklin T., tech. coord. Woody plant seed manual, [Online]. Washington, DC: U.S. Department of Agriculture, Forest Service (Producer) 48 CAPITULO 1 Distribution and performance of populations of the invasive species Ailanthus altissima on Mediterranean Protected Areas1 1 Manuscrito enviado Autores: Soraya Constán-Nava, Andreu Bonet 49 50 ABSTRACT If we are to improve the control and eradication of invasive species within protected natural areas, we need to know their distribution and degree of invasion in relation to environmental conditions. A. altissima is a tree from China and North Vietnam that has invaded numerous ecosystems around the world. Our work aimed to analyze the occurrence of A. altissima in diverse land use types in a Site of European Community Importance with a Mediterranean climate, as well as determining the influence of several environmental variables on its performance. To do this, we located and measured the area of all populations in a whole natural protected area (22757 ha, including surrounding lands with roads). The distribution of such populations was related to land use type. To study the performance of these A. altissima populations according to environmental conditions, we selected 99 populations and measured their area and stem density. We also measured the aspect, slope, altitude, habitat and presence/absence of roads as environmental and land-use variables of interest. Our results show that the distribution of A. altissima in the studied area was highly associated with roadsides, built-up areas and pathsides. Our study suggests that this species uses these anthropogenic land use types to further its colonization to such surrounding habitats as pine forests, riparian forests and scrublands. Our results agree with previous literature and indicate that this species uses roadsides as ecological corridors to reach habitats that it could not otherwise invade, such as oak forests. The mean population area was larger on pronounced slopes and moister north-slope aspects. Stem density, however, was mainly affected by habitat, with denser populations in scrublands/forests than in the other habitats studied (agricultural fields, urban areas, riparian forests). Our study highlights the importance of considering landscape when analyzing the invasion of a natural area by an exotic species. By quantifying the degree of invasion according to different land use types, it may be possible to develop adequate management strategies, prioritizing habitats with more conservational importance or those more prone to invasion. In the case of A. altissima under Mediterranean conditions, we suggest focusing first on controlling their population of anthropogenic areas to prevent further expansion. 51 52 INTRODUCTION I nvasive species are causing numerous ecological and economic impacts in most ecosystems worldwide (Vitousek et al. 1997; Mack et al. 2000; Simberloff 2001). Protected areas, including those with Mediterranean environments, are not free from this global problem and have been successfully colonized by many invasive species (Drake 1988; Luken 1988; Luken and Thieret 1997; Broncano et al. 2005). In these areas, the control of invasive species has been prioritized in management plans in order to conserve native species and ecosystems (Luken and Thieret 1997; Pauchard et al. 2003; Andreu et al. 2009). Early detection of alien species is necessary when prevention is not enough to avoid their invasion of protected areas, (Pauchard et al. 2003; Paurchard and Alabarck 2004; Rout et al. 2011). When analyzing the distribution and performance of alien plant populations according to environmental factors and the sensitivity of the different habitats to an invasion, it is important to determine the risk and current impact of said invasion and to develop focused control actions (Daehler et al. 2004; Hulme 2006; Hortal et al. 2010). Moreover, analysis of alien plant distribution should include not only environmental information, but also landscape composition and structure, which may thus reveal the effect of significant differences in land management (Pauchard and Alaback 2004; Vilà and Ibáñez 2011). Undoubtedly, increasing our knowledge about how invasive species colonize and grow in different environments will enhance the efficiency of management strategies in protected areas by prioritizing the most vulnerable areas to invasion, or by identifying the ecological corridors that accelerate such invasion (Hansen and Clevenger 2005; Hulme 2006; Gulezian et al. 2010; Säumel and Kowarik 2010). As an example, linear habitats such as roads and rivers are known to act as ecological corridors enhancing the dispersal of invasive species into adjacent ecosystems (Pyšek and Prach 1994; Forman and Alexander 1998; Parendes and Jones 2000; Säumel and Kowarik 2010). Furthermore, a high degree of disturbance, such as that found in anthropogenic systems, is known to increase the sensitivity of a given area to an invasion (Vilà and Ibáñez 2011). Ailanthus altissima (Mill.) Swingle is a tree originates from China and North Vietnam and was introduced into Europe in the 18th century. It has successfully invaded several habitats with a Mediterranean climate including roadsides, old fields and pine, oak and riparian forests (Kowarik 1983; Constán-Nava et al. 2007; Kowarik and Säumel 53 2007). A. altissima is a hardy invasive species due to two main ecological characteristics: 1) its ability to reproduce both sexually and through resprouting (Little 1974; Bory and Clair-Maczulaijtys 1980; Kowarik 1995; Kowarik and Säumel 2007), and 2) its herbicidal and allelopathic compounds, which make it highly competitive compared to native plants (Heisey 1990, 1996; Heisey and Heisey 2003). Previous studies indicate that A. altissima grows in different soil types and at different altitudes (with a altitude limit at about 1000 m.a.s.l.), grows better in well-lit environments, and varies its performance among habitats, with larger populations on roadsides and old fields than in temporary streams (Kota el al. 2007; Traveset et al. 2008; Moore and Lacey 2009). This study was designed to evaluate the distribution and performance of Ailanthus altissima, regarding land use types and the environmental (both ecological and anthropogenic) conditions found throughout an entire Mediterranean protected area. To do so, we located all the populations of A. altissima in the study area, measured their performance (area and stem-density) and established the environmental characteristics for each population. The main hypotheses were that A. altissima invades several habitats, principally linear transport corridors, where its performance is greater and its growth is influenced by environmental variables such as altitude (Kowarik and Säumel 2007; Traveset et al. 2008). MATERIAL AND METHODS STUDY SITE The study was conducted at the Site of European Community Importance (hereafter SCI; Directive 92/43/EEC) Serres de Mariola i el Carrascar de la Font Roja in southeastern Spain. This conservation area stretches over 19,945.9 ha and consists of two Natural Parks with their corresponding buffer zones for protection: Sierra de Mariola (16,926 ha; 38º 44´ 1´´ N, 0º 35´ 30´´ O) and Carrascal de la Font Roja (6,301 ha; 38º 38´ 51´´ N, 0º 32´ 46´´ O) situated between north-western Alicante and southwestern Valencia, Spain. We included the roads surrounding this protected area in our study because they are considered to be the habitats most prone to invasion by A. altissima (Kowarik and Säumel 2007), giving a final studied area of 22,757 ha. The climate is Mediterranean, with a mean annual precipitation of 408-493 mm and a mean annual temperature ranging from 13-14.5 ºC (Baradello and Bañeres de Mariola 54 meteorological stations located in the study area, at 788 and 729 m.a.s.l.; data from period 2005-2010). The soils are xerorthents on limestone (Soil Survey Staff 2006), with the presence of impermeable clays. The study area includes diverse habitats such as deciduous forests (Acer granatense Boiss., Fraxinus ornus L., Quercus faginea Lam.), oak forests (Quercus ilex subsp. ballota (Desf.) Samp.), Aleppo pine forests (Pinus halepensis Mill.), riparian forests (Salix sp, Ulmus minor Mill.) and scrublands (Genista scorpius (L.) DC., Juniperus sp. pl, Quercus coccifera L.). Invasion of the studied area by A. altissima started at least 75 years ago (Natural Park staff, personal comm.), because of its use as an ornamental tree on private property and for roadside and railway restoration. DISTRIBUTION ANALYSIS All the populations of A. altissima present in the study area were located and marked using a global positioning system (GARMIN GPS 12; Fig. 1). In the field, we assigned a land use type (roadside, pathside, built-up area, agricultural fields, oak forests, etc.) and used the GPS to measure the area of each population. These areas were later introduced as polygons into a Geographic Information System (ArcGis 9.2; ESRI 2008) to create a new layer (hereafter A. altissima layer). To determine the degree of invasion according to conservation designations, the layer of A. altissima was crossed with Conservation designation layers (Source: Conselleria de Infraestructuras, Territorio y Medio Ambiente). Layer crossing was conducted using the ArcGis 9.2 join function, which permits analysis of the total invaded area in the different categories within the other layers included (Peña 2006). The invaded area in both Natural parks was compared using the χ2 test. PERFORMANCE ANALYSIS We randomly selected a set of 99 populations from those described above and measured their area and stem density. To measure stem density, we applied two methodologies because of logistic constraints: we counted and measured the root collar diameter (henceforth RCD) of all the stems in 54 randomly selected populations and for the rest of the populations we randomly located three 2 × 2 m plots and counted and measured the RCD of all shoots within them. Both methodologies were used for 12 of these populations and the results gathered were highly correlated (r = 0.73; P < 0.001). Moreover, we used these 12 populations to establish a correction factor for the number 55 of stems obtained for the large populations and thus homogenized the data obtained with both methodologies. We do not therefore expect any influence on our results or conclusions as a result of the differential methodology used depending on population area. Stem density (stems/m2) and the distribution of size classes were estimated using this sampling procedure. The following environmental variables were recorded for each population: habitat (grouped in agricultural fields, forests/scrublands, riparian forests, urban areas; based on Traveset et al. 2008), altitude, slope (low, medium, high), aspect (South, North), and presence/absence of roads. Separate multiple regressions for each performance variable used (population area and stem-density) were conducted to determine which environmental variables (habitat, altitude, slope, aspect and presence/absence of roads) were most important with regard to the performance of A. altissima. Population area and density were log10 transformed to meet normality and homoscedasticity assumptions. Frequency analysis was carried out on RCD. Multiple regression analyses were conducted using SPSS v.15 (SPSS Inc., Chicago, IL, USA). RESULTS DISTRIBUTION ANALYSIS A total of 280 populations of A. altissima were located in the study area, with a total invaded area of 5.36 ha, making up 0.02 % of the study area (Table 1). Differences in the invaded area were found between the Sierra de Mariola and Carrascal de la Font Roja Natural Parks (Table 1). Table 1 Invaded area (ha) and % invaded by A. altissima according to the conservation designation of the study area Conservation designation Area (ha) Invaded area (ha) % invaded Total study area SCI 22,757 19,946 5.36 4.89 0.020 0.020 Sierra de Mariola N.P. 16,926 0.74* 0.004 Carrascal de la Font Roja N.P. 6,301 5.36* 0.080 2 *Significant differences in invaded area between both Natural Parks, χ > 7.88, P < 0.005 The occurrence of A. altissima was principally associated with roadsides, built-up areas and pathsides (Fig. 1, Table 2). From these areas, the invasive species encroached into the surrounding natural and anthropogenic habitats. For example, A. altissima 56 frequently invaded pathsides from built-up areas (ca. 3,000 m2). From roadsides, the invasive species encroached principally upon pine forests (more than 8,000 m2) and riparian forests (more than 10,000 m2), and, despite low-frequency invasion of scrubland, this is where the greatest invaded area was seen (over 13,000 m2). From pathsides, it invaded approximately 3,000 m2 of pine forests. A. altissima was also frequent on railway lines, with 4,321.6 m2 of invaded area. Isolated populations were found in old fields and in oak, pine and riparian forests. Table 2 Presence of A. altissima populations in different land use types in the study area. N: number of populations; %: percentage of the total invaded area; Min. occupied area: minimum occupied area; max occupied area: maximum occupied area 16.4 Min occupied Max occupied area (m2) area (m2) 0.80 795.8 Total occupied area (m2) 2,765 11 2 36 2 6 6 3.90 0.70 12.9 0.70 2.10 2.10 0.20 6.50 0.70 72.1 1.10 32.9 534.6 79.70 514.7 506.8 226.2 198.9 731.5 86.20 1,927 578.9 474.5 810.0 48 17.1 0.20 235.4 1,431 5 3 1 28 10 2 1.80 1.10 0.40 10.0 3.60 0.70 3.10 3.10 45.0 0.80 4.00 5,665 197.9 87.00 45.00 1,506 4,887 74,37 454.2 107.6 45.00 8,124 10,325 13,102 Land use type N % Built-up area From built-up area Encroaching into agricultural fields Encroaching into green spaces Encroaching into pathsides Encroaching into oak forests Encroaching into pine forests Encroaching into scrublands 46 Roadsides From roadsides Encroaching into agricultural fields Encroaching into old fields Encroaching into oak forests Encroaching into pine forests Encroaching into riparian forests Encroaching into scrublands Pathsides From pathsides Encroaching into pine forests Encroaching into riparian forests Encroaching into scrublands 32 11.4 0.20 883.5 2,876 4 1 3 1.40 0.40 1.10 50.0 20.1 54.1 1,907 20.10 135.3 2,777 20.10 260.6 Railway line 16 5.70 0.10 1,841 4,322 Forests Oak forests Pine forests Riparian forests 1 1 1 0.40 0.40 0.40 4.20 50.7 325.8 4.20 50.70 325.8 4.20 50.70 325.8 Abandoned quarries Agricultural fields Old fields Total 2 12 1 280 0.70 4.30 0.40 100 626 0.20 557 737.5 28.70 557.0 1,363 107.8 557.0 53,628 57 Figure 1 Distribution of A. altissima (red circles) in the study area (grey lines: roads; green line: abandoned railway line). Above left: pathsides, above right: railway line, below left: roadsides, below right: built-up area 58 PERFORMANCE ANALYSIS Multiple regression analyses showed that aspect and slope affected the area of A. altissima populations (Table 3) whilst the rest of the variables entered were not significant (P > 0.05). Slope aspect was the most important predictor, with population areas significantly greater on North than on South-sloping aspects. Slope was a second order predictor, with larger populations on pronounced (high) slopes than on the rest. In contrast to population area, habitat was the only significant predictor variable for stemdensity (Table 3), with denser populations in scrubland/forests than in the other habitats included. Table 3 Results of multiple regressions analysing the effect of environmental variables on A. altissima performance Analysis of variance 2 R 2 Area (m ) df 0.43 97 Density (number of shoots/m2) 0.21 88 Predictor variable F-value P 11.11 <0.0001 4.25 0.04 t P Aspect 4.11 <0.0001 Slope 2.18 0.03 Habitat -2.06 0.04 RCD presented an asymmetric distribution, ranging from 0.003 cm to 58.93 cm, with the major frequency being lower than 10 cm (Fig. 2). 12000 Min 0.003 cm Max 58.93 cm Mean 1.001 cm 10000 Total shoots 8000 6000 4000 2000 0 0 10 20 30 40 50 60 RCD (cm) Figure 2 Frequency distribution of the basal diameter (RCD) of A. altissima shoots in the study area (n = 11950) DISCUSSION Similarly to previous studies (Kowarik 1983; Hulme 2004; Kowarik and Säumel 2007), the occurrence of Ailanthus altissima was principally associated with anthropogenic disturbances, mainly linear transport corridors and built-up areas. The much higher abundance of populations of A. altissima in these areas can be explained by the common use of this invasive species for ornamental purposes and the fact that it was recommended for use in landscape restoration until recently (Ruiz de la Torre et al. 1990; Valladares et al. 2011). From this initial colonization, many of the populations could have expanded due to road traffic acts as a secondary dispersal mechanism for A. altissima, thus transporting the seeds over long distances (Kowarik and von der Lippe 2006; Kowarik and von der Lippe 2011). This secondary dispersal explains the presence of isolated populations along road or pathsides, where the initial populations could be dispersed by wind or vehicles (Kota 2005; Kowarik and von der Lippe 2006; Kowarik and von der Lippe 2011). As in other parts of the Mediterranean Basin (Kowarik 1983), A. altissima colonized oak forests and scrubland close to roads (Table 2). Paths or roads are disturbances which cross administrative boundaries in different ways, for example, between private or public properties, or between city areas and permit the entrance of invasive species which could not access the areas in any other way (Timmins and Williams 1991; Tyser and Worley 1992). Furthermore, transport corridors such as roads are often placed close to administrative boundaries and are altered and artificial areas (Forman and Moore 1992; Landres et al. 1998), which are known to affect alien species spreading into natural areas, increasing their colonization in adjacent habitats (Tyser and Worley 1992; Spellerberg 1998; Parendes and Jones 2000; Pauchard and Alaback 2004), as well as being regarded as a severe threat to the native species in many nature reserves (Usher 1988; Spellerberg 1998). A. altissima was present in built-up areas, both abandoned and non-abandoned, where it was introduced as an ornamental tree. The invasive species appeared forming dense stands in the abandoned areas, where population size could have increased over time since abandonment (Domènech et al. 2005; Vilà and Ibáñez 2011). On the other hand, A. altissima has also invaded non-abandoned built-up areas, which could have been caused by the high propagule pressure from ornamental trees (to 325,000 samaras per tree, Little 1974; Bory and Clair-Maczulajtys 1980) and also because ineffective 60 control measures applied to new shoots (Such as mechanical removal, S. Constán-Nava, pers. observ.) may have increased its density (Hoshovsky 1988; Bory et al. 1991; Constán-Nava et al. 2010). The