UNIVERSIDADE ESTADUAL PAULISTA "JÚLIO DE MESQUITA FILHO INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (ZOOLOGIA) TESE DE DOUTORADO CHARACINAE (ACTINOPTERYGII: CHARACIFORMES: CHARACIDAE): IDENTIFICAÇÃO MOLECULAR E ESTUDO DAS RELAÇÕES FILOGENÉTICAS ENTRE ESPÉCIES CAMILA DA SILVA DE SOUZA BOTUCATU-SP 2022 UNIVERSIDADE ESTADUAL PAULISTA INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (ZOOLOGIA) CHARACINAE (ACTINOPTERYGII: CHARACIFORMES: CHARACIDAE): IDENTIFICAÇÃO MOLECULAR E ESTUDO DAS RELAÇÕES FILOGENÉTICAS ENTRE ESPÉCIES Discente: Camila da Silva de Souza Orientador: Dr. Claudio de Oliveira Co-orientador: Dr. Bruno Francelino de Melo Tese apresentada ao Programa de Pós-Graduação em Ciências Biológicas (Zoologia) do Instituto de Biociências de Botucatu, Universidade Estadual Paulista “Júlio de Mesquita Filho”, como parte dos requisitos para a obtenção do título de Doutorado. BOTUCATU-SP 2022 AVISO Esta tese é parte dos requerimentos necessários à obtenção do título de Doutor em Ciências Biológicas, Zoologia, e como tal, não deve ser vista como uma publicação no senso do Código Internacional de Nomenclatura Zoológica (apesar de disponível publicamente sem restrições). Desta forma, quaisquer informações inéditas, opiniões e hipóteses, bem como nomes novos, mudanças taxonômicas não estão disponíveis na literatura zoológica. Pessoas interessadas devem estar cientes de que referências públicas ao conteúdo deste estudo, na sua presente forma, somente devem ser feitas com a aprovação prévia do autor. NOTICE This thesis is a partial requeriment for the PhD degree in Biological Sciences with emphasis in Zoology and, as such, should not be considered as a publication in the sense of the International Code of Zoological Nomenclature (although it is available without restrictions). Therefore, any new information, opinions, and hypotheses, as well as new names, taxonomic changes ae not available in the zoological literuature. Interested people are advised that any public reference to this study, in its current form, should only be done after previous acceptance of the author. Esta pesquisa foi financiada com recursos da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - Processo Número: 2017/06551-0 e 2019/04602-1 "It seems to me that the natural world is the greatest source of excitement; the greatest source of visual beauty; the greatest source of intellectual interest. It is the greatest source of so much in life that makes life worth living." (David Attenborough) À vida AGRADECIMENTOS Deixo meus sinceros agradecimentos às instituições e pessoas que de forma direta ou indireta, contribuíram para a concretização deste trabalho. Ao auxílio financeiro concedido pelo processo nº 2017/06551-0 e 2019/04602-1, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Ao Dr. Claudio de Oliveira, pela orientação acadêmica em todas as fases do projeto, e às oportunidades oferecidas desde a iniciação científica, essenciais para a minha formação. Ao Dr. Bruno F. Melo pela orientação, amizade e imensurável motivação! Ao Dr. Ricardo Benine pela oportunidade em realizar o estágio de docência sob sua orientação e pelas discussões e ensinamentos. Ao Dr. George M.T. Mattox e Dr. Mônica Toledo-Piza pelas grandiosas colaborações e conhecimento compartilhado. Ao Dr. Brian L. Sidlauskas, por ter me acolhido e orientado na Oregon State University - USA durante o doutorado-sanduíche e pelos valiosos exemplos e ensinamentos sobre a vida profissional e pessoal. Ao Dr. Adamm Summers, pela oportunidade de obter dados para o meu projeto do doutorado- sanduíche no Friday Harbor Laboratories - University of Washington - USA. Ao Dr. Thaddaeus Buser, Álvaro Cortés, Hakan Aydogan e Jonathan Huie pela ajuda e colaboração durante o doutorado-sanduíche. À Dr. Luz Ochoa e Dr. Nadayca Mateussi pela amizade e por compartilharem seus conhecimentos para a execução desse trabalho e terem sido minhas maiores referências próximas da representatividade feminina na ictiologia. À Dr. Rafaela Ota e ao Dr. Victor Tagliacollo, Dr. Fernando Carvalho e Dr. Fernando Jerep por todos os ensinamentos e sugestões na última etapa deste trabalho. A todos integrantes e ex-integrantes