PÂMELA LAVOR ROLIM ______Dissertação De Mestrado Natal/RN, Março De 2013

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE BIOCIÊNCIAS PROGRAMA DE PÓS-GRADUAÇÃO EM SISTEMÁTICA E EVOLUÇÃO

GENÉTICA E BIOLOGIA REPRODUTIVA DE Vriesea minarum (BROMELIACEAE): EM BUSCA DE ESTRATÉGIAS DE CONSERVAÇÃO NO QUADRILÁTERO FERRÍFERO, MINAS GERAIS

PÂMELA LAVOR ROLIM ______Dissertação de Mestrado Natal/RN, março de 2013

PÂMELA LAVOR ROLIM

GENÉTICA E BIOLOGIA REPRODUTIVA DE Vriesea minarum (BROMELIACEAE): EM BUSCA DE ESTRATÉGIAS DE CONSERVAÇÃO NO QUADRILÁTERO FERRÍFERO, MINAS GERAIS

Dissertação a ser apresentada à coordenação do Programa de Pós Graduação em Sistemática e Evolução da Universidade Federal do Rio Grande do Norte, como parte dos requisitos para obtenção do título de Mestre em Sistemática e Evolução.

Orientador: Profº Dr. Leonardo de Melo Versieux.

Natal, 2013

PÂMELA LAVOR ROLIM

GENÉTICA E BIOLOGIA REPRODUTIVA DE Vriesea minarum (BROMELIACEAE): EM BUSCA DE ESTRATÉGIAS DE CONSERVAÇÃO NO QUADRILÁTERO FERRÍFERO, MINAS GERAIS

Dissertação apresentada ao Programa de Pós- graduação em Sistemática e Evolução da Universidade Federal do Rio Grande do Norte, em cumprimento às exigências para obtenção do título de Mestre em Sistemática e Evolução.

Aprovada em: 11 de março de 2013.

Comissão Examinadora:

______Dra. Vânia Cristina Rennó Azevedo – EMBRAPA/CENARGEN

______Dr. Fábio de Almeida Vieira – UFRN

______Dr. Leonardo de Melo Versieux – UFRN (orientador)

Dedico este trabalho a todos aqueles que acreditaram em mim e que nunca me deixaram desistir.

AGRADECIMENTOS Gostaria de agradecer a CAPES por conceder o auxílio financeiro para desenvolvimento deste projeto e ao Instituto Estadual de Florestas (IEF) pela autorização de pesquisa na Parque Estadual da Serra do Rola-Moça (PESRM). Da mesma forma, agradeço antes de todos, a Deus, pois apenas ele sabe todas as dificuldades e atribulações pelas quais já passei até hoje, e ele sempre foi meu esteio, meu abrigo, meu refúgio e força nos momentos em que mais precisei. Em segundo lugar, e não menos importante, agradeço a minha família: meu pai, minha mãe, minhas irmãs (Priscila e Paloma), meu cunhado e meus grandes amores: minhas sobrinhas Giovanna e Giulia. Para muitos, a família talvez seja a base de agradecimento, mas para mim, eles são os principais responsáveis por eu ter conseguido chegar aonde cheguei: graças ao apoio de meu pai e minha mãe, sempre me dediquei aos estudos e, mesmo quando enfrentando dificuldades e privações, eles se faziam presentes da melhor forma possível e me apoiaram e me impulsionaram para frente, quando nem eu tinha mais forças para a luta. Minhas irmãs contribuíram igualmente, mas de formas diferentes: foi na tentativa de amadurecimento que mais cresci e, mesmo que inconscientemente, elas acabaram crescendo e também fazendo parte de mim. Minhas sobrinhas são meus olhos no futuro: vejo nelas o que um dia fui e a cada dia tento ser um exemplo de dedicação e coragem que talvez elas possam seguir. Ao meu cunhado, que mesmo longe, ainda ajudava da melhor forma possível e talvez, o seu menor esforço, o de fazer minha família feliz, já se tornou mais que suficiente para me fazer bem. Gostaria de agradecer também a todos os meus amigos (Juliana, Bruna, Yuri e Franscico), que mesmo após tantos anos de separação, ainda se mostram presentes e me apoiam em qualquer momento ou decisão. De igual forma, agradeço à minha tia Zulmira, que me acolheu como filha e me ajudou durante todas as minhas coletas, sendo “toda” a minha família diante do desconhecido. Agradeço também a todos os que me ajudaram durante minha passagem por Minas Gerais, em especial aos alunos da UFMG, que tentaram me auxiliar ao máximo possível, em especial o doutorando Flávio do Carmo, que foi fundamental para meus trabalhos de coleta: obrigada não só por sua ajuda em campo, mas também por partilhar sua experiência e conhecimento sobre as cangas mineiras.

Agradeço aos funcionários do Parque Estadual da Serra do Rola-Moça, que me auxiliaram no campo e me receberam de braços abertos para o desenvolvimento de minha pesquisa: em especial Cláudio Costa, que além de um colega, tornou-se um amigo. Agradeço aos alunos, funcionários e ao Profº. responsável pelo Laboratório Molecular de Plantas, Dr. Cássio van den Berg, na Universidade Estadual de Feira de Santana, que não tiveram apenas a paciência de me ensinar o trabalho de laboratório: partilharam comigo suas experiências, vivências, anseios e sonhos e incentivos para prosseguir. Agradeço, também, a Maria Cristina, que além de “tutora”, tornou-se uma grande amiga, a quem ainda hoje dedico minhas orações. Agradeço à Profª Dra. Claudia Maria Jacobi e ao Prof. Dr. Cássio van den Berg, por terem acolhido a mim e ao meu projeto em seus laboratórios, e por terem tido a paciência em me ensinar, trocar experiências e partilhar seus grandes conhecimentos: muito obrigada pela parceria. Agradeço ao Programa de Pós-Graduação em Sistemática e Evolução, aos seus coordenadores, Profs. Iuri Baseia e Bruno Goto, a todo o corpo docente, pelas excelentes disciplinas oferecidas e à Secretária Gisele de Melo, por múltiplos apoios. Agradeço a Profª Alice Calvente, pela ajuda em campo e nas elucidações de dúvidas; e ao Profº Dr. Sérgio Maia Queiroz Lima e ao Dr. Anderson Vilasboas de Vasconcellos, por toda a ajuda teórica e prática, durante a obtenção dos resultados e pela excelente disciplina de genética de populações e filogeografia. Por fim, há duas pessoas que gostaria de agradecer especialmente: 1º, ao meu orientador, Profº Dr. Leonardo de Melo Versieux, que aceitou me orientar, mesmo sem me conhecer, e propôs este projeto, o qual veio a ser um grande desafio e ao mesmo uma grande chance de aprendizado. Obrigada por confiar e acreditar em mim e por tornar-se meu parceiro mais fiel. Em 2º lugar, e não menos importante, ao Dônis, que no longo desses dois anos tornou- se muito mais que meu companheiro ou colega de profissão: tornou-se meu melhor amigo. Obrigada por me apoiar e me dizer as palavras certas quando precisei e também por se calar quando era necessário; obrigada por nunca me abandonar, mesmo frente a tantos meses de distância, fruto de nosso oficio; e obrigada também por tentar fazer de todos os meus dias os melhores possíveis, tornando esta uma das melhores épocas da minha vida! Obrigada a todos!

RESUMO Vriesea minarum é uma espécie de bromélias rupícola, com populações naturalmente fragmentadas, restrita a região do Quadrilátero Ferrífero, em Minas Gerais, Brasil. É uma espécie ameaçada, que está sofrendo com a perda de habitat devido ao crescimento das cidades e às atividades de mineração. O conhecimento da variabilidade genética em populações de plantas é um dos principais ramos de genética da conservação, associando dados genéticos para as estratégias de conservação, enquanto que o conhecimento sobre a biologia reprodutiva pode ajudar na compreensão de aspectos fundamentais da história de vida, distribuição e estratégias de sobrevivência das plantas. Assim, o estudo da diversidade, riqueza, estrutura genética e biologia reprodutiva das populações de V. minarum podem contribuir para o desenvolvimento de ações de conservação. O capítulo 1 apresenta a transferabilidade de 14 loci de microssatélites para V. minarum. Entre os resultados desse capítulo, destaca-se o sucesso da transferabilidade de 10 loci de microssatélites descritos para outras espécies de Bromeliaceae, sendo todos eles polimórficos. No capítulo 2, é apresentada a genética de 12 populações de V. minarum que se distribuem por todo o Quadrilátero Ferrífero. Os resultados obtidos mostram pouca estruturação entre as populações (Fst = 0,088), mas com diferentes valores de riqueza (média = 2.566) e diversidade genética (média = 0.635) para todas as populações; o coeficiente de endogamia foi alto (Gis = 0.376). Estes dados podem ser resultado da ação de polinizadores e/ou dispersão de sementes eficientes, já que as populações são naturalmente fragmentadas. No capítulo 3, é estudada a biologia reprodutiva e morfologia floral de uma população de V. minarum, situada no Parque Estadual da Serra do Rola-Moça, Minas Gerais. Como resultado, foi possível identificar que a espécie possui floração de janeiro a março; com flores que duram dois dias; síndrome mista de polinização; sendo primariamente alógama, mas também tem capacidade para ser auto fecundada. Espera-se que dados obtidos nos capítulos 1, 2 e 3 sirvam como base para outros estudos com espécies de campos rupestres ferruginosos, já que até o presente momento, não é de nosso conhecimento a existência de registros de outras pesquisas feitas com espécies endêmicas do Quadrilátero Ferrífero, que busquem conciliar o entendimento da genética, com os dados da biologia reprodutiva, tendo como alvo a conservação da biodiversidade neste hábitat altamente ameaçado pela mineração. Torna-se crucial uma análise cuidadosa para a criação de novas áreas de proteção, para conservação das populações da espécie. Palavras-chave: Biologia e morfologia floral; campos rupestres ferruginosos; canga, genética de populações; microssatélites; mineração; transferabilidade.

ABSTRACT Vriesea minarum is a rupiculous bromeliad species, with naturally fragmented populations, restricted to the Iron Quadrangle, Minas Gerais, Brazil. It is a threatened species, which is suffering from habitat loss due to the growth of cities and mining activities. The knowledge of genetic variability in plant populations is one of the main branches of conservation genetics, linking genetic data to conservation strategies while the knowledge about plant reproductive biology can aid in understanding key aspects of their life story, as well as in the comprehension of their distribution and survival strategies. Thus, the study of diversity, richness, and genetic structure, as well as the reproductive biology of populations of V. minarum can contribute to the development of conservation actions. Chapter 1 presents the transferability of 14 microsatellite loci for V. minarum. Among the results of this chapter, we highlight the successful transferability of 10 microsatellite loci described for other species of Bromeliaceae, all of which are polymorphic. In Chapter 2, we present the genetic analyses of 12 populations of V. minarum that are distributed throughout the Iron Quadrangle. We used the 10 microsatellite loci tested in Chapter 1. The results show a low population structuring (Fst = 0.088), but with different values of genetic richness (mean = 2.566) and gene diversity (mean = 0.635) for all populations; and a high inbreeding coefficient (Gis = 0.376). These may be the result of pollinators action and/or efficient seed dispersal, thus allowing a high connectivity among populations of naturally fragmented outcrops. The reproductive biology and floral morphology of a population of V. minarum, located in the Parque Estadual da Serra do Rola-Moça, are studied in Chapter 3. This reserve is the only public environmental protection area where the species occurs. As a result of field experiments and observations, we found that the species has its flowering period from January to March, with flowers that last for two days and that it has a mixed pollination syndrome. It is primarily alogamous, but also has the capacity to be self-ferilized. It is expected that data obtained in chapters 1, 2 and 3 serve as basis for other studies with species from the ferruginous rocky fields, since until now, to our knowledge, there are no other survey of endemic species from the Iron Quadrangle, seeking to merge the genetic knowledge, with the data of the reproductive biology, with the ultimate aim of biodiversity conservation. Considering the great habitat loss for the species by mining, it becomes crucial to analyze the creation of new protected areas for its conservation.

Key words: Canga; ferruginous rocky fields; genetic and reproductive biology; microsatellites; mining; population genetics; transferability

SUMÁRIO 1 INTRODUÇÃO GERAL ...... 9 1.1 PRINCÍPIOS DA GENÉTICA DA CONSERVAÇÃO ...... 9 1.2 QUADRILÁTERO FERRÍFERO: IMPORTÂNCIA ECOLÓGICA FRENTE À EXPLORAÇÃO ECONÔMICA ...... 10 1.3 BROMELIACEAE ...... 12 1.4 GENÉTICA DE POPULAÇÕES EM BROMELIACEAE ...... 14 1.5 BIOLOGIA REPRODUTIVA EM BROMELIACEAE ...... 16 2 OBJETIVOS ...... 18 2.1 OBJETIVO GERAL: ...... 18 2.2 OBJETIVOS ESPECÍFICOS: ...... 18 3 CAPÍTULO 1 ...... 19 4 CAPITULO 2 ...... 30 5 CAPITULO 3 ...... 63 6 CONCLUSÃO GERAL ...... 90 6.1 IMPLICAÇÕES PARA A CONSERVAÇÃO ...... 90 7 REFERENCIAS BIBLIOGRÁFICAS ...... 92 8 ANEXOS ...... 99 8.1 NORMAS DAS REVISTAS ...... 99

1 INTRODUÇÃO GERAL

1.1 PRINCÍPIOS DA GENÉTICA DA CONSERVAÇÃO

Atualmente, muito se tem falado sobre a grande devastação que a ação humana tem causado nos ambientes naturais. Nestes termos, nos deparamos com um cenário em que muitas espécies estão sendo extintas da natureza. Diante desta preocupação, que é a crescente perda da biodiversidade mundial, há algumas décadas, mais precisamente na década de 80 (século XX), uma nova área de pesquisa passa a ganhar força no meio científico: a biologia da conservação (FRANKHAM, 2005). A biologia da conservação é uma área multidisciplinar, que integra conceitos ecológicos, demográficos, biológicos, sociais, políticos e, atualmente, genéticos, na tentativa de unir esforços na luta pela preservação da biodiversidade, por meio da definição de unidades e prioridades para estratégias de manejo, assim como desenvolvimento de modelos demográficos e estudo sobre valor adaptativo de populações naturais ou em cativeiro (FRANKHAM, 1995; EIZIRIK, 1996; HEDRICK, 2001). A biologia da conservação vem crescendo como área de pesquisa principalmente através do uso de dados da genética das populações das espécies, buscando sempre conservar ao máximo a variabilidade genética das populações, que se reflete em suas capacidades de evoluírem e se adaptarem ao meio (MORITZ, 2002; STOCKWELL et al., 2003). Através de diferentes metodologias empregando-se dados moleculares, é possível descobrir e compreender a diferenciação genética populacional gerada por fatores como isolamento reprodutivo, a deriva genética ou adaptação local, sendo importante esse reconhecimento para tratamento das populações como um legado evolutivo único para a espécie (NARUM, 2006). A genética da conservação utiliza-se também de dados moleculares, sendo que, em sua maioria, são os mesmos empregados em estudos de genética de populações, tais como AFLPs (Polimorfismo de Comprimento de Fragmento Amplificado), RAPDs (DNA Polimórfico Aleatoriamente Amplificado), SNP (Polimorfismo de Nucleotídeo Único), ISSRS (Inter Simples Sequência Repetida), SSR (Simples Sequência Repetida ou microssatélites) e sequenciamento de DNA, que são os mais comuns tanto para animais quanto para plantas (DESALLE; AMATO, 2004). Da mesma forma, análises estatísticas clássicas de genética de população também foram inicialmente utilizadas para mensurar os níveis de variação genética entre populações (como as estatísticas F ou coeficientes de endogamia de

Wright - Fst), sendo muito criticadas (e.g. MEIRMANS; HEDRICK, 2011), passando então outros métodos de análise da genética de populações a serem utilizados para conservação, como o Fst refinado (AMOVA - análise de variância molecular) e análises baseadas na teoria da coalescência (DESALLE; AMATO, 2004). De acordo com DeSalle e Amato (2004), a maior importância do uso da genética da conservação vem de sua capacidade de ajudar a melhor entender os padrões e processos genéticos e evolutivos (como depressão por endogamia, reprodução efetiva, tamanho populacional, níveis de variação genética e fluxo gênico) de espécies ameaçadas, permitindo uma melhor descrição dos processos que deram origem aos atuais estados de ameaça destas populações ou espécies. Nesse contexto, torna-se amplamente discutido na literatura sobre genética da conservação que, a perda de habitat, o aumento da fragmentação ou redução no tamanho das populações de uma dada espécie, pode levar a erosão genética, diminuindo assim sua capacidade de adaptação e as suas chances da sobrevivência (e.g. ELLSTRAND; ELLAN, 1993; FRANKHAM, 1995; 2005; WOODWORTH et al., 2002; CHUNG; CHUNG, 2008; PETIT et al., 1998; SEGELBACHER et al., 2010). Assim sendo, o entendimento dos fatores envolvidos na história de vida de uma população, principalmente os genéticos, é o primeiro passo se tentar garantir sua continuada sobrevivência e manutenção de seu potencial evolutivo no presente e no futuro (MORITZ, 2002; STOCKWELL et al., 2003; DING et al., 2008), sendo estes os princípios, no qual, se baseiam o presente trabalho.