colonization of surrounding habitats from anthropogenic areas could be caused by the possible presence of gaps, such as oak forests which could have facilitated the entrance of the species (Davies 1944; Kowarik 1995; Knapp and Canham 2000; Kota 2005). Land use type has an influence on invasion, with anthropogenic areas being the most susceptible. Nevertheless, the surrounding landscape configuration is also very important with regard to the presence and establishment of alien species (Pauchard and Alaback 2004; Vilà and Ibáñez 2011). This landscape configuration could explain the A. altissima invasion of the different habitats, for example, the many populations that have expanded from road or pathsides into nearby pine forests, or those populations extending from built-up areas into surrounding agricultural fields. The degree of invasion of A. altissima from road and pathsides to surrounding habitats varied (Table 2). This may due to the suitability of the habitats, which could have an influence on the success of the invasion (Hansen and Clevenger 2005), and also due to the habitat, where seed dispersion differs between habitats according to the degree of disturbance (Landerberg et al. 2007). A. altissima invaded riparian forests, as has been found in other studies (Kowarik 1983; Lepart and Debussche 1991). Riparian forests were mainly found on roadsides, and water presence combined with seed dispersion by water, wind and traffic could aid its colonization (Kowarik and von der Lippe 2006; Kota 2005; Kaproth and McGraw 2008; Kowarik and von der Lippe 2011). Presence/absence of roads was not a variable predictor of A. altissima performance (considered in terms of population area and stem-density), despite the fact that the invasive species is very frequently found at roadsides. Our results show that, despite roads explaining the distribution and colonization of A. altissima, these areas do not explain the growth of such populations. In this sense, environmental variables that were good predictors of the performance of the invasive species were found (Traveset et al. 2008). First, the population area was greater at shady sites. Previous studies are contrasted. Despite A. altissima has been reported as a shade-intolerant species (Miller 1990; Facelli and Pickett 1991; Knapp and Canham 2000), Espenschied-Reilly and Runkle (2008) did not find any significant effect of this aspect on the presence of A. altissima, and Kota et al. (2007) found that A. altissima grew more in shady sites as from the second growth. The population area was also greater on steep slopes. This 61 could be due to light resources and microsite conditions on areas with steep slopes favouring the expansion of the invasive species (Le Maitre et al. 1996; Kohama et al. 2006; Kowarik and Säumel 2007). Our results indicated that altitude had no influence on population size, in contrast to previous studies (Traveset et al. 2008), except for its absence at > 1,200 m (Kowarik and Säumel 2007). This could be due to the control measures of the invasive species counteracting the effect of altitude. Secondly, habitat was the predictor variable for stem-density, with the density being greater in forests/scrublands than in the other habitats. In these habitats, control measures based on mechanical methods have been more frequently applied to A. altissima than in the other habitats (for example, in riparian forests, control measures have not yet been applied) based on management plans (Decreto 76/2001; Decreto 121/2004). Consequently, density increased (Hoshovsky 1988; Bory et al. 1991; Constán-Nava et al. 2010). A wide range of various sizes of A. altissima was found in the study, from seedlings and small resprouts to adult trees, with a much higher frequency of young shoots. Despite A. altissima being recommended for the landscape restoration of road slopes, the annual obligatory roadside cleaning (Real Decreto-ley 11/2005) using mechanical or chemical methods that do not involve the correct herbicide could increase invasive species stem density and reduce size class (Hoshovsky 1988; Bory et al. 1991; Meloche and Murphy 2006; Constán-Nava et al. 2010). A. altissima invaded a larger area in Carrascal de la Font Roja than in Sierra de Mariola. These differences could be explained by the high density of roads and paths and tourist vehicles in the former, combined with greater use of the invasive species in built-up areas. Our results highlight the importance of understanding the degree of invasion and spatial distribution for improving control on different levels of conservation. Furthermore, these results highlight the importance of reducing the impact and growth of A. altissima in anthropogenic areas, and of avoiding the construction of transport corridors close to habitats that are especially sensitive to invasion. Therefore, obligatory control actions should be applied, prioritizing habitats of conservation interest (oak and riparian forests; Decreto 76/2001; Decreto 121/2004). Potential models of the current distribution of A. altissima (Albrigth et al. 2010) in natural protected areas should be included in management plans because they could help to identify areas potentially prone to invasion. 62 CONCLUSIONS A. altissima is related with human disturbances by means invades surrounding habitats, including conservation interest areas such as oak forests, and threatening native species in the natural area. Moreover, the performance of the species was found to be affected by environmental variables (aspect, slope and habitat). Our results highlight the importance of identifying the location and degree of invasion in protected areas and of taking into account the composition and configuration of the landscape when developing conservation strategies for protected areas. ACKNOWLEDGEMENTS S. Soliveres provided helpful comments on an early version of this manuscript. We also thank Language Centre (University of Alicante) for improving the English of this manuscript. We are grateful for the collaboration of the staff at the Natural Parks and all private landowners and for the permits granted. C. Constán, A. Constán, M.J. Nava, E. Pastor, N. Vizcaíno, S. Soliveres, J. Monerris, M. Herrera, J. Acosta and the other collaborators helped with the fieldwork. E. Pastor, Q. Rubio and S.M. Catalán helped with the Geographic Information System. 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Science 277: 494–499 66 APÉNDICE FOTOGRÁFICO Foto 1 Invasión de A. altissima en una masía abandonada dentro de la LIC Foto 2 Población de A. altissima invadiendo matorral a partir de uso ornamental Foto 3 Individuos de A. altissima creciendo dentro de carrascal Foto 4 Individuos de A. altissima invadiendo pinar 67 68 CAPITULO 2 Genetic variability modulates the effect of habitat type and environmental conditions on early invasion success of Ailanthus altissima in Mediterranean ecosystems2 2 Este capitulo está en 2ª revisión en Biological Invasions Autores: Soraya Constán-Nava, Andreu Bonet 69 70 ABSTRACT At the early stages of an invasion by an exotic species, there are diverse environmental and genetic factors that limit its expansion. Ailanthus altissima is a tree from China and northern Vietnam which has become an invasive species in numerous ecosystems around the word. Our objective was to identify the relative effect of both genetic and environmental factors and how they interact with the emergence and early establishment of this invasive tree under Mediterranean conditions. To achieve this, seed germination and early establishment from different maternal sources were analyzed under contrasted environmental conditions in a series of experiments, using both laboratory and field approaches. Seed germination and early survival were affected by environmental factors such as habitat type, the percentage of bare soil and climatic conditions (rainfall pulses), although the influence of these factors changed depending on the maternal source. Our study reveals that the genetic component affected not only the performance of A. altissima, it also modulated its response to environmental factors, which seemed to be the main drivers of germination and early establishment for this species. Our results highlight the importance of considering both genetic and environmental factors when studying plant invasion risk and success, and may be helpful in predicting and reducing the spread of this species in Mediterranean ecosystems. 71 72 INTRODUCTION T he introduction of invasive plant species and their colonization depend not only on the ecological traits of the invader species but also on the conditions of both habitat and climate, resulting in varying distributions of invasive species in different types of habitat (Gleadow and Rowan 1982; East et al. 1999; Koop 2004; Lloret et al. 2005). Previous studies show that disturbed habitats such as human-created environments are vulnerable to plant invasion because of the lack of competition or gap abundance (Elton 1958; Baker 1986; Hobbs and Huenneke 1992; Bruno et al. 2003). Less disturbed habitats such as protected areas have also been successfully colonized by many invasive species (Drake 1988; Luken 1988; Hiebert et al. 1993; Vitousek et al. 1997). Moreover, these habitats are not free of plant invasions (Broncano et al. 2005; Traveset et al. 2008; Constán-Nava et al. 2010), and the factors allowing such invasions are poorly understood. When a plant has dispersed to a given area, there are numerous factors that can enhance or limit its probability of success (Keddy 1992; Baskin and Baskin 1998; Zamora et al. 2008). There exist environmental factors such as climatic conditions, habitat type (defined by plant community, such as open areas with low vegetation growth, open woodlands and forests, and which also represent, to a certain extent, a degree of increasing disturbance), and the abiotic and biotic interactions within these habitat types (e.g. litter depth, abundance of herbivores, etc.; Williams et al. 1990; Bewley and Black 1994; Facelli 1994; Koop 2004). In addition to these external, environmental factors, genetic factors also influence plant germination and early establishment. These genetic factors derive from the mother tree, the mother tree’s interaction with the environment (seed position in plant, climatic conditions, etc.) and paternal genetic contributions (Roach and Wulff 1987; Baskin and Baskin 1998; Bischoff et al. 2006). Thus, all these environmental and genetic factors, and their interactions, can influence the success of a plant species when colonizing a given habitat in very complex ways. Studying such factors separately may therefore result in misleading conclusions, and more comprehensive approaches are needed to improve our knowledge of the relative importance of such factors and how their interaction influences plant establishment. These comprehensive approaches are especially needed for invasive species, as they may be essential for predicting alien plant invasion and 73 developing management techniques to prevent such invasions in natural ecosystems, including protected areas (Mack et al. 2000; Rejmánek 2000). Ailanthus altissima (Mill.) Swingle is a tree from China and northern Vietnam, where it grows under temperate conditions. This species has invaded several ecosystems out of its original range, chiefly because of its ornamental use and in landscape restoration (Kowarik and Säumel 2007). A. altissima is a dioecious species which produces a large number of samaras assisted by wind and water dispersion; it also grows quickly and has a high ability to resprout and form dense clonal stands (Kowarik 1995; Kowarik and Säumel 2007, 2008; Kaproth and McGraw 2008; Säumel and Kowarik 2010). This species has successfully colonized several habitat types under Mediterranean conditions, such as roadsides, old fields, and pine, oak and riparian forests (Kowarik 1983; Danin 2000; Constán-Nava et al. 2007; Kowarik and Säumel 2007). Germination and the early establishment of A. altissima are known to occur in many soil types and be enhanced by a high light availability and low altitudes (>1000m), although they do not seem to depend on the habitat types (Mihulka 1998; Kota et al. 2007; Vilà et al. 2008; Moore and Lacey 2009) or source tree (Kota et al. 2007; Delgado et al. 2009). However, the combined effect on the early stages of this invasive species and the interaction between genetic and environmental factors, whether or not they are related to habitat type, are poorly understood. In this study, we aimed to evaluate the influence of both environmental and genetic factors on the emergence and early establishment of Ailanthus altissima under Mediterranean conditions by using a series of experiments including both field and growth chamber approaches. Growth chamber experiments evaluated the effects of temperature, seed storage time and genetic effects on the germination and viability of A. altissima seeds. In the field experiment, we considered six contrasted Mediterranean habitat types and analyzed the germination and early establishment of A. altissima, examining the differences among these habitats, the environmental attributes (whether or not they were related to the habitat type) and climatic conditions during the study period. Our main hypothesis was that environmental, not genetic factors affect the germination rate of A. altissima (Kota et al. 2007; Delgado et al. 2009). To our knowledge, this is the first comprehensive approach evaluating the joint effects of environmental and genetic factors and their interactions on A. altissima success. 74 MATERIAL AND METHODS STUDY SITE The study was conducted in the Carrascal de la Font Roja Natural Park, in southeast Spain. The climate is Mediterranean, with a mean annual precipitation of 407 mm during the study period (2006-2010) and mean monthly temperatures ranging from 4.4º C in January to 24.9º C in July (Baradello meteorological station, representative of the climate in the study area and located between 1.4 and 5.5 km from the plots used in the field study, 38º 41´ 90´´ N, 00º 31´ 31´´ W, 788 m.a.s.l.). Soils are xerorthents on limestone (Soil Survey Staff 2006), with the presence of impermeable clays. The presence of A. altissima in this Natural Park was caused by its use in roadside restoration and as an ornamental plant. The species was first planted in private properties located throughout the park at least 75 years ago (Natural Park staff, personal comm). A. altissima went on to infest various habitats in the Natural Park, such as pine forests, scrubland, oak forests and riparian forests. Currently, 5.4 ha of the park have been invaded by the species and management plans are now being applied to control its spread (Constán-Nava et al. 2007; 2010). VIABILITY TEST In October 2006, seeds of A. altissima were collected from 12 maternal sources randomly selected from disturbed areas (not ornamental trees) located along the boundary of the Natural Park and at a minimum distance of 500 m from one another. These seeds were stored separately in paper bags in the dark and under room conditions with a temperature of 17-20º C for two months. Because A. altissima seeds are viable after up to two years of storage, we did not expect their germination ability to be affected by this storage time (Appendix 1; Little 1974; Hildebrand 2006). We compared differences in seed viability among the maternal seed sources by applying the tetrazolium test (ISTA 1966) to four replicates of 50 seeds per each maternal seed source (totaling 200 seeds per maternal source). The seeds were dissected and imbibed for 24 h with distilled water and then soaked with 2,3,5,-triphenol tetrazolium chloride (TTC) at 1% for 24 h in dark conditions. Seeds containing living embryos were stained red or pink and were counted as viable. Treated seeds with no stained contents were considered non-viable. 75 TEMPERATURE AND GENETIC EFFECTS We developed a full factorial experiment to assess the effect of different temperatures and maternal seed sources on A. altissima germination. As seeds are the result of maternal and paternal genetic contributions and of interaction with the environment (Roach and Wulff 1987; Baskin and Baskin 1998; Bischoff et al. 2006), hereinafter the effect of the maternal seed source is referred to as a genetic effect. In October 2007, A. altissima seeds were collected from the 12 maternal sources (the same trees used for the viability test, except for four of them, which were new and are marked with a comma in Results to distinguish them from the others). All seeds were germinated under the same photoperiod (16/8 h light/dark), but under three contrasted temperature regimes (15º, 20º and 30º C). Temperatures ranging between 20º and 30º C are considered optimal for the species studied (Little 1974, Graves 1990). The temperature of 15º C was selected to contrast with the 20º and 30º C temperatures, and as an approximation of the mean annual temperature in the study area (14º C, Ninyerola et al. 2005). Four replicates of 20 non-stratified seeds were established for each maternal source and temperature (80 seeds x 12 maternal sources x 3 temperatures; 2,880 seeds in total). These seeds were treated with a sodium hypochlorite solution (2%) for ten minutes to avoid fungal infection and were rehydrated with distilled water for 24 hours. Each replicate was then placed on a 9 cm Petri dish containing two layers of filter paper. Filter papers were kept soaked throughout the experimental period (31 days). Petri dishes were randomly moved to avoid position effects in the germination chamber (the same chamber was used for the three temperature regimes but at different times, so no chamber effect was expected). Seed germination was monitored daily and, when the radicle was visible, it was considered germinated and removed from the Petri dish. SEED GERMINATION AND EARLY SEEDLING ESTABLISHMENT UNDER FIELD CONDITIONS Six habitat types in the Natural Park were selected, defined by plant community and following a decreasing disturbance gradient: local roadside (RS), pathside (PS), early old fields (OLD), south-facing slopes pine forest (SPF), north-facing slopes pine forest (NPF), and open oak forests (OF). RS had steep slopes. PS habitats were uncommon in the study site and contained species from nearby habitats (such as NPF). NPF included shade-tolerant species and a high density of bryophytes, whereas SPF presented more heliophyllus species. SPF was the only studied habitat type with a south aspect. OF 76 included some deciduous species. OLD had previously been almond crop terraces, and after being abandoned were colonized by shrubby species and several herb species. In autumn 2008, seeds from A. altissima were collected from 11 of the 12 maternal sources selected in 2007 (one tree was excluded because it did not produce fruit) and stored separately in paper bags in the dark under room conditions. In March 2009, three sites were selected by habitat type (n = 18 sites) covering a wide range of altitudes (from 670 to 1150 m.a.s.l.; see Table 1). Although A. altissima has invaded most of the selected habitats, no reproductive individuals of this species were present closer than 200 m from any of the selected sites, avoiding uncontrolled seed dispersion in the study sites (Landenberger et al. 2007). In each site, we randomly marked two 1 m × 1 m plots, at a distance of 1-4 m from each other, resulting in six plots in each habitat type, all on gentle slopes (except at RS, which had steep slopes) and north-facing slopes (except SPF sites). Each plot was divided into 12 quadrats of 25 cm × 33 cm; in each quadrat, 12 non-stratified seeds of a given seed source were buried at a depth of 1 cm and at a distance of <5 cm apart (the last quadrat always remained empty because we had no seeds from the 12th mother tree). Overall, 132 seeds were planted in each plot, making for a total of 4,752 seeds. We acknowledge that the distance among the seeds could be insufficient to avoid early competition among seedlings. However, due to the high growth rate and the extensive root system of A. altissima, avoiding this effect would have required large plots, which was not viable in the Natural Park. Seeds were buried with the minimum disturbance possible to avoid removing structural attributes characteristic of each plot. The number of emerging seedlings and their survival was recorded every two to four weeks during the study period (spring 2009-summer 2010). Upon completion of the study, all A. altissima seeds and seedlings were removed from the study sites to avoid further invasion. To assess habitat characteristics that are important for seed germination, such as light availability, litter layer depth and competition with other species (Tilman 1988), we measured total cover (%) of litter, bare soil, perennial herbs, shrubs and trees at each of the six plots per habitat type immediately after seeding (Table 1). Five measurements of litter depth (cm) were also taken in each plot. Although microclimatic measurements taken within each plot would have been better, we believe that measuring these attributes is a good surrogate for the availability of light, the condition of the soil and water competition experienced by the seedlings (Raich 1989; Bartuszeyige et al. 2007; Pérez-Ramos et al. 2010). 