História evolutiva dos lagartos anões (Lygodactylus, Gekkonidae) no continente Sul Americano

Texto

(1)História evolutiva dos lagartos anões (Lygodactylus, Gekkonidae) no continente Sul Americano. Flávia Mól Lanna. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE BIOCIÊNCIAS DEPARTAMENTO DE ECOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM ECOLOGIA. Adrian Antonio Garda (Orientador) Fernanda de Pinho Werneck (INPA) (Coorientadora) Marcelo Coelho Miguel Gehara (AMNH – USA) (Coorientador). Natal/2017.

(2) FLÁVIA MÓL LANNA. HISTÓRIA EVOLUTIVA DOS LAGARTOS ANÕES (Lygodactylus, Gekkonidae) NO CONTINENTE SUL AMERICANO. Dissertação apresentada ao Programa de Pós-Graduação em Ecologia da Universidade Federal do Rio Grande do Norte como parte das exigências para obtenção do Grau de Mestre. ORIENTADOR: Adrian Antonio Garda COORIENTADORES: Fernanda de Pinho Werneck Marcelo Coelho Miguel Gehara Natal/2017.

(3) Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Leopoldo Nelson - -Centro de Biociências - CB. Lanna, Flávia Mól. História evolutiva dos lagartos anões (Lygodactylus, Gekkonidae) no continente Sul Americano / Flávia Mól Lanna. Natal, 2017. 79 f.: il. Dissertação (Mestrado) - Universidade Federal do Rio Grande do Norte. Centro de Biociências. Programa de Pós-Graduação em Ecologia. Orientador: Prof. Dr. Adrian Antonio Garda. Coorientadora: Profa. Dra. Fernanda de Pinho Werneck. Coorientador: Dr. Marcelo Coelho Miguel Gehara. 1. Caatinga - Dissertação. 2. Chaco - Dissertação. 3. Complexo de espécies - Dissertação. 4. Florestas Tropicais Sazonalmente Secas - Dissertação. 5. Hipótese do Arco Pleistocênico Dissertação. 6. Rio São Francisco - Dissertação. I. Garda, Adrian Antonio. II. Werneck, Fernanda de Pinho. III. Gehara, Marcelo Coelho Miguel. IV. Universidade Federal Do Rio Grande do Norte. V. Título. RN/UF/BSE-CB. CDU 574.

(4) FLÁVIA MÓL LANNA. HISTÓRIA EVOLUTIVA DOS LAGARTOS ANÕES (Lygodactylus, Gekkonidae) NO CONTINENTE SUL AMERICANO Dissertação apresentada ao Programa de Pós-Graduação em Ecologia da Universidade Federal do Rio Grande do Norte como parte das exigências para obtenção do Grau de Mestre. Data da defesa: 21 de fevereiro de 2017. BANCA EXAMINADORA. ___________________________ Dr. Adrian Antonio Garda Presidente/Orientador | UFRN. ___________________________. ___________________________. Dr. Fabrícius Maia Chaves Bicalho Domingos. Dra. Simone Nunes Brandão. Membro externo | UnB. Membro externo | UFRN.

(5) “Na vida, não vale tanto o que temos, nem tanto importa o que somos. Vale o que realizamos com aquilo que possuímos e, acima de tudo, importa o que fazemos de nós”. – Chico Xavier –.

(6) AGRADECIMENTOS Mais uma etapa está sendo concluída, outra página virada deste livro de histórias em que cada capítulo é um desafio encerrado com um final feliz, cheio de personagens que vou levar pra sempre comigo. Foram tantas pessoas importantes nesses dois anos de mestrado que fica até difícil agradecer o ombro amigo e os conselhos de todo mundo da forma como merecem. Agradeço primeiramente ao meu orientador Adrian Garda pela confiança depositada em mim, pela oportunidade de realizar esse projeto, paciência nos momentos de aperto, apoio e ensinamentos do início ao fim desses processo. Ao meu coorientador Marcelo Gehara pela prontidão em responder minhas dúvidas, pelas horas e horas discutindo as análises e pela crucial direção nos momentos de dúvida. À minha coorientadora Fernanda Werneck por confiar à mim este projeto, por todo ensinamento durante este período, pela recepção em Manaus e todo o suporte laboratorial no INPA e na BYU. À vocês três em conjunto pela especial oportunidade de realizar a coleta de dados nos Estados Unidos, uma experiência incrivelmente enriquecedora profissionalmente, culturalmente e pessoalmente. Ao Guarino Colli por ter viabilizado toda a viagem para os Estados Unidos e idas a campo, além das doações dos tecidos. Ao Miguel Rodrigues pelo auxílio às idas a campo e doações dos tecidos. À Eliana Oliveira e ao Gabriel Costa pelas sugestões na qualificação. Ao Jack Sites Jr. por ter me recebido com muito carinho em seu laboratório na Brigham Young University..

(7) Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pelos dois anos de bolsa e ao programa de Pós-Graduação em Ecologia/UFRN pelo suporte. Ao amigos do LAR-UFRN (David Lucas Röhr, Eliana Oliveira, Emanuel Fonseca, Felipe Camurugi, Felipe Magalhães, Marilia Lion, Ricardo Marques, Ricardo Rodrigues, Sarah Mângia, Vinícius São-Pedro e Willianilson Pessoa) pelas discussões de trabalho, pelos momentos de diversão, idas a campo, papos cabeça e algumas poucas piadas boas. Obrigada pessoal! Aos amigos do Sites Lab (César Aguilar, Derek Tucker, Juan Santos, Luciano J. Ávila, Mariana Morando, Perry L. Wood e Randy Klabacka) pela recepção agradável, toda a ajuda no laboratório, piqueniques e passeios por lugares maravilhosos de Provo e região. Aos amigos do INPA que me receberam de braços abertos, me auxiliaram no laboratório e me apresentaram a região (Alan Filipe, Gabriela Farias, Erik Choueri, Jéssica dos Anjos e, em especial, Lídia Martins). Ao pessoal da república Tanquetão (Marina Vergara, Carol Vergara, Jessé Ramos, Édina Vergara, Cíntia Pinheiro (Tida), Clarinha, Natália Pires (Pocas), Nicolas Penna, Maysa Gomes, Gustavo Paterno, Alexsander Hada, Felipe Camurugi e Morena), por todas as festas, noites de músicas, brincadeiras, comilanças, descontrações nos momentos de tensão, amizade, carinho e noites passadas em claro em batalhas de outra realidade. Aos amigos de Natal e Minas Gerais ainda não citados acima, com os quais compartilhei bons momentos e uma gostosa amizade. Um especial obrigado aos grandes amigos Vinícius São-Pedro e Eliana Oliveira pelo carinho e companheirismo. À toda minha família, que apesar da saudade, respeitou minha ausência e distância. Agradeço principalmente ao meu pai (Eduardo) e minha mãe (Teresa), que sempre me apoiaram.

(8) e me incentivaram a seguir meus sonhos, e também ao meu irmão (Arthur), que sempre esteve presente apesar de distante durante esse tempo. Vocês são o meu alicerce! O apoio de vocês foi fundamental pro meu crescimento. E por último, mas de forma alguma menos importante, agradeço ao El (Emanuel Fonseca), meu amor, meu companheiro, parceiro em todas as aventuras. Te agradeço por estar sempre ao meu lado e por superarmos juntos os desafios que encontramos e que nos dispomos a enfrentar. Sem você tudo isso não teria sido possível. Obrigada de coração!.

(9) SUMÁRIO. LISTA DE TABELAS ....................................................................................................................... I LISTA DE FIGURAS ........................................................................................................................ II. RESUMO ........................................................................................................................................ V ABSTRACT .................................................................................................................................... VII. INTRODUÇÃO GERAL.................................................................................................................... 1. CHAPTER I: Out of Africa: a cryptic speciation history of a small traveler gecko in South America .................................................................................................................... 11 ABSTRACT ..................................................................................................................................... 13 INTRODUCTION ............................................................................................................................. 14 MATERIAL AND METHODS ........................................................................................................... 17 Taxon sampling....................................................................................................................... 17 Sequencing .............................................................................................................................. 17 Phylogenetic relationships ...................................................................................................... 18 Divergence time estimates ...................................................................................................... 18 Species delimitation ................................................................................................................ 19 RESULTS ....................................................................................................................................... 20 Genetic data and phylogenetic analyses ................................................................................. 20 Divergence times and species delimitation ............................................................................. 20.

