Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L.

Departamento de Botánica Facultade de Bioloxía Universidade de Santiago de Compostela Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Celestino Quintela Sabarís Tese de Doutoramento, Marzo 2011 ISBN 978-84-9887-752-6 (Edición Digital PDF) Dra. M. ISABEL FRAGA VILA, PROFESORA TITULAR DE UNIVERSIDADE DO DEPARTAMENTO DE BOTÁNICA DA FACULTADE DE BIOLOXÍA DA UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, INFORMA: Que a presente memoria titulada “Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L." presentada por D. Celestino Quintela Sabarís para optar ó Grao de Doutor en Bioloxía, foi realizada baixo a miña dirección no Departamento de Botánica da Universidade de Santiago de Compostela. E considerando que representa traballo de Tese de Doutoramento, autorizo a súa presentación ante o Tribunal correspondente. E para que así conste, asino a presente en Santiago de Compostela a 22 de Marzo de 2011. Vº e Prace da Directora, O Doutorando, Asdo.: Dra. M. Isabel Fraga Vila Asdo.: D. Celestino Quintela Sabarís Aos meus pais e avós, responsáveis polo autor A Berta e Martinho, que me lembram cada dia o que é importante na vida A Bibi. RESUMO EN GALEGO i Introdución e antecedentes i Obxectivos e tarefas desenvolvidas vi Síntese e conclusións viii Bibliografía x 1Introduction and objectives 1 1.1 The measurement and determination of tolerance . . . . . . . . . . . . . . . . . 6 1.2 The physiology of tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.3 Population genetics and evolution of tolerance . . . . . . . . . . . . . . . . . . . 10 1.4 Cistus ladanifer L., an interesting pseudometallophyte . . . . . . . . . . . . . 17 1.4.1 Taxonomy, description, breeding system and seed dispersal . . . 17 1.4.2 Competitive traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 1.4.3 Relation to soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 1.5 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 1.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2Chloroplast DNA phylogeography of the shrub Cistus ladanifer L. (Cistaceae) in the highly diverse Western Mediterranean region 31 Abstract 33 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.2.1 The Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.2.2 Plant sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.2.3 DNA extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Contents 2.2.4 Microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.2.5 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Acknowledgements 47 2.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer L have multiple origins 51 Abstract 53 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 3.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3.2.1 Plant and soil sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3.2.2 Soil chemical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3.2.3 DNA extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.2.4 Microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.2.5 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.3.1 Soil characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.3.2 Genetic diversity and structure . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Acknowledgements 69 3.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 4Heavy metal accumulation in leaves of divergent chloroplast lineages of the pseudometallophyte Cistus ladanifer L. Implications for phytostabilization 73 Abstract 75 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 4.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 4.2.1 Study species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 4.2.2 Plant and soil sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.2.3 Chemical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.2.4 Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 v relacións simbióticas (ectomicorrizas) con C. ladanifer (Comandini et al. 2006). Pero o máis importante entre todas estas características interesantes é o feito de que C. ladanifer é un pseudometalófito. As subespecies ladanifer e africanus teñen establecido con éxito poboacións en áreas serpentínicas ou en entulleiras de mina onde, nalgúns casos, son a especie dominante. A presenza de C. ladanifer sobre solos metalíferos, e a acumulación de metais en follas desta planta está reflectida nunha ampla produción científica. Nalgúns destes traballos (Alvarenga et al. 2004, Pratas et al. 2005, Murciego Murciego et al. 2006), a esteva foi descrita como especie indicadora ou mesmo acumuladora de As, Sb e Zn, e Mn, Sb e W, respectivamente. Estas referencias proporcionan información útil sobre a esteva e os metais, aínda que, ao ser estudos a nivel local ou rexional, mesmo empregando diferentes metodoloxías de mostraxe e cuantificación de metais, é difícil facer comparacións entre eles. No relativo á tolerancia de C. ladanifer aos metais, só temos constancia de tres traballos científicos previos, cada un deles usando diferentes enfoques. • Alados et al. (1999), usando unha análise de estabilidade no desenvolvemento, demostraron a adaptación de C. ladanifer aos solos serpentínicos de Málaga (S de España). Estes autores presentan a hipótese de que o baixo requerimento de Ca2+ por parte desta especie pode ser unha vantaxe na colonización de áreas serpentínicas. Nesta mesma liña, Ater et al. (2000) cuantificaron unha ratio Mg/Ca elevada en follas de C. ladanifer crecendo en zonas serpetínicas do norte de Marrocos. • Kidd et al. (2004) someteron a plántulas de 5 de poboacións de solos metalíferos e non metalíferos do NE de Portugal a experimentos de tolerancia ao Cd, Co, Cr, Mn, Cu, Ni, Pb e Zn en cultivo hidropónico. Observaron patróns de tolerancia e acumulación de metais específicos para cada poboación, e estimaron que mesmo poboacións non-metalícolas posuían unha certa tolerancia aos metais pesados. • Santos et al. (2009) procuraron actividade diferencial de enzimas antioxidantes en plantas de C. ladanifer dunha zona de minas abandonadas en SE Portugal, pero non atoparon ningunha variación relacionada especificamente cos metais. Nunha Tese de Doutoramento de elevado interese, Díez-Lázaro (2008) tratou a optimización do uso de Cistus ladanifer para procesos de fitorremediación. Encontrou que a adición de fertilizantes e a acidificación do solo melloraron o crecemento e a extracción de Mn e Zn por plantas desta especie procedentes do Nordeste de Portugal. Ademais, estableceu que a esteva podería ser utilizado para a fitoextracción de Zn en solos con contido baixo-medio deste metal. Finalmente, o efecto beneficioso desta planta é subliñado por Simões et al. (2009), quen estimaron que pode producir máis de 4.600 kg de follada·ha-1·ano-1, o que mellora a calidade do solo e pode promover a rexeneración da vexetación ao facilitar a colonización dunha área por esRESUMO vi Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. pecies con requerimentos máis esixentes. En resumo, C. ladanifer posúe unha serie de características (adaptación á seca, baixo requerimento de nutrintes, tolerancia a metais en certas poboacións) que a fan especialmente útil para a recuperación de áreas degradadas na rexión mediterránea. Ademais, é unha especie nativa desta rexión rica en biodiversidade e, polo tanto, o seu uso non implica efectos prexudiciais sobre os ecosistemas circundantes producido polo emprego de especies exóticas e invasoras (Méndez e Maier 2008, e referencias citadas nese traballo). Alén disto, C. ladanifer constitúe unha especie modelo interesante para o estudo do proceso de colonización de áreas metálicos por plantas. OBXECTIVOS E TAREFAS DESENVOLVIDAS: Dentro deste marco, e co obxectivo de mellorar o coñecemento sobre Cistus ladanifer e as súas relacións con metais, desenvolveuse unha campaña de recollida de mostras de planta (follas, sementes) e de mostras de solo en diferentes localidades do rango desta especie (Rexión Mediterránea Occidental, principalmente Península Ibérica e Norte de Marrocos). Buscouse cubrir diferentes tipos de material xeolóxico, dando especial atención ás áreas metalíferas (solos serpentínicos de Trás-Os-Montes, Málaga e Rif; áreas mineiras do SW da Península Ibérica), resultando nun total de 33 poboacións. O material recolectado empregouse nunha serie de investigacións, desenvolvidas a fin de tratar os seguintes temas: • Un primeiro paso para inferir os efectos dos metais sobre a xenética da especie é entender a interacción de procesos que determinan a súa filoxeografía ou “paisaxe xenética”. Dentro dos diferentes tipos de marcadores moleculares, aqueles baseados no ADN do cloroplasto (cpADN, ben sexan microsatélites do cloroplasto-cpSSRs ou PCR-RFLPs) teñen sido amplamente empregados en estudos de filoxeografía de plantas (Petit et al. 2003, Magri et al. 2007) ao posuír unha serie de características útiles: - Nas anxiospermas, o cpADN polo xeral hérdase por vía materna, polo que a súa dispersión realízase soamente através das sementes, resultando nunha mellor estruturación xeográfica das súas variedades. - Ao herdarse por vía materna, os patróns xeográficos do cpADN non se ven influídos polo fluxo de pole entre poboacións. - Ademais, o cpADN é haploide, polo que o seu tamaño efectivo de poboación é menor que o dos xenes nucleares (diploides). Destes xeito, a diferenciación por deriva xenética pode ser máis forte (Comes e Kadereit 1998) e fenómenos como colos de botella xenéticos poden ser detectados de xeito máis doado (Echt et al. 1998) co cpADN que cos xenes nucleares. Por exemplo, os marcadores cpSSR detectaron que as poboacións de Silene paradoxa medrando en entulleiras de minas de cobre sufriran unha redución na súa diversidade xenética (Mengoni et al. 2001), en canto un estudo vii previo con RAPD sobre esas mesmas poboacións fallara na detección desa baixa diversidade (Mengoni et al. 2000). - Finalmente, os microsatélites do cloroplasto son considerados marcadores neutros, é dicir, todos os seus alelos (variantes) teñen efectos iguais sobre o individuo que os transporta. Así, os cpSSRs proporcionar unha información independente da selección que nos permite separar os efectos da filoxeografía dos efectos da contaminación por metais. É por isto que desenvolvemos un estudo a grande escala da filoxeografía de C. ladanifer empregando cpSSRs (capítulo 2). • Posteriormente cuantificamos, empregando fluorescencia de raios X (XRF), ICP-masas e espectrometría de absorción atómica (AAS), os contidos totais e extraíbeis de metais nos solos de 33 poboacións procedentes de case todo o rango de C. ladanifer. En base a esta información, clasificamos esas poboacións como metalícolas (que medran en solos con contidos elevados de metais, abreviado M) e non-metalícolas (que medran en solos “normais”, abreviado NM). O tipo de poboación integrouse coa información filoxeográfica dos cpSSRs (ver capítulo 3) para respostar ás seguintes preguntas: As poboacións M teñen unha orixe mono ou polifilética? A colonización de áreas metalíferas provoca perda de diversidade xenética? • Un seguinte paso foi cuantificar a acumulación de metais pesados en follas de esteva recollidas no campo e procedentes das 33 poboacións consideradas no capítulo anterior; para o que empregamos XRF, ICP-OES e ICP-masas. Dado que a nivel xenético unha especie non é unha mestura homoxénea de alelos, senón que adoita estar dividida en subgrupos, no caso de que as poboacións M teñan unha orixe evolutiva múltiple, é posíbel que existan diferencias nas estratexias de resposta aos metais (Gonnelli et al. 2001, Nyberg Berglund et al. 2003). Consideramos este aspecto ao computar índices de bioacumulación de metais en cada poboación e avaliar o efecto do tipo de solo (metalífero ou non metalífero) e a información filoxeo gráfica sobre os patróns de acumulación (contidos totais e relativos de metais nas follas) (ver capítulo 4). • Alén da resposta en acumulación en campo, desenvolvemos experimentos de invernadoiro en condicións de cultivo hidropónico para avaliar a tolerancia de C. ladanifer aos metais Co, Ni e Zn (ver capítulo 5). Estimamos a tolerancia en base a medidas de crecemento (lonxitude da raíz mais longa, lonxitude do caule e incremento de número de follas), biomasa (peso seco) e eficiencia fotosintética (“yield”). Transformamos estas variábeis en medidas relativas computadas como unha porcentaxe dos valores obtidos para as plantas control, segundo a proposta orixinal de Wilkings (1978) para o crecemento da raíz. Así, eliminamos a maior parte da variación nas respostas non relacionadas cos tratamentos con metais. • Un último paso, interesante para o desenRESUMO viii Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. volvemento de futuras investigacións, é a identificación de marcadores potencialmente ligados á tolerancia a solos metalíferos. Isto é especialmente necesario en plantas non-modelo, como C. ladanifer. Abordamos este tema aproveitando as posibilidades de desenvolvemento de escáneres do xenoma (“genome scans”) dos AFLP (siglas do inglés: Amplified Fragment Length Polymorphism) (Vos et al. 1995). Estes marcardores moleculares fornecen información do xenoma nuclear e permiten obter centos de marcadores potencialmente non ligados dunha especie determinada sen ter un coñecemento previo das secuencias do seu ADN. Os AFLPs teñen sido empregados en organismos non-modelo (Bonin et al. 2007, e referencias dese traballo). En concreto, utilizáronse para detectar loci potencialmente implicados na adaptación a diversas condicións ambientais en plantas (Namroud et al. 2008, Parisod e Christin 2008, Poncet et al. 2010), e mesmo adaptación a metais pesados en poboacións do pseudometalófito Arabidopsis halleri (Meyer et al. 2009). Nesta tese de doutoramento, optimizamos un protocolo de análise de C. ladanifer con AFLP, obtendo un elevado número de marcadores. Posteriormente, comparamos estes marcadores coa información dispoñíbel sobre os solos (pH, ratios Ca:Mg e contidos de metais pesados), mediante a aplicación de Ecuacións de Estimación Xeralizadas (GEE, en inglés) (ver capítulo 6). As GEE, que son unha extensión dos modelos lineares xeralizados (Carl e Kuhn 2007), permiten analizar os modelos de distribución de alelos e estimar as variábeis ambientais que teñen unha maior influencia sobre eses modelos de distribución. Ademais, as GEE permiten introducir e corrixir a posíbel autocorrelación entre as mostras procedentes dunha mesma poboación ou dunha mesma liñaxe cloroplastidial. Finalmente, realizamos unha comparación das inferencias da estrutura xenética das poboacións de Cistus ladanifer obtidas con AFLPs (marcadores nucleares, diploides, de herdanza biparental e que poden sufrir recombinación) e cpSSRs (marcadores cloroplastidiais, haploides, de herdanza materna, que non sofren recombinación e entón hérdanse como bloques-haplotipos). SÍNTESE E CONCLUSIÓNS: A seguir presentamos unha síntese final e conclusións elaboradas en base aos resultados obtidos a partir dos experimentos expostos nos parágrafos anteriores: Grazas á análise a grande escala de Cistus ladanifer empregando microsatélites de cloroplasto (cpSSRs), inferimos que a diversidade xenética desta especie con grande rango de distribución está na realidade estructurada en dúas (ou tres, dependendo da metodoloxía estatística empregada) liñaxes ou clusters principais. Atendendo aos datos de pole presentes na bibliografía, estes conxuntos son o resultado da recolonización posglacial da Rexión Mediterránea Occidental a partir de refuxios illados e situados no norte de Marrocos e no sudoeste e sueste da Península Ibérica. Así mesmo, temos que ix concluír que as poboacións metalícolas desta especie son o resultado de múltiples e independentes procesos de colonización. Curiosamente, a colonización de áreas metalíferas non deixou pegadas xenéticas (na forma de colos de botella xenéticos ou efecto fundador) relacionadas co tipo de solo. Este feito constitúe un primeiro apoio á tolerancia a metais pesados como unha característica ‘constitutiva’ (presente en toda a especie) da esteva. Hai un gran número de estudos locais e rexionais sobre a acumulación de metais pesados por C. ladanifer. A aplicación dos coñecementos filoxeográficos permitiunos separar, nun estudo a nivel de toda a especie, os efectos da liñaxe cloroplastidial e do solo. Ao compararmos as poboacións metalícolas (M) e non-metalícolas (NM), non observamos diferenzas entre elas nos contidos foliares de diferentes metais, agás Ni. Unha explicación plausible é a aparición, nas poboacións M, de mecanismos de restricción da acumulación de metais. A pesar da afirmación anterior, atopamos diferentes patróns de acumulación de metais pesados entre as poboacións M de liñaxes cloroplastidiais diferentes. Este fenómeno, xa observado noutras especies de pseudometalófitos, reflicte e serve de apoio a unha historia de evolución independente das poboacións M, que evolucionaron en paralelo dentro de liñaxes que se mantiveron illadas desde o Último Máximo Glacial (que rematou aprox. 20.000 anos antes do presente). Con todo, e a pesar da esteva rexeitar claramente a acumulación dos metais Co, Cr e Pb, temos notado diferenzas significativas na resposta a outros metais entre as poboacións a nivel individual. Disto podemos derivar que calquera procedemento de fitoestabilización que implique o uso desta especie debe ser precedido por unha investigación que permita a caracterización dos seus ecotipos locais respecto dos metais pesados, a fin de evitar unha transferencia de metais (mediada pola esteva) do solo cara a rede trófica do ecosistema. Os experimentos de tolerancia a Co, Ni e Zn, en condicións de cultivo hidropónico, revelaron que cada metal afecta de xeito diferente á esteva. Ademais, os efectos de cada metal son congruentes cos patróns de acumulación/exclusión que observamos para cada metal a partir de mostras tomadas no campo. Así, en futuras análises de tolerancia a metais en plantas sería útil coñecer a estratexia de resposta aos metais pesados dunha determinada especie, a fin de determinar o mellor parámetro (estimador de tolerancia) a ser medido. Nas condicións do noso experimento non foron observadas diferenzas entre as poboacións M e NM para a meirande parte das variábeis resposta (crecemento, biomasa, fluorescencia clorofílica). Este feito pode ser interpretado como un segundo apoio para a tolerancia a metais como un carácter constitutivo de C. ladanifer. Unha outra vez, as diferentes liñaxes cloroplastidiais implican diferentes patróns ou mecanismos de resposta aos metais. Con todo, e dado o efecto dos tratamentos con metais sobre as variábeis resposta, suxerimos que pre-adaptacións á RESUMO x Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. escaseza de nutrientes ou ao estrés hídrico, en lugar dunha verdadeira tolerancia aos metais, poden ter facilitado a colonización de solos metalíferos por C. ladanifer. Os marcadores AFLP, aínda posuíndo diferentes propiedades que os cpSSRs (xenoma diploide de herdanza biparental fronte a xenoma haploide de herdanza materna) forneceron as mesmas inferencias sobre a filoxeografia da especie. Así mesmo, os AFLPs non demostraron unha influencia do tipo de solo sobre a diversidade xenética e a diferenciación entre poboacións desta especie. O procedemento de GEE resultou ser unha ferramenta estatística útil que nos permitiu analizar en conxunto os datos moleculares e a información sobre o solo. De acordo coas evidencias proporcionadas por diferentes autores sobre as esixencias nutricionais de C. ladanifer, a relación Ca: Mg (un dos factores de estrés máis importantes nas áreas serpentínicas, Brady et al. 2005) non tivo ningún efecto sobre a distribución de marcadores AFLP. Porén, verificamos que, entre todas as variábeis consideradas, o contido de Mn en solos ten o efecto máis forte na distribución de alelos. De feito, temos detectado unha banda cun posíbel papel na tolerancia ao alto contido de Mn no solo, aínda que precisamos futuras investigacións que permitan estimar o valor adaptativo da mesma. Como apartado adicional, suxírense algunhas posíbeis liñas de futuras investigacións, que se verían favorecidas polos coñecementos derivados desta tese de doutoramento: i) estudo da implicación de simbioses planta-microorganimos (fungos ectomicorrízicos, rizobacterias) na tolerancia; ii) estudos sobre a colonización de áreas metalíferas a nivel da xenética da paisaxe e iii) integración dos datos filoxeográficos en estudos de quimioecoloxía (variación nos exudados foliares da esteva). BIBLIOGRAFÍA: Alados CL, Navarro T, Cabezudo B (1999) Tolerance assessment of Cistus ladanifer to serpentine soils by developmental stability analysis. Plant Ecology 143:51-66 Alvarenga PM, Araújo MF, Silva JAL (2004) Elemental uptake and root-leaves transfer in Cistus ladanifer L. growing in a contaminated pyrite mining area (Aljustrel-Portugal). Water Air and Soil Pollution 152:81-96 Antonovics J, Bradshaw AD, Turner RG (1971) Heavy metal tolerance in plants. 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New Phytologist 161:199-209 Parisod C, Christin PA (2008) Genome-wide association to fine-scale ecological heterogeneity within a continuous population of Biscutella laevigata (Brassicaceae). New Phytologist 178:436–447 Pérez-Fernández MA, Rodríguez-Echeverría S (2003) Effect of smoke, charred wood, and nitrogenous compounds on seed germination of ten species from woodland in central-western Spain. Journal of Chemical Ecology 29:237–251 Pérez-García F (1997) Germination of Cistus ladanifer seeds in relation to parent material. Plant Ecology 133:57–62 Petit RJ, Aguinagalde I, Beaulieu JL, Bittkau C, Brewer S, Cheddadi R, Ennos R, Fineschi S, Grivet D, Lascoux M, Mohanty A, Müller-Starck G, Demesure-Musch B, Palmé A, Martín JP, Rendell S, Vendramin GG (2003) Glacial refugia: hotspots but not melting pots of genetic diversity. Science 300:1563-1565 Pollard AJ, Powell KD, Harper FA, Smith JAC (2002) The genetic basis of metal hyperaccumulation in plants. 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SpringerVerlag, Berlin, pp 313-344 Wilkings DA (1978) The measurement of tolerance to edaphic factors by means of root growth. New Phytologist 80:623-633 Wu L (1990) Colonization and establishment of plants in contaminated sites. In: Shaw AJ (ed) Heavy Metal Tolerance in Plants: Evolutionary Aspects. CRC Press, Boca Raton, pp 269-284 RESUMO 7 final yield on any species. Measuring this is difficult and laborious, so a growth parameter which is assumed to be correlated with fitness has to be used (Macnair 1993). The first and most commonly used parameter to characterise tolerance to heavy metals is the tolerance index (TI), which is calculated as: TI = Response to metal treatment / Response under control conditions Although the TI has received some criticism (Macnair 1990, Macnair 1993) it is still frequently employed, since it allows elimination of most of the variation in plant responses unrelated to metal treatment (Meyer et al. 2010). Usually, the response is estimated by measuring root growth in a hydroponic culture (Wilkins 1978), but other variables such as shoot growth, leaf length or biomass are also used (reviewed in Köhl and Lösch 2004). As an alternative to these growth measures, several biomarkers, that is, biochemical, physiological or morphological changes owing to metal exposure have been used (plasmolysis capacity, pollen viability, seed germination, photosynthesis, respiration) and others have been tested in recent years (phytochelatin synthesis, ATP concentration, stress proteins…) (Köhl and Lösch 2004). There are a wide variety of test designs for the determination of tolerance. Experiments can be developed sequentially (the same plant is first cultivated in control conditions and then subjected to a treatment with the metal) or in parallel (responses to control and to metal treatment are measured at the same time in different plants). It can be a single or multiple-concentration test, the latter using arithmetic or geometric series of metal concentrations (Schat and Ten Bookum 1992). Its duration may vary from around 2 days (short-term root elongation tests) to several weeks (long-term growth tests). In addition, the plants can be cultivated in hydroponic systems (with good reproducibility of nutrient and metal contents, accessibility to roots, easy harvesting of plants) or in solid media (whose cultivation conditions show a close resemblance to those in the field). Given this wide diversity of approaches, as Macnair (1993) stated, “the 'correct' test to use must be determined by the judgement of worker, based on experience of the species and metal under study”. 1.2 The physiology of tolerance In one of the basic papers in this field, Baker (1981) ranked tolerant plants in three categories (accumulator, indicator and excluder) according to their patterns of response to increasing quantities of heavy metals in soils (Fig. 1.2). Thus, an excluder shows low shoot levels of metals over a wide range of external concentrations up to a critical soil value above which the mechanism breaks down and unrestricted transport results. In contrast, accumulators concentrate metals in aerial plant parts from low or high soil levels, whereas ‘indicators’ show a proportional relationship between metal levels in INTRODUCTION 8 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. the soil and accumulation in aerial plant parts. An additional category comprises the hyperaccumulators: plants that behave similarly to accumulators, but with a higher accumulation factor. Several processes and mechanisms, both at the cellular and at whole organism level or even involving microorganisms from the rhizosphere, underlie these three general patterns. At the cellular level, Hall (2002) cites the main mechanisms of tolerance (summarized in Fig. 1.3). Rather than developing proteins that can resist heavy metal effects, these mechanisms are aimed at preventing the build-up of toxic concentrations at the cytosol: First, the role of rhizosphere organisms in tolerance has been demonstrated (reviewed in Kidd et al. 2009). Ectomycorrhizal fungi (which are exclusive to woody plants) develop a hyphae sheath that envelopes the root surface (and actually enters the intercellular spaces of the root cortex) and can reduce the inflow of metals to the plant host (point 1, Fig. 1.3). Moreover, the rhizosphere bacteria (mainly Plant Growth Promoting RhizobacteriaPGPR) also provoke an increasing tolerance, given their role in the change in availability of metals in the rhizosphere and, especially, by secreting Plant Growth Promoting substances (referred as PGPs in point 2, Fig. 1.3). Turning to the plant's own mechanisms, some of them act at the extracellular level. We refer to the binding of metals to the cell wall and, especially, immobilisation of metals outside the plant by binding to metal-chelating molecules exuded by roots (point 3, Fig. 1.3). E.g. Thlaspi arvense exposed to Ni increased the exudation of citrate and histidine by roots. These molecules form chelates with Ni and prevent its absorption by roots (Salt et al. 2000). Other strategies to control the internal concentration of metals rely on the plasma membrane, mainly through reduction of the influx across the membrane by the modification of metal transporters (point 4, Fig. 1.3) e.g. arsenate tolerance in Holcus lanatus is related to the presence of an altered phosphate-arsenate uptake system (Meharg and Macnair 1990). The active efflux of metals through the plasma membrane to the apoplast has been proposed as another as another process involved in tolerance (point 5, Fig. 1.3). This Figure 1.2: Classification of plant species on the basis of metal uptake. A: excluder. B: accumulator. C: hyperaccumulator. D: ‘indicator’. Modified from Baker (1981) and Greger (2004). 9 mechanism is common in bacteria but with little direct evidence in plants (Hall 2002). For instance, metal transporter AtHMA4 has been described in Arabidopsis thaliana. This transporter may play a role in the translocation of Zn from root to shoot and it may be also involved in tolerance to Zn and Cd (Mills et al. 2005). The remaining mechanisms are fully intracellular. They include, on one side. the chelation of metals by different organic ligands: aminoacids, organic acids and the cysteine-rich polypeptides Metallothioneins (MTs) and Phytochelatins (PCs) (point 6, Fig. 1.3). The formed chelates can then be removed from the cytosol by efflux outside the cell (e.g. complexation of Ni with histidine and transport to shoots) or pumped to the vacuole. This is the final step in the detoxification of Cd by PCs (point 8, Fig. 3). However, free metals can be also sequestered in the vacuoles by different metal transporters in the tonoplast (point 9, Fig. 1.3). For instance, Zn tolerance in Silene vulgaris is mediated by theses uptake systems (Chardonnens et al. 1999). The last mechanism of tolerance Figure 1.3: Summary of cellular mechanisms of metal tolerance in plants. M: metal atoms. PCs: phytochelatins. MTs: metallothioneins. HSPs: heat-shock proteins. PGPs: plant growth promoting substances. Bacteria are conventionally represented with a flagellum without any speculation on their biological status. Modified from Hall (2002). INTRODUCTION 10 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. to metals implies the repair and protection of plasma membranes under stressful conditions (point 7, Fig. 1.3). Heat Shock Proteins (HSPs) are the molecules mainly involved in this process, acting as chaperones in normal protein folding and assembly, as well as being involved in the protection and repair of proteins under metal stress. Some of the mechanisms cited above are specific to the response against a particular metal, e.g. Cd detoxification by PCs; although the response to other trace metals may involve several mechanisms within the same cell or cells belonging to different tissues or organs. In this way, the redistribution of metals within the plant may play an important role in tolerance, depending on the general pattern of response (sensu Baker 1981). Thus, in excluder species mechanisms to reduce the absorption of metals were predominant, and intracellular detoxification processes will occur mostly at the level of the roots. In contrast, in metal accumulators the main strategy is the transportation of metals to the aboveground plant parts and their accumulation in non-sensitive places. This mechanism is especially important in the extreme case of metal hyperaccumulating plants. For instance in Stackhousia tryonii (Ni hyperaccumulator) and Thlaspi praecox (Cd / Zn hyperaccumulator) metals are activelly transported to aerial parts and accumulated preferentially in the vascular and epidermal leaf tissue, away from the photosynthetically active leaf tissues (Bhatia et al. 2004, Vogel-Mikuš et al. 2008, respectively). 