77 Table 1 Environmental variables measured in the studied sites (Mean ± SE of the two plots/site). Legend: RS: Roadside; PS: path side; NPF: North facing aspect pine forests; SPF: South facing aspect pine forests; OF: open oak forests; OLD: old fields Bare soil Herb Shrub Tree cover Litter Habitat Site Litter (%) (%) cover (%) cover (%) (%) depth (cm) Altitude 670 RS 1 7.5±2.5 15±5 12.5±2.5 67.5±2.5 0±0 0.14±0.02 750 2 12.5±2.5 0±0 87.5±2.5 0±0 0±0 0.37±0.03 730 3 50±25 2.5±2.5 72.5±2.5 0±0 45±15 1.21±0.85 810 PS 4 5±0 7.5±2.5 5±0 82.5±2.5 10±10 0.06±0.02 1160 5 57.5±32.5 22.5±22.5 0±0 50±20 95±5 0.28±0.24 860 6 25±5 25±5 0±0 50±10 0±0 0.49±0.21 770 NPF 7 20±10 20±10 0.25±0.25 60±20 50±30 0.72±0.02 790 8 1±1 4±4 0±0 95±5 7.5±7.5 0.01±0.01 810 9 7.5±2.5 0±0 0±0 92.5±2.5 47.5±32.5 0.44±0.26 880 SPF 10 20.5±19.5 5±5 0±0 70±30 0±0 0.19±0.05 910 11 17.5±2.5 15±0 0±0 67.5±2.5 20±5 0.24±0 910 12 27.5±17.5 25±15 0±0 47.5±2.5 0±0 0.19±0.07 1050 OF 13 1±0 47.5±47.5 0±0 52.5±47.5 50±50 0.04±0.02 920 14 8±7 70±10 0±0 22±3 0±0 0.08±0.06 1000 15 7.5±2.5 15±15 0±0 77.5±17.5 100±0 1.19±0.09 760 OLD 16 6±4 7±6 62.5±17.5 20±10 10±0 0.13±0.13 1150 17 0±0 32.5±2.5 0±0 67.5±2.5 0±0 0±0 796 18 0±0 70±5 12.5±2.5 17.5±2.5 7.5±7.5 0±0 STATISTICAL ANALYSES Differences in viability between A. altissima maternal sources were assessed using onefactor analysis of variance (ANOVA) with “maternal source” as a random factor with 12 levels. Germination percentage at the laboratory experiment was analyzed using a two-way mixed model with temperature and maternal seed source as fixed and random factors, respectively. According to the effects of the time and climatic factors on germination percentage in the field experiment, we tried to integrate all the maternal, habitat-related and other environmental factors in an analysis with maternal seed source, habitat, site, and sampling period. In order to avoid overhead problems using an overly large matrix, we analyzed our data in different subsets. Firstly we averaged germination rate by habitat type or site and tested for differences in germination times among habitats and sites, and for the effect of current environmental conditions (rainfall and temperature) on germination. To achieve this, the analysis included sampling period (time) and habitat type (fixed factors), and site (nested within habitat type); monthly temperature and precipitation in the previous month were introduced as covariates. Second, germination percentage was analyzed using a three-way mixed model, 78 introducing the maternal seed source as a random factor, habitat type as a fixed factor and site as a nested factor. This allowed us to test for differences in germination among maternal sources or habitat types, to test for the effect of other environmental factors not related to habitat type (the “Site” effect), and also to assess for interactions between maternal and environmental factors (both habitat type and others). All these analyses were performed using the semi-parametric PERMANOVA approach (Anderson 2001; McArdle and Anderson 2001; Anderson and ter Braak 2003). Differences among habitats in litter cover, bare soil, trees and perennial herbs were also analyzed using PERMANOVA, with habitat type and site as fixed and random factors, the latter nested within the former. Litter depth and shrub cover were excluded because they were correlated to the other variables (Spearman’s Rho coefficient ≥ 0.5, P < 0.01). The other measured variables were entered into the model as they did not correlate to one other, meaning that PERMANOVA assumptions were not violated. All PERMANOVA analyses were performed using Euclidean distance to create the resemblance matrix, and a type III sum of squares (except for the analysis with covariates, where we used a type I sum of squares), permutation of residuals under a reduced model (except for the analysis of viability, which included one factor and the unrestricted permutation of raw data was used), and 9999 permutations were used to calculate pseudo-F and P-values. The Kaplan–Meier procedure (Fox 1993) was conducted to model differences in A. altissima germination curves among the three temperatures assayed (factor) and maternal seed sources (strata) in the growth chamber experiment, and among the different habitat types (factor) and maternal seed sources (strata) in the field experiment. Survival curves for the latter experiment were also analyzed with this procedure. Comparisons of the cumulative germination/survival curves among different maternal sources were tested using non-parametric Log-Rank tests (Pyke and Thompson 1986) in both the growth chamber and the field experiment. A regression tree was performed as an exploratory analysis to assess for the most important environmental factors: bare soil, perennial herb, shrub and tree cover, and litter depth on the germination percentage of A. altissima (Appendix 2). As this exploratory analysis only revealed bare soil to be important, it was analyzed using a linear regression. All statistical analyses were performed using PERMANOVA+ for the PRIMER statistical package for Windows (PRIMER-E Ltd., Plymouth Marine Laboratory, UK), 79 except for germination and survival curves, and linear regression, which were analyzed using SPSS v.15 (SPSS Inc., Chicago, IL, USA). RESULTS VIABILITY AND GERMINATION TESTS Seed viability ranged from 45.5 ± 6% to 90 ± 5% (mean ± SE) and was significantly different among maternal seed sources (ANOVA: F11,47 = 9.46, P < 0.001). Both maternal seed source and temperature affected germination rates in the growth chamber experiment (Fig. 1). A significant maternal seed source × temperature interaction was found (Fig. 1). In general, most maternal seed sources showed highest and lowest germination rates at 15º C (17.4 ± 2.7%) and 30º C (3.65 ± 0.9%), respectively. GERMINATION AND SEEDLING SURVIVAL UNDER FIELD CONDITIONS Three germination pulses occurred during the entire study period, two in spring (2009 and 2010) and one in autumn (2009), with the latter showing the highest germination values. Germination percentage was significantly affected by the time, the site and the rainfall of the previous month, but not by the mean month temperature or habitat type (Fig. 2). Significant interactions were observed between time × habitat type and time × site, i.e. germination varied throughout the time among habitat types and sites. In spring 2009, seeds only germinated at RS, PS and NPF, and showed low mean germination rates (<1% in all cases, Fig. 2B.). A predation event occurred with a 2.3% seed predation rate at one OLD site in spring 2009. In both autumn 2009 and spring 2010, seeds germinated in all assayed habitats except for RS, where no germination was registered in autumn and which showed the lowest germination rates the following spring. The highest germination rates were found at SPF and OF in autumn 2009, and at OF in spring 2010. 80 80 15 ºC 1 2´ 3 4 5´ 6 7´ 8 9 10´ 11 12 60 40 20 0 80 Germination (% ) 20 ºC 60 40 20 0 80 T: F 1,108 = 6.5; P = 0.0023 MS: F 11,108 60 30 ºC = 11.6; P = 0.0001 T × MS: F 22,108 = 6.4; P = 0.0001 40 20 0 0 5 10 15 20 25 30 Day Figure 1 Germination curves of A. altissima seeds from 12 different maternal seed sources (showed with different color dots) after 31 days in a growth chamber at 15 ºC, 20 ºC and 30 ºC. Data are mean percentage ± SE (n = 4). Statistical results for the PERMANOVA are given (T: temperature, MS: maternal source) 81 180 T min T max T mean Rainfall (mm) 140 30 120 20 100 80 10 60 40 0 Mean temperature (ºC) 160 40 A 20 0 16 B Roadside Path side North facing slopes pine forests South facing slopes pine forests Oak forests Old fields Germination (%) 14 12 10 T: F 8 = 0.07; P = 0.791 1,270 P: F 1, 270 = 3.8; P = 0.0541 Ti: F = 14.9; P = 0.0001 12, 270 6 H: F = 1.2; P = 0.3707 5, 270 S: F 4 2 12, 270 Ti × H: F = 2.1; P = 0.0159 70, 270 Ti × S: F = 1.6; P = 0.011 168, 270 = 1.4; P = 0.0162 0 J F M A M J J A S O N D J F M A M J 2010 2009 Month and year Figure 2 Monthly rainfall (grey bars) and temperature (black circles) (Baradello meteorological station, representative of the climate in the study area) (A) and germination percentage curves (mean ± SE, n = 3) of A. altissima at the six different habitat types studied (B) during the study period. Statistical results for the PERMANOVA are given (T: temperature, P: precipitation, Ti: time, H: habitat, S: site) Final germination percentages differed significantly among maternal source and site but not among habitat types, and a significant maternal source × habitat type interaction was found, with seeds from each maternal source differing in their germination rates depending on the habitat type (Fig.3A). Differences between germination curves among habitat types and maternal seed sources were found (log– rank test, P < 0.001). There were no significant differences among maternal seed sources in separated analyses for each sampling period, except in November 2009 (Pseudo-F= 5.44; P < 0.001). Although all seedlings died during the study period, 82 differences between survival curves were found between habitats and maternal seed sources (log-rank test, P < 0.001, Fig. 3B). (A) 30 Roadside H: F 5,198 20 Path side = 1.1; P = 0.3609 MS: F 10,198 = 4.2; P = 0.0001 S: F12, 198= 8.2; P = 0.0001 H × MS: F = 1.4; P = 0.0781 10 50,198 MS × S: F 120,198 = 0.6; P = 0.9984 Germination (%) 0 30 North facing slopes pine forests South facing slopes pine forests 20 10 0 30 20 10 Oak forests Old fields 1 2´ 3 4 6 7´ 8 9 10´ 11 12 0 J F M A M J J AS O N D J F M A M J 2009 J F M A M J J AS O N D J F M A M J 2010 Month and year 83 2009 2010 (B) 100 Roadside Pathside 80 2´ 3 6 7´ 8 9 10´ 11 12 1 6 7´ 8 10´ 11 12 60 40 20 Survival (%) 0 100 South facing slopes pine forests North facing slopes pine forests 80 1 2´ 3 4 6 8 9 10´ 11 12 60 40 20 1 2´ 3 4 7´ 8 9 10´ 11 12 0 100 Oak forests 80 Old fields 1 2´ 3 4 6 7´ 8 9 10´ 11 12 60 40 20 1 2´ 3 4 6 7´ 8 9 10´ 11 12 0 0 1 2 3 4 0 1 2 3 4 Month after emergence Figure 3 Germination (A) and survival (B) percentages curves (mean ± SE, n = 3) of A. altissima seeds from 11 maternal sources at each habitat type throughout the field experiment study period. Statistical results for the PERMANOVA are given (H: habitat, MS: maternal source, S: site) RS showed the poorest survival rates, with all seedlings dead after a month (Fig.4). The highest survival duration was found in PS and OLD habitats, where seedlings survived until the fourth month. Maternal seed source significantly affected seedling survival in all habitats, except for RS (Fig.3B). 84 100 Roadside Path side North facing slopes pine forests South facing slopes pine forests Oak forests Old fields Survival (%) 80 60 40 20 0 0 1 2 3 4 Months after emergence Figure 4 Survival percentage curves (mean ± SE, n = 3) of A. altissima seedlings after emergence at the six different habitat types studied Table 2 Two-way nested model results of the environmental variables (cover of litter, bare soil, trees and perennial herbs) according to the habitat type and site using PERMANOVA Source Habitat Site (Habitat) Res Total *P < 0.10; **P < 0.05 df 5 12 18 35 MS 7443.6 4276.6 1272 F 1.74 3.36 P 0.0641* 0.0001** Significant differences were found in environmental variables among habitat types and site (Table 2). Linear regression analysis showed a positive relationship between bare soil cover and A. altissima germination (Fig. 5). 85 35 F = 54.901, df = 1 R2 = 0.618, P < 0.0001 30 Germination (%) 25 20 15 10 5 0 0 20 40 60 80 100 Bare soil (%) Figure 5 Linear regression analysis of A. altissima germination rate vs bare soil cover. Statistical results are given DISCUSSION The comprehensive approach used in this study, using both laboratory and field experiments and evaluating a wide array of factors influencing germination and early establishment of A. altissima, highlights the importance of both genetic and environmental factors in modulating A. altissima germination, as well as the existence of interactions between these factors. Seed germination and early survival varied among maternal sources, and were affected by environmental factors such as the site, the percentage of bare soil and rainfall pulses. Furthermore, the interaction between genetic and environmental factors influenced the early success of A. altissima, as demonstrated by the contrasting responses found for each maternal source depending on temperature (laboratory experiment) or other environmental conditions, such as those represented by the different sites or habitat types tested (field experiment), which suggests that both habitat preference and optimal environment conditions (and therefore the effect of external factors) vary depending on the genetic source. 86 ENVIRONMENTAL FACTORS AFFECT A. ALTISSIMA GERMINATION AND SURVIVAL In accordance with previous studies (Kota et al. 2007; Vilà et al. 2008), we found a low germination percentage under field conditions (although we repeated the field experiment for three years, as no seeds germinated until the third year). The low viability and germination recorded for this species by our work and by other studies are at odds with the high degree of invasion of A. altissima in many habitats across the globe (Kowarik and Säumel 2007). A possible explanation for these contrasting results is the high fecundity of the species, with a single tree able to produce a large number of seeds (since 325,000 samaras per tree; Little, 1974; Bory and Clair-Maczulajtys 1980). We found that temperature had a significant effect on the germination of A. altissima, with lower temperatures rendering higher germination rates (Fig. 1). This result contrasts with previous studies (Little 1974, Graves 1990) and with our own results in the field. Emergent pulses in the field were related to rainfall pulses but not to temperature (Fig. 2). The most plausible explanation for these contrasting results is that the temperature in the field fluctuated (both daily and seasonally) and is not fixed; germination cannot therefore be related to a unique value of temperature. Conversely, and in contrast with previous studies using the same species (Vilà et al. 2008), we found significant differences in germination curves among the different habitat types tested, which are related to the differences found according to the environmental variables (Table 1). The habitat type with the lowest germination and early survival rates was roadside (RS), which could be related to the scarce water availability and low fertility in soils often found in these areas (Bochet and Garcia-Fayos 2004; García-Palacios et al. 2010). Roadsides are ecosystems commonly invaded by A. altissima under Mediterranean conditions (Kowarik 1983; Danin 2000; Constán-Nava et al. 2007; Traveset et al. 2008). In spite of our results, the high degree of invasion in these areas may be due chiefly to the use of this species in roadside restoration and because these ecosystems act as dispersal corridors, where a large number of seeds can easily reach the area, aiding its invasion (Vilà and Pujadas, 2001; Kowarik and von deer Lippe 2006; Kowarik and Säumel 2007; Kowarik and von deer Lippe 2011). Our results also show that the effect of habitat type on the germination rate changed depending on the seasons or the microenvironmental characteristics of each site inside the habitat. The field experiment revealed that 61% of the variance in the seed germination rate was explained by the percentage of bare soil in the plot. This 87 highlights how important areas with high bare soil cover are for A. altissima germination, particularly in habitats with relatively low stress (such as open oak forests), as reported previously for this and other invasive species (e.g. Burke and Grime 1996; Bartuszevige et al. 2007; Kota et al. 2007). This dependence on bare soil could be explained by the lower competition with other plants and the lower litter presence in these areas, which may have both direct negative effects (a lower availability of resources and a physical hindrance of seed emergence) and indirect effects by increasing insect herbivory (Facelli and Pickett 1991; Facelli 1994). Similarly, we found 2.3% of seeds were predated in old fields, possibly caused by arthropods or small mammals (Facelli 1994; Ostfeld et al. 1997). The effect of the site on A. altissima germination could be