(10) DISCUSSION................................................................................................................................... 21 Monophyly of Lygodactylus in South America ...................................................................... 21 Pleistocenic Arc Hypothesis ................................................................................................... 22 Cryptic diversity...................................................................................................................... 23 CONCLUSIONS ............................................................................................................................... 25 ACKNOWLEDGEMENTS ................................................................................................................. 25 REFERENCES ................................................................................................................................. 26 TABLES ......................................................................................................................................... 35 FIGURES ........................................................................................................................................ 38. SUPPORTING INFORMATION ................................................................................................. 41 Tables ................................................................................................................................ 41 Figures ............................................................................................................................... 42 References ......................................................................................................................... 43. CHAPTER II: The role of the São Francisco River on the diversification of a dwarf gecko endemic to the semiarid Caatinga, Northeastern Brazil........................................... 44 ABSTRACT ..................................................................................................................................... 46 INTRODUCTION ............................................................................................................................. 47 MATERIAL AND METHODS ........................................................................................................... 49 Sample collection and sequencing .......................................................................................... 49 Gene tree ................................................................................................................................. 50 Assignment of genetic lineages .............................................................................................. 51 Haplotype network and DNA polymorphism ......................................................................... 52.

(11) Species tree estimation ............................................................................................................ 52 Phylogeographic reconstruction.............................................................................................. 53 Testing the SFR barrier ........................................................................................................... 54 RESULTS ....................................................................................................................................... 55 Assignment of lineages ........................................................................................................... 55 Gene trees, species tree, haplotype network and DNA polymorphism .................................. 56 Phylogeographic reconstruction.............................................................................................. 56 Testing the SFR as a barrier .................................................................................................... 57 DISCUSSION................................................................................................................................... 57 CONCLUSIONS ............................................................................................................................... 59 ACKNOWLEDGEMENTS ................................................................................................................. 59 REFERENCES ................................................................................................................................. 60 TABLES ......................................................................................................................................... 66 FIGURES ........................................................................................................................................ 68. SUPPORTING INFORMATION ................................................................................................. 72 Tables ................................................................................................................................ 72 Figures ............................................................................................................................... 75 References ......................................................................................................................... 77.

(12) LISTA DE TABELAS CAPÍTULO I: Table 1: Samples used in this study with respective voucher, localities number (associated with Figure 1) and GenBank number (if available) .............................................................................. 35. Table 2: Genetic distance (uncorrelated p-distance) among South American Lygodactylus species suggested by SpedeSTEM. Upper values are from ND2 (mtDNA) and lower values are from RAG1 (nuDNA) marker ....................................................................................................... 37. Table S1: Information about markers and PCR protocols used in this study .............................. 41. CAPÍTULO II: Table 1: Genetic statistics of northern and southern lineages of Lygodactylus klugei (according to GMYC results) for each locus .................................................................................................. 66. Table 2: Genetic distance (p-distance) between and within northern and southern lineages (according to GMYC results) for the four markers ...................................................................... 67. Table S1: Information about Lygodacylus samples used in this study, with locality numbers and coordinates .................................................................................................................................... 72. Table S2: Information about gene, primers, and PCR protocols used in this study .................... 74. I.

(13) LISTA DE FIGURAS CAPÍTULO I: Figure 1: Distribution map of South American Lygodactylus samples. The biomes of the open diagonal are in a gray scale: Chaco (CH) in dark gray, Cerrado (CE) in gray and Caatinga (CA) in black, highlighting for Seasonally Dry Tropical Forests enclaves in Cerrado also in black. Orange dots represents L. klugei samples, green dot Lygodactylus sp. 1 (Santo Inácio), blue dot Lygodactylus sp. 2 (Condeúba), light red Lygodactylus sp. 3 (São Domingos), and dark red L. wetzeli ........................................................................................................................................... 38. Figure 2: Maximum Likelihood concatenated tree with the individuals gathered in five highly supported groups (pp = 100). Node numbers correspond to 1000 ML bootstrap values. Different colors represent distinct groups .................................................................................................... 39. Figure 3: Bayesian species tree and divergence times for South American Lygodactylus. The African Lygodactylus were collapsed only on the representation for better visualization of the tree. For the whole species tree, see Supporting Information, Figure S1. The node numbers correspond to posterior probability. Nodes with posterior probability higher than 99% are marked with an asterisk (*). Outgroup 1 correspond to L. angularis, outgroup 2 to L. chobiensis and L. kimhowelli, and outgroup 3 to the other 15 species of African Lygodactylus used here. Abbreviations: Pli, Pliocene; P, Pleistocene; BA, Bahia State; GO, Goiás State ......................... 40. Figure S1: Bayesian species tree and divergence time for Lygodactylus. Complete tree encompassing the 18 African species. Abbreviations: Pli, Pliocene; P, Pleistocene .................... 42. II.

(14) CAPÍTULO II: Figure 1: Map of the sample localities of Lygodactylus klugei and its related lineage according GMYC results. Pink circles correspond to northern lineage. Green circles correspond to southern lineage. São Francisco River is represented in blue and its paleocurse is represented in grey in a dotted line...................................................................................................................................... 68. Figure 2: Lineage assignment based on the Generalized Mixed Yule Coalescent (GMYC) method. (A) gene tree generated by GMYC. The two most probable groups are showed in red, with one individual not allocated for any of them. (B) heat map represents Bayesian implementations of the GMYC (bGMYC). Darker colors indicate lower probability of grouping while lighter colors indicate higher probability. Probabilities higher than 50% were used to assign the lineages......................................................................................................................... 69. Figure 3: Haplotype network for (A) ND4, (B) DMXL1, (C) DNAH3, and (D) PRLR markers according to their respective Bayesian gene tree. The size of each circle is proportional to the haplotype frequency. The small blue correspond to the number of mutational steps. Pink circles represent northern lineage and green circles represent southern lineage (according to GMYC results) ........................................................................................................................................... 70. Figure 4: Bayesian spatiotemporal diffusion of mtDNA for Lygdactylus klugei in six time frames. Lighter shades represent older diffusion events and darker shades represent younger diffusion events ............................................................................................................................. 71. III.

(15) Figure S1: Estimated gene trees for ND4 (A), DMXL1 (B), DNAH3 (C), and PRLR (D). Individuals are highlighted according to GMYC results. Individuals in pink correspond to northern lineage and green correspond to southern lineage. Nodes with posterior probability higher than 95% are marked with an asterisk (*) ......................................................................... 75. Figure S2: Genetic structure of Lygodactylus klugei based on nuclear markers performed in Structure. (A) probability of individuals' assignment to each population (green and red). (B) map with localities colored according Structure population assignment; Green triangles correspond to population 1 and red circles correspond to population 2; Blue line correspond to the São Francisco River and A correspond to São Francisco River paleocourse ...................................... 76. IV.

(16) RESUMO Quais os processos e mecanismos responsáveis pela diversificação das espécies? Essa é uma questão antiga que tem sido revolucionada com o avanço tecnológico, computacional e metodológico, e tem sido agora compreendida de uma forma que antes não era possível. A filogeografia é uma multidisciplina que utiliza ferramentas derivadas da biogeografia, filogenia molecular e genética de populações para entender o contexto da distribuição dos genes no tempo e espaço. O presente estudo utiliza análises filogenéticas e filogeográficas para inferir os processos determinantes na diversificação de lagartos do gênero Lygodactylus nas Florestas Tropicais Sazonalmente Secas (FTSS) da América do Sul. No primeiro capítulo nós investigamos as relações entre os Lygodactylus Sul Americanos, buscando entender a influência do Arco Pleistocênico em sua diversificação e se essas espécies representam um grupo monofilético. Através de análises filogenéticas e de delimitação de espécie, nós recuperamos o monofiletismo do grupo quando comparado com as espécies Africanas e reconhecemos L. klugei como um complexo de espécies crípticas. Nós sugerimos o aumento de duas para cinco espécies de Lygodactylus na América do Sul. O tempo de divergência entre L. klugei e as espécies candidatas endêmicas das FTSSs não foi congruente com a hipótese do Arco Pleistocênico. Porém, a fragmentação das FTSS pode ter influenciado na divergência de L. wetzeli e uma espécie candidata endêmica de um enclave de FTSS no Cerrado (São Domingos, região do Vale do Paranã). No segundo capítulo investigamos a diversificação dentro da Caatinga, testando o papel do rio São Francisco (RSF) como barreira geográfica nesse bioma. Utilizamos um lagarto endêmico dessa região (L. klugei) como modelo de estudo. Nós delimitamos as possíveis linhagens, investigamos as relações filogenéticas entre elas, a história de difusão espaçotemporal e, para testar a hipótese do rio (barreira para fluxo gênico), nós utilizamos uma análise. V.