do Laboratório de Biologia e Genética de Peixes, em especial a Angélica Dias, Beatriz Dorini, Bruno Mora, Cristiane Shimabukuro-Dias, Cristhian Conde, Diogo Freitas, Daniela Oliveira, Duílio Zerbinato, Érica Serrano, Fabilene Gomes, Fabio Roxo, Gabriel Costa e Silva, Guilherme J. da Costa Silva, Junior Flausino, Lais Reia, Maria Ligia, Natalia Mendez, Sâmia Mouallem, Silvana Melo e Renato Devidé, por todos os momentos de descontração, incentivo e colaboração. As grandes amigas Aline Veríssimo, Carolina Magalhães, Laura Borgatto e Milene Pereira, por todo companheirismo e motivação ao longo da vida acadêmica e pessoal. À Eric. V. Ywamoto por ter chegado na minha vida no começo desse projeto e ser um dos motivos de eu seguir em frente e finalizá-lo. Meus sinceros agradecimentos por toda ajuda, companheirismo e por sempre acreditar em mim, muitas vezes mais do que eu mesma. A toda minha família que são fonte de motivação e inspiração e em especial a Giulia, Mirella, Miguel, Bruna, Tiago, David, Felipe, Cinthia, Rosa e William. E, finalmente, deixo meu eterno agradecimento aos meus pais, Valdomiro e Silvana, pelo amor, suporte e por sempre incentivarem minhas aventuras pelo mundo em busca de experiências e sabedoria. RESUMO Characinae é uma das subfamílias mais diversas de Characidae com 85 espécies válidas amplamente distribuídas pelas América do Sul e Central. A subfamília é representada por espécies de pequeno a médio porte que adotam diferentes estratégias alimentares, tais como carnivoria, onivoria e lepidofagia. As relações filogenéticas dentro de Characinae e desta com outras subfamílias de Characidae têm sido objeto de alguns estudos morfológicos, porém, não há, até o momento, hipóteses de relacionamento incluindo uma significativa representatividade de espécies de Characinae usando caracteres morfológicos ou moleculares. Além disso, os estudos publicados ainda apresentam incongruências nas relações intergenéricas e interespecíficas. Neste contexto, o presente trabalho teve como objetivos testar as hipóteses de relações filogenéticas da subfamília Characinae através de análises filogenômicas utilizando os elementos ultraconservados (UCEs) e construir um banco de dados genéticos de DNA barcode para identificar geneticamente as espécies de Characinae. Os resultados das análises filogenéticas corroboram a hipótese morfológica em relação à monofilia de Characinae e revelam novas hipóteses de relações intergenéricas e interespecíficas. Com base na filogenia obtida, analisamos origem e diversificação, assim como a evolução do tamanho e formato do corpo. Os resultados das identificações moleculares reconheceram uma diversidade antes subestimada que deverá contribuir para ampliação do conhecimento sobre a diversidade das espécies de Characinae. PALAVRAS-CHAVE: DNA Barcode, Biodiversidade, Filogenia, Sistemática, Taxonomia, Peixes. ABSTRACT Characinae is one of the most speciose subfamilies of Characidae and widely distributed throughout South and Central America. The subfamily contains small to medium-sized species that adopt distinct feeding strategies as carnivory, omnivory and lepidophagy. Phylogenetic relationships within the Characinae and with other subfamilies have been subject of a few morphological studies, although none hypothesis included a representative number of Characinae species using either morphological or molecular characters. Furthermore, the proposed hypotheses still present inconsistencies at intergeneric and interspecific levels. In this context, the present study aimed to test the hypotheses of phylogenetic relationships of the subfamily Characinae through phylogenomic analyzes using ultraconserved elements (UCEs) and to generate a barcode DNA genetic database for molecular identification of Characinae. The results of the phylogenetic analyses corroborate the morphological hypothesis about the monophyly of Characinae and reveal new hypotheses of intergeneric and interspecific relationships. Based on the phylogeny obtained, we analyzed the origin, diversification, and the evolution of body size and body shape. The results of molecular identification recognized a previously underestimated diversity that should contribute to the expansion of knowledge about the diversity of Characinae. Keywords: DNA Barcode, Biodiversity, Phylogenomics, Systematics, Taxonomy, Fish. SUMÁRIO INTRODUÇÃO GERAL ............................................................................................................. 