also related to microenvironmental factors not included in this field study, such as soil moisture or light availability (the latter was measured indirectly using vegetation cover, but was not statistically significant), which have been found to be relevant to the success of A. altissima (Kota et al. 2007; González-Muñoz et al. 2011). GENETIC FACTORS INFLUENCE THE PERFORMANCE OF A. ALTISSIMA AND MODULATE ITS RESPONSE TO ENVIRONMENTAL FACTORS Although variations in seed germination between maternal sources have been found in numerous species (e.g. Baskin and Baskin 1998), no previous studies have found any effect of this factor on the germination of A. altissima (only on seed weight; Kota et al. 2007; Delgado et al. 2009). However, our results, both under field and laboratory conditions, show that genetic factors do affect the germination ability of A. altissima. These contrasting results might be influenced by the higher number of maternal sources and seeds used in this study in comparison with previous ones (12 vs. 6 genetic sources). The survival and growth rates of A. altissima are known to differ among provenances (Feret 1985), which clearly suggests some genetic component affecting A. altissima performance and adding confidence to our results. Genetic effects are those resulting from the maternal tree and how it interacts with the environment, as well as those from the different paternal contributions, as the variation within the seeds could be ascribed to the fact that different seeds were started by pollen from different trees (Roach and Wulff 1987; Baskin and Baskin 1998; Bischoff et al. 2006). In this sense, environmental factors such as differences in the availability of water, nutrients or light for the maternal tree could explain changes in 88 germination rates of seeds coming from the same maternal source, but collected in different years (e.g. seeds from maternal sources 3 or 12; see Fig. 1 and Fig. 3A), but may also be related to the paternal genetic contributions, which could differ from one year to another. Knowledge of the genetic and genotypic diversity of A. altissima in the Mediterranean area of Europe (Dallas et al. 2005) could help in this sense. Significant differences were found in the survival curves of A. altissima seedlings among habitat types and maternal sources. Our results suggest that, on the one hand, habitat type per se could be influencing the survival of A. altissima due to the differences in environmental factors, as discussed above. On the other hand, the differences among maternal sources suggest that genetic factors also affect the survival ability of the invasive species. Regardless of the maternal source, however, all seedlings died, during either winter or summer, most likely due to the low winter temperatures (Kowarik and Säumel 2007) or the summer drought that is typical in Mediterranean environments (Maestre et al. 2001). This suggests that climatic conditions are more important than the genetic component and that they could be the main limiting factor for the invasive success of this species. However, this conclusion must be considered with care, as only maternal individuals from one type of environment were used in this study, and further research including sources from contrasting environments is needed to confirm or reject this hypothesis. CONCLUSIONS In this study we used a comprehensive approach that included the effect of environmental factors at different scales: 1) local temperature and rainfall, represented by the climate data, 2) habitat-related environmental conditions and 3) microenvironmental conditions, represented by our “site” effect and the attributes measured at each plot, genetic factors and the interaction among them on the invasive success of A. altissima under Mediterranean conditions. Our study reveals that the genetic component not only affected the performance of A. altissima, it also modulated its response to environmental factors, which seemed to be the main drivers of germination and early establishment for this species. These results may help to predict the invasion success of A. altissima in contrasted habitats under Mediterranean conditions, and therefore to better reduce the spread of this species in Mediterranean ecosystems. 89 ACKNOWLEDGEMENTS S. Soliveres and two anonymous reviewers provided helpful comments and improvements on an early version of this manuscript. We also thank Language Centre (University of Alicante) and C. Beans for improving the English of this manuscript. We are grateful to the staff of the Font Roja Natural Park (Generalitat Valenciana), Alcoy Council and J.L. Ferrándiz (landowner) for the permits provided and their collaboration. We would also like to thank C. Constán, A. Constán, M.J. Nava, G. Plaza, E. Pastor, N. Vizcaíno and the other collaborators who helped during the laboratory and fieldwork. We thank J. Huesca for the technical support, M.J. Baeza and V.M. Santana for their comments and M.J. Anderson for her statistical suggestions. This research and SCN fellowship were supported by the projects GV06/029 founded by the Generalitat Valenciana and the ESTRES (063/SGTB/2007/7.1) and RECUVES (077/RN08/04.1) founded by the Spanish Ministry for the Environment. 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After the application of this treatment, we located the seeds on Petri dishes in a growth chamber during 31 days under optimum conditions (20º C, 16/8 h light/dark photoperiod and moist filter paper; Little 1974, Graves 1990). Petri dishes were randomly moved to avoid position effects in the germination chamber. The seeds were examined daily and when the radicle was visible were considered germinated and removed from the Petri dishes. Results are shown below. 100 Seed germination (%) 80 2005 2006 2007 60 40 20 0 0 5 10 15 20 25 30 35 Day Figure 6 Germination curves of A. altissima seeds from 3 different years of recollection (showed with different dots) after 31 days in a growth chamber. Data are mean percentage ± SE, n = 4) Germination rates were not significantly different between the three times of storage (P > 0.05), being of 12 ± 2.5% in seeds of 2007, 19 ± 6.4% in seeds of 2006 and 20 ± 3.8% in seeds of 2005. 95 Appendix 2: Regression tree A regression tree (De’ath and Fabricius 2000; Crawley 2007) was performed to analyze the effect of bare soil, perennial herbs, shrubs and trees cover, and litter depth on A. altissima germination percentage. This method allows introducing correlated predictors and detecting non-linear responses. The tree was pruned to improve parsimony using 10-fold cross-validation (De’ath and Fabricius 2000). This analysis was done using the Tree package (Oksanen et al. 2005) for R software (R Development Core Team 2009). Figure 7 Regression tree model for A. altissima germination. Split values for the predictor used are shown in each branch. Terminal nodes show the mean value for each group of the response variable introduced and the number of cases in each node (between parenthesis; n = 36 cases for the tree). The general fit of the model (D2, percentage of variance explained by the model), extracted from the null deviance (Deviance root), and the deviance of the final chosen tree after 10-fold cross-validation (Deviance tree) are shown The regression tree assessing the effect of environmental variables explained ~55% of the variance in seed germination, and pinpointed the percentage of bare soil as the best predictor for seed germination. References (except those in the main text) Crawley MJ (2007) The R Book. John Wiley & Sons, Ltd., Chichester, UK De’ath G, Fabricius KE (2000) Classification and regression trees: a powerful yet simple technique for ecological data analysis. Ecology 81: 3178–3192 Oksanen J, Kindt R, O’Hara RB (2005) Vegan: Community Ecology Package. R. package Version 1.6–10 96 APÉNDICE FOTOGRÁFICO Foto 1 Semilla de A. altissima teñida mediante test de Tetrazolio Foto 2 Experimento con semillas de A. altissima bajo condiciones controladas en cámara de germinación Foto 3 Parcela de 1 × 1 m para el estudio de la germinación de A. altissima en campo (en pinar de solana) Foto 4 Plántula de A. altissima germinada en experimento de campo 97 98 CAPITULO 3 Direct and indirect effects of invasion by the alien tree Ailanthus altissima on riparian plant communities and ecosystem multifunctionality3 3 Manuscrito enviado Autores: Soraya Constán-Nava, Santiago Soliveres, Rubén Torices, Lluís Serra, Andreu Bonet 99 100 ABSTRACT The effects of invasive species on biodiversity are well-known and their effect on separate ecosystem functions and soil attributes has gained interest more recently. However, most of the existing studies focus on species richness, ignoring better indicators of biodiversity and better predictors of ecosystem functioning such as the diversity of evolutionary histories or phylodiversity. Moreover, and in spite of the well known relationship between them, no previous study has separated the direct effect of alien plants on multiple ecosystem functions simultaneously (multifunctionality) from the indirect effect mediated by the effect of alien plants on biodiversity. This latter issue is deeply necessary for improving management plans and developing effective conservation strategies for natural ecosystems undergoing alien plant invasions. We aimed to analyze the direct and indirect effects, mediated or not by its effect on biodiversity, of Ailanthus altissima -an invasive tree that reduces species diversity and some ecosystem functions in natural ecosystems worldwide- on ecosystem multifunctionality of riparian habitats under Mediterranean climate. For doing this, we measured vegetation attributes (species richness and cover, phylodiversity), soil functions (plant biomass and soil enzyme activities) and soil attributes (pH, EC, OM, P) in plots infested by A. altissima and in control (non invaded) ones. Our results show that plant species richness, phylodiversity and multifunctionality were reduced under the presence of A. altissima. The effect of the alien plant on multifunctionality was indirect, mediated mainly by its effect on phylodiversity and, to a less extent, on species richness. Species composition and plant cover, but not soil attributes, were also affected by the invasive plant. We provide an easy methodology to tease apart direct and indirect effect of alien species on ecosystem multifunctionality by using observational data. This may help to center restoration and conservation efforts either on the elimination of invasive species and introduction of native ones, in case of existing solely indirect effects, such as in our case, or to include soil restoration measures to recover ecosystem services and functions in case of existing both direct and indirect effects of the alien species on ecosystem multifunctionality. 101 102 INTRODUCTION I nvasive plant species are a major threaten for biodiversity and ecosystem functioning in many ecosystems around the world (Vitousek et al. 1997; Liao et al. 2008; Vilà et al. 2011). The last years have seen an increasing interest on the effects of the invasive species on species composition, community structure or ecosystem functioning of invaded habitats (Williamson 1998, 2001; Simberloff et al. 2003; Weber 2003). Previous research shows that plant invasions affect the diversity of plants (Richardson et al. 1989; Vilà et al. 2011; Hejda et al. 2009) or animals (Maerz et al. 2005; Watling et al. 2011), modify soil properties (Vilà et al. 2006; Truscott et al. 2008), and alter nutrient cycles (Vitousek and Walker 1989; Ehrenfeld 2003; Castro-Díez et al. 2009), or native microbial communities and their associated ecosystem processes (Wolfe and Klironomos 2005; van der Putten et al. 2007; Weidenhamer and Callaway 2010). Previous studies focus separately on the effect of invasive plants either on biodiversity or on ecosystem functioning, ignoring the well-known and tight relationship between both ecosystem attributes. Many ecosystem functions are known to increase with higher species richness (e.g. Tilman et al. 1997; Zavaleta et al. 2010; Maestre et al. in press); and, therefore, a reduction of diversity or alteration of the composition and structure of vegetation -both effects documented in invaded ecosystems- can result in an indirect decline of ecosystem functioning (Hooper et al. 2005; Richardson et al. 2007). The presence of invasive species can also directly alter ecosystem functions by several mechanisms such as releasing allelopathic compounds, altering the nutrient balance, or by soil salinisation and impoverishment (e.g. Vitousek 1986; Callaway and Aschehoug 2000; Levine et al. 2003), to name a few. Thus, we are still far from a full mechanistic understanding on the nature of the effect of alien plants on ecosystems, since the directness or indirectness of their effect on ecosystem functioning, mediated or not by their effect on biodiversity, is poorly understood. Knowing the nature of the effects of invasive species on ecosystem functions and services may help to improve management plans and restoration strategies for those ecosystems undergoing invasion by alien species. In this sense, if invasive species produces indirect effects in ecosystem functioning, management plans should focus mainly on removing the invasive species and introducing, or enhancing the establishment of, native ones. However, if invasive species produce both direct and 103 indirect effects, management plans should include, additionally, actions for restoring other ecosystem properties, such as soil fertility and functions. Most of the existing studies about the effect of invasive plants on biodiversity focus just on the number of species as a measure of biodiversity ignoring better indicators of biodiversity and better predictors of ecosystem functioning, such as the diversity of evolutionary histories or phylogenetic diversity (Forest et al. 2007; Maherali and Klironomos 2007). Numerous and important ecological traits are preserved through evolutionary times and, therefore, phylogenetic diversity (hereafter phylodiversity) is thought to be a good surrogate of the diversity of functional forms present in a given community (Prinzing et al. 2001; Webb et al. 2002). Species assemblages containing phylogenetically diverse species are more likely to be able to provide high levels of different ecosystem functions and services than those communities formed by functionally similar ones either by complementarity in the use of resources, interspecific positive interactions or the existence of trade-offs in important ecological traits featuring different species (Cardinale et al. 2007; Zavaleta et al. 2010). Indeed, phylodiversity may be a better indicator of biodiversity since it is more related to important ecosystem functions than other more commonly used measurements, such as species richness; and therefore, can provide critical information to understand the functional effects of biodiversity loss (Forest et al. 2007; Maherali and Klironomos 2007; Cadotte et al. 2008). While the study of the ecosystem functions and the services they provide has been a major topic in ecology during the last years (reviewed in Hooper et al. 2005), most of these studies focus on single functions or processes. However, the effect of a given factor (i.e. invasive species) on ecosystem functioning must be studied considering together multiple ecosystem functions (multifunctionality; Hector and Bagchi 2007; Zavaleta et al. 2010; Maestre et al. in press). Studying multiple ecosystem functions simultaneously is crucial since different ecosystem processes or services may be affected by a given factor, but in different directions (Zavaleta et al. 2010). Therefore, analyzing them separately could result in contradictory results and misleading conclusions. Invasive species, for example, could increase C sequestration and plant productivity, and reduce soil erosion in the midterm, due to their high growth rates (Liao et al. 2008; Vilà et al. 2011). However, they may also simultaneously reduce nutrient cycling, soil fertility or ecosystem resilience due to their elevated nutrient uptake, the reduction in species diversity or the releasing of allelopathic compounds 104 (Liao et al. 2008; Weidenhamer and Callaway 2010). These contradictory effects make necessary to study the effect of alien plants on multiple ecosystem functions simultaneously to avoid misleading conclusions on the net effect of these species on natural ecosystems. This is especially relevant considering the reduction of species richness commonly found in ecosystems undergoing alien species invasion (Vilá et al. 2011). Since it is known that the minimum number of species to maintain ecosystem functioning increase with the number of functions considered (Hector and Bagchi 2007; Zavaleta et al. 2010), the indirect impact of alien species on ecosystem functioning could be, therefore, much higher than previously suggested by studies focused in single functions or processes. We aimed to analyze the direct and indirect effects, mediated or not by its effect on biodiversity, of the invasive tree Ailanthus altissima on ecosystem functionality of riparian habitats under Mediterranean climate. We measured vegetation and soil attributes, together with measurements of different ecosystem functions in invaded and non invaded riparian forests by the exotic species. Our main hypotheses were 1) A. altissima reduces not only the species richness, but also the phylodiversity of neighbor vegetation, 2) The effects of A. altissima on vegetation diversity indirectly reduce ecosystem multifunctionality, and 3) A. altissima reduction on ecosystem multifunctionality is not only indirect, mediated by its effect on plant diversity, but also directly by its known effects on nutrient cycles and soil properties. MATERIAL AND METHODS STUDY AREA The study was conducted in riparian forests of water courses and streams localized in the Site of European Community Importance (Directive 92/43/CEE) SCI Serres de Mariola i el Carrascar de la Font Roja, in southeast Spain (X 713000, Y 4288000 UTM). This area is one of the ones with highest diversity of the Mediterranean Basin, considered as Mediterranean Hotspot and one of the 25 Earth Hotspot (Myers et al. 2000; Médail and Quézel 1999). Furthermore, this area is part of one of the refugee areas for flora after climatic change in the Mediterranean since Pleistocene (Serra et al. 2003; Médail and Diadema 2009), so the presence of invasive species could cause high negative effects. The climate is Mediterranean, with mean annual precipitation and temperature of 647 mm and 14.7 ºC, respetively (Bocairent meteorological station 105 located in the study area, at 641 m.a.s.l.; data from period 1985-2006 for temperature and 1966-2006 for precipitation; Rivas Martínez et al. 