(17) de migração. Nós recuperamos duas linhagens estruturadas de acordo com o RSF: uma ao norte e outra ao sul do rio. A divergência dessas linhagens ocorreu à 295 mil anos atrás, congruente com a mudança do curso do RSF para seu atual curso. Não encontramos influência do paleocurso do RSF na estruturação de L. klugei.. Palavras-chave: Caatinga, Chaco, complexo de espécies, Florestas Tropicais Sazonalmente Secas, hipótese do Arco Pleistocênico, hipótese do rio, rio São Francisco. VI.

(18) ABSTRACT Which processes and mechanisms are responsible for species diversification? This old question has been revolutionized with technological, computational and methodological advancements, and is now being understood in a way that was previously not possible. Phylogeography is a multidiscipline that uses tools derived from biogeography, molecular phylogeny, and population genetics to understand the context of gene distribution in time and space. The present study uses phylogenetic and phylogeographic analyses to infer determinant processes in the diversification of the lizard genus Lygodactylus in Seasonally Dry Tropical Forests (SDTF) in South America. In the first chapter we investigate the relationships among South American Lygodactylus species, seeking to understand the influence of the Pleistocenic Arc on its diversification and whether these species represent a monophyletic group. Through phylogenetics and species delimitation analyses we recovered the monophyly of the group in relation to African species and recognized L. klugei as a cryptic species complex. We suggest that Lygodactylus in South America actually comprises five species instead of two. The divergence time among L. klugei and candidate species endemic to SDTFs was not congruent with the Pleistocenic Arc Hypothesis. However, we suggest that the fragmentation of SDTFs likely influenced the divergence of L. wetzeli, and of a candidate species endemic to a SDTF enclave within the Cerrado biome (São Domingos, Vale do Paranã region). In the second chapter we investigate the diversification within the Caatinga, testing the role of the São Francisco River (SFR) as a prominent geographic barrier. We used a lizard endemic to this region (L. klugei) as study model. We delimited the existent lineages, investigated the genetic relationships between them, the spatio-temporal diffusion history, and used a migration analysis to test the riverine hypothesis (barrier to gene flow). We recovered two lineages structured in respect to the SFR: a northern and a southern one. Lineage divergence. VII.

(19) occurred 295 kya, congruent with the course change of the SFR to its current position. We found no influence of the paleo-SFR on L. klugei structure.. Keywords: Caatinga, Chaco, Pleistocenic Arc Hypothesis, riverine hypothesis, São Francisco River, Seasonally Dry Tropical Forests, species complex. VIII.

(20) INTRODUÇÃO GERAL O continente Sul-Americano apresenta uma enorme diversidade de espécies. Entretanto, sua história evolutiva ainda é pouco conhecida (Turchetto-Zolet, et al. 2013). A compreensão dos processos que levaram à diversificação das espécies Sul-Americanas foi intensificada na última década com o surgimento de ferramentas, como a filogeografia, que tornaram o teste de hipóteses alternativas mais viáveis (Riddle, et al. 2008). A filogeografia é a interface da filogenia molecular, a biogeografia e a genética de populações no estudo da distribuição espacial dos genes no espaço e no tempo entre espécies próximas ou populações de uma mesma espécie (Avise 2000). Análises filogeográficas vem sendo consideradas importantes para testar hipóteses biogeográficas e identificar diferentes linhagens evolutivas, como por exemplo barreiras geográficas separando populações e até mesmo espécies (Carnaval, et al. 2009; Fitzpatrick, et al. 2009; Shepard and Burbrink 2008; Thomé, et al. 2010). Inicialmente, muita atenção foi dada aos biomas de florestas úmidas da América do Sul (Turchetto-Zolet, et al. 2013), e apenas recentemente os biomas de vegetação aberta começaram a ser investigados e os processos que levaram à diversificação das espécies nessa região estão sendo melhor compreendidos (Gamble, et al. 2012; Magalhaes, et al. 2014; Recoder, et al. 2014; Werneck, et al. 2012; Werneck, et al. 2015). As áreas de formações abertas estão distribuídas na forma de uma diagonal na América do Sul, ligando a região Nordeste do Brasil ao Paraguai, Argentina e Bolívia, englobando os biomas Caatinga, Cerrado e Chaco (Werneck 2011). Apesar de existirem espécies compartilhadas por esses três biomas, cada um tem sua biota característica com espécies endêmicas. Inicialmente, pesquisadores sugeriram que a Caatinga era um bioma com uma baixa. 1.

(21) diversidade de espécies, não possuindo uma fauna característica, onde suas espécies estavam amplamente distribuídas pela Diagonal de Formações Abertas (Mares, et al. 1981; Pennington, et al. 2000; Vanzolini 1988, 1974, 1976; Werneck 2011). Atualmente, mesmo ainda sendo pouco estudada, sabe-se que a diversidade nessa região é muito maior do que afirmada anteriormente, o que mostra que esses estudos se basearam em coletas pouco representativas e amostragem geográfica insuficiente do bioma (Rodrigues 2003). A Caatinga, um bioma exclusivamente brasileiro, está distribuído principalmente no nordeste do Brasil, se estendendo também ao norte de Minas Gerais, na região que segue o Rio São Francisco e o médio Rio Jequitinhonha (Prado 2003). A Caatinga apresenta um clima quente e seco durante a maior parte do ano, com um curto período de chuvas no inverno, que coincide com o solstício de verão (Prado 2003). Sua vegetação é predominantemente decídua e espinhosa, podendo variar de florestas altas e secas à afloramentos rochosos com cactos, bromélias e arbustos baixos (Prado 2003). A produtividade primária, diferentemente das florestas tropicais úmidas, é maior no inverno por ser a estação chuvosa e, consequentemente, o período de maior crescimento vegetal (Pennington, et al. 2006). A Caatinga é a maior área contínua de Florestas Tropicais Sazonalmente Secas (FTSS) (Werneck, et al. 2012). As FTSSs englobam esse bioma e também formações com características semelhantes e que possuem: média de temperatura anual de 17º C, livre de geadas, com duas estações bem definidas (seca – chuvosa) e precipitação entre 200 e 2000 mm anuais (Murphy and Lugo 1986). Na região Neotropical, essa unidade fitogeográfica ocorre descontinuamente desde a América do Sul até a América do Norte (Pennington, et al. 2009). Alguns remanescentes isolados de FTSS ocorrem como enclaves no Cerrado, em áreas com condições edáficas favoráveis (Silva and Bates 2002; Werneck and Colli 2006).. 2.

(22) De acordo com a hipótese do Arco Pleistocênico, as FTSSs atualmente representam uma pequena porção do contínuo que já foram um dia (Pennington, et al. 2004). Esse contínuo é conhecido como Arco Pleistocênico (Prado and Gibbs 1993), e atualmente está fragmentado e possui três grandes núcleos: Caatinga (Brasil), Missiones (rio Paraguai-Paraná) e Montes subandinos (Bolívia e Argentina) (Pennington, et al. 2000; Prado 2000). A suposta formação do Arco Pleistocênico teria possibilitado a difusão de espécies endêmicas das FTSSs. Adicionalmente, a ruptura do arco em manchas isoladas teria assim produzido condições favoráveis à especiação alopátrica, aumentando a diversidade de espécies endêmicas das FTSSs (Pennington, et al. 2000). Evidências sugerem que o Arco Pleistocênico tenha alcançado tamanho máximo durante o último máximo glacial no Pleistoceno (Prado and Gibbs 1993). Entretanto, Werneck, et al. (2011) sugerem que ele possa ter ocorrido anteriormente, entre o final do Plioceno e o início do Pleistoceno. Outra hipótese de diversificação para a Caatinga envolve a atuação dos grandes rios. Os rios são considerados importantes agentes de especiação, atuando, por exemplo, como uma barreira ao fluxo gênico entre populações (Garda and Cannatella 2007; Ribas, et al. 2012; Wallace 1854; Werneck, et al. 2015). O Rio São Francisco é o rio perene de maior extensão da Caatinga e é conhecido por estruturar populações e até mesmo espécies em suas margens opostas (Faria, et al. 2013; Passoni, et al. 2008; Rodrigues 2003). Evidências geomorfológicas indicam que o paleocurso desse rio diferia consideravelmente do curso atual, sendo responsável pela diversificação de algumas espécies (Nascimento, et al. 2013; Werneck, et al. 2015). As paleodunas do Médio do São Francisco, formadas durante o Quaternário, contém um grande número de espécies de lagartos endêmicos, muitos dos quais restritos a uma ou outra margem do Rio São Francisco (Rodrigues 2003; Werneck, et al. 2012; Werneck, et al. 2015). Todos esses fatores. 3.

