RESSALVA Atendendo solicitação da autora, o texto completo desta tese será disponibilizado somente a partir de 4/11/2024 Priscila Santos Carvalho Systematics and Biogeography of the Hydropsini tribe (Serpentes: Xenodontinae) São José do Rio Preto 2022 Priscila Santos Carvalho Systematics and Biogeography of the Hydropsini tribe (Serpentes: Xenodontinae) Tese apresentada como parte dos requisitos para obtenção do título de Doutora em Biologia Animal, junto ao Programa de Pós-Graduação em Biodiversidade, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto. Financiadora: CAPES Orientador: Prof. Dr. Diego José Santana Silva São José do Rio Preto 2022 Carvalho, Priscila Santos C331s Systematics and biogeography of the Hydropsini tribe (Serpentes: Xenodontinae) / Priscila Santos Carvalho. -- , 2022 144 f. : tabs., mapas Tese (doutorado) - Universidade Estadual Paulista (Unesp), Instituto de Biociências Letras e Ciências Exatas, São José do Rio Preto, Orientador: Diego José Santana Silva 1. América do Sul. 2. Dipsadidae. 3. Filogenia molecular. 4. Filogeografia. 5. Sistemática. I. Título. Priscila Santos Carvalho Systematics and Biogeography of the Hydropsini tribe (Serpentes: Xenodontinae) Tese apresentada como parte dos requisitos para obtenção do título de Doutora em Biologia Animal, junto ao Programa de Pós-Graduação em Biodiversidade, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto. Financiadora: CAPES Comissão Examinadora Profa. Dra. Fernanda de P. Werneck INPA – Manaus, AM Profa. Dra. Thaís Barreto Guedes UNICAMP – Campinas, SP Profa. Dra. Renata Magalhães Pirani UNR – Reno, USA Profa. Dra. Sara Ruane FMNH – Chicago, USA Prof. Dr. Diego José Santana UFMS – Campo Grande, MS Orientador São José do Rio Preto 04 de novembro de 2022 Às mulheres. Agradecimentos O Diego é um orientador ímpar, um ser humano do bem, engraçado, acolhedor, nerd e querido amigo. Há uns meses atrás ele me emprestou um livro chamado "O dia do Curinga". Que livro do caralho! E várias vezes o enxerguei naquelas citações. Ele é um curinga na vida de muitas pessoas. Me espelho muito nele pra me tornar uma pesquisadora melhor. Diego cuida do laboratório muito bem e mantém o ambiente saudável, alegre, vivo, onde podemos falar sobre tudo e todos. Sempre me senti respeitada, valorizada e querida. Diego, obrigada por cada palavra amiga, pelos ensinamentos, pela paciência comigo e por me respeitar como cientista e mulher. Você é o meu curinga. Agradeço aos curadores das coleções científicas e colegas que cederam as amostras de tecidos ou sequências, possibilitando a execução do meu trabalho. Muito obrigada, Adrian A. Garda (UFRN), Ana L. C. Prudente (MPEG), Frank Burbrink (AMNH), Carla Bessa e Vanessa Arzamendia (FHUC), Gustavo H. C. Vieira e Fagner R. Delfim (CHUFPB), Selvino N. de Oliveira (CHUFSC), Guarino Colli (CHUNB), Diego Baldo (CONICET-UNaM), Felipe Grazziotin (IBSP), Christopher Austin (LSUMZ), Miguel R. Treffaut (USP), Paulo Passos (MNRJ), Selma Torquato (MUFAL), Reuber Brandão (UnB), Omar Torres-Carvajal (PUCE), Santiago C. Fischer (PUC-RS), Rejane M. L. da Silva (UFBA-NOAP), Paulo Garcia (UFMG), Felipe Curcio (UFMT), Luiz R. R. Rodrigues (UFOPA), Laura Verrastro e Márcio B. Martins (UFRGS), Vanda Ferreira (UFMS), Gregory Watkins-Colwell (YPM), Pedro M. S. Nunes e Pedro I. Simões (CHUFPE), María E. López e Enrique Arbeláez-Cortés (IAvH), Yaneth Muñoz-Saba (ICN), Martha P. R. Pinilla (MHN-UIS), Fernando Rojas-Runjaic, Hugo E. Cabral, Henrique Folly, Jorge A. D. Pérez e Leandro A. Silva. Também gostaria de agradecer ao Ricardo Marques, Albedi A. Cerqueira Jr. e Fernando Rojas-Runjaic por ter cedido as fotos usadas no capítulo 1 e na apresentação. Quero agradecer também a galera que entrou nessa loucura, assim como eu, de estudar essas cobrinhas incríveis. Nathalie Citeli, Antonio Moraes-da-Silva, Albedi A. Cerqueira Jr. e os professores, Reuber Brandão, Pedro M. S. Nunes, Felipe Curcio e Paulo Passos. Bora time resolver os babados desses bichos maravilhosos. Agradeço ao Ricardo Koroiva pela ajuda no laboratório de Biologia Molecular, ao Felipe Camurugi pela ajuda nas análises moleculares, a Eliana de Oliveira pelas conversas sobre ciência e a vida, por ter me ajudado desde meu mestrado e por estar sempre disponível a sanar dúvidas sobre métodos filogeográficos. Agradeço imensamente ao Don Shepard pelo apoio na bancada, por preparar minhas placas para o sequenciamento e por ter topado participar dessa empreitada. Aos meus amigos Bruna Carvalho, Jéssica Laurentino, Paulo Ricardo, Cirlene da Cunha e Reinaldo Lima por sempre me incentivarem a seguir meus sonhos, por toda amizade, carinho, conversas loucas. Vocês são fodas! Contem sempre comigo, Aos queridos amigos que compartilhei tantos momentos no MZUSP: Ana Bottallo, Marcela Brasil, Renata Fadel, Renata Montalvão, Rafael Henrique, Raissa Siqueira, Paola Sánchez e Diego Cavalheri. Obrigada por todas as conversas, risadas, viagens e pelo carinho. Vocês fazem parte de uma fase muito importante da minha vida. Aos imensos e queridos amigos do laboratório Mapinguari. Com certeza meus dias foram mais alegres, cheio de bagunça e gritaria com vocês: Sarah Mângia, Felipe Camurugi, Eliana Oliveira, Carla Guimarães, Hugo Cabral, Juan Cuestas-Carrilo, Beatriz Vasconcelos, Ibrahim Nehemy, Márcia Müller, Henrique Nogueira, Diego Cavalheri, Nuno (João Emílio), Sean Keuroghlian-Eaton, Leonardo Castro, Henrique Oliveira. O que seria do Mapinguari sem os estagiários? Valeu demais, Lauany Serafim, Ana Alice Cabral, Rafaela Machado e Pílade Filho, vocês me salvaram em vários momentos nessa reta final insana. Ao longo dessa trajetória eu fiz amizades que nem