2007). Soils are xerorthents on limestone (Soil Survey Staff 2006), with the presence of impermeable clays. Riparian habitats are dynamic and complex presenting regular floods which favor species movement (Forman and Godron 1986; Pyšek and Prach 1994). These regular floods, together with human disturbances, make these areas vulnerable to invasion by exotic plants (Hood and Naiman 2000; Holmes et al. 2005; Pyšek et al. 2010). Indeed, riparian areas act as seed transport corridors that promote the expansion of invasive species (Thébaud and Debussed 1991; Pyšek and Prach 1993; 1994; Säumel and Kowarik 2010). Vegetation in riparian ecosystems is especially important for numerous ecological processes, such as providing habitat for wildlife, stabilizing riverbanks, filtering sediments and nutrients into stream and influencing soil properties (e.g. Forman and Godron 1986; Décamps 1993; Tabacchi et al. 2000). Therefore, the presence of invasive species in such ecosystems is likely to alter ecosystem functions indirectly by reducing the high levels of plant diversity they hold. Moreover, invasive plants are also likely to alter ecosystem multifunctionality directly by the bank destabilization, lowering of water tables, salinisation of soil and change channel capacity for flood flow (e.g. Mooney and Drake 1986; Hulme and Bremner 2006). Ailanthus altissima (Mill.) Swingle (Simaroubaceae), our target species, is a deciduous tree from China and North Vietnam which has became in an invasive species around the world since the 18th century due to its use as ornamental and landscape restoration (Kowarik and Säumel 2007). It has occupied numerous ecosystems in the Mediterranean Basin, as disturbed urban areas, old fields, pine and oaks forests, or riparian communities (Kowarik 1983; Constán-Nava et al. 2007; Kowarik and Säumel 2007). A. altissima is known to affect ecosystem functioning and vegetation composition, structure and dynamics (Lawrence et al. 1991; Vilà et al. 2006; Motard et al. 2011). Previous research has shown that A. altissima reduces plant diversity (Vilà et al. 2006; Motard et al. 2011), alters N cycling (Castro-Díez et al. 2009), slows down litter decomposition, or increases the total N, organic C, C/N ratio and pH in the soils it colonizes (Vilà et al. 2006; Gómez-Aparicio and Canham 2008; Godoy et al. 2010; but see Castro-Díez et al. 2011). The importance of plants for multiple ecosystem functions and the sensitivity to invasion of riparian ecosystems, together with the well-known reduction in both diversity and several ecosystem functions promoted by A. altissima, 106 make this study area and species a suitable target to study both direct and indirect effects of invasive plants on ecosystem multifunctionality. EXPERIMENTAL DESIGN We established ten 10 m × 10 m control plots (without A. altissima) and ten 10 m × 10 m plots infested by the invasive species (cover of A. altissima >85%) in July 2008 and 2009. We randomly placed forty 0.5 × 0.5 m quadrats within each plot and we visually estimated plant cover separated by species. To complete the sampling of the species richness, we covered the whole plot then to look for other species present in the plot but not detected within the quadrats. In each plot, we also measured leaf area index (LAI) using a LAI 2000 plant canopy analyzer (Li-Cor, Inc., Lincoln, NE). In half of the sampled quadrats (n = 20), we also estimated litter depth at three different sampling points within each quadrat, and the cover of litter, bare soil and rock. In five of the sampled quadrats, we removed all the aerial plant biomass of both shrub and herb strata and transported it to the laboratory where was oven-dried until constant weight at 50 ºC. Tree biomass was not removed because of the important ecological role and conservation value that riparian trees hold in the study area. Nevertheless, no important implications in our conclusions are expected since we did not find significant differences in tree cover (as estimated with LAI at heights > 1.5 m), but only on tree species composition, between invaded and not invaded plots. In each plot, we took a composed sample from five different soil cores of 30 cm depth. Soil samples were oven-dried during 72 h at 50 ºC in the laboratory and sieved to obtain the fraction < 2 mm, which was used for the analyses described below. ANALYSIS OF SOIL FUNCTIONS AND ATTRIBUTES Our measurements of important ecosystem functions covered surrogates of both the C and P cycle (β-glucosidase and acid phosphatase soil enzymatic activities) together with the ability of plants for C fixation (biomass measurements, described above). Both enzyme activities were estimated by using the methodology described in Tabatabai (1982; β-glucosidase) and Tabatabai and Bremner (1969; acid phosphatase). The functional variables measured in this study are known to be strongly related to key ecosystem processes, such as ecosystem productivity (plant biomass; Tilman 1988; Flombaum and Sala 2008), Carbon cycling (β-glucosidase; Tabatabai 1982) and 107 Phosphorus cycling (phosphatase; e.g. Sinsabaugh et al. 2008, Maestre and Puche 2009). Apart from the ecosystem functioning variables (see above), structural soil variables were measured. We analyzed pH, electric conductivity (EC), organic matter and available phosphorus as key soil attributes. Electric conductivity (EC) and pH were measured in 1: 2.5 mass: volume soil and water suspension (CMA 1973). Organic matter (OM) was measured by the Walkley–Black acid digestion method (CMA 1973). Available phosphorus, a good surrogate of P availability in soils for plants and microbes (Bardgett 2005), was analyzed by NaHCO3 extraction (Watanabe and Olsen 1965). This component was included as structural soil variable because many of the reactions that govern the availability of P are geochemical, rather than biological (i.e. in calcareous soils, such as the ones studied here, phosphates combine with calcium becoming unavailable for plants; Bardgett 2005). MEASUREMENT OF THE EVOLUTIONARY RELATIONSHIPS We assembled a phylogenetic tree for the 115 species included in this study using Phylomatic function available at Phylocom 4.1 software (Webb et al. 2008). All the families in our dataset matched the family names of the angiosperm megatree used in Phylomatic (R20100701.new), which was based on the APG III phylogenetic classification of flowering plant orders and families (Angiosperm Phylogeny Group 2009). Phylogenetic relationships were further resolved based on data from various published molecular phylogenies (Apiaceae [Downie et al. 2000], Asteraceae [Funk et al. 2009], Dipsacales [Winkworth et al. 2008], Lamiids [Bell et al. 2010], Fabaceae [Steele et al. 2010], Poaceae [Bouchenak-Khelladi et al. 2008], Rosaceae [Potter et al. 2007]). After assembling the phylogenetic tree, we adjusted its branch lengths with the help of the Phylocom BLADJ algorithm, which fixes the age of internal nodes based on clade age estimates, whereas undated internal nodes in the phylogeny are spaced evenly (Webb et al. 2008). Thus, BLADJ is a simple tool that fixes the root node of a phylogeny at a specified age, as well as other nodes for which age estimates are available. It sets all other branch lengths by placing the nodes evenly between dated nodes, as well as between dated nodes and terminals (of Age 0). We search for divergence time estimations in the TimeTree database (Hedges et al. 2006; http://www.timetree.org). TimeTree uses a tree-based (hierarchical) system to identify 108 all published molecular time estimates bearing on the divergence of two chosen taxa (e.g. species), compute summary statistics, and present the results. We mainly used this database to fix the ages of internal nodes on our phylogenetic hypothesis, completing TimeTree results with other published sources when this database did not provide any date (Lavin et al. 2005; Besnard et al. 2009; Bremer and Eriksson 2009, Bell et al. 2010; Bouchenak-Khelladi et al. 2010; Torices 2010; Wang et al. 2010). The procedure described above resulted in the fixation of 80 nodes (representing more than 70% of internal nodes of our tree). DATA REDUCTION We avoided conducting an elevated number of analyses derived from the high number of response variables measured by summarizing our dataset using different methodologies. First, we organized soil variables not directly related with ecosystem functioning (pH, EC, OM, litter cover, and available phosphorus) and reduced them to a single synthetic variable by using principal component analysis (PCA). Prior to the PCA, we standardized (sensu Anderson et al. 2008) the five variables, substracting the mean and dividing by its standard deviation. With this a priori data transformation, we homogenized units for all the variables and therefore reduced the higher influence of a given variable just by having different units (i.e. parts per million instead of percentage; Anderson et al. 2008). The first axis of the PCA conducted explained 85.2% of the variation in the data (eigenvalue = 58) and therefore this first axis was used to infer the effects of A. altissima on soil attributes (hereafter, we will refer to this first PCA axis as “soil attributes”). This first axis was highly related to available phosphorus (eigenvector = 0.718) and litter cover (eigenvector = -0.695), but not to pH, EC or OM (eigenvectors = -0.044, -0.008 and 0.012, respectively). Secondly, to summarize variables related to ecosystem functioning, we calculated a multifunctionality index (M), recently proposed by Maestre et al. in press (see below). The use of this index help us to improve two aspects of our results: 1) reduces the three variables (biomass and β-glucosidase and acid phosphatase activities) to a single one, therefore reducing multiple testing, and 2) allows us to assess for the effect of A. altissima on the ability of the ecosystem to maintain multiple functions simultaneously, rather than conducting separate analyses for each function. The multifunctionality index (M) used was based on the calculation of the Z-scores of the three functions measured. The multifunctionality index for each plot was the average Z109 score for these three functions. The use of Z-scores allows measuring all functions on a common scale, regardless of their units, has good statistical properties (normal distribution, mean and variance not related), and is highly correlated with other multifunctionality indices proposed in the literature (Maestre et al. in press). Apart from calculating the number of species in each plot, to allow for comparisons with other studies, we also calculated the phylodiversity of such plots. A substantial number of indices have been proposed to evaluate different aspects of phylodiversity (see Helmus et al. 2007; Vamosi et al. 2009; Pausas and Verdú 2010; for recent reviews). Among them, the most used and accepted are the Mean Phylogenetic Distance (MPD), the Mean Nearest Taxon Distance (MNTD), the Phylogenetic Species Variability (PSV) and the Phylogenetic Species Evenness (PSE; see Webb et al. 2002; Helmus et al. 2007; Kraft et al. 2007). In our case, all of them were strongly correlated (r > 0.5 in all cases) and yielded similar results regarding the effect of A. altissima invasion on them and their relationships with ecosystem multifunctionality. For simplicity, we only show here the results regarding PSE. We selected this index because it incorporates both phylogenetic and species abundance information, its variability is less sensitive to sample size and does not depend on species richness (Helmus et al. 2007). The maximum value of PSE is 1, corresponding with a community formed by evolutionarily independent species (the so-called “star phylogeny”) and with equal abundances of those species (complete evenness). Since PSE can be more related to evenness in the community than to its phylogenetic pattern (Helmus et al. 2007), we also briefly comment on the results of the Phylogenetic Species Variability index (PSV) to aid with the interpretation. STATISTICAL ANALYSES To assess for the differences in plant species composition and cover between invaded and non invaded plots, ordination methods were carried out. Based on the length of the main gradient in the data, estimated by an indirect gradient analysis (Detrended Correspondences Analysis, DCA: ter Braak and Smilauer 2002) redundancy analysis (RDA) was identified as appropriate method. Analyses of DCA and RDA were carried out using CANOCO for Windows v 4.5 (ter Braak and Smilauer 2002). We included species cover, and presence/absence of A. altissima, litter depth, cover of litter, cover of bare soil and cover of rock as environmental variables. A forward selection procedure by means of a Monte Carlo permutation test was carried out in order to test the significance of the 110 environmental variables. Significance of just the first and then all canonical axes were tested by the distribution-free Monte Carlo test (999 permutations). We analyzed the effect of A. altissima invasion on species richness, phylodiversity (PSE), soil attributes, and ecosystem multifunctionality (M) separately. For doing this, we used ANOVAs with A. altissima invasion as a fixed factor with two levels (control and invaded). We ran separated models for each of the four ecosystem attributes described above, which were introduced as response variables. We also evaluated the relationship between M (dependent variable) and either species richness or PSE (predictors) by using linear regressions. Apart from looking at the indirect effect of A. altissima on multifunctionality (M), mediated by its effect on species richness or phylodiversity (the ANOVA analysis described above), we aimed to assess for the direct effect of A. altissima on such variable. For doing this, we used the residuals of the linear regressions between either species richness or PSE and multifunctionality, described above, as a response variable for an ANOVA with A. altissima invasion as a fixed factor. The use of the residuals of the linear regressions between species richness or PSE and M allowed us to account for the direct effect of A. altissima on M independently of the effect of the invasive species on either the species richness or the phylodiversity, respectively. Although we justified above the rationale for the inclusion of each measured variable as either structural or functional, we acknowledge that other of the measured variables could be considered as functional rather than structural (e.g. phosphorus availability or soil organic matter; Maestre et al. in press). For this reason, we recalculated the M index using also phosphorus availability as a functional variable and the results gathered were very similar (see results below). All the variables used accomplished analyses assumptions and no transformations were needed. All the analyses were carried out using SPSS 13.0 for Windows (Chicago, Illinois, USA), excepting the Principal Component Analysis (PCA). The latter was conducted by using Primer v. 6 statistical package for Windows (PRIMER-E Ltd., Plymouth Marine Laboratory, UK). We calculated PSV and PSE indexes using the Picante package (Kembell et al. 2010) for R version 2.10.1 (R Development Core Team 2009). 111 RESULTS Species composition of understory and plant cover in riparian communities was affected by invasion of A. altissima and litter accumulation (RDA axes I and II speciesenvironmental variables correlation: 0.98 and 0.77, respectively; cumulative percentage variance 94.5 %) (Table 1). Table 1 Results of the forward selection procedure on selected environmental variables using Monte Carlo Permutation Test of the RDA analysis on species cover Variable λA P F Presence/absence of A. altissima 0.65 33.25 0.001 % litter 0.08 5.13 0.004 litter depth 0.03 0.122 1.74 % stone 0.02 0.190 1.49 % bare soil 0.01 0.742 0.53 The invasion of A. altissima significantly reduced species richness (25 ± 2 vs 15 ± 2 plant species in control vs invaded plots, respectively; Table 2) and phylodiversity (PSE = 0.29 ± 0.02 vs 0.14 ± 0.02 in control vs invaded plots, respectively). The latter results show an increase in phylogenetic clustering on the invaded communities. Since results from the PSE index were almost identical to those of the PSV index (results not shown), this suggests that the results found are driven by the effects of A. altissima on the phylodiversity rather than on the evenness among understory species. These negative effects of A. altissima on richness and phylodiversity also extended to ecosystem multifunctionality (M index = 0.29 ± 0.08 vs -0.29 ± 0.15 in control vs invaded plots, respectively) but not to soil attributes, which were unaffected by A. altissima invasion (Table 2). Both species richness and phylodiversity were significantly and positively related to ecosystem multifunctionality (Fig. 1). In our case, phylodiversity was a better indicator of multifunctionality than species richness (25% of the variance explained vs 17% explained by species richness). The latter results were robust regardless of the phylodiversity indicator used (e.g. PSV vs M: R2 = 0.22; P = 0.038; MPD vs M: R2 = 0.24; P = 0.028). The ANOVAs conducted using A. altissima invasion as a fixed factor and the residuals of the linear regressions between species richness or phylodiversity and ecosystem multifunctionality revealed that the negative effect of A. altissima was caused indirectly, mediated by its effect on phylodiversity and, to a less extent, species 112 richness (Table 2). When filtering the effect of species richness on M, the effect of A. altissima on the latter was importantly reduced (a three-fold reduction in the F when DIRECT EFFECT Table 2 Summary of A. altissima invasion effects on the different ecosystem attributes measured. The F-statistic and the P-value for A. altissima invasion effect, and also the amount of variance (R2) explained by the model are shown. Since some of the variables used were derived from the simplification of multiple data (i.e. soil attributes and multifunctionality; see main text), an interpretation of the results has been added for clarity. “Direct” and “indirect effect” labels mean that the raw data or the residuals from linear regressions were used in the analyses, respectively. In the latter case, the predictor variable used in the linear regression (with multifunctionality as a response) is shown between parentheses. Soil attributes = First axis of the Principal Components Analysis performed with soil pH, EC, OM, available P and litter cover. Phylodiversity = Results from the Phylogenetic Species Evenness index used. Multifunctionality = M index constructed by averaging the Z-scores of the three functional variables used (glucosidase and phosphatase enzymatic activities, plant biomass) Ecosystem attribute F1,18 R2 P-value Interpretation Soil attributes 0.85 0.05 0.369 No effect on soil pH, EC, OM or available P Species richness 12.7 0.41 0.002 A. altissima reduces species richness and phylodiversity Phylodiversity (PSE) 29.4 0.62 <0.0001 A. altissima reduces ecosystem Multifunctionality EFFECT INDIRECT Multifunctionality (species richness) Multifunctionality (PSE) 11.3 0.39 0.004 3.23 0.15 0.089 multifunctionality. However, this effect is mediated mainly by its effect on phylodiversity and, to a less extent, on 1.36 0.07 0.26 species richness analyzing residuals vs raw data; Table 2) and turned from highly to marginally significant. This change was more important when filtering by the effect of phylodiversity on M. In the latter case, the effect of A. altissima on M turned to not significant at all when analyzing the residuals and the F was reduced almost 10 times (Table 2). When using the M index including P availability as a functional variable, the results were very similar (effect of A. altissima on the raw index: F1,18 = 4.14; P = 0.057; R2 = 0.2; relationship between M and species richness: R2 = 0.16; P = 0.077; relationship between M and phylodiversity: R2 = 0.19; P = 0.054). 