14 JUSTIFICATIVA ......................................................................................................................... 21 OBJETIVOS ................................................................................................................................. 22 CAPÍTULO 1. Phylogenomic analysis of the Neotropical fish subfamily Characinae using ultraconserved elements (Teleostei: Characidae) ...................................................................... 29 Introduction ..................................................................................................................................... 31 Materials and Methods ................................................................................................................... 36 Results and Discussion ................................................................................................................... 42 Bibliography ................................................................................................................................... 54 Supplementary information ............................................................................................................ 63 CAPÍTULO 2. Evolution and ecomorphology of the subfamily Characinae (Actinopterygii: Characiformes: Characidae) ....................................................................................................... 83 Introduction ..................................................................................................................................... 85 Materials and Methods ................................................................................................................... 87 Results ............................................................................................................................................ 98 Discussion ..................................................................................................................................... 105 Bibliography ................................................................................................................................. 108 Supplementary information .......................................................................................................... 115 CAPÍTULO 3. Molecular identification and description of a new species of Phenacogaster (Characidae: Characinae) ........................................................................................................... 119 Introduction ................................................................................................................................... 120 Materials and Methods ................................................................................................................. 122 Results .......................................................................................................................................... 124 Discussion ..................................................................................................................................... 134 Bibliography ................................................................................................................................. 137 Supplementary information .......................................................................................................... 140 CAPÍTULO 4. Molecular species delimitation of the Cynopotamini genera Acestrocephalus, Cynopotamus, and Galeocharax (Teleostei: Characidae: Characinae) ................................... 145 Introduction ................................................................................................................................... 146 Materials and Methods ................................................................................................................. 