imaginava. Sempre me sinto querida por você Ma (Marcella Souza), mesmo há centenas de quilômetros de distâncias, quando eu voltar em Juiz de Fora, você poderia me emprestar aquele vestido maravilho todo estampado com cobras lindas? hahahahaha. Que surpresa maravilhosa você, Beatriz Vasconcelos, ter escolhido justamente o Mapinguari pra continuar sua trajetória rumo ao desconhecido mundo do jacaré-paguá. Bia, nem sei o que escrever sobre você, já estou com os olhos marejados, mas sei que seu abraço é o mais aconchegante. Você é uma amiga do caralho! Obrigada por tudo. Te amo! O final da minha trajetória no doutorado se mistura muito com a história da Carlinha. Enfrentamos juntas esses momentos alegres e conturbados. Carlota Joaquina, que fase, né amiga? Hahahaha. Que jornada maluca, mas cheia de risada, Pepinha, Queimadinha, gritaria nós vivemos. Outra surpresa maravilhosa que o Mapinguari me deu. Você é pau pra toda obra hahaha. Me leva logo pra conhecer Viçosa e comer a comida da sua mãe. Te amo! Karol Ceron e Renata Fadel, mais conhecidas como Cacatua berrante e Maridinha hahahahaha. Eu sei, a Karol vai berrar quando ler isso. Vocês também foram a minha família em Campo Grande. Dou risada sozinha quando lembro da nossa convivência. Estou morrendo de saudade de vocês. Amo vocês! Vanessinha, minha companheira de leitura. Agora poderemos discutir com mais frequência nossas aventuras pelos maravilhosos livros que navegamos. Obrigada por ser uma amiga tão parceira. Te amo! Peetinha (Sarah Mângia), minha amiga e irmã que amo tanto que chega a doer. Obrigada por ser meu porto seguro, minha família em Campo Grande, por ter me dado o prazer de ser tia das maravilhosas, Farofinha e Mentirinha. Ainda bem que sobrevivemos aquele campo bizarro na Amazônia e agora podemos rir juntas daquela história e de tantas outras coisas. Te amo! Agradeço a minha família por sempre me apoiar e acreditar nos meus sonhos. Obrigada Mãe, por ser uma mulher tão forte e batalhadora. Amo vocês, Sandra, Alexandre, Nilo, Dani e Júnior. Um agradecimento especial a minha irmã maravilhosa Daniela, que é um ser humano incrível, obrigada irmã por tudo! Você é o meu espelho. Por muito tempo eu achei que estava infeliz, sem perspectiva, não sabia onde as minhas escolhidas estavam me levando. Mas percebi que estava muito frustrada com o momento histórico que estamos vivendo. Porém, também percebi que amo muito o que faço e que por mais que esse processo foi desgaste emocional e fisicamente eu ficava empolgada com cada artigo novo que lia. Gostaria que o processo no início tivesse sido diferente e que esse atual momento político no Brasil não tivesse me desgastado tanto. Portanto, gostaria de agradecer cada estudante, professore, pesquisadore, cientista que resiste a cada dia na expectativa de um mundo melhor. Eu quero deixar claro aqui que sou completamente contra esse atual governo genocida, ecocida, misógino, machista, preconceito, neofascista. O simples ato de estudar e fazer ciência no Brasil é uma resistência. #FORABOLSONARO. Agradeço ao programa de pós-graduação em Biodiversidade (antigo Biologia Animal) da Universidade Estadual Paulista, os coordenadores do PPG Francisco Langeani e Antonio Carlos Lofego e aos funcionários da secretária por todo suporte e apoio, principalmente nesse momento pandêmico. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. Ano passado eu morri, mas esse ano eu não morro. (Sujeito de sorte - Belchior) Respeita a existência ou espere resistência. (Noite inteira - Pitty) Nada em biologia faz sentido exceto à luz da evolução. (Theodosius Dobzhansky, 1973) Resumo A região Neotropical é caracterizada pela alta diversidade biológica e pela presença de grandes bacias hidrográficas, que exercem papel importante no padrão de distribuição e evolução da biota. Tal diversidade está também associada, dentre vários fatores, ao soerguimento final dos Andes a partir do Mioceno. Apesar dos recentes avanços na sistemática de serpentes, a diversidade de várias linhagens neotropicais permanece pouco compreendida. Aqui, abordamos a sistemática e biogeografia de Hydropsini (Pseudoeryx, Hydrops e Helicops), e apresentamos uma análise filogenética com base na maior amostragem molecular feita até o momento, cerca de 83% da diversidade da tribo (22 das 26 espécies), incluindo 1.080 sequencias de dois genes mitocondriais (16S e Cytb) e quatro nucleares (Cmos, NT3, BDNF e R35). Dividimos a tese em três capítulos. No primeiro capítulo inferimos a filogenia de Hydropsini, cujos resultados mostraram que os três gêneros são válidos e recuperamos a monofiletismo da tribo. A inclusão de mais espécies resultou em relacionamentos contrastantes quando comparados com inferências filogenéticas anteriores. No segundo capítulo, com base na filogenia datada do capítulo 1, conduzimos uma estimativa de área ancestral, a fim de investigar a história biogeográfica dessas serpentes aquáticas. Revelamos que o cenário ancestral mais provável para a diversificação de Hydropsini foi a região amazônica, por volta de 21 milhões de anos, no início do Mioceno. Discutimos como o dinamismo da paisagem durante o Mioceno na região amazônica teve um grande impacto na diversificação de Hydropsini, influenciado principalmente pelo sistema Pebas. Por fim, no terceiro capítulo, nos baseamos nos achados sobre o complexo de Helicops leopardinus (He. leopardinus, He. modestus, He. infrataeniatus e He. tapajonicus) do primeiro capítulo e estudamos a filogeografia deste complexo para compreendermos sua diversidade e estruturação genética em níveis mais recentes de diversificação. O complexo de Helicops leopardinus se originou durante o Pleistoceno (~1,2 Mya), e mostramos que este complexo pode representar uma única espécie com cinco agrupamentos geneticamente estruturados com fluxo gênico de forma desigual entre eles. A diferenciação genética do complexo de He. leopardinus é explicada principalmente pela interação da distância geográfica, variação climática e bacias hidrográficas. Fornecemos novas propostas sobre padrões e processos de diversificação para um complexo de espécies de cobras aquáticas amplamente distribuído ao longo da América do Sul. Palavras-chave: América do Sul. Dipsadidae. Distribuição geográfica. Filogenia molecular. Filogeografia. Sistemática. Xenodontinae. Abstract The Neotropical region is characterized by high biological diversity and the presence of large hydrographic basins, which play an important role in the distribution pattern and evolution of the biota. Such diversity is also associated, among several factors, with the final uplift of the Andes during the Miocene. Despite recent advances in snake systematics, the diversity of several Neotropical lineages remains poorly understood. Here, we address the systematics and biogeography of Hydropsini (Pseudoeryx, Hydrops e Helicops) and present a phylogenetic analysis based on the largest molecular sampling to date, about 83% of the tribe’s diversity (22 of 26 species), including 1,080 sequences of two mitochondrial genes (16S and Cytb) and four nuclear (Cmos, NT3, BDNF, and R35). We organized the thesis into three chapters. In the first chapter, we inferred the phylogeny of Hydropsini, and our results showed that the three genera are valid and we recovered the monophyly of the tribe. The inclusion of more species resulted in contrasting relationships when compared to previous phylogenetic inferences. In the second chapter, based on the dated phylogeny from chapter 1, we conducted an ancestral area estimate in order to investigate the biogeographic history of these aquatic snakes. We reveal that the most likely ancestral scenario for Hydropsini diversification was the Amazon region, around 21 million years ago, in the early Miocene. We discuss how the landscape dynamism during the Miocene in the Amazon region had a great impact on the diversification of Hydropsini, mainly influenced by the Pebas system. Finally, in the third chapter, based on the findings on the Helicops leopardinus complex (He. leopardinus, He. modestus, He. infrataeniatus and He. tapajonicus) from the first chapter, we carried out a phylogeography for these snakes to understand their diversity and genetic structure. The Helicops leopardinus complex originated during the Pleistocene (~1.2 Mya), and we show that this complex may represent a single species with five genetically structured clusters with uneven gene flow between them. The genetic differentiation of the He. leopardinus complex is mainly explained by the interaction of geographic distance, climatic variation, and watersheds. We provide new proposals on diversification patterns and processes for a complex of aquatic snake species widely distributed across South America. Keywords: South America. Dipsadidae. Geographic distribution. Molecular phylogeny. Phylogeography. Systematics. Xenodontinae. FIGURE CAPTIONS CHAPTER 1 Figura 1 – Sampling coverage used in this study for Pseudoeryx, Hydrops, and Helicops throughout South America. 31 Figura 2 – Bayesian phylogenetic inference of Hydropsini based on mtDNA (16S and Cytb) and nuDNA (Cmos, NT3, BDNF, and R35) genes using BEAST. Posterior probabilities (PP) and bootstrap (BS) values are ≥ 0.95 and ≥ 70%, above and below, respectively (we present only well-supported node values). Asterisks (*) indicate different phylogenetic relationships found in the ML tree. Clade A: Hydropsini tribe; Clade B: Pseudoeryx; Clade C: Hydrops; Clade D: Hy. relictualis; Clade E: Hy. caesurus; Clade F: Hy. martii; Clade G: Hy. triangularis; Clade H: Helicops; Clade I: He. leopardinus group; Clade J: He. leopardinus complex; Clade K: He. trivittatus; Clade L: He. carinicaudus group; Clade M: He. danieli; Clade N: He. hagmanni group; Clade O: He. pastazae complex; Clade P: He. polylepis; Clade Q: He. angulatus group. Photos: DJSantana (P. plicatilis, He. angulatus); FJMRojas-Runjac (P. relictualis); AACerqueira Junior (Hy. casesurus); Moraes-da-Silva et al. 2019 (He. boitata) and CHdeONogueira (He. carinicaudus L1). 33 Figura 3 – Bayesian phylogenetic inference of Pseudoeryx and Hydrops based on mtDNA (16S and Cytb) and nuDNA (Cmos, NT3, BDNF, and R35) genes using BEAST. Posterior probabilities (PP) values are ≥0.95 (we present only well-supported node values). Clade B: Pseudoeryx; Clade C: Hydrops; Clade D: Hy. relictualis; Clade E: Hy. caesurus; Clade F: Hy. martii; Clade G: Hy. triangularis. L: indicates distinct lineages. 34 Figura 4 – Bayesian phylogenetic inferences based on mtDNA (16S and Cytb) and nuDNA (Cmos, NT3, BDNF, and R35) genes using BEAST. Posterior probabilities (PP) values are ≥ 0.95 (we present only well-supported node values). (A) Helicops leopardinus group and He. leopardinus complex. Clade I: He. leopardinus group; Clade J: He. leopardinus complex. (B) Helicops trivittatus and He. carinicaudus group. Clade K: He. trivittatus; Clade L: He. carinicaudus group. (C) Helicops danieli, He. hagmanni group and He. pastazae complex. Clade M: He. danieli; Clade N: He. hagmanni group; Clade O: He. pastazae complex. 36 Figura 5 – Bayesian phylogenetic inference of He. polylepis, He. angulatus group and He. pastazae complex based on mtDNA (16S and Cytb) and nuDNA (Cmos, NT3, BDNF, and R35) genes using BEAST. Posterior probabilities (PP) values are ≥0.95 (we present only well-supported node values). Clade P: He. polylepis; Clade Q: He. angulatus group. L: indicates distinct lineages. 