113 1,0 A 0,8 0,6 2 R = 0.17; P = 0.071 0,4 0,2 0,0 Multifunctionality index -0,2 -0,4 -0,6 -0,8 -1,0 0 5 10 15 20 25 30 35 40 Species richness 1,0 B 0,8 0,6 0,4 2 R = 0.25; P = 0.026 0,2 0,0 -0,2 -0,4 Control Invaded -0,6 -0,8 -1,0 0,0 0,1 0,2 0,3 0,4 Phylodiversity (PSE) Figure 1 Relationships between species richness (A) or phylodiversity (B) and ecosystem multifunctionality. Results from the linear regressions are shown in each panel (n = 20). To aid interpretation, the particular relationships for control and invaded plots are, for species richness vs multifunctionality, R2 = 0.39; P = 0.071 and R2 = 0.09; P = ns, respectively. For the relationship between phylodiversity and multifunctionality results were: R2 = 0.49; P = 0.025 for control and R2 = 0.06; P = ns for invaded plots. 114 DISCUSSION The novelty of our study settles in that is one of the few existing ones addressing the effect of invasive species on other measures of biodiversity than species richness, and on multiple ecosystem functions simultaneously. To our knowledge, this is also the first study separating direct and indirect (mediated by its effect on biodiversity) effects of invasive plants on ecosystem functioning. Our main findings were that plant species richness and phylodiversity, together with ecosystem multifunctionality, were reduced under the presence of Ailanthus altissima. The reduction of ecosystem multifunctionality was indirectly mediated by the decline in phylodiversity and, to a less extent, in species richness. This suggests that mainly with the removal of the invasive species and the reintroduction of native ones, with no other actions needed, important functions and services could be re-established. We speculate that a more rapid increase of phylodiversity, and therefore on ecosystem multifunctionality, would be found if the reintroduction of native species would include those taxa more distantly related with the remaining ones. Among these functions, and at the light of previous research (Strauss et al. 2006; Diez et al. 2008; Zavaleta et al. 2010) is likely that more phylodiverse communities might prevent future invasions. Our results support the first hypothesis since A. altissima reduced not only species richness, but also the phylodiversity of neighbor vegetation. Moreover, we found an alteration of species cover and composition in invaded plots. Although the studies regarding the effect of invasive species on biodiversity show some controversy (e.g. Sax and Gaines 2003; Meffin et al. 2010), our results are in the lines of those developed in riparian habitats (e.g. Gerber et al. 2008; Gaertner et al. 2009; Davies 2011) or with A. altissima as their target species (Vilà et al. 2006; Motard et al. 2011). A more detailed view of our results suggests an even more important reduction of A. altissima on plant biodiversity than the one suggested by looking just at species richness or phylodiversity. For example, we found other invasive species, such as Robinia pseudoacacia L., in the plots invaded by A. altissima but not in the control ones. It is known that the presence of invasive species accelerates invasions of other alien species and amplify their effects on native communities (invasional meltdown; see Simberloff and Von Holle 1999; Richardson et al. 2000; Pyšek and Richardson 2010). Moreover, among the species only present in the control plots, we found rare species of high conservation interest, such as Cephalanthera damasonium (Mill.) Druce (protected 115 species with one of the best populations in the area, Decreto 70/2009; Serra et al., 2006). Other species of interest were found out, but near, of the sampled plots (Himantoglossum hircinum (L.) Spreng., Populus canescens (Aiton) Sm., Potamogeton coloratus Hornem. or Zannichellia peltata Bertol.); since they were near to plots invaded by Ailanthus altissima, increasing the area invaded by A. altissima could represent a potential threaten for these species too. These results highlight the importance of preventing the invasion of exotic plants in habitats with conservation interest, as the riparian forests studied here. Restoration measures including A. altissima removal could favor the colonization of native species and help to preserve richness (especially in case of endangered species). Our study is one of the few existing ones that show how phylodiversity is a better indicator of ecosystem functioning than other more commonly used measured of biodiversity, such as species richness (Forest et al. 2007; Maherali and Klironomos 2007; Cadotte et al. 2008) and, to our knowledge, is the first one showing this relationship using multiple ecosystem functions simultaneously. We found a reduction of phylodiversity in invaded plots, which could have negative effects on multiple ecosystem functions and services. Thus, our results highlight that management plans, apart from removing A. altissima, should include strategies for the reintroduction of the lost native species, especially of those taxa more distantly related to the remaining ones and protected native species. On the other hand, the entrance of the invasive species may be influenced by the phylogenetic structure of the host community (Vacher et al. 2010), with alien species more distantly related to the host community more likely to end as invasive ones in such communities (Strauss et al. 2006; Diez et al. 2008). Although it has not been studied before, joining the results of both lines of research suggest that conserving more phylogenetically diverse species assemblages should increase the resilience of such communities to future invasions. Communities assembled by more functionally diverse species (i.e more phylodiverse) are more likely to 1) sustain higher levels of ecosystem functions such as resilience and resistance to plant invasions (Zavaleta et al. 2010) and 2) include among these species some taxa closely related to possible invaders, therefore preventing them to establish and invade the host communities (Strauss et al. 2006; Diez et al. 2008). Our results also sustain the second hypothesis since the effects of A. altissima on vegetation diversity indirectly reduced ecosystem multifunctionality. Although overall our results clearly show a positive relationship between either species richness or 116 phylodiversity and ecosystem multifunctionality, we found that this relationship changed when considering invaded or control plots separately (Fig. 1). There was a negative relationship between multifunctionality and phylodiversity or richness when we included only non invaded plots (blue dots in Fig. 1) and there was no relation when including only invaded ones (red dots). The most plausible explanation for these results that we might think of is that the high species richness or phylodiversity of the noninvaded plots could include functionally redundant species and a higher soil nutrient uptake. It must be noted that most of the measured functions included in this study are mostly related to nutrient cycling in soils and plant productivity, ignoring a number of other important ecosystem functions. These particular functions may be favored by some particular species, showing elevated growth rates or complementarity in the use of resources (Cardinale et al. 2007), making the rest of species functionally redundant for these particular functions but increasing more complex interspecific networks thus enhancing coexistence and facilitating biodiversity maintenance (Bascompte et al. 2006). However, although less diverse assemblages may be able to increase the performance of one particular function or process, more diverse communities are necessary to sustain relatively high levels of multiple ecosystem functions simultaneously (Zavaleta et al. 2010). The lack of relationship in the invaded plots seems more likely driven by a high statistical noise in our data, probably derived from different times since A. altissima invasion (Vilà and Ibañez 2011), or the presence of other invasive species (Appendix 1). Regardless of the particular mechanisms behind the differential biodiversity-functioning relationship between invaded and non-invaded plots, we clearly show a reduction of ecosystem multifunctionality with the presence of A. altissima. Although A. altissima has a rapid growth (Zasada and Little 2002), and may increase the soil fertility and C fixation (Vilà et al. 2006; Gómez-Aparicio and Canham 2008), our results show a net negative effect of A. altissima on the productivity of herbs and shrubs and on C and P cycling. Conversely, and contradicting our third hypothesis, we found that the effect of A. altissima on multifunctionality was not direct, but indirectly mediated by its effect on diversity. The reduction of plant diversity may cause important changes in plant productivity and nutrient storage and cycling (Tilman et al. 1997; Hooper and Vitousek 1998; Cardinale et al. 2007), these were the functions covered with our measurements and this may explain the overwhelming importance of these indirect effects in our study. Previous experimental studies, however, has shown direct effects of A. altissima 117 on several ecosystem functions and soil attributes (litter decomposition and pH, Godoy et al. 2010; N cycle, Castro-Díez et al. 2011); evidences also found with observational studies (pH, organic C, C/N ratio, Vilà et al. 2006; Gómez-Aparicio and Canham 2008). These contrasting results could be explained by the differences in the target habitat or the soil functions between previous research and our study, or because we failed in interpreting A. altissima as a cause rather than a consequence of the differences between invaded and non-invaded plots (Levine et al. 2003). Although it is known that habitat characteristics can influence on A. altissima invasion (e.g. Traveset et al. 2008; Constán-Nava and Bonet under review), this latter explanation is unlikely. Firstly, habitat characteristics were very similar among plots since we only worked in riparian communities, we did not find differences in important soil attributes, and management and disturbance levels of these plots has been exactly the same since the study area has been a protected area for over 100 years. Secondly, A. altissima competiveness with native species by means of allelopathic and herbicide substances (Heisey 1990, 1996; Lawrence et al. 1991), or by its extended root system (Motard et al. 2011) may take precedence over the initial habitat characteristics. Therefore, the difference in the degree of invasion between control and invaded plots are more likely to be caused by dispersal limitation than by initial differences among the plots. Invaded plots were closer than control ones to roadsides, a well-known dispersal corridor for this species (Kowarik and von der Lippe 2006; 2011), supporting this statement. All these evidences suggest that the reduction found in invaded plots on species richness, plant cover, phylodiversity and ecosystem multifunctionality are a consequence, and not a cause, of the presence of A. altissima in such plots. Nevertheless, the contrasting results found with previous studies call for future research including manipulative approaches and the measurement of multiple ecosystem functions simultaneously to conclude if the invasive species alters directly or indirectly ecosystem functioning. CONCLUSIONS Our study is the first one showing how phylodiversity and ecosystem multifunctionality were reduced under the presence of an invasive species. Plots infested by Ailanthus altissima showed lower phylogenetic diversity and species richness, which indirectly decreased ecosystem multifunctionality. Our study highlights the importance of taking into account both direct and indirect effects of invasive species for improving 118 management strategies in natural ecosystems. Moreover, we provide an easy methodology to tease apart these direct and indirect effects by using observational data. This may help to focus restoration and conservation efforts either on the elimination of invasive species and introduction of native ones when indirect effects prevail, such as in our case; or, alternatively, to include soil reclaiming or other restoration practices aimed to recover the loss of ecosystem services and functions in case of existing both direct and indirect effects of the alien species on ecosystem multifunctionality. ACKNOWLEDGEMENTS We thank to Carrascal de la Font Roja and Sierra de Mariola Natural Parks staff, MJ. Nava, A. Constán, E. Pastor, A. Dávila and the rest of collaborators for their help in the fieldwork. F.T. Maestre, Y. Valiñani, A. Sanz and P. 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Winkworth RC, Bell CD, Donoghue MJ (2008) Mitochondrial sequence data and Dipsacales phylogeny: mixed models, partitioned Bayesian analyses, and model selection. Mol Phylogenet Evol 46: 830–843 Wolfe BE, Klironomos JN (2005) Breaking new ground: soil communities and exotic plant invasion. BioScience 55: 477-487 Zasada JC, Little S (2002) Ailanthus altissima (P. Mill.) Swingle. In: Bonner, Franklin T., tech. coord. Woody plant seed manual, [Online]. Washington, DC: U.S. Department of Agriculture, Forest Service (Producer) Zavaleta ES, Pasari JR, Hulvey KB, Tilman GD (2010) Sustaining multiple ecosystem functions in grassland communities requires higher biodiversity. Proc Natl Acad Sci 107: 1443−1446 125 Appendix 1: list of species in control (non invaded) and invaded by A. altissima in Mediterranean riparian forests Control Species Life form* Abundance§ Adiantum capillus-veneris L. Agrimonia eupatoria L. subsp. eupatoria Allium sphaerocephalon L. Andryala integrifolia L. Arctium minus (Hill) Bernh. Aristolochia paucinervis Pomel Asparagus acutifolius L. Asperula aristata subsp. scabra (J. and C. Presl) Nyman Bituminaria bituminosa (L.) C. H. Stirt. Brachypodium phoenicoides Roem. and Schult. Buglossoides arvensis (L.) I. M. Johnst. Carex flacca Schreb. subsp. serrulata (Biv.) Greuter Carex mairii Coss. and Germ. Catananche caerulea L. Celtis australis L. Centaurea aspera L. subsp. aspera Cephalanthera damasonium (Mill.) Druce Cichorium intybus L. Cirsium monspessulanum (L.) Hill. subsp. ferox (Coss.) Talavera Cistus albidus L. Convolvulus arvensis L. Corylus avellana L. Crataegus monogyna Jacq. Daphne gnidium L. Daucus carota L. subsp. carota Dittrichia viscosa (L.) Greuter Epilobium sp. Equisetum ramosissimum Desf. Euphorbia sp. Festuca arundinacea Schreb. subsp. fenas (Lag.) Arcang. Ficus carica L. Foeniculum vulgare Mill. subsp. piperitum (Ucria) Cout. Fraxinus ornus L. Genista scorpius (L.) DC. Hedera helix L. Helichrysum italicum subsp. serotinum (Boiss.) P. Fourn. Hypericum perforatum L. Juglans regia L. Juncus subnodulosus Schrank Marrubium vulgare L. Matthiola fruticosa (L.) Maire Medicago minima (L.) L. Medicago suffruticosa Ramond ex DC. Mentha suaveolens subsp. suaveolens (L.) Hudson Mercurialis tomentosa L. Piptatherum miliaceum (L.) Coss. subsp. miliaceum Origanum vulgare L. subsp. virens (Hoffmanns. and Link) Bonnier and Layens G H G T/H H G C/N C/H C/H H/G T G H H M H G H H N G M Me N H C H G C M C M M M C C C CC C C M M E CC R C C C CC R C C CC CC H Me H M N P C H M G H C T C/M H C H H M C C M C C C C M M C C C C C M C M 126 CC Osyris alba L. N C Pallenis spinosa (L.) Cass. H C Pinus halepensis Mill. M CC Plantago lanceolata L. H C Polygala monspeliaca L. T M Populus nigra L. M CC Potentilla reptans L. H C Prunus domestica L. M R Prunus spinosa L. N/Me M Pulicaria dysenterica (L.) Bernh. H C Quercus coccifera L. Me/N CC Quercus ilex L. subsp. ballota (Desf.) Samp. M C Rhamnus alaternus L. N/Me CC Rosa canina L. P R Rosmarinus officinalis L. N CC Rubia peregrina L. subsp. peregrina P C Rubus ulmifolius Schott P CC Salix atrocinerea Brot. Me M Salvia verbenaca L. H M Sambucus nigra L. Me M Scabiosa atropurpurea L. H M Scirpus holoschoenus L. G CC Scrophularia auriculata L. subsp. valentina (Rouy) Ortega Oliv., Serra, Herrero H C & Muñoz Garm. Silybum marianum (L.) Gaertn. H M Smilax aspera L. P C Solanum dulcamara L. P M Sonchus maritimus L. subsp. aquatilis (Pourr.) Nyman H C Torilis sp. T Trachelium caeruleum L. H M Trifolium repens L. H C Trifolium sp. Ulex parviflorus Pourr. N C Ulmus minor Mill. M C Verbascum sp. H Viburnum tinus L. Me M Vicia sp. Viola alba Besser H M *C: chamaephyte, P: phanerophyte, G: geophyte, H: hemicryptophyte, M: macrophanerophyte, Me: mesophanerophyte, N: nanophanerohyte, T: therophyte § CC: very common, C: common, E: exotic, M: medium abundance, R: rare, RR: very rare 127 Invaded by A. altissima Species Life form* Abundance§ Agrostis stolonifera L. Ailanthus altissima (Mill.) Swingle Allium sphaerocephalon L. Asparagus acutifolius L. Asperula aristata subsp. scabra (J. and C. Presl) Nyman Avena sp. Bituminaria bituminosa (L.) C. H. Stirt. Brachypodium phoenicoides Roem. and Schult. Brachypodium retusum (Pers.) Beauv. Bromus diandrus Roth Bromus sp. Bryonia dioica Jacq. Buglossoides arvensis (L.) I. M. Johnst. Carex flacca Schreb. Celtis australis L. Cichorium intybus L. Convolvulus arvensis L. Conyza sumatrensis (Retz.) E. Walker Crataegus monogyna Jacq. Cynoglossum cheirifolium L. Daphne gnidium L. Daucus carota L. subsp. carota Equisetum ramosissimum Desf. Eryngium campestre L. Ficus carica L. Foeniculum vulgare Mill. subsp. piperitum (Ucria) Cout. Galium aparine L. Galium spurium L. Geranium rotundifolium L. Hedera helix L. Humulus lupulus L. Juncus subnodulosus Schrank Lonicera etrusca G. Santi Marrubium vulgare L. Medicago sativa L. Mentha sp. Mentha suaveolens subsp. suaveolens (L.) Hudson Oxalis corniculata L. Pallenis spinosa (L.) Cass. Parietaria judaica L. Piptatherum miliaceum (L.) Coss. subsp. miliaceum