(23) reforçam o papel do Rio São Francisco como uma das mais importantes barreiras para diversificação de espécies na Caatinga. Assim, tanto a formação e a fragmentação do Arco Pleistocênico como a presença do Rio São Francisco e suas mudanças de curso ao longo do tempo devem ter contribuído para a formação e a manutenção da biodiversidade da Caatinga. O presente trabalho teve como objetivo testar essas hipóteses por meio de técnicas filogeográficas. Para tanto, nós utilizamos um gênero de lagarto da família Gekkonidae, uma família cosmopolita composta por lagartos pequenos e com pele muito delicada, onde a maioria das espécies possui hábitos noturnos (Vanzolini, et al. 1980). Das 1103 espécies dessa família, apenas oito ocorrem no Brasil, duas delas pertencendo ao gênero utilizado aqui, Lygodactylus (Uetz 2016). Lygodactylus contém aproximadamente 64 espécies, das quais apenas duas ocorrem na América do Sul (Gamble et al., 2011). Lygodactylus wetzeli (Smith, Martin & Swain, 1977) ocorre no Brasil no estado do Mato Grosso do Sul e também no Paraguai e na Bolívia (Smith, et al. 1977; Uetz 2016), apresentando distribuição restrita ao Chaco (Vanzolini 1974, 1976). Enquanto isso, Lygodactylus klugei (Smith, Martin & Swain, 1977) ocorre no Brasil, é endêmico da Caatinga, mas até então poderia ser encontrado também em enclaves de FTSS no Cerrado, no estado de Goiás, considerado assim endêmico das FTSS (Rodrigues 2003; Werneck and Colli 2006). Ambas espécies são de pequeno porte (podendo atingir pouco mais de 5cm), diurnas, arborícolas e insetívoras (Galdino, et al. 2011; Smith, et al. 1977). Nós utilizamos as espécies Sul-Americanas de Lygodactylus para compreender os processos responsáveis pela diversificação na diagonal de formações abertas, mais especificamente testando: (i) a hipótese do Arco Pleistocênico; e (ii) o papel do rio São Francisco como barreira. A dissertação foi dividida em dois capítulos. No primeiro, buscamos entender. 4.

(24) melhor a relação do gênero Lygodactylus no continente Sul-Americano e investigar (i) se o tempo de divergência entre L. klugei da Caatinga e dos enclaves de FTSS no Cerrado corresponde à época em que teria ocorrido a fragmentação do Arco Pleistocênico, (ii) se os Lygodactylus na América do Sul representam um complexo de espécies, e (iii) se esse grupo é monofilético indicando um único evento de colonização a partir das linhagens africanas. No segundo capítulo focamos na Caatinga e utilizando o L. klugei, restrito à Caatinga, buscamos (i) compreender os processos que levaram à diversificação dessa espécie, e (ii) o papel do rio São Francisco para a estruturação populacional e genealógica.. Referências bibliográficas Avise JC. 2000. The History and Formation of Species. Harverd University press, Cambridge, Massachusetts. Carnaval AC, Hickerson MJ, Haddad CFB, Rodrigues MT, Moritz C 2009. Stability predicts genetic diversity in the Brazilian Atlantic Forest hotspot. Science (Washington D C) 323: 785–789. Faria MB, Nascimento FF, Oliveira JAd, Bonvicino CR 2013. Biogeographic determinants of genetic diversification in the mouse opossum Gracilinanus agilis (Didelphimorphia: Didelphidae). Journal of Heredity 104: 613–626. Fitzpatrick SW, Brasileiro CA, Haddad CFB, Zamudio KR 2009. Geographical variation in genetic structure of an Atlantic Coastal Forest frog reveals regional differences in habitat stability. Molecular Ecology 18: 2877–2896.. 5.

(25) Galdino CAB, Passos DC, Zanchi D, Bezerra CH 2011. Lygodactylus klugei (NCN). Sexual dimorphism, habitat, diet. Herpetological Review 42: 275–276. Gamble, T., Bauer, A.M., Colli, G.R., Greenbaum, E., Jackman, T.R., Vitt, L.J., Simons, A.M., 2011. Coming to America: multiple origins of New World geckos. Journal of Evolutionary Biology 24, 231–244. Gamble T, Colli GR, Rodrigues MT, Werneck FP, Simons AM 2012. Phylogeny and cryptic diversity in geckos (Phyllopezus; Phyllodactylidae; Gekkota) from South America's open biomes. Molecular Phylogenetics and Evolution 62: 943-953. Garda AA, Cannatella DC 2007. Phylogeny and biogeography of paradoxical frogs (Anura, Hylidae, Pseudae) inferred from 12S and 16S mitochondrial DNA. Molecular Phylogenetics and Evolution 44: 104–114. doi: 10.1016/j.ympev.2006.11.028 Magalhaes ILF, Oliveira U, Santos FR, Vidigal THDA, Brescovit AD, Santos AJ 2014. Strong spatial structure, Pliocene diversification and cryptic diversity in the Neotropical dry forest spider Sicarius cariri. Molecular Ecology 23: 5323-5336. doi: 10.1111/mec.12937 Mares MA, Willig MR, Steilein KE, Lacher-Jr TE 1981. The mammals of northeastern Brazil: a preliminary assessment. Annals of Carnegie Museum 50: 81–137. Murphy PG, Lugo AE 1986. Ecology of Tropical Dry Forest. Annual Review of Ecology and Systematics 17: 67–88. Nascimento FF, Lazar A, Menezes AN, Durans AdM, Moreira JC, Salazar-Bravo J, D'Andrea PS, Bonvicino CR 2013. The role of historical barriers in the diversification processes in. 6.

(26) open vegetation formations during the Miocene/Pliocene using an ancient rodent lineage as a model. PLoS One 8: 1–13. Passoni JC, Benozzati ML, Rodrigues MT 2008. Phylogeny, species limits, and biogeography of the Brazilian lizards of the genus Eurolophosaurus (Squamata: Tropiduridae) as inferred from mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 46: 403–414. Pennington RT, Lavin M, Oliveira-Filho A 2009. Woody plant diversity, evolution, and ecology in the tropics: perspectives from Seasonally Dry Tropical Forests. Annual Review of Ecology,. Evolution,. and. Systematics. 40:. 437–457.. doi:. 10.1146/annurev.ecolsys.110308.120327 Pennington RT, Lavin M, Prado DE, Pendry CA, Pell SK, Butterworth CA 2004. Historical climate change and speciation: neotropical seasonally dry forest plants show patterns of both Tertiary and Quaternary diversification. Philosophical Transactions of the Royal Society of London B Biological Sciences 359: 515–537. Pennington RT, Lewis GP, Ratter JA. 2006. An Overview of the Plant Diversity, Biogeography and Conservation of Neotropical Savannas and Seasonally Dry Forests. In. Neotropical Savannas and Seasonally Dry Forests: Plant Diversity, Biogeography, and Conservation. p. 1–30. Pennington RT, Prado DE, Pendry CA 2000. Neotropical seasonally dry forests and Quaternary vegetation changes. Journal of Biogeography 27: 261–273.. 7.