1.2 QUADRILÁTERO FERRÍFERO: IMPORTÂNCIA ECOLÓGICA FRENTE À EXPLORAÇÃO ECONÔMICA

A região do Quadrilátero Ferrífero (QF), que compreende uma área de aproximadamente 7.000 km² na parte centro-sul do Estado de Minas Gerais e no extremo sul da serra do Espinhaço, é conhecida no âmbito nacional e internacional por suas características econômicas e histórico-culturais, por conta de sua grande riqueza em recursos minerais (CARVALHO FILHO et al., 2010). O QF constitui-se com uma enorme unidade geotectônica pertencente ao Cráton do São Francisco, com algumas das rochas mais antigas do Escudo Brasileiro; apresentando gigantescos depósitos de minério de ferro (29 bilhões de toneladas que contêm cerca de 50-

65% de ferro), além de ouro, diamantes, entre outros minerais (CHAVES et al., 1998; HARTMANN et al., 2006). Por ser essa formação geológica antiga e complexa, o QF mostra-se como um ambiente altamente heterogêneo. Tal heterogeneidade é documentada também em sua biota, onde a vegetação é formada por um mosaico de fitofisionomias, principalmente os campos rupestres ferruginosos, nos quais são observadas as “cangas” - afloramentos de minério de ferro que podem formar carapaças rígidas no nível do solo e que formam autênticas “ilhas de ferro” nos topos das serras, em altitudes que variam de 900 a 1.900m (JACOBI; CARMO, 2008a; GARCIA et al., 2009). Em todo o mundo, ambientes como esse são ricos em espécies endêmicas de plantas, muitas vezes bem adaptadas às altas concentrações de metais, e que são conhecidas como metalófitas (PORTO; SILVA, 1989; TEIXEIRA; LEMOS-FILHO, 1998; ROSIÈRE; CHEMALE, 2000; BUTCHER et al., 2009). Da mesma forma, no QF, a grande diversidade advém do fato das cangas constituírem um reduto para diversas espécies que apresentam adaptações fisiológicas, morfológicas e reprodutivas aos seus vários hábitats (JACOBI et al., 2007; JACOBI; CARMO, 2008a). Como um reflexo disto, estudos recentes apontam para a ocorrência de 1.109 espécies distribuídas em 456 gêneros, em 115 famílias de plantas vasculares no QF, sendo que destas, 36 espécies foram encontradas apenas nas cangas da região (JACOBI; CARMO, 2012). Além da riqueza biológica, Carmo et al. (2012) destacam que cerca de 20% das cavernas inventariadas no Brasil formaram-se em geossistemas ferruginosos, onde podem ser encontradas paleotocas, artefatos de cerâmica e pedra, além de desenhos de antigos povos indígenas. Assim, além de toda essa importância cultural e biológica, as cangas também possuem uma função ecológica importantíssima: elas permitem a recarga dos lençóis freáticos por funcionarem como esponjas, transferindo a água da chuva para as nascentes e formando um sistema de aquíferos, que chegam a armazenar até 4 bilhões de m3 de água, que abastecem a região metropolitana de Belo Horizonte (CARMO et al., 2012). No entanto, apesar dessa imensa riqueza, a região do QF, por ser um dos principais polos de extração de minério de ferro no mundo, possui ecossistemas entre os mais ameaçados e menos estudados no Brasil, já que a forte atividade mineradora acaba alterando todo o meio ambiente, com grandes impactos para toda a biodiversidade da região (JACOBI; CARMO, 2008b).

Este cenário de destruição é ainda mais grave por conta das dificuldades de implementação de programas de recuperação de áreas degradadas, já que há muitas lacunas no conhecimento sobre a biologia das espécies de cangas, levando ao uso de espécies exóticas nas tentativas de reabilitação (GARCIA et al., 2009). De acordo com Jacobi e Carmo (2012) hoje apenas 2% da área do Quadrilátero está dentro de áreas protegidas, sendo que nos últimos anos, cerca de 40% das áreas de canga já foram perdidas, expondo ainda mais as diversas espécies da flora, especialmente as endêmicas, ao risco da extinção. No entanto, apesar deste cenário, aparentemente tem havido um aumento na preocupação mundial com as necessidades de preservação da biodiversidade nas áreas de exploração de minérios, com a criação de políticas públicas de proteção em alguns países. Além disso, foi estabelecida a política de “orientação de boas práticas para a mineração e biodiversidade” criada pelo Conselho Internacional de Mineração e Metais (ICMM), que visa à conservação e o planejamento do uso da terra pelas mineradoras; e que deveria servir como modelo para as políticas ambientais brasileiras frente ao crescente aumento da devastação e perda irreversível dos ecossistemas pela atividade mineradora (JACOBI et al., 2011).

1.3 BROMELIACEAE

Bromeliaceae é uma das famílias de angiospermas mais diversa em termos morfológicos, ecológicos e em riqueza de espécies sendo bem distribuída (e quase que endêmica) na região dos neotrópicos (GIVNISH et al., 2011). Atualmente Bromeliaceae inclui 3.248 espécies em 58 gêneros (LUTHER, 2010), classificados em oito subfamílias (GIVNISH et al., 2007; 2011). Muitos autores possuem apontamentos controversos sobre a origem da família Bromeliaceae, mas acabam por concordar que sua origem é antiga, mas sua radiação e diversificação são bem recentes (BENZING, 2000a). Givnish et al. (2011) concluem através de filogenias moleculares datadas que as bromélias teriam surgido no Escudo das Guianas, cerca de 100 milhões de anos (Ma), com suas linhagens mais modernas começando a divergir há apenas 19 Ma, e se difundindo por todo o novo mundo há 16-13 Ma, com uma dispersão secundária para a África a 9.3 Ma. Givnish et al. (2011) mostram ainda que a distribuição atual das Bromeliáceas é fruto de dois grandes processos de radiação: a do núcleo das Tillandsioideae, originado nos Andes a cerca de 14,2 Ma; e a das Bromelioideae, no escudo brasileiro (com sua origem na região da

Serra do Mar) a cerca de 9,1 Ma, explicando então, grande parte da imensa diversidade e riqueza de espécies na América do Sul. A família apresenta uma enorme diversificação em termos de estruturas morfológicas e fisiológicas para adaptação aos mais diversos habitats (de florestas úmidas a desertos) onde podem crescer como epifítas, rupículas ou terrestres (SMITH; DOWNS, 1974). Entre as adaptações mais conspícuas observadas nas bromélias, permitindo que cresçam em habitats díspares, ressalta-se: formação de uma roseta acumuladora de água; folhas espessas, coriáceas, com reserva de água em um parênquima aqüífero; presença de tricomas foliares que substituem as raízes para absorção de água e nutrientes; fotossíntese CAM; síndromes de polinização e dispersão de sementes ajustas ao ambiente em que se encontram, entre outras (BENZING, 2000a; VERSIEUX et al., 2010a). Dessa forma, não é surpresa que as bromélias superem a maioria das outras famílias em vários aspectos, principalmente em riqueza de espécies e, consequentemente, em importância biológica, sendo elas tão bem adaptadas aos mais diversos habitats, em especial aqueles de maior estresse hídrico e adversidade, como os ambientes xéricos dos afloramentos rochosos (BENZING, 2000a; VERSIEUX et al., 2010a) e, no presente caso, os afloramentos de minério de ferro ou cangas. Dentro do importante papel ecológico desempenhado pelas bromélias, destaca-se que muitos organismos vivem ou dependem direta ou indiretamente da água que pode se acumular entre as folhas ou que as bromélias podem facilitar o estabelecimento de outras espécies vegetais mais sensíveis, durante a colonização de ambientes extremos (BENZING, 2000b; SCARANO, 2002). Deste modo, a conservação das bromélias tem um efeito secundário muito positivo na preservação de vários outros organismos; nos ambientes de afloramentos rochosos, a água retida entre as bases das folhas de várias espécies de bromélias é extremamente importante para manutenção de condições toleráveis por outros organismos (BENZING, 2000b). Os ambientes caracterizados pela presença de solos lateríticos, tais como as cangas, apresentam severas restrições ao estabelecimento de formas de vida vegetais, seja pela alta concentração de determinados metais, seja por fatores ligados à permeabilidade do substrato e à condição de estresse hídrico daí decorrente (PORTO; SILVA, 1989). No entanto, os afloramentos de canga são extremamente ricos em espécies vegetais, muitas delas endêmicas, e constituem-se como um ambiente de grande heterogeneidade topográfica (ROSIÈRE; CHEMALE, 2000).

A região do Quadrilátero Ferrífero (QF) é uma das mais importantes em termos de riqueza de espécies de bromélias com um elevado número de espécies endêmicas e ameaçadas de extinção (VERSIEUX; WENDT, 2007; VERSIEUX et al., 2008; 2010b). A espécie objeto de estudo da presente dissertação pertence ao gênero Vriesea, que é o mais rico em espécies no Brasil, com distribuição predominante em áreas úmidas da mata atlântica e que apresenta um marcado endemismo em áreas montanhosas (e.g. VERSIEUX; WENDT, 2007; VERSIEUX, 2011; GOMES-DA-SILVA, 2012). Vriesea pertence à subfamília Tillandsioideae, que é facilmente reconhecida pelas folhas sem espinhos e por ter frutos capsulares, com sementes plumosas e anemocóricas (SMITH; DOWNS, 1977). Vriesea minarum L.B. Sm. é, endêmica do QF, com ocorrência em 12 municípios diferentes ao redor de Belo Horizonte, mas sempre restrita aos afloramentos rochosos de minério de ferro, acima de 1.000 m de altitude (VERSIEUX, 2011). Suas populações vêm sofrendo perdas significativas (VERSIEUX; WENDT, 2007) e atualmente encontra-se listada como ameaçada (categoria EN da IUCN) pelos critérios adotados pelo Centro Nacional de Conservação da Flora (CNCFLORA, 2013).

1.4 GENÉTICA DE POPULAÇÕES EM BROMELIACEAE

O conhecimento de como a diversidade genética está estruturada em um contexto espacial, tanto dentro de uma, quanto em várias populações, pode contribuir significativamente para a compreensão da história evolutiva e dinâmica populacional das espécies (MOREIRA et al., 2010). Esta estruturação pode ser definida como uma distribuição de alelos ou genótipos no espaço ou no tempo, sendo ela o resultado da ação (em conjunto) de processos como mutação, migração, deriva e seleção, além de fatores como sistema reprodutivo; morfologia floral; mecanismo de polinização; dispersão de sementes; fenologia; ciclo de vida; tempo de reprodução; estágio sucessional; distribuição geográfica, entre outros (LOVELESS; HANDRICK, 1984). Dessa forma, existem diferentes técnicas de biologia molecular que permitem o estudo e detecção da variabilidade e estruturação em populações (FERREIRA, 2001), sendo essas informações extremamente importantes, pois podem servir como base para a utilização e conservação dos recursos genéticos disponíveis (GUERRA et al., 1998; BERT et al., 2002). Muitos estudos de genética de populações têm sido realizados com populações naturais de bromélias do Brasil nos últimos anos (e.g. CAVALLARI et al., 2006; BARBARÁ et al.,

2007; 2009; PAGGI et al., 2007, 2008, 2010, 2012; PALMA-SILVA et al., 2004, 2007, 2009, 2011; ZANELLA et al., 2012). Sendo que muito destes estudos empregaram os mais diferentes tipos de marcadores moleculares nos mais diversos ambientes. Sgorbati et al. (2004) utilizaram análises com AFLP para investigar a estruturação genética de Puya raimondii (a maior bromélia que existe), endêmica dos Andes, e encontraram uma baixíssima variação entre as populações (4%) e devido seu reduzido número de populações, indicaram a urgência em se conservar tal espécie. Já estudos focados em espécies dos campos rupestres ainda são raros. Cavallari et al. (2006) utilizaram RAPD para estudar espécies de Encholirium (E. biflorum, E. pedicellatum e E. subsecundum) endêmicas de campo rupestre em MG, e inferiram, a partir das análises genéticas, uma baixa capacidade de dispersão para as sementes e observaram uma grande variabilidade genética nos perfis de RAPD no nível dos indivíduos (encontraram perfis em número semelhante aos das amostras para cada espécie). Barbará et al. (2007; 2009) apresentam dados para o gênero Alcantarea (A. geniculata, A. imperialis, A. glaziouana e A. regina ) em inselbergs na mata atlântica, a partir de marcadores de microssatélites, constatando um baixo fluxo gênico e uma moderada a alta diferenciação entre as populações de cada espécie (Fst = 0.111; 0.434; 0.217 e 0.195, respectivamente). Palma-Silva et al. (2011), também usando microssatélites, encontraram resultados interessantes para se discutir o processo de rápida especiação em Bromeliaceae, estudando bromélias rupícolas de afloramentos rochosos do gênero Pitcairnia (P. albiflos e P. staminea). Segundo esses autores, a hibridização e introgressão desempenharam um papel fundamental na radiação adaptativa observada nessa família, especialmente em espécies proximamente relacionadas que ocorrem em simpatria em afloramentos rochosos (PALMA- SILVA et al., 2011). Os microssatélites representam regiões instáveis do genoma, que estão sob alterações mutacionais, geralmente adições ou deleções de um número integral de repetições, com taxa de mutação muito mais elevada do que aquelas observadas em sequências de DNA não repetitivo (JARNE; LAGODA, 1996), estando distribuídos ao longo de sequências codificantes e não codificantes (SCHLÖTTERER; TAUTZ, 1992). Dessa forma, esses marcadores moleculares podem ser utilizados para resolver questões de diferentes ordens (genética e evolutiva) e esclarecer dúvidas quanto ao sistema de cruzamento e capacidade de colonização de populações (BONEH et al., 2003; BARBARÁ et al., 2007; PALMA-SILVA, 2008).

Assim, os estudos de genética de populações, utilizando-se das mais diversas ferramentas (moleculares), passam a ter uma grande importância na preservação destas mesmas populações, já que é possível principalmente detectar a redução no tamanho populacional, dado importante, já que pode levar à diminuição da diversidade genética dentro da mesma ou causar uma mudança na genética da população (diminuição de heterozigotos) (FRANKHAM et al., 2002). Da mesma forma, se as populações pequenas e isoladas perderem a sua diversidade genética devido a acelerada endogamia e deriva genética, a capacidade reprodutiva e viabilidade dos indivíduos dentro das populações podem ser reduzidas e sua capacidade de adaptação às mudanças ambientais pode ser comprometida, o que pode levar a extinção dessas populações (JEONG et al., 2010; NAMOFF et al., 2011).

1.5 BIOLOGIA REPRODUTIVA EM BROMELIACEAE

Diversos atributos florais, como formato, cor, odor, e até disponibilidade de néctar, estão muitas vezes intimamente relacionados à atração de polinizadores (ARAÚJO et al., 2009); sendo que as características existentes na interação entre planta e polinizador podem ser um resultado direto da evolução floral (ROCCA; SAZIMA, 2013). Dessa forma, dados sobre a biologia floral das plantas tornam-se de extrema importância por fornecerem os primeiros indícios para elucidação de sua biologia reprodutiva, auxiliando no entendimento de síndromes de polinização, tipos de sistema reprodutivo e até padrões de distribuição das espécies. No entanto, a despeito de sua importância e da grande diversidade presente em Bromeliaceae, poucos dados estão disponíveis na literatura sobre a biologia reprodutiva de espécies de bromélias (e.g., MARTINELLI, 1994; 1997; VARASSIN; SAZIMA, 2000; WENDT et al., 2001; 2002; MATALLANA et al., 2010) ou sobre as estratégias utilizadas por espécies da subfamília Tillandsioideae para a dispersão de propágulos (e.g., PAGGI et al., 2010). Em geral, as Bromeliaceae são autocompatíveis e as sementes podem ser dispersas por diferentes agentes, como o vento, a água (e.g. enxurradas sobre afloramentos rochosos) ou por animais, no caso dos frutos bacáceos (HOLST; LUTHER, 2004; MATALLANA et al., 2010). Nos campos rupestres, trabalhos voltados à compreensão das estratégias de polinização e dispersão, ainda que com outros grupos de angiospermas, também são raros (e.g., BORBA

17 et al., 1999) e, em áreas especificamente de canga (campo rupestre ferruginoso) são ainda mais escassos (e.g., JACOBI; ANTONINI, 2008). Entretanto, estudos de biologia floral e reprodutiva são extremamente importantes para se compreender e discutir os dados de estruturação genética de plantas (e.g., BARBARÁ et al., 2007; 2009). A família Bromeliaceae é uma das poucas onde a polinização por vertebrados é mais frequente que a polinização por insetos, já que, de acordo com alguns autores, essa última seria mais eficiente (PROCTOR et al., 1996). Dessa mesma forma, o gênero Vriesea é caracterizado por apresentar síndromes ornitófilas e quiropterófilas. Nesse gênero, quando os beija-flores (família Trochilidae) são os principais vetores de pólen, as flores costumam ser tubulares, diurnas, amarelas, com brácteas de cores fortes e contrastantes, de nenhum odor, e apresentam produção de néctar em longos períodos (e.g. ARAÚJO et al. 1994; ROCCA; SAZIMA, 2013). Já quando são os morcegos os principais polinizadores, as flores tendem a ser campanuladas, noturnas, com odor semelhante ao do alho, com nectários muito desenvolvidos, coloração pouco conspícua, apresentam anteras inclinadas para apenas um dos lados da flor (e.g. SAZIMA et al., 1995). Em ambos os casos, os nectários septais tendem a ser bem desenvolvidos, sendo o néctar aí produzido uma importante recompensa aos polinizadores, principalmente os vertebrados. A presença de nectários septais uma das características únicas pertencentes a família Bromeliaceae, se comparada às demais famílias da ordem Poales (SAJO et al., 2004). Outro aspecto reprodutivo que a muito intriga os pesquisadores em Bromeliaceae, \e a combinação de modos de reprodução, já que há uma tendência dentro da família de se combinar os dois tipos: a reprodução sexuada, através da produção de sementes; e a reprodução assexuada, com propagação clonal (genetes) (VOSGUERITCHIAN; BUZATO, 2006), sendo que a escolha pelo tipo de reprodução pode estar ou não ligada a combinação de disponibilidade de polinizadores com a sazonalidade do ambiente. Diante de tantas questões, muitos estudos ainda se fazem necessários, principalmente no que tangem a espécie V. minarum. Por tratar-se de uma espécie com grande ameaça de extinção, os conhecimentos acerca de sua biologia reprodutiva podem auxiliar no melhor entendimento de sua estruturação genético-espacial, e consequentemente, na tentativa de se traçarem metas efetivas para sua preservação.