Plantago lanceolata L. Polygala monspeliaca L. Populus nigra L. Prunus spinosa L. Quercus coccifera L. Quercus ilex L. subsp. ballota (Desf.) Samp. Rhamnus alaternus L. Robinia pseudoacacia L. Rosa canina L. Rubia peregrina L. subsp. peregrina Rubus ulmifolius Schott G M/Me G C/N C/H C E C C C C/H H/G H/G T C CC CC C H T G M H G T Me H N H G H Me H T T T P P G P H H M C C E C CC M C C C CC CC CC C C C M CC C RR M M C CC H H H H H H T M N/Me Me/N M N/Me M P P P C C C CC C C M CC M CC C CC E R C CC 128 Sambucus nigra L. Me M Silybum marianum (L.) Gaertn. H M Smilax aspera L. P C Solanum dulcamara L. P M Torilis arvensis (Huds.) Link subsp. neglecta (Spreng.) Thell. T C Torilis sp. T Trifolium sp. Ulmus minor Mill. M C Urtica urens L. T CC Viburnum tinus L. Me M Vicia sp. *C: chamaephyte, P: phanerophyte, G: geophyte, H: hemicryptophyte, M: macrophanerophyte, Me: mesophanerophyte, N: nanophanerohyte, T: therophyte §CC: very common, C: common, E: exotic, M: medium abundance, R: rare, RR: very rare 129 Appendix 2: Evolutionary relationships between the sampled species 130 APÉNDICE FOTOGRÁFICO Foto 1 Bosque de ribera mediterráneo presente en el área de estudio Foto 2 Presencia de A. altissima en bosque de ribera dentro del área de estudio 131 132 CAPITULO 4 Long-term Ailanthus control of the altissima: invasive Insights tree from Mediterranean protected forests4 4 Manuscrito publicado en: Forest Ecology and Management 206 (6), pp 1058-1064 Autores: Soraya Constán-Nava, Andreu Bonet, Estrella Pastor, Maria José Lledó 133 134 ABSTRACT Ailanthus altissima is an invasive tree species which has colonized numerous ecosystems and affected ecosystem processes worldwide. Despite its importance as an invasive species and the high economic costs incurred from its spread, there is a lack of long-term management planning for its control. Although mechanical disturbance is commonly applied, the effect that this treatment may have exhausting its resprouting ability and also its joint effect with phytochemical treatments are poorly understood, especially in Mediterranean environments. We tested three treatments (plus a control) aimed to reduce A. altissima growth in Mediterranean forests throughout 5 years of study. The treatments (one cut stump, double cut stump and cut stump with glyphosate application) were repeated annually. General plant performance (biomass, height and resprout-type density) was measured yearly during the study. Water potential and stomatal conductance were also measured at the end of the study to evaluate particular ecophysiological factors which might affect the response of A. altissima to assayed treatments, together with leaf area index. Our results show that only the cut stump with glyphosate application treatment was able to reduce the long-term growth and spread of A. altissima. The treatments applied favoured collar sprout growth in response to disturbance events (treatments) opposite to the control, where new sprouts grew mainly from the root. Treated resprouts displayed ecophysiological changes depending on the assayed treatment. To our knowledge, this is the only study testing the long-term effect of both physical disturbance and phytochemical application on A. altissima growth. Our study further refines our knowledge on the effects of repeating both commonly and newly used treatments, improving our management techniques to reduce the presence and growth of this invasive tree. 135 136 INTRODUCTION A ilanthus altissima (Mill.) Swingle (Simaroubaceae) is a deciduous tree native to China and North of Vietnam that has developed into an invasive species expanding on all continents except Antarctica (Kowarik and Säumel 2007). It is a significant invasive plant in the Mediterranean Basin, where it mainly occurs on disturbed urban sites, oldfields, and along roadsides (Kowarik and Säumel 2007). This expansion has been caused by its use in roadside restoration and as an ornamental species (Kowarik and Säumel 2007). A. altissima exhibits rapid establishment and growth, with a high rate of sexual reproduction (Little 1974; Bory and ClairMaczulaijtys 1980) and the ability to resprout rapidly and form dense clonal stands after disturbance (Kowarik 1995; Kowarik and Säumel 2007). A. altissima has strong competitive effects on other species due to the presence of allelopathic compounds (Heisey 1990, 1996; Heisey and Heisey 2003; Carter and Fredericksen 2007), which affects ecosystem functioning and vegetation dynamics and structure (Lawrence et al. 1991; Vilà et al. 2006). Its control is therefore desired, particularly in protected areas (Castroviejo et al. 2003; Meggaro and Vilà 2002). The main method commonly used to eliminate A. altissima is mechanical removal by means of the complete removal of aerial biomass once a year (hereafter onecut stump treatment; Hoshovsky 1988; Hunter 2000). This is problematic, however, due to the plant’s tendency to resprout following disturbance (Hoshovsky 1988; Bory et al. 1991). The tendency to resprout is a highly efficient strategy in response to the loss of above-ground biomass following disturbance (Midgley 1996; Bellingham and Sparrow 2000; Bond and Midgley 2001). While one cut stump treatment once a year may be an unsuccessful control method, continued long–term stump cutting, especially after this species spends some of its reserves during its annual growth pulse, could conceivably lead to the depletion of the reserves in this resprouting species and therefore compromise the chances of regeneration (Vilà and Terradas 1995; Canadell and LópezSoria 1998). Although never tested, mechanical treatments applied twice per year (hereafter double cut stump treatment) during the growing period could therefore improve the success of control methods, particularly in the long–term success. Alternatively, recent research in temperate environments has identified the fact that the joint application of mechanical and chemical (herbicide) treatments (hereafter cut stump with herbicide application treatment) is more effective at eradicating this species than 137 mechanical treatment alone (Meloche and Murphy 2006). These joint methods have not, however, been tested under Mediterranean conditions. The mid– and long–term effects of repeated application of these treatments on this resprouting species are virtually unknown, and therefore studies to assess the long–term prognosis for control are needed in order to test the usefulness of these techniques. The invasion of A. altissima into Mediterranean ecosystems has occurred despite the fact that it has not evolved within a climate of persistent summer droughts and unpredictable soil water, characteristics of this Mediterranean climate. Theoretically, therefore, A. altissima should be unable to outcompete native woody species, which have evolved structural and physiological mechanisms to cope with these environmental constraints (Levitt 1980). However, the water-saving mechanism (i.e., simultaneous leaves water loss and root hydraulic conductance reduction) found on seedlings of A. altissima has been suggested to be related to its wide expansion in Mediterranean areas (Trifilò et al. 2004). Control treatments applied to reduce this species presence may affect resource uptake by the new resprouts after perturbation to overcompensate biomass loss. This would lead to soil water depletion and a derived increase in competition with native vegetation. Alternatively, control treatments could negatively affect the physiology of the invasive species resprouts (Meloche and Murphy 2006), causing changes in the response of A. altissima to drought due to stomatal alterations. Also potential positive feedback effects between the direct (biomass loss, poisonous effect) and indirect (reduced water use efficiency, loss of competitive ability against native plants) negative effects on A. altissima performance, could reduce the competitive ability of this species against native vegetation because of a reduction of its performance and occupied area (Huston 1999) and therefore lead to more successful control of this species in Mediterranean environments. Because of these possible complex and at times counter-intuitive effects, it is important to increase our knowledge about the traits that allow A. altissima to invade Mediterranean environments -in particular its water use strategy- and how control treatments affect these adaptations and therefore its competitive ability against native species. This is critical if we are to understand its potential effects on Mediterranean ecosystems, how it competes with native woody plants, and how we can work towards preventing it from becoming a dominant woody invader. In this paper we aimed to determine the best strategy to reduce the long–term invasion of A. altissima at Mediterranean forests. We tested the effects of three factors 138 combining mechanical and chemical treatments (one cut stump per year, double cut stump per year, and one cut stump with herbicide application per year) on the growth of Ailanthus altissima. The double cut stump treatment was considered an easier alternative to herbicide application as the study area is as protected area used for conservation. Our main hypotheses were: i) The one cut stump treatment per year will be the least efficient treatment, because of an increase of A. altissima resprout growth and survival during the following years (Bory et al. 1991; Meloche and Murphy 2006). This increase could implicate ecophysiological changes in the species which allow it to grow and maintain itself in the plant community, increasing its competitive ability against native species (e.g., high stomatal conductance), ii) The double cut stump treatment per year and one cut stump with herbicide application treatment per year will be the most efficient methods, because they will affect negatively to the invasive species. The first will reduce aboveground growth of resprouts as a result of a reduction of root reserves as demonstrated in native resprouting species (Vilà and Terradas 1995; Canadell and López-Soria 1998). The second will reduce plant performance and will produce morphological alterations caused by the herbicide. The reduction of A. altissima resprouts in both treatments will be jointed to ecophysiological changes which will affect negatively to the species. This will reduce its competitive ability with native species. MATERIAL AND METHODS STUDY SITE The study was carried out in Carrascal de la Font Roja Natural Park in the northwest of Alicante Province (SE Spain). Elevations range from 600 to 1356 m above sea level. Soils are limestone with the presence of impermeable clays. The climate is Mediterranean and is characterised by cold and wet winters, and a marked summer drought. Mean monthly temperatures range from 4.4 ºC in January to 24.9 ºC in July. During the five year study period, total annual rainfall ranged from 199.3 mm to 636 mm. The Natural Park includes different ecosystems such as deciduous forests (with Quercus faginea Lam., Fraxinus ornus L., Acer granatense Boiss.), holm oak forests (Quercus ilex subsp. ballota (Desf.) Samp.), Aleppo pine forests (Pinus halepensis Mill.) and scrublands (with Genista scorpius (L.) DC., Ulex parviflorus Pourr., Cistus albidus L., Cistus clusii Dunal, Rosmarinus officinalis L., Quercus coccifera L., 139 Daphne gnidium L). Ailanthus altissima was first introduced at the Natural Park as an ornamental garden plant and as a stabilising species used in motorway slope restoration (Constán-Nava et al. 2007). Later A. altissima began to invade different vegetation areas, infesting mainly pine forests and scrubland, but its presence is also important in areas of conservation interest, such as oak and river forests (Constán-Nava et al. 2007). EXPERIMENTAL DESIGN In summer of 2005, we randomly selected 12 populations of Ailanthus altissima (see a detailed description in Table 1), which were located along the roads crossing the natural park in the north-facing slope. The distance between nearest populations ranged from 0.2 km to 2.8 km. All populations came from resprouts because of the repeated application of annual stump cutting on them during more than 10 years. Biomass removal stopped two years before the study. Three treatments (plus a control, i.e., no herbicide or mechanical treatment) were applied in a completely randomised design (n = 3) across the 12 populations, selecting 3 populations for each treatment. The different treatments applied were: one cut stump, double cut stump, and one cut stump with herbicide (glyphosate) application (see below). Each treatment was repeated annually on the overall shoots composing each population, during the 2005-2008 period. In each of these populations, we randomly selected three 2 m × 2 m subplots to conduct the monitoring explained below. Selection of subplots in each treated (and control) populations was done avoiding border effect and other soil heterogeneity factors (e.g. stones) and to ensure that all resprouts in the treated area derived from roots that have no green shoot to supply carbohydrates immediately after cut. One cut stump treatment (hereafter 1CT) consisted of cutting all the resprouts in July. Double cut stump treatment (hereafter 2CT) included the one cut treatment plus a second cut stump on September, when A. altissima resprouts after the first cut stump. Cut stump with glyphosate application treatment (hereafter CHT) was based on applying the 1CT treatment combined with an immediate application of glyphosate on the cut section of the resprout. We used a paintbrush to prevent any impact on surrounding vegetation. Glyphosate was used because of its effectiveness on cut stumps and because its use is allowed in the Natural Park under their management plans. The glyphosate treatment was applied late in the growing season (July) when the root system is most affected by the herbicide (Hoshovsky 1988). 140 Table 1 Characteristics of A. altissima populations assigned to the different treatments of the experiment. Ai: invaded area; RCD: root collar diameter (mean ± ES) Treatment Population Ai (m2) Control 1CT 2CT CHT 1 2 3 1 2 3 1 2 3 1 2 3 286.1 147 927 45.8 59.3 92.4 55.9 910.6 858.8 62.5 910.6 2058.3 Total number RCD (mm) of shoots 951 11.7 ± 0.2 314 15.2 ± 0.5 1043 7.7 ± 0.2 86 13.7 ± 1.5 181 16.8 ± 1.0 214 9.1 ± 0.6 130 24.1 ± 1.7 275 9.8 ± 0.3 2412 6.3 ± 0.1 72 11.7 ± 0.9 275 11.5 ± 0.6 1066 9.5 ± 0.2 Vegetation Oak forest Pine forest Pine forest Oak forest Pine forest Pine forest Pine forest Pine forest Pine forest Pine forest Pine forest Pine forest Altitude (m a.s.l.) 1050 710 750 1040 900 710 950 800 910 920 800 740 PLANT PERFORMANCE SURVEY During the five years of the study, we measured height and root collar diameter (hereafter RCD) of all resprouts in the subplots. We used allometric relationships among biomass, height and RCD of resprouts growing under natural conditions (n = 100) to estimate biomass on our plots. The RCD was strongly related to dry weight (biomass = 0.0508 × RCD2.8427, R2 = 0.96, P < 0.001), so was used as a field–based surrogate of plant biomass. The density of resprouts growing on the 2 m × 2 m subplots was measured each year following application of the necessary treatments. As disturbance events affect the origin of the new sprouts (Del Tredici 2001), three resprout types were defined: stem (adventitious resprout on a cut section the previous year), collar resprout (growing in a lateral of a previous year cut section) and root resprout (growing in a distance ≥ 5 cm from a cut section the previous year) (Hoshovsky 1988; Bory et al. 1991), in order to test the effects of applied treatments on each resprout type. In the last year of the study (2009), we measured leaf area index (LAI) in each subplot in summer using a LAI 2000 plant canopy analyzer (Li-Cor, Inc., Lincoln, NE). Leaf water potential was also determined on freshly cut leaves of five resprouts of each treatment (plus control) at pre-dawn (Ψpd) and midday (Ψmd) of spring (May) and summer (July) of 2009 using a Schölander chamber (Soil Moisture Equipment Corp., Santa Barbara, California, USA). Stomatal conductance to water vapour (gs) was carried out on five different resprouts (five leaves per resprout) in the morning and midday of spring (May) and summer (July) with a portable infrared gas analyser (LI-6400, LI141 COR Inc., Lincoln, Nebraska, USA). gs measurements were made in full sunlight, recording data after a period of stabilization when the coefficient of variation was < 5%. STATISTICAL ANALYSES Effect of treatments on A. altissima biomass, height, RCD, density of different resprout types and total resprout density, Ψ and gs were analyzed by repeated measures analysis of variance (RM ANOVA), using as input the mean value from the three subplots of each population (n = 3). In all RM ANOVA models performed, Greenhouse-Geisser correction was used when data did not satisfy sphericity assumptions. In these models “treatment” was consider as the between-subjects factor and “time” (sampling date) as the within-subjects factor. In Ψ and gs models, “hour of the day” was considered as the within-subjects factor. Interactions between time and treatment were found in all variables (see below). As these interactions may lead to misinterpretations in the effect of applied treatments (Quinn and Keough 2002), separated analysis of variance (ANOVA) were performed for each sampling date in order to disentangle the effect of each treatment upon these variables at each sampling date. Tukey´s HSD post-hoc test was used to test for significant differences among treatments. Data were log10– transformed, when necessary, to satisfy ANOVA assumptions. Analyses of variance were performed to assess for differences on biomass, height, and total resprout density between populations prior to the application of the treatments (2005). In all cases, no differences were found between populations prior to the application of treatments (P > 0.05). Differences in LAI between treatments were tested with the non-parametric Kruskal-Wallis test. All statistical analyses were performed using SPSS v.15 (SPSS Inc., Chicago, IL, USA). RESULTS PLANT GROWTH SURVEY The combined herbicide plus mechanical treatment (CHT) reduced biomass, RCD and height by about 90% one year after application (2006) and over the following three years (Table 2). No other treatments differed from the control. The 2CT treatment reduced plant height by more than 50% from the second year (2007) onwards and the 1CT treatment reduced plant height by 60%, but only during the last two years. 