(27) Prado DE. 2003. As Caatingas da América do Sul. In: Leal IR, Tabarelli M, da Silva JMC, editors. Ecologia e Conservação da Caatinga. Editora Universitária UFPE. p. 3–73. Prado DE 2000. Seasonally dry forests of tropical South America: from forgotten ecosystems to a new phytogeographic unit. Edinburgh Journal of Botany 57: 437–461. doi: 10.1017/S096042860000041X Prado DE, Gibbs PE 1993. Patterns of species distributions in the dry seasonal forests of South America. Annals of the Missouri Botanical Garden 80: 902–927. Recoder RS, Werneck FDP, Teixeira M, Jr., Colli GR, Sites JW, Jr., Rodrigues MT 2014. Geographic variation and systematic review of the lizard genus Vanzosaura (Squamata, Gymnophthalmidae), with the description of a new species. Zoological Journal of the Linnean Society 171: 206–225. Ribas CC, Maldonado-Coelho M, Smith BT, Cabanne GS, d'Horta FM, Naka LN 2012. Towards an integrated historical biogeography of the neotropical lowland avifauna: combining diversification analysis and landscape evolution. Ornitologia Neotropical 23: 187–206. Riddle BR, Dawson MN, Hadly EA, Hafner DJ, Hickerson MJ, Mantooth SJ, Yoder AD 2008. The role of molecular genetics in sculpting the future of integrative biogeography. Progress in Physical Geography 32: 173–202. doi: 10.1177/0309133308093822 Rodrigues MT. 2003. Herpetofauna da Caatinga. In: Leal IR, Tabarelli M, Silva JMC, editors. Ecologia e Conservação da Caatinga. Brasil: Universidade Federal de Pernambuco. p. 181– 236.. 8.

(28) Shepard DB, Burbrink FT 2008. Lineage diversification and historical demography of a sky island salamander, Plethodon ouachitae, from the Interior Highlands. Molecular Ecology 17: 5315–5335. doi: 10.1111/j.1365-294X.2008.03998.x Silva JMC, Bates JM 2002. Biogeographic patterns and conservation in the South American Cerrado: a tropical savanna hotspot. Bioscience 52: 225–233. Smith HM, Martin RL, Swain TA 1977. A new genus and two new species of south american geckos (Reptilia: Lacertilia). Papéis Avulsos de Zoologia 30: 195–213. Thomé MTC, Zamudio KR, Giovanelli JGR, Haddad CFB, Baldissera Jr. FA, Alexandrino J 2010. Phylogeography of endemic toads and post-Pliocene persistence of the Brazilian Atlantic Forest.. Molecular Phylogenetics and Evolution. 55: 1018–1031. doi:. 10.1016/j.ympev.2010.02.003 Turchetto-Zolet AC, Pinheiro F, Salgueiro F, Palma-Silva C 2013. Phylogeographical patterns shed light on evolutionary process in South America. Molecular Ecology 22: 1193–1213. doi: 10.1111/mec.12164 Uetz P 2016. The Reptile Database. Disponível em http://www.reptile-database.org. Vanzolini PE. 1988. Distributional Patterns of South American Lizards. In:. Heyer WR,. Vanzolini PE, editors. Proceedings of a Workshop on Neotropical Distribution Patterns. Academia Brasileira de Ciências, Rio de Janeiro, Brasil. p. 317–342. Vanzolini PE 1974. Ecological and geographical distribution of lizards in Pernambuco, Northeastern Brasil (Sauria). Papéis Avulsos de Zoologia 18: 61–90.. 9.

(29) Vanzolini PE 1976. On the lizards of a cerrado-caatinga contact: evolutionary and zoogeographical implications (Sauria). Papéis Avulsos de Zoologia 29: 111–119. Vanzolini PE, Ramos-Costa AMM, Vitt LJ. 1980. Repteis da Caatinga. Academia Brasileira de Ciências, Rio de Janeiro. Wallace AR 1854. On the Monkeys of the Amazon. Journal of Natural History Series 2 14: 451– 454. doi: 10.1080/037454809494374 Werneck FP 2011. The diversification of eastern South American open vegetation biomes: Historical biogeography and perspectives. Quaternary Science Reviews 30: 1630–1648. doi: 10.1016/j.quascirev.2011.03.0 09 Werneck FP, Colli GR 2006. The lizard assemblage from seasonally dry tropical forest enclaves in the Cerrado biome, Brazil, and its association with the Pleistocenic Arc. Journal of Biogeography 33: 1983–1992. Werneck FP, Costa GC, Colli GR, Prado DE, Sites JW, Jr. 2011. Revisiting the historical distribution of Seasonally Dry Tropical Forests: new insights based on palaeodistribution modelling and palynological evidence. Global Ecology and Biogeography 20: 272–288. Werneck FP, Gamble T, Colli GR, Rodrigues MT, Sites JW, Jr. 2012. Deep diversification and long-term persistence in the South American 'Dry Diagonal': integrating continent-wide phylogeography and distribution modeling of geckos. Evolution 66: 3014–3034.. 10.

(30) Werneck FP, Leite RN, Geurgas SR, Rodrigues MT 2015. Biogeographic history and cryptic diversity of saxicolous Tropiduridae lizards endemic to the semiarid Caatinga. BMC Evolutionary Biology 15: 1–24.. 11.

(31) – CHAPTER I –. OUT OF AFRICA: A CRYPTIC SPECIATION HISTORY OF A SMALL TRAVELER GECKO IN. SOUTH AMERICA. Manuscript to be submitted to Molecular Phylogenetics and Evolution. 12.

(32) Out of Africa: a cryptic speciation history of a small traveler gecko in South America. Flávia M. Lanna1*, Fernanda P. Werneck2, Marcelo Gehara3, Emanuel M. Fonseca1, Guarino R. Colli4, Jack W. Sites Jr5, Miguel T. Rodrigues6, Adrian A. Garda7. 1. Programa de Pós-Graduação em Ecologia, Universidade Federal do Rio Grande do. Norte, Campus Universitário, Lagoa Nova, 59078-900, Natal, RN, Brazil. 2. Coordenação de Biodiversidade, Programa de Coleções Científicas Biológicas,. Instituto Nacional de Pesquisas da Amazônia (INPA), 69067–375, Manaus, Amazonas, Brazil. 3. American Museum of Natural History, Department of Herpetology, 79th St. Central. Park West, New York, NY 10024. 4. Departamento de Zoologia, Universidade de Brasília, 70910–900 Brasília, DF, Brazil.. 5. Department of Biology and Bean Life Science Museum, Brigham Young University,. Provo, UT 84602. 6. Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo,. 05508–090, São Paulo, SP, Brazil. 7. Departamento de Botânica e Zoologia, Centro de Biociências, Universidade Federal do. Rio Grande do Norte, Campus Universitário, Lagoa Nova, 59078-900, Natal, RN, Brazil. * Corresponding author. E-mail address: flaviamollanna@gmail.com (F.M. Lanna). 13.

(33) Abstract. The Pleistocenic Arc Hypothesis (PAH) posits that South American Seasonally Dry Tropical Forests (SDTF) were interconnected during Pleistocene glacial periods, enabling the expansion of species ranges that were subsequently fragmented, promoting speciation. The lizard genus Lygodactylus occurs in Africa, Madagascar, and South America and represents an interesting case of unbalanced diversity and distribution across these continents. While many African Lygodactylus species are recognized as complexes of cryptic species, the only two species described in South America have a disjoint distribution occurring in SDTFs and the Chaco biomes. We use a phylogenetic approach based on mitochondrial (ND2) and nuclear (RAG-1) markers and a sampling encompassing the known range of South American Lygodactylus to investigate if: (i) divergence timing of L. klugei is congruent with the PAH; (ii) the species currently recognized correspond to species complexes; and (iii) South American Lygodactylus are monophyletic. Species delimitation analysis suggested the existence of five species, two of which correspond to the already described taxa, and three representing new candidate species. Divergence times among L. klugei and the other species endemic to the SDTFs were not congruent with the PAH. However, fragmentation of the once broader and continuous SDTFs likely influenced the divergence of L. wetzeli and Lygodactylus sp. 3 (from São Domingos region). Our molecular results corroborate the monophyly of South American Lygodactylus.. Keywords: Caatinga, Chaco, dwarf gecko, Lygodactylus, monophyly, Pleistocenic Arc Hypothesis, species complex, STDF.. 14.