1.3 Population genetics and evolution of tolerance The study of population genetics has served to unveil the genetic bases of tolerance to heavy metals. In addition, metal-contaminated sites, either natural outcrops or mining deposits, may be considered as ecological islands which provide the opportunity to investigate the establishment and differentiation of plant populations under severe selection pressure (Lefèbvre and Vernet 1990). Based on their presence or absence in metalliferous areas, plant species have been classified into three categories (Fig. 1.4). These categories are often related to the occurrence of genotypes tolerant to metals at the species level (Pollard et al. 2002): - Strict (or obligate) non-metallophytes (species A in Fig. 1.4). Species restricted to soils with low metal concentrations, which is the case for great majority of plants. - Strict (or obligate) metallophytes, also called eumetallophytes (species D in Fig. 1.4). These are taxa which grow exclusively on metalliferous soils. All the members of such taxa which have a tolerance to heavy metals are included Some species in this group are endemic to mine spoils or to serpentine soils, such as Viola calaminaria and Alyssum serpyllifolium, respectively. - Facultative metallophytes, also called pseudometallophytes (species B and C in Fig. 1.4). Species with populations grow- 11 ing on metalliferous and non-metalliferous soils belong to this group. Within the pseudometallophytes we can distinguish two sub-types: on one side, those species with constitutive (or species-wide) tolerance, that is, all genotypes are metal tolerant, even in those populations growing on soils with low metal contents (species C on Fig. 1.4); and on the other side, those species with metal-tolerant races or ecotypes that have evolved during the colonisation of metalliferous areas by populations where tolerant genotypes were at low frequencies (species B on Fig. 1.4) (Pollard et al. 2002). To complete this relatively simple scheme, we add that several authors have stressed that plants which are, irrespective of their tolerance to metals, pre-adapted to any of the harsh edaphic conditions of metalliferous soils (nutrient shortage or imbalance, drought, high insolation, etc.) could colonize these soils more readily (Brady et al. 2005; and references therein), so, as Wu (1990) outlined, it is possible to colonize metalliferous soils without the evolution of tolerance. In addition, it is important to consider the ‘cost of tolerance’, that is, the fact that metallicolous plants are at a Figure 1.4: Patterns of plant metal tolerance (upper x-axis) and frequency distribution of plant genotypes on metal-containing soils exemplified for four contrasting species. A: strict non-metallophyte. D: obligate eumetallophyte. B: pseudometallophyte with ecotypic tolerance. C: pseudometallophyte with specieswide tolerance. Note that in some cases metal-tolerant genotypes are growing on non-metalliferous soils (shaded area). From Pollard et al. (2002). INTRODUCTION 12 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. disadvantage in non-metalliferous soils. This physiological cost may explain the general absence of metallophytes in nonmetalliferous substrates. Recent findings indicate that the cost of tolerance is not an effect of the tolerance genes, but is due to adaptation to the previously mentioned conditions or even to higher susceptibility to herbivores; it is therefore more precise to talk about the ‘cost of adaptation to metalliferous environment’ (Macnair et al. 2000, Dechamps et al. 2008 and references therein). Given the within-species differences in tolerance to metals found in pseudometallophytes, these kinds of plants allow us to investigate the genetics and physiology of the differences between the tolerant and non-tolerant plants in a way that is generally not possible with the edaphic endemics (Macnair 1993). In addition, neighbouring metallicolous and non-metallicolous populations of pseudometallophytes are highly relevant models for the study of local adaptation in plants (Linhart and Grant 1996). Other interesting properties of pseudometallophytes are their high capacity to adapt to a wide range of adverse soil conditions, together with their usually higher bio-mass production and their good competitiveness in soils with moderate metal toxicity; they may therefore be useful for phytoremediation technologies (Poschenrieder et al. 2001). On the basis of several experimental crosses between selected lines for tolerance and sensitivity to heavy metals within pseudometallophyte species, Macnair (1993) demonstrated that tolerance is a trait controlled by a small number (usually one) of major genes with main effects. This assertion is congruent with the results obtained by Wu et al. (1975), who indicated that evolution of tolerance can arise in a single generation. Moreover, minor genes, present in greater numbers and less effective than major genes, have been discovered. These minor genes would be responsible for the variation in tolerance levels observed within and among metal-tolerant populations (Macnair et al. 2000). In addition, minor genes are hypostatic to major genes; that is, their effect on tolerance can be only observed if major genes are present. Another interesting finding is that the co-tolerance (that is, the fact that tolerance to one metal confers a tolerance to other metals) has not been demonstrated; this fact contradicts the possible physiological role of nonspecific systems of metal tolerance, such as PCs, MTs and organic acids (Macnair et al. 2000). The ease of growing, selecting and crossing herbaceous species in the laboratory has caused a certain bias in genetic studies of tolerance to herbs (Macnair 1993). Complementary to these laboratory studies, other lines of research employed population biology and genetics as tools for identifying evolutionary and genetic factors involved in tolerance (Pauwels et al. 2008a). Indeed, these population-based approaches are useful to study the origin and evolution of metal tolerance in woody pseudometallophyte species, with longer life cycles. 13 From a evolutionary point of view, researchers tried to address three major questions: i) Does colonisation of metalliferous areas imply a reduction in genetic diversity?, ii) Do metallicolous (M) populations share a common ancestry or are they the result of local colonization events? and iii) Have soil conditions promoted a significant genetic isolation between population types? These three questions are ultimately related to the frequency of tolerance genes and also to the physiological cost of tolerance. If tolerance to heavy metals is not common in non-metallicolous (NM) populations of a certain pseudometallophyte species, it is expected that the metallicolous (M) population will be founded by a low number of mother plants. Thus, a founder effect (which implies a reduction of genetic diversity in M population and also a significante genetic differentiation between M and NM populations) may occur (Lefèbvre and Vernet 1990). The different selection forces in metalliferous and non-metalliferous soils contribute to maintain the genetic differentiation between M and NM populations (Linhart and Grant 1996), whereas the gene flow between neighbouring M and NM populations may contribute towards homogenizing them. The differentiation between edaphic types can be promoted by the onset of barriers for gene flow between M and NM populations preventing the dilution of metal tolerance by pollen coming from non-tolerant populations (Lefèbvre and Vernet 1990). Some of these processes include increasing self-fertility in M populations, divergence in flowering time between M and NM populations, pollenstigma incompatibiliy or hybrid sterility (Lefèbvre and Vernet 1990, Vekemans and Lefèbvre 1997). In order to summarize the research carried out on the influence of the colonisation of metalliferous areas on the genetic structure of pseudometallophyte species, we have elaborated a table (Table 1.1), in which we present a review of papers on this topic arranged according to their year of publication. This table shows a progressive change in the molecular markers used in genetic studies (from isozymes and allozymes to DNA based markers), and also an increase in the number of populations and the geographic range considered. These transitions are caused by the greater information provided by DNA compared to allozymes (markers very prone to homoplasy) and mainly for the need to separate the historical processes (phylogeography) from the selective processes really involved in the colonisation of M areas. As Staton et al. (2001) underline, the inference of the phylogeography of a species makes possible a better understanding of the effect of metal pollution on the genetic structure of populations, avoiding spurious correlations resulting from historical or demographic processes. Among the DNA-based markers, those obtained from the chloroplast genomes (either chloroplast microsatellites –cpSSRor chloroplast PCR-RFLP) are of special interest when studying colonisation patterns, and thus they have been widely used to infer the history of plant populaINTRODUCTION 14 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Table 1.1: Summary of the effects of the colonisation of metalliferous areas on the genetic diversity and/or the population genetic structure of different pseudometallophyte species. Reference Species Markers N of Pops Colonisation of M areas NM M (s) M (h) Reduction Diversity Genetic differentiation Origin Wu et al. (1975) Agrostis stolonifera Isozymes 2 9 No n.e. n.e. Ducousso et al. (1990) Arrhenatherum elatius Allozymes 3 3 No n.e. n.e. Westerbergh and Saura (1992) Silene dioica Isozymes 9 8 No No Multiple Bush and Barret (1993) Deschampsia cespitosa Isozymes 8 10 Yes Yes Multiple Vekemans and Lefèbvre (1997) Armeria maritima Allozymes 9 1 8 Yes No Multiple Lehmann (1997) Calamagrostis epigejos Isozymes 2 2 No n.e. n.e. Koch et al. (1998) Thlaspi caerulescens Isozymes 13 15 No No Multiple Nordal et al. (1999) Lychnis alpina Isozymes 1 2 Yes n.e. Multiple Mengoni et al. (2000) Silene paradoxa RAPD 1 52 No Yes (mine vs. serpentines) Multiple Mengoni et al. (2001)† S. paradoxa cpSSR 1 52Yes (mine pops) No Multiple Nkongolo et al. (2001) D. cespitosa RAPD 2 7 n.e. No Multiple n.e.: not estimated; † this paper analysed the same populations as Mengoni et al. (2000) 15 Table 1.1 (continued) Reference Species Markers N of Pops Colonisation of M areas NM M (s) M (h) Reduction Diversity Genetic differentiation Origin Nyberg Berglund and Westerbergh (2001) Cerastium alpinum Isozymes 19 12 No No Multiple Dubois et al. (2003) T. caerulescens Allozymes 7 7 No Yes (in one region) n.e. Pauwels et al. (2005) Arabidopsis halleri (cpDNA) PCR-RFLP 14 14 No No Multiple Mengoni et al. (2006) Onosma echioides AFLP 3 5 No No n.e. Baumbach and Hellwig (2007) A. maritima AFLP 12 10 No No Multiple Deng et al. (2007) Sedum alfredii RAPD 2 5Yes Yes n.e. Jiménez-Ambriz et al. (2007) T. caerulescens nuSSR 3 3 No No n.e. Pauwels et al. (2008)‡ A. halleri (cpDNA) PCR-RFLP 50 14 No No Multiple n.e.: not estimated; ‡ this paper used the same populations as Pauwels et al. (2005), as well as additional NM populations. INTRODUCTION 16 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. tions (Petit et al. 2003, Magri et al. 2007). CpDNA is generally maternally inherited in angiosperms, its dispersion is therefore carried out through seeds only. Thus, they are not influenced by pollen flow among M and NM populations. In addition, the effective population size for haploid cpDNA is smaller than diploid nuclear genes, so the differentiation due to genetic drift may be stronger (Comes and Kadereit 1998) and phenomena like genetic bottlenecks may be more easily detected (Echt et al. 1998). For instance, cpSSR markers detected a reduction in genetic diversity within copper-mine populations of Silene paradoxa where RAPD markers failed (Mengoni et al. 2001). Moreover, chloroplast microsatellites are considered to be neutral markers, that is, all their alleles (variants) have equal effects on the individual carrying them. Thus, cpSSRs provide a selectionindependent framework which allows us to separate the effects of phylogeography from the effects of metal pollution. As regards genetic diversity, a common trend across species is not found. In general, a founder effect has been detected in studies with populations on mining areas. This points to the effect of time together with the fact that most of the presented papers rely on markers related to nuclear DNA. Whereas the populations on mine tailings have supposedly been founded recently, the origin of populations on serpentine outcrops is generally older. A longer time implies a greater pollen flow from neighbouring non-metallicolous populations that could have increased genetic diversity and masked the putative founder effect. This is reflected by the aforementioned papers by Mengoni et al. (2000) and Mengoni et al. (2001) on Silene paradoxa. In addition, several authors proposed that the increase in genetic diversity inferred in M populations of clonal grasses (e.g. Arrhenatherum elatius or Calamagrostis epigejos) is a sum of soil heterogeinity together with low intraspecific competition in polluted deposits, which allows the coexistence of a higher number of clones than in non-metalliferous soils (Ducousso et al. 1990, Lehmann 1997). Only two pseudometallophytes have been studied with chloroplast markers: Silene paradoxa (Mengoni et al. 2001) and Arabidopsis halleri (Pauwels et al. 2005, Pauwels et al. 2008) with constrasting results. Pauwels et al. (2005) proposed that the colonisation of metal-polluted environments is associated with a genetic bottleneck in species with populational tolerance (as S. paradoxa), whereas in species with constitutive (or “specieswide”) tolerance (such as A. halleri) the effect of a bottleneck may not be detected. Genetic differentiation between population types has been detected in few works, and has mainly been related to geographic distances between M and NM populations than to a true effect of metals in soil (Dubois et al. 2003), or to a limited sampling that has made it impossible to distinguish between phylogeographic and selective effects (Bush and Barret 1993, Deng et al. 2007). As a rule, then, we may 23 the study of the process of colonisation of metalliferous areas by plants. Within this framework, and with the aim of improving the knowledge about Cistus ladanifer and its relationships with metals, we have developed a series of investigations using populations sampled from nearly the entire distribution area, in order to deal with the following topics: - In order to infer the effects of the metals on the genetics of the species, it is first of all essential to understand the interplay of processes that creates its phylogeography, or genetic landscape. Using neutral maternally-inherited markers (cpSSRs) we inferred the phylogeography of Cistus ladanifer (Chapter 2). - Then we integrated the soil type (metalliferous or non-metalliferous) and the phylogeographic information in a population-genetics approach to the tolerance of Cistus ladanifer to metals: are the metallicolous populations monoor polyphyletic? Is the colonisation of metalliferous areas accompanied by a reduction in genetic diversity? (Chapter 3). - A species range is not homogeneous, but it is often subdivided in into genetic subgroups. If metallicolous populations have evolved independently within different subgroups, it is interesting to infer whether the parallel evolution resulted in similar or different strategies of tolerance (exclusion, accumulation?). We assessed this theme, within the framework provided by cpSSR, through the analysis of field-collected soils and C. ladanifer leaves (Chapter 4) and through hydroponic-based experiments of tolerance to Co, Ni and Zn (Chapter 5). - As a basis for future research, the identification of markers potentially linked to tolerance to metalliferous soils is especially needed in non-model plants such as C. ladanifer. We address this topic applying generalized estimating equations (GEE) to AFLP markers and data of total metal contents in soils (Chapter 6). We also compared the information on population genetics provided by AFLP markers (from nuclear DNA) and cpSSRs. 1.6 References Alados CL, Navarro T, Cabezudo B (1999) Tolerance assessment of Cistus ladanifer to serpentine soils by developmental stability analysis. Plant Ecology 143:51-66 Alloway BJ (1995) Heavy Metals in Soils, 2nd ed. Blackie Academic and Professional, Glasgow. 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SpringerVerlag, Berlin, pp 313-344 Westerbergh A, Saura A (1992) The effect of serpentine on the population structure of Silene dioica (Caryophyllaceae). Evolution 46:1537-1548 Wilkings DA (1978) The measurement of tolerance to edaphic factors by means of root growth. New Phytologist 80:623-633 Wu L (1990) Colonization and establishment of plants in contaminated sites. In: Shaw AJ (ed) Heavy Metal Tolerance in Plants: Evolutionary Aspects. CRC Press, Boca Raton, pp 269-284 Wu L, Bradshaw AD, Thurman DA (1975) The potential for evolution of heavy metal tolerance in plants. III. The rapid evolution of copper tolerance in Agrostis stolonifera. Heredity 34:165-187 INTRODUCTION Chloroplast DNA phylogeography of the shrub Cistus ladanifer L. (Cistaceae) in the highly diverse Western Mediterranean region Chapter 2 This chapter reproduces the published paper: Quintela-Sabarís C, Vendramin GG, CastroFernández D, Fraga MI (2011) Chloroplast DNA phylogeography of the shrub Cistus ladanifer L. (Cistaceae) in the highly diverse Western Mediterranean region. Plant Biology 13:391400 Previous page: General view of the vegetation in the Despeñaperros Gorge area (Sierra Morena, Jaén province, S of Spain). Here, Cistus ladanifer subsp. ladanifer coexists with Quercus ilex and Juniperus oxycedrus plants. (Photo: C. Quintela-Sabarís) 39 dom permutations was performed with the matrix of pair-wise genetic differentiation between populations, (ΦST ⁄ (1 – ΦST)), and a matrix of the ln (geographic distance) with the Arlequin 3.11 software (Excoffier et al. 2005). Third, we used a simulated annealing procedure implemented in the spatial analysis of molecular variance (SAMOVA) algorithm (Dupanloup et al. 2002) to define groups of populations that are geographically homogeneous and maximally differentiated from each other. The program repeatedly seeks the composition of a user-defined number, K, of groups of geographically adjacent populations that maximises ΦCT, the proportion of total genetic variance due to differences among groups of populations. The program was run for 10,000 iterations for K values from K = 2 to K = 15 from each of 100 random initial conditions. Within each of the groups defined by SAMOVA, separate AMOVA analyses were performed to partition the genetic diversity at intraand inter-population levels. Fourth, we tested for the presence of genetic barriers among Figure 2.2: Cistus ladanifer cpSSR haplotype map. Grey-shaded area indicates the present natural distribution of C. ladanifer. The same patterns are used for haplotypes as in Fig. 2.1. In order to obtain a proper view of haplotype distribution, some radial graphs were displaced from their original position (arrows connect them with their original position). Population codes (see Table 2.1) are presented next to each radial graph. cpDNA PHYLOGEOGRAPHY 40 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Source of variation d.f. SS Variance components % of total variance P (a) Whole data set (ΦST = 0.66) Among populations 37 212.70 0.57 66.35 < 0.0001 Within populations 327 94.31 0.29 33.65 Total 364 307.01 0.86 (b) SAMOVA results (ΦCT = 0.67) Among SAMOVA groups 3 164.19 0.82 66.83 < 0.0001 Among pops. within SAMOVA groups 34 48.51 0.12 9.69 < 0.0001 Within populations 327 94.31 0.29 23.48 < 0.0001 Total 364 307.01 1.23 (c) Rif group (ΦST = 0.42) Among populations 5 7.28 0.12 41.59 < 0.0001 Within populations 56 9.77 0.17 58.41 Total 61 17.05 0.30 (d) Betic group (ΦST = 0.06) Among populations 5 0.52 0.00 6.18 N.S. Within populations 53 3.34 0.06 93.82 Total 58 3.86 0.07 (e) Western group (ΦST = 0.32) Among populations 24 40.71 0.15 32.18 < 0.0001 Within populations 209 65.20 0.31 67.82 Total 233 105.91 0.46 (f) Geographic AMOVA (ΦCT = 0.25) Iberian Peninsula vs. N Morocco 1 35.12 0.26 25.07 0.003 Among populations within groups 36 177.58 0.48 46.97 < 0.0001 Within populations 327 94.31 0.29 27.96 < 0.0001 Total 364 307.014 1.03 Table 2.2: Analysis of molecular variance (AMOVA) of Cistus ladanifer (a) considering the whole data set, (b) SAMOVA groups (K = 4), (c to e) separate analyses for three groups defined by SAMOVA analysis and (f) hierarchical geographic AMOVA comparing groups of populations to the north and south of the Strait of Gibraltar. d.f. = degrees of freedom, SS = sum of squared deviation, P = level of probability of obtaining a more extreme component estimate by chance alone. n.s. = not significant (α = 0.05). populations using the Monmonier algorithm implemented in the BARRIER 2.2 software (Manni et al. 2004). Virtual points were added to the original tessellation ⁄ triangulation in order to indicate the presence of the Mediterranean Sea barrier and to enable connections to be established among populations from the South of France and central Iberian Peninsula. The distances used were pair-wise ΦST (Michalakis and Excoffier 1996). Statistical confidence for the predicted barriers 41 was obtained by resampling individuals within populations in order to obtain 100 bootstrap replicates of each genetic distance matrix. A hierarchical AMOVA was then performed in order to assess the partitioning of variance between the Iberian Peninsula and North Morocco. In addition, the genetic differences among populations were visualised through a principal coordinate analysis (PCoA) performed using GenAlEx 6.3 (Peakall and Smouse 2006) and a Euclidean genetic haploid distance similar to the genetic binary distance described by Huff et al. (1993). 2.3 Results The two polymorphic microsatellites, ccmp2 (three size variants) and ccmp3 (six size variants), were combined into 11 different haplotypes among the 365 individuals analysed (Fig. 2.1). The number of haplotypes per population ranged from 1 to 4, while the mean effective number of haplotypes for the 38 populations was 1.58 (with a range of variation between 1.00 and 2.78). In addition, the average HE and D2 SH values were 0.32 (0.00–0.71) and 0.40 (0.00–5.33), respectively (Table 2.1). None of the three diversity indices used was correlated with latitude: we can find depauperated populations (with diversity values = 0.00) and also diverse populations throughout the range of C. ladanifer. Three haplotypes (H1, H6 and H11) were singletons, while H2 was unique to one population in southern Spain (EMA). More than 30% of the plants shared the same haplotype (H4). Two haplotypes (H4 and H8) were present on both sides of the Strait of Gibraltar, whereas haplotype H7 only appeared in populations from southeast Spain (Fig. 2.2). Median-joining analysis resulted in a complex haplotype network, with three loops (Fig. 2.1). Haplotype H8 occupies a central place within the three loops and connects the common haplotypes, H3 and H7. Although a phylogeographic structure was not detected by the permutation analysis (GST not significantly different from RST), a strong genetic structure was observed in C. ladanifer populations, with high values of both GST (0.60 ± 0.06) and RST (0.57 ± 0.12). The AMOVA analysis also revealed a high and significant value for inter-population differentiation (ΦST = 0.66) (Table 2). The isolation by distance test showed that among-population differentiation increased significantly with the ln (geographical distance) (Mantel test; P < 0.05), although the regression accounted for only a very low proportion of the total variance (r2 = 0.013). The SAMOVA analyses indicated distinct groups of genetically defined geographic areas; when K = 2, a group that comprised populations from the Betic area (EAC, EGR, ESA, ESB, ESP, ETO) was separated from the rest of the populations (ΦCT = 0.61). In analyses where K = 3, an additional partition was identified that subdivided the second group into two areas: one comprising populations from the South of France, the whole of the Iberian Peninsula (except the Betic area) and a population from North Morocco; the other comprising populations of C. ladanifer subsp. africanus from the Rif area (ΦCT = 0.64). When K = 4, a cpDNA PHYLOGEOGRAPHY 42 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. new group comprising a single population from the southwest Iberian Peninsula (EMA) was identified (ΦCT = 0.67). With value of K between 5 and 15, ΦCT values did not increase significantly, and in most cases the newly defined groups comprised single populations. Thus, SAMOVA analysis suggested the presence of three main geographic groups: one from North Morocco (hereafter Rif group), a group from the southeast Iberian Peninsula (hereafter Betic group) and a widespread group that occupies an area throughout the range of C. ladanifer (hereafter Western group); a fourth group is also present comprising a single population on the southwest coast of Spain (Huelva). The degree of population differentiation differed within each group (Table 2.2). Despite the short geographic distances among populations, the Rif group had the highest value of inter-population differentiation (ΦST = 0.42), whereas the widespread Western group had a lower differentiation value (ΦST = 0.32). No significant differentiation among populations within the Betic group was observed (Table 2.2). The analysis using pair-wise Figure 2.3: BARRIER results. Ten barriers were computed with ΦST pairwise distance matrices. The statistical support for each barrier (computed from the basis of 100 random matrices) is showed as line thickness. Only those barriers with a frequency higher than 50% were represented. The value of each barrier (ΦST) is indicated with a colour code (green, yellow, red). Populations are indicated by grey dots. The population code is indicated next to each dot (see Table 2.1). In some cases the population code is connected with an arrow to its dot. 43 ΦST distances in BARRIER software partially corroborated the SAMOVA analysis (Fig. 2.3). Although several barriers were inferred throughout the distribution area of C. ladanifer, the strongest genetic boundaries (with both high ΦST as well as high bootstrap support) were found in North Morocco (with barriers isolating single populations) and especially around the Betic area of the Iberian Peninsula. We stress that the BARRIER analyses separating the Iberian Peninsula and North Morocco were not inferred, although AMOVA analysis revealed that 25% of molecular variance was explained by genetic differences between populations north and south of the Strait of Gibraltar (Table 2). Principal coordinate analysis (PCoA) extracted two axes that explained 100% of genetic variation (see Supplementary Material S 2.1). Plotting the populations along these axes revealed a grouping that matches the SAMOVA results: three groups (Betic, Rif and Western) were obtained, whereas the EMA population was plotted next to the populations from the Western cluster. This grouping has a certain taxonomic relevance. Population EBE, which belongs to C. ladanifer subsp. africanus is plotted in an intermediate position between Western and Rif group, whereas the two populations of subsp. sulcatus were placed together with populations of subsp. ladanifer. 