142 The density of shoots of A. altissima did not differ significantly between treatments, despite the 1CT and 2CT tended to increase gradually over time compared with the control and CHT tended to reduce the number of shoots (Table 2). Table 2 Biomass, RCD, height and density of A. altissima (mean ± ES, n = 3) for each treatment and year of study (ANOVA and Tukey´s test, P < 0.05). Treatments are: Control; 1CT: one cut stump treatment; 2CT: double cut stump treatment; CHT: cut stump with glyphosate application treatment. Different letters indicate significant differences between treatments Treatment 2005 Biomass (gr m-2 ) Control 1318 ±603 a 1CT 1879 ±1205 a 2CT 2262 ±1825 a CHT 828 ±246 a RCD (mm) Control 11.7 ±2.2 a 1CT 14.8 ±4.0 a 2CT 14.5 ±3.6 a CHT 12.8 ±1.1 a Height (m) Control 1.2 ±0.3 a 1CT 1.3 ±0.3 a 2CT 1.4 ±0.4 a CHT 1.0 ±0.2 a Density (num m-2) Control 8 ±2 a 1CT 4 ±0 a 2CT 8 ±3 a CHT 5 ±1 a 2006 2007 2008 2009 2422 ±1042 a 283 ±85 ab 341 ±144 ab 36 ±25 b 3330 ±1511 a 802 ±463 ab 414 ±163 ab 115 ±42 b 3129 ±1366 a 235 ±81 ab 534 ±55 a 45 ±30 b 3560 ±1656 a 160 ±69 a 206 ±101 a 15 ±15 b 14.3 ±3.2 a 7.4 ±1.3 ab 7.7 ±0.5 ab 4.4 ±0.9 b 15.5 ±3.4 a 7.6 ±1.8 ab 7.1 ±0.2 ab 4.5 ±0.3 b 15.7 ±3.1 a 5.7 ±1.6 bc 6.9 ±0.7 ab 2.4 ±0.7 c 17.3 ±4.3 a 6.2 ±2.1 a 6.0 ±0.3 a 1.4 ±0.5 b 1.3 ±0.4 a 0.6 ±0.1 ab 0.6 ±0.2 ab 0.2 ±0.0 b 1.5 ±0.3 a 0.7 ±0.2 ab 0.6 ±0.0 b 0.3 ±0.0 b 1.4 ±0.3 a 0.5 ±0.1 b 0.5 ±0.0 b 0.1 ±0.0 b 1.6 ±0.5 a 0.5 ±0.1 b 0.4 ±0.0 b 0.07 ±0.0 b 8 ±2 a 8 ±1 a 10 ±3 a 3 ±1 a 6 ±1 a 11 ±2 a 13 ±5 a 6 ±3 a 7 ±1 a 14 ±4 a 16 ±6 a 5 ±2 a 7 ±1 a 12 ±6 a 11 ±4 a 2 ±1 a Temporal changes differed in relation to treatment on stem resprouts type density (Time × Treatment interaction: F9,24 = 2.82, P = 0.02; Table 3). 1CT and 2CT had significantly more stem resprouts than control and CHT which were reduced over time. There were more collar resprouts on the 1CT and 2CT treatments than the control (F3,8 = 6.41, P = 0.01, Table 3), but the combined (CHT) treatment did not differ from any of the treatments. The density of collar resprouts also changed significantly over time (F3,24 = 7.04, P < 0.001, Table 3), but not time × treatment interaction was found. There were no significant differences in density of root resprouts over time between treatments. The LAI of trees in the combined (CHT) treatment was significantly less than that of trees in the other treatments (Fig. 1). 143 Table 3 Resprout density of different types (mean ± ES. n = 3) by treatment from the period study (ANOVA and post hoc Tukey´s test, P < 0.05). Treatments are: Control; 1CT: one cut stump treatment; 2CT: double cut stump treatment; CHT: cut stump with glyphosate application treatment. Different letters indicate significant differences between treatments Resprout type density (num m-2) Stem Collar Root Treatment 2006 2007 2008 2009 Control 1CT 2CT CHT Control 1CT 2CT CHT Control 1CT 2CT CHT 0 ±0 a 3 ±1 b 2 ±1 b 0 ±0 a 0 ±0 a 4 ±1 b 5 ±1 b 2 ±1 ab 0 ±0 a 1 ±1 a 3 ±2 a 1 ±1 a 0 ±0 a 2 ±1 ab 3 ±1 b 0.4 ±0 ab 0 ±0 a 6 ±1 ab 6 ±3 b 2 ±1 ab 0 ±0 a 3 ±1 a 4 ±2 a 3 ±3 a 0 ±0 a 0 ±0 a 1 ±1 a 0 ±0 a 0 ±0 a 10 ±2 ab 11 ±5 b 5 ±2 ab 1 ±0 a 3 ±2 a 4 ±1 a 0 ±0 a 0 ±0 a 0.2 ±0 b 0 ±0 a 0 ±0 a 0 ±0 a 7 ±1 b 7 ±2 b 2 ±1 ab 0 ±0 a 1 ±1 a 4 ±2 a 0 ±0 a 6 a Leaf area index 5 4 a 3 2 a 1 b 0 Control 1CT 2CT CHT Treatment Figure 1 Leaf area index (LAI; mean ± SE. n = 3) of A. altissima. Treatments are: Control; 1CT: one cut stump treatment; 2CT: double cut stump treatment; CHT: cut stump with glyphosate application treatment. Different letters indicate significant differences (KruscalWallis test) ECOPHYSIOLOGICAL MEASUREMENTS Ailanthus altissima showed marked seasonal changes, both in the morning and at midday and relative to the treatments in Ψ (RM ANOVA: Time, F1,16 = 37.34, P < 0.001, Moment of the day, F1,16 = 345.47, P < 0.001, Treatment, F3,16 = 9.63, P = 0.001). Also, A. altissima showed changes on gs (RM ANOVA: Time, F1,14 = 167.64, P 144 < 0.001, Moment of the day, F3,42 = 94.97, P < 0.001, Treatment, F3,14 = 21.12, P < 0.001). In May, Ψpd of resprouts under the 1CT and CHT treatments were lower than the 2CT, but none was significantly different from the control (Table 4). gs in the morning was higher for CHT resprouts than the other treatments (F3,16 = 81.62, P < 0.001, Table 4). At midday, Ψ of the control resprouts was lower than the other treatments which had all declined substantially. All treated resprouts showed higher gs at midday than control. In July, Ψ pd declined in the 1CT resprouts, but the opposite response was found on the other treatments (Table 4). There were no differences in Ψmd between treatments. 1CT and 2CT showed lower gs in the morning than control and CHT (Table 4). All treatments substantially reduced gs at midday, excepting CHT resprouts. Table 4 Leaf water potential (Ψ) and leaf conductance to water vapour (gs) of each treatment measured in the morning and midday on May and July (mean ± ES). Different letters indicate significant differences between treatments (ANOVA and post hoc Tukey´s test. n = 5). Treatments are: Control; 1CT: one cut stump treatment; 2CT: double cut stump treatment; CHT: cut stump with glyphosate application treatment Ψ (-MPa) Control 1CT 2CT CHT gs (mmol m-2 s-1) Control 1CT 2CT CHT May Morning Midday July Morning Midday 0.5 ± 0.03 ab 0.5 ± 0.02 b 0.4 ± 0.04 a 0.6 ± 0.01 b 2.1 ± 0.2 a 1.4 ± 0.1 b 1.2 ± 0.2 b 1.6 ± 0.1 b 0.6 ± 0.04 a 1.2 ± 0.2 b 0.8 ± 0.1 a 0.6 ± 0.02 a 2.3 ± 0.1 a 2.1 ± 0.2 a 2.0 ± 0.2 a 1.8 ± 0.1 a 155.1 ± 18.3 a 72.3 ± 5.6 b 169.4 ± 10.3 a 417.1 ± 25.3 c 45.8 ± 7.9 a 222.8 ± 31.4 c 120.0 ± 6.6 b 194.7 ± 17.6 c 122.6 ± 16.02 a 55.5 ± 13.5 b 57.7 ± 5.8 b 152.9 ± 22.7 a 5.9 ± 1.0 a 33.8 ± 6.7 ab 4.7 ± 0.2 a 71.7 ± 20.3 b DISCUSSION Long–term control of A. altissima resprouting was efficient when using the combined herbicide and cutting treatment (CHT), mainly because of its reduction on aboveground growth. Treatments only based on cut stump did not reduce the invasive species presence significantly. Despite root removal is a commonly used technique to reduce growth of resprouting trees, we did not consider this technique in our study because of its high cost and predictable impact on soil surface, besides the use of heavy machinery is limited by the natural park management plans and therefore this methodology is not 145 applicable in the study area. Overall, our results indicate that CHT repeated annually reduced biomass of A. altissima performance at long-term (this treatment reduced the invasive tree biomass, leaf index area and also affected its ecophysiological traits). These effects could reduce its competitive ability with native species as suggested by the fact that natural recolonization by native species was observed after five years of treatment application. EFFECTS OF THE TREATMENTS ON ABOVEGROUND GROWTH In our study, neither single nor double cut stump treatments (1CT, 2CT) significantly reduced resprout density, as evident in a number of other studies developed at temperate areas (Bory et al. 1991; Burch and Zedaker 2003; Meloche and Murphy 2006). These treatments did not reduce biomass, though resprout height was reduced after three years of repeated annual cutting. A trade-off between stem height and stem number has been described in sprouting species following disturbance (Midgley 1996; Kruger et al. 1997; Vesk et al. 2004), consistent with the response we observed in A. altissima. Thus the probability of survival of this invasive species is greater given its tendency to recovering lost biomass through resprouting (Malanson and Trabaud 1988; Bellingham and Sparrow 2000; Bond and Midgley 2001). Rainfall could have been responsible for the species tendency to resprout in this study (Riba 1998), particularly the high spring rainfall during 2007-2008 in the study site. In our study, the combined herbicide and cutting treatment (CHT) failed to reduce resprout density, in contrast with previous studies (Meloche and Murphy 2006). However, although results were non-significant, this treatment finally removed resprouts from more than half of the subplots by repeated application of the CHT treatment. This reduced plant height and biomass, which suggests that more prolonged, persistent application of herbicide and stem cutting may result in total control, though further studies are needed to test this formally. The resprouting response of A. altissima could be influenced by topographic conditions, such as aspect, which could influence both water and nutrient availability (López-Soria and Castell 1992; Gracia 2000). The north-facing slope on which our subplots were located could explain resprouting in the CHT treatments in spite of marked reductions in biomass. Only the CHT treatment reduced LAI, through reduced biomass and resprout height. The reduction in A. altissima growth under this treatment could promote the persistence and colonization of native species over invasive species. 146 For example, we found natural recovery of native species such as Thymus vulgaris L. subsp. vulgaris, Brachipodium retusum (Pers.) P. Beauv., Cistus albidus L., or seedlings of Quercus ilex L., Viburnum tinus L.and Pinus halepensis Mill in plots undergoing this treatment but not the rest of assayed techniques(S. Constán-Nava, pers. observ.). This could indicate a reduction in competition for light and the production of allelopathic compounds through lower plant biomass and LAI. All three treatments altered plant resource allocation. The majority of new resprouts in the control treatment were root resprouts, which promote the colonization of new ground. This is a common behaviour in sprouting species, and could involve the development of adventitious roots that become autonomous from the main parent plant (Del Tredici 2001). However, all disturbance treatments favoured the growth of collar resprouts, which is a common response to frequent disturbance (Del Tredici 2001). The sprouting collar is an active specialized organ of regeneration and rejuvenation, resulting in the proliferation of a large numbers of sprouts (Bory et al. 1991; Del Tredici 2001). In contrast, the low number of stem resprouts growth could be due to an inefficient mechanism for resprouting species, comparing with collar type which comes from active organ. EFFECTS ON ECOPHYSIOLOGICAL TRAITS Rehydration and stomatal conductance of control plants were higher in spring than summer which indicates higher water availability in spring. Adaptations consisting on the progressive stomatal closure in response to increasing summer drought are known from seedlings of this species (Trifilò et al. 2004), and may be a mechanism for improving water use efficiency under Mediterranean climate. Our results corroborate that A. altissima present high plasticity to Mediterranean drought by means of a watersaving strategy (Levitt 1980). This strategy allows growing and developing the invasive species during drought periods (Levitt 1980). The high leaf water potential at pre-dawn found on A. altissima plants, mainly during summer drought, could indicate a better rehydration of the invasive species than native ones. For example, Fraxinus ornus, a native deciduous forests tree, showed more negative leaf water potential values (-2.56 ± 0.56 MPa) than A. altissima (-0.6 ± 0.04 MPa) under the same environmental conditions (S. Constán-Nava, unpublished data). This high rehydration of A. altissima could allow for better growth and improve its competitive abilities against native species under drought. 147 Substantial ecophysiological changes were detected on treated resprouts. All treated resprouts showed high rates of stomatal conductance during spring, especially at midday. High stomatal conductance could help the invasive species to recover and grow after disturbance events. This could explain the lack of biomass reduction under 1CT and 2CT treatments. The regulation of stomatal conductance is not only affected by changes in environmental factors, but also endogenous effects are important (Schulze 1986; Meinzer et al. 2001). High stomatal conductance were registered on CHT resprouts, maybe caused by internal effects derived from negative effects on leaf morphology produced by the herbicide (S. Constán-Nava, pers. observ.). The stomatal conductance of resprouts of all assayed treatments were higher than in control plants due to a better hydration (with Ψmd > -1.6 MPa). A plausible explanation for this result is the difference on size. That is, control plants showed hydration related with its tree structure, in contrast with the young resprout structure of treated plants. In summer midday, all treated resprouts tended to close the stomata, such as control plants, excepting CHT resprouts. The latter had high stomatal conductance despite being at summer drought. This could damage its water status and reduce its overall performance, which agree with the rest of our results on CHT resprouts. CONCLUSIONS Despite numerous methods to control the invasive tree Ailanthus altissima that have been previously tested (e.g. Meloche and Murphy 2006), none of them has been tested or monitored along long-term periods and under Mediterranean conditions. We tested three different treatments on resprout type, resprout density, and plant growth of A. altissima over a five year period. Our results indicated that joint cut stump and herbicide application is the only effective treatment in the long-term to reduce A. altissima in Mediterranean forests. Contrary to our expectations, the double cut stump treatment did not reduce the invasive species growth, and had similar results than the single stump treatment. Passive management on A. altissima resprouts should consider all negative ecological effects of the species on ecosystem diversity and function. Methods involving the physical removal of stumps (cutting) are ineffective, even where stumps were cut again the following years (our study shows results from five years of monitoring, but this particular treatment has been applied in the study area during 10 148 years without any success in A. altissima eradication). Although the combination of mechanical and chemical treatments did not reduce the number of resprouts over a fiveyear period, they did reduce resprout biomass, height and leaf area index. A combined herbicide cutting treatment should be included in plans of management for Mediterranean protected areas and the technique should be monitored over the longterm to assess for its real success and to ensure native species recolonization and ecosystem recovery. Since stump removal and glyphosate application reduces the competitive ability of the invasive species, it should be used together with species afforestation restoration programmes to improve the success of A. altissima control. ACKNOWLEDGEMENTS D. Eldridge, S. Soliveres and two anonymous reviewers provided helpful comments and improvements on earlier versions of this manuscript. Also thank Font Roja Natural Park personnel (in special, Abraham Santonja†) and all collaborators and volunteers for their help in the fieldwork. This research and SCN PhD fellowship were supported by the project (GV06/029) founded by Generalitat Valenciana, ESTRES Project (063/SGTB/2007/7.1) and RECUVES Project (077/RN08/04.1) founded by the Spanish Ministerio de Medio Ambiente, and BAHIRA CICYT project (CGL2008-03649/BTE) founded by the Spanish Ministerio de Ciencia y Tecnología. Font Roja Natura UA Scientific Station (ECFRN UA), depending on the Office of the Vice President for Research, Development and Innovation (VIDI) of the University of Alicante, supported also this research. 149 REFERENCES Bellingham PJ, Sparrow AD (2000) Resprouting as a life history strategy in woody plant communities. Oikos 89: 409–416 Bond WJ, Midgley JJ (2001) Ecology of sprouting in woody plants: the persistence niche. Ecol Evol 16 : 45-51 Bory G, Clair-Maczulajtys D (1980) Production, dissémination et polyphormisme des semences d’Ailanthus altissima (Mill.) Swingle, Simarubacées. 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Am J Bot 82: 1241-1248 Vilà M, Tessier M, Suehs, CM, Brundu G, Carta L et al (2006) Local and regional assessments of the impacts of plant invaders on vegetation structure and soil properties of Mediterranean islands. J. Biogeogr 33: 53–861 151 APÉNDICE FOTOGRÁFICO Foto 1 Parcela de 2 × 2 m para determinar la mejor estrategia de control sobre A. altissima Foto 2 Aplicación de herbicida sobre tocones recién cortados de A. altissima Foto 3 Población de A. altissima tras 4 años de tratamiento de dos desbroces anuales Foto 4 Parcela sin A. altissima tras 4 años de tratamiento de desbroce y herbicida 152 AGRADECIMIENTOS Quiero ser breve pues han sido unos cuantos añitos y no quiero enrollarme más, ni mirar hacia detrás para evitar emociones,… así que, mis agradecimientos van a A. Bonet (director de tesis), a la Estación Científica Font Roja Natura (Vicerrectorado de Investigación, Desarrollo e Innovación, Universidad de Alicante), a tod@s mis amig@s (cada uno de ellos saben quienes son ☺), a los colaboradores, y a toda la gente buena que he tenido el placer de conocer y de tratar estos años. A mi familia (mascotas incluidas), por TODO. A Santi, por estar siempre ahí y por hacer mejor cada segundo de mi VIDA. Así pues, INFINITAS GRACIAS a tod@s. Proyectos GV06/029. Generalitat Valenciana. España Recuves (077/RN08/04.1). Ministerio de Medio Ambiente. España Estrés (063/SGTB/2007/7.1). Ministerio de Medio Ambiente. España Entidades colaboradoras Conselleria d’Infraestructures, Territori i Medi Ambient CEMACAM Font Roja-Alcoy Gerencia de Medi Ambient. Alcoy 153 154 AFILIACIÓN DE LOS COAUTORES Andreu Bonet Jornet Departamento de Ecología, Universidad de Alicante, 03080, Alicante, Spain. Instituto Multidisciplinar para el Estudio del Medio Ramón Margalef. Universidad de Alicante, 03080 Alicante, Spain. E-mail: andreu@ua.es Santiago Soliveres Codina Área de Biodiversidad y Conservación, Departamento de Biología y Geología, Escuela Superior de Ciencias Experimentales y Tecnología, Universidad Rey Juan Carlos, 28933 Móstoles, Spain. E-mail: santiago.soliveres@urjc.es Lluís Serra Laliga Generalitat Valenciana, Conselleria d’Infraestructures, Territori i Medi Ambient, SS. TT. d’Alacant. C/Churruca nº 29, 03071 Alacant, Spain. E-mail: serralaliga@yahoo.es Rubén Torices Blanco Centro de Ecologia Funcional. Departamento de Ciências da Vida. Universidade de Coimbra. 3001–455. Coimbra, Portugal. E-mail: rubentorices@gmail.com 155 Estrella Pastor Llorca Estación Científica Font Roja Natura UA. Universidad de Alicante, s/n 03801 Alcoy, Spain. E-mail: estrella.pastor@ua.es María José Lledó Solbes Departamento de Ecología, Universidad de Alicante, 03080, Alicante, Spain. E-mail: mj.lledo@ua.es 156 Reunido el Tribunal que suscribe en el día de la fecha, acordó otorgar, por Tesis Doctoral de Don/Doña. Alicante, de la calificación de de . El Secretario, El Presidente 157 a la . 158 UNIVERSIDAD DE ALICANTE Comisión de Doctorado La presente Tesis de Don/Doña. ha sido registrada con el nº del registro de entrada correspondiente. Alicante, de de . El Encargado del Registro, 159