(34) 1. Introduction The Neotropics are considered one of the most diverse regions on Earth (Antonelli and Sanmartín, 2011; Myers et al., 2000), and different diversification processes were responsible for this great biodiversity through time (Rull, 2011). The interest to understand diversification drivers increased during the past decades with the increase of molecular-based biogeographic approaches (Riddle et al., 2008). Studies aimed to unveil the processes responsible for Neotropical biodiversity patterns have mostly focused on wet biomes, such as the Atlantic Forest and the Amazon rainforest (Turchetto-Zolet et al., 2013). Still, a large portion of the region is covered by the less studied open formations, which have also been subject to some of the same evolutionary forces that drove speciation on forested areas and other regional determinants (Turchetto-Zolet et al., 2013; Werneck, 2011). The glaciation periods during the Pleistocene likely promoted expansion of the open areas, connecting otherwise isolated fragments (Prado and Gibbs, 1993). The current fragmented distribution of Seasonally Dry Tropical Forests (SDTFs), for example, has been used as evidence for a previously more widespread distribution of this dominium (Prado, 1991; Prado and Gibbs, 1993). The SDTFs are forest formations that occur in frost-free tropical regions, marked by a highly seasonal rainfall and severe droughts (less then 1800 mm/year) (Murphy and Lugo, 1986). They usually occur on fertile soils with low levels of aluminum and moderate to high pH (Pennington et al., 2006). Most of the vegetation is deciduous, loosing more than half of arboreal cover during the dry season (Murphy and Lugo, 1986; Pennington et al., 2006). SDTFs have a patched distribution throughout the Neotropical region, and in South America the largest remaining areas represent three nuclei: Caatinga (northeastern Brazil), Misiones (along Paraguay-Paraná rivers), and Piedmont (northwestern Argentina and southwestern Bolivia). 15.

(35) (Prado and Gibbs, 1993; Pennington et al., 2000). Some small and isolated patches occur in favorable soil conditions, as enclaves within the Cerrado savanna biome in central Brazil (Silva and Bates, 2002; Werneck and Colli, 2006). The Caatinga biome (the largest nucleus of SDTF in South America) was initially considered to have low diversity, no characteristic fauna and no endemic species, sharing its biota with the two other biomes of the diagonal of open formations - Cerrado and Chaco (Mares et al., 1981; Vanzolini, 1974, 1976, 1988). Currently, the idea of the Caatinga as a poor biome was abandoned and the number of recognized endemic species has increased in the past years (Carvalho et al., 2016; Leal et al., 2003; Recoder et al., 2014). This erroneous interpretation was based in unrepresentative collections and insufficient geographic sampling in this biome (Rodrigues, 2003). The Pleistocenic Arc Hypothesis (PAH) posits that the disjoint distribution of presentday SDTFs results from the fragmentation of a previously more extensive and uninterrupted formation that reached its maximum extension during the dry-cool Last Glacial Maximum (LGM) period of late Pleistocene (Prado, 1991; Prado and Gibbs, 1993). The SDTFs would have then retracted during subsequent humid-warm periods causing allopatric speciation by vicariance, what would explain the presence of endemic species on remaining patches (Pennington et al., 2000). However, environmental niche models projected to the past did not recovered an expansion of SDTFs during LGM (Werneck et al., 2011). Thus, a different time of expansion was proposed, where the Pleistocenic Arc would have occurred during the early Pliocene/lower Pleistocene, with the fragmentation of SDTFs occurring before the LGM (Werneck et al., 2011). Indeed, the few papers using molecular data to test the PAH found that. 16.

(36) plant species isolated in SDTF patches diverged before the LGM (Caetano et al., 2008; Collevatti et al., 2012; Pennington et al., 2004). The genus Lygodactylus comprises 64 species, 62 occurring in Africa and Madagascar and two in South America (Uetz, 2016). These Gekkonidae lizards are small, cryptic, arboreal, and diurnal (Galdino et al., 2011; Vitt, 1995). Although African Lygodactylus had received much attention in the past years (Castiglia and Annesi, 2011; Malonza et al., 2016; Mezzasalma et al., 2017; Puente et al., 2005; Röll et al., 2010; Travers et al., 2014), South American Lygodactylus have been poorly studied, and no molecular study involving the two species was ever performed. South American Lygodactylus have a disjoint distribution and similar morphologies. Lygodactylus wetzeli (Smith, Martin & Swain, 1977) occurs in the State of Mato Grosso do Sul in Brazil, Paraguay, and Bolivia, and is mostly restricted to the Chaco (Uetz, 2016; Vanzolini, 1974, 1976), a biome with strong seasonality (high temperatures on summer and frosts on winter), compact soils with poor drainage, rainfall from 1000 mm/year to 500 mm/year and floods during the summer (Pennington et al., 2000; Prado, 1993). Lygodactylus klugei (Smith, Martin & Swain, 1977) has a widespread distribution in the Caatinga biome (Rodrigues, 2003; Smith et al., 1977), also occurring on enclaves of SDTF inserted in the Cerrado biome (the Brazilian savannas) (Werneck and Colli, 2006). The presence of L. klugei on SDTFs enclaves and its absence on the Cerrado biome may indicate previous connections between the Caatinga and SDTFs enclaves. Indeed, this species was considered endemic of SDTFs of the Pleistocenic Arc (Werneck and Colli, 2006). Many of the African Lygodactylus formed complexes of cryptic species before being properly investigated (Malonza et al., 2016; Portik et al., 2013; Röll, 2005; Röll et al., 2010; Travers et al., 2014). Likewise, many lizards from the Caatinga are now. 17.

(37) considered part of cryptic species complexes (Oliveira et al., 2015; Recoder et al., 2014; Werneck et al., 2012; Werneck et al., 2015). Herein we investigate the potential cryptic diversity of South American Lygodactylus and the possible influence of the Pleistocenic Arc in its diversification. We used a molecular phylogenetic approach to test the following hypotheses: (i) the diversification between L. klugei from Caatinga and L. klugei from SDTFs enclaves will agree with the time originally (LGM) or subsequently (early Pliocene–late Pleistocene) suggested for the PAH; (ii) L. klugei corresponds to a complex of cryptic species; (iii) South American Lygodactylus are monophyletic.. 2. Material and Methods 2.1 Taxon sampling We sequenced a total of 25 individuals of South American Lygodactylus encompassing eight localities: one in the Chaco, one in a SDTFs enclave within the Cerrado biome, and six in the Caatinga (Figure 1). These individuals are deposited in three zoological collections: Coleção Herpetológica da Universidade Federal do Rio Grande do Norte (UFRN), Coleção Herpetológica da Universidade de Brasilia (CHUNB), and Coleção Herpetológica do Museu de Zoologia da Universidade de São Paulo (MZUSP). To improve species tree calibration and test monophyletism in this group, we used sequences of 18 African Lygodactylus species available in GenBank (Table 1).. 2.2 Sequencing We extracted genomic DNA from muscle, liver, finger or tail tissues preserved in 95– 100% ethanol, with a DNA Purification Kit (Wizard®, Promega). We amplified ND2. 18.

(38) (mitochondrial marker - mtDNA) and RAG1 (nuclear marker - nuDNA) using a standard polymerase chain reaction (PCR) technique. We chose these markers based on published phylogenetic data for Lygodactylus (Travers et al., 2014). For PCR protocols and markers details see Supporting Information (Table S1). We purified PCR products using polyethylene glycol (PEG 8000), prepared sequencing reactions using BigDye terminator kit v.3.1 (Applied Biosystems), precipitated the products with EDTA/Ethanol, and produced sequences using an ABI 3130xl sequencer (Applied Biosystems) at INPA Sequencing Center (Laboratório Temático de Biologia Molecular, Instituto Nacional de Pesquisas da Amazônia, Manaus/AM, Brazil). We assembled, edited for ambiguous bases, and aligned sequences using Muscle algorithm (Edgar, 2004) in Geneious v8.1.7 (Biomatters). We used PHASE v2.1.1 (Stephens et al., 2001; Stephens and Wiens, 2003) to determine the pair of alleles with higher probability for nuclear sequences.. 2.3 Phylogenetic relationships To recover phylogenetic relationships of South American Lygodactylus we inferred a Maximum Likelihood (ML) concatenated tree combining both markers using a Randomized Axelerated Maximum Likelihood approach in RAxML v7.2.6 (Stamatakis, 2014). The same model of nucleotide substitution (GTR+Gamma) was assigned for both markers, as this is the option the program offers. Two hundred independent searches and one thousand bootstrap replicates were used to assess nodal support.. 2.4 Divergence time estimates To infer divergence times among South American Lygodactylus we considered the concatenated tree topology that separated samples in five groups (see Results, Figure 2). We. 19.