37 Figure S1 – Maximum likelihood (ML) phylogenetic inference of Hydropsini tribe based on concatenated mtDNA (16S and Cytb) and nuDNA (Cmos, NT3, BDNF, and R35). Numbers below nodes are bootstrap values (BS) ≥ 70% (we present only well- supported node values). 58 Figure S2 – Geographic distribution of Pseudoeryx and Hydrops samples from this study along six watersheds across South America. 61 Figure S3 – Geographic distribution of Helicops leopardinus group and He. leopardinus complex samples from this study along six watersheds across South America. 62 Figure S4 – Geographic distribution of Helicops trivittatus and He. carinicaudus group samples from this study along of four watersheds across South America. 63 Figure S5 – Geographic distribution of Helicops danieli, He. hagmanni group, Helicops pastazae complex and He. polylepis samples from this study along five watersheds across South America. 64 Figure S6. Geographic distribution of Helicops angulatus group samples from this study along 10 watersheds across South America. 65 CHAPTER 2 Figure 1. Ancestral geographic scenario of the Hydropsini tribe reconstructed under DEC+j model by BioGeoBEARS. The biogeographic areas defined based on the combination of Neotropical ecoregions, with the basin hydrographic, and distribution pattern of Hydropsini species. Single capital letters and colors indicate different biogeographic units used in this study. Mixed letters and colors represent combinations of such units. 78 Figure S1 – Dated Species Tree (*BEAST) of the Hydropsini based on mtDNA (16S and Cytb) and nuDNA (Cmos, NT3, BDNF and R35). Values above the nodes indicate posterior probabilities. The bars represent the 95% HDP. 94 CHAPTER 3 Figure 1 – Geographic distribution of Helicops leopardinus complex samples (H. leopardinus, H. infrataeniatus, H. modestus, and H. tapajonicus) along the major hydrobasins across South America. White symbols indicate samples not used in the BAPS analysis. 110 Figure 2 – Phylogenetic relationships of Helicops leopardinus complex and divergence times by Bayesian Inference in BEAST based on mtDNA 16S and Cytb concatenated genes. Numbers below nodes are posterior probability values; nodes without values indicate PP < 0.95. Bars represent the 95% Highest Posterior Density (HPD) interval for divergence dates. Taxon name, voucher number, and locality are indicated for each terminal. 114 Figure 3 – Bayesian Analysis of Population Structure (BAPS) results: A) spatial clustering of individuals. B) Admixture based on mixture clustering. Each color represents one cluster (among the five clusters) generated by the mitochondrial and nuclear gene dataset. 115 Figure 4 – Network of five clusters indicating the gene flow among them by weighted arrows. 116 Figure 5 – Haplotype networks for each locus within of Helicops leopardinus complex. The haplotype colors correspond to the clusters recovered by BAPS, and the size of its area is proportional to its frequency. Black circles represent unsampled haplotypes and the dashes represent mutational steps. 116 TABLE LIST CHAPTER 1 Table S1 – Taxa, vouchers, locality data, genetic markers and GenBank accession numbers used in this study. Absent acronyms in Sabaj (2022): MTR or MRT: researcher field number Miguel Trefaut U. Rodrigues (University of São Paulo, USP); A: researcher field number Vanessa Arzamendia (Faculty of Humanities and Sciences, National University del Litoral); AAGARDA: researcher field number Adrian Antonio Garda; AF: researcher field number Antoine Fouquet; GGU: researcher field number Giussepe Gagliardi Urrutia (Universidad Nacional de la Amazonía Peruana, Facultad de Ciencias Biológicas); LW: field number of the Federal University of Western Pará; MAP and MAP-T: field and tissue number of the Mapinguari laboratory at the Federal University of Mato Grosso do Sul; MHNBA: Bahia Natural History Museum voucher; RABRANDÃO: researcher field number Reuber Brandão (University of Brasilia) SB: project field number scales of biodiversity; VLF: researcher field number Vanda Lucia Ferreira (Federal University of Mato Grosso do Sul. Other museum acronyms follow Sabaj (2022). L: lineages. 66 Table S2 – List of outgroup taxa and GenBank accession numbers of specimens used in this study. 67 Table S3 – PartitionFinder 2 model of nucleotide substitution. Best-fitting partitioning scheme model of nucleotide substitution for 16S, Cytb, Cmos, NT3, BDNF and R35 genes. 69 CHAPTER 2 Table 1 – Summary of the temporal and spatial main events that influenced the dispersal/vicariance for the Most Recent Common Ancestor (MRCA) of Hydropsini lineages. (A) Transandean; (B) Northwest Amazonia; (C) Southeast Amazonia; (D) Northeast Diagonal; (E) Southwest Diagonal and (F) Atlantic Forest. We follow the stratigraphy International Chronostratigraphic Chart (ICS). 