2 OBJETIVOS

2.1 OBJETIVO GERAL:

· Verificar as relações genéticas interpopulacionais de Vriesea minarum no Quadrilátero Ferrífero (sul da serra do Espinhaço), estado de Minas Gerais, a fim de se traçar metas para sua conservação.

2.2 OBJETIVOS ESPECÍFICOS:

· Avaliar a diversidade e estrutura genética interpopulacionais, usando marcadores moleculares de microssatélites; · Quantificar o grau de diferenciação genética entre as populações e correlacionar distâncias genéticas às distâncias geográficas entre as populações. · Buscar padrões biogeográficos para as populações dentro do QF, que subsidiem propostas de conservação da espécie in situ; · Discutir processos biológicos que poderiam estar influenciando os padrões genético- espaciais observados, especialmente caracteres ligados à polinização e capacidade de dispersão das sementes para a espécie.

3 CAPÍTULO 1

Transferability of 10 nuclear microsatellite primers to Vriesea minarum L.B.Sm

(Bromeliaceae), a narrowly endemic and threatened species from Brazil

Manuscrito aceito no Brazilian Journal of Botany

Transferability of 10 nuclear microsatellite primers to Vriesea minarum (Bromeliaceae), a narrowly endemic and threatened species from Brazil

P. Lavor, C. van den Berg and L. M. Versieux1

Transferability of Bromeliaceae microsatellite primers

1P. Lavor and L. M. Versieux

Programa de Pós-Graduação em Sistemática e Evolução, Laboratório Botânica Evolutiva, Departamento de Botânica, Ecologia e Zoologia, Universidade Federal do Rio Grande do Norte, Lagoa Nova, Natal, RN, 59072- 970, Brazil. e-mail: [email protected].

C. van den Berg Laboratório de Biologia Molecular de Plantas, Universidade Estadual de Feira de Santana, BR-116, Km 03, Feira de Santana, Bahia, 44031-460, Brazil

Transferability of 10 nuclear microsatellite primers to Vriesea minarum (Bromeliaceae), a narrowly endemic and threatened species from Brazil

Abstract - Vriesea minarum is an endemic rupiculous bromeliad species, with naturally fragmented populations, restricted to the Iron Quadrangle, in Minas Gerais, Brazil. It is a threatened species, which is suffering from habitat loss due to the growth of cities and mining activities. Thus, it is extremely important to know its genetic structure in order to set strategies for its in situ and ex situ conservation. Here we tested 14 nuclear microsatellite primers (SSRs) for one population of V. minarum in order to search for polymorphisms and evaluated the transferability of previously developed primers. We succeeded in the amplification of 10 loci in which we also found polymorphisms. The expected and observed heterozygosity found here is similar to other Bromeliaceae population genetics studies. Our results show the great potential in working with these co-dominant markers for V. minarum, which may help in developing conservation actions for the species in the future.

Key words: Conservation Genetics, Cross-amplification, Iron Quadrangle, Metallophytes,

SSR.

Introduction

Vriesea minarum L.B.Sm. (Bromeliaceae), is a endemic species of bromeliad, restricted to the Iron Quadrangle region, Minas Gerais, Brazil (Versieux & Wendt 2007, Jacobi et al.

2007) and its populations are naturally fragmented. This metallophyte species has been suffering with increasing in mining activities and the growth of cities, what has caused the decline in its area of occupancy and quality of habitat (Versieux 2011). Thus, some studies have considered this species as endangered (Versieux & Wendt 2007, Versieux 2011).

Currently, V. minarum is classified as threatened inside the EN (Endangered category of the

IUCN) by the Brazilian Official Plant Red List (CNCFlora, 2013).

Therefore, it is necessary to know the genetic structure of V. minarum populations in order to have a conservation plan for the species. Studies of conservation genetics in Bromeliaceae have provided a rich source of primers for microsatellite (SSR) loci. The SSR primers may be transferred to different plant species depending on the extent to which the primer sites flanking SSRs are conserved among related taxa, and the stability of the SSR throughout evolution (Power et al. 1996). This study aims to optimize a set of previously published microsatellite loci primers for V. minarum.

Material and Methods

Leaf samples from twenty individuals were collected in Pedra Rachada, in Sabará municipality (Fig 1-3). The genomic DNA was extracted following Doyle & Doyle (1990).

Fourteen SSR loci previously described were tested: Vriesea gigantea Gaud. (Palma-Silva et al. 2007 - loci: VgB10, VgC01, VgF01, VgF02, VgG02, VgG03, and VgG5), Tillandsia fasciculata Sw. and Guzmania monostachya Rusby (Boneh et al. 2003 - loci: e6, p2p19, e19, and CT5), Pitcairnia albiflos Herb. (Paggi et al. 2008 - locus: PaZ01) and Fosterella rusbyi

(Mez) L.B.Sm. (Wohrmann et al. 2012 - loci: ngFos_6 and ngFos_22).

Three primers were used for each SSR locus: a forward SSR-specific primer with the M13 tail at its 5′ end, a reverse locus-specific primer, and a universal M13 (5'

CACGACGTTGTAAAACGAC 3') primer labelled with four fluorescent dyes (Applied

Biosystems matrix DS-33, with 6-FAM, VIC, NED, and PET). The amplifications were done using the GeneAmp PCR system 9700 (Applied Biosystems) thermal cyclers and

Swift.maxpro (Esco).

For seven loci (VgB10, VgC01, VgF02, VgG03, VgG5, and ngFos_6 ngFos_22) the following "touchdown" program was used: 95 °C for 3 min, then 10 cycles of 94 °C for 30 s,

58 °C decreasing to 48 °C at 1 °C per cycle for 30 s, 72 °C for 30 s followed by 35 cycles of

94 °C for 30 s, 48 °C for 30 s, 72 °C for 30 s, followed by a final extension of 30 min at 72 °C before cooling down to 4 °C. For five SSR loci (PaZ01, e6, p2p19, e19 and CT5) the program used was: 94 °C for 3 min, 40 cycles of 94 °C for 20 s, 51 °C for 40 s, 72 °C for 20 s, followed by a final extension of 30 min at 72 °C before cooling down to 4 °C. And for the two remaining SSR loci (VgF01 and VgG02) the program was: 94 °C for 3 min, 40 cycles of

94 °C for 20 s, 54 °C for 40 s, 72 °C for 20 s, followed by a final extension of 30 min at 72 °C before cooling down to 4 C°. For all the programs we added 8 cycles of 94 °C for 1 min, 53

°C for 1 min, 72 °C for 1 min before the final extension to incorporate the M13 tail.

Microsatellite alleles were resolved on an ABI 3130XL Genetic Analyzer with a 50 cm capillary array (Applied Biosystems) and were precisely sized using GeneMapper 4.0

(Applied Biosystems). GenAlex 6.5 software was used to calculate the estimates of allelic diversity and test for departure from Hardy-Weinberg equilibrium (HWE). The inbreeding coefficient (Fis) was estimated by Fstat version 2.9.3 software (Goudet, 1995). The

Microchecker software was used for detection and correction of null alleles (Brookfield 1 estimator). The polymorphic information content (PIC) was calculated using PICcalc

(http://w3.georgikon.hu/pic/english/default.aspx).

Results and Discussion

Of the 14 primers tested in the present study, 10 showed amplifications. All of these 10 loci were polymorphic, displaying an average allele number of 6.4 (2-13). The average observed heterozygosity (Ho) was of 0.432 and the average expected heterozygosity (He) was of 0.579. Six loci showed null alleles. The mean of the inbreeding coefficient (Fis) was 0.225.

Deviations from Hardy-Weinberg equilibrium were found in five loci, where levels of observed heterozygosity were lower than expected (Table 1).

The values of observed and expected heterozygosity are within the values found in other studies of Bromeliaceae, such as: Palma-Silva et al. (2009) for V. gigantea (Ho = 0.424 and

He = 0.714); Barbará et al. (2007a) for Alcantarea imperialis (Carrière) Harms (Ho = 0.362 and He = 0.615) e A. geniculata (Wawra) J.R. Grant (Ho = 0.357 and He = 0.429); Zanella et al. (2011) for Bromelia antiacantha Bertol. (Ho = 0.369 and He = 0.746). All of these studies indicate an observed heterozygosity smaller than the expected, due to a deficit of heterozygotes. The same pattern is observed in this study with V. minarum. The inbreeding coefficient was high probably because V. minarum is partially self-compatible (Lavor 2013, in prep.), it also has a developed clonal growth (Versieux, 2011) and the inbreeding coefficient value found here is also similar to those found in other Bromeliaceae (e.g. Palma-Silva et al.

2011; Zanella et al. 2011).

We consider the level of transferability obtained here for V. minarum as high, since 10

(~70%) out of 14 primers pairs provided positive results. Apparently, the ability to transfer primers seen in this study is not related to the phylogenetic position of the taxon in which the original microsatellite was isolated. Vriesea minarum is placed within the Tillandsioideae subfamily, and we transfered primers developed for the Pitcairnioideae subfamily (Fosterella and Pitcairnia) that worked well. In contrast, four primers that were isolated from other species of Tillandsioideae did not amplify.

This successful cross-amplification in Bromeliaceae could perhaps be one indication of the great adaptive radiation within the family, leading to rapid speciation and to low levels of divergence in DNA sequences, allowing the SSRs to be transferred among species of the same or different subfamilies (Barbará et al. 2007b).

Thus, given the high number of bromeliad species threatened of extinction in Brazil the transferring of previously developed markers may save time and eliminate part of the costs, allowing more studies in population genetics to be done. This will certainly draw up concrete targets for conservation in the future.

Acknowledgements

We thank CAPES for the first author’s M.Sc. fellowship and FAPESB for financial support (PNX0014/2009). CVDB thanks CNPq for his fellowship (PQ-1D). We thank two anonymous reviewers for their comments, F. F. Carmo for field assistance, and M. C. López-

Roberts for training in molecular laboratory techniques.

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Zanella CM, Bruxel M, Paggi GM, Goetze M, Buttow MV, Cidade FW, Bered F.2011.

Genetic structure and phenotypic variation in wild populations of the medicinal tetraploid

species Bromelia antiacantha (Bromeliaceae). American Journal of Botany 98: 1511-1519.

Figure Legends

Figures 1-3 - Specimens of Vriesea minarum in habitat: (1) population, (2) Blooming individual (3) detail of the inflorescence. Bars = 10 cm (1,2); 5 cm (3) (Voucher: Lavor 13939, UFRN herbarium).

Figures 1-3

Table 1 - Characteristics of the 10 primers transfered for one population of Vriesea minarum.

1 Organism Locus Reference Size PB Ta (°C) A He Ho Fis

Tillandsia fasciculata and Guzmania e6 Boneh et al. 2003 134-148 51 3 0.471 0.500 -0.035 monostachya Tillandsia fasciculata and Guzmania e19 Boneh et al. 2003 129-137 51 2 0.054 0.056 0.000 monostachya Tillandsia fasciculata and Guzmania p2p19 Boneh et al. 2003 204-222 51 8 0.798 0.643 0.230 monostachya Fosterella rusbyi ngFos_6 Wöhrmann et al. 2012 140-188 58-48 2 0.142 0.154 -0.043

Fosterella rusbyi ngFos_22 Wöhrmann et al. 2012 170-232 58-48 6 0.695 0.368 0.357**

Vriesea gigantea VgB10 Palma-Silva et al. 2007 132-174 58-48 12 0.864 0.684 0.206

Vriesea gigantea VgC01 Palma-Silva et al. 2007 230-270 58-48 13 0.843 0.444 0.379***

Vriesea gigantea VgF02 Palma-Silva et al. 2007 130-174 58-48 8 0.776 0.200 0.625***

Vriesea gigantea VgG03 Palma-Silva et al. 2007 219-233 58-48 3 0.397 0.389 0.048***

Vriesea gigantea VgG05 Palma-Silva et al. 2007 168-216 58-48 7 0.754 0.882 -0.140***

Ta = annealing temperature; A number of alleles; Ho observed heterozygosity; He expected heterozygosity; Fis inbreeding coefficient;

1Departures from Hardy–Weinberg Equilibrium are indicated by asterisks: **P<0.01, ***P<0.001.

4 CAPITULO 2

Population genetics of the endemic and endangered Vriesea minarum L.B.Sm.

(Bromeliaceae) in the Iron Quadrangle, Espinhaço Mountain Range, Brazil

Manuscrito a ser submetido ao American Journal of Botany

Lavor, P et al.

POPULATION GENETICS OF THE ENDEMIC AND ENDANGERED Vriesea

minarum L.B.Sm. (BROMELIACEAE) IN THE IRON QUADRANGLE, ESPINHAÇO

MOUNTAIN RANGE, BRAZIL1

Pâmela Lavor2,3, Cássio van den Berg4, Claudia M. Jacobi5, Flávio F. Carmo5, Leonardo M.

Versieux3

3 Programa de Pós-Graduação em Sistemática e Evolução, Laboratório Botânica Evolutiva, Departamento de Botânica, Ecologia e Zoologia, Universidade Federal do Rio Grande do Norte, Lagoa Nova, Natal, RN, 59072- 970, Brazil. 4 Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Av. Transnordestina. s.n., Feira de Santana, Bahia, 44036-900, Brazil. 5 Departamento de Biologia Geral, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

1Manuscript received ______; revision accepted ______. 2 E-mail for correspondence:[email protected].

The authors thank CAPES for a scholarship to the first author; FAPESB for financial support (PNX0014/2009); CNPq for a fellowship to CVDB (PQ-1D) and CMJ (PQ 2). CMJ thanks the Brazilian National Council for Scientific and Technological Development (CNPq) for a Research Productivity Scholarship. We thank two anonymous reviewers for their comments; MSc López-Roberts for training in molecular laboratory techniques and Dr. Sérgio Maia Queiroz Lima, by theoretical help. 32

ABSTRACT

Premise of the study: The knowledge of genetic variability in plant populations is one of the main branches of conservation genetics, linking genetic data to conservation strategies. Thus, the study of the structure, richness and genetic diversity of populations of V. minarum becomes crucial to set goals for its effective conservation. V. minarum is a species of bromeliad endemic to the Iron Quadrangle region (southeastern Brazil), which is now in the category of Endangered due to habitat loss particularly by mining (which is the main source of wealth in the region).

Methods: To study the genetics of 12 populations of V. minarum, 10 microsatellite loci transferred from other species of Bromeliaceae were used and statistical analyses to compare and describe the variability were performed.

Key results: The 10 polymorphic loci had an average number of alleles of 13; the observed

(Ho) and expected heterozygosity (He) were 0.424 and 0.628, respectively. Our results show a low population structure (Fst = 0.088), but with different values of richness (mean = 2.566) and genetic differentiation (mean = 0.635) for all populations; high inbreeding coefficient

(Gis = 0.376). These may be the result of a great efficiency by pollinators to carry pollen and/or efficient seed dispersal, thus leading a high connectivity among populations of these fragmented outcrops.

Conclusions:However, despite these results, considering the great habitat loss for species (by mining), it becomes crucial to analyze the creation of new protected areas for conservation of the species, particularly to protect population that have unique genotypes.

Key words: Bromeliaceae; Population genetics; Iron Quadrangle; Vriesea minarum.

INTRODUCTION

The Iron Quadrangle (IQ) region in southeastern Brazil is one of the most relevant centers of Bromeliaceae species richness, showing a high number of endemic and endangered taxa (Versieux and Wendt, 2007; Versieux et al., 2008, 2010). Despite their ecological importance and frequency in areas of rocky outcrops where many other plant species would have difficulty to grow, several bromeliad species are threatened by habitat loss derived from human activities. Studies to understand the patterns and processes distribution of these species are urgently needed in order to set goals for their conservation.

Vriesea minarum L.B.Sm. is a bromeliad endemic to the Iron Quadrangle region, a mountain area of about 7,200 km2 located at the southern portion of the Espinhaço Mountain

Range, in southeastern of Brazil (Versieux, 2011). The region is worldwide renown for its importance for mining, and it is inserted between two global biodiversity hotspots: the

Atlantic Forest and Brazilian Cerrado (Mittermeier et al., 2004).

The peaks of IQ mountains are topped with granite, quartzite and, predominantly, banded iron formations and ironstone outcrops (or “cangas”) and they may reach up to 1,850 m.a.s.l (Jacobi et al., 2007; Jacobi and Carmo, 2012). The region presents temperate highland tropical climate with dry winters - according to Köppen classification - characterized by a dry winter and wet summer (Jacobi and Carmo, 2012). The Cangas form extensive plates which are the result of the millions of years of weathering of banded iron formations, being constituted basically by hematite (Fe2O3) and goethite (FeOOH) (Spier et al., 2007; Shuster et al., 2012).