(39) expected a divergence time among the sister African Lygodactylus and the South American Lygodactylus to be of approximately 25 million years (My) (Gamble et al., 2011), and the diversification timing within the South American Lygodactylus to fall within the Pleistocene if a Pleistocene Arc fragmentation promoted speciation of this genus. We estimated a time calibrated Bayesian species tree using *Beast implemented in BEAST 1.8.2 (Drummond et al., 2012) using the five groups recovered by the gene tree as species assignments (See Results). We calibrated the *Beast tree using a mitochondrial mutation rate of 1.15% per million years as suggested for geckos and other lizards (Arnold et al., 2008). We conducted three independent runs of 3x108 generations, sampling every 3x104 steps, with a Yule speciation process prior and an uncorrelated lognormal relaxed clock. To calibrate the molecular clock, we set up mtDNA ucld.mean parameter using normal prior (mean: 0.0115; standard deviation: 0.002). In addition, for the nuDNA ucld.mean we used a default gamma prior and for ucld.stdev we used an exponential prior (mean: 0.5). We checked convergence among runs and effective sample sizes above 200 using Tracer v1.6 (Drummond and Rambaut, 2007). We used TreeAnnotator (Drummond et al., 2012) to calculate a maximum clade credibility tree, excluding the first one thousand trees as burn-in.. 2.5 Species delimitation In order to test if groups recovered with high support in the concatenated gene tree fit as candidate species, we used a species delimitation analysis, with SpedeSTEM 2 (Ence and Carstens, 2010). SpedeSTEM estimates a ML species tree from mitochondrial and nuclear gene trees to compute AIC (Akaike Information Criterion) scores of different models based on a line of evidence (e.g. molecular). We generated gene trees required for this analysis in BEAST 1.8.2. 20.

(40) (Drummond et al., 2012) and used JModelTest 2 (Darriba et al., 2012) to determine the nucleotide substitution models. We calculated nucleotide diversity for each gene and subsequently the mean between them as an approximation of θ (Theta). To account for a possible influence of theta on incomplete lineage sorting, we used a variation of θ/4 and 4θ of theta values. We tested 52 models, with k (number of species) varying from 1 to 5. For this analysis we used only South American species and Lygodactylus angularis as an outgroup based on the phylogenetic relationships with South American group (see Supporting information, Figure S1). We also estimated genetic distances among groups as the uncorrected p-distance for both mitochondrial and nuclear markers using Mega 7.0 (Kumar et al., 2016).. 3. Results 3.1 Genetic data and phylogenetic analyses We obtained sequences from 67 specimens (25 for South American and 42 for African Lygodactylus). Final aligned sequences comprise 540 base pairs (bp) for ND2 and 302 bp for RAG1. The ML concatenated tree recovered with high bootstrap support the monophyly of Lygodactylus from South America (Figure 2). Five major groups were identified within South American Lygodactylus: L. klugei (encompassing L. klugei from four sampling localities in the Caatinga), Lygodactylus Santo Inácio (population from north Bahia State, Brazil), Lygodactylus Condeúba (population from south Bahia State, Brazil), L. wetzeli (encompassing L. wetzeli individuals from Paraguay) and Lygodactylus São Domingos (population from STDF enclaves within the Cerrado in São Domingos, Goiás State, Brazil; previously identified as L. klugei).. 21.

(41) 3.2 Divergence times and species delimitation The monophyly of South American Lygodactylus observed in the concatenated tree was confirmed by the Bayesian species tree (Figure 3). Divergence between African and South American species of Lygodactylus occurred at 29 Mya (18–44 My HPD interval) (Figure 3). Clades within South American Lygodactylus also had high nodal support values (all posterior probabilities > 0.99). The divergence time among these groups varied from 1.9 Mya to 22.8 Mya (Figure 3). SpedeSTEM analysis recognized five species as the most likely model under three values of θ (θ=0.05, 0.2 and 0.8) and the genetic distances among these groups varied from 3.8 – 33.3% for ND2 and 0.3 – 3% for RAG1 (Table 2). Taking into consideration the genetic distances and divergence times estimated between the closest groups and the results from the species delimitation analysis, we suggest three new candidate species of Lygodactylus in South America: Lygodactylus sp. 1 (Santo Inácio), Lygodactylus sp. 2 (Condeúba) and Lygodactylus sp. 3 (São Domingos).. 4. Discussion 4.1 Monophyly of Lygodactylus in South America Our results corroborate the monophyly and existence of cryptic species in South American Lygodactylus. Previous phylogenies involving this group are scarce and have used only one individual of L. klugei (Gamble et al., 2011; Pyron et al., 2013). Because no sample of L. wetzeli or the candidate species identified herein were included in such analyses, it was impossible to ascertain if South American species were monophyletic in respect to African species. If not, the region either experienced multiple colonization events or some species would have arrived before the separation of Africa and South America.. 22.

(42) Although L. klugei and L. wetzeli are not sister species, the South American clade is monophyletic, corroborating a single colonization event of South America from African Lygodactylus. Gamble et al. (2011) suggested that the first lineage of Lygodactylus reached South America by a trans-Atlantic dispersal around 25 Mya. The divergence time between South American and African Lygodactylus estimated using all five species identified was around 29 Mya (HPD 95%: 18 – 44 Mya), corroborating the hypothesis of Gamble et al. (2011).. 4.2 Pleistocenic Arc Hypothesis Lygodactylus klugei was considered to occur across the Caatinga biome and SDTFs enclaves within Cerrado. For such reason, it was considered an appropriate taxon to test the Pleistocenic Arc Hypothesis (Werneck and Colli, 2006). However, based on our species delimitation results, we suggest that Lygodactylus from SDTFs enclaves is actually a new species (Lygodactylus sp. 3), and its divergence time does not support the PAH. Divergence times among candidate species and L. klugei occurred during the Miocene (at least 8 Mya), before the hypothesized existence of the Pleistocenic Arc (Prado and Gibbs, 1993; Werneck et al., 2011). The diagonal of open formations extends from northeastern Brazil to northwestern Argentina, encompassing three South American biomes: Caatinga, Cerrado, and Chaco (Mayle, 2004; Pennington et al., 2000). An historical connection between Caatinga and Chaco has been previously suggested (Vanzolini, 1974), but subsequent studies have shown that SDTFs endemic species are different from Chaco endemics, and hence excluded the Chaco from the Pleistocenic Arc (Colli, 2005; Pennington et al., 2000; Prado, 1993; Prado and Gibbs, 1993). Also, palaeodistribution modeling of the SDTFs never predicted stable areas of SDTFs in areas currently occupied by the Chaco (Werneck et al., 2011). Conversely, phylogenetic relationships. 23.

(43) recovered in our study suggest a historical connection between lineages/species from the Chaco and SDTFs enclaves. The divergence time between Lygodactylus sp. 3 (São Domingos) and L. wetzeli dates to the late Pleistocene. The original hypothesis suggested the occurrence of the Pleistocene Arc during the Last Glacial Maximum (Prado, 1991; Prado and Gibbs, 1993). Based on paleomodeling, Werneck et al. (2011) suggested that the Pleistocene Arc might have existed in the early Pliocene/lower Pleistocene, and that SDTFs were fragmented during the LGM. Our results match with the timing suggested by Werneck et al. (2011) for the Pleistocenic Arc. Few studies dated divergence times to test the PAH based on SDTF’s endemic taxa (Collevatti et al., 2012; Pennington et al., 2004). Divergence times similar to the one recovered here were found for SDTFs endemic trees (Pennington et al., 2004). Authors concluded that the fragmentation of SDTFs that once formed the Pleistocene Arc probably had no influence on the biogeographic pattern detected. Nevertheless, they did not rule out possible effects of Pleistocene climatic changes on speciation within the different SDTF nuclei (Pennington et al., 2004).. 4.3 Cryptic diversity We propose the existence of three undescribed species of Lygodactylus in South America. Cryptic species have also been identified for other lizard species within the open diagonal (Domingos et al., 2014; Guarnizo et al., 2016; Recoder et al., 2014; Rodrigues, 2003; Werneck et al., 2015). Indeed, this pattern seems to be recurrent, and the underestimation of the biodiversity of the open diagonal is becoming even more evident, especially for the Caatinga. Indeed, according to a phylogeography study of L. klugei (FML, in prep.), two candidate species and L. klugei are endemic or occur mostly in the Caatinga. Once treated as a SDTF endemic (Werneck and Colli, 2006), L. klugei is mostly restricted to the Caatinga, with. 24.