78 Table 2 – Comparison of the BioGeoBEARS model for Hydropsini within six areas based on the log-likelihood (LnL) and the Akaike information criterion (AIC); N, parameters number; d, dispersion rate; e, extinction rate; J, relative probability of speciation between founding events. The best model is shown in bold. 81 Table S1 – Taxa, vouchers, locality data, genetic markers and GenBank accession numbers used in Carvalho 2022 (PSC 2022). L: lineages. Absent acronyms in Sabaj (2022): MTR or MRT: researcher field number Miguel Trefaut U. Rodrigues (University of São Paulo, USP); A: researcher field number Vanessa Arzamendia (Faculty of Humanities and Sciences, National University del Litoral); AAGARDA: researcher field number Adrian Antonio Garda; AF: researcher field number Antoine Fouquet; GGU: researcher field number Giussepe Gagliardi Urrutia (Universidad Nacional de la Amazonía Peruana, Facultad de Ciencias Biológicas); LW: field number of the Federal University of Western Pará; MAP and MAP-T: field and tissue number of the Mapinguari laboratory at the Federal University of Mato Grosso do Sul; MHNBA: Bahia Natural History Museum voucher; RABRANDÃO: researcher field number Reuber Brandão (University of Brasilia) SB: project field number scales of biodiversity; VLF: researcher field number Vanda Lucia Ferreira (Federal University of Mato Grosso do Sul. 95 CHAPTER 3 Table 1. Model selection of what variables influence population genetic diversity using redundancy analyses (RDA) for Helicops leopardinus complex. 117 Table S1 – Taxa, vouchers, locality data, genetic markers and GenBank accession numbers used in this study. Absent acronyms in Sabaj (2022): MTR or MRT: researcher field number Miguel Trefaut U. Rodrigues (University of São Paulo, USP); A: researcher field number Vanessa Arzamendia (Faculty of Humanities and Sciences, National University del Litoral); AAGARDA: researcher's field number Adrian Antonio Garda; AF: researcher field number Antoine Fouquet; GGU: researcher field number Giussepe Gagliardi Urrutia (Universidad Nacional de la Amazonía Peruana, Facultad de Ciencias Biológicas); LW: field number of the Federal University of Western Pará; MAP and MAP-T: field and tissue number of the Mapinguari laboratory at the Federal University of Mato Grosso do Sul; MHNBA: Bahia Natural History Museum voucher; RABRANDÃO: researcher field number Reuber Brandão (University of Brasilia) SB: project field number scales of biodiversity; VLF: researcher field number Vanda Lucia Ferreira (Federal University of Mato Grosso do Sul. 132 Table S2 – PartitionFinder 2 model of nucleotide substitution. Best-fitting partitioning scheme model of nucleotide substitution for 16S, Cytb, Cmos, NT3, BDNF, and R35 genes. 132 Table S3 – Specimens, vouchers, and geographic coordinate of samples used in Bayesian Analysis of Population Structure (BAPS) and which spatial clusters they belong. In bold the specimens that were admixture. 133 Table S4 – Genetic statistics for each locus for cluster Bayesian Analysis of Population Structure (BAPS) of Helicops leopardinus complex. L: length in base pairs (bp); N: sample size; S: number of polymorphic sites; H: number of haplotypes (number of exclusive haplotypes in this lineage); Hd: haplotype diversity; π: nucleotide diversity. neutrality tests Tajima’s D and Fu's F. * Phased sequences; ** sampling were not enough to estimate these parameters; *** genetic variation was not enough to estimate these parameters. 135 SUMMARY 1 GENERAL INTRODUCTION 18 2 CHAPTER 1: Phylogeny and systematic of Hydropsini (Serpentes: Dipsadidae: Xenodontinae) 31 2.1 Introduction 25 2.2 Material and Methods 30 2.3 Results 32 2.4 Discussion 38 2.5 Conclusions 49 2.6 References 51 2.7 Supplementary information 58 3 CHAPTER 2: Spatial-temporal evolution of water snakes in South America driven by Pebas System during the Early Miocene 72 3.1 Introduction 73 3.2 Material and Methods 75 3.3 Results 77 3.4 Discussion 81 3.5 Conclusions 84 3.6 References 85 3.7 Supplementary information 94 4 CHAPTER 3: Phylogeography of Helicops leopardinus complex (Serpentes: Dipsadidae: Xenodontinae) 106 4.1 Introduction 107 4.2 Material and Methods 109 4.3 Results 113 4.4 Discussion 117 4.5 Conclusions 122 4.6 References 122 4.7 Supplementary information 132 5 Final considerations 138 References general introduction 138 18 1. General introduction Hydropsini: A brief systematic history With over 3,900 species encompassing a diversity of morphologies, ecologies, and lifestyles (arboreal, fossorial, terrestrial, aquatic) (Vitt & Caldwell 2013), snakes represent about 35% of squamate diversity (Uetz 2022) and approximately 30% of global snake diversity occurs in the Neotropical region (Guedes et al. 2018). Within the extensive Neotropical diversity of snakes, the family Dipsadidae is one of the largest radiations of Caenophidia (“Advanced Snakes”) (>750 species; sensu Grazziotin et al., 2012). Dipsadids were considered restricted to the New World, but recent placement of Stichophanes and Thermophis as sister to Dipsadidae expanded their distribution into the Old World (e.g., Figueroa et al. 2016). Dipsadidae is morphologically supported by two putative hemipenial synapomorphies (body calyces on the asulcate surface of the lobes and lateral enlarged spines disposed on the sides of the hemipenial body) and molecularly (Zaher et al. 2009). Among dipsadids, the tribe Hydropsini belongs to Xenodontinae Bonaparte, 1845, and is composed by three genera: Helicops Wagler, 1828 (21 species); Hydrops Wagler, 1830 (3 spp); and Pseudoeryx Fitzinger, 1826 (2 spp). This tribe is endemic to South America, occur from Trinidad to Uruguay, and their species are strongly associated and adapted to the aquatic environment (Moraes-da-Silva et al. 2019). Among the variety of lifestyles in snakes, about 5% have aquatic habitats (Pauwels et al. 2008) (e.g., members of Acrochordidae, Boidae, Colubridae, Dipsadidae, Elapidae, Homalopsidae, Natricidae and Viperidae). Thereby, snakes have independently invaded the aquatic environment many times (Pauwels et al. 2008). Interestingly, the Oriental and Neotropical regions have a great hydrological richness and are the two largest regions of aquatic or semiaquatic freshwater snake diversity (Pauwels et al. 2008). Some morphological specializations in aquatic/semiaquatic snakes include