2.4 Discussion Chloroplast microsatellites have been widely used in phylogeographic studies (Magri et al. 2007, Fady et al. 2008, Pardo et al. 2008). Although some criticisms of cpSSRs arose due to problems with homoplasy, the level of homoplasy has been considered to be low enough to permit population genetic analysis (Provan et al. 1999a). Even when homoplasy has been identified, it was considered ‘moderate’ and its potential for invalidating results can be disregarded (Cuenca et al. 2003). Estoup et al. (2002), using simulations, concluded that the large amount of variability at microsatellite loci often largely compensates for homoplasious evolution, and cases in which size homoplasy may be a problem are related to high mutation rates. No estimate of mutation rates in Cistus is available but the lower mutation rate of chloroplast microsatellites compared to nuclear microsatellites (Provan et al. 1999b) suggests that size homoplasy may not be a major problem for studies at within-species level. In this work C. ladanifer populations exhibited levels of genetic differentiation (GST = 0.60) that are similar to the mean and median values for maternally inherited markers in angiosperms (Petit et al. 2005a). Most of the molecular variance occurs between each of the four population groups, which exhibit high frequencies of haplotypes H2 (EMA), H7 (Betic group), H8 (Rif group) and H4 and H5 (Western group). From these haplotypes, it is possible that H8 is the more ancestral, since it has the largest number of connections with other haplotypes and is found in a more central position in the network (Posada and Crandall 2001). Furthermore, H8 gave rise to H7 and H4 haplotypes, cpDNA PHYLOGEOGRAPHY 44 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. which are the most frequent in the Betic and Western group of populations, respectively. These facts could point to the origin of the Betic and Western groups of C. ladanifer as a result of independent colonisation events from North Africa to the Iberian Peninsula, as proposed by Guzmán and Vargas (2009) based on chloroplast and nuclear sequence analysis. The chronology of these colonisations is not clear, although the presence of C. ladanifer pollen in sites from the Betic area before the Last Glacial Maximum (around 90,000 years BP, Pons and Reille 1988, Fig. 2.4) and in southern Portugal during the Late Glacial (around 12000 years BP, Fletcher et al. 2007, Fig. 2.4) might indicate that it could have occurred during the Middle Pleistocene, a few million years after the opening of the Strait of Gibraltar at the end of the Messinian. Since opening (ca. 5.33 Ma, Hsü et al. 1977), the sea barrier of the Strait (14-km wide and about 400-m deep) has been maintained even during glacial maxima, although the lower sea level allowed emergence of different islands that reduced the width of the sea channels (Collina-Girard 2001): C. ladanifer should have been able to cross the Strait of Gibraltar using the emerging islands as stepping stones. The isolation effect caused by the Strait has left some traces on the genetic structure of C. ladanifer populations. First, three of the four groups of populations inferred by SAMOVA are present only on one side of Figure 2.4: Sites and chronology of different deposits where samples of C. ladanifer pollen were recovered. Numbers next to each point indicate the chronology (years before present) of the first occurrence of C. ladanifer pollen in each site. The bibliographic sources of pollen data are reported in Supplementary material S 2.2. 45 the Strait. Second, only two haplotypes (H3 and H8) are present both in Morocco and in the Iberian Peninsula. Finally, differentiation across the Strait accounts for 25% of the total molecular variance. However, the filtering effect of the Strait must have been lower than expected for a species like C. ladanifer, whose seeds tend to fall in a radius of 40 cm from the mother plant canopy (Bastida and Talavera 2002), given that the BARRIER analysis suggests that the Rif and Betic mountain ranges have acted as more effective barriers for seed flow than the Strait of Gibraltar. The patterns of differentiation across the Strait of Gibraltar are diverse among different plant species. In a recent review, Rodríguez-Sánchez et al. (2008) found that establishment, rather than dispersal, may act as a key factor in genetic differentiation across the Strait. Thus, in spite of poor dispersal abilities, the high establishment potential of C. ladanifer, a species that can produce thousands of seeds per year that maintain viability for several years (3-year-old seeds have germination as high as 80%; personal observation, C. QuintelaSabarís) could explain the relatively low effect of the Strait of Gibraltar on the genetic structure of its populations. In addition to the effect of the Strait, the high physiographic diversity of the northern part of Morocco (Rif Mountain ranges) and of the southern part of the Iberian Peninsula (river valleys and Betic ranges) created the geographic context for isolation during glacial periods, with the maintenance of pockets of Mediterranean taxa on south-facing slopes and in river gorges (Thompson 2005). Thus, in North Morocco (Rif region) and the south of Spain, four different clusters of populations are present. These clusters reflect the processes of post-glacial recolonisation of the Western Mediterranean area by C. ladanifer from its putative refugia. In the Rif region, we inferred a high degree of population differentiation and several barriers that delineate single populations (Fig. 2.3). This is congruent to an ancient presence of the species in this region and the effect of the Rif Mountains as barriers to seed flow. Regarding the Iberian Peninsula, our data suggest the occurrence of several independent glacial refugia instead of a single refugium area: first, the occurrence of three different clusters of populations (according to SAMOVA and PCoA), one of which is made up of population EMA with a unique haplotype at high frequency (0.6) on the southwest coast of Spain (this population is highly differentiated from Betic and Western clusters); second, the important genetic boundaries revealed by BARRIER analysis around the Betic region and especially in the Algeciras area (southernmost tip of the Iberian Peninsula) may indicate an area of contact between lineages expanding from different glacial refugia. In contrast with the high differences among populations in the southern Iberian Peninsula, only the Western group of populations is found in the northern part of the C. ladanifer natural range, suggesting that refugia in the southwest Iberian Peninsula were probably the only contributors to northward colonisation of this shrub. Populations in this area were cpDNA PHYLOGEOGRAPHY 46 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. in a ‘leading edge’ position, since the lack of high mountain ranges and presence of siliceous soils in the southwest of the Iberian Peninsula favoured their expansion. The post-glacial expansion may have occurred rapidly, as illustrated by the early occurrence of C. ladanifer pollen in the centre of Portugal or in the Spanish Northern Meseta around 10,000–8000 BP (Van der Knaap and Van Leeuwen 1997, Franco-Mú gica et al. 2001, see Fig. 2.4). The presence of the related chlorotypes H4 and H5 in nearly all the populations of the Western cluster, together with the relatively low degree of population differentiation estimated among this group of populations, also support the rapid expansion model suggested by pollen data. A stronger population genetic structure would be expected in a species with seeds dispersed mainly by barochory (Duminil et al. 2007), so the possible effect of endozoochory by red deer (Cervus elaphus; Malo and Suárez 1998) and even human activities should be taken into account as possible homogenising factors. C. ladanifer plays an important ecological role as coloniser in disturbed areas, so its expansion could be favoured by humaninduced disturbances; thus the increase of C. ladanifer pollen recorded in several palaecological records is accompanied by other anthropogenic indicators, such as the increase of pollen from ruderal species (Van der Schriek et al. 2007) or even the decrease of Olea and evergreen oak pollen as a result of forest clearing by fire (Ló pez Sáez et al. 2007). Moreover, and related to changes in land use and microclimate variations, the presence of C. ladanifer pollen varied through different periods (e.g., reduction in C. ladanifer pollen in northeast Spain accompanied by an intense expansion of agriculture and forest cultivation from the 16th century AD; Ló pez-Sáez et al. 2009), a fact that could be linked to local extinctions ⁄ expansions of this plant that might have contributed to blurring of the original population genetic structure. Moreover, C. ladanifer has been commercially exploited for centuries because of its fragrant resin (labdanum). The presence of C. ladanifer pollen in mummified remains from the 4th century AD found in Lyon (Girard and Maley 1999) as well as in cesspits from the 14th and 15th centuries in Flanders (Deforce 2006), is explained by its use as a cosmetic as well as in medicine. The intense use of C. ladanifer may have favoured human-mediated long-distance dispersal of this species in areas outside its natural distribution range. This may have occurred with C. ladanifer populations in the northeast Iberian Peninsula and southern France, where the hypothesis of artificial introduction of this species was formulated (Demoly and Montserrat 1993). Indeed, the populations analysed in southern France are fixed for the same chlorotype of populations as in the western part of the Iberian Peninsula. In contrast to expansion of the Western cluster, populations in the Betic area were encompassed by mountain ranges that acted as important barriers to the north, and may have expanded to the west, until they contacted other clusters of popula- 47 tions in the Algeciras area. Some authors (e.g., Petit et al. 2005b) proposed that colonisation of new territories might result in accelerated rates of molecular evolution and haplotype diversification, whereas the stable populations would retain ancestral characters. This could be an explanation for the lack of genetic structure, with fixation of haplotype H7 (which is directly related to the most ancestral haplotype H8) in almost all populations of this cluster. The Betic area has been identified as one of 10 hotspots of plant diversity in the Mediterranean Basin (Médail and Quézel 1997). In addition, at least two putative glacial refugia for plants have been identified in the southeast Iberian Peninsula (Médail and Diadema 2009). 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Journal of Biogeography 34:1505–1517 López Sáez JA, López García P, López-Merino L, Cerrillo Cuenca E, González Cordero A, Prada Gallardo A (2007) Origen Prehistórico de la Dehesa en Extremadura: una Perspectiva Paleoambiental. Revista de Estudios Extremeños 63:493–510 López-Sáez JA, López-Merino L, Mateo MA, Serrano O, Pérez-Díaz S, Serrano L (2009) Palaeoecological potential of the marine organic deposits of Posidonia oceanica: a case study in the NE Iberian Peninsula. Palaeogeography, Palaeoclimatology, Palaeoecology 271:215–224 Magri D, Fineschi S, Bellarosa R, Buonamici A, 55 Cistus ladanifer L. (gum rockrose) is a woody, semi-deciduous shrub growing in a wide range of latitudes, altitudes and climatic conditions in the Western Mediterranean region (South of France, Iberian Peninsula and North of Algeria and Morocco) (Demoly and Montserrat 1993). Its populations constitute early successional stages adapted to disturbances in Mediterranean ecosystems, in particular fires (Bastida and Talavera 2002). It is a pseudometallophyte that has established populations over different types of bedrock material (granites, schists, slates, etc.) and has also colonized different ultramafic areas in N Morocco (Bni Bouchra) (Ater et al. 2000), S Spain (Málaga) (Alados et al. 1999), NE Portugal (Trás-Os-Montes) (Díez Lázaro et al. 2006, Freitas et al. 2004a) and diverse mine tailings in Central to South-Western Iberian Peninsula (Murciego et al. 2007, Freitas et al. 2004b). C. ladanifer is an entomophyllous, obligatory outcrossing species, bearing a gametophytic mechanism of incompatibility (Talavera et al. 1993). It is the major component of shrublands in oligotrophic acid soils in the western half of the Iberian Peninsula (Rivas-Martínez 1979). Three subspecies have been described based on leaf traits (Demoly and Montserrat 1993). Two subspecies, Cistus ladanifer subsp. ladanifer and subsp. africanus, are widespread and they have colonized M (ultramafic) areas, although only subsp. ladanifer is found also in mine tailings from the Iberian Peninsula. Finally C. ladanifer subsp. sulcatus (formerly C. palhinhae) is restricted to limestone derived soils on the coast of the southwestern tip of Portugal. In this work, we have analysed 33 Cistus ladanifer populations sampled throughout the species distribution range using chloroplast (cp) DNA markers (microsatellites, SSRs). These markers are of special interest when studying colonisation patterns. Chloroplast DNA is generally maternally inherited in angiosperms, whose dispersion is therefore mediated by seeds only. In addition, the effective population size for haploid cpDNA is smaller than diploid nuclear genes, so the differentiation due to genetic drift can be stronger (Comes and Kadereit 1998) and phenomena like genetic bottlenecks can be more easily detected (Echt et al. 1998). For instance, cpSSR markers detected a reduction in genetic diversity within M populations of Silene paradoxa where RAPD markers failed (Mengoni et al. 2001). These characteristics justify the wide use of cpDNA in order to infer the population history of plant populations (Petit et al. 2003, Magri et al. 2007). Once the phylogeography of the species has been inferred, a better understanding of the effect of metal pollution on the genetic structure of populations is possible, avoiding spurious correlations resulting from historical or demographic processes (Staton et al. 2001). This is especially interesting in C. ladanifer, since it colonizes M areas at different latitudes (from N Morocco to NE Portugal) in a region whose physiographic (spatial heterogeneity) and climatic diversity offers complex phylogeographic patterns (Gómez and ORIGIN OF METALLICOLOUS POPULATIONS 56 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Lundt 2007). In the present paper, the following main questions were addressed: Do M populations of C. ladanifer have the same origin? Did M populations of Cistus ladanifer suffer a reduction in diversity? Do differences exist in the demographic effects of the colonisation of M areas along a latitudinal gradient? 3.2 Material and Methods 3.2.1 Plant and soil sampling Thirty-three Cistus ladanifer populations covering almost the entire distribution natural range of this species were sampled. The subspecies growing in each site was identified on the basis of morphological traits. We included metallicolous (M) populations from different geographic areas: ultramafic outcrops of Bni Bouchra (N of Morocco), Málaga (SE of Spain) and Trásos-Montes (NE Portugal), and M populations growing on mine tailings from the centre of the Iberian Peninsula (Table 3.1). After the analyses of phyto-available trace metals, two populations (EAL and EDE), growing near highways, were included in the M group (see Fig. 3.1). In each population, a longitudinal transect was established. Ten plants separated by at least 5 m were selected along the transect and their ripe fruits were collected. Seeds were sown and seedlings grown in a laboratory. One seedling per mother plant was selected for the subsequent analyses. Young plants were frozen in liquid nitrogen and conserved at −20°C until DNA extraction. In addition, in each site one (or two) soil samples were collected from 5 to 15 cm in depth. Each soil sample was airdried and sieved through a 2 mm-mesh. 3.2.2 Soil chemical analyses Sieved soil subsamples were milled in an agatha mortar to achieve homogeneity. Total amounts of Cr, Cu, Mn, Ni, Pb and Zn in soils were quantified in solid subsamples with Energy-Dispersive X-Ray Fluorescence spectrometry (EDXRF). Other subsamples were digested with HNO3 for the quantification of Co contents with Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) element analysis. In order to determine the concentration of heavy metals potentially available for plants, 10 g of dried soil was mixed with an extraction solution (Ammonium Acetate 0.5 M + EDTA 0.02 M + Acetic Acid 0.5 M, buffered at pH 4.65) (Lakanen and Erviö 1971) in a ratio of Table 3.1: Within-population haplotypic diversity estimates. First column includes population name and code. Second column indicates Cistus ladanifer subspecies: afr: subsp. africanus; lad: subsp. ladanifer; sul: subsp. sulcatus. Geographic coordinates are given in decimal degrees. M: metallicollous population; (u): ultramafic area; (m): mine tailing; (h): highway affected area. NM: non-metallicollous population; N.S.: number of plants surveyed in each population; HE: Nei’s (1987) haplotypic diversity; r(7): haplotypic richness after rarefaction to the uniform sample size of 7 (El Mousadik and Petit, 1996);D2 SH: average genetic distances among individuals (Vendramin et al. 1998); Overall Mean: mean for the whole set of populations ± standard deviation values. 57 Population (Code) Subsp Long Lat Soil Type Substratum N.S. HEr(7) D2 SH Ketama (MKE) afr 4.64º W 34.95º N NM Schists 10 0.38 1.40 0.22 Bni Hadifa (MBH) afr 4.17º W 35.02º N NM Sandstones 10 0.2 0.70 0.1 Bab Tazaa (MBT) lad 5.24º W 35.08º N NM Micaschists 10 0.2 0.70 0.1 El Jebha (MEJ) afr 4.64º W 35.18º N NM Sandstone 10 0 0 0 East Bni Bouchra (MSII) afr 4.89º W 35.29º N M (u) Serpentinised peridotite 12 0.7 2.16 0.58 West Bni Bouchra (MSI) afr 4.90º W 35.30º N M (u) Serpentinised peridotite 10 0.53 1 0.27 Tanger (MTA) afr 5.93º W 35.78º N NM Sandstone 10 0 0 0 Almodóvar (EAL) lad 5.65º W 36.16º N M (h) Clays close to a road 10 0 0 0 Benalup (EBE) afr 5.73º W 36.32º N NM Sandstone 10 0.64 2.33 0.76 Sierra Bermeja (ESB) lad 5.18º W 36.48º N M (u) Serpentinised peridotite 10 0 0 0 Sierra Palmitera (ESP) lad 5.07º W 36.60º N M (u) Serpentinised peridotite 9 0.42 1.56 0.5 Sierra de Tolox (ETO) lad 4.93º W 36.68º N M (u) Serpentinised peridotite 10 0.2 0.70 0.1 Grazalema (EGR) lad 5.27º W 36.78º N NM Decarbonated limestone 10 0 0 0 Sierra de Aguas (EAC) lad 4.79º W 36.84º N M (u) Serpentinised peridotite 10 0 0 0 São Vicente (PSV) sul 8.98º W 37.03º N NM Limestone 10 0.2 0.70 0.1 Burgau (PBU) sul 8.78º W 37.07º N NM Limestone 9 0.39 0.97 0.19 Mazagón (EMA) lad 6.84º W 37.15º N NM Sand deposits 10 0.62 1.87 1.78 Corte Figueira (PCF) lad 8.03º W 37.39º N NM Schists 9 0.39 0.97 0.19 Aljustrel (PAL) lad 8.18º W 37.88º N M (m) Pyrite mine tailing 9 0.64 1.78 0.53 Cardeña (ECA) lad 4.36º W 38.28º N NM Granite 10 0.71 1.93 0.89 Despeñaperros (EDE) lad 3.51º W 38.39º N M (h) Quartzites, close to the highway. 10 0.64 1.70 0.46 El Guijo (EGJ) lad 4.77º W 38.52º N NM Slates 10 0.47 0.99 0.23 La Codosera (ECO) lad 7.08º W 39.19º N M (m) Sb mine tailing 10 0.56 1 0.28 Valdecaballeros (EVC) lad 5.34º W 39.33º N NM Sedimentary material (gravels, clays) 10 0.71 2.39 0.71 Vela (PVE) lad 7.29º W 40.43º N NM Granite 9 0 0 0 Ciudad Rodrigo (ECR) lad 6.49º W 40.63º N NM Quartzites 10 0.36 0.93 0.18 Guadarrama (EGU) lad 4.10º W 40.68º N NM Granite 10 0.2 0.70 0.1 Fuente Saúco (EFS) lad 5.51º W 41.19º N NM Sandstone and conglomerates 10 0.64 1.70 0.46 Macedo dos Cavaleiros (PMC) lad 6.82º W 41.52º N M (u) Serpentinised peridotite 10 0 0 0 Ricobayo (ERB) lad 5.81º W 41.70º N NM Quartzites and filites 10 0.53 1 0.27 Samil (PSA) lad 6.75º W 41.78º N M (u) Serpentinised peridotite 7 0 0 0 Bragança (PBR) lad 6.87º W 41.85º N M (u) Dunite 10 0.53 1 0.27 Monte Furado (EMF) lad 7.20º W 42.39º N NM Schists 10 0 0 0 Overall mean ± SD 0.31 ± 0.27 0.91 ± 0.77 0.27 ± 0.36 ORIGIN OF METALLICOLOUS POPULATIONS 58 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. soil:extraction solution of 1:5. The suspension was shaken for 30 min, after which it was allowed to stand for at least half an hour and was then filtered through paper (Albet DP 145). The filtrate was stored cold to be analysed with an atomic absorption spectrophotometer (AAS). The following available trace metals were determined with AAS: Co, Cr, Cu Mn, Ni, Pb and Zn. 3.2.3 DNA extraction DNA was extracted from 100 mg of frozen leaves using Dneasy® Plant Mini Kit (QIAGen), following the manufacturer’s indications. In several cases an additional wash with 500 μl of absolute ethanol was needed in order to remove secondary compounds from the DNA extracts. 3.2.4 Microsatellite analysis In an initial screening, universal cpSSR primers ccmp1 to ccmp10 (Weising and Gardner 1999) and Fagaceae cpSSR primers cmcs 1 to 14 (Sebastiani et al. 2004) were tested on a subset of 30 samples from 6 geographically distant populations. Only primers ccmp1, ccmp2, ccmp3, ccmp5, ccmp10 and cmcs1 yielded consistent amplifications, of which ccmp1, ccmp5, ccmp10 and cmcs1 were monomorphic. The two polymorphic cpSSRs were then used to amplify all samples of the 33 populations. Amplification reactions were performed in 12.5 μl total volume using 10 ng of template DNA, 1× reaction buffer (Promega, Madison, WI, USA) containing 1.5 mM of MgCl2, 0.2 μM of each primer, 0.2 mM of each dNTP, 1% of bovine serum albumin, and 0.5 U of GoTaq® DNA Polymerase (Promega). DNA was amplified with the following thermal profile: one denaturation cycle of 4 min at 95°C, followed by 25 cycles each consisting of 95°C for 30 s, 50°C for 30 s, 72°C for 30 s with a final extension step at 72°C for 8 min. PCR products were loaded on a capillary automatic sequencer MegaBACE 1000 (GE Healthcare Biosciences). MegaBACE ET400 (GE Healthcare Biosciences) was used as size standard. Fragment lengths were determined using the MegaBACE FRAGMENT PROFILER software version 1.2 (GE Healthcare Biosciences). 3.2.5 Data analysis A Principal Component Analysis (PCA) was performed in order to aggregate populations according to (1) total contents of metals Co, Cr, Cu, Mn, Ni, Pb and Zn in soils (referred here as CoT, CrT, CuT, MnT, NiT, PbT and ZnT) or (2) Ammonium Acetate/EDTA metal extractable contents (referred here as CoE, CrE, CuE, MnE, NiE, PbE and ZnE). Values below detection limits were recorded as 0.1 μg.g−1 for statistical analysis. With the PCA, we reduced the dimensionality of the data, retaining, in our case, two first Principal Components (PCs) that contribute most to the variance of soils. A Varimax rotation was applied to the PCAs in order to simplify the interpretation of the extracted Principal Components. Phylogenetic relationships among haplotypes were inferred with NETWORK 4.5 (Fluxus Technology Ltd. at www. fluxus-engineering.com) using the median 59 joining (MJ) method (Bandelt et al. 1999). We applied the three criteria (frequency, topology and geography) proposed by Pfenninger and Posada (2002) in order to remove loops or ambiguities in the haplotype network. Different parameters of genetic diversity within populations were estimated: (1) the haplotypic diversity (HE = [n/(n-1)][1-Σpi 2], (where n is the number of individuals analysed in a population and pi is the frequency of the i-th haplotype in a population; Nei 1987), (2) the haplotypic richness r(n), which is obtained after rarefaction to a uniform sample size of n (in our study, the value of n was fixed at 7, the lowest size of the analysed populations), as described in El Mousadik and Petit (1996), and (3) the D2 SH measure, as defined by Vendramin et al. (1998), which takes into account the difference in the number of repeats among the different cpDNA haplotypes considered. In addition, the distribution of the pairwise cpSSR repeat length differences among individual plants, totalled over all two cpSSR loci within an individual plant, was plotted to compare different patterns. Genetic differentiation among populations was estimated by the analysis of molecular variance (AMOVA, Excoffier et al. 1992) using Arlequin software (version 3.11; Excoffier et al. 2005). The significance of the values was computed by a permutation test from 10,000 permutated matrices. The AMOVA was based on distances between cpSSR haplotypes, calculated as the sum of the squared number of repeat differences between two haplotypes: dxy = Σ[axi – ayi]2 (where axi and ayi are the number of repeats for the ith locus in haplotypes x and y). This gives ΦST, an analogue of Slatkin’s RST (Slatkin 1995) for population differentiation (Michalakis and Excoffier 1996). The Cavalli-Sforza and Edwards distances based on haplotype frequencies were used to construct a Neighbour Joining (NJ, Saitou and Nei 1987) dendrogram with the Populations software (O. Langella, UMR de Génétique Végétale, Ferme du Moulon, Gif/Yvette, France). To test for node robustness, bootstrapping was performed on individuals using 1,000 resamplings. In order to infer population genetic structure, Bayesian analysis using a spatial clustering model implemented in BAPS software version 5.2 was performed (Corander et al. 2008). These authors have shown that the spatial model improves the statistical power to detect the underlying population structure when dealing with a low number of loci. The spatial clustering of groups model was run using each population, with known coordinates, as the unit to be clustered. We initially fixed k (the number of clusters) from 2 to 25. We then selected the value of k that had the minimum log marginal likelihood and re-ran the analysis 100 times to obtain the optimal partition of populations. A neighbour-joining tree was then constructed (Saitou and Nei 1987) with the Kullback–Leibler divergence matrix provided as output with BAPS. This matrix can be used as a measure of relative genetic distance between the BAPS-identiORIGIN OF METALLICOLOUS POPULATIONS 60 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. fied clusters (BAPS 5.2 manual distributed with the program). Taking into account the grouping performed by BAPS, we (1) compared the levels of intra-population diversity of M and NM populations using an analysis of variance (ANOVA) and (2) tested the differentiation of M and NM populations with additional separate AMOVA analyses (Excoffier et al. 1992). With this two stepapproach (BAPS and ANOVA/AMOVA) we tried to extract first the effect of phylogeography and then analyse the effect of colonisation of metallicolous areas within phylogeographic homogeneous groups of populations, thus avoiding confounding effects of phylogeography on the effect of metal pollution over population genetic structure. Isolation-by-distance patterns between populations were tested considering all populations and then considering M and NM populations separately. A Mantel test with 10,000 random permutations was performed with the matrix of pairwise genetic differentiation between populations, NM Pop M Pop Human-polluted M Pop Ultramafic Pop Type PC1 (54% of total variance) 4,02,00,0 PC2 (38%of total variance) 6,0 4,0 2,0 0,0 A PC1 (46% of total variance) 6,04,02,00,0 PC2(39%of total variance) 4,0 2,0 0,0 B EAL EAL EDE EDE PBR PBR 1,00,0 1,0 0,0 ZnT PbT NiT MnT CuT CrT CoT 1,0 0,0 1,0 0,0 ZnE PbE NiE MnE CuE CrE CoE MSII ESP ETO PMC PSA MSIEAC ESB PAL ECO MSII ESP ETO PMC PSA MSI EAC ESB PAL ECO PC 1 PC 2 PC 2 PC 1 Figure 3.1: Principal Component Analysis (PCA) results. Populations are ordinated according to the total (Fig. 1a) or Ammonium Acetate/ EDTA extractable (Fig. 1b) quantities of heavy metals in their soils. The percentage of variance explained by each axis is reported. Circles indicate non-metallicolous (NM) populations, whereas triangles and squares indicate metallicolous (M) populations from ultramafic areas and humanpolluted soils, respectively. The codes of M populations are also indicated. In the upper right-hand corner of each graph the loading of each metal on each of the PCs is reported. 61 using ΦST/(1 - ΦST), and a matrix of the logarithmically (ln) transformed geographic distance. AMOVA and Mantel tests were computed with the Arlequin software version 3.11 (Excoffier et al. 2005). 3.3 Results 3.3.1 Soil characteristics The results of the analyses of total and Ammonium Acetate/EDTA extractable metals in soils for each population are presented in the Supplementary Material S 3.1. On the basis of these data, we conducted two Principal Component Analyses (PCA). The PCA performed on total metal contents (Fig. 3.1a) extracted two principal components (PCs) that explained 92% of total variance. The first Principal Component (PC1; 54% of total variance) was mainly composed of the total contents of Co, Cr, Ni, and Mn, suggesting that this axis is related to the ultramafic nature of soils. The second PC (PC2; 38% of total variance) was related to the degree of pollution due to human activities, since the metals with higher loadings were Cu, Pb and Zn. The PCA based on extractable metals showed a similar pattern (Fig. 3.1b). Two PCs explaining 85% of total variance were extracted. In this case, the first PC (PC1; 46% of total variance) was related to human pollution (extractable Cu, Pb and Zn were the main contributors to this PC) whereas the second PC (PC2; 39% of total variance) reflected the ultramafic origin of soils, since the extractable contents of Co, Cr and Ni contributed significantly in explaining the observed variance. The manganese content showed similar loadings on both axes. According to the defined Principal Components, in both cases (total or extractable metal contents) NM populations were plotted in a dense swarm placed mainly in the negative values on both axes, whereas the M populations showed high scores along one of the PCs, depending on their nature (ultramafic area or human-polluted site) (Fig 3.1a and b). On the other hand, three populations (EAL, EDE and PBR) showed differences between the two PCAs. Population PBR, whose bedrock material are dunites (a type of peridotite) is clearly separated from the NM populations based on total metal contents. Thus, it is plotted along Figure 3.2: Median Joining (MJ) network of Cistus ladanifer cpSSR haplotypes. Haplotypes are identified with the same colours as in Fig.3.3. Circles: haplotypes present both in M and NM populations. Triangles: haplotypes exclusive to NM populations. Square: haplotype present only in M population. Symbols’ sizes are proportional to the absolute haplotype frequency in the whole sample. Arrows indicate the connections between haplotypes that can be removed following the criteria of Pfenninger and Posada (2002). ORIGIN OF METALLICOLOUS POPULATIONS 62 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. the axis of ultramafic populations in Fig 3.1a. In contrast, in Fig 3.1b (PCA based on extractable metal contents) this population is plotted with the NM populations. This population was treated as M because of the high total contents of metals Cr, Mn and Ni (944, 1,578 and 1,151 μg.g−1, respectively) in the analysed soil samples. The two other populations (EAL and EDE) were initially considered as NM (taking into account bedrock material and total metal contents, see Supplementary Material S 3.1 and Fig. 3.1a). Nevertheless, when performing the PCA on Ammonium Acetate/EDTA extractable metal contents, these two populations were separated from the group of NM populations (Fig 3.1b). These populations, affected by highway traffic, were consequently classified as M. EDE has an extractable Pb of more than 100 μg.g−1, whereas EAL possesses in its soil moderate extractable contents of all the analysed metals. 