(44) some localities in neighbor biomes such as the Atlantic Forest (our sample 1, from Espírito Santo, Rio Grande do Norte, for example). Lygodactylus sp. 2 is known only from Condeúba in southwestern Bahia State, northeast Brazil. This species occurs in microhabitats similar to other Lygodactylus from the Caatinga, albeit at higher elevations (680 m against 15–550 m for L. klugei across most of the Caatinga). The Caatinga of Southern Bahia is poorly studied, and further fieldwork is necessary to clarify the distribution of this new species and potential processes involved in the diversification of Lygodactylus and perhaps other species. Quaternary sand dunes of São Francisco River (SFR - the largest perennial river in the Caatinga) are a key center of endemism for Caatinga vertebrates (Barreto et al., 2002; LencioniNeto, 1994; Nascimento et al., 2013; Rocha, 1995; Rodrigues and Juncá, 2002). The sand dunes are desert-like formations that have a characteristic composition, making it a very different landscape from the rest of the Caatinga (Rodrigues, 1996). Accordingly, Lygodactylus sp. 1 is apparently endemic to one of these Quaternary dunes which, together, contain impressive levels of endemism for squamates in the Caatinga (Passoni et al., 2008; Rodrigues, 1996; Rodrigues, 2003; Werneck et al., 2015). Approximately 37% of lizards and amphisbaenians and 16% of snakes found in the Caatinga are endemic to these sand dunes (Rodrigues, 2003). Although such paleodunes were dated to the Quaternary, their size suggest that the semiarid condition in this area may date back to the Tertiary (Barreto et al., 2002). Despite this uniqueness, the SFR sand dunes are still unprotected by formal protected areas. Despite the occurrence of Lygodactylus in SDTFs enclaves, this genus is absent from the adjacent Cerrado biome (Colli, 2005; Werneck and Colli, 2006). Indeed, the Central Brazilian Plateau (CBP) geographically separates L. klugei and L. wetzeli. A previous hypothesis suggested that the uplift of the CBP was responsible for a vicariant speciation in Lygodactylus,. 25.

(45) followed by differentiation in the Caatinga and Chaco, and subsequent extinction in Cerrado (Vanzolini, 1963). Our results show that L. klugei and L. wetzeli are not sister species and their divergence time is older than the uplift of the CBP. Lygodactylus sp. 3 (São Domingos) shares a more recent common ancestor with L. wetzeli and has diverged more recently than the uplift of the CBP. Hence, there is no evidence for a role of the CBP on the diversification of Lygodactylus.. 5. Conclusions South American Lygodactylus is monophyletic and originated from a single colonization event into the New World from an African ancestor around 29 Mya. South American Lygodactylus represents a complex of cryptic species, including three endemic or associated to the Caatinga species (L. klugei, Lygodactylus sp. 1, and Lygodactylus sp. 2), one related to the Chaco (L. wetzeli), and one species endemic to a SDTFs enclave within Cerrado (Lygodactylus sp. 3). Although there is no evidence that the PAH accounts for the diversification of L. klugei, we suggest an influence of SDTFs fragmentation on the split of the ancestor of L. wetzeli and Lygodactylus sp. 3.. Acknowledgments We are grateful to researchers that worked in collaboration with us at AAG, GRC, and MTR laboratories for help with fieldwork and samples donation. We also thank Felipe Magalhães and Willianilson Pessoa for help with fieldwork. We are grateful to Erik Choueri for laboratorial assistance. FML and EMF thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their graduate scholarships. FPW thanks National Science Foundation. 26.

(46) DDIG award (DEB-1210346), Science Without Borders Program from CNPq (#374307/2012-1), CAPES/Fulbright (#15073722–2697/06–8), and the Partnerships for Enhanced Engagement in Research (PEER) program for financial support. GRC thanks Coordenação de Apoio à Formação de Pessoal de Nível Superior (CAPES), CNPq, Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF), and PEER program for financial support. MTR thanks Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2003/10335-8 and 2011/50146-6), CNPq, and Dimensions of Biodiversity Program [FAPESP (BIOTA, 2013/50297-0), NSF (DOB 1343578), and NASA]. AAG thanks CNPq (563352/2010-8, 552031/2011-9, 431433/2016-0 and 457463/2012-0) and CAPES (23038.005577/2012-28 and 23038.009565/2013-53) for financial support.. References Antonelli, A., Sanmartín, I., 2011. Why are there so many plant species in the Neotropics? Taxon 60, 403–414. Arnold, E.N., Vasconcelos, R., Harris, D.J., Mateo, J.A., Carranza, S., 2008. Systematics, biogeography and evolution of the endemic Hemidactylus geckos (Reptilia, Squamata, Gekkonidae) of the Cape Verde Islands: based on morphology and mitochondrial and nuclear DNA sequences. Zoologica Scripta 37, 619–636. Barreto, A.M.F., Suguio, K., Oliveira, P.E., Tatumi, S.H., 2002. Campo de Dunas Inativas do Médio Rio São Francisco, BA. Sítios geológicos e paleontológicos do Brasil, Departamento Nacional de Produção Mineral-DNPM.. 27.

(47) Caetano, S., Prado, D., Pennington, R.T., Beck, S., Oliveira Filho, A.T., Spichiger, R., Naciri, Y., 2008. The history of Seasonally Dry Tropical Forests in eastern South America: inferences from the genetic structure of the tree Astronium urundeuva (Anacardiaceae). Molecular Ecology 17, 3147–3159. Carvalho, A.L.G., Sena, M.A., Peloso, P.L.V., Machado, F.A., Montesinos, R., Silva, H.R., Campbell, G., Rodrigues, M.T., 2016. A new Tropidurus (Tropiduridae) from the semiarid Brazilian Caatinga: evidence for conflicting signal between mitochondrial and nuclear loci affecting the phylogenetic reconstruction of South American collared lizards. American Museum Novitates, 1–66. Castiglia, R., Annesi, F., 2011. The phylogenetic position of Lygodactylus angularis and the utility of using the 16S rDNA gene for delimiting species in Lygodactylus (Squamata, Gekkonidae). Acta Herpetol 6, 35–45. Collevatti, R.G., Terribile, L.C., Lima-Ribeiro, M.S., Nabout, J.C., Oliveira, G., Rangel, T.F., Rabelo, S.G., Diniz-Filho, J.A.F., 2012. A coupled phylogeographical and species distribution modelling approach recovers the demographical history of a Neotropical seasonally dry forest tree species. Molecular Ecology 21, 5845–5863. Colli, G.R., 2005. As origens e a diversificação da herpetofauna do Cerrado. In: Scariot, A., Souza-Silva, J.C., Felfili, J.M. (Eds.), Cerrado: Ecologia, Biodiversidade e Conservação, Brasília: Ministério do Meio Ambiente, pp. 247–264. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9, 772.. 28.

(48) Domingos, F.M.C.B., Bosque, R.J., Cassimiro, J., Colli, G.R., Rodrigues, M.T., Santos, M.G., Beheregaray, L.B., 2014. Out of the deep: Cryptic speciation in a Neotropical gecko (Squamata, Phyllodactylidae) revealed by species delimitation methods. Mol Phylogenet Evol 80, 113–124. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7, 214. Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution 29, 1969–1973. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 1792–1797. Ence, D.D., Carstens, B.C., 2010. SpedeSTEM: a rapid and accurate method for species delimitation. Molecular Ecology Resources 11, 473–480. Galdino, C.A.B., Passos, D.C., Zanchi, D., Bezerra, C.H., 2011. Lygodactylus klugei (NCN). Sexual dimorphism, habitat, diet. Herpetological Review 42, 275–276. Gamble, T., Bauer, A.M., Colli, G.R., Greenbaum, E., Jackman, T.R., Vitt, L.J., Simons, A.M., 2011. Coming to America: multiple origins of New World geckos. Journal of Evolutionary Biology 24, 231–244. Guarnizo, C.E., Werneck, F.P., Giugliano, L.G., Santos, M.G., Fenker, J., Sousa, L., D'Angiolella, A.B., Dos Santos, A.R., Strussmann, C., Rodrigues, M.T., DoradoRodrigues, T.F., Gamble, T., Colli, G.R., 2016. Cryptic lineages and diversification of an endemic anole lizard (Squamata, Dactyloidae) of the Cerrado hotspot. Mol Phylogenet Evol 94, 279–289.. 29.