position of nostrils on the snout top, valvular nostrils, dorsolaterally oriented eyes (Pauwels et al. 2008; Segall et al. 2016). This morphology adaptations are observed, irrespective of the phylogenetic relationships among species of snakes from different families, suggesting that the aquatic environment does indeed drive the evolution of morphology in snakes, thus driving the evolutionary trajectory of this group of animals (Segall et al. 2016). Among the current taxonomy of Hydropsini, Pseudoeryx has two valid species: P. plicatilis and P. relictualis with allopatric distributions. Though, P. plicatilis presents two subspecies P. p. plicatilis and P. p. mimeticus both with cis Andean distribution. Meanwhile, P. relictualis is a trans Andean species, occurring in Lake Maracaibo, Venezuela (Schargel et al. 2007). Hydrops contains three species: Hy. triangularis, Hy. martii and Hy. caesurus. 19 Hydrops martii and Hy. triangularis have widely overlapping geographic distributions, and are often found in syntopy, especially along the central part of the Amazon basin. Meanwhile, Hy. Caesurus is endemic in the La Plata basin (Argentina, Paraguay and Brazil) (von May et al. 2019). Hydrops differs from Helicops and Pseudoeryx in its smooth dorsal scales (keeled in Helicops) and in its maxillary diastema and color pattern with transverse bands (no diastema and longitudinal lines or dots in Pseudoeryx) (Roze 1957a, b). Lastly, Helicops is the most diverse genus of Hydropsini with 21 known species, diagnosed by the presence of nude flounces on the lobes of the hemipenis (Zaher 1999), and differs from Hydrops and Pseudoeryx by the presence of keels on the dorsal scales, absent in both latter taxa (Roze 1957a, b; Moraes-da-Silva et al. 2019). Helicops has species with a wide and restricted distribution throughout South America (Nogueira et al. 2019) and most occur in the Amazon region (e.g., He. petersi, He. pastazae, He. yacu), while He. leopardinus and He. angulatus are widely distributed. In this genus, there are two species with restricted trans Andean distribution (Moraes-da-Silva et al. 2019; 2021; Citeli et al. 2022). Many species within this tribe have undefined type localities, which makes taxonomic decisions difficult. Some species are only known from a few specimens or their type specimens, as many specimens were collected when tissue collection for molecular analysis was not yet standard practice (e.g., He. yacu, He. hogei). Currently, only 22 specimens have been sequenced representing 12 species out of the 26 recognized. There have been several taxonomic rearrangements in recent decades, mainly due to morphological differences in hemipenis between the three genera (see below) (Roze 1957b; Rossman 1973). Since the three genera share a single internasal scale, eyes and nostrils located in a dorsal position, Roze (1957a, b) indicated a phylogenetic affinity between them, and considered such traits as adaptations of these genera to aquatic habits. The author also suggested that Hydrops and Helicops would be more closely related due to dentition characteristics, qualitative (color pattern) and quantitative (dorsal scales) features, and that Pseudoeryx would be an sister clade of this clade. However, Rossman (1973) based sole on hemipenial evidence considered that such characteristics would be convergent adaptations of the aquatic habit and rejected the hypothesis of Roze (1957a, b). Although he did not place the genera into any family, the author suggested that new evidence beyond the hemipenis might alter his conclusion. Within Xenodontinae, Pseudoeryx and Hydrops are the only genera that do not have hemipenial characteristics that would diagnose them as belonging to this subfamily (Zaher 1999). Despite this, the relationship of the three genera was supported by sharing the presence of a highly 20 developed adductor mandible externus superficialis muscle at their point of origin (Zaher 1999). The taxonomic history of Pseudoeryx and Hydrops is confusing and complex, mainly because the species and subspecies have been described from qualitative and quantitative variations of a limited number of specimens (Roze 1957b; Mertens 1965; Albuquerque 2000; Albuquerque & Lema 2008). Furthermore, in Pseudoeryx the lack of information on type localities makes it difficult to solve taxonomic problems. The two currently subspecies of P. plicatilis (P. p. plicatilis and P. p. mimeticus) show great overlap in qualitative and quantitative characters and occur in sympatry at some areas (Dixon & Soini 1986; Cunha & Nascimento 1993; Schargel et al. 2007). Such overlaps seems to indicate that the subspecies of P. plicatilis do not represent distinct evolutionary lineages and merely depict artificial clusters of geographic/individual variation (Schargel et al. 2007), although, so far this issue has not been resolved. Variations in hemipenial morphology in three specimens of P. plicatilis were observed and there is a possibility that some populations may represent different species (Zaher 1999). Roze (1957b) performed a taxonomic review of Hydrops and described two subspecies for Hy. martii (Hy. m. martii and Hy. m. callostictus) and six for Hy. triangularis (Hy. t. triangularis, Hy. t. fasciatus, Hy. t. venezuelensis, Hy. t. neglectus, Hy. t. bassleri, Hy. t. bolivianus), based on morphological characters. Almost 50 years later, Albuquerque (2000) and Albuquerque & Lema (2008) reviewed the genus with a larger sample and found that there was a great overlap between the subspecies of both Hy. triangularis and Hy. martii, and placed the subspecies of each taxon in synonymy with the nominal species. As it presents the greatest diversity within the tribe, Helicops presents the greatest taxonomic issues. There is polymorphism of several quantitative and qualitative data, and most variables overlap between several species, both in taxa with restricted and wide distributions (Kawashita-Ribeiro et al. 2013; Moraes-da-Silva et al. 2019, 2021; Costa et al. 2016). Briefly, Rossman (1976) coined the pastazae complex (He. pastazae and He. petersi) and the “polylepis section” (He. pastazae, He. petersi, He. polylepis and He. yacu). Soon after, Rossman and Abe (1979) discussed the possible synonymy of He. yacu with He. pastazae, since two additional specimens of He. yacu were collected from the upper Amazon region that resembled He. pastazae. Rossman (2002) reported that he “believes” that He. hogei would be a junior synonym of He. scalaris based on morphology. From the 2000s until now, there have been five descriptions of new species of Helicops (Kawashita-Ribeiro et al. 2013; Costa et al. 2016; Moraes-da-Silva et al. 2019, 2021, 2022). However, several widespread taxa, such as He. angulatus, He. leopardinus, He. hagmanni and He. polylepis 21 may represent species complexes and have never had their taxonomic status tested. Therefore, the relationships between Helicops taxa need strong investigations considering integrative approaches, and with larger sampling data. Our understanding of snake phylogenetic relationships has significantly improved in recent years due to the advancement and development of molecular genetics (Zaher et al. 2009; Pyron et al. 2013; Figueroa et al. 2016; Zaher et al. 2019). Molecular phylogenies confirmed the monophyly of the tribe, establishing its validity, although the intergeneric and interspecific relationships remain unresolved, given that taxonomic and geographic sampling remains limited (e.g., Moraes-da-Silva et al., 2019, 2021, Murphy et al. 2020). With the recent discoveries of new species of Helicops, Costa et al. (2016) and subsequent works began to use a molecular phylogeny approach to validate such species and verify their phylogenetic positioning. From these phylogenies with low taxonomic and geographic sampling, a phylogenetic perspective on the tribe began to be elucidated. Summarily, in the description of He. nentur, Costa et al. (2016) revealed phylogenetic affinity of this taxa with He. carinicaudus, and which was corroborated by subsequent studies (Moraes-da-Silva et al., 2019, 2021, Murphy et al. 2020). Moraes-da-Silva et al., 2019, 2021 described the species He. boitata and He. phantasma, respectively. Such studies recovered the first as a sister rate of He. carinicaudus and He. nentur, while He. phantasma was closely related to He. leopardinus. Moraes-da-Silva et al. (2019) presented for the first time the close relationship of He. leopardinus with He. infrataeniatus and He. modestus. They still revealed He. polylepis as sister taxa of the clade formed by He. gomesi and He. angulatus. Moraes-da- Silva et al. (2021) also included He. hagmanni in this new study that was recovered as a sister taxon of the clade formed by He. polylepis, He. gomesi and He. angulatus with low support value. Murphy et al. (2020) pointed out that He. angulatus is paraphyletic with respect to He. gomesi and revalidated Helicops cyclops Cope, 1868 to solve the problem. However, the study presents some taxonomic inconsistencies that need to be reviewed. Despite the panorama presented here, molecular phylogenetic studies so far have used a limited sample of the diversity of the tribe, in addition to using only species and samples occurring in Brazil. Evolutionary approach The molecular approach, combined with advances in bioinformatics, have helped to understand the evolution of biota, aiding in the development and knowledge of systematics, taxonomy and biogeography (Posada & Crandall 2001; Hickerson et al. 2010; Burbrink & Gehara 2018). Phylogeographic studies focus on the patterns and processes that determine the geographic distribution of genealogical lineages, trying to correlate, for example, geoclimatic 22 events, geographic distance, topography (historical and contemporary events) with the evolutionary history of lineages (Avise 2012; Knowles 2009). Such studies can help in the delimitation of species, in the recognition of new and cryptic species, as well as in the understanding of events such as extinction, dispersal, vicariance, and the demographic history of a species or population (e.g., Ledo et al. 2017; Dal Vechio et al. 2019; Carvalho et al. 2020; Damasceno et al. 2021). Phylogeographic studies of aquatic snakes from other regions such as Asia and North America have revealed that the evolutionary trajectory of the species does not depend only on geomorphological and climatic processes, but also intrinsic traits of each species. Therefore, species phylogenetically and ecologically related show different phylogeographic patterns (e.g., Guiher & Burbrink 2008; Brandley et al. 2010; Lukoschek et al. 2011; McCartney-Melstad et al. 2012; Carvalho et al. in press). Besides, comparative observations are determined by shared ancestry, and testing evolutionary hypotheses requires focusing on closely related taxa (Pyron 2015). Methods based on estimates of reconstruction of ancestral areas demand dated trees to connect the spatial and temporal dimensions (e.g., Ronquist 1997; Matzke 2014). The history of the uplift of the Andes includes several elevation phases that triggered a long and complex history of the landscape and river systems linked to the evolutionary history of the South American biota (e.g., Wesselingh & Salo 2006; Hoorn et al., 