149 Results .......................................................................................................................................... 151 Discussion ..................................................................................................................................... 155 Bibliography ................................................................................................................................. 158 Supplementary information .......................................................................................................... 163 CAPÍTULO 5. Molecular identification of species of the tribe Characini (Teleostei: Characidae: Characinae) ............................................................................................................ 168 Introduction ................................................................................................................................... 170 Materials and Methods ................................................................................................................. 172 Results .......................................................................................................................................... 174 Discussion ..................................................................................................................................... 179 Bibliography ................................................................................................................................. 182 Supplementary information .......................................................................................................... 188 14 INTRODUÇÃO GERAL Subfamília Characinae Characinae Eigenmann, 1910 representa a terceira maior subfamília de Characidae (Teleostei: Characiformes) (Fricke et al., 2022) e sensu Mattox & Toledo-Piza, 2012 compreende 85 espécies (Fricke et al., 2022) agrupadas em sete gêneros: Acanthocharax Eigenmann, 1912, Acestrocephalus Eigenmann 1910, Charax Scopoli 1777, Cynopotamus Valenciennes 1850, Phenacogaster Eigenmann 1907, Galeocharax Fowler 1910 e Roeboides Günther, 1864 (Tabela 1). As espécies de Characinae são conhecidas popularmente como peixes-cachorra, peixes-cigarra, dentudos e tetra- vidros (glass tetras), dentre outros nomes (Géry 1977; Mattox & Toledo-Piza 2012) e estão amplamente distribuídas pela região Neotropical desde o sul do México até afluentes da bacia dos rios Paraguai e Uruguai na Argentina, com ocorrência principalmente em rios de fluxo lento associados com a vegetação marginal (Lucena & Menezes 2003; Mattox et al., 2017). A maioria das espécies são facilmente reconhecidas devido ao formato alto do corpo, especialmente na região anterior onde uma gibosidade é característica, embora esta esteja ausente em Phenacogaster e Acestrocephalus (Lucena & Menezes 2003). São espécies de pequeno a médio porte que apresentam diferentes hábitos alimentares, como a carnivoria adquirida pela maioria dos gêneros, a onivoria presente nas espécies do gênero Phenacogaster e a lepidofagia nas espécies de Roeboides, que apresentam modificações especializadas como dentes mamiliformes externos para esse tipo de habito alimentar (Géry 1977; Sazima & Machado 1982; Sazima 1984; Lucena & Menezes 2003). As espécies de Characinae não exercem grande importância para a economia pesqueira, mas apresentam importância para a pesca de subsistência de populações ribeirinhas e é de interesse da aquariofilia, sendo reconhecidas como espécies com potencial ornamental (Venere & Garutti 2001; Hercos et al., 2009). 