Ironstone outcrops are rapidly losing their area to iron ore mining activities, and its removal is threatening the survival of different plant species adapted to this very peculiar environment, which urges for conservation measurements (Jacobi et al., 2007; Versieux and

Wendt, 2007; Jacobi and Carmo, 2008; Jacobi and Carmo, 2012). In this scenario, V.

34 minarum stands as threatened, and is currently listed as endangered by the Brazilian official plant species’ red list (CNCFlora, 2013). It is officially protected only within the Rola-Moça

State Park (PESRM), with only a few records in small private reserves (Versieux, 2011).

Knowledge of how genetic diversity is structured in a spatial context, either within a population or among multiple populations, can contribute greatly to our understanding of species evolutionary history and population dynamics (Moreira et al., 2010). This structure can result from the combination of a number of factors, such as breeding system, floral morphology, mode of reproduction, pollination mechanisms, seed dispersal, phenology, life cycle, time of reproduction, successional stage, and geographic range among others (Loveless and Hamrick, 1984).

Additionally, understanding the historical-evolutionary processes responsible for the distribution patterns of genetic variation in plant populations, such as those occurring in the

Iron Quadrangle, is still a completely unexplored research field. The Espinhaço mountain have one of the richest floras in the world with hundreds of endemic species (Giulietti et al.,

1997). Knowing the patterns of distribution of genetic diversity for endemic plants of this region may help to conserve the biodiversity in this area, as shown for similar environments in other parts of the world (Butcher et al., 2009).

The phylogenetic knowledge of the large genus Vriesea is still fragmentary and far from satisfactory. However some data are available and may be used to create hypotheses to be tested with a population genetics approach. Recent phylogenetic hypotheses suggest that the

Espinhaço Range species of Vriesea are derived from Atlantic rainforest ancestors (Versieux et al., 2012). Most Vriesea species occur along and are restricted to the Atlantic rainforest domain. Vriesea prefers mesophytic habitats and, even along the Espinhaço Range, species grow preferably on the top of mountain ranges where mist and fog are constantly formed, or as epiphytes. According to Versieux and Wendt (2007), the southern Espinhaço Range shares

35 taxa of bromeliads with the forested areas of the core Atlantic rainforest that grows in the southeastern Minas Gerais up to close to the coast. This similarity would be caused by the proximity, allowing dispersion between them.

As V. minarum belongs to a phenetically delimited group of rupicolous yellow-flowered species that occurs from Rio de Janeiro (RJ) to central and north Espinhaço range in MG, we could hypothesize that the colonization of the Iron Quadrangle by V. minarum ancestors occurred from the south or southeastern border (Hypothesis 1: south/southeast to west and north stepping stone model, assuming that migration occurred from RJ). If this is correct we could expect that south/southeastern populations are older and may have more genetic variability if compared to populations from the western border, which are possibly younger and less differentiated. A second hypothesis is that V. minarum showed in the past a broader distribution within the Iron Quadrangle and, due to recent anthropic effects, its populations are now fragmented and isolated on mountain tops.

The aims of the present work were: (1) to assess the intra and interpopulation genetic diversity and structure of V. minarum using microsatellites markers (SSR); (2) to quantify the degree of genetic differentiation among populations; (3) to uncover biogeographic patterns within the Iron Quadrangle and propose conservation of the species, and (4) to discuss the biological processes that could have influenced such patterns.

MATERIAL AND METHODS

Population sampling and DNA extraction - Leaf samples were collected between January and February of 2012 in ten different locations totaling 12 different populations across the

Iron Quadrangle, Minas Gerais state, Brazil (Fig. 1; Table 2). The region has most rains concentrated from November to January and the dry season goes from June to August (Meyer et al., 2004). Populations were previously known by herbaria records (Versieux, 2011) or were searched in feasible places, particularly at the top of mountain ranges (1,300-1,600

36 m.a.s.l.) where iron ore deposits form outcrops. The minimum distance between populations was 1.7 km (Pop. Rola-Moça 1 to Pop. Rola-Moça 2) and the maximum was 92.5 km (Pop.

Itatiaiuçu to Lavras Novas) (Fig. 1).

Vriesea minarum is a rupicolous species, which reproduces by clonal growth through the production of shoots on the base of the mother-plant or on the axil of older leaves, as well as by sexual reproduction by seeds (Versieux, 2011 Fig. 2). To avoid sampling clones or close relatives, leaves were collected at a minimum distance of 5 m between plant rosettes.

All individuals were georeferenced with a GPS, but as the species is oficially threatened we do not provide exact location of each population sampled in this paper. Only in Itatiaiuçu the 5 m distance could not be strictly followed due to the small population size (personal observation) and the low number of individuals remaining along this sampling site (six individuals). In most of the populations visited at least 16 individuals were sampled (Table 2).

Leaf samples measuring approximately 3 × 3 cm2 were cut from the middle portion of healthy leaves using sterilized scissors and immediately stored in plastic bags containing silica gel for dehydration. Herbarium specimens used as vouchers for populations surveyed were stored in the Herbarium of the Federal University of Rio Grande do Norte (UFRN). The genomic DNA was extracted from a total of 206 individuals following the protocol of Doyle and Doyle (1990).

Microsatellite analysis - Fourteen SSR loci previously described were tested during a preliminary screening (Lavor et al. submitted): Vriesea gigantea Gaud. (Palma-Silva et al.,

2007 - loci: VgB10, VgC01, VgF01, VgF02, VgG02, VgG03, and VgG5), Tillandsia fasciculata Sw. and Guzmania monostachya Rusby (Boneh et al., 2003 - loci: e6, p2p19, e19, and CT5), Pitcairnia albiflos Herb. (Paggi et al., 2008 - locus: PaZ01) and Fosterella rusbyi

(Mez) L.B.Sm. (Wöhrmann et al., 2012 - loci: ngFos_6 and ngFos_22).

Three primers were used for each SSR locus: a forward SSR-specific primer with the

M13 tail at its 5′ end, a reverse locus-specific primer, and a universal M13 primer labelled with four fluorescent dyes (Applied Biosystems) with 6-FAM, VIC, NED, and PET). The amplifications were done using GeneAmp PCR system 9700 (Applied Biosystems) and Swift

Maxpro (Esco) thermocyclers.

For seven loci (VgB10, VgC01, VgF02, VgG03, VgG5, and ngFos_6 ngFos_22) the following "touchdown" program was used: 95 °C for 3 min, then 10 cycles of 94 °C for 30 s,

58 °C decreasing to 48 °C at 1 °C per cycle for 30 s, 72 °C for 30 s followed by 35 cycles of

94 °C for 30 s, 48 °C for 30 s, 72 °C for 30 s, by 08 cycles of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 1 min (to incorporate the M13 tail), followed by a final extension of 30 min at

72 °C before cooling down to 4 °C. For five SSR loci (PaZ01, e6, p2p19, e19 e CT5) the program used was: 94 °C for 3 min, 40 cycles of 94 °C for 20 s, 51 °C for 40 s, 72 °C for 20 s, by 08 cycles of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 1 min (for incorporation of the tail M13), followed by a final extension of 30 min at 72 °C before cooling down to 4 °C. And for the two remaining SSR loci (VgF01 and VgG02) the program used was: 94 °C for 3 min,

40 cycles of 94 °C for 20 s, 54 °C for 40 s, 72 °C for 20 s, by 08 cycles of 94 °C for 1 min,

53 °C for 1 min, 72 °C for 1 min (for incorporation of the tail M13), followed by a final extension of 30 min at 72 °C before cooling down to 4 °C.The microsatellite alleles were resolved on a ABI 3130XL Genetic Analyzer (Applied Biosystems) and they were precisely sized using the GeneMapper 4.0 (Applied Biosystems) software. The microsatellite data were tested for genotyping errors resulting from stuttering, short allele dominance and null alleles using software micro-checker 2.2.3 (Brookfield 1 estimator) (van Oosterhout et al., 2004).

Statistical analysis - Genetic diversity - Levels of genetic diversity within populations and loci were described by calculating the following parameters: A – Number of alleles; Rs - allelic richness; Gd - gene diversity; Apr - number of private alleles; Ho - observed

38 heterozygosity; and He - expected heterozygosity. All genetic diversity parameters were estimated with the softwares GenAlEx 6.5 (Peakall and Smouse, 2012), PopGene 1.31 software (Yeh et al., 1999) and Fstat version 2.9.3 (Goudet, 1995).

For test of Hardy-Weinberg Equilibrium, the software Genepop (on line) version 4.3

(Raymond and Rousset 1995) was used. For estimate the inbreeding coefficient Fis (Weir &

Cockerham 1984) in populations was used software Fstat version 2.9.3 (Goudet, 1995).

Genetic structure within and among populations – For measures of genetic differentiation Fst

(Wright, 1965), Gst, G”st and Jost’s D (Meirmans and Hedrick, 2011) and estimate inbreeding coefficient (Gis) (Nei and Cheeser, 1983) was used the software GenAlEx 6.5 (Peakall and

Smouse, 2012). The significance was tested by resampling with 1,000 permutations.

The principal coordinate analysis (PCO) was done using software GenAlEx 6.5 (Peakall and Smouse, 2012) to infer genetic variation among individuals and populations.

An unrooted phenogram of Nei’s unbiased genetic distances (1978) was constructed using the Neighbor Joining (NJ) method as implemented in Paup 4.0 (Swofford, 2002). For the bootstrap (BS) analysis, 1,000 matrix replicates were generated in MSA (Dieringer and

Schlötterer, 2003) and used to build an unrooted NJ trees and the major rule bootstrap consensus tree in Paup 4.0 (Swofford, 2002).

The Analysis of Molecular Variance (AMOVA) was used to test groupings revealed by the neighbor-joining tree (PRac=Pedra Rachada; SPie=Serra da Piedade / SIta=Serra do

Itatiaiuçu, MTam=Morro do Tamanduá / SCal=Serra da Calçada / SRM1=Serra do Rola-

Moça 1, SRM2=Serra do Rola-Moça 2, MSer=Marinho da Serra, SOBr=Serra do Ouro

Branco, LNov=Lavras Novas, SGan2=Serra da Gandarela 2 and SGan1=Serra da Gandarela

1). The AMOVA was performed with software GenAlEx 6.5 (Peakall and Smouse, 2012) the significance was estimated using R-statistics and tested using 10,000 permutations.

Bayesian structure analysis - The Bayesian clustering method implemented in the program

STRUCTURE 2.3.4 (Pritchard et al., 2000) was used to search for population structure. To determine the most likely number of clusters (K) present in the data, we conditioned our data to various values of K ranging from 1 to 12. The analyses were carried out under the admixture model (Q) assuming dependent allele frequencies and using a burn-in period of

50,000 replicates and 500,000 Markov chain Monte Carlo replicates, and 10 iterations per K to confirm stabilization of the summary statistics (Pritchard et al., 2000). Alpha and Lambda were fixed in 1.0. To determine the most likely number of clusters (K), the method of Evanno et al. (2005) was employed, based upon the ad hoc measure ΔK, in software on line

STRUCTURE HARVESTER v0.6.93 (Earl and vonHoldt, 2012).

Isolation-by-distance - The Mantel test (Mantel, 1967) was conducted in the GenAlEx 6.5 software (Peakall and Smouse, 2012) using the straight line distance among populations measured in GoogleEarth based in our GPS collection points associated to the Nei’s unbiased genetic distances (Nei, 1978). The statistical significance was tested using 10,000 permutations.

RESULTS

Variation across loci - The 10 polymorphic loci had an average number of alleles (A) of 13, ranging from three (NgFos_6) to 22 (VgB10 and VgC01) alleles per locus. The observed (Ho) and expected heterozygosity (He) for all loci calculated for all populations ranged from 0.146 to 0.663 and 0.231 to 0.930, respectively (Table 1). For only two loci (E6 and ngFos_6) the departure Hardy-Weinberg Equilibrium was not significant. The genetic differentiation among the 12 populations, was low (mean Fst = 0.088, Gst = 0.036, G”st = 0.098 and Jost’s D

= 0.062, all significant (P value < 0,01), Table 1). The mean of inbreeding coefficient was high (Gis = 0.376) (P value < 0,01). The mean of allelic richness (Rs) and gene diversity (Gd) in locus were 2.678 and 0.618, respectively (table 1).

Patterns of genetic variability across populations - Private alleles are distributed in all populations, ranging from 1 (e.g., Pop. 6) to 4 (e.g., Pop 1). Observed and expected heterozygosity per population varied from 0.297 to 0.475 and 0.533 to 0.620, respectively.

The departure from the Hardy-Weinberg Equilibrium was significant (p< 0.01) for populations all. The mean allelic richness (Rs) was 2.566 and gene diversity (Gd) was 0.635

(Table 2 and Fig. 3).

Patterns of population divergence and gene flow - A significant Mantel correlation was observed between genetic (Nei’s non-biased distance) and geographical distance (r² = 0.170;

P < 0.05), suggesting lower isolation by distance (Fig. 4).

The PCO with all individuals indicated that, despite the existence of some genetically deviant individuals, the whole data set seems to behave as a large, unstructured population.

(Fig 5).

Despite that the neighbor-joining phenogram of 12 populations based on Nei’s unbiased genetic distances revealed a distinct geographic structure among populations. At least three clusters could be recognized, supported by bootstrap values (50, 60 and 67%) (Fig. 6): Two populations from the Piedade mountain range complex (Serra da Piedade and Pedra Rachada) clustered separately from the remaining as well as the populations from the center/western portion (Serra do Itatiaiuçu, Morro do Tamanduá, and Serra da Calçada); the remaining populations formed a single weakly supported cluster (Fig. 6).

Hierarchical AMOVA detected significant differentiation at distinct levels, in presence of differentiation among regions (1%), followed among Populations (4%), with further differentiation occurring within the Individual level (48%) (Table 3).

Genomic admixture analysis with STRUCTURE 2.3.4 identified K = 2 as the most likely number of genetic clusters (Fig. 7).

DISCUSSION

Landscape Genetics - Many studies of population genetics have been conducted with natural populations of Brazilian bromeliads in recent years (e.g. Cavallari et al., 2006; Barbará et al.,

2007, 2009; Paggi et al., 2007, 2008, 2010, 2012; Palma-Silva et al., 2004, 2007, 2009, 2011).

In the region of Minas Gerais, more specifically its rocky fields, these studies are still scarce

(e.g. Cavallari et al., 2006; Ribeiro, 2009). Regarding ironstone rocky fields, there are currently no reported works of population genetics of species endemic to the region.

The values of observed and expected heterozygosity in loci are within the values found in other studies of Bromeliaceae, such as Barbará et al. (2007) with two species, Alcantarea imperialis (Ho = 0,362 and He = 0,615) and A. geniculata (Ho = 0,357 and He = 0,429);

Palma-Silva et al. (2009) for Vriesea gigantea (Ho = 0,424 and He = 0,714) and Zanella et al.

(2011) found for Bromelia antiacantha (Ho = 0,335 e He = 0,573). In all these works the observed heterozygosity is lower than the expected, due to a deficit of heterozygotes and the same pattern is seen here in V. minarum.

For V. minarum, our data reveal as well as a low structure (Mean Fst = 0088) compared to distinct works of population genetics of other Bromeliaceae using microsatellite (Table 4).

The Fst values found for V. minarum populations are far below those found for Alcantarea

(the sister genus) species: A. geniculata (Fst = 0.111) and A. imperialis (0.434) (Barbará et al.,

2007); A. glaziouana (0.217) and A. regina (0.195) (Barbará et al., 2009); V. gigantea (0.211)

(Palma-Silva et al., 2009); Pitcainia geyskesii (0.156) (Boisselier-Dubayle et al., 2010), P. albiflos (0.336) and P. staminea (0.260) (Palma-Silva et al., 2011); Bromelia antiacantha

(0.224) (Zanella et al., 2011) (Table 4).

In contrast, our inbreeding coefficient for V. minarum (Gis = 0.376) is larger than in most of these other works, except of B. antiacantha (Fis = 0.431) (Zanella et al. 2011) (Table

4). These data are consistent with other studies of self-compatible species from inselberg

(Palma-Silva et al., 2009).

These values of Fst and inbreeding coefficient may be related to multiple factors, such as mating system type, species distribution range and distance among populations. For V. minarum, we assumed a mixed reproductive strategy (outcrossing + self-compatible, see

Lavor et al., 2013 in prep.) and its distribution is within the range found for all of these species that we used for comparisons (Table 4).

Patterns of diversity in Vriesea minarum – For V. minarum, some of the genetic diversity parameters, were lower than those found in other Bromeliaceae studies, such as the mean allelic richness (Rs) which was 2.56 versus 6.25 for Alcantarea geniculata, 5.25 for A. imperialis (Barbará et al. 2007), 6.13 for A. glaziouana, and 4.36 for A. regina (Barbará et al., 2009); 2.83 for Vriesea gigantea (Palma-Silva et al., 2009), and 3.20 for Pitcairnia albiflos and 3.71 P. staminea (Palma-Silva et al. 2011). However, these values can be differentiated as a result transferred loci, some alleles which may not have been identified.

In the same way, the values found in this work for number of alleles and private alleles are also lower than those found in the species mentioned above, however, all these parameters are shown unevenly distributed within populations (Fig. 3).

These values, combined with greater differentiation at the level of individual, found in

AMOVA (48%) (Table 4), the lack of clusters in the PCO (Fig. 5) and low positive correlation between genetic and geographic distances of populations in V. minarum (Fig. 4), could possibly be explained by either seed or pollen dispersal efficiency.

A recent study with one species of the genus Vriesea showed that seed dispersal can occur only at a short distance (Paggi et al., 2010), although seeds are plumose and dispersed by wind, as all Tillandsioideae subfamily members (Madison, 1977; Benzing, 1990). Based on our results we believe that seed dispersal for V. minarum is more effective and not directly

43 comparable to the results of Paggi’s et al. (2010) study, in which most seeds were trapped closer to the dispersing plant. These authors studied V. gigantea, an epiphyte from the

Atlantic forest understory. In our case, V. minarum is restricted to rocky terrain of open environments, generally above 1,000 m.a.s.l, where a high incidence of winds, can improve their dispersion (being high winds are common along major mountain ranges of Minas

Gerais) (Nimer, 1979).

Besides that, the usually low stature of the rupiculous vegetation offer less obstacles to dispersion than a forested area. Additionally fruit ripening occurs from May to October

(Versieux, 2011), corresponding to the dry season thus avoiding drenching and clustering of the plumose seeds within the capsule and/or close to the dispersing plant.

Alternatively, the lack of genetic structure can also be explained by the pollinator behavior and by historical colonization processes. Although data on the reproductive biology of V. minarum are still preliminary, ongoing field observations suggest hummingbird pollination due to the time of anthesis in the early morning and the tubular and odorless yellow corolla (Lavor et al. in prep.). But bat pollination is not excluded, since flowers last two days, do not close at night, have stigmatic receptivity only in the late afternoon, and nectar is also produced during the night.

Also, this taxon has been traditionally placed in the section Xiphion, which is characterized by dull-colored flowers and bracts, and bat pollination (Smith and Downs,

1977). Bats are considered good pollen carriers and may guarantee pollen dispersal among separated outcrops (Martins and Gribel, 2007). Manual pollination treatments (Lavor et al.,

2013 in prep.) suggest that the species is mainly an out-crosser, which would also help to explain the lack of structure among populations and the low Fst values.

This lack of structure could be explained from the historical biogeography perspective, considering a possible scenario where a recent fragmentation among populations of V.

44 minarum have occurred, concomitant to the rapid loss of many iron outcrops in recent years along the Quadilátero Quadrangle (Jacobi and Carmo, 2012). This historical change could explain groups found within the NJ tree (supported by bootstrap values) (Fig. 6) and the two groups of Bayesian analysis (Fig. 7). Although some populations may be separate today, in the recent past time they were interconnected by intermediary outcrops that were mined recently.

Since we found high values of inbreeding (Gis = 0.376) and deviations of almost all loci and all populations from Hardy-Weinberg Equilibrium a possible explanation is that in the absence or shortage of pollinators, the V. minaruam would be forced do selfing (since it is self-compatible) (Lavor et al., 2013, in prep.) or reproduce by clonal growth (Versieux, 2011).

Conservation - The study of population genetics is of utmost importance because information about the genetic variability within and among populations can serve as a basis for the use and conservation of available genetic resources (Handa, 1998). Accurate identification of populations or portions of a species’ range that contain the greatest allelic variation, and hence evolutionary potential, can assist in the designation of priority areas for protection and helps capture genetic variation to be used in reintroduction and ex-situ preservation programs

(Moritz, 1994; Holtsford and Hancock, 1998; Frankham et al., 2002; Moreira et al., 2010).

The floristic diversity of the Iron Quadrangle outcrops, in particular ironstones, was overlooked until recently and their protection is challenging because of the escalating habitat loss by iron ore mining (Jacobi et al., 2007, 2008, 2011). As indicated by Versieux (2011), there are records of the species in only one state reserve: the Rola-Moça State Park, but even within this reserve there are known cases of illegal collecting and other threats to the conservation of V. minarum (Biodiversitas, 2007). The type locality (Serra da Piedade, Caeté) is a Catholic Sanctuary that also do not offer a secure proctection to the taxon (Versieux,

2011).

All other populations of V. minarum where we collected (e.g. Pico do Itatiauçu, Serra do Gandarela, Serra da Pedra Rachada and Serra da Moeda), are not under legal protection and therefore are exposed to many risks. A big fire in 2011 devastated a large area of Serra da

Moeda, in the region of Marinho da Serra, decimating a large percentage of the population that we sampled. In the same year, a fire also affected the populations of the Parque Estadual da Serra do Rola-Moça, leaving intact only a single population that survived within the 10% of the park that did not burn.

In addition, a more effective risk occurs due to mining, which according to Jacobi and

Carmo (2012), has consumed in the last four decades 40% of the total area of the cangas by

46 mines of iron ore extraction in the region. This habitat loss process is going so fast that even the population sampled here (year 2011) in the municipality of Igarapé, has been totally lost and no longer exists.

Although the populations of PESRM are the only ones that are officially protected, they did not show enough variability to make us fill confident that the survival of this species in the long term is assured. Other unprotected populations, namely those from Pedra Rachada,

Morro do Tamanduá or Gandarela 2, had the largest numbers of private alleles (Table 2), indicating that they are unique. Moreover, because they are in areas that are in the process of release for mining, the Serra da Gandarela populations should have special attention, since they are the ones with the largest number of individuals (by simple visual measure), appearing to be the best preserved of all the populations investigated.

Thus, it would be important to consider the creation of new conservation units for these populations, which today occur surrounded by mining areas and creating germplasm banks with these populations.

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Tables Table 1 - Measures of genetic diversity in 10 microsatellite loci for 12 population of Vriesea minarum. Locus A Rs Gd Ho He Fst Gst G''st Dest Gis E6 6 1.639 0.295 0.270 0.325 0.107* 0.074* 0.112* 0.035* 0.131ns

E19 11 1.482 0.232 0.146 0.231 0.065 0.021 0.029 0.007 0.307

ngFos_6 3 1.499 0.232 0.231 0.257 0.102 0.068 0.095 0.024 0.059ns

ngFos_22 13 2.857 0.768 0.540 0.762 0.072 0.014 0.058 0.044 0.376

P2p19 12 2.838 0.750 0.550 0.764 0.096** 0.049** 0.196** 0.151** 0.308

VgB10 22 3.602 0.904 0.663 0.930 0.077** 0.034** 0.368** 0.343** 0.314

VgC01 22 3.554 0.885 0.602 0.920 0.093** 0.046** 0.405** 0.373** 0.383

VgF02 17 3.590 0.945 0.397 0.928 0.086 0.004 0.061 0.057 0.696

VgG03 8 1.883 0.441 0.294 0.421 0.081 0.036 0.068 0.030 0.337

VgG05 17 2.800 0.737 0.550 0.742 0.102** 0.050** 0.185** 0.139** 0.345

Overall/Mean 13 2.678 0.618 0.424 0.628 0.088** 0.036** 0.098** 0.062** 0.376**

A = number of alleles; Rs = allelic richness; Gd = gene diversity; Ho = observed heterozygosity; He = expected heterozygosity; Fst = genetic differentiation among populations; Gst = Analog of Fst, adjusted for bias; G''st = Hedrick's standardized Gst; Dest =

Jost's estimate of differentiation; Gis = Inbreeding coefficient; ns = not significant (p value:

>0.05) from departure Hardy–Weinberg Equilibrium. P values: *P<0.05, **P<0.01,

***P<0.001.

Table 2 - Locality of provenance followed by the municipality (Mun.) in the state of Minas Gerais, approximate elevation, number of samples collected for each population and measures of genetic diversity for the 12 populations studied in all loci of Vriesea minarum.

Locality Population Elevation N A Apr Rs Gd Ho 1He Pedra-Rachada – Mun. Sabará PRac 1,300 m 20 6 4 2.520 0.600 0.469 0.578*** Santuário Estadual Nossa Senhora de Piedade – Mun. Caeté Spie 1,600 m 20 6 3 2.532 0.615 0.448 0.560*** Serra do Ouro Branco – Mun. Ouro Branco SOBr 1,400 m 20 6 3 2.578 0.633 0.423 0.588*** Parque Estadual da Serra do Rola-Moça 1 – Mun. Nova Lima SRM1 1,500 m 19 6 2 2.518 0.603 0.354 0.565*** Serra da Calçada – Mun. Brumadinho SCal 1,500 m 20 7 3 2.618 0.677 0.444 0.608*** Marinho da Serra – Mun. Moeda MSer 1,500 m 16 5 1 2.444 0.598 0.397 0.529** Pedra Grande - Serra do Itatiaiuçu – Mun. Igarapé Sita 1,200 m 6 3 1 2.573 0.693 0.297 0.533** Morro do Tamanduá – Mun. Nova Lima MTam 1,500 m 20 6 4 2.685 0.639 0.386 0.599*** Lavras Novas – Mun. Ouro Preto LNov 1,400 m 16 5 2 2.586 0.613 0.336 0.562*** Serra da Gandarela 1 – Mun. Rio Acima SGan1 1,600 m 19 5 3 2.518 0.612 0.400 0.567*** Serra da Gandarela 2 - Mun. Rio Acima SGan2 1,600 m 20 6 4 2.666 0.677 0.475 0.620*** Parque Estadual da Serra do Rola-Moça 2 – Mun. Nova Lima SRM2 1,500 m 10 5 1 2.548 0.671 0.444 0.565***

N = sample size; A = number of alleles; Apr = number of private alleles; Rs = allelic richnes; Gd = gene diversity; Ho = observed heterozygosity; He = expected heterozygosity. 1Departures from Hardy–Weinberg Equilibrium are indicated by asterisks: *P<0.05, **P<0.01, ***P<0.001

Table 3 - Analysis of molecular variance (AMOVA) of 12 population of Vriesea minarum, for details of clusters regions see material and methods.

Source of variation Df Sum of square Variation % P

Among regions 3 500.686 518.870 1% < 0.05

Among Populations 8 935.045 1601.704 4% < 0.001

Among Individuals 194 12.463 21.642 48% < 0.001

Within Individuals 206 4.317 20.960 47% < 0.001

Table 4 – Data about mating system, population structure, geographical distribution and mean of distance among populations (km) for many species of Bromeliaceae studied with SSR.

Distance (Min.-Máx.) Mating Fst Fis Species Geographical distribution among populations Reference system mean mean (Km) Alcantarea geniculata Out 0.111 0.094 Rio de Janeiro, Brazil 0.4 – 2.4 Barbará et al., 2007 Alcantarea imperialis Out 0.434 0.099 Rio de Janeiro, Brazil 7 – 100 Barbará et al., 2007 Alcantarea glaziouana Out 0.217 0.156 Rio de Janeiro, Brazil 3.5 – 47 Barbará et al., 2009 Alcantarea regina Out 0.195 -0.051 Rio de Janeiro, Brazil 1.5 Barbará et al., 2009 Vriesea gigantea Mix 0.211 0.273 South and Southeast Brazil 34 – 1600 Palma-Silva et al., 2009 Pitcainia geyskesii ND 0.156 0.125 French Guyana and Suriname 1.2 – 292.2 Boisselier-Dubayle et al., 2010 Bromelia antiacantha Out 0.224 0.431 Southeastern Brazil 2 – 679 Zanella et al., 2011 Pitcairnia albiflos Out 0.336 0.109 Rio de Janeiro, Brazil 1.70 – 17 Palma-Silva et al., 2011 Pitcairnia staminea Aut 0.260 0.240 Rio de Janeiro, Brazil 1.70 – 29 Palma-Silva et al., 2011

Aut = Autogamous; Mix = Mixed; ND = Not determined; Out = Outcrossing; (Adapted fromZanella et al., 2012).

Figure legends

Figure 1 - Featured for iron formations (BIF + cangas) in the Iron Quadrangle, highlighting location of populations collected from Vriesea minarum (modified from Garcia et al., 2009).

Figure 2 - Specimens of Vriesea minarum in habitat: (a) Blooming individual found in

Lavras Novas, municipality of Ouro Preto, MG (b) Fruiting individual with mining area in the background in mine of Tamanduá, municipality of Nova Lima, MG (Photos P. Lavor).

Figure 3 - Relation between allelic richness, number of alleles, alleles privates and gene diversity of Vriesea minarum (For population abbreviations, see Table 2).

Figure 4 – Relationship between genetic divergence, based on Nei’s non-biased distance

(1978) for nuclear microsatellites, and geographical distances in kilometers among 12 populations of Vriesea minarum (Mantel test correlation: r² = 0.170; P > 0.05).

Figure 5 – The principal coordinate analysis for 12 populations of Vriesea minarum (For population abbreviations, see Table 2).

Figure 6 - NJ dendrogram of genetic distance based on Nei’s non-biased distance (1978).

Unrooted phenogram of Nei’s unbiased genetic distances (1978) constructed using the

Neighbor Joining (NJ) (For population abbreviations, see Table 2).

Figure 7 - Bayesian analysis of 206 individuals from 12 populations of Vriesea minarum, occurring in the Iron Quadrangle, southeastern Brazil, based on 10 SSR loci, of

STRUCTURE software. Graphic representation of the different genetic pools for K = 2.

Populations are separated by vertical bars (For population abbreviations, see Table 2).

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

5 CAPITULO 3

Reproductive biology of Vriesea minarum L.B.Sm., an endangered rupicolous Bromeliaceae,

endemic to the Iron Quadrangle ferruginous rocky fields, Eastern Brazil

Manuscrito a ser submetido à Revista Flora

Reproductive biology of Vriesea minarum L.B.Sm., an endangered rupicolous Bromeliaceae, endemic to the ironstone outcrops of Southeastern Brazil

Pâmela Lavor1, Claudia M. Jacobi2, Flávio F. Carmo2, Cássio van den Berg3, Leonardo M. Versieux1

1Programa de Pós-Graduação em Sistemática e Evolução, Laboratório Botânica Evolutiva, Departamento de Botânica, Ecologia e Zoologia, Universidade Federal do Rio Grande do Norte, Lagoa Nova, Natal, RN, Brazil. e-mail for correspondence: [email protected]. 2Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. 3Laboratório de Biologia Molecular de Plantas, Universidade Estadual de Feira de Santana, BR-116, Km 03, Feira de Santana, Bahia, Brazil.

Abstract – Understanding the issues involved in the reproduction of an endangered species may contribute to the development of conservation actions. Here we investigated the floral biology, reproductive system, germination of seed and floral morphology of Vriesea minarum, a bromeliad endemic to ironstone outcrops (known as canga) of Minas Gerais, southeastern Brazil. The species has diurnal anthesis, flowers last for two days, and produce a considerable amount of nectar (mean = 0.023 ml ± 0.02 SD) with sugar concentration of mean

= 13% ± 9.5 SD, suggesting a mixed (hummingbird + bat) pollination syndrome. The production and germination of seeds by cross-pollination and selfing showed that the species is predominantly alogamous, but also partially self-compatible. Your seeds are small (length 6 mm ± 0.06 SD; N = 5), filiform, brown, bearing whitish feathery appendages along the base, with germination initiating at 12-13 days after imbibition. These results suggest that V. minarum is well adapted to the ferruginous rocky field habitat where the species occurs. Thus, due to habitat loss to mining and studies to understand the strategies of pollination and dispersal in canga environments are still scarcer is extremely important understanding the reproductive biology of V. minarum in order to add data to aid in conserving this threatened species.

Key words - reproductive biology; pollination syndrome; seed germination; Vriesea minarum; endangered species, Espinhaço Range.

Introduction

Bromeliaceae Juss. contains ca. 3,248 species in 58 genera (Luther, 2010), which are grouped into eight subfamilies distributed mainly in the Neotropical region (Givnish et al.,

2007). The subfamily Tillandsioideae encompasses ca. 1,300 species distributed in ten genera

(Barfuss et al., 2005; Luther, 2010), among which Vriesea Lindl. is the second largest, with ca. 280 species (Luther, 2008; Gomes-da-Silva and Costa, 2011), and grouped into sections

Xiphion and Vriesea (Smith and Downs, 1977). These sections are distinguished by the presence of red to yellow floral bracts, oblong petals, and flowers with diurnal anthesis in the former; and brown floral bracts, obovate petals, and flowers with nocturnal anthesis in the latter (Smith and Downs, 1977; Gomes-da-Silva et al., 2012).

Knowledge about the reproductive biology of a species, especially if it is endangered, is of fundamental importance, since aspects related to plant fertility are relevant in assessing population viability (Paggi et al . 2007).

Vriesea minarum L.B. Sm. is an endemic species from the Iron Quadrangle (IQ), a region that lies on the southern portion of the Espinhaço mountain range in Minas Gerais

(MG), harboring a rich and endangered vegetation, particularly bromeliads (Versieux, 2005;

Versieux and Wendt, 2006; Jacobi et al., 2007; Versieux et al., 2008). Throughout the IQ, a characteristic type of rock outcrop, constituted of iron-rich hardpan, consisting of fragments of hematite cemented by limonite and locally known as "canga" (Dorr II, 1969), is observed along the top of the mountains. Due to massive loss to mining, cangas are among the most threatened and least studied Brazilian ecosystems (Jacobi et al., 2007; Jacobi and Carmo,

2008) thus deserving special attention to all aspects of its rich biota.

Recent studies have shown the risk that many taxa of Bromeliaceae from MG are exposed to, especially those endemic to the IQ (Versieux and Wendt, 2007). Most bromeliads

66 are well adapted to water stress and can dominate many rocky outcrops; and many ironstone outcrops have rich plant communities, especially of lithophytes, including V. minarum.

Due to habitat loss to mining, V. minarum and other locally endemic species from the

IQ cangas are threaten by extinction and included as endangered in the Red List of Brazilian

Endangered Plant Species (Versieux, 2012; CNCFlora, 2013).

Along the entire Espinhaço mountain range rocky fields, studies to understand the strategies of pollination and dispersal within groups of angiosperms are rare (e.g. Borba et al.,

1999; Franceschinelli et al., 2006) and in canga environments such studies are even scarcer

(e.g., Jacobi and Antonini, 2008).

Few data are available in the literature about the reproductive biology of bromeliad species (eg, Martinelli, 1994, 1997; Varassin and Sazima, 2000; Wendt et al., 2001, 2002;

Matallana et al., 2010) or on the strategies used by the species of the subfamily

Tillandsioideae to disperse propagules (e.g. Paggi et al., 2010). Considering this, the present study is extremely important for understanding the reproductive biology of V. minarum in order to better comprehend the species, its distribution, add data to conservation genetics studies with plants from this area and aid in conserving this threatened species.

Material and methods

Study area - The study was conducted between February and March 2012 in the Rola Moça

State Park (PESRM, 20º02’40”S 43º59’48”W; Fig. 1), Minas Gerais, Brazil. The climate in the region, according to Köppen’s classification, corresponds to type Cw (highland tropical) with rainy summers (November through January) and dry winters (June through August),

(Pinheiro & Baptista 1998). Its vegetation is characterized by a mosaic of phytophysiognomies, ranging from rock outcrops plant communities to gallery forests and cerrado – the Brazilian savanna. The species occurs in an area of ferruginous rocky field, more specifically ironstone outcrops, or canga, (Jacobi et al., 2007; Guarçoni et al., 2010).

The population studied is located next to the Park headquarters (20º02’40.8’’S e

43º59’47.5’’W, 1480 m).

Vriesea minarum is a rupicolous plant, 35-70 cm tall when flowering, bearing a rosette of 19-34 cm tall that varies from tubular to infundibuliform; the inflorescence can be simple or compound, with secund to distichous flowers; the sepals are elliptic, obtuse and yellow, sometimes purplish-yellow along the base; the petals are ligulate, obtuse, erect to spreading, yellow, bearing a pair of ligulate appendages on the base; the stamens are exserted and the anthers are linear, dorsifixed near the base; the pistil is also exserted and has a convoluted blade type stigma (Versieux, 2011).

Floral biology - The morphological characterization of flowers of V. minarum was performed by observation and photographic documentation of the ex situ components from floral verticils (calyx, corolla, androecium and gynoecium) and glands, completing the descriptions made by Versieux (2011).

Daily visits to observe and monitor the floral biology of eight genets separated by a minimum distance of 5 meters were carried out from March 6 to March 27, 2012. We observed the time of anthesis and floral senescence, flower duration (in days), period of pollen availability, and number of open flowers per day per genet. Stigmatic receptivity was tested three times a day (8:00 am, 12:00 and 5:00 pm) with hydrogen peroxide, using a 10x magnification hand lens to observe the formation of bubbles (Dafni et al., 2005). Nectar volume was monitored at three different periods (morning, noon and late afternoon) by removing all nectar accumulated at the base of the ovary with a graded microsyringe

(Hamilton). Sugar concentration was analyzed with the aid of a hand-held refractometer

0-50% Brix (Bernardello et al., 1991). Flores de diferentes indivíduos (n = 8) foram acondicionadas em tubos falcon por 24 horas e depois verificadas (através do olfato) quanto a presença ou ausência de odor.

Focal observations were conducted during the day, along one week, totaling 12 hours of direct observation, noting the time and number of floral visitors (and collection of possible visitors) in eight flowering individuals.

Breeding system – A series of treatments were performed, with the aid of pollinator and pollen exclusion nets, totaling 59 flowers. The treatments were: (1) manual self-pollination: to test for self-compatibility, unopened flowers were bagged, and on the next day, after anthesis, were pollinated with their own pollen and bagged again (n=12); (2) manual cross-pollination: to test cross-fertilization, unbagged flowers were pollinated with pollen from other flowers

(preferably from plants distant at least 5 m apart to avoid collecting pollen from relatives) and bagged (n=12); (3) spontaneous self-pollination: to test the need of pollinator vectors, buds were bagged and no artificial pollination was performed (n=16); (4) to test the effective contribution of pollinators in the system, senescent flowers were bagged, serving as a control treatment (n=19).

In May 2012, the bagged fruits of V. minarum (already considered mature due to the color and shape of the capsules) were collected and the seeds of each fruit were counted under stereomicroscope.

Seed morphology and post-seminal development - The seeds of each treatment were germinated in Petri plates with filter paper soaked in distilled water, and placed under natural light at room temperature for 30 days, where the start of germination was measured by the emission of the radicle. Daily observations were made to verify the emergence and growth of the radicle and other vegetative organs of the seedling.

Results

Observations and floral biology – In the PESRM the flowering period (measured by the start to end of the inflorescence production) of V. minarum is from January to March. During the first month there was the emergence of the inflorescence peduncle and, in the second month,

69 flower buds were already visible in an immature inflorescence. On the third month the inflorescences were mature and presented two overlapping phenophases (flower buds and open flowers) (Fig. 2).

Inflorescence maturation occurs from base toward the apex, with the opening of a single flower per day (Fig. 2a). Flowers last two days and have diurnal anthesis (after 8:00 am), remaining open during the first night. Although observations were not carried out during the night, we deduced that flowers remain opened due our last observation at 6:00 pm until our first observations the next morning at 6:00 am, the flowers are still open.

The first sign of anthesis is the spreading of the petals apexes (Fig. 2b). After that, the stigma emerges, but is not yet receptive (Fig. 2c), followed by the appearance of the stamens, which in a next moment will be more exposed and with the anthers completely exserted (Fig.

2d).

The anthers appeared close to petal apex a few hours after anthesis, starting to release pollen after 9:00 am, with the release of pollen throughout the day until the early hours of the following morning.

The stigma receptivity was checked only at the end of the first day (around 4:00 pm)

(Fig. 2e) until the morning of second day.

Flower senescence start on the morning of the second day. Senescence in V. minarum flowers is characterized by the loss of the yellow color of the sepals and darkening of the petals (Fig. 2f).

Nectar is accumulated at the base of the corolla, and production starts around 8:00 am(mean = 0.023 ml ± 0.02 SD, N = 3). There was a general trend of decrease (Fig. 3), with end of the production around 2:00 pm of the second day. Both mean nectar volume and sugar concentration were higher on the first day.

Similarly, the mean sugar concentration in the nectar was higher on the first day (mean

= 13% ± 9.5 SD, N = 3). On the second day the average sugar concentration was 9%.

Similarly as with production, there is a general trend of decreasing sugar concentration throughout the day (Fig. 3). When nectar was removed the flower restored the same average amount of nectar throughout the day and its output coincides with the dehiscence of tecas to release pollen.

Flowers have no perceivable odor after being kept in sealed vials.

Unfortunately no floral visitors or pollinators were observed, although many different species of pollinators (hummingbirds, butterflies, bees and beetles) in other plant species that were flowering in the same area could be noticed. In only one individual there was the occurrence of an unidentified ant which circulated throughout the axis of the inflorescence, and got into the flowers sporadically without touching stamen and stigma nor causing damage to the floral parts.

The flowers of V. minarum have trimerous perianth (three sepals and three petals), with petal appendages in six number (two per petal) (Fig. 4c). The ovary is superior (Fig. 4g) and 3-locular (Fig. 4b and d). Nectaries are infralocular, withseptal nectaries well developed

(Fig. 4a). The anthers are dorsifixed and have longitudinal dehiscence (Figs. 4e, f). The stigma is type convolute blade (Fig. 4h).

Reproductive biology - The number of fruit set per treatment was 18 in the control; 11 in spontaneous selfing; 9 in manual self-pollination and 5 in manual cross-pollination (Table 1).

The number of seeds for fruit was 4,156 in the control treatment; 2,396 in the spontaneous self-pollination; 1,797 in the manual self-pollination and1,318 in manual cross- pollination (Table 1).

The largest number of germinated seeds occurred in treatments with manual cross- pollination (207 = 54%), following of spontaneous self-pollination (139 = 36%), while the

71 manual self-pollination treatment was 38 and none of the seeds from the control treatment germinated (Table 1).

Seed morphology and post-seminal development - V. minarum has small seeds (length 6 mm

± 0.06 SD; N = 5), filiform, brown, bearing whitish feathery appendages along the base (Fig.

5a). The germination was initiated at 12-13 days after imbibition (Fig. 5b).The radicle appears between the cotyledon and the basal coma. A characteristic post-seminal in V. minarum is the presence of the undeveloped hypocotyl. The first leaf appears five days after the radicle emergence (Fig. 5c), and it takes five more days to the formation of the second leaf (Fig. 5d).

After the appearance of the third leaf, leaves develop so intertwined that the shape of a miniature rosette, typical of Bromeliaceae, can be noticed (Fig. 5e).

Discussion

Observations and floral biology – The reproductive phase observed here for V. minarum in

PESRM coincides with the rainy season, corroborating Versieux’s data (2011). Nevertheless, the flowers only started to open in March. In other areas of the IQ the blooming of V. minarum appears to be longer, from December to March based on herbaria records (Versieux

& Wendt 2006; Versieux 2011).

The lack of visitors to V. minarum may be explained by the atypical climatic conditions in 2012. An above-the-mean amount of rain in January was followed by a severe drought in

February and March, which was reflected in failure of buds to open and a limited number of flowers available. On the other hand, other shrubs and herbs had numerous flowers and could be more attractive to potential pollinators.

Sick (1984) suggested that the diversification of the families Bromeliaceae and

Trochilidae (family of hummingbirds) has proceeded in parallel, because the latter contains the most important pollinators for bromeliads (Machado and Semir, 2006). This idea has been supported by several studies that link hummingbirds as pollen vectors of about 85% of

72 bromeliads in several communities investigated, the other species being visited by bats and, to a lesser extent, bees (Machado and Semir, 2006).

Despite the absence of visitors during our observations, the basic characteristics of the flowers of V. minarum suggest a mixed pollination syndrome. Mixed pollination systems are characteristic of several Mesoamerican Bromeliaceae species, with some species of Tillandsia presenting flowers that last for almost 48 hours, a strategy to allow access of pollinators that are active during the night (Benzing 2000).

The flower has typical ornitophily features, such as exposed flowers and inflorescence, tubular and odorless flowers, diurnal anthesis, conspicuous or contrasting colors among floral parts, nectar sugar concentration lower, stigma and anthers away from the nectaries and positioned to optimize the contact with birds, and absence of a landing platform (Machado and Semir, 2006). Marques & Pires (2008) observed visits of birds to V. minarum (at that time identified as V. citrina) in Serra da Piedade, 41 km from PESRM. These authors also noted that above 1,500 m. a. s. l. in the area of Serra da Piedade ornithophily was a trend among the

Vriesea species. Other morphologically similar species were also indicated as ornithophilous, as is the case of V. marceloi Versieux & Mota, from Serra do Caraça (Versieux & Mota,

2012). Versieux (2011) also made personal observations of hummingbird visits to V. minarum.

On the other hand, flowers have a duration of two days, do not close during the night, and the stigma becomes receptive in the late afternoon, suggesting visits by nocturnal pollinators, possibly bats. Bats are the main pollinators in several bromeliad genera, including

Vriesea, Section Xiphion (e.g. Vogel, 1969; Sazima et al., 1989, 1995; Martinelli, 1997;

Kessler and Kromer, 2000; and Benzing, 2000b), although most bat-pollinated of Vriesea species have wide-mouthed flowers and stamens positioned on one side of the flower (Sazima et al., 1995). Moth pollination is also a possibility to be further tested in V. minarum, though

73 pollination by insects is not very common in Bromeliaceae and, when present, is made mostly by bees (Araujo et al., 2004) or butterflies (Varassin and Sazima, 2000; Kaehler et al., 2005).

A recent population genetics study of the species (Lavor et al. in prep.) indicated low structuring, attributed either to efficient seed or pollen dispersal. Connectivity among the populations would be better explained if bats were the main pollinators, since they can travel great away to feeding sites (up to 38 km), whereas many species of hummingbirds are territorial, thus restricting cross-pollination distances (Proctor et al., 1996). Nevertheless, the low levels of gene flow obtained with the rupicolous and closely related genus Alcantarea lead Barbará et al. (2007) to suggest that bats do not contribute so much to promote cross- pollination between inselberg populations.

Reproductive biology – There are many characteristics that may influence the proportions of selfing and outcrossing breeding systems in Bromeliaceae. Protandry is the main means of promoting outcrossing in self-compatible species (Benzing, 2000), as occurs with V. minarum. This strategy was observed by Martinelli (1994) in 17 Vriesea species pollinated by hummingbirds and bats in southeastern Brazil Atlantic rainforest.

The larger number of germinated seeds per fruit produced by manual cross-pollination treatment suggests that the species is predominantly allogamous. However, the number of seeds produced by both manual and spontaneous self-pollination (Table 1) indicates that the species is partially self-compatible and capable of reproducing in case of pollinator shortage.

For V. minarum the reproductive pattern is similar to observed of the bromeliad species studied for Martinelli (1994) and Mantallana et al. (2010), what presented cross- fertization and characteristics for the outcrossing breeding system (being an advantage when pollinator availability is large). However, in their works the bromeliads with self fertilization were more abundant.

Morphology of seeds and post-seminal development – The seeds of V. minarum were morphologically similar to those from other species Tillandsioideae. This subfamily is characterized mainly by the coma, the flight apparatus allowing anemochory, which is also used to segregate among genera, since it is quite variable in the Tillandsioideae (Benzing,

2000). This structure increases the surface/volume ratio, reducing speed of the fall during dispersal by air streams or winds in the dry season (Pereira et al., 2008).

Seed germination time is considered intermediate according to Pereira et al. (2008) classification, and similar with that described for other Tillandsioideae (Pereira et al., 2008), as well as their post-seminal characteristics, such as presence of the undeveloped hypocotyl.

Vriesea minarum had a smaller germination rate (of 2,785 viable seeds, 384 germinated

= 14%) at ambient temperature (around 25-30 °C) in comparison with other Tillandsioideae

(Maques 2002). The lack of germination in seeds may be due to factors such as selectivity to the conditions of temperature and luminosity, coupled with the loss of seed viability.

Conservation - In terms of ecological importance, the family Bromeliaceae is remarkable and the amount of scientific literature available about the floral biology, phenology and reproductive systems is still quite scarce (Rios et al., 2010) if compared to the richness and dominance of Bromeliaceae taxa in many habitats.

This knowledge is therefore fundamental, since the bromeliads are biodiversity promoters and help in the establishment and maintenance of a whole chain of organisms

(Santana and Machado, 2010).

Thus, given the current stage of the danger that V. minarum is exposed to, it is suggested that the creation of more protected areas should be integrated with ex situ conservation strategies, such as a seed bank. Different techniques are available to store and preserve seeds in germplasm banks as an efficient method for long-term conservation of plant genetic resources (Pereira et al., 2010; Aoyama et al., 2012).

Acknowledgements

We thank CAPES for the first author’s M.Sc. fellowship and FAPESB for financial support (PNX0014/2009). CVDB thanks CNPq for his fellowship (PQ-1D). CMJ thanks the

Brazilian National Council for Scientific and Technological Development (CNPq) for a

Research Productivity Scholarship. The Rola Moça State Park, and the availability of personnel assistance to collect and observation of specimens, the State Forest Institute (IEF).

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Tables

Table 1 – Results for the reproductive system treatments of Vriesea minarum in the State Park of Serra do Rola-Moça.

Treatment Nº of fruits Nº of seeds for fruits Nº of seeds germinated spontaneous self-pollination 11 2396 139 manual cross-pollination 5 1318 207 manual self-pollination 9 1797 38 Control 18 4156 0

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure Legends

Figure 1 – Map of Parque Estadual da Serra do Rola-Moça (PESRM) in the Iron Quadrangle (IQ)

Minas Gerais, Brazil, indicating the study area (canga) (modified from Garcia et al., 2009).

Figure 2 - Specimen of Vriesea minarum growing on typical ironstone habitat. (a) individual blooming, (b) flower bud, (c) stigma protruding, (d) stamens with closed anthers, (e) fully open flower, (f) senescent flower. Bars: (a) = 10 cm, (b – f) = 1 cm.

Figure 3 - Nectar production (ml) and nectar concentration (%) in Vriesea minarum flowers.

Figure 4 - Flower morphology of Vriesea minarum. (a) longitudinal section of V. minarum showing the well-developed septal nectaries; (b) transversal section of the ovary showing the locules; (c) petal appendages; (d) ovules; (e) anther in front view; (f) anther in side view; (g) superior ovary; (h) convolute blade stigma. Bars = 1 mm.

Figure 5 - Post seminal development of Vriesea minarum. (a) seed of V. minarum; (b) initiation of germination; (c) the first leaf appearance and radicle emergence; (d) formation of the second leaf;

(e) appearance of the third leaf. Bars = 2 mm.

6 CONCLUSÃO GERAL

V. minarum apresenta uma baixa estruturação genética e pouco diferenciação entre as populações, havendo uma baixa correlação entra a distância genética e a distância geográfia das mesmas; da mesma forma, apresenta um alto coeficiente de endogamia, além de diferentes níveis de diversidade genética, onde algumas populações (Gandarela 2, Pedra Rachada e Tamanduá) apresentam os maiores índices de diversidade. Estes dados podem ser fruto de dois processos que podem estar agindo em conjunto: o modo de distribuição geográfica e a biologia reprodutiva encontrada na espécie. Em termo de distribuição, as populações de V. minarum encontram-se hoje fragmentadas ao longo do Quadrilátero Ferrífero; no entanto, este státus de fragmentação pode ser um processo recente, devido a grande perda dos hábitats (cangas) nos quais a espécie se encontra. É sabido que, nas ultimas décadas, a atividade de mineração tem aumentado significativamente e com isso, cangas que antes existiam e que haviam registros de ocorrencia de V. minarum, já não mais existem, e dessa forma, cria uma lacuna de acesso que as populações teriam para uma conectividade. Dessa forma, as populações hoje encontram-se isoladas, mas seu tempo de divergencia, ainda é muito pouco para possivel estruturação. Da mesma forma, o tipo de reprodução encontrado na espécie (sexuada e assexuada), associada a uma gama maior de polinizadores (devido a um sistema misto de polinização que poder ser tanto por beija-flor como por morcego), e o fato da espécie ser predominantemente alógama, mas também parcialmente auto-compatível, e ter suas sementes dispersas pelo vento, auxilia em sua maior conectividade, mas também nos altos indices de endogamia. Estes aspectos seriam estratégias da espécie para evitar competição pelos polinizadores, além de auxiliar na possível falta dos mesmos. Assim, V. minarum, encontraria-se como uma espécie que investe nas mais variadas formas para sobrevivência, sendo estas vitais em para sua sobrevivência, principalmente frente a grande destruição antrópica a qual está exposta atualmente.

6.1 IMPLICAÇÕES PARA A CONSERVAÇÃO

V. minarum constitui-se hoje como uma espécie altamente ameaçada e com nenhum programa para sua preservação. Esta realidade torna-se ainda mais preocupante quando se nota o grau acelerado de devastação e perda de habitat pela mineração: das 12 populações amostradas para o estudo, apenas três encontram-se em área de preservação (duas no Parque Estadual da Serra do Rola-Moça e uma no Santuário de Nossa Senhora da Piedade); quatro estão em áreas com licenças

91 aprovadas para mineração, com lavras já abertas (populações da Gandarela, no município de Rio Acima; Morro do Tamanduá, no município Nova Lima e Serra da Calçada, no município de Brumadinho) e as demais, encontram-se em áreas onde o fogo é também um fator de risco (como nas populações do Marinho da Serra, no município de Moeda; Pedra Rachada, em Sabará e no Parque Estadual da Serra do Rola-Moça, onde o tamanho da população foi reduzido a apenas poucos indivíduos por grandes queimadas nos anos anteriores). Estas perdas são fatais para a uma espécie de distribuição tão restrita. Sua genética e biologia reprodutiva são apenas alguns dos aspectos que tentamos desvendar, mas que não denotam toda a riqueza que a espécie pode ter, já que as plantas dos campos rupestres ferruginosos são as que apresentam uma imensa gama de poder de adaptação, frente a tantas adversidades que as cangas lhes proporcionam. Dessa forma, o estudo da genética de V. minarum nos mostra dois cenários: que as populações possuem um grande poder de dispersão de sementes e/ou que o pólen é levado entre populações distintas, colaborando para o padrão de estruturação observado. Ou se trata de uma única população mais antiga, com ampla distribuição por todo Quadrilátero Ferrífero. Os dados aqui apresentados permitiram compreender a estruturação genética das populações e indicar ações práticas que devem ser seguidas pelo poder público para maximizar a proteção tanto em número de indíviduos quanto em genótipos. Os resultados obtidos com os experimentos de campo e observações da biologia floral nos permitiram conhecer melhor a morfologia, o desenvolvimento pós-seminal e as características que se relacionam com a síndrome da espécie. Foi constatado que a planta possui um síndrome mista, talvez para maximizar o uso dos potenciais polinizadores. Não foram vistos visitantes florais, o que demandará por novas observações no campo devido ao clima atípico que se desenvolveu no ano de 2012. Assim, este trabalho abrangeu as mais diferentes áreas do conhecimento para compilar o máximo de conhecimento para esta espécie tão ameaçada, Vriesea minarum, para quem sabe agora, munido de todas essas informações, medidas de conservação sejam realmente traçadas, e que o meio científico e a sociedade saibam o quanto se faz necessário, e urgente, o melhor entendimento sobre a biologia de todas as espécies do Quadrilátero Ferrífero, para que no futuro não se lamente a perda de toda essa biodiversidade.

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involved in species cohesion and reproductive isolation in Neotropical inselbergs. Molecular Ecology, v. 20, p. 3185-3201, 2011. PETIT, R.J.; EL MOUSADIK, A.; PONS, O. Identifying Populations for Conservation on the Basis of Genetic Markers. Conservation Biology, v. 12, p. 844-855, 1998. PORTO, M.L.; SILVA, M.F.F. Tipos de vegetação metalófila em áreas da Serra de Carajás e de Minas Gerais. Acta Botanica Brasilica, v. 3, p. 13-21, 1989. PROCTOR M.; YEO, P.; LACK, A. The Natural History of Pollination. Portland, Oregon: Timber Press, p. 480, 1996. ROCCA, M.; SAZIMA, M. Quantity versus quality: identifying the most effective pollinators of the hummingbird-pollinated Vriesea rodigasiana (Bromeliaceae). Plant Systematics and Evolution, v. 299, p. 97-105, 2013. ROSIÈRE, C.A.; CHEMALE, F. Brazilian iron formations and their geological setting. Revista Brasileira de Geociências, v. 30, p. 274-278, 2000. SAJO, M.G.; RUDALL, P.J.; PRYCHID, C.J. Floral anatomy of Bromeliaceae, with particular reference to the evolution of epiginy and septal nectaries in commelinid monocots. Plant Systematics and Evolution, v. 247, p. 215-231, 2004. SAZIMA, M.; BUZATO, S.; SAZIMA, I. Polinização de Vriesea por morcegos no sudeste brasileiro. Bromélia, v. 2, p. 29-37, 1995. SCARANO, F.R. Structure, function and floristic relationships of plant communities in stressful habitats marginal to the Brazilian Atlantic rainforest. Annals of Botany, v. 90, p. 517-524. 2002. SCHLÖTTERER, C.; TAUTZ, D. Slippage synthesis of simple sequence DNA. Nucleic Acids Research, v. 20, p. 211-215, 1992. SEGELBACHER, G.; CUSHMAN, S.A.; EPPERSON, B.K.; FORTIN, M.-J.; FRANCOIS, O.; HARDY, O.J.; HOLDEREGGER, R.; TABERLET, P.; WAITS, L.P.; MANEL, S. Applications of landscape genetics in conservation biology: Concepts and challenges. Conservation Genetics, v. 11, p. 375-385, 2010. SGORBATI, S.; LABRA, M.; GRUGNI, E.; BARCACCIA, G.; GALASSO, G.; BONI, U.; MUCCIARELLI, M.; CITTERIO, S.; IRAMATEGUI, A.B.; GONZALES, L.V.; SCANNERINI, S. A survey of genetic diversity and reproductive biology of Puya raimondii (Bromeliaceae), the endangered queen of the Andes. Plant Biology, v. 6, p. 222-230, 2004. SMITH, L.B.; DOWNS, R.J. Bromeliaceae (Tillandsioideae). Flora Neotropica Monograph, v.14, p. 663–1492. 1977. STOCKWELL, C.A.; HENDRY, A.P.; KINNISON, M.T. Contemporary evolution meets conservation biology. Trends in Ecology & Evolution, v. 18, p. 94-101, 2003.

TEIXEIRA, W.A.; LEMOS-FILHO, J.P. Metais pesados em folhas de espécies lenhosas colonizadoras de uma área de mineração de ferro em Itabirito, Minas Gerais. Revista Árvore, v. 22, p. 381-388, 1998. VARASSIN, I.G.; SAZIMA, M. Recursos de Bromeliaceae utilizados por beija-flores e borboletas em mata atlântica no sudeste do Brasil. Boletim do Museu de Biologia Mello Leitão, v. 11/12, p. 57-70, 2000. VERSIEUX, L.M. Brazilian plants urgently needing conservation: the case of Vriesea minarum (Bromeliaceae). Phytotaxa, v. 28, p. 35-49, 2011. VERSIEUX, L.M.; WENDT, T. Bromeliaceae diversity and conservation in Minas Gerais, Brazil. Biodiversity and Conservation, v. 16, p. 2989-3009, 2007. VERSIEUX, L.M.; WENDT, T.; LOUZADA, R.B.; WANDERLEY, M.G.L. Bromeliaceae da Cadeia do Espinhaço. Megadiversidade, v. 4, p. 98-110, 2008. VERSIEUX, L.M.; ELBL, P.M.; WANDERLEY, M.G.L.; MENEZES, N.L.D. Alcantarea (Bromeliaceae) leaf anatomical characterization and its systematic implications. Nordic Journal of Botany, v. 28, p. 385-397, 2010a. VERSIEUX, L.M.; LOUZADA, R.B.; VIANA, P.L.; MOTA, N.; WANDERLEY, M.G.L. An illustrated checklist of Bromeliaceae from Parque Estadual do Rio Preto, Minas Gerais, Brazil, with notes on phytogeography and one new species of Cryptanthus. Phytotaxa, v. 10, p. 1-16, 2010b. VOSGUERITCHIAN, S.B.; BUZATO, S. Reprodução sexuada de Dyckia tuberosa (Vell.) Beer (Bromeliaceae, Pitcairnioideae) e interação planta-animal. Revista Brasileira de Botânica, v. 29, p. 433-442, 2006. WENDT, T.; CANELA, M.B.F.; DE FARIA, A.P.G.; RIOS, R.I. Reproductive biology and natural hybridization between two endemic species of Pitcairnia (Bromeliaceae). American Journal of Botany, v. 88, p. 1760-1767, 2001. WENDT, T.; CANELA, M.B.F.; KLEIN, D.E.; RIOS, R.I. Selfing facilitates reproductive isolation among three sympatric species of Pitcairnia (Bromeliaceae). Plant Systematics and Evolution, v. 232, p. 201-212, 2002. WOODWORTH, L.M.; MONTGOMERY, M.E.; BRISCOE, D.A.; FRANKHAM, R. Rapid genetic deterioration in captive populations: causes and conservation implications. Conservation Genetics, v. 3, p. 277-288, 2002. ZANELLA, C.M..; JANKE, A.; PALMA-SILVA, C.; KALTCHUK-SANTOS, E.; PINHEIRO, F.G.; PAGGI, G.M.; SOARES, L.E.S.; GOETZE, M.; BÜTTOW, M.V.; BERED, F. Genetics, evolution and conservation of Bromeliaceae. Genetics and Molecular Biology, v.35, p. 1020-1026, 2012.

8 ANEXOS 8.1 NORMAS DAS REVISTAS 8.1.1 Brazilian Journal of Botany Manuscripts should be double-spaced and with consecutive page numbering. Use Word for Windows 6.0 or latest version, A4 paper, and only 2 cm margins; font - Times New Roman, size 12. Place only one space between words and do not hyphenate them. Do not use tabulation (Tab key). Do not use bold or underline. Restrict italics only for scientific names or words in Latin. Manuscript format First page - Title: concise and informative (in bold); authors' full names (in capital letters); Affiliation and complete address as a footnote, corresponding author and respective e-mail; running title. Second page - ABSTRACT (include title, in English), RESUMO (include title, in Portuguese), Key words (up to 5, in English). Abstract and Resumo should have up to 250 words each. Text - Start a new page according to the following sequence: Introduction, Material and methods, Results, Discussion, Acknowledgements and References. Cite each figure and table in the text in numeric order. Present references according to the following examples: Smith (1960) / (Smith 1960); Smith (1960, 1973); Smith (1960a, b); Smith & Gomez (1979) / (Smith & Gomez 1979); Smith et al. (1990) / (Smith et al. 1990); (Smith 1989, Liu & Barros 1993, Araujo et al. 1996, Sanches 1997). In taxonomic papers, cite botanic material in detail in the following sequence: place and date of collection, collector's name and number, and herbarium abbreviation, according to the samples below: BRAZIL: Mato Grosso: Xavantina, s.d., H.S. Irwin s.n. (HB 3689). São Paulo: Amparo, 23/12/1942, J.R. Kuhlmann & E.R. Menezes 290 (SP); Matão, ao longo da BR 156, 8/6/1961, G. Eiten et al. 2215 (SP, US). References to unpublished results or submitted papers should appear as follows: (S. E. Sanchez, unpublished data). Provide numbers and units as follows: Numbers up to nine should be written in full, except if they are followed by units or indicate tables or figures. For decimal numbers, use a comma in articles in Portuguese and Spanish (10,5 m) or a dot in papers in English (10.5 m). Separate units from values by placing a space (except for percentages, degrees, minutes and seconds); use abbreviations whenever possible.

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For composed units, use exponentiation, not bars (Example: mg.day-1 instead of mg/day, µmol.min-1 instead of µmol/min). Do not insert spaces to move to the next line in case the unit does not fit in the same line. Do not insert figures in the text file. References - Indicate in pencil, next to the reference, the page where it was mentioned. Adopt the format used in the examples as follows: ZAR, J.H. 1999. Biostatistical analysis. Prentice-Hall, New Jersey. YEN, A.C. & OLMSTEAD, R.G. 2000. Phylogenetic analysis of Carex (Cyperaceae): generic and subgeneric relationships based on chloroplast DNA. In Monocots: Systematics and Evolution (K.L. Wilson & D.A. Morrison, eds.). CSIRO Publishing, Collingwood, p.602-609. BENTHAM, G. 1862. Leguminosae. Dalbergiae. In Flora brasiliensis (C.F.P. Martius & A.G. Eichler, eds.). F. Fleischer, Lipsiae, v.15, pars 1, p.1-349. DÖBEREINER, J. 1998. Função da fixação de nitrogênio em plantas não leguminosas e sua importância no ecossistema brasileiro. In Anais do IV Simpósio de Ecossistemas Brasileiros (S. Watanabe, coord.). ACIESP, São Paulo, v.3, p.1-6. FARRAR, J.F., POLLOCK, C.J. & GALLAGHER, J.A. 2000. Sucrose and the integration of metabolism in vascular plants. Plant Science 154:1-11. Cite dissertations or thesis only in exceptional cases, when the information provided is essential for a good understanding of the paper, and when they have not been published as scientific papers. In this case, use the format below: SANO, P.T. 1999. Revisão de Actinocephalus (Koern.) Sano - Eriocaulaceae. Tese de doutorado, Universidade de São Paulo, São Paulo. Do not cite meeting abstracts. Tables Use Word for Windows' design resources and table formatting. Avoid abbreviations (except for units). Tables should be placed in separate pages, their titles inserted in the upper part, according to the example: Table 1. Total flavonoids and total phenol production (% of dry weight) in leaves of Pyrostegia venusta. Do not insert vertical lines; use horizontal lines only to stress the header and close the table. For tables covering more than one page, add "(cont.)" in the upper left corner of the following page(s), and repeat the header. Figures Submit a set or original figures in black and white and three high-resolution copies.

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The space available for illustrations (plates with photographs or drawings, charts, maps, diagrams) is 15 x 21 cm the most, including the space required for the legend. Any figure exceeding the size established will be refused. Digitalised files must be accompanied by a hard copy (printed in glossy paper or photo paper) of the figure. Charts or other figures fitting into a single column (7.2 cm) will be reduced; therefore, be sure that numbers or font size remain visible after reduction. Font type and size, both in the legend and in the chart, should be the same used in the text. Insert each figure in a separate page. Type all figure legends (numbered sequentially), in another page. Use bar scale to indicate size. Scales should be placed on the bottom of the page on the left hand side. The right hand side must contain figure number. Details of the manuscript organisation can be found in the last pages of every issue. For further information, refer to the journal's latest issue. The paper's final acceptance date will only be revealed after approval by the Editorial Board, both in terms of scientific merit and graphic format. The paper's final version, accepted for publication, should be sent in printed copy and in diskette, duly identified. 8.1.2 American Journal of Botany Manuscript Content 1.Title Page Place a running head 2.5 cm (1 in) below the top of the page with the surname of the FIRST author (followed, as appropriate, with the surname of a sole co-author, or with et al. if there are three or more authors) and a short title. The manuscript title for research papers should be specific and informative, conveying the key findings of the research in an active voice. Center boldfaced title written with sentence-style capitalization, followed by superscript 1 (for footnote 1, to appear on footnote page). In most cases, Latin binomials in a title should be followed by the name of the family in parentheses. Below the title, list authors: each author’s first name, middle initial, surname. On the next line, give affiliation and unabbreviated address. If authors have different affiliations and addresses, add a superscript number after each author’s name to indicate the footnoted address. Include another footnote superscript number to indicate the author for correspondence. 2. Footnote Page Include the following footnote: 1Manuscript received ______; revision accepted ______.

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Place brief acknowledgments, if desired, as a separate paragraph, using the following style: “The author(s) thank(s)…”. For brevity, do not use first names. Include grant acknowledgments here. Other footnotes (e.g., e-mail for correspondence) are permitted: match footnote numbers with those on the title page. 3. Abstract Page AJB requires structured abstracts for manuscript submission. The abstract is 250 words or less, written in the following structured format: Premise of the study (why the work was done, what major questions of plant biology are addressed, and why it is important to the broad AJB readership) Methods Key results Conclusions (what major points should the reader take from this article) Note that the abstract will be used in an RSS feed and thus should capture the interest of the general botanical community as well as the specialists and include the most important contribution of this paper. Avoid references; if essential, cite parenthetically with journal name, volume number, pages, and year. Provide a list of 3–10 “Key words” that will be used for the volume index. Capitalize proper nouns, place in alphabetical order, and separate by semicolons. 4. Text In the first paragraph of the introduction, include the theoretical or conceptual basis for your work in a context accessible to the diverse botanical readership that AJB attracts. Include a summary of conclusions and a take-home message for the generally informed reader in the DISCUSSION. Center main headings and capitalize all letters: MATERIALS AND METHODS, RESULTS, and DISCUSSION. Indent subheadings at the start of a paragraph; capitalize only the first word and proper nouns and adjectives. Second-level headings—(boldface italic followed by an em dash) Third-level headings—(italic followed by an em dash) Fourth-level headings—(regular text followed by an em dash) In MATERIALS AND METHODS add name, city, spelled-out state (if in USA), and country of manufacturers/suppliers after brand names. If statistical analyses are used, include statistical values in the RESULTS either in the text or within tables. Include the statistic value, degrees of freedom, and p-value for each result reported

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(e.g., for a t-test report “t = 32.41, df = 1, P = 0.03“ for an ANOVA report “F5, 23 = 26.45, P less than 0.001" [note two df-values as subscripts with F]). Use P for significance, and p for probability. Common Latin words (e.g., in vivo, sensu lato) are not italicized. Footnotes are not used in the text. 5. Literature Cited Verify all entries against original sources. Double check that all references in the manuscript text are in the Literature Cited and vice-versa and that they agree in spelling and year. Literature citations in text—Cite references in chronological order (oldest first); within a given year, order them alphabetically (e.g., Jones and Gil, 1999, 2006; Ashton et al., 2007; Brown, 2007; Jackson, 2005, 2008). Single author: Jones (2008) or (Jones, 2008). Two authors: Jones and Gil (2008) or (Jones and Gil, 2008). More than two authors: Jones et al. (2008) or (Jones et al., 2008). Manuscripts accepted for publication but not yet published: Jones (in press) or (Jones, in press). Include “In press” citations in LITERATURE CITED (shown later). Unpublished data and manuscripts (e.g., submitted, in prep.) and personal communication: (F. Jones, Institution, unpublished data [or unpublished manuscript or personal observation]). These are not included in LITERATURE CITED. References listed in LITERATURE CITED—List citations in alphabetical order by author. Single-author titles precede multi-authored titles by the same senior author, regardless of date. List works by the same author(s) chronologically, beginning with earliest date of publication. Spell out all author(s)’ names. Use “a”, “b” (determined alphabetically) for works with the same author(s) and year citation. For multi-authored works, list the first seven authors and then “et al.”— unless there are only eight authors and then list all eight. Type author names in citations in upper and lower case or in large and small caps, not in all caps. For formatting examples (note spacing, capitalization, italics, etc.), go to http://www.botany.org/ajb/ajb_Lit_Cited_Instructions.pdf. 6. Tables – include in manuscript file and place immediately after Literature Cited Tables need to be formatted using the Table feature in Word or in a spreadsheet such as Excel. Number tables with Arabic numerals followed by a period. Capitalize first word of title; all others, except proper nouns, are lowercase; spell out names of genera and abbreviations on first mention; place period at end. Include study organism (species or group) and geographic location in each caption when appropriate. Place explanatory notes and define all abbreviations below the table after the heading “Note:” or “Notes:”. Place footnotes after the Notes.

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Every column must have an appropriately placed heading (esp. the first at left—the stub head), with appropriate subheadings. In the body of the table, capitalize the first word of each entry (and proper nouns); do not use vertical lines between columns; indicate footnotes by lowercase superscript letters. If the use of color in a table is essential, please contact the Editorial Office at [email protected]. 7. Appendices – include in manuscript file and place immediately after the tables If voucher and gene accession information support the study, list these in Appendix 1, which will be published in the print and online versions. Provide an appendix title, and a sentence-style row of headings for the data. For each taxon sampled, include specimen voucher information and/or gene accession numbers, separated by commas. To save space, the taxa can be run together in a paragraph. See a current issue or http://www.amjbot.org/content/98/6/1049.full for an example. Additional appendices may be included. AJB encourages online-only publication of extensive appendices, as well as other supplemental materials that support the article but are best presented electronically (see “Online Supplemental Materials” below). 8. Figure Legends – include in manuscript file and place immediately after the Appendices (or after the tables if there are no appendices) Each figure legend must be complete and informative so that reference to the text is not necessary to understand the content of the figure. Abbreviations should be defined unless they are standard convention. Place legends as separate paragraphs following the appendices. For figures with multiple lettered panels, a general title for the figure should be followed by a description of each panel (e.g., Fig. 5. Relationship between… (A) All fruits. (B) Fruits less than 0.5 mm.). When applicable, study organism (species or group) or geographic location, and define scale bar (e.g., Bar = 0.1 µm). For micrographs, include pertinent information such as magnification and type of section, stain, optics, or special techniques. Any nonlinear adjustment to photographs must be detailed. Define all symbols and abbreviations either in a key within the figure or in the legend; if defined in an earlier legend, the appropriate figure or table may be cited. Place figure abbreviations in alphabetical order and format as follows: c, cell; n, nucleus. 9. Figures/Illustrations - upload as separate files (do not include in the manuscript file) For details and illustrated examples, see http://www.botany.org/ajb/AJB_Digital_Art_Guidelines.pdf. A figure checklist is also available at http://www.botany.org/ajb/AJB_Figure_Checklist.pdf. TIFF or EPS formats are preferred for color and black and white photographs, drawings, and graphs.

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Prepare figures at the final size desired: 1 column (8.9 cm [3.5 in]), 1.5 column (12.7-15.3 cm [5-6 in]), or 2 columns (18.4 cm [7.25 in]) wide and less than the length of the page (23 cm [9 in]). Low-resolution files may be initially uploaded/submitted for the review process. Once your manuscript has been tentatively accepted, printer-quality (high-resolution) figures are required. See “Tips for Large Files” below. Figure Manipulations Certain types of electronic manipulations of micrographs and other digital images may not be ethically acceptable. Images that will be compared with each other must be acquired and processed under the same conditions. Manipulations such as background subtraction or white-balancing should be explained in the Materials and Methods section. Note that a selected area within an image may not be altered or enhanced; the entire image must be treated the same. Linear adjustments to contrast, brightness, or color must be applied to an entire image or plate equally (or explained). Detail nonlinear adjustments in the legend. Always keep original raw data files for documentation upon request. Resolution for Final Figures Line art (black lines and text, including phylogenetic trees): 1000-1200 dpi. Halftone/grayscale (images with shades of gray, such as black and white photographs): a minimum of 300 dpi. Color: a minimum of 300 dpi. Use RGB mode (not Indexed Color Mode). [Note: Do not send color files if images are to be printed in black and white.] Combination art (grayscale image with type): 600-900 dpi. Grayscale images should have the whitest area of the image set at a 2% highlight value, while the blackest area of the image should be set to a 98% shadow value. Include the screen and printer font files for any text that has been added to the figure. Use PC or Mac versions of Adobe Postscript fonts. To avoid font problems, convert all type to curves or paths. Format and Style Use consistent style, font, and font size (between 6 and 10 pt.) for all figures. Use of standard fonts (Times New Roman, Helvetica) gives better results. For figures with multiple elements (photos, drawings, or graphs), group elements in a rectangle or square and label the top left corner of each element with a capital letter (e.g., A, B). Keep elements close together for best use of space. Photographs in a composite plate should each be numbered and separated by a thin line or blank space.

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Label axes; include Standard International (SI) Units of measure in parentheses; capitalize only the first letter of the first word (e.g., “Stem growth (%)”). Axis label should be c. 0.2 cm from units on axis, but no more than 0.5 cm; x- and y-axis labels should be equidistant from axes. Use abbreviations consistently in the text and figures. For magnified illustrations, provide a scale bar defined in either the figure itself or at the end of the legend. Cover image and caption You are invited to submit one or more color photographs (or artificially colorized photomicrographs) to be considered for a cover illustration. The image must be at least 300 ppi and in portrait format slightly larger than 21.6 cm wide × 28 cm high (8.5 x 11 in). Submit the file(s) online with your original submission or revised manuscript. Also include a brief caption that describes the image, scientific name and authority of any organism, photographic technique, image manipulation, and the major result of the research. For micrographs, include pertinent information such as magnification and type of section, stain, optics, or special techniques. The legend should do more than just describe the image itself: it should "tell a story" by explaining why the image is important to entice the reader to search for the full article. See http://www.amjbot.org/content/vol95/issue4/cover.shtml for an example. Tips for Large Files Files >5 MB may be slow (or impossible) to upload on most servers. When saving graphics, LZW compression (Save As/Option) may be used to reduce file size. If your image is line-art and all pixels are either black or white, first convert the image to grayscale mode, then convert to bitmap mode at 1200 dpi, then save with LZW compression. If your image is black and white with gray portions, convert the image to grayscale mode, then save with LZW compression.(If you have any confusion about bitmap mode for line-art, your Digital Art Guidelines contain examples of image types with suggested resolutions. Alternatively, the Editorial Office may direct you to upload the files to an FTP site or send them via e-mail through http://www.YouSendIt.com. Online Supplemental Materials Authors may wish to augment their manuscripts with online supplemental materials (e.g., large data sets, three-dimensional reconstructions, simulations, real-time movies, color photographs). Upload these appendices as separate files with the initial manuscript submission. Include a header on each file using this format: Smith et al.—American Journal of Botany 99. 2012. – Data Supplement S1 – Page 1”. Name online supplements Appendix S1, Appendix S2, etc, in the order in which they appear in the text, regardless of whether they are tables, figures, text, other media, or a combination thereof. In the manuscript, after the mention of an online appendix, include the following: “(see Supplemental Data with the online version of this article)”.

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Note that if authors wish to submit long DNA sequence appendices as supplemental material, they should select the "DNA sequences (online-only supplemental)" option on Editorial Manager. This ensures that lengthy appendices are not built into the reviewers' PDF, but are still accessible to the reviewers. Abbreviations, Units, and Symbols See a recent Table of Contents page for commonly used abbreviations. Do not begin a sentence, heading, or title with an abbreviation. Abbreviate figure as “Fig.” or “Figs.” Use the following abbreviations with numerals without spelling out at first use: h, min, s, yr, mo, wk, d, cm, mm, DNA, cpDNA, RNA, dNTP. Designate temperature as in 30°C (use the degree sign, not zero or the letter o). Numbers: write out one through nine unless a measurement, a designator, or in a range (e.g., four petals, 3 mm, 6 yr, 5–11 species, day 2). Use % instead of percent with numerals; 1000 instead of 1,000: 10 000 instead of 10,000; 0.13 instead of .13. Use Standard International (SI) units throughout the text, figures, and tables. Use the word mass (kg, g, mg) correctly; weight is reported in newtons (N). Use either a solidus for one unit in the denominator (e.g., kg/m2) or a negative exponent with multiplier dot (e.g., kg•m-2•d-1) for two or more units in the denominator. Use L for liter (mL for milliliter). Include a space before and after all operation signs (e.g., =, +) with equations and definitions; use an en dash (width of two hyphens) for minus sign. 8.1.3 Flora 1.Manuscripts should be written in English or German; publication in English is recommended. Publication in French or Spanish is possible in exceptional cases by appointment of the editor-in- chief. Authors not using their mother tongue are strongly advised to have the text reviewed by a native speaker before submission. Manuscripts should be submitted in final form and prepared in accordance with the journal’s accepted practice, form and content. Manuscripts should be checked carefully to exclude the need for corrections in proof. They should be typed double spaced throughout, on one side of the paper only and with wide margins. 2.The first page (title page) should contain the full title of the paper, the full name(s) and surname(s) of the author(s), name of laboratory where the study was carried out, and the address (incl. e-mail) of the author(s). 3.Each manuscript must be preceded by an English title and an English abstract which presents briefly the major results and conclusions of the paper. In case of not-English written papers this summary must be more extensive as normal and may be as long as maximally 1½ printed pages. Immediately following the abstract, up to six English key words should be supplied indicating the

108 scope of the paper. Legends of figures and tables must be given also in English in the case of non- English papers. 4.Papers should be written as concise as possible; as a rule, the total length of an article must not exceed 10 printed pages; exceptions are possible only upon explicite consent of the editors. The main portion of the paper should preferably be divided into four sections: Introduction, Materials and methods, Results, and Discussion, followed by Acknowledgements (if necessary) and References. Each section and sub-section must bear a heading. 5.Text marking: Names of Authors should not be written in capitals. Scientific names up to the genus are to be written in italics (Viola alba subsp. alba); plant community names are not to be printed in italics (Seslerietum, but Sesleria-slope). The SI-System of units must be used wherever possible. 6.The beginning of a paragraph should be indented. The section “References”, captions for illustrations and tables will be printed in small print (petit). 7.Each table should be typed on a separate sheet of paper resp. on a separate page of a file. Tables should be numbered consecutively in Arabic numerals, e.g. “Table 1, Table 2”, etc., and attached to the end of the text. Tables should be supplied with headings, kept as simple as possible. 8.Figures (including photographic prints, line drawings and maps) should be numbered consecutively in Arabic numerals, e.g. “Fig. 1, Fig. 2”, etc. and attached to the text after the tables. Legends for figures should be listed consecutively on a separate page. Plan all figures to suit a column width of 8.8 cm or a page width of 18.2 cm. Figures, in particular photographs, may be combined to a maximum plate size of 18.2 cm x 24.3 cm. Submit illustration files separately from text files. Files for full color images must be in a RGB color space for online publication (e.g. at ScienceDirect). Usally, the RGB fi les will be converted to the CMYK color space for the print process. Elsevier recommends that only TIFF, EPS or PDF formats are used for electronic artwork. MS Office files (Word, Excel and Powerpoint) are also accepted. Journal quality reproduction will require greyscale and color fi les at resolutions yielding approximately 300 dpi. Bitmapped line art should be submitted at resolutions yielding 600-1200 dpi. 9.Photographs should be black-and-white, high-contrast, sharp glossy prints of the original negative and in a square or rectangular format. Free colour reproduction. If, together with your accepted article, you submit usable colour figures then Elsevier will ensure, at no additional charge, that these figures will appear in colour on the web (e.g., ScienceDirect and other sites) regardless of whether or not these illustrations are reproduced in colour in the printed version. Colour figures can be printed only if the costs are covered by the author (€ 250.00 (+VAT) for first colour figure, € 200.00 (+VAT) for every following colour figure)..

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Magnification of microphotographs should be indicated by a scale bar. Inscriptions, marks, and scale bars should preferably be drawn neatly in black ink in an appropriate size on the face of the illustrations, or appropriate fonts should be used when preparing the electronic fi le. When several pictures are used to produce a single plate, please ensure that they fi t each other in size, are of equal contrast, and that they correspond to the caption in number and description. 10.Line drawings (incl. maps) should be large enough in all their details to permit a suitable reduction. Important points to note are thickness of lines, size of inscriptions, size of symbols, adequate spacing of shaded and dotted areas. 11.Figures and tables should always be mentioned in the text in numerical order. The author should mark in the margin of the manuscript where figures and tables are to be inserted. 12.When quoting references in the text, the following format should be used: Meyer (1999) resp. (Meyer, 1999), Meyer and Smith (1995) resp. (Meyer and Smith, 1995) or Meyer et al. (1990) resp. (Meyer et al., 1990). Several papers by the same author(s) published in the same year should be differentiated in the text, and in the list of references, by a, b, c following the year of publication. “et al.” should be used in the text in the case of more than two authors. Quotations of references from different authors within one pair of brackets must be separated by semicolons, commata are to be put between the years of publication of papers of the same author: (Meyer, 1992, 1999; Meyer and Smith, 1995; Jones et al., 1998a, b). References should be listed alphabetically. Listings of several works by the same author should be grouped in chronological order. Then, papers of this author each with another one will follow according to the alphabetical order of the second author names, papers with three and more authors (“et al.” in the text) will then be arranged again in the chronological order. The style to be used is shown in the following examples: a.Papers published in periodicals: Akhalkatsi, M., Wagner, J., 1996. Reproductive phenology and seed development of Gentianella caucasea in different habitats in the Central Caucasus. Flora 191, 161–168. Zotz, G., Patiño, S., Tyree, M.T., 1997. CO2 gas exchange and the occurrence of CAM in tropical woody hemiepiphytes. Flora 192, 143–150. b.Books: Takhtajan, A., 1959. Die Evolution der Angiospermen. G. Fischer, Jena. c.Papers published in multiauthor books: Mathes, U., Feige, G.B., 1983. Ecophysiology of lichen symbiosis. In: Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H. (Eds.), Physiological Plant Ecology. II. Responses to the Chemical and Biological Environment. Encyclopedia of Plant Physiology. New Series, vol. 12C, Springer, Berlin-Heidelberg-New York, pp. 423-467.

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The titles of books and papers in periodicals should always be quoted completely and exactly. Titles of periodicals should be abbreviated according to the usual rules listed e.g. in the World List of Scientifi c Periodicals or in Biological Abstracts. The number of the volume should be given in Arabic numerals. When papers are cited which were originally published in languages which use alphabets other than Latin (e.g. Russian Cyrillic etc.), then the author, title of the paper and the periodical name itself must be transliterated using standards like ISO 1 or ISO 2 (cf. Taxon 30: 168–183).