2010a, b). Therefore, geological events and landscape remodeling on a continental scale have a strong influence on dispersal and vicariance processes, which reflects on the distribution patterns of organisms (Lundberg et al. 1998). Among the most important of these events are marine introgressions, the formation of the Pebas and Acre systems, changes in drainage connectivity and fusion, and the formation of the current transcontinental Amazon River drainage (Wesselingh & Salo 2006; Hoorn et al., 2010a, b; Rosa et al. 2003). The dispersal of freshwater species requires corridors of aquatic habitat connecting basins, and the range limits of most of these species coincide with the limits of watersheds, although many others have distributions that extend beyond the limits of watersheds between basins (Albert & Reis 2011). However, for species that are not restricted to the aquatic environment, as in the case of aquatic or semiaquatic snakes, which can also forage on the terrestrial environment, hydrographic limits are not necessarily barriers (Carvalho et al. 2020; Carvalho et al. in press). Furthermore, species with a wide distribution are less likely to show phylogeographic breaks caused by geographic barriers (e.g., Avise 2012) and may still show greater genetic differentiation due to local adaptation (Kremer et al. 2012; Berthouly-Salazar et al. 2013). 23 Despite the panorama presented, the molecular phylogenetic studies presented so far for Hydropsini used a limited sample of the diversity of the tribe, using only species and samples that occur in Brazil. Furthermore, given its widely and narrowly distributed taxa and aquatic habitat, Hydropsini is an excellent model to study the spatial distribution of aquatic snake diversity in the South American region. Therefore, here we address the systematics and biogeography of Hydropsini, presenting for the first time a robust multi loci (two mtDNA, four nuDNA) molecular phylogenetic analysis including 1080 sequences from 22 of the 26 species. We also discuss the origin and diversification of the tribe based on a dated tree and geological events. Main results We have divided the thesis into three chapters. In the first chapter we inferred the phylogenetic relations of Hydropsini, which our results showed that the three genera are valid and we recover the monophyly of the tribe. In addition, we have redefined new groupings based on phylogenetic evidence. We recovered paraphyletic lineages in He. carinicaudus, He. leopardinus complex, He. pastazae and He. angulatus and discussed new relationships and taxonomic issues. Within Helicops, the inclusion of the majority of species had an impact on previous proposed phylogenetic positions. In the second chapter, based on the phylogeny of chapter 1, we performed an ancestral area estimation, in order to investigate the biogeographic history of these aquatic snakes. We reveal that the most likely ancestral scenario for Hydropsini diversification was the Amazon region, around 21 Mya in the early Miocene. The mountain ranges of Colombia and Venezuela acted as a vicariant barrier separating the cis and trans Andean taxa. Furthermore, colonization to adjacent watersheds further to east was also caused by distinct dispersal events. We discuss how landscape dynamism during the Miocene in the Amazon region had a great impact on the diversification of Hydropsini, mainly influenced by the Pebas system. Lastly, in the third chapter we based on the findings for Helicops leopardinus complex (He. leopardinus, He. modestus, He. infrataeniatus and He. tapajonicus) in the first chapter, we performed a phylogeography of this species complex in order to better understand its diversity and genetic structure. Helicops leopardinus complex originated during the Pleistocene (~1.2 Mya). We found that this complex may represent a single species with five genetically structured clusters, which show gene flow unevenly shared among them. The genetic differentiation of the He. leopardinus complex is mainly explained by the interaction of geographic distance (IBD), climatic variation (IBE), and 24 hydrographic basins. We provided new insights about diversification patterns and processes for a species complex of a broadly distributed group of watersnakes along South America. 138 5 Final considerations The gaps in knowledge about the patterns and processes of South American water snakes are significant due to the lack of scientific attention these taxa receive. We address the systematics and biogeography of Hydropsini (Pseudoeryx, Hydrops e Helicops) based on the largest molecular sampling to date, about 83% of the tribe’s diversity (22 of 26 species). The inclusion of more species and specimens resulted in contrasting relationships when compared to previous phylogenetic inferences. We highlight the problematic taxonomy of the tribe and the possibility of new species or synonymizations, which would reflect in the current knowledge of the systematic of Hydropsini. Therefore, to establish a congruent classification based on monophyletic clades we recommend that all these species have integrative taxonomic revisions to elucidate the cryptic diversity. Furthermore, we propose that the landscape dynamism during the Miocene in the Amazon region had a great impact on the origin and diversification of Hydropsini, mainly influenced by the Pebas system, which possible for Hydropsini to disperse to adjacent basins, driven by adequate conditions of long duration (time) in a large region (area). Moreover, we presented the first phylogeographic study of a South American water snake. 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