15 Na sistemática Characinae tem uma notória importância por conter Charax, o gênero-tipo da família e da ordem. Apesar dessa notoriedade a história taxômica de Characinae foi baseada por muito tempo a agrupamentos que incluíram representantes da família Characidae (Eigenmann 1910, 1912; Myers 1960; Géry 1966, 1977; Weitzman & Vari 1987; Lucena 1998, Lucena & Menezes 2003). A última definição de agrupamento foi proposto por Lucena & Menezes, 2003 que incluiu 12 gêneros Acanthocharax; Acestrocephalus; Charax; Cynopotamus; Galeocharax; Gnathocharax Fowler 1913; Heterocharax Eigenmann 1912; Hoplocharax Géry 1966; Lonchogenys Myers 1927; Phenacogaster; Priocharax Weitzman & Vari 1987; Roeboides) baseados no formato do corpo, presença de mais de 20 dentes cônicos na maxila, pseudotímpano na frente da primeira costela pleural e nadadeira peitoral larval em espécimes de até 41,0 mm de comprimento. Mirande (2009, 2010) baseado em dados filogenéticos morfológicos e da literatura recuperou a monofilia de Characinae incluindo oito dos 12 gêneros (sensu Lucena & Menezes, 2003): Acanthocharax, Acestrocephalus, Charax, Cynopotamus, Galeocharax, Phenacogaster, Priocharax e Roeboides. Adicionalmente, propôs três gêneros, Bryconexodon Géry, 1980, Exodon Müller & Troschel 1844 e Roeboexodon Géry 1959 como pertencente a Characinae formando um clado-irmão de todos os gêneros remanescentes da subfamília. No entanto, mais recentemente com um conjunto mais amplo de táxons e caracteres, reinterpretou esses resultados e atribuiu Bryconexodon, Exodon e Roeboexodon para sua própria subfamília, Exodontinae (Mirande 2019: 296). Mattox & Toledo-Piza (2012) realizaram o primeiro estudo filogenético de Characinae baseado em caracteres morfológicos. Seus resultados indicaram Characinae irmã de Tetragonopterus Cuvier, 1816 e internamente restringida ao clado (Phenacogaster ((Charax+Roeboides) (Acanthocharax (Cynopotamus (Acestrocephalus+Galeocharax))))). Priocharax é recuperado numa politomia composta por Lonchogenys, Heterocharax e Hoplocharax dentro de Heterocharacinae. Os autores ainda propõem/redefinem as tribos Phenacogasterini (Phenacogaster), Characini (Charax e 16 Roeboides) e Cynopotamini (Acanthocharax, Acestrocephalus, Cynopotamus e Galeocharax). Os demais gêneros propostos por Lucena & Menezes, 2003 foram incluídos em Heterocharacinae e constituíram a tribo Heterocharacini (Gnatocharax (Lonchogenys (Heterocharax+Hoplocharax))) irmã de Roestini (Roestes Günther 1864 + Gilbertolus Eigenmann 1907) e distante dos Characinae (sensu Lucena & Menezes 2003). Outros estudos de Characiformes também incluíram representantes de Characinae usando dados moleculares (Javonillo et al., 2010; Oliveira et al., 2011; Tagliacollo et al., 2012; Melo et al., 2016; Betancur‐ R et al., 2019) e análise de evidência total (Mirande 2019). Estes estudos concordam sobre a relação entre Characinae e Tetragonopterinae (Javonillo et al., 2010; Oliveira et al., 2011; Tagliacollo et al., 2012; Melo et al., 2016; Mirande, 2019; Betancur‐ R et al., 2019); no entanto, os estudos moleculares não corroboram a monofilia de Cynopotamus (Oliveira et al., 2011; Tagliacollo et al., 2012; Melo et al., 2016) e de Roeboides (Betancur‐R et al., 2019). Adicionalmente, Acanthocharax é hipotetizado como o grupo irmão de (Charax+Roeboides) ou (Cynopotamus (Acestrochephalus+Galeocharax)) em estudos morfológicos (Lucena 1998; Mattox & Toledo-Piza 2012), mas ainda não foram incluídos em estudos moleculares. Relações interespecíficas em Characinae Hipóteses de relações interespecíficas em Characinae ainda são escassas e restritas às análises morfológicas, revisões taxonômicas ou descrições de espécies. Phenacogaster, um dos gêneros mais diversos, ainda necessita de descrições de muitas espécies e da definição de subgrupos (Lucena & Malabarba 2010). Das 23 espécies válidas, quatro (P. beni Eigenmann 1911, P. microstictus Eigenmann 1909, P. pectinata (Cope 1870) e P. suborbitalis Ahl 1936) apresentam ampla distribuição e compõem o complexo P. pectinata (Lucena & Malabarba 2010). Além delas, várias espécies não descritas compõem este grupo (Lucena & Malabarba 2010) e a dificuldade em 17 diagnosticar essas espécies tem sido explicada pela ampla distribuição geográfica e ausência de diferenças morfológicas consistentes (Géry 1972; Lucena 2003; Lucena & Malabarba 2010). Lucena (1998) propõe quatro subunidades de Roeboides: grupo dispar (R. dispar Lucena 2001), grupo microlepis (R. araguaito Lucena, 2003, R. margareteae Lucena 2003, R. microlepis (Reinhardt 1851) e R. myersii Gill 1870), grupo affinis [R. affinis (Günther, 1868), R. biserialis (Garman, 1890), R. descalvadensis Fowler 1932, R. numerosus Lucena 2000, R. oligistos Lucena 2000, R. paranensis Pignalberi 1975 (= R. descalvadensis), R. prognathus (Boulenger 1895) (=R. affinis), R. thurni Eigenmann 1912 (=R. affinis), R. xenodon (Reinhardt 1851) e R. sazimai Lucena 2007)]) e grupo guatemalensis (R. bouchellei Fowler 1923, R. carti Lucena 2000, R. dayi (Steindachner 1878), R. dientonito Schultz 1944, R. guatemalensis (Günther 1864), R. ilsea Bussing 1986, R. occidentalis Meek & Hildebrand 1916 e R. loftini Lucena 2011). O grupo microlepis é hipotetizado como mais relacionado às espécies transandinas do grupo guatemalensis (Lucena 2000). Alternativamente, Mattox & Toledo-Piza (2012), mostraram a relação (R. dientonito ((R. affinis + R. descalvadensis) (R. myersii (R. occidentalis + R. xenodon))))). Recentemente publicada, a revisão de Charax inclui a redescrição de todas as espécies além da descrição de C. delimai Menezes & Lucena 2014 e a sinonimização de C. unimaculatus Lucena 1989 em C. michaeli Lucena 1989 (Menezes & Lucena 2014). A única hipótese de relacionamento é apresentada baseada em caracteres morfológicos e inclui quatro das 17 espécies (Mattox & Toledo-Piza 2012). Esta filogenia recupera a relação ((C. condei + C. stenopterus) (C. gibbosus + C. pauciradiatus)). Cynopotamus contém 12 espécies (Fricke et al., 2022) e apenas a relação (C. xinguano Menezes 2007 (C. gouldingi Menezes 1987 (C. kincaidi (Schultz 1950) (C. juruenae Menezes 1987 + C. tocantinenses Menezes 1987))) foi hipotetizada através de caracteres morfológicos (Mattox & Toledo-Piza 2012). Dados moleculares não recuperaram a monofilia de Cynopotamus, sendo C. 18 kincaidi irmão de Roeboides guatemalensis e C. venezulae (Schultz 1944) irmão de Acestrocephalus+Galeocharax (Oliveira et al., 2011). Acestrocephalus teve cinco espécies descritas recentemente num total de oito (Menezes 2006) e a única hipótese existente consiste na relação (A. acutus Menezes 2006 (A. sardina (Fowler 1913) (A. pallidus Menezes 2006 + A. stigmatus Menezes 2006))) (Mattox & Toledo-Piza 2012). Galeocharax foi recentemente revisado (Giovannetti et al., 2017) e apenas três espécies foram consideradas como válidas (G. gulo (Cope 1870), G. goeldii (Fowler 1913), G. humeralis (Valenciennes 1834), e com G. knerii (Steindachner 1879) considerada sinônima de G. gulo. A filogenia morfológica de Characinae contendo todas as espécies de Galeocharax não resolve as relações, apresentando uma politomia (Mattox & Toledo-Piza 2012). Nenhuma filogenia molecular entre espécies desses gêneros, nem estudos aplicando técnicas de filogenômica foram realizadas em Characinae até presente momento. DNA Barcode Estudos baseados em identificação molecular usando a metodologia DNA barcode (Hebert et al., 2003) tem revelado números subestimados de espécies antes não reconhecidas e vem se mostrando resolutivos para questões taxonômicas em diferentes grupos de peixes neotropicais, tais como Characidae (Pereira et al., 2011; Silva et al. ,2013, Barreto et al., 2018), Lebiasinidae (Benzaquem et al., 2015), Loricariidae (Costa-Silva et al., 2015; de Borba et al., 2019; Fagundes et al., 2020), Serrasalmidae (Machado et al., 2018; Mateussi et al., 2020; Ota et al., 2020) e Curimatidae (Melo et al., 2016b). O uso dessa técnica pode então ser promissor para Characinae podendo possivelmente revelar uma diversidade ainda não reconhecida, uma vez que não há estudos de identificação molecular para Characinae e os estudos recentes têm identificado novas espécies (Menezes & Lucena 2014; Lucena, Antonetti & Lucena 2018; Guimarães, Brito, Ferreira & Ottoni 2018; Lucena 19 & Lucena 2019) principalmente em Roeboides e Phenacogaster que existem espécies de ampla distribuição e em Phenacogaster que apresenta o complexo P. pectinata e outras espécies ainda não descritas (Lucena & Malabarba 2010). Com isso, o DNA barcoding foi utilizado no presente estudo de Charcinae a fim de elucidar as unidades taxonômicas que os compõem e no direcionamento dos estudos filogenômicos. Elementos ultraconservados (UCEs) Os elementos ultraconservados (ultraconserved elements - UCEs) são regiões do genoma extremamente conservadas e assim compartilhadas entre grupos pertencentes a linhagens muito distintas, como, por exemplo, aves e humanos (Bejerano et al., 2004). Eles foram descritos por