3.3.2 Genetic diversity and structure The two polymorphic microsatellites ccmp2 and ccmp3 yielded 3 and 5 size variants, respectively. According to the nature of these microsatellites (both are mononucleotide repeats; Weising and Gardner 1999) the sizes of alleles varied by one base-pair. The variants found in each microsatellite locus combined into 10 different haplotypes (see Supplementary Material S 3.2 for the definition of each haplotype). They are connected through a complex haplotype network, with 3 loops and no missing haplotypes (Fig. 3.2). Only two haplotypes (H3 and H7) are present both in the Iberian Peninsula and North of Morocco. Haplotypes H2 to H4 are distributed across the Iberian Peninsula, whereas haplotypes H8 to H10 are found in North of Morocco and H6 is restricted to south-eastern Spain (Betic Area) (Fig. 3.3). Three haplotypes (H1, H5 and H10), found on the tips of the network, were exclusive to one population type (M or NM) (Fig. 3.2). Two of these haplotypes (H5 and H10) were singletons, whereas H1 is exclusive to NM population EMA from south-western Spain. The geographic distribution and the frequency of haplotypes, together with a criterion of topology (Pfenninger and Posada 2002) were employed to remove one of the edges in each of the 3 loops inferred in the haplotype network and thus resolve the uncertainties in the network (Fig. 3.2). Bayesian analysis yielded an ideal grouping with 8 clusters (Fig. 3.4), of which clusters 1, 3, 4, 6, and 7 included both M and NM populations. The NJ tree grouped these 8 clusters again in two diverging lineages of populations. The first lineage (hereafter referred to as ‘South lineage’) comprises populations in which H6 to H10 haplotypes are dominant (clusters 7, 8, 6 and 2), whereas the other lineage (hereafter referred to as ‘North lineage’) includes those populations with a high frequency of H1 to H5 haplotypes (clusters 4, 1, 3 and 5). These lineages have a taxonomic support, since the ‘South lineage’ comprises populations of C. ladanifer subsp africanus, together with populations 63 of C. ladanifer subsp. ladanifer growing in the Betic area, whereas the ‘North lineage’ is composed of populations of C. ladanifer subsp. sulcatus and C. ladanifer subsp ladanifer from the Iberian Peninsula, plus one population of subsp. ladanifer from the N of Morocco. The AMOVA analysis performed on the whole sample of populations indicate that most of the molecular variation is found among populations (ΦST=0.69, Table 3.2a), that is a value similar to the mean value for angiosperms (Petit et al. 2005). Differentiation between edaphic types were not significant either when the whole set of populations (33 pops) was considered (Table 3.2b), nor when separate AMOVA analyses within each of the Figure 3.3: Geographic distribution of Cistus ladanifer cpSSR haplotypes. Colour patterns used are the same as in Fig 3.2. In order to obtain a proper view of haplotype distribution, radial graphs were displaced from their original position (arrows connect them with their original position). Next to each graph the population code is indicated (see Table 3.1). Metallicolous (M) populations are noted as white dots, non-metallicolous (NM) ones as black dots. ORIGIN OF METALLICOLOUS POPULATIONS 64 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. lineages (North and South, Table 3.2c and d) were performed. The NJ tree further confirmed that M and NM populations did not constitute distinct genetic groups (Fig. 3.5). M populations appear dispersed among NM populations, forming clusters that are partially congruent with BAPS results. In most cases, M populations are clustered with geographically close NM populations and were genetically distant to M populations from other geographic areas; on the other hand populations PMC, PSA and PVE (from the NE and C of Portugal) were clustered with population MBT from the N of Morocco. No isolation-by-distance pattern was detected by Mantel’s tests, irrespectively of whether we considered M populations (P = 0.12), NM populations (P = 0.23) or the whole set of populations (P = 0.23). The number of haplotypes per population ranged from 1 to 4. The different estimators of within population diversity (HE, r(7) and D2 SH) displayed overall mean values of 0.31±0.27, 0.91±0.77 and 0.27±0.36, respectively (Table 3.1). The Analysis of Variance (ANOVA) showed no significant differences of diversity among M and NM populations considering either the whole set of populations or the lineages defined by BAPS (North, South) separately (Table 3.3). In addition, there were no qualitative differences among mismatch distributions from M and NM populations (see Supplementary Material S 3.3). 3.4 Discussion Over the last 50 years pseudometallophytes have been studied as models of microevolution and of regional differentiation among plants. The main aim of these works has been, on the one hand, to test whether the colonisation of areas with heavy metals may result in genetically depauperated populations (as a result of 0 0.5 1 1.5 2 2.5 3 3.5 4 Cluster 7 (MBH, MKE, , MTA) MSI, MSII Cluster 8 (MEJ) Cluster 6 ( , EGR, ) EAC ESB, ESP, ETO Cluster 2 (EBE) Cluster 4 (ECA, , ECR, , EFS, EGJ, ERB, EVC, , PCF) ECO EDE PAL, PBR Cluster 1 ( , EGU, EMF, PBU) EAL Cluster 3 (MBT, , PSV, PVE) PMC, PSA Cluster 5 (EMA) Figure 3.4: BAPS-based clustering and relationships among clusters based on the Kullback–Leibler divergence matrix. Clusters of populations were constructed with a spatial clustering model (Corander et al. 2008). Codes in brackets indicate populations included within each cluster. Population codes are the same as in Table 3.1. M populations are indicated in bold type. Horizontal scale bar indicates Kullback-Leibler distances among clusters. 71 between alleles with special reference for microsatellite loci. 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The rapid evolution of copper tolerance in Agrostis stolonifera. Heredity 34:165–178 Heavy metal accumulation in leaves of divergent chloroplast lineages of the pseudometallophyte Cistus ladanifer L. Implications for phytostabilization Chapter 4 Previous page: General view of an old pyrite mine tailing in Aljustrel (Alentejo, S Portugal). In this tailing we quantified (total contents): 752 mg·kg-1 As; 374 mg·kg-1 Cu; 1098 mg·kg-1 Mn; 2347 mg·kg-1 Pb and 633 mg·kg-1 Zn. Cistus ladanifer subsp. ladanifer has naturally colonised this tailing and now it is the dominant species. (Photo: C. QuintelaSabarís) Heavy metal accumulation in leaves of divergent chloroplast lineages of the pseudometallophyte Cistus ladanifer L. Implications for phytostabilization Keywords: Accumulation, Cistus ladanifer, heavy metals, pseudometallophyte ABSTRACT Cistus ladanifer is a shrub which grows in different kinds of soils (including serpentine outcrops and human-polluted sites) in the Western Mediterranean area. Chloroplast DNA analyses have inferred that metallicolous populations of this plant have arisen through distinct foundation events within two different postglacial recolonisation lineages. We have quantified the levels of Co, Cr, Cu, Mn, Ni, Pb and Zn in soils and in leaves of C. ladanifer plants from 33 populations, covering almost its entire natural range. In addition, we have computed the ratio between metal contents in leaves and in soils, as a measure of accumulation abilities. Through a nested analysis of variance (nANOVA) we tried to evaluate whether the population type (metallicolous vs. non-metallicolous) or the chloroplast lineage (‘North’ vs. ‘South’) differ in their metal contents and accumulation patterns. Our results show that, on a broad scale, metallicolous populations have higher Ni contents than non-metallicolous ones (p<0.001) and lineage ‘North’ higher Mn contents than lineage ‘South’ (p<0.001). In addition, Cistus ladanifer clearly rejects the accumulation of Co, Cr and Pb in its leaves, whereas for the other metals there were different accumulation patterns between lineages and population types. The implications of our results in the use of Cistus ladanifer for phytostabilization procedures are discussed. 4.1 Introduction Areas with high contents of heavy-metals in soils are the result of natural processes (weathering of ultramafic rocks) or human activities (mining and industrial activities, atmospheric deposition, excessive use of agrochemicals or even traffic emissions). Several technologies have been employed in order to remediate human polluted soils, the most of them based on expensive mechanical soil treatments that sometimes include soil removal and replacement. In recent years, phytoremediation, that is, the use of different plant species for soil remediation, has been proposed as an environmentally friendly technology that in addition has a lower economic cost than traditional approaches (Padmavathiamma and Li 2007). Plant growth is inhibited in the most severely contaminated sites, so human and animal exposure to heavy metals can be increased by the migration of contaminated soil (erosion, dispersal by wind) or leaching into groundwater (Ruttens et al. 2006). In sites with high and multi-elemental contamination, phytostabilization (the use of native or introduced plants to transform soil metals to less toxic forms, but not remove the metal from the soil, Chaney CHAPTER 4 76 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Table 4.1: Description of Cistus ladanifer populations included in this work. Population (Code) Lineage Type Substratum Vegetation Long Lat Sierra de Aguas (EAC) South M (u) Serpentinised peridotite Open shrubland with dispersed Pinus pinea and P. halepensis trees 4.79º W 36.84º N Almodóvar (EAL) North M (h) Clays close to a road Grassland with dispersed C. ladanifer plants 5.65º W 36.16º N Benalup (EBE) South NM Sandstone Cleared Quercus suber forest with Phyllirea angustifolia, Calicotome villosa and C. ladanifer 5.73º W 36.32º N Cardeña (ECA) North NM Granite Open Quercus ilex forest with C. ladanifer and Pistacia lentiscus 4.36º W 38.28º N La Codosera (ECO) North M (m) Sb mine tailing Shrubland dominated by C. ladanifer with Dittrichia viscosa 7.08º W 39.19º N Ciudad Rodrigo (ECR) North NM Quartzites Dense matorral with isolated Quercus ilex trees 6.49º W 40.63º N Despeñaperros (EDE) North M (h) Quartzites, close to the highway. Dense Cistus ladanifer shrubland with Quercus ilex and Juniperus oxycedrus 3.51º W 38.39º N Fuente Saúco (EFS) North NM Sandstone and conglomerates Shrubland with Quercus ilex 5.51º W 41.19º N El Guijo (EGJ) North NM Slates Open dehesa with Quercus ilex and isolated C. ladanifer plants 4.77º W 38.52º N Grazalema (EGR) South NM Decarbonated limestone Shrubland with C. ladanifer and C. monspelliensis 5.27º W 36.78º N Guadarrama (EGU) North NM Granite Dense shrubland with isolated Quercus ilex 4.10º W 40.68º N Mazagón (EMA) South NM Sand deposits Eucalyptus globulus and Pinus pinea plantations 6.84º W 37.15º N Monte Furado (EMF) North NM Schists Dense shrubland with Quercus ilex 7.20º W 42.39º N Ricobayo (ERB) North NM Quartzites and filites Open shrubland with Quercus ilex 5.81º W 41.70º N Sierra Bermeja (ESB) South M (u) Serpentinised peridotite Dense shrubland with scattered Pinus pinaster 5.18º W 36.48º N Sierra Palmitera (ESP) South M (u) Serpentinised peridotite Open matorral with Quercus coccifera and Ulex sp. 5.07º W 36.60º N Sierra de Tolox (ETO) South M (u) Serpentinised peridotite Open matorral with scattered Pinus pinaster and Ulex sp. 4.93º W 36.68º N Valdecaballeros (EVC) North NM Sedimentary material (gravels, clays) Dense shrubland with Pinus pinaster 5.34º W 39.33º N Bni Hadifa (MBH) South NM Sandstones Open Pinus halepensis forest 4.17º W 35.02º N Bab Tazaa (MBT) North NM Micaschists Open shrubland with dispersed Cistus plants 5.24º W 35.08º N El Jebha (MEJ) South NM Sandstone Open Pinus halepensis forest 4.64º W 35.18º N Ketama (MKE) South NM Schists Dense shrubland with evergreen oaks 4.64º W 34.95º N Chloroplast lineage and population type following Quintela-Sabarís et al. (2010). Longitude and latitude are expressed in decimal degrees. 77 et al. 1997) is the most suitable method of remediation (Kidd et al. 2009). As the plants cover the soil surface, they prevent erosion, reduce water percolation and increase biodiversity (Brown et al. 2005). In addition, the biological activities and the production of organic matter by the vegetation may contribute to metal immobilization (Vangrosveld et al. 2009). Identification and characterization of plant species capable of growing and surviving in polluted areas could be very helpful in developing phytostabilization technologies. Ideal plant species should have a rapid growth rate and dense root and shoot systems. Moreover, this species should not accumulate metals into above-ground tissues to prevent wildlife exposure and surface contamination. In addition, the plants used in phytostabilization work in the Mediterranean region should be adapted to water stress in order to cope with a long dry summer season (Frérot et al. 2006). Given the fact that the introduction of alien (and potentially) invasive species could produce detrimental effects on the surrounding ecosystems (Méndez and Maier, 2008; and references therein), the use of native plants for local flora in phytostabilization procedures should be a priority. PATTERNS OF METAL ACCUMULATION IN LEAVES Table 4.1: (continued) Population (Code) Lineage Type Substratum Vegetation Long Lat East Bni Bouchra (MSII) South M (u) Serpentinised peridotite Open matorral with Halimium atriplicifolium, Pistacia lentiscus and Tetraclinis articulata 4.89º W 35.29º N West Bni Bouchra (MSI) South M (u) Serpentinised peridotite Open matorral with Pistacia lentiscus, Phillyrea latifolia and Tetraclinis articulata 4.90º W 35.30º N Tanger (MTA) South NM Sandstone Open Pinus pinaster forest 5.93º W 35.78º N Aljustrel (PAL) North M (m) Pyrite mine tailing Dense matorral dominated by C. ladanifer and Lavandula stoechas 8.18º W 37.88º N Bragança (PBR) North M (u) Dunite Dense shrubland with Pinus pinaster and Genista sp. 6.87º W 41.85º N Burgau (PBU) North NM Limestone Dense shrubland with Chamaerops humilis and Pistacia lentiscus 8.78º W 37.07º N Corte Figueira (PCF) North NM Schists Quercus suber ‘Montado’ with dense cover of Cistus ladanifer 8.03º W 37.39º N Macedo dos Cavaleiros (PMC) North M (u) Serpentinised peridotite Dense shrubland with Quercus ilex 6.82º W 41.52º N Samil (PSA) North M (u) Serpentinised peridotite Open matorral with Alyssum serpyllifolium and Quercus ilex 6.75º W 41.78º N São Vicente (PSV) North NM Limestone Open shrubland with Pistacia lentiscus and Juniperus phoenicea 8.98º W 37.03º N Vela (PVE) North NM Granite Pinus pinaster plantation 7.29º W 40.43º N Chloroplast lineage and population type following Quintela-Sabarís et al. (2010). Longitude and latitude are expressed in decimal degrees. 78 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. populations with independent origins might show different patterns of response to heavy metals, as shown in Silene paradoxa (Gonnelli et al. 2001), Cerastium alpinum (Nyberg Berglund et al. 2003) or Thlaspi caerulescens (Assunção et al. 2003). The aim of this study was to analyse the Co, Cr, Cu, Mn, Ni, Pb and Zn leaf contents of field-collected plants from numerous metallicolous and nonmetallicolous populations of C. ladanifer from two diverging chloroplast lineages in order to explore the extent and variability of metal accumulation in natural populations of the species and thus determine if any populations are more useful for phytostabilization procedures. To compare the metal accumulation abilities of plants originating from metalliferous and nonmetalliferous soils, ratios between metal content in leaves and in soils were calculated (Bert et al. 2002). More precisely, the following questions were addressed: (i) Do the metallicolous (M) and nonmetallicolous (NM) populations differ in metal accumulation? and (ii) Do the M and NM populations from different chloroplast lineages show differences in metal accumulation patterns? Cistus ladanifer L. (Cistaceae) is a woody, semideciduous shrub found growing in a wide range of latitudes, altitudes, climatic conditions and soil types in the Western Mediterranean region. Its populations constitute early successional stages adapted to disturbances operating in Mediterranean ecosystems, especially fire (Bastida and Talavera 2002). Moreover, this species retains potentially active leaves through the summer drought (Núñez-Olivera et al. 1996). It has been found that natural populations of this plant can produce up to 4,600 kg of dry matter ha-1.year-1 of litterfall that improves soil quality (Simões et al. 2009). In addition, it develops dense root and shoot systems that can limit the erosion of soil (Martín Bolaños and Guinea López 1949). This species is a pseudometallophyte. Thus, it is present in non-metalliferous soils and also in metalliferous areas (ultramafic outcrops and also mine tailings) from the North of Morocco to NW of the Iberian Peninsula (Alvarenga et al. 2004, Ater et al. 2000, Batista 2003, Díez-Lázaro et al. 2004, Freitas et al. 2004, Pratas et al. 2005). In these sites, C. ladanifer behaves as an indicator, or even an accumulator, of different heavy metals. In previous work using chloroplast DNA markers, (Quintela-Sabarís et al. 2010) we inferred that metallicolous populations of this plant have arisen through distinct foundation events within two different postglacial recolonisation lineages. Given the fact that variations in tolerance and accumulation capacity are genetically controlled, metallicolous Table 4.2: Heavy metal contents in leaves of Cistus ladanifer. The mean ± SE is presented for each population. Last three rows indicate the overall mean, maximum and minimum contents measured in the leaves. Values are expressed in µg.g-1 of dry weight. Population codes in bold type indicate those which we considered as metallicolous (M). Population codes are the same than Table 4.1. 79PATTERNS OF METAL ACCUMULATION IN LEAVES Pop Co Cr Cu Mn Ni Pb Zn EAC 1.6 ± 0.9 24.1 ± 5.6 10.7 ± 2.4 60.5 ± 7.7 36.9 ± 5.2 1.2 ± 0.8 75.2 ± 9.8 EAL 5.5 ± 4.5 28.8 ± 9.2 13.4 ± 2.9 734.1 ± 544.2 20.0 ± 5.4 0.4 ± 0.5 123.5 ± 27.7 EBE 5.8 ± 4.5 23.1 ± 3.4 11.0 ± 2.3 316.3 ± 107.6 11.8 ± 1.9 0.5 ± 0.4 89.8 ± 31.2 ECA 2.3 ± 0.9 25.5 ± 12.3 8.9 ± 2.1 539.5 ± 323.9 10.2 ± 2.9 0.9 ± 0.4 105.5 ± 107.9 ECO 2.8 ± 2.1 25.5 ± 12.2 12.4 ± 3.7 1688.5 ± 1324.6 19.3 ± 5.9 4.2 ± 2.3 88.2 ± 15.0 ECR 2.4 ± 2.0 23.0 ± 4.2 13.3 ± 3.6 1078.1 ± 799.6 17.1 ± 5.5 0.1 ± 0.1 86.1 ± 9.1 EDE 5.7 ± 2.7 24.9 ± 8.1 14.9 ± 3.4 865.1 ± 253.2 15.9 ± 2.9 0.7 ± 0.5 97.1 ± 18.0 EFS 3.7 ± 3.0 21.1 ± 5.2 15.5 ± 4.4 1135.4 ± 374.9 32.3 ± 2.8 0.2 ± 0.3 122.1 ± 36.5 EGJ 3.3 ± 1.6 21.7 ± 2.7 11.1 ± 1.7 914.6 ± 462.6 18.6 ± 4.6 1.2 ± 0.4 95.4 ± 9.7 EGR 0.8 ± 0.3 22.7 ± 3.3 14.9 ± 4.2 310.4 ± 100.5 17.8 ± 4.2 0.1 ± 0.1 100.3 ± 15.7 EGU 0.7 ± 0.4 20.8 ± 3.1 10.8 ± 2.4 523.8 ± 257.4 10.2 ± 2.8 0.5 ± 0.8 102.3 ± 20.2 EMA 2.6 ± 1.6 28.4 ± 9.2 37.4 ± 6.9 264.3 ± 88.5 14.8 ± 1.7 0.7 ± 0.6 128.7 ± 33.9 EMF 2.5 ± 0.6 25.8 ± 4.1 12.8 ± 4.4 344.5 ± 235.8 18.0 ± 4.8 0.3 ± 0.3 93.9 ± 26.4 ERB 4.7 ± 1.0 29.4 ± 8.5 8.3 ± 0.8 483.0 ± 149.5 18.0 ± 2.7 0.2 ± 0.4 143.4 ± 86.0 ESB 7.8 ± 8.9 29.3 ± 11.0 8.5 ± 1.7 275.6 ± 331.9 50.0 ± 16.6 0.3 ± 0.3 79.0 ± 30.7 ESP 1.5 ± 0.8 31.2 ± 7.1 8.3 ± 1.3 117.5 ± 39.9 58.8 ± 17.9 0.1 ± 0.2 76.6 ± 14.3 ETO 2.0 ± 0.7 27.3 ± 7.3 6.6 ± 0.9 98.7 ± 14.2 75.7 ± 7.5 0.3 ± 0.3 63.3 ± 6.6 EVC 2.3 ± 1.3 30.2 ± 6.5 8.6 ± 2.3 805.2 ± 343.9 14.4 ± 3.1 0.3 ± 0.3 108.6 ± 32.7 MBH 3.2 ± 1.7 23.8 ± 5.3 13.5 ± 3.8 336.9 ± 80.2 12.6 ± 2.0 0.3 ± 0.4 86.3 ± 19.1 MBT 3.8 ± 1.6 25.4 ± 4.0 10.5 ± 3.2 1179.0 ± 573.4 18.7 ± 4.4 0.4 ± 0.4 93.5 ± 28.1 MEJ 1.5 ± 1.1 30.8 ± 15.4 8.5 ± 0.5 264.0 ± 148.1 12.5 ± 4.6 0.1 ± 0.2 92.7 ± 13.8 MKE 4.7 ± 2.5 25.5 ± 6.4 12.6 ± 2.8 554.9 ± 120.1 15.7 ± 2.5 0.2 ± 0.2 85.6 ± 20.8 MSI 1.0 ± 0.7 26.2 ± 7.6 8.2 ± 1.1 51.6 ± 27.4 47.1 ± 6.8 0.5 ± 0.5 66.1 ± 9.1 MSII 1.2 ± 0.2 25.3 ± 3.8 7.3 ± 1.1 59.3 ± 25.3 53.7 ± 12.8 0.5 ± 0.4 73.8 ± 8.3 MTA 1.5 ± 0.7 25.2 ± 4.8 12.5 ± 4.1 78.8 ± 37.5 10.9 ± 1.9 0.2 ± 0.2 83.9 ± 27.7 PAL 6.7 ± 2.4 27.2 ± 8.0 26.6 ± 3.2 980.7 ± 385.0 25.7 ± 6.4 1.7 ± 0.9 288.0 ± 94.5 PBR 2.1 ± 1.5 25.7 ± 4.0 9.3 ± 2.7 232.6 ± 43.5 74.5 ± 13.8 0.3 ± 0.4 85.1 ± 16.2v PBU 0.6 ± 0.5 23.3 ± 4.9 9.8 ± 2.5 174.5 ± 169.7 8.3 ± 1.4 0.4 ± 0.4 56.8 ± 14.6 PCF 5.4 ± 2.5 22.4 ± 6.5 12.2 ± 2.9 1485.8 ± 414.8 18.7 ± 2.9 0.2 ± 0.2 57.5 ± 8.0 PMC 2.6 ± 2.0 24.7 ± 7.1 12.5 ± 1.4 308.1 ± 338.6 99.6 ± 41.6 0.2 ± 0.3 87.0 ± 9.5 PSA 1.5 ± 0.3 50.0 ± 9.1 8.1 ± 0.8 186.2 ± 62.2 93.1 ± 28.3 0.3 ± 0.3 77.5 ± 18.4 PSV 0.3 ± 0.2 24.5 ± 5.2 10.8 ± 2.2 52.4 ± 15.2 9.7 ± 2.4 0.3 ± 0.3 78.2 ± 34.1 PVE 1.9 ± 1.1 20.0 ± 4.7 9.6 ± 2.3 809.6 ± 518.3 10.2 ± 2.3 0.1 ± 0.1 71.9 ± 20.8 Overall Mean 2.9 26.2 12.1 517.1 29.4 0.5 95.7 Max. 25.1 61.12 46.13 3952.3 141.3 8.1 423.7 Min. 0.03 11.32 4.43 20.71 6.1 0 35.2 80 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. 4.2 Material and methods 4.2.1 Study species Cistus ladanifer is an entomophyllous, obligatory outcrossing species, bearing a gametophytic mechanism of incompatibility (Talavera et al. 1993). It is the major component of shrublands in oligotrophic acid soils in the western half of the Iberian Peninsula (Rivas-Martínez 1979). Three subspecies have been described based on leaf traits (Demoly and Montserrat 1993). Two subspecies, Cistus ladanifer subsp. ladanifer and subsp. africanus, are widespread and they have colonized metalliferous (ultramafic) areas, although only subsp. ladanifer is found also in mine tailings from the Iberian Peninsula. Finally C. ladanifer subsp. sulcatus (formerly C. palhinhae) is restricted to limestone-derived soils on the coast of the south-western tip of Portugal. 4.2.2 Plant and soil sampling Thirty-three Cistus ladanifer populations covering almost the entire natural range of this species (and its three subspecies) were sampled. In previous work (QuintelaSabarís et al. 2010), on the basis of soil metal contents (either Total or Ammonium Acetate/Acetic Acid/EDTA extractable contents) we have classified these populations as metallicolous (M) and non-metallicolous (NM). M populations included those growing on ultramafic outcrops in Bni Bouchra (N of Morocco), Málaga (SE of Spain) and Trás-os-Montes (NE Portugal), or on human-polluted soils, such as mine tailings, or areas affected by highway traffic. In addition, using chloroplast microsatellites (cpSSRs) we have inferred that M and NM populations belong to two independent lineages (‘North’ and ‘South’) that were isolated during last Glacial period (Quintela-Sabarís et al. 2010). In each population, a longitudinal transect was established, along which six plants, separated by at least 5m, were selected. We collected 5 branches with leaves from each of the selected plants. At the laboratory, leaves were separated from stems, and they were cleaned with fresh water, rinsed twice with distilled-deionised water and dried at 60ºC for two days. In addition, in each site soil samples were collected from 5-15 cm in depth. Each soil sample was air-dried and sieved through a 2mm-mesh. 4.2.3 Chemical analyses Dried leaves and soil samples were ground to achieve homogeneity. Soil Total and extractable metal contents were quantified previously (Quintela-Sabarís et al. 2010). The amounts of Cr, Cu, Mn, Ni, Pb and Zn in leaves were quantified in solid subsamples with Energy-Dispersive XRay Fluorescence spectrometry (EDXRF). Other subsamples were digested with HNO3 for the quantification of Co contents with Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) element analysis. Any value lower than the detection limit was recorded as 0.1 μg•g−1 for statistical analyses (Bert et al. 2002). 87 work with populations of Cistus ladanifer from NE Portugal (Díez-Lázaro et al. 2006, Kidd et al. 2004). However, most of these studies were all based in hydroponic cultures with controlled metal contents, so we have to be cautious when comparing our results with those. Deram et al. (2007) proposed two explanations for the higher accumulation in NM populations. On the one side, NM populations could develop heavy metal active cellular mechanisms involved in the detoxification of heavy metals. On the other side, M populations could restrict better metal accumulation in aerial parts. Kidd et al. (2004) found that NM populations of C. ladanifer with low tolerance to Co accumulate more than 3,000 µg.g-1 Co in shoots but they showed toxicity symptoms when subjected to a 500µM Co treatment; in contrast, the populations with more tolerance to Cu or Ni in hydroponic cultures also showed a low accumulation in aerial parts. Therefore, it appears that in the case of C. ladanifer, the most suitable explanation is that of restricted metal accumulation in M populations. However, there were also differences among M populations. If we consider the ratios computed with extractable metals, which should be more realistically related to plant accumulation abilities, we found that M populations from lineage ‘North’ showed greater accumulation rates than ‘South’ for the metals Co, Cr, Mn and specially Ni; whereas M populations from lineage ‘South’ possessed a greater accumulation rate for Cu. This phenotypic diversity can be viewed as a result of multiple and independent events of colonisation in areas with heavy metals within diverging chloroplast lineages (Quintela-Sabarís et al. 2010). Similarly, serpentine populations of Cerastium alpinum, which originated within different recolonisation lineages, developed different growth responses to Ni and Mg (Nyberg-Berglund et al. 2004). The ideal plant species for use in phytostabilisation procedures in an area like the Mediterranean region should be a native plant tolerant to both drought and metal stress, and with an extensive root system. In addition, this species should not accumulate metals into above-ground tissues, in order to prevent further transfer into the food chain and thus reduce human or animal access to contaminants (Frérot et al. 2006). Given these requirements and the ample phenotypic diversity we have observed in Cistus ladanifer, Metallicolous populations from lineage ‘South’ are the best suited for use in phytostabilization procedures in soils polluted with Co, Cr, Mn or Ni, whereas the M populations from lineage ‘North’ are better for use in Cu polluted soils. However, the considerable differences in the response to those metals among populations means that any remediation procedure should be preceded by a survey that allows the characterization of local ecotypes of Cistus ladanifer in relation to heavy metals. Acknowledgements Dr. Javier Díez Lázaro, Dr. Eduardo García-Rodeja and Dr. Petra Kidd provided useful comments that improved PATTERNS OF METAL ACCUMULATION IN LEAVES 88 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. the quality of the text. Miguel González Szamocki revised the English usage. 4.5 References Alloway BJ (1995) Heavy Metals in Soils 2nd ed. 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Universidade de Coimbra, Portugal Pratas J, Prasad MNV, Freitas H, Conde L (2005) Plants growing in abandoned mines of Portugal are useful for biogeochemical exploration of arsenic, antimony, tungsten and mine reclamation. Journal of Geochemical Exploration 85:99-107 Quintela-Sabarís C, Vendramin GG, Castro-Fernández D, Fraga MI (2010) Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer have multiple origins. Plant and Soil 334:161-174 Rengel Z (2004) Heavy Metals as Essential Nutrients. In: Prasad MNV, Hagemeyer J (eds) Heavy Metal Stress in Plants: From Molecules to Ecosystems, 2nd ed. Springer-Verlag, Berlin, pp 271-294 Ruttens A, Mench M, Colpaert JV, Boisson J, Carleer R, Vangronsveld J (2006) Phytostabilization of a metal contaminated sandy soil. I: Influence of compost and/or inorganic metal immobilizing soil amendments on phytotoxicity and plant availability of metals. Environmental Pollution 144:524-532 Simões MP, Madeira M, Gazarini L (2009) Ability of Cistus L. shrubs to promote soil rehabilitation in extense oak woodlands of Mediterranean areas. Plant and Soil 323:249-265 Vangrosveld J, Herzig R, Weyens N, Boulet J, Adriaensen K, Ruttens A, Thewys T, Vassilev A, Meers E, Nehnevajova E, van der Lelie D, Mench M (2009) Phytoremediation of contaminated soils and groundwater: lessons from the field. Environmental Science and Pollution Research 16:765-794 PATTERNS OF METAL ACCUMULATION IN LEAVES Effect of population type and chloroplast lineage on the response of Cistus ladanifer L. (Cistaceae) to metals: a hydroponic culture analysis Chapter 5 Previous page: Hydroponically-grown plantlets from different populations of Cistus ladanifer. They have been just transferred to plastic buckets in order to perform tests of tolerance to heavy metals. (Photo: C. Quintela-Sabarís) Effect of population type and chloroplast lineage on the response of Cistus ladanifer (Cistaceae) to metals: a hydroponic culture analysis Keywords: Chlorophyll fluorescence, Cistus ladanifer, growth rates, heavy metals, hydroponic culture, tolerance ABSTRACT Cistus ladanifer is a pseudometallophyte plant whose metallicolous (M) populations belong to two diverging chloroplast lineages. Previous studies showed that tolerance to heavy metals could be a characteristic of this plant species. However, it is possible that there are differences in tolerance to metals between population types and/or chloroplast lineages. In order to check these issues, twenty five populations of this species were grown in hydroponic cultures and their tolerance to the trace metals Co, Ni and Zn was assessed. Growth rates, dry weights and photosystem II fluorescence were measured and transformed into measures of relative tolerance. These data were compared using nested Analysis of Variance and correlation analyses. Co caused reduction in root growth, whereas Ni and Zn influenced root and shoot growth and also photosynthetic performance. In contrast, the relative increase in the number of leaves was not affected by any treatment. In most cases, the tolerance to metals was similar between population types and chloroplast lineages. This fact indicates that tolerance to heavy metals could be a common trait in this species. Finally, in view of our findings, we suggest interesting lines for future research, such as the preadaptation of C. ladanifer to metals or the possible role of root symbioses in tolerance. CHAPTER 5 5.1 Introduction Heavy metals have toxic effects on plant metabolism, such as the production of reactive oxygen species, the blocking of essential functional groups in biomolecules and the displacement of essential metal ions (Schutzendubel and Polle 2002), which subsequently produce reduction in growth, variation in photosynthesis or even death. Due to these toxic effects, soils with high contents of these elements, either of natural or human origin, are areas with low vegetation cover and, in the case of human-polluted sites, can constitute environmental problems due to the leaching of metals into groundwater or the migration of contaminated soil by erosion and/or dispersal by wind (Ruttens et al. 2006). Some plant species have developed mechanisms for metal tolerance (e.g. chelation of metals in the cytosol by phytochelatins, (Goldsbrough 2000)) whose objective is to avoid the build-up of toxic concentrations at sensitive sites within the plant cell (Hall 2002). This tolerance can be ‘constitutive’ (=’species-wide’; i.e. common to all the individuals of a given species) or ‘population-specific’ (i.e. the tolerance to heavy metals have evolved only in certain 94 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. populations of a given species) (Pollard et al. 2002). Different research has indicated that tolerance to heavy-metals in plants; i.e., the ability to grow and survive in soils toxic to other plants, is a characteristic governed by a low number of major-genes, which are also modified by a large number of minor-genes (reviewed in Macnair et al. (2000)). As a consequence of the genetic determination of this characteristic, a variation in responses to heavy metals is expected and observed within different species (e.g. Zn tolerance in Arabidopsis halleri, Pauwels et al. (2006)). This variation in responses is clearer in those species whose metallicolous populations have evolved independently, as shown in Silene paradoxa (Gonnelli et al. 2001), Cerastium alpinum (Berglund et al. 2004) or Thlaspi caerulescens (Assunção et al. 2003). Cistus ladanifer L. (Cistaceae) is a woody, semideciduous shrub from the Western Mediterranean Area (from the South of France to North of Morocco and Algeria) (Demoly and Montserrat 1993). This plant is a pseudometallophyte; thus, it is present in non-metalliferous soils (mainly derived from acid rocks) and it also has colonised metalliferous areas (serpentine outcrops and mine tailings) throughout its distribution area (Alvarenga et al. 2004, Ater et al. 2000, Batista 2003, Díez Lázaro et al. 2006, Freitas et al. 2004, Pratas et al. 2005). Using cpSSR markers, we have inferred that the metallicolous populations of this species (i.e., those present on metalliferous soils) have arisen within two diverging chloroplast lineages (Quintela-Sabarís et al. 2010). On the basis of similar genetic diversity values between metallicolous (M) and non-metallicolous (NM) populations, we proposed that tolerance to heavy metals could be a ‘constitutive’ character in C. ladanifer. In the present paper, we investigated the tolerance of C. ladanifer to the following heavy metals: Co, Ni and Zn, in 25 populations throughout its whole range of distribution. Previous work has assessed the tolerance of C. ladanifer to different heavy metals, but only in five populations from NE of Potugal (Kidd et al. 2004). Here, we asked whether tolerance to heavy metals is present both in M and NM populations of C. ladanifer and whether differences exist in tolerance to these metals between population types (metallicolous vs. non-metallicolous) and/or chloroplast lineages. Specifically, we estimated tolerance by measuring growth, biomass and photochemical efficiency. 5.2 Material and methods 5.2.1 Populations studied Twenty-five populations of C. ladanifer sampled throughout its natural geographic distribution area were included in this work. These populations were classified in two ‘soil types’: Metallicolous (M) and Non-metallicolous (NM) (see supplementary material S 3.1 and S 5.1), based on previous soil analyses (Quintela-Sabarís et al. 2010). M populations include plants growing in serpentine outcrops from Morocco, Portugal and Spain and also populations developed on mine tailings from 95 Portugal and Spain. In addition, previous analyses of chloroplast microsatellites (cpSSR) revealed that these populations come from two chloroplast lineages that spread independently throughout the N of Morocco and SE of the Iberian Peninsula (‘South’ lineage) and the rest of the Iberian Peninsula (‘North’ lineage) after the Last Glacial Maximum (Quintela-Sabarís et al. 2010). (Table 5.1). In each population, a longitudinal transect was established at random. 10 plants separated by at least five meters were selected along this transect and their ripe fruits were collected. 5.2.2 Plant material and growth conditions Samples of seeds from each population were subjected to a dry-heating treatment (100ºC for 30 minutes) in order to break the physical dormancy and increase germination percentages (Perez-Garcia 1997). The treated seeds were then sown in Petri dishes with sterilized acid-washed sand and moisturized with distilled water. Seeds were placed in a culture chamber under the following conditions: 16/8 h light/ darkness, day/night temperature 20/15 ºC, RH 70%, and PPFD 190 μmol·m−2·s−1. Four weeks after germination (when the plantlets reached a four-leaf stage), they were carefully transferred to polystyrene sheets suspended in a nutritive solution with low concentrations of trace metals (control solution) at pH 4.5. This solution, optimized for Cistus ladanifer by Kidd et al. (2004), had the following composition (μM): 2000 CaCl2.2H2O, 1000 MgSO4.7H2O, 250 NH4NO3, 50 KH2PO4, 200 NaOH, 150 KCl, 25 ZnSO4.7H2O, 20 MnSO4.H2O, 15 CuSO4.5H2O, 15 FeEDTA, 10 H3BO3, 0.0143 (NH4)6Mo7O24.4H2O. Stock solutions of 100-(macronutrients) and 1000-strength (micronutrients) were made up and diluted appropriately. Culture solutions were continuously aerated with an aquarium air pump and changed every week. After 42 d in this solution, seedlings of similar size were selected and randomly placed into thirty 6 Lplastic buckets, each holding 10 seedlings. At this stage seedlings were transferred to a greenhouse (20–28 ºC) with natural light, where they continued to grow until the end of the experiment. 5.2.3 Treatment with metals and ecophysiologycal measurements Due to space limitations, the 25 populations were grouped in batches of five populations each. A total of 10 successive experiments (two for each batch) were carried out, replicating the same growth conditions previously described. In each batch, five buckets of C. ladanifer plants (each containing two replicates for each population) were randomly allocated to one of the following treatments with trace metals: cobalt (Co), nickel (Ni) and zinc (Zn), and three controls. Treatment concentrations and chemical form in which the metal was added were chosen following Kidd et al. (2004) (see Table 5.2 for details). Solutions were completely replaced every three days to TOLERANCE TO METALS Co, Ni AND ZnTOLERANCE TO METALS Co, Ni AND Zn 96 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Table 5.1: List of Cistus ladanifer populations included in our analyses. For each population, the lineage (North, South) and the type (M, NM) are indicated. Population (Code) Lineage Type Substratum Vegetation Long Lat Sierra de Aguas (EAC) South M (u) Serpentinised peridotite Open shrubland with dispersed Pinus pinea and P. halepensis trees 4.79º W 36.84º N Benalup (EBE) South NM Sandstone Cleared Quercus suber forest with Phyllirea angustifolia, Calicotome villosa and C. ladanifer 5.73º W 36.32º N Cardeña (ECA) North NM Granite Open Quercus ilex forest with C. ladanifer and Pistacia lentiscus 4.36º W 38.28º N La Codosera (ECO) North M (m) Sb mine tailing Shrubland dominated by C. ladanifer with Dittrichia viscosa 7.08º W 39.19º N Ciudad Rodrigo (ECR) North NM Quartzites Dense matorral with isolated Quercus ilex trees 6.49º W 40.63º N El Guijo (EGJ) North NM Slates Open dehesa with Quercus ilex and isolated C. ladanifer plants 4.77º W 38.52º N Mazagón (EMA) South NM Sand deposits Eucalyptus globulus and Pinus pinea plantations 6.84º W 37.15º N Monte Furado (EMF) North NM Schists Dense shrubland with Quercus ilex 7.20º W 42.39º N Ricobayo (ERB) North NM Quartzites and filites Open shrubland with Quercus ilex 5.81º W 41.70º N Sierra Bermeja (ESB) South M (u) Serpentinised peridotite Dense shrubland with scattered Pinus pinaster 5.18º W 36.48º N Sierra Palmitera (ESP) South M (u) Serpentinised peridotite Open matorral with Quercus coccifera and Ulex sp. 5.07º W 36.60º N Sierra de Tolox (ETO) South M (u) Serpentinised peridotite Open matorral with scattered Pinus pinaster and Ulex sp. 4.93º W 36.68º N Bab Tazaa (MBT) North NM Micaschists Open shrubland with dispersed Cistus plants 5.24º W 35.08º N Ketama (MKE) South NM Schists Dense shrubland with evergreen oaks 4.64º W 34.95º N East Bni Bouchra (MSII) South M (u) Serpentinised peridotite Open matorral with Halimium atriplicifolium, Pistacia lentiscus and Tetraclinis articulata 4.89º W 35.29º N West Bni Bouchra (MSI) South M (u) Serpentinised peridotite Open matorral with Pistacia lentiscus, Phillyrea latifolia and Tetraclinis articulata 4.90º W 35.30º N Aljustrel (PAL) North M (m) Pyrite mine tailing Dense matorral dominated by C. ladanifer and Lavandula stoechas 8.18º W 37.88º N Bragança (PBR) North M (u) Dunite Dense shrubland with Pinus pinaster and Genista sp. 6.87º W 41.85º N Burgau (PBU) North NM Limestone Dense shrubland with Chamaerops humilis and Pistacia lentiscus 8.78º W 37.07º N Corte Figueira (PCF) North NM Schists Quercus suber ‘Montado’ with dense cover of Cistus ladanifer 8.03º W 37.39º N Martinchel (PMA) North NM Sedimentary material (gravels, clays) Dense Pinus pinaster plantation 8.29º W 39.52º N Chloroplast lineage and population type following Quintela-Sabarís et al. (2010). Longitude and latitude are expressed in decimal degrees. 103 We may speculate that findings point to two possible explanations (which are not mutually-exclusive): Firtsly, a preadaptation of Cistus ladanifer to soils rich in heavy metals. Macnair (1987) suggested that plant species that are preadapted for any of the harsh conditions of metal rich soils (such as water stress or nutrient shortage) successfully colonize these regions more easily; moreover, Taylor and Levy (2002) detected preadaptation to low Ca:Mg ratios (one of the selective factors of serpentine soils) in a variety of Phacelia dubia that was endemic to granite outcrops. In the case of C. ladanifer, different authors provide arguments which support the hypothesis of preadaptation: NúñezOlivera et al. (1996) uncovered a high leaf plasticity in C. ladanifer, which implies an intrinsic adaptation to scarcity of water and nutrients in this species; Alados et al. (1999) suggested that the abilities of C. ladanifer inhabiting acidified soils with low Ca concentrations allowed the plants to perform well in serpentine soils in spite of the presence of heavy metals; finally, Kidd et al. (2004) observed that plants from non-metallicolous population of C. ladanifer maintained high growth rates in presence of Zn in hydroponic cultures. A second hypothesis that should be explored in future research, partially linked to that of preadaptation, is the possibility that C. ladanifer colonised metalliferous areas through collaboration with soil micro-organisms such as fungi and rhizobacteria, given that ectomycorrhizal fungi can provide protection and improve the tolerance to heavy metals in host species (Jentchske et al. 2000). Along these lines, Ramos Solano et al. (2006) have detected different species of plantgrowth promoting rhizobacteria (PGPR) in C. ladanifer roots, most of them involved in P-mobilisation and production of siderophores. In addition, more than 30 fungal species have been recorded as establishing symbiotic relations (ectomycorrhiza) with C. ladanifer in the literature (Comandini et al. 2006). Thus, the negative effects of heavy metals we observed in the plants under metal-treatments could be caused by the lack of these root symbionts in the conditions of hydroponic culture (Epstein and Bloom 2005). In summary, our investigation revealed that Co, Ni and Zn produced different toxic effects in C. ladanifer plants growing in hydroponics. In most cases the response to metals is similar between population types or chloroplast lineages, a fact that points to the tolerance to heavy metals as a common trait in this species. Moreover, interesting lines for future research, such as the occurrence of preadaptive traits in this species or the role of root symbioses in tolerance to metals, are suggested. Acknowledgements Dr. Julia Sánchez-Vilas provided useful comments that improved the manuscript. Miguel González Szamocki revised and improved the English usage 5.5 References Alados CL, Navarro T, Cabezudo B (1999) Tolerance assessment of Cistus ladanifer to serpentine soils by developmental stability analysis. Plant Ecology 143:51–66 TOLERANCE TO METALS Co, Ni AND Zn 104 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Alvarenga PM, Araújo MF, Silva JAL (2004) Elemental uptake and root-leaves transfer in Cistus ladanifer L. growing in a contaminated pyrite mining area (Aljustrel-Portugal). Water, Air and Soil Pollution 152:81-96 Assunção AGL, Bookum WM, Nelissen HJM, Vooijs R, Schat H, Ernst WHO (2003) Differential metal-specific tolerance and accumulation patterns among Thlaspi caerulescens populations originating from different soil types. New Phytologist 159: 411-419 Ater M, Lefèbvre C, Gruber W, Meerts P (2000) A phytogeochemical survey of the flora of ultramafic and adjacent normal soils in North Morocco. 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Springer-Verlag, Berlin, pp 434-454 Macnair MR, Tilstone GH, Smith SE (2000) The genetics of metal tolerance and accumulation in higher plants. In: Terry N, Bañuelos G (eds) Phytoremediation of contaminated soil and water. CRC Press, Boca Raton, Florida, pp 235-250 Mysliwa-Kurdziel B, Prasad MNV, Strzalka K (2004) Photosynthesis in Heavy Metal Stressed Plants. In: Prasad MNV, Hagemeyer J (eds) Heavy Metal Stress in Plants: From Molecules to Ecosystems, 2nd ed. Springer-Verlag, Berlin, pp 146-181 Nordal I, Haraldsen KB, Ergon A, Eriksen AB 105 (1999) Copper resistance and genetic diversity in Lychnis alpina (Caryophyllaceae) populations on mining sites. Folia Geobotanica 34:471–481 Núñez-Olivera E, Martinez-Abaigar J, Escudero JC (1996) Adaptability of leaves of Cistus ladanifer to widely varying environmental conditions. Functional Ecology 10:636–646 Nyberg Berglund AB, Dahlgren S, Westerbergh A (2004) Evidence for parallel evolution and site-specific selection of serpentine tolerance in Cerastium alpinum during the colonization of Scandinavia. New Phytologist 161:199-209 Pauwels M, Frérot H, Bonnin I, Saumitou-Laprade P (2006) A broad-scale analysis of population differentiation for Zn tolerance in an emerging model species for tolerance study: Arabidopsis halleri (Brassicaceae) Journal of Evolutionary Biology 19:1838-1950 Pérez-García F (1997) Germination of Cistus ladanifer seeds in relation to parent material. Plant Ecol 133:57-62 Pollard AJ, Powell KD, Harper FA, Smith JAC (2002) The genetic basis of metal hyperaccumulation in plants. Critical Reviews in Plant Sciences 21:539-566 Pratas J, Prasad MNV, Freitas H, Conde L (2005) Plants growing in abandoned mines of Portugal are useful for biogeochemical exploration of arsenic, antimony, tungsten and mine reclamation. Journal of Geochemical Exploration 85:99-107 Quintela-Sabarís C, Vendramin GG, Castro-Fernández D, Fraga MI (2010) Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer have multiple origins. Plant and Soil 334:161-174 Ramos Solano B, Pereyra de la Iglesia MT, Probanza A, Lucas García JA, Megías M, Gutierrez Mañero FJ (2006) Screening for PGPR to improve growth of Cistus ladanifer seedlings for reforestation of degraded mediterranean ecosystems. Plant and Soil 287:59-68 Ruttens A, Mench M, Colpaert JV, Boisson J, Carleer R, Vangronsveld J (2006) Phytostabilization of a metal contaminated sandy soil. I: Influence of compost and/or inorganic metal immobilizing soil amendments on phytotoxicity and plant availability of metals. Environmental Pollution 144:524-532 Schütznedübel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. Journal of Experimental Botany 372:1351-1365 Simões MP, Madeira M, Gazarini L (2009) Ability of Cistus L. shrubs to promote soil rehabilitation in extense oak woodlands of Mediterranean areas. Plant and Soil 323:249-265 Taylor SI, Levy F (2002) Responses to soils and a test of preadaptation to serpentine in Phacelia dubia (Hydrophyllaceae). New Phytologist 155:437447 Wilkings DA (1978) The measurement of tolerance to edaphic factors by means of root growth. New Phytologist 80:623-633 TOLERANCE TO METALS Co, Ni AND Zn AFLP analysis of the pseudometallophyte Cistus ladanifer: a comparison with cpSSRs and an exploratory genome scan to investigate loci associated to soil variables Chapter 6 Previous page: Screenshot of electropherograms of different C. ladanifer plants genotyped with AFLP markers. Red peaks correspond to the molecular weight marker, whereas green peaks indicate loci amplified with the primer pair: NED-EcoRIAGG/ MseICAG. (Image: C. Quintela-Sabarís) AFLP analysis of the pseudometallophyte Cistus ladanifer: a comparison with cpSSRs and an exploratory genome scan to investigate loci associated to soil variables Keywords: Amplified Fragment Length Polymorphism, chloroplast microsatellites, Cistus ladanifer, Generalized Estimating Equations, heavy metals ABSTRACT Cistus ladanifer is a pseudometallophyte which grows in different metal-rich substrates from the Western Mediterranean area. We have analysed 33 populations of this species with AFLPs in order to i) assess the genetic patterns of colonisation of metalliferous areas by C. ladanifer obtained with an AFLP genome scan and to compare them with previous cpSSRs results and ii) identify loci potentially linked to tolerance to metalliferous soils. AFLP results were partially congruent with previous cpSSR results, revealing no influence of soil type on the genetic diversity and differentiation of this species. However, some differences arose, mainly due to different properties (mutation rate, ploidy level, inheritance, …) inherent in each marker. Then we used Generalized Estimating Equations models to test the correlation between allele distribution and soil data. These regression analyses showed that the total soil contents of Mn has an important effect on allele distribution in Cistus ladanifer, the strongest in relation to the other soil variables we also have analysed. Moreover, we report the detection of a particular allele with a possible role in tolerance to high Mn concentrations in soils. CHAPTER 6 6.1 Introduction Soils with high concentrations of metals (metalliferous soils) are toxic to most plants and other living organisms (Shaw et al. 2004). Moreover, soils polluted by human activities, particularly those activities related to the production of metals (mine tailings, smelting areas ...) constitute a threat to the environment and public health: they usually have a sparse (or even absent) plant cover and thus metals can leach into groundwater or the polluted soil can be dispersed by wind which then affects productive agricultural land or natural reserves (Ruttens et al. 2006, Tordoff et al. 2000). However, the metalliferous areas also may be considered as ecological islands in which different metal-tolerant plant species are found. Thus, soils with high contents of heavy metals provide the opportunity to study the establishment and differentiation of plant populations under severe selection pressure (Lefèbvre and Vernet 1990). Among metal-tolerant species we may distinguish between strict metallophytes (or eumetallophytes), whose populations only grow on metalliferous substrates, and pseudometallophytes (or facultative metallophytes), which can grow both in metalliferous and non-metal- 110 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Table 6.1: Description of Cistus ladanifer populations studied. Pop (Code) Lineage Type Substratum Vegetation Long Lat N Sierra de Aguas (EAC) South M (u) Serpentinised peridotite Open shrubland with scattered Pinus pinea and P. halepensis 4.79º W 36.84º N 7 Almodóvar (EAL) North M (h) Clays close to a road Grassland with dispersed C. ladanifer plants 5.65º W 36.16º N 8 Benalup (EBE) South NM Sandstone Cleared Quercus suber forest with Phyllirea angustifolia, Calicotome villosa and C. ladanifer 5.73º W 36.32º N 8 Cardeña (ECA) North NM Granite Open Quercus ilex forest with C. ladanifer and Pistacia lentiscus 4.36º W 38.28º N 9 La Codosera (ECO) North M (m) Sb mine tailing Shrubland dominated by C. ladanifer with Dittrichia viscosa 7.08º W 39.19º N 8 Ciudad Rodrigo (ECR) North NM Quartzites Dense matorral with isolated Quercus ilex trees 6.49º W 40.63º N 9 Despeñaperros (EDE) North M (h) Quartzites, close to the highway. Dense Cistus ladanifer shrubland with Quercus ilex and Juniperus oxycedrus 3.51º W 38.39º N 8 Fuente Saúco (EFS) North NM Sandstone and conglomerates Shrubland with Quercus ilex 5.51º W 41.19º N 7 El Guijo (EGJ) North NM Slates Open dehesa with Quercus ilex and isolated C. ladanifer plants 4.77º W 38.52º N 8 Grazalema (EGR) South NM Decarbonated limestone Shrubland with C. ladanifer and C. monspelliensis 5.27º W 36.78º N 9 Guadarrama (EGU) North NM Granite Dense shrubland with isolated Quercus ilex 4.10º W 40.68º N 9 Mazagón (EMA) South NM Sand deposits Eucalyptus globulus and Pinus pinea plantations 6.84º W 37.15º N 9 Monte Furado (EMF) North NM Schists Dense shrubland with Quercus ilex 7.20º W 42.39º N 8 Ricobayo (ERB) North NM Quartzites and filites Open shrubland with Quercus ilex 5.81º W 41.70º N 5 Sierra Bermeja (ESB) South M (u) Serpentinised peridotite Dense shrubland with scattered Pinus pinaster 5.18º W 36.48º N 9 Sierra Palmitera (ESP) South M (u) Serpentinised peridotite Open matorral with Quercus coccifera and Ulex sp. 5.07º W 36.60º N 8 Sierra de Tolox (ETO) South M (u) Serpentinised peridotite Open matorral with scattered Pinus pinaster and Ulex sp. 4.93º W 36.68º N 9 Valdecaballeros (EVC) North NM Sedimentary material (gravels, clays) Dense shrubland with Pinus pinaster 5.34º W 39.33º N 7 Bni Hadifa (MBH) South NM Sandstones Open Pinus halepensis forest 4.17º W 35.02º N 8 Bab Tazaa (MBT) North NM Micaschists Open shrubland with dispersed Cistus plants 5.24º W 35.08º N 7 El Jebha (MEJ) South NM Sandstone Open Pinus halepensis forest 4.64º W 35.18º N 9 Ketama (MKE) South NM Schists Dense shrubland with evergreen oaks and Cistus laurifolius 4.64º W 34.95º N 7 West Bni Bouchra (MSI) South M (u) Serpentinised peridotite Open matorral with Pistacia lentiscus, Phillyrea latifolia and Tetraclinis articulata 4.90º W 35.30º N 8 Lineage and Type were defined on the basis of analyses published in Quintela-Sabarís et al. 2010. Lineage: cpSSR lineage (‘North’, ‘South’); Type: M (metallicolous), NM (non-metallicolous). Geographic coordinates are given in decimal degrees. N: number of plants genotyped with AFLP in each population. 111 Table 6.1: (continued) Pop (Code) Lineage Type Substratum Vegetation Long Lat N East Bni Bouchra (MSII) South M (u) Serpentinised peridotite Open matorral with Halimium atriplicifolium, Pistacia lentiscus and Tetraclinis articulata 4.89º W 35.29º N 7 Tanger (MTA) South NM Sandstone Open Pinus pinaster forest 5.93º W 35.78º N 8 Aljustrel (PAL) North M (m) Pyrite mine tailing Dense matorral dominated by C. ladanifer and Lavandula stoechas 8.18º W 37.88º N 7 Bragança (PBR) North M (u) Dunite Dense shrubland with Pinus pinaster and Genista sp. 6.87º W 41.85º N 8 Burgau (PBU) North NM Limestone Dense shrubland with Chamaerops humilis and Pistacia lentiscus 8.78º W 37.07º N 8 Corte Figueira (PCF) North NM Schists Quercus suber ‘Montado’ with dense cover of Cistus ladanifer 8.03º W 37.39º N 6 Macedo dos Cavaleiros (PMC) North M (u) Serpentinised peridotite Dense shrubland with Quercus ilex 6.82º W 41.52º N 6 Samil (PSA) North M (u) Serpentinised peridotite Open matorral with Alyssum serpyllifolium and Quercus ilex 6.75º W 41.78º N 7 São Vicente (PSV) North NM Limestone Open shrubland with Pistacia lentiscus and Juniperus phoenicea 8.98º W 37.03º N 7 Vela (PVE) North NM Granite Pinus pinaster plantation 7.29º W 40.43º N 9 Lineage and Type were defined on the basis of analyses published in Quintela-Sabarís et al. 2010. Lineage: cpSSR lineage (‘North’, ‘South’); Type: M (metallicolous), NM (non-metallicolous). Geographic coordinates are given in decimal degrees. N: number of plants genotyped with AFLP in each population. liferous soils (Pollard et al. 2002). Due to the aforementioned duality of metalliferous areas, pseudometallophytes are interesting organisms since i) they constitute highly relevant models to study local adaptation in plants (Linhart and Grant 1996); and ii) they usually possess, along with metal tolerance, several traits (high adaptability to adverse soil conditions, high biomass production, good competitiveness…) that may make them useful for phytoremediation technologies (Poschenrieder et al. 2001). The population genetics of different pseudometallophyte species has been investigated in order to identify evolutionary and genetic factors involved in tolerance (Westerbergh and Saura 1992, Vekemans and Lefèbvre 1997, Mengoni et al. 2001, Jiménez-Ambriz et al. 2007, Pauwels et al. 2008). These studies mainly tried to identify whether metallicolous populations suffered a founder effect during the colonisation of metalliferous areas or to determine if metallicolous populations of a particular species share a common ancestry or whether they are the result of local colonization events. Their results show that metallicolous populations are usually the result of local evolution. However, regarding the occurrence of founder effect, a common trend across species was not found. Pauwels et al. (2005) proposed that AFLPs, PHYLOGEOGRAPHY AND GENOME SCAN 112 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. the colonisation of metal-polluted environments is associated with a genetic bottleneck in species with populational tolerance (e.g. Silene paradoxa, Mengoni et al. 2001), whereas in species with constitutive (or 'specieswide') tolerance (such as Arabidopsis halleri, Pauwels et al. 2005) the effect of a bottleneck may not be detected. Among the molecular markers applied to non-model pseudometallophytes, those based on maternally-inherited chloroplast DNA (such as chloroplast microsatellitescpSSR), are useful to infer the phylogeography of a species, making possible a better understanding of the effect of metal pollution on the genetic structure of its populations (Staton et al. 2001). However, chloroplast markers are mainly neutral and seldom related to metal tolerance. In contrast, AFLP markers (Vos et al. 1995), which have predominantly a nuclear origin (Meudt and Clarke 2007) can be applied to any organism without previous knowledge of sequences. Thus, this kind of marker has allowed genome scans to be applied to non-model organisms (Bonin et al. 2007). The use of genome scans along with environmental data allows the detection of those markers potentially linked to adaptive loci (Holderegger et al. 2008) among many markers (such as AFLP). Thus, AFLP loci with potential ecological relevance have been identified in Arabis alpina (Poncet et al. 2010), or, interestingly, Meyer et al. (2009) have inferred loci putatively involved in tolerance to heavy metals in metallicolous (M) and non-metallicolous (NM) populations of the pseudometallophyte Arabidopsis halleri. Cistus ladanifer L. (Cistaceae) is a pseudometallophyte shrub native to the Western Mediterranean region (from S of France to the N of Morocco and Algeria, Demoly and Montserrat 1993). In addition to its adaptability to disturbances occurring in Mediterranean areas, mainly fires (Pérez-García 1997) and water and light stress (Martín Bolaños and Guinea López 1949, Núñez-Olivera et al. 1996), this species has been described as interesting for phytostabilisation procedures and also for phytoextraction of Zn in soils with low to medium contents of this metal (DíezLázaro 2008). In a previous paper we analysed 33 NM and M (serpentine and mines) Cistus ladanifer populations with cpSSR markers, from almost its entire distribution area (Quintela-Sabarís et al. 2010). We inferred that M populations evolved in parallel within two independent chloroplast lineages. In this work, we re-analyse the same 33 populations with AFLP markers with two main aims: i) to assess and to compare the genetic structure and the patterns of colonisation of metalliferous areas by C. ladanifer obtained using AFLPs (nuclear markers, dispersed by pollen and seeds) and cpSSRs (maternally-inherited, and thus dispersed only through seeds (Guzmán and Vargas 2009)) analyses, and ii) to identify AFLP loci potentially associated to tolerance to metalliferous soils in this species. We addressed the latter topic through the analysis of correlation be- 119 puted based on Nei’s genetic distance, D (after Lynch and Milligan 1994), using the AFLP-SURV. The genetic distances were validated by a bootstrap procedure with 1000 replications. Each bootstrapped matrix was used to compute a NeighbourJoining (NJ, Saitou and Nei 1987) dendrogram with the program NEIGHBOR. The program CONSENSE allowed us to construct a consensus dendrogram on the basis of 1000 NJ dendrograms. NEIGHBOR and CONSENSE belong to the package PHYLIP (v. 3.6, Felsenstein 2004). We computed the correlation between pairwise D matrix (obtained with AFLP) with (a) the matrix of genetic distance obtained with cpSSR markers (based on Cavalli-Sforza and Edwards distances, Quintela-Sabarís et al. 2010), and with (b) a matrix of pairwise geographic distances (expressed as natural logarithm of Km, Ln(km)) using the Mantel matrixcorrespondence test (Mantel 1967), which is included in the module MXCOMP from NTSYS-pc (v. 2.11L; Rohlf, 2002). We set 10000 permutations to validate the results. In order to infer population genetic structure, Bayesian analysis using a spatial clustering model implemented in BAPS software version 5.4 was performed (Corander et al. 2008). The spatial clustering of groups model was run using each population, with known coordinates, as the unit to be clustered. We initially fixed k (the number of clusters) from 2 to 33. Afterwards, we selected the value of k that had the minimum log marginal likelihood and repeated the analysis 100 times to obtain the optimal population structure. The use of spatial information increases the power to correctly detect the underlying population structure (Bonin et al. 2007). The results of the mixture analysis were then used to perform an admixture analysis, following the protocol by Corander and Marttinen (2006). We used the following settings: (1) minimal size of clusters. five individuals; (2) 200 iterations to estimate the admixture coefficients for the individuals; (3) 300 simulated reference individuals from each population, and (4) 20 iterations to estimate the admixture coefficients for the reference individuals. According to Corander and Marttinen (2006) BAPS performs equally well or even better than the widely used program STRUCTURE (Pritchard et al. 2000) with a 400-fold speed advantage. The population genetic structure was further explored using a locus-by locus Analysis of Molecular Variance (AMOVA, Excoffier et al. 1992), implemented with the Arlequin 3.5 software (Excoffier et al. 2005). This test allowed for estimation of variance components between individuals within populations, between populations within groups and among groups. The groups were defined either on the basis of soil analyses or on the basis of BAPS-clustering. Variance components and Φ statistics were estimated for each locus and then combined to produce synthetic estimators of Φ statistics. The significance values were computed by a permutation test from 20000 permuted matrices. Finally, we computed the pollento-seed migration ratio(r = mp/ms) followAFLPs, PHYLOGEOGRAPHY AND GENOME SCAN 120 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. ing Petit et al. (2005). We estimated the genetic subdivision at nuclear markers (ΦSTb) using Arlequin 3.5 software (Excoffier et al. 2005), whereas the subdivision at maternally inherited markers (ΦSTm) was obtained from a previous study (QuintelaSabarís et al. 2010). 6.2.6 Data analysis: detection of ecologically relevant loci using GEE We used generalized estimating equations (GEE) in order to detect alleles that were correlated to soil variables. GEE are an extension of generalized linear models (Carl and Kuhn 2007), which may consider autocorrelation between samples by including an additional variance component directly into the independent data model’s estimating equation to accommodate correlated data. This method allows us to consider that neighbouring individuals within chloroplast lineages are genetically more similar than individuals belonging to different chloroplast lineages which, as we inferred in a previous work (Quintela-Sabarís et al. 2010), may be isolated from each other from the Last Glacial Maximum at least. As we dealt with binary data, we used a logit-link and binomial error distribution to correlate allele occurrence for each AFLP locus per sampling location to quadratic polynomials of environmental variables. To consider the variety of response curve shapes other than a linear response (Legendre and Legendre 1998) we used quadratic polynomials. In order to select the best GEE models, we used the quasi-likelihood information criterion (QIC) adapted by Pan (2001). The best model is that with the lowest QIC. All combinations of variables were investigated for each locus. Finally, we tested 128 GEE (from the null model to the full model with seven variables in second-order polynomials) on our data set. All GEE models were calculated using the R package geepack (Yan and Fine 2004), and an implementation developed by Poncet et al. (2010) for the QIC calculation in R (R Development Core Team 2007). For each selected model, we also tested each regression coefficient using the Wald test. To avoid a high rate of false positives due to multiple tests, significance levels were calculated for each regional data set by minimizing the false discovery rate (FDR; Storey 2002). P-value rejection thresholds were adjusted so as to have less than one false positive locus per data set and were estimated using the R package qvalue. 6.3 Results A total of 109 and 112 different fragments were amplified with primer combinations A and B, respectively. However, after the filtering process (error rates and size ranges) 110 markers were finally considered (50 obtained with combination A and 60 with combination B). The total error rate using both combinations was 0.055 (Table 6.2). 6.3.1 Genetic diversity and differentiation The values of genetic diversity within populations are presented in Table 6.3. The Mann-Whitney tests did not reveal significant differences in diversity between M 121 and NM populations, neither when considering the whole set of populations nor each chloroplast lineage separately (Table 6.4). We found high and significant linear correlations among the three diversity estimates computed on AFLP data. Interestingly, the estimates of genetic diversity based on AFLP and cpSSR were not correlated (Figure 6.1). The matrix of Nei’s D genetic distances among populations is available in Supplementary Material S 6.2. As previously obtained with chloroplast markers, the consensus dendrogram revealed a clustering more related to phylogeography than to soil type (Fig. 6.2). A group of populations of C. ladanifer subsp. africanus from the North of Morocco is defined (marked in green colour in Fig. Figure 6.3: Localities for the 33 sampled populations of Cistus ladanifer (for details of each population see Table 6.1). Black circles indicate NM populations whereas white circles indicate M populations. The graphs next to each population indicate the proportional assignment of individuals from each population to the four different clusters as detected in an admixture analysis of AFLP data conducted with the program Bayesian Analysis of Population Structure (BAPS). Every vertical coloured bar corresponds to an individual. A vertical bar is split into several colours when there is evidence for admixture. The BAPS-defined clusters are: cluster 1 (red), cluster 2 (blue), cluster 3 (yellow) and cluster 4 (green). Only significant admixtures (significance level 5%) are showed. A NJ dendrogram showing the relationships among each of those clusters is presented on the upper right corner. NJ dendrogram is based on Kullback-Leibler distances among clusters. AFLPs, PHYLOGEOGRAPHY AND GENOME SCAN 122 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Table 6.5: Summary of analysis of molecular variance (AMOVA) of Cistus ladanifer (a) considering the whole data set, (b) be tween population types (M vs. NM), (c) between BAPS-defined groups (K = 4) and (d to g) separate analyses between population types for each of the four groups defined by SAMOVA analysis. SS = sum of squared deviation, P = level of probability of obtaining a more extreme component estimate by chance alone. n.s. = not significant (5% level). Analysis Source of variation SS Variance components % Total Variance P (a) Whole data set (ΦST= 0.35) Among populations 836.74 2.62 35.47 < 0.0001 Within populations 1126.61 4.77 64.53 Total 1963.35 7.39 (b) M vs NM Between population types (ΦCT = 0.01) 40.87 0.11 1.52 0.02 Among pops. within population types 795.87 2.57 34.44 < 0.0001 Within populations 1126.61 4.77 64.04 < 0.0001 Total 1963.35 7.45 (c) BAPS mixture results Among BAPS-defined clusters (ΦCT = 0.31) 515.69 2.50 31.04 < 0.0001 Among pops. within clusters 321.05 0.77 9.62 < 0.0001 Within populations 1126.61 4.77 59.33 < 0.0001 Total 1963.35 8.04 (d) Cluster 1 Between population types (ΦCT = 0.01) 12.68 0.06 1.06 n.s. Among pops. within population types 117.23 0.74 13.72 < 0.0001 Within populations 435.09 4.57 85.22 < 0.0001 Total 565.00 5.37 (e) Cluster 2 Between population types (ΦCT = -0.03) 9.97 -0.14 -2.84 n.s. Among pops. within population types 66.39 1.01 20.12 < 0.0001 Within populations 229.43 4.16 82.71 < 0.0001 Total 305.79 5.03 (f) Cluster 3 Between population types (ΦCT = 0.002) 13.03 0.01 0.16 n.s. Among pops. within population types 52.45 1.11 22.63 < 0.0001 Within populations 159.39 3.78 77.21 < 0.0001 Total 224.87 4.90 (g) Cluster 4 Between population types (ΦCT = 0.01) 9.77 0.07 1.00 n.s. Among pops. within population types 39.52 0.15 2.19 0.04 Within populations 302.70 6.78 96.81 0.01 Total 351.99 7.01 123 6.2). In addition, another cluster groups all the populations from South and SE of the Iberian Peninsula (Betic Area, marked in blue in Fig. 6.2). Populations from the rest of Iberian Peninsula are arranged in two other clusters. Population MBT from the North of Morocco, which belongs to C. ladanifer subsp. ladanifer is located next to populations of the same subspecies from Iberian Peninsula. Each of the four defined clusters includes both M and NM populations. The congruency between AFLP and chloroplast markers is also supported by the fact that a Mantel test (1967) revealed that genetic distances among populations based on AFLP or on cpSSR were positively correlated (the value of correlation factor r was 0.5688; P 0.0001). However, the population EAL showed differences between AFLP and cpSSR: on the basis of AFLP it is placed in a cluster with other populations from the Betic area, but if we consider cpSSR this population is more linked to populations from SW to the North of Iberian Peninsula (Fig. 6.2). Mantel test revealed a low but significant correlation between matrices of genetic and geographic distances between populations (r = 0.3923; P < 0.0001). Bayesian mixture analysis inferred four clusters of populations (k = 4; Log(marginal likelihood) of optimal partition: -6 099.8331) which fit the four clusters identified in NJ consensus dendrogram (Fig. 6.3). The degree of admixture among clusters is low, and it occurs mainly in populations from the Iberian Peninsula. It is remarkable the fact that no individual plant from population MBT (C. ladanifer subsp. ladanifer from N of Morocco) showed indications of admixture with the north African cluster 3 (marked in yellow). In contrast population EBE (C. ladanifer subsp. africanus from S of Iberian Peninsula) presents an indication of admixture with the cluster 3. A with the consensus dendrogram, each of the four defined clusters includes both M and NM populations (Fig. 6.3). The AMOVA analysis inferred that most of the molecular variance occurs at the intra population level (64.53%; Table 6.5a), and a ΦSTb value of 0.35 (P < 0.0001). AMOVA also revealed a significant partitioning of 31.04% of molecular variance among the four clusters defined by BAPS (ΦCT = 0.31; P < 0.0001; Table 6.5c). In contrast, the comparisons between population types at the global level (Table 6.5b) revealed that less than 2% of molecular variance occurs between M and NM populations. The differentiation between population types became non-significant when the AMOVA analyses where performed within each BAPS cluster (Table 6.5d to 6.5g). 6.3.2 Detection of relevant loci using GEE GEE were used to correlate allele frequencies to soil variables correcting for chloroplast lineages. The threshold for significance minimizing the false discovery rate was estimated as 0.0031. Sixteen loci (14.5% of total loci) were significantly correlated to one or two soil variables (Fig. 6.4), giving a total of 21 significant AFLPs, PHYLOGEOGRAPHY AND GENOME SCAN 124 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. relationships (Supplementary Material S 6.3). MnT and SbT were the soil variables with most influence, since each of them represented 28.6% of detected significant correlations. NiT and ZnT accounted for 14% each. No locus was significantly related to Ca:Mg ratio. In order to prune possible false positives, we further explored these 21 relationships by i) performing separate Mann-Whitney U tests to detect significant differences in soil variables between the group of plants where a certain band was absent or present, and ii) observing the geographic distribution of the presences/ absences of each locus. The Mann-Whitney U tests showed significant differences for only 8 loci (5 of them related to MnT), whereas in the case of the loci A492 and B346 they were marginally significant (0.05 < P < 0.10) (Table 6.6). The distribution maps of band presences in relation to soil variables show that several bands (e.g. A183, B293, B320, B485 in relation to PbT,…) appear mainly in populations with low metal contents, and thus, those bands may not really be related to metals. However, two bands (B401 and B485) are present mainly in populations with higher soil contents of manganese. The map of B401 presences is presented in Fig. 6.5, whereas the maps for the other loci are in Supplementary Material S 6.4. 6.4 Discussion 6.4.1 Comparison of AFLP and cpSSR inferences of genetic diversity and differentiation In this work we present a species-wide analysis of C. ladanifer using a genome scan of AFLP markers. We have obtained an estimation of genetic differentiation (ΦST = 0.35) which is much higher than data for other species (above the 3rd quartile in data from 77 species, Petit et al. 2005) and is congruent with the trends inferred by Nybom (2004) from species with similar life history traits (perennial, outcrosser, barochorous and from early-mid successional status). Moreover, we estimated a quite low pollen/seed migration ratio, which indicates that the dispersal of seeds have accounted for a large component of the historical gene flow between populations (above the 45%). In fact, Metcalfe and 0 5 10 15 20 First var Second var pH Mn T Ni T Pb T Sb T Zn T Figure 6.4: Number of AFLP loci significantly correlated with soil variables in the generalized estimating equations (GEE) applying a correlation for chloroplast lineage. Sixteen bands (14.5% of obtained loci) were correlated with one variable, 5 of which were also correlated with a second variable. Different fill patterns represent pH and Total soil contents of Mn, Ni, Pb, Sb and Zn. 125 Kunin (2006) reported that pollination success in isolated C. ladanifer plants dropped to 0 when the distance to the nearest neighbour was around 3.5 m. This near equal contribution of seed and pollen flow may explain the fact that, in spite of the different properties (such as mutation rate, ploidy level, homoplasy, inheritance) of AFLPs and cpSSRs, the matrices of inter-population genetic distances based on each type of markers were significantly correlated. As a consequence of matrix correlations, similar NJ and Bayesian clusterings were obtained with both markers. The only discrepancy between markers was observed in the population EAL, located in a contact zone between different glacial lineages (Quintela-Sabarís et al. 2010). We may consider this case as an example of the aforementioned properties of maternally-inherited cpDNA (e.g. Comes and Kadereit 1998), which conserve the history of colonisation, whereas in the case of nuclear markers this history is blurred by recombination or by pollen flow from nearby populations. Similarities in population clustering also implies that AFLPs tell the same story as cpSSRs about the origin of the metallicolous (M) populations of C. ladanifer: they are the result of multiple and independent colonisation events that, in addition, did not imply any genetic differentiation related to soil type, as indicated by the AMOVA results within each BAPSTable 6.6: Summary of results of the Mann-Whitney test on potentially environmental related loci detected by GEE. Only those loci with significant (or near significant) results are presented. U: Mann-Whitney statistic. ma and mp refer to the median value of the related variable for the group of plants where the AFLP marker was absent or present, respectively. na and np refer to the number of plants where each AFLP marker was absent or present, respectively. P: probability of obtaining a more extreme U value than that presented by chance alone. Locus Related Variable U manampnpP (2-tailed) A183 SbT 5302.0 0.014 189 0.012 81 < 0.001 A189 pH 1101.5 6.30 250 6.99 50 < 0.001 A339 NiT 2626.5 45.99 240 27.86 30 0.016 A492 NiT 1417.5 45.99 255 18.69 15 0.092 B293 MnT 1273.0 532.7 252 241.9 16 0.013 B320 MnT 2677.0 532.7 238 424.8 30 0.026 B346 SbT 3688.0 0.012 229 0.014 39 0.082 B391 MnT 742.5 478.4 258 2805.0 10 0.001 B401 MnT 2614.5 454.9 233 1577.5 35 0.001 B485 MnT 1065.0 478.4 252 2191.3 16 0.002 B485 PbT 997.0 17.75 252 11.59 16 0.001 AFLPs, PHYLOGEOGRAPHY AND GENOME SCAN 126 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. defined cluster. Instead, and given the significance of the Mantel test on geographic and genetic distances, the genetic differentiation may be explained better as a result of historical processes (i.e. location and isolation of glacial refugia) together with isolation-by-distance. In this case, the significant difference between M and NM populations revealed by species-wide AMOVA can be interpreted as a spurious result produced by a “covariation” of phylogeography and soil type: each type of population has a different relative weight within each cluster inferred by BAPS. Hence, and following Staton et al. (2001), we have to underline the need to understand the phylogeography of a species as a prerequisite to correctly interpreting the effect of stress factors (heavy metals, in our case) on its genetic structure. Similarly, studies which applied AFLPs to other pseudometallophytes also revealed the lack of an effect of soil type on genetic differentiation (e.g. Cerastium velutinumGustafson et al. 2003; Onosma echiodesMengoni et al. 2006; Armeria maritimaBaumbach and Hellwig 2007). From a taxonomic point of view, and like previous cpSSR results (QuintelaSabarís et al. 2010), the populations from S and SE Andalusia (Betic area) actually form a cluster separated from the other populations, nevertheless they are identified as belonging to subsp. ladanifer on the basis of leaf morphology. In contrast, the populations of the subsp. sulcatus (formerly Cistus palhinhae), despite showing several morphological differences, do not form a single cluster and are instead grouped together with populations of subsp. ladanifer. Regarding subsp. sulcatus, our results are in contrast with those obtained by Carlier et al. (2008), who, analysing populations from the Algarve region (S of Portugal) with AFLPs and ISSRs, inferred a genetic differentiation between subsp. ladanifer and subsp. sulcatus. Our differences may be explained by the fact that Carlier et al. (2008) analysed DNA samples bulked for each population and also because the ISSR markers had an important effect, since they showed some alleles exclusive to one of the two subspecies considered. However, they obtained a low differentiation between subspecies (Dice index 0.98; Carlier et al. 2008). Overall, these results point to the need for a taxonomic revision of the intraspecific taxa in C. ladanifer, especially in order to recognize the value and unique genetic features of Betic populations of this species. As indicated by the AMOVA analyses, most of the genetic variation revealed by AFLPs occurs at the intrapopulation level, as would be expected for nuclear markers in an obligate-outcrossing species such as C. ladanifer (Loveless and Hamrick 1984, Nybom 2004). The assessment of genetic diversity within populations may suffer bias due to the dominant nature of AFLP markers. In order to overcome this possible bias, we have used SMC, the best metric for dominant markers in diploid species (Kosman and Leonard 2005), and we complemented SMC with two allele frequency-based diversity 127 estimators (PPL and Hj), computed using a robust Bayesian approach (Zhivotovsky 1999). Thus, we have obtained reliable estimators of genetic diversity, giving the similarities and significant correlations among the three indices. As we previously inferred with cpSSRs, AFLPs detected no differences in genetic diversity related to soil type, a trend that seems to be common in the analyses of pseudometallophytes with nuclear-DNA markers (Mengoni et al. 2000, Mengoni et al. 2006, Baumbach and Hellwig 2007; but also some exceptions, e.g. Deng et al. 2007). This fact points to a lack of selective constraints on metallicolous populations of C. ladanifer and, thus, it may be an additional support on our consideration of tolerance to heavy metals as a constitutive trait in this species (Quintela-Sabarís et al. 2010). However, we have to keep in mind that we are working with more than one hundred (putatively) independent and neutral markers, so possible selective effects in few markers may be diluted by the other neutral ones. Ribeiro et al. (2002) found a correlation of genetic diversity estimations obtained with either AFLPs or cpSSRs in Pinus pinaster. These authors attributed this congruence to the fact that in this species gene flow through pollen (cpDNA is paternally-inherited in P. pinaster) is more important than marker-specific factors. In the case of C. ladanifer, the maternallyinherited cpDNA is not influenced by pollen flow, so the lack of correlation among the estimates of within-population genetic diversity obtained with AFLP markers and those obtained with cpSSRs, may be caused by the previously mentioned different properties of each kind of markers and the different effect of processes (such as genetic drift, recombination…) on them. 6.4.2 Detection of relevant loci using GEE We have applied generalized estimating equations (GEE) to our data in order to reveal loci potentially related to soil variables (pH, Ca:Mg ratio, but especially heavy metal contents in soils). Contrary to other methods for detecting loci under selection, which usually rely on prior assumptions about population structure, migration and mutation rates, and also involve the estimation of FST from allele frequencies (see Bonin et al. 2007, and references therein), GEE directly correlate the allele distribution of each marker independently with environmental variation to produce ‘allele distribution models’. Thus, these models are also individual-based and hence insensitive to biases caused by low sample size (Holderegger et al. 2008), an issue especially interesting for our study, with a medium number of plants per population. Although we are analysing the relationships between soil conditions and AFLP, other two main factors may include “noise” on our analyses: phylogeography and climatic variation. We have corrected the possible influence of phylogeography by introducing a correcting factor based on the previous information about chloroplast lineages (Quintela-Sabarís et al. 2010). Regarding the climate, PCA analyses on AFLPs, PHYLOGEOGRAPHY AND GENOME SCAN 128 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. 19 bioclimatic variables from each population revealed no clines or patterns on our populations (data not shown), so we may conclude that the allele distribution models mainly reflect the influence of soil variables. Regarding soil variables, it is remarkable that we did not detected any loci related to Ca:Mg ratio, which points to a lack of selective effect of this soil variable on Cistus ladanifer. High levels of Mg in relation to Ca are considered one of the main stress factor to plants growing on serpentine soils (e.g. Brady et al. 2005). This unexpected result is congruent with previous reports signalling the low Ca requirements of C. ladanifer as a competitive trait allowing this plant growing in serpentine soils (Alados et al. 1999), and the high Mg requirements of C. ladanifer plants growing on serpentine soils from N of Morocco(Ater et al. 2000). The opposite situation to Ca:Mg ratio is MnT, the total content of manganese in soils, which was shown to be the most influential of the soil variables. Its effect is even higher than other variables with a similar variation range (up to several thousand mg·kg-1 in soil; e.g. NiT and PbT), and may be related to the higher mobility in soils of Mn compared to Ni or Pb (Friedland 1990). Manganese is a micronutrient essential to plant metabolism. It has functions in photosynthesis and also a protective role against oxidative stress (Epstein and Bloom 2005), but it may be toxic at high concentrations, provoking chlorosis Figure 6.5: Geographic distribution of loci B401, potentially related to MnT. The geographic distribution of the band presence overlays the spatial variation of MnT. The diameter of each circle is proportional to the value of MnT in each population. The colour of the circle represent for each population the frequency (f) of plants in which the AFLP-fragment is present: blue (f = 0); green (0 < f ≤ 0.50); yellow (0.50 < f ≤ 0.75) and red (0.75 < f). 35 The use of molecular markers has enabled clarification of the post-glacial migration of plant species for which there are very limited fossil pollen records, such as Ilex aquifolium and Hedera species (Grivet and Petit 2002, Rendell and Ennos 2003). Chloroplast DNA (cpDNA) is inherited through the maternal line in C. ladanifer (Guzmán and Vargas 2009), and thus reflects only the effect of seed flow and seed dispersal. In addition, the effective population size for haploid cpDNA is smaller than for diploid nuclear genes, so that differentiation through genetic drift may be stronger (Comes and Kadereit 1998). These characteristics justify the wide use of cpDNA to infer the history of plant populations (Petit et al. 2003, Magri et al. 2007, Petit and Vendramin 2007). We analysed chloroplast microsatellite (cpSSR) variation in C. ladanifer throughout its distribution range in order to: (i) locate its putative Quaternary refugia and reconstruct its recolonisation patterns in the highly heterogeneous Iberian Peninsula; and (ii) assess the role of geographic features such as mountain ranges, rivers and the Strait of Gibraltar as barriers to seed flow and expansion of the species. Whenever possible, molecular evidence is complemented with pollen data from bibliographic references. 2.2 Material and Methods 2.2.1 The Species Cistus ladanifer is an entomophyllous, obligatory outcrossing species, with a gametophytic mechanism of self-incompatibility (Talavera et al. 1993). An individual plant can produce more than 100,000 small, long-lived seeds each year (Bastida and Talavera 2002), which mainly fall beneath the mother plant canopy (Malo and Suárez 1998, Bastida and Talavera 2002). The seeds have a physical dormancy mechanism that can be interrupted by fire, high temperatures (PérezGarcía 1997) and smoke and nitrogenous salts (Pérez-Fernández and RodrıíguezEcheverría 2003). The post-fire recovery of plants in the Cistaceae is accomplished by massive seedling emergence during the first post-fire year (Ferrandis et al. 1999), which allows rapid regeneration of original populations (2 years after experimental burning, C. ladanifer covers 40% of the original area; Calvo et al. 2005). Although the main dispersal strategy of this species is barochory, different authors have described endozoochory by red deer (Malo and Suárez 1998) and sheep (Manzano et al. 2005) as other mechanisms of longdistance seed dispersal. Three C. ladanifer subspecies (africanus, ladanifer and sulcatus) have been described based on leaf traits (Demoly and Montserrat 1993). Subspecies ladanifer and africanus are present in the Iberian Peninsula and in North Africa, whereas subspecies sulcatus is restricted to limestone-derived soils on the southwest Iberian coast. A recent work (Guzmán and Vargas 2009) has dated the origin of C. ladanifer and divergence of its different subspecies in the Upper Pleistocene. cpDNA PHYLOGEOGRAPHY 36 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. 2.2.2 Plant sampling Thirty-eight C. ladanifer populations, covering almost the entire natural range of the species and its three subspecies, were sampled (Table 2.1). A longitudinal transect was established at each population. Ten plants separated by at least 5 m were selected along the transect and their ripe fruits were collected. Seeds were sown in Petri dishes and grown to seedling stage in the laboratory. One seedling per mother plant was selected for the subsequent analyses. Young plants were frozen in liquid nitrogen and conserved at )20 C until DNA extraction. In three populations (ESA, FVI and FVII), DNA was extracted from field-collected mature leaves from at least nine mother plants. 2.2.3 DNA extraction DNA was extracted from 100 mg of frozen leaves with a Dneasy® Plant Mini Kit (Qiagen, Hilda, Germany), following the manufacturer’s instructions. In several cases, an additional wash with 500 μl of absolute ethanol was necessary to remove secondary compounds from the DNA extracts. 2.2.4 Microsatellite analysis In an initial screening, universal cpSSR primers ccmp1 to ccmp10 (Weising and Gardner 1999) and Fagaceae cpSSR primers cmcs 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13 and 14 (Sebastiani et al. 2004) were tested on a subset of 30 samples from six geographically distant populations. Only primers ccmp1, ccmp2, ccmp3, ccmp5, ccmp10 and cmcs1 yielded consistent amplifications, of which ccmp1, ccmp5, ccmp10 and cmcs1 were monomorphic. The two polymorphic cpSSRs were then used to amplify all samples of the 38 populations. Amplification reactions were performed in a total volume of 12.5 μl containing 0.5 U GoTaq®DNA Polymerase (Promega, Madison, WI, USA), under standard reaction conditions. DNA was amplified under the following thermal profile: one denaturation cycle of 4 min at 95 C, followed by 25 cycles each consisting of 95 C for 30 s, 50 C for 30 s, 72 C for 30 s, with a final extension step at 72 C for 8 min. PCR products were loaded onto a 96 capillary automatic sequencer MegaBACE 1000 (GE Healthcare, Uppsala, Sweden). MegaBACE ET400 (GE Healthcare) was used as size standard. Fragment lengths were determined with the MegaBACE Fragment Profiler software, version 1.2 (GE Healthcare). Table 2.1: Populations included in this work. The country of origin is shown next to population name. FR: France; MO: Morocco; PT: Portugal; SP: Spain. Subsp: C. ladanifer subspecies. afr: subsp. africanus. lad: subsp. ladanifer. sul: subsp. sulcatus. Latitude and longitude are shown as decimal degrees; N.S.: number of plants surveyed in each population. The last three columns indicate different indices of within-population haplotypic diversity: Ne: effective number of haplotypes; HE: Nei’s (1987) haplotypic diversity; D2 SH: average genetic distances between individuals (Vendramin et al. 1998); Mean: mean ± standard deviation values. 37 Population/Country Code Subsp Lat Long N.S. NeHED2 SH Ketama /MO MKE afr 34.95º N 4.64º W 10 1.52 0.38 0.22 Bni Hadifa /MO MBH afr 35.02º N 4.17º W 10 1.22 0.20 0.10 Bab Tazaa /MO MBT lad 35.09º N 5.24º W 10 1.22 0.20 0.10 El Jebha /MO MEJ afr 35.18º N 4.64º W 10 1.00 0.00 0.00 Bni Bouchra/MO MSI afr 35.30º N 4.90º W 10 1.92 0.53 0.27 Bni Bouchra /MO MSII afr 35.30º N 4.89º W 12 2.77 0.70 0.58 Tanger /MO MTA afr 35.78º N 5.93º W 10 1.00 0.00 0.00 Almodóvar /SP EAL lad 36.16º N 5.65º W 10 1.00 0.00 0.00 Benalup de Sidonia /SP EBE afr 36.32º N 5.73º W 10 2.38 0.64 0.76 Sierra Bermeja /SP ESB lad 36.48º N 5.18º W 10 1.00 0.00 0.00 Sierra Palmitera /SP ESP lad 36.60º N 5.07º W 9 1.59 0.42 0.50 Tolox /SP ETO lad 36.68º N 4.93º W 10 1.22 0.20 0.10 Grazalema /SP EGR lad 36.78º N 5.27º W 10 1.00 0.00 0.00 Sierra de Aguas /SP EAC lad 36.84º N 4.79º W 10 1.00 0.00 0.00 Sierra Alhamilla /SP ESA lad 36.99º N 2.30º W 10 1.00 0.00 0.00 São Vicente cape /PT PSV sul 37.03º N 8.98º W 10 1.22 0.20 0.10 Burgau /PT PBU sul 37.07º N 8.78º W 9 1.53 0.39 0.19 Mazagón /SP EMA lad 37.15º N 6.84º W 10 2.27 0.62 1.78 Corte Figueira /PT PCF lad 37.39º N 8.03º W 9 1.53 0.39 0.19 Aljustrel /PT PAL lad 37.88º N 8.18º W 9 2.31 0.64 0.53 Cardeña /SP ECA lad 38.28º N 4.36º W 10 2.78 0.71 0.89 Despeñaperros /SP EDE lad 38.39º N 3.51º W 10 2.38 0.64 0.46 El Guijo /SP EGJ lad 38.52º N 4.77º W 10 1.72 0.47 0.23 La Codosera /SP ECO lad 39.19º N 7.08º W 10 2.00 0.56 0.28 Valdecaballeros /SP EVC lad 39.33º N 5.34º W 10 2.78 0.71 0.71 Martinchel /PT PMA lad 39.52º N 8.29º W 3 1.80 0.67 5.33 Vela /PT PVE lad 40.44º N 7.29º W 9 1.00 0.00 0.00 Ciudad Rodrigo /SP ECR lad 40.63º N 6.49º W 10 1.47 0.36 0.18 Sierra Guadarrama /SP EGU lad 40.68º N 4.10º W 10 1.22 0.20 0.10 Fuente Saúco /SP EFS lad 41.19º N 5.51º W 10 2.38 0.64 0.46 Macedo dos Cavaleiros /PT PMC lad 41.52º N 6.82º W 10 1.00 0.00 0.00 Ricobayo /SP ERB lad 41.70º N 5.81º W 10 1.92 0.53 0.27 Samil /PT PSA lad 41.78º N 6.75º W 7 1.00 0.00 0.00 Bragança /PT PBR lad 41.85º N 6.87º W 10 1.92 0.53 0.27 Monte Furado /SP EMF lad 42.39º N 7.20º W 10 1.00 0.00 0.00 Bárcena /SP EBA lad 42.57º N 6.56º W 10 1.85 0.51 0.72 La Bouverie /FR FVI lad 43.48º N 6.66º E 9 1.00 0.00 0.00 La Bouverie /FR FVII lad 43.49º N 6.66º E 9 1.00 0.00 0.00 Mean ± SD 1.58 ± 0.59 0.32 ± 0.27 0.40 ± 0.90 cpDNA PHYLOGEOGRAPHY 38 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. 2.2.5 Data analysis Genetic diversity within populations was assessed as the effective number of haplotypes (Ne = 1/Σpi 2), the haplotypic diversity (HE = [n/(n-1)][1-Σpi 2], where n is the number of individuals analysed in a population and pi is the frequency of the ith haplotype in a population; Nei 1987) and the D2 SH measure, as defined by Vendramin et al. (1998). The latter measure takes into account the difference in number of repeats among the different cpDNA haplotypes considered. The correlation between population genetic diversity parameters and latitude was assessed using the Spearman’s correlation index. Phylogenetic relationships among haplotypes were inferred with Network 4.5 (Fluxus Technology Ltd. at http://www. fluxus-engineering.com/sharenet.htm) by the median joining (MJ) method (Bandelt et al. 1999). Genetic differentiation among populations was estimated by analysis of molecular variance (AMOVA; Excoffier et al. 1992) with Arlequin software (version 3.11; Excoffier et al. 2005). The significance of the values was computed by a permutation test with 10,000 permutated matrices. The AMOVA was based on distances between cpSSR haplotypes, calculated as the sum of the squared number of repeat differences between two haplotypes: dxy = Σ[axi – ayi]2 (where axi and ayi are the number of repeats for the ith locus in haplotypes x and y). This gives ΦST, an analogue of Slatkin’s RST (Slatkin 1995) for population differentiation (Michalakis and Excoffier 1996). The possible presence of geographic structure was evaluated with several tests. First, we tested for the presence of a phylogeographic structure by comparing differentiation of unordered alleles (GST) and ordered alleles (RST) with the Permut CpSSR 2.0 software (Pons and Petit 1996; http:// www.pierroton.inra.fr/genetics/labo/ Software). One thousand random permutations of haplotype identities were carried out, while maintaining haplotype frequencies and the matrix of pair-wise haplotype differences as in the original study (Burban et al. 1999). If RST, which takes into account genetic differences between the haplotypes, is significantly higher than GST, this indicates the presence of a phylogeographical structure (Pons and Petit 1996), i.e., closely related haplotypes are more often found in the same geographical area than would be expected by chance. Second, we tested for a pattern of isolation by distance. A Mantel test with 10,000 ranFigure 2.1: Median Joining (MJ) network for Cistus ladanifer cpSSR haplotypes. Axes indicate the size of ccmp2 and ccmp3 variants for each haplotype. Thin lines in each of the three loops indicate links that could be removed following the coalescence theory predictions (Crandall and Templeton 1993). Haplotypes are indicated with the same patterns as in Fig.2.2 39 dom permutations was performed with the matrix of pair-wise genetic differentiation between populations, (ΦST ⁄ (1 – ΦST)), and a matrix of the ln (geographic distance) with the Arlequin 3.11 software (Excoffier et al. 2005). Third, we used a simulated annealing procedure implemented in the spatial analysis of molecular variance (SAMOVA) algorithm (Dupanloup et al. 2002) to define groups of populations that are geographically homogeneous and maximally differentiated from each other. The program repeatedly seeks the composition of a user-defined number, K, of groups of geographically adjacent populations that maximises ΦCT, the proportion of total genetic variance due to differences among groups of populations. The program was run for 10,000 iterations for K values from K = 2 to K = 15 from each of 100 random initial conditions. Within each of the groups defined by SAMOVA, separate AMOVA analyses were performed to partition the genetic diversity at intraand inter-population levels. Fourth, we tested for the presence of genetic barriers among Figure 2.2: Cistus ladanifer cpSSR haplotype map. Grey-shaded area indicates the present natural distribution of C. ladanifer. The same patterns are used for haplotypes as in Fig. 2.1. In order to obtain a proper view of haplotype distribution, some radial graphs were displaced from their original position (arrows connect them with their original position). Population codes (see Table 2.1) are presented next to each radial graph. cpDNA PHYLOGEOGRAPHY 40 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Source of variation d.f. SS Variance components % of total variance P (a) Whole data set (ΦST = 0.66) Among populations 37 212.70 0.57 66.35 < 0.0001 Within populations 327 94.31 0.29 33.65 Total 364 307.01 0.86 (b) SAMOVA results (ΦCT = 0.67) Among SAMOVA groups 3 164.19 0.82 66.83 < 0.0001 Among pops. within SAMOVA groups 34 48.51 0.12 9.69 < 0.0001 Within populations 327 94.31 0.29 23.48 < 0.0001 Total 364 307.01 1.23 (c) Rif group (ΦST = 0.42) Among populations 5 7.28 0.12 41.59 < 0.0001 Within populations 56 9.77 0.17 58.41 Total 61 17.05 0.30 (d) Betic group (ΦST = 0.06) Among populations 5 0.52 0.00 6.18 N.S. Within populations 53 3.34 0.06 93.82 Total 58 3.86 0.07 (e) Western group (ΦST = 0.32) Among populations 24 40.71 0.15 32.18 < 0.0001 Within populations 209 65.20 0.31 67.82 Total 233 105.91 0.46 (f) Geographic AMOVA (ΦCT = 0.25) Iberian Peninsula vs. N Morocco 1 35.12 0.26 25.07 0.003 Among populations within groups 36 177.58 0.48 46.97 < 0.0001 Within populations 327 94.31 0.29 27.96 < 0.0001 Total 364 307.014 1.03 Table 2.2: Analysis of molecular variance (AMOVA) of Cistus ladanifer (a) considering the whole data set, (b) SAMOVA groups (K = 4), (c to e) separate analyses for three groups defined by SAMOVA analysis and (f) hierarchical geographic AMOVA comparing groups of populations to the north and south of the Strait of Gibraltar. d.f. = degrees of freedom, SS = sum of squared deviation, P = level of probability of obtaining a more extreme component estimate by chance alone. n.s. = not significant (α = 0.05). populations using the Monmonier algorithm implemented in the BARRIER 2.2 software (Manni et al. 2004). Virtual points were added to the original tessellation ⁄ triangulation in order to indicate the presence of the Mediterranean Sea barrier and to enable connections to be established among populations from the South of France and central Iberian Peninsula. The distances used were pair-wise ΦST (Michalakis and Excoffier 1996). Statistical confidence for the predicted barriers 41 was obtained by resampling individuals within populations in order to obtain 100 bootstrap replicates of each genetic distance matrix. A hierarchical AMOVA was then performed in order to assess the partitioning of variance between the Iberian Peninsula and North Morocco. In addition, the genetic differences among populations were visualised through a principal coordinate analysis (PCoA) performed using GenAlEx 6.3 (Peakall and Smouse 2006) and a Euclidean genetic haploid distance similar to the genetic binary distance described by Huff et al. (1993). 2.3 Results The two polymorphic microsatellites, ccmp2 (three size variants) and ccmp3 (six size variants), were combined into 11 different haplotypes among the 365 individuals analysed (Fig. 2.1). The number of haplotypes per population ranged from 1 to 4, while the mean effective number of haplotypes for the 38 populations was 1.58 (with a range of variation between 1.00 and 2.78). In addition, the average HE and D2 SH values were 0.32 (0.00–0.71) and 0.40 (0.00–5.33), respectively (Table 2.1). None of the three diversity indices used was correlated with latitude: we can find depauperated populations (with diversity values = 0.00) and also diverse populations throughout the range of C. ladanifer. Three haplotypes (H1, H6 and H11) were singletons, while H2 was unique to one population in southern Spain (EMA). More than 30% of the plants shared the same haplotype (H4). Two haplotypes (H4 and H8) were present on both sides of the Strait of Gibraltar, whereas haplotype H7 only appeared in populations from southeast Spain (Fig. 2.2). Median-joining analysis resulted in a complex haplotype network, with three loops (Fig. 2.1). Haplotype H8 occupies a central place within the three loops and connects the common haplotypes, H3 and H7. Although a phylogeographic structure was not detected by the permutation analysis (GST not significantly different from RST), a strong genetic structure was observed in C. ladanifer populations, with high values of both GST (0.60 ± 0.06) and RST (0.57 ± 0.12). The AMOVA analysis also revealed a high and significant value for inter-population differentiation (ΦST = 0.66) (Table 2). The isolation by distance test showed that among-population differentiation increased significantly with the ln (geographical distance) (Mantel test; P < 0.05), although the regression accounted for only a very low proportion of the total variance (r2 = 0.013). The SAMOVA analyses indicated distinct groups of genetically defined geographic areas; when K = 2, a group that comprised populations from the Betic area (EAC, EGR, ESA, ESB, ESP, ETO) was separated from the rest of the populations (ΦCT = 0.61). In analyses where K = 3, an additional partition was identified that subdivided the second group into two areas: one comprising populations from the South of France, the whole of the Iberian Peninsula (except the Betic area) and a population from North Morocco; the other comprising populations of C. ladanifer subsp. africanus from the Rif area (ΦCT = 0.64). When K = 4, a cpDNA PHYLOGEOGRAPHY 42 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. new group comprising a single population from the southwest Iberian Peninsula (EMA) was identified (ΦCT = 0.67). With value of K between 5 and 15, ΦCT values did not increase significantly, and in most cases the newly defined groups comprised single populations. Thus, SAMOVA analysis suggested the presence of three main geographic groups: one from North Morocco (hereafter Rif group), a group from the southeast Iberian Peninsula (hereafter Betic group) and a widespread group that occupies an area throughout the range of C. ladanifer (hereafter Western group); a fourth group is also present comprising a single population on the southwest coast of Spain (Huelva). The degree of population differentiation differed within each group (Table 2.2). Despite the short geographic distances among populations, the Rif group had the highest value of inter-population differentiation (ΦST = 0.42), whereas the widespread Western group had a lower differentiation value (ΦST = 0.32). No significant differentiation among populations within the Betic group was observed (Table 2.2). The analysis using pair-wise Figure 2.3: BARRIER results. Ten barriers were computed with ΦST pairwise distance matrices. The statistical support for each barrier (computed from the basis of 100 random matrices) is showed as line thickness. Only those barriers with a frequency higher than 50% were represented. The value of each barrier (ΦST) is indicated with a colour code (green, yellow, red). Populations are indicated by grey dots. The population code is indicated next to each dot (see Table 2.1). In some cases the population code is connected with an arrow to its dot. 43 ΦST distances in BARRIER software partially corroborated the SAMOVA analysis (Fig. 2.3). Although several barriers were inferred throughout the distribution area of C. ladanifer, the strongest genetic boundaries (with both high ΦST as well as high bootstrap support) were found in North Morocco (with barriers isolating single populations) and especially around the Betic area of the Iberian Peninsula. We stress that the BARRIER analyses separating the Iberian Peninsula and North Morocco were not inferred, although AMOVA analysis revealed that 25% of molecular variance was explained by genetic differences between populations north and south of the Strait of Gibraltar (Table 2). Principal coordinate analysis (PCoA) extracted two axes that explained 100% of genetic variation (see Supplementary Material S 2.1). Plotting the populations along these axes revealed a grouping that matches the SAMOVA results: three groups (Betic, Rif and Western) were obtained, whereas the EMA population was plotted next to the populations from the Western cluster. This grouping has a certain taxonomic relevance. Population EBE, which belongs to C. ladanifer subsp. africanus is plotted in an intermediate position between Western and Rif group, whereas the two populations of subsp. sulcatus were placed together with populations of subsp. ladanifer. 2.4 Discussion Chloroplast microsatellites have been widely used in phylogeographic studies (Magri et al. 2007, Fady et al. 2008, Pardo et al. 2008). Although some criticisms of cpSSRs arose due to problems with homoplasy, the level of homoplasy has been considered to be low enough to permit population genetic analysis (Provan et al. 1999a). Even when homoplasy has been identified, it was considered ‘moderate’ and its potential for invalidating results can be disregarded (Cuenca et al. 2003). Estoup et al. (2002), using simulations, concluded that the large amount of variability at microsatellite loci often largely compensates for homoplasious evolution, and cases in which size homoplasy may be a problem are related to high mutation rates. No estimate of mutation rates in Cistus is available but the lower mutation rate of chloroplast microsatellites compared to nuclear microsatellites (Provan et al. 1999b) suggests that size homoplasy may not be a major problem for studies at within-species level. In this work C. ladanifer populations exhibited levels of genetic differentiation (GST = 0.60) that are similar to the mean and median values for maternally inherited markers in angiosperms (Petit et al. 2005a). Most of the molecular variance occurs between each of the four population groups, which exhibit high frequencies of haplotypes H2 (EMA), H7 (Betic group), H8 (Rif group) and H4 and H5 (Western group). From these haplotypes, it is possible that H8 is the more ancestral, since it has the largest number of connections with other haplotypes and is found in a more central position in the network (Posada and Crandall 2001). Furthermore, H8 gave rise to H7 and H4 haplotypes, cpDNA PHYLOGEOGRAPHY 44 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. which are the most frequent in the Betic and Western group of populations, respectively. These facts could point to the origin of the Betic and Western groups of C. ladanifer as a result of independent colonisation events from North Africa to the Iberian Peninsula, as proposed by Guzmán and Vargas (2009) based on chloroplast and nuclear sequence analysis. The chronology of these colonisations is not clear, although the presence of C. ladanifer pollen in sites from the Betic area before the Last Glacial Maximum (around 90,000 years BP, Pons and Reille 1988, Fig. 2.4) and in southern Portugal during the Late Glacial (around 12000 years BP, Fletcher et al. 2007, Fig. 2.4) might indicate that it could have occurred during the Middle Pleistocene, a few million years after the opening of the Strait of Gibraltar at the end of the Messinian. Since opening (ca. 5.33 Ma, Hsü et al. 1977), the sea barrier of the Strait (14-km wide and about 400-m deep) has been maintained even during glacial maxima, although the lower sea level allowed emergence of different islands that reduced the width of the sea channels (Collina-Girard 2001): C. ladanifer should have been able to cross the Strait of Gibraltar using the emerging islands as stepping stones. The isolation effect caused by the Strait has left some traces on the genetic structure of C. ladanifer populations. First, three of the four groups of populations inferred by SAMOVA are present only on one side of Figure 2.4: Sites and chronology of different deposits where samples of C. ladanifer pollen were recovered. Numbers next to each point indicate the chronology (years before present) of the first occurrence of C. ladanifer pollen in each site. The bibliographic sources of pollen data are reported in Supplementary material S 2.2. Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer L have multiple origins Chapter 3 This chapter reproduces the published paper: Quintela-Sabarís C, Vendramin GG, CastroFernández D, Fraga MI (2010) Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer L have multiple origins Plant and Soil 334:161-174 Previous page: Cistus ladanifer subsp. africanus growing in a degraded Tetraclinis articulata forest in the Bni Bouchra ultramafic area (Rif, N of Morocco). (Photo: C. QuintelaSabarís) Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer L have multiple origins Keywords: Chloroplast microsatellites; Cistus ladanifer; genetic diversity; heavy metals; ultramafic areas ABSTRACT Cistus ladanifer L. (Cistaceae) is a Mediterranean shrub covering different kinds of soils in the Western Mediterranean area. This species has colonised several metalliferous areas (serpentine outcrops as well as human-polluted sites) throughout its distribution range, and is therefore an interesting species to study the possible effects on genetic diversity and differentiation produced by the colonisation of areas polluted with heavy metals. The genetic structure of 33 natural populations distributed across its entire natural distribution range (Morocco, Portugal and Spain) and growing on either metalliferous or non-metalliferous soils was investigated using chloroplast microsatellites. Population genetic parameters were estimated and genetic groups were identified using Bayesian inference. In addition, we compared the genetic diversity and differentiation among metallicolous and non-metallicolous populations within each Bayesian-defined group. The cpSSR data suggested that metallicolous populations of Cistus ladanifer have arisen through multiple independent evolutionary origins within two different chloroplast lineages. Evidence that the soil type provoked genetic bottlenecks in metallicolous populations or genetic differentiation among metallicolous and non-metallicolous populations was not observed. Historical factors are the main cause of the present genetic structure of C. ladanifer. The nature of tolerance to heavy metals as a species-wide trait in this shrub is discussed. 3.1 Introduction Sites with high heavy metal contents in soils, either of natural origin (such as weathering of ultramafic bedrocks) or generated by anthropic activities (mining and industrial activities, atmospheric deposition, excessive use of agrochemicals or even highway traffic) (Padmavathiamma and Li 2007) are interesting areas for plant researchers due to their specific soil conditions and distinctive flora. Although some metals, such as Cu or Zn, are essential for their development, the occurrence of high contents of heavy metals has several toxic effects on plants: binding to proteins and alteration of their structure, displacement of essential elements resulting in deficiency effects or even promoting the formation of free radicals (Hall 2002). Metal toxicity, together with deficiency in nutrient contents, yield areas that are usually shallow with rocky soils and low moisture and with scarce plant cover (Brady et al. 2005). Thus, serpentine outcrops and mine deposits act as edaphic discontinuities in mainland regions, which have been defined as ecological or ‘edaphic islands’ (Lefèbvre and Vernet 1990). When colonising these ‘islands’, plant populations have to cope with several environmental constraints (metCHAPTER 3 54 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. als, dryness, isolation, etc.) that can leave their imprints on the genetic structure of plant populations. For instance, it has been proposed that plant populations in metalliferous areas can suffer a founder effect, which would significantly reduce their genetic diversity (Lefèbvre and Vernet 1990). Moreover, in some plant species, populations growing in metalliferous soils have shown significant genetic differentiation with respect to metal tolerance from neighbouring populations in ‘normal’ soils, even with the occurrence of substantial gene flow (Vékemans and Lefèbvre 1997, Linhart and Grant 1996 and references therein). Deng et al. (2007) using RAPD markers found significant genetic differentiation between mine populations and uncontaminated populations of the pseudometallophyte Sedum alfredii. Plants growing in metalliferous soils can be either exclusive metallophytes (plants restricted to metalenriched habitats) or facultative metallophytes (also called pseudometallophytes), that is, species having both metallicolous (growing in metalliferous soils) and non-metallicolous populations (Wu 1990). Thus, pseudometallophytes are interesting species when studying the potential influence of environmental constraints on patterns of genetic diversity (Linhart and Grant 1996). Several works have investigated the distribution of neutral genetic diversity within and between metallicolous populations (M) and non-metallicolous populations (NM) of different pseudometallophyte plant species (mainly herbaceous or undershrubs), using either isozymes (Wu et al. 1975, Ducousso et al. 1990, Westerbergh and Saura 1992, Bush and Barrett 1993, Vekemans and Lefèbvre 1997, Nordal et al. 1999, Nyberg Berglund and Westerbergh 2001) or DNA based markers (Mengoni et al. 2000, 2001, 2006, Pauwels et al. 2005). In most cases, similar values of within-population diversity were estimated in M and NM populations, although in some works a diversity decrease was observed in M populations of Deschampsia cespitosa (Bush and Barrett 1993), Lychnis alpina (Nordal et al. 1999) and copper mine (but not serpentine) populations of Silene paradoxa (Mengoni et al. 2001). Another subject that researchers have focused on, is the origin of the M populations. Analyses of diverse pseudometallophytes (Bush and Barrett 1993, Vekemans and Lefèbvre 1997, Mengoni et al. 2001, Pauwels et al. 2005) support the theory that geographically distant M populations (but even at short distances of hundreds of meters; Al-Hiyali et al. 1988) could have evolved independently from neighbouring NM populations; that is, M and NM populations do not constitute different phylogenetic lineages, and genetic distances among N and NM populations are a function of geographic distances between them. These findings underline the importance of studying historical factors and population-genetic processes in order to dissect the effects of the demographic processes (such as patterns of migration or bottlenecks) from those related to selective processes (Staton et al. 2001). 55 Cistus ladanifer L. (gum rockrose) is a woody, semi-deciduous shrub growing in a wide range of latitudes, altitudes and climatic conditions in the Western Mediterranean region (South of France, Iberian Peninsula and North of Algeria and Morocco) (Demoly and Montserrat 1993). Its populations constitute early successional stages adapted to disturbances in Mediterranean ecosystems, in particular fires (Bastida and Talavera 2002). It is a pseudometallophyte that has established populations over different types of bedrock material (granites, schists, slates, etc.) and has also colonized different ultramafic areas in N Morocco (Bni Bouchra) (Ater et al. 2000), S Spain (Málaga) (Alados et al. 1999), NE Portugal (Trás-Os-Montes) (Díez Lázaro et al. 2006, Freitas et al. 2004a) and diverse mine tailings in Central to South-Western Iberian Peninsula (Murciego et al. 2007, Freitas et al. 2004b). C. ladanifer is an entomophyllous, obligatory outcrossing species, bearing a gametophytic mechanism of incompatibility (Talavera et al. 1993). It is the major component of shrublands in oligotrophic acid soils in the western half of the Iberian Peninsula (Rivas-Martínez 1979). Three subspecies have been described based on leaf traits (Demoly and Montserrat 1993). Two subspecies, Cistus ladanifer subsp. ladanifer and subsp. africanus, are widespread and they have colonized M (ultramafic) areas, although only subsp. ladanifer is found also in mine tailings from the Iberian Peninsula. Finally C. ladanifer subsp. sulcatus (formerly C. palhinhae) is restricted to limestone derived soils on the coast of the southwestern tip of Portugal. In this work, we have analysed 33 Cistus ladanifer populations sampled throughout the species distribution range using chloroplast (cp) DNA markers (microsatellites, SSRs). These markers are of special interest when studying colonisation patterns. Chloroplast DNA is generally maternally inherited in angiosperms, whose dispersion is therefore mediated by seeds only. In addition, the effective population size for haploid cpDNA is smaller than diploid nuclear genes, so the differentiation due to genetic drift can be stronger (Comes and Kadereit 1998) and phenomena like genetic bottlenecks can be more easily detected (Echt et al. 1998). For instance, cpSSR markers detected a reduction in genetic diversity within M populations of Silene paradoxa where RAPD markers failed (Mengoni et al. 2001). These characteristics justify the wide use of cpDNA in order to infer the population history of plant populations (Petit et al. 2003, Magri et al. 2007). Once the phylogeography of the species has been inferred, a better understanding of the effect of metal pollution on the genetic structure of populations is possible, avoiding spurious correlations resulting from historical or demographic processes (Staton et al. 2001). This is especially interesting in C. ladanifer, since it colonizes M areas at different latitudes (from N Morocco to NE Portugal) in a region whose physiographic (spatial heterogeneity) and climatic diversity offers complex phylogeographic patterns (Gómez and ORIGIN OF METALLICOLOUS POPULATIONS 56 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Lundt 2007). In the present paper, the following main questions were addressed: Do M populations of C. ladanifer have the same origin? Did M populations of Cistus ladanifer suffer a reduction in diversity? Do differences exist in the demographic effects of the colonisation of M areas along a latitudinal gradient? 3.2 Material and Methods 3.2.1 Plant and soil sampling Thirty-three Cistus ladanifer populations covering almost the entire distribution natural range of this species were sampled. The subspecies growing in each site was identified on the basis of morphological traits. We included metallicolous (M) populations from different geographic areas: ultramafic outcrops of Bni Bouchra (N of Morocco), Málaga (SE of Spain) and Trásos-Montes (NE Portugal), and M populations growing on mine tailings from the centre of the Iberian Peninsula (Table 3.1). After the analyses of phyto-available trace metals, two populations (EAL and EDE), growing near highways, were included in the M group (see Fig. 3.1). In each population, a longitudinal transect was established. Ten plants separated by at least 5 m were selected along the transect and their ripe fruits were collected. Seeds were sown and seedlings grown in a laboratory. One seedling per mother plant was selected for the subsequent analyses. Young plants were frozen in liquid nitrogen and conserved at −20°C until DNA extraction. In addition, in each site one (or two) soil samples were collected from 5 to 15 cm in depth. Each soil sample was airdried and sieved through a 2 mm-mesh. 3.2.2 Soil chemical analyses Sieved soil subsamples were milled in an agatha mortar to achieve homogeneity. Total amounts of Cr, Cu, Mn, Ni, Pb and Zn in soils were quantified in solid subsamples with Energy-Dispersive X-Ray Fluorescence spectrometry (EDXRF). Other subsamples were digested with HNO3 for the quantification of Co contents with Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) element analysis. In order to determine the concentration of heavy metals potentially available for plants, 10 g of dried soil was mixed with an extraction solution (Ammonium Acetate 0.5 M + EDTA 0.02 M + Acetic Acid 0.5 M, buffered at pH 4.65) (Lakanen and Erviö 1971) in a ratio of Table 3.1: Within-population haplotypic diversity estimates. First column includes population name and code. Second column indicates Cistus ladanifer subspecies: afr: subsp. africanus; lad: subsp. ladanifer; sul: subsp. sulcatus. Geographic coordinates are given in decimal degrees. M: metallicollous population; (u): ultramafic area; (m): mine tailing; (h): highway affected area. NM: non-metallicollous population; N.S.: number of plants surveyed in each population; HE: Nei’s (1987) haplotypic diversity; r(7): haplotypic richness after rarefaction to the uniform sample size of 7 (El Mousadik and Petit, 1996);D2 SH: average genetic distances among individuals (Vendramin et al. 1998); Overall Mean: mean for the whole set of populations ± standard deviation values. 57 Population (Code) Subsp Long Lat Soil Type Substratum N.S. HEr(7) D2 SH Ketama (MKE) afr 4.64º W 34.95º N NM Schists 10 0.38 1.40 0.22 Bni Hadifa (MBH) afr 4.17º W 35.02º N NM Sandstones 10 0.2 0.70 0.1 Bab Tazaa (MBT) lad 5.24º W 35.08º N NM Micaschists 10 0.2 0.70 0.1 El Jebha (MEJ) afr 4.64º W 35.18º N NM Sandstone 10 0 0 0 East Bni Bouchra (MSII) afr 4.89º W 35.29º N M (u) Serpentinised peridotite 12 0.7 2.16 0.58 West Bni Bouchra (MSI) afr 4.90º W 35.30º N M (u) Serpentinised peridotite 10 0.53 1 0.27 Tanger (MTA) afr 5.93º W 35.78º N NM Sandstone 10 0 0 0 Almodóvar (EAL) lad 5.65º W 36.16º N M (h) Clays close to a road 10 0 0 0 Benalup (EBE) afr 5.73º W 36.32º N NM Sandstone 10 0.64 2.33 0.76 Sierra Bermeja (ESB) lad 5.18º W 36.48º N M (u) Serpentinised peridotite 10 0 0 0 Sierra Palmitera (ESP) lad 5.07º W 36.60º N M (u) Serpentinised peridotite 9 0.42 1.56 0.5 Sierra de Tolox (ETO) lad 4.93º W 36.68º N M (u) Serpentinised peridotite 10 0.2 0.70 0.1 Grazalema (EGR) lad 5.27º W 36.78º N NM Decarbonated limestone 10 0 0 0 Sierra de Aguas (EAC) lad 4.79º W 36.84º N M (u) Serpentinised peridotite 10 0 0 0 São Vicente (PSV) sul 8.98º W 37.03º N NM Limestone 10 0.2 0.70 0.1 Burgau (PBU) sul 8.78º W 37.07º N NM Limestone 9 0.39 0.97 0.19 Mazagón (EMA) lad 6.84º W 37.15º N NM Sand deposits 10 0.62 1.87 1.78 Corte Figueira (PCF) lad 8.03º W 37.39º N NM Schists 9 0.39 0.97 0.19 Aljustrel (PAL) lad 8.18º W 37.88º N M (m) Pyrite mine tailing 9 0.64 1.78 0.53 Cardeña (ECA) lad 4.36º W 38.28º N NM Granite 10 0.71 1.93 0.89 Despeñaperros (EDE) lad 3.51º W 38.39º N M (h) Quartzites, close to the highway. 10 0.64 1.70 0.46 El Guijo (EGJ) lad 4.77º W 38.52º N NM Slates 10 0.47 0.99 0.23 La Codosera (ECO) lad 7.08º W 39.19º N M (m) Sb mine tailing 10 0.56 1 0.28 Valdecaballeros (EVC) lad 5.34º W 39.33º N NM Sedimentary material (gravels, clays) 10 0.71 2.39 0.71 Vela (PVE) lad 7.29º W 40.43º N NM Granite 9 0 0 0 Ciudad Rodrigo (ECR) lad 6.49º W 40.63º N NM Quartzites 10 0.36 0.93 0.18 Guadarrama (EGU) lad 4.10º W 40.68º N NM Granite 10 0.2 0.70 0.1 Fuente Saúco (EFS) lad 5.51º W 41.19º N NM Sandstone and conglomerates 10 0.64 1.70 0.46 Macedo dos Cavaleiros (PMC) lad 6.82º W 41.52º N M (u) Serpentinised peridotite 10 0 0 0 Ricobayo (ERB) lad 5.81º W 41.70º N NM Quartzites and filites 10 0.53 1 0.27 Samil (PSA) lad 6.75º W 41.78º N M (u) Serpentinised peridotite 7 0 0 0 Bragança (PBR) lad 6.87º W 41.85º N M (u) Dunite 10 0.53 1 0.27 Monte Furado (EMF) lad 7.20º W 42.39º N NM Schists 10 0 0 0 Overall mean ± SD 0.31 ± 0.27 0.91 ± 0.77 0.27 ± 0.36 ORIGIN OF METALLICOLOUS POPULATIONS 58 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. soil:extraction solution of 1:5. The suspension was shaken for 30 min, after which it was allowed to stand for at least half an hour and was then filtered through paper (Albet DP 145). The filtrate was stored cold to be analysed with an atomic absorption spectrophotometer (AAS). The following available trace metals were determined with AAS: Co, Cr, Cu Mn, Ni, Pb and Zn. 3.2.3 DNA extraction DNA was extracted from 100 mg of frozen leaves using Dneasy® Plant Mini Kit (QIAGen), following the manufacturer’s indications. In several cases an additional wash with 500 μl of absolute ethanol was needed in order to remove secondary compounds from the DNA extracts. 3.2.4 Microsatellite analysis In an initial screening, universal cpSSR primers ccmp1 to ccmp10 (Weising and Gardner 1999) and Fagaceae cpSSR primers cmcs 1 to 14 (Sebastiani et al. 2004) were tested on a subset of 30 samples from 6 geographically distant populations. Only primers ccmp1, ccmp2, ccmp3, ccmp5, ccmp10 and cmcs1 yielded consistent amplifications, of which ccmp1, ccmp5, ccmp10 and cmcs1 were monomorphic. The two polymorphic cpSSRs were then used to amplify all samples of the 33 populations. Amplification reactions were performed in 12.5 μl total volume using 10 ng of template DNA, 1× reaction buffer (Promega, Madison, WI, USA) containing 1.5 mM of MgCl2, 0.2 μM of each primer, 0.2 mM of each dNTP, 1% of bovine serum albumin, and 0.5 U of GoTaq® DNA Polymerase (Promega). DNA was amplified with the following thermal profile: one denaturation cycle of 4 min at 95°C, followed by 25 cycles each consisting of 95°C for 30 s, 50°C for 30 s, 72°C for 30 s with a final extension step at 72°C for 8 min. PCR products were loaded on a capillary automatic sequencer MegaBACE 1000 (GE Healthcare Biosciences). MegaBACE ET400 (GE Healthcare Biosciences) was used as size standard. Fragment lengths were determined using the MegaBACE FRAGMENT PROFILER software version 1.2 (GE Healthcare Biosciences). 3.2.5 Data analysis A Principal Component Analysis (PCA) was performed in order to aggregate populations according to (1) total contents of metals Co, Cr, Cu, Mn, Ni, Pb and Zn in soils (referred here as CoT, CrT, CuT, MnT, NiT, PbT and ZnT) or (2) Ammonium Acetate/EDTA metal extractable contents (referred here as CoE, CrE, CuE, MnE, NiE, PbE and ZnE). Values below detection limits were recorded as 0.1 μg.g−1 for statistical analysis. With the PCA, we reduced the dimensionality of the data, retaining, in our case, two first Principal Components (PCs) that contribute most to the variance of soils. A Varimax rotation was applied to the PCAs in order to simplify the interpretation of the extracted Principal Components. Phylogenetic relationships among haplotypes were inferred with NETWORK 4.5 (Fluxus Technology Ltd. at www. fluxus-engineering.com) using the median 59 joining (MJ) method (Bandelt et al. 1999). We applied the three criteria (frequency, topology and geography) proposed by Pfenninger and Posada (2002) in order to remove loops or ambiguities in the haplotype network. Different parameters of genetic diversity within populations were estimated: (1) the haplotypic diversity (HE = [n/(n-1)][1-Σpi 2], (where n is the number of individuals analysed in a population and pi is the frequency of the i-th haplotype in a population; Nei 1987), (2) the haplotypic richness r(n), which is obtained after rarefaction to a uniform sample size of n (in our study, the value of n was fixed at 7, the lowest size of the analysed populations), as described in El Mousadik and Petit (1996), and (3) the D2 SH measure, as defined by Vendramin et al. (1998), which takes into account the difference in the number of repeats among the different cpDNA haplotypes considered. In addition, the distribution of the pairwise cpSSR repeat length differences among individual plants, totalled over all two cpSSR loci within an individual plant, was plotted to compare different patterns. Genetic differentiation among populations was estimated by the analysis of molecular variance (AMOVA, Excoffier et al. 1992) using Arlequin software (version 3.11; Excoffier et al. 2005). The significance of the values was computed by a permutation test from 10,000 permutated matrices. The AMOVA was based on distances between cpSSR haplotypes, calculated as the sum of the squared number of repeat differences between two haplotypes: dxy = Σ[axi – ayi]2 (where axi and ayi are the number of repeats for the ith locus in haplotypes x and y). This gives ΦST, an analogue of Slatkin’s RST (Slatkin 1995) for population differentiation (Michalakis and Excoffier 1996). The Cavalli-Sforza and Edwards distances based on haplotype frequencies were used to construct a Neighbour Joining (NJ, Saitou and Nei 1987) dendrogram with the Populations software (O. Langella, UMR de Génétique Végétale, Ferme du Moulon, Gif/Yvette, France). To test for node robustness, bootstrapping was performed on individuals using 1,000 resamplings. In order to infer population genetic structure, Bayesian analysis using a spatial clustering model implemented in BAPS software version 5.2 was performed (Corander et al. 2008). These authors have shown that the spatial model improves the statistical power to detect the underlying population structure when dealing with a low number of loci. The spatial clustering of groups model was run using each population, with known coordinates, as the unit to be clustered. We initially fixed k (the number of clusters) from 2 to 25. We then selected the value of k that had the minimum log marginal likelihood and re-ran the analysis 100 times to obtain the optimal partition of populations. A neighbour-joining tree was then constructed (Saitou and Nei 1987) with the Kullback–Leibler divergence matrix provided as output with BAPS. This matrix can be used as a measure of relative genetic distance between the BAPS-identiORIGIN OF METALLICOLOUS POPULATIONS 60 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. fied clusters (BAPS 5.2 manual distributed with the program). Taking into account the grouping performed by BAPS, we (1) compared the levels of intra-population diversity of M and NM populations using an analysis of variance (ANOVA) and (2) tested the differentiation of M and NM populations with additional separate AMOVA analyses (Excoffier et al. 1992). With this two stepapproach (BAPS and ANOVA/AMOVA) we tried to extract first the effect of phylogeography and then analyse the effect of colonisation of metallicolous areas within phylogeographic homogeneous groups of populations, thus avoiding confounding effects of phylogeography on the effect of metal pollution over population genetic structure. Isolation-by-distance patterns between populations were tested considering all populations and then considering M and NM populations separately. A Mantel test with 10,000 random permutations was performed with the matrix of pairwise genetic differentiation between populations, NM Pop M Pop Human-polluted M Pop Ultramafic Pop Type PC1 (54% of total variance) 4,02,00,0 PC2 (38%of total variance) 6,0 4,0 2,0 0,0 A PC1 (46% of total variance) 6,04,02,00,0 PC2(39%of total variance) 4,0 2,0 0,0 B EAL EAL EDE EDE PBR PBR 1,00,0 1,0 0,0 ZnT PbT NiT MnT CuT CrT CoT 1,0 0,0 1,0 0,0 ZnE PbE NiE MnE CuE CrE CoE MSII ESP ETO PMC PSA MSIEAC ESB PAL ECO MSII ESP ETO PMC PSA MSI EAC ESB PAL ECO PC 1 PC 2 PC 2 PC 1 Figure 3.1: Principal Component Analysis (PCA) results. Populations are ordinated according to the total (Fig. 1a) or Ammonium Acetate/ EDTA extractable (Fig. 1b) quantities of heavy metals in their soils. The percentage of variance explained by each axis is reported. Circles indicate non-metallicolous (NM) populations, whereas triangles and squares indicate metallicolous (M) populations from ultramafic areas and humanpolluted soils, respectively. The codes of M populations are also indicated. In the upper right-hand corner of each graph the loading of each metal on each of the PCs is reported. 67 populations of a given species seems to be common in pseudometallophytes (Mengoni et al. 2001, Nyberg Berglund and Westerbergh 2001, Vekemans and Lefèbvre 1997), even at a scale of hundreds of metres (Al-Hiyali et al. 1988). Interestingly, we found M populations within two chlorotype lineages while, for example, in Arabidopsis halleri, a model pseudometallophyte, all M populations, of independent origins, come from one chlorotype lineage only (north of the Alps) (Pauwels et al. 2005, 2008). Most of the works on genetic structure of pseudometallophytes did not reveal evidence of genetic bottlenecks in M populations (for review, see Vekemans and Lefèbvre 1997, Mengoni et al. 2000). These results were mainly based on nuclear markers (isozymes or RAPDs), so the possible founder effect at nuclear loci may be eroded by subsequent pollen flow from neighbouring NM populations which could increase genetic variation in M populations. In contrast, chloroplast markers are (generally) maternally inherited, so they will only reflect the effect of seed flow. To our knowledge, only two other studies have analysed pseudometallophytes using chloroplast markers, obtaining contrasting results: Mengoni et al. (2001) detected a founder effect in Silene paradoxa populations growing on copper mine deposits, whereas Pauwels et al. (2005) did not find any difference in genetic diversity between M and NM populations of Arabidopsis halleri. To explain these different patterns of diversity between S. paradoxa and A. halleri, Pauwels et al. (2005) proposed that the colonisation of metalpolluted environments is associated with a genetic bottleneck in species with populational tolerance (that is, species in which metal tolerance is found only in those populations growing on metalliferous soils), whereas in species with constitutive (or “specieswide”) tolerance (such as A. halleri) the effect of a bottleneck may not be detected. In the case of Cistus ladanifer, once we had extracted the phylogeographHEr(7) D2 SH M NM Sig. M NM Sig. M NM Sig. Cluster defined by BAPS South 0.308 0.203 n.s. 0.903 0.738 n.s. 0.242 0.180 n.s. North 0.339 0.387 n.s. 0.783 1.061 n.s. 0.220 0.371 n.s. Whole data 0.325 0.332 n.s. 0.838 0.964 n.s. 0.230 0.314 n.s. Table 3.3: ANOVA results. The table presents the mean values for M (Metallicolous) and NM (Non-Metallicolous) of within-population diversity indexes for each cluster of populations (defined after Bayesian analysis), and for the whole set of populations. HE: Nei’s (1987) haplotypic diversity. r(7): allelic richness (El Mousadik and Petit, 1996) with a fixed rarefaction size of 7. D2 SH: average genetic distances among individuals (Vendramin et al. 1998). Sig.: significance value. n.s.: not-significant (α value= 0.05). ORIGIN OF METALLICOLOUS POPULATIONS 68 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. ic effect through the Bayesian analysis, we did not detected any significant differentiation nor significant differences in the genetic diversity between edaphic types, even in the M populations of more recent origin, i.e. populations growing on mine tailings from the Iberian Peninsula. These inferences may suggest that M populations were founded recently by a high number of individuals, or that the foundation events are antique but in the presence of a significant seed flow (cpDNA is maternally inherited in Cistus ladanifer; Guzmán and Vargas 2009) from neighbouring NM populations that masked the effect of genetic bottlenecks and limited genetic differentiation between edaphic types. Both hypotheses assume that the metalliferous areas do not exert selective pressures on Cistus ladanifer; however, it should be stressed that variation of neutral markers, like cpSSRs, cannot be related to variation of adaptive traits such as tolerance to heavy metals (Le Corre and Kremer 2003), as it occurred in Thlaspi caerulescens (Jiménez-Ambriz et al. 2007). Thus, genetic differentiation between M and NM populations at genes related to metal-tolerance can be significantly underestimated. Nevertheless, as discussed by Vekemans and Lefèbvre (1997), after a bottleneck the number of alleles at neutral loci increases as a result of new mutations, when population size increases. This phenomenon can be observed in the pseudometallophyte Silene paradoxa (Mengoni et al. 2001): 8 populations (5 serpentine outcrops, 2 copper mines, 1 nonmetallicolous) separated up to 205 km showed 13 cpSSR haplotypes exclusive to one population (of 27 haplotypes detected) and no haplotypes were shared by all populations. In contrast, in the case of C. ladanifer only a singleton was exclusive of M populations. This fact suggests that the inferred levels of diversity cannot be caused by mutations after a bottleneck following the colonisation of metalliferous areas. To conclude, following Pauwels et al. (2005) and considering the multiple origins of M populations and the lack of bottlenecks, we propose that the tolerance to heavy metals could be a characteristic of Cistus ladanifer, even though this plant is more commonly found in non-metalliferous areas, as already observed in Thlaspi montanum (Boyd and Martens 1998) or in Andropogon virginicus (Gibson and Risser 1982). Indeed, our hypothesis is supported by the results obtained by Kidd et al. (2004), who observed that nonmetallicolous populations from North of Portugal showed high tolerance to Cu and Zn in hydroponic cultivation. Taking into account our findings on Cistus ladanifer and those obtained by other authors, we believe that this plant could be very useful in the recovering of degraded soils in the Mediterranean region, also considering that it has other interesting traits (Frérot et al. 2006). First, C. ladanifer is adapted to water stress (Nuñez-Olivera et al. 1996) so it can cope with a long, dry summer season. Second, it has a high growth rate and productivity (Patón et al. 1998) when conditions 69 permit, developing dense root and shoot systems that can limit soil erosion. And third, it is a native species of Western Mediterranean flora; therefore, its use in phytoremediation would not imply any potential threats to ecosystems deriving from the use of alien species (Méndez and Maier 2008; and references therein), an important issue in the biodiversity-rich Mediterranean area. Acknowledgments We thank Olivier Langella for providing us with a compiled version of the Populations software; and Dr. Margarida Ribeiro and Dr. Rita Costa for their help in the first phases of this work. Alessio Mengoni and two anonymous referees provided useful comments that improved this paper. 3.5 References Alados CL, Navarro T, Cabezudo B (1999) Tolerance assessment of Cistus ladanifer to serpentine soils by developmental stability analysis. 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In this tailing we quantified (total contents): 752 mg·kg-1 As; 374 mg·kg-1 Cu; 1098 mg·kg-1 Mn; 2347 mg·kg-1 Pb and 633 mg·kg-1 Zn. Cistus ladanifer subsp. ladanifer has naturally colonised this tailing and now it is the dominant species. (Photo: C. QuintelaSabarís) Heavy metal accumulation in leaves of divergent chloroplast lineages of the pseudometallophyte Cistus ladanifer L. Implications for phytostabilization Keywords: Accumulation, Cistus ladanifer, heavy metals, pseudometallophyte ABSTRACT Cistus ladanifer is a shrub which grows in different kinds of soils (including serpentine outcrops and human-polluted sites) in the Western Mediterranean area. Chloroplast DNA analyses have inferred that metallicolous populations of this plant have arisen through distinct foundation events within two different postglacial recolonisation lineages. We have quantified the levels of Co, Cr, Cu, Mn, Ni, Pb and Zn in soils and in leaves of C. ladanifer plants from 33 populations, covering almost its entire natural range. In addition, we have computed the ratio between metal contents in leaves and in soils, as a measure of accumulation abilities. Through a nested analysis of variance (nANOVA) we tried to evaluate whether the population type (metallicolous vs. non-metallicolous) or the chloroplast lineage (‘North’ vs. ‘South’) differ in their metal contents and accumulation patterns. Our results show that, on a broad scale, metallicolous populations have higher Ni contents than non-metallicolous ones (p<0.001) and lineage ‘North’ higher Mn contents than lineage ‘South’ (p<0.001). In addition, Cistus ladanifer clearly rejects the accumulation of Co, Cr and Pb in its leaves, whereas for the other metals there were different accumulation patterns between lineages and population types. The implications of our results in the use of Cistus ladanifer for phytostabilization procedures are discussed. 4.1 Introduction Areas with high contents of heavy-metals in soils are the result of natural processes (weathering of ultramafic rocks) or human activities (mining and industrial activities, atmospheric deposition, excessive use of agrochemicals or even traffic emissions). Several technologies have been employed in order to remediate human polluted soils, the most of them based on expensive mechanical soil treatments that sometimes include soil removal and replacement. In recent years, phytoremediation, that is, the use of different plant species for soil remediation, has been proposed as an environmentally friendly technology that in addition has a lower economic cost than traditional approaches (Padmavathiamma and Li 2007). Plant growth is inhibited in the most severely contaminated sites, so human and animal exposure to heavy metals can be increased by the migration of contaminated soil (erosion, dispersal by wind) or leaching into groundwater (Ruttens et al. 2006). In sites with high and multi-elemental contamination, phytostabilization (the use of native or introduced plants to transform soil metals to less toxic forms, but not remove the metal from the soil, Chaney CHAPTER 4 76 Evolutionary origin and ecophysiology of metallicolous populations of Cistus ladanifer L. Table 4.1: Description of Cistus ladanifer populations included in this work. Population (Code) Lineage Type Substratum Vegetation Long Lat Sierra de Aguas (EAC) South M (u) Serpentinised peridotite Open shrubland with dispersed Pinus pinea and P. halepensis trees 4.79º W 36.84º N Almodóvar (EAL) North M (h) Clays close to a road Grassland with dispersed C. ladanifer plants 5.65º W 36.16º N Benalup (EBE) South NM Sandstone Cleared Quercus suber forest with Phyllirea angustifolia, Calicotome villosa and C. ladanifer 5.73º W 36.32º N Cardeña (ECA) North NM Granite Open Quercus ilex forest with C. ladanifer and Pistacia lentiscus 4.36º W 38.28º N La Codosera (ECO) North M (m) Sb mine tailing Shrubland dominated by C. ladanifer with Dittrichia viscosa 7.08º W 39.19º N Ciudad Rodrigo (ECR) North NM Quartzites Dense matorral with isolated Quercus ilex trees 6.49º W 40.63º N Despeñaperros (EDE) North M (h) Quartzites, close to the highway. Dense Cistus ladanifer shrubland with Quercus ilex and Juniperus oxycedrus 3.51º W 38.39º N Fuente Saúco (EFS) North NM Sandstone and conglomerates Shrubland with Quercus ilex 5.51º W 41.19º N El Guijo (EGJ) North NM Slates Open dehesa with Quercus ilex and isolated C. ladanifer plants 4.77º W 38.52º N Grazalema (EGR) South NM Decarbonated limestone Shrubland with C. ladanifer and C. monspelliensis 5.27º W 36.78º N Guadarrama (EGU) North NM Granite Dense shrubland with isolated Quercus ilex 4.10º W 40.68º N Mazagón (EMA) South NM Sand deposits Eucalyptus globulus and Pinus pinea plantations 6.84º W 37.15º N Monte Furado (EMF) North NM Schists Dense shrubland with Quercus ilex 7.20º W 42.39º N Ricobayo (ERB) North NM Quartzites and filites Open shrubland with Quercus ilex 5.81º W 41.70º N Sierra Bermeja (ESB) South M (u) Serpentinised peridotite Dense shrubland with scattered Pinus pinaster 5.18º W 36.48º N Sierra Palmitera (ESP) South M (u) Serpentinised peridotite Open matorral with Quercus coccifera and Ulex sp. 5.07º W 36.60º N Sierra de Tolox (ETO) South M (u) Serpentinised peridotite Open matorral with scattered Pinus pinaster and Ulex sp. 4.93º W 36.68º N Valdecaballeros (EVC) North NM Sedimentary material (gravels, clays) Dense shrubland with Pinus pinaster 5.34º W 39.33º N Bni Hadifa (MBH) South NM Sandstones Open Pinus halepensis forest 4.17º W 35.02º N Bab Tazaa (MBT) North NM Micaschists Open shrubland with dispersed Cistus plants 5.24º W 35.08º N El Jebha (MEJ) South NM Sandstone Open Pinus halepensis forest 4.64º W 35.18º N Ketama (MKE) South NM Schists Dense shrubland with evergreen oaks 4.64º W 34.95º N Chloroplast lineage and population type following Quintela-Sabarís et al. (2010). Longitude and latitude are expressed in decimal degrees. 83 Taking into account the factor Type, significant differences between M and NM populations of Ni content in leaves were found (Fig. 4.2). In addition, the ratios leaf: soil total for all the metals, except Pb, were significantly higher in NM populations. The interaction Lineage × Type, revealed significant differences only for the characters ZnL and CoL:CoT (Fig. 4.3). ZnL in M populations of lineage ‘North’ was higher than NM populations from that lineage, whereas in lineage ‘South’ the M populations had a ZnL value lower than NM ones. In the case of CoL:CoT, the group of M populations have lower values than NM populations for both lineages. However, the accumulation of metals varied considerably degree among populations, since for all the characters analysed the factor ‘Population’ nested within the interaction Lineage × Type showed significant values (in CrL, P < 0.01; in the remaining characters, P < 0.001). The combined analysis of the quantities of metals in leaves resulted in a PCA where the M populations from human-polluted sites are placed next to other NM populations along PC1 (which is mainly made up of ZnL, CuL and CoL), whereas the M populations from ultramafic areas are placed apart from the other populations, with negative scores on the first PC and positive scores on the second PC (mainly made up of CrL and NiL) (Fig. 4.4a). When considering all the ratios leaf: total soil, M populations (with ratio values usually lower than, or near, 1) are grouped in the negative scores on PC1. In contrast, NM populations are more scatFigure 4.2: ANOVA results for the factor ‘Type’. Asterisks indicate statistically significant differences. * P < 0.05; ** P<0.01; *** P<0.001. Means for each group were log-transformed for graphical purposes. PATTERNS OF METAL ACCUMULATION IN LEAVES [Document text truncated for crawler view.]