Bejerano et al. (2004), que encontraram 481 segmentos maiores de 200 pares de bases que eram absolutamente (100%) conservados em regiões ortólogas de humanos, ratos e camundongos e altamente conservados nos genomas de galinhas e cães (95–99%, respectivamente). Estudos posteriores mostraram que os UCEs também estão presentes em diversos outros organismos, como outros vertebrados, insetos e fungos (Siepel et al., 2005; Faircloth et al., 2012). O papel dos UCEs no genoma ainda não está esclarecido (Dermitzakis et al., 2005), embora os UCEs tenham sido associados com regulação gênica ou desenvolvimento (Sandelin et al., 2004; Woolfe et al., 2004) e se tem assumido que os UCEs são importantes pela sua natureza extremamente conservada entre grupos muito distantes filogeneticamente. Os UCEs são identificados nos organismos pelo alinhamento de vários genomas e por regiões desse alinhamento com áreas com conservação de sequências muito altas (95–100%) e filtradas utilizando critérios específicos como o comprimento das sequências (e.g. Bejerano et al., 2004). Suas sequências conservadas permitem uma fácil identificação e alinhamento entre genomas e a premissa de contínua variabilidade nas sequências que flanqueiam cada UCE sugere que eles podem ser um tipo 20 de “fóssil molecular”, retendo um sinal de história evolutiva em diversas escalas de tempo, dependendo da distância da região central dos UCEs (Faircloth et al., 2012). Faircloth et al. (2012) introduziram os UCEs como uma nova classe de marcadores moleculares em estudos filogenéticos através do enriquecimento de bibliotecas genômicas contendo centenas ou milhares de loci, utilizando sequenciamento de nova geração (Faircloth et al., 2012). Como as sequências de UCEs são altamente conservadas elas são utilizadas para o anelamento de sondas (probes), a partir das quais as sequências flanqueadoras dos UCEs são lidas. A presença de regiões com diferentes níveis de variabilidade tem tornado esta técnica muito promissora no campo da sistemática filogenética (Pennisi 2013). Além disso, os UCEs têm sido utilizados em muitos níveis de comparação entre organismos, de populações até grandes grupos (McCormack et al., 2012, 2013; Crawford et al., 2012; Smith et al., 2014; Starrett et al., 2016). Em peixes o primeiro estudo foi realizado por Faircloth et al. (2013). Nesse estudo foram sequenciados 500 loci de cerca de 30 espécies de Actinopterygii e as filogenias obtidas mostram nós altamente resolvidos em todos níveis (recentes e antigos). Os resultados suportaram as relações entre Amia e Lepisosteus (Holostei) e revelaram que os Elopomorpha e depois os Osteoglossomorpha são as primeiras linhagens a divergir entre as linhagens dos teleósteos. Em sequência, outros trabalhos foram realizados mostrando os UCEs como promissor e excelente marcador molecular para estudos filogenéticos: Amarsipidae (Harrington et al., 2016), Characiformes (Chakrabarty et al., 2017); Acanthomorpha (Alfaro et al., 2018), Loricariidae (Roxo et al., 2019), Trichomycteridae (Ochoa et al., 2020), Serrasalmidae (Mateussi et al., 2020) e Heptapteridae (Silva et al., 2021). Em comparação com os estudos baseados em marcadores moleculares multi-locus o uso dos UCEs tem se mostrado bastante eficiente. A razão para este rápido crescimento está ligada a diferentes características de sua abordagem, como a obtenção de dados de eventos de divergência 21 recente e antiga (Crawford et al., 2012; Harvey et al., 2016; Manthey et al., 2016) e alto custo- benefício em função do tempo e baixo custo dada a grande quantidade de dados gerados. JUSTIFICATIVA Várias questões no âmbito da sistemática de Characinae ainda permanecem não resolvidas como levantadas acima e aqui sumarizadas: 1) a posição de Acanthocharax ainda é incerta, devido à falta de amostragem para evidência molecular; 2) a posição de Priocharax ainda não foi testada utilizando dados moleculares; 3) a monofilia de Cynopotamus foi rejeitada pelas hipóteses moleculares de Oliveira et al. (2011), Tagliacollo et al. (2012) e Melo et al. (2016) e de Roeboides em Betancur‐R et al. (2019); 4) as relações interespecíficas em cada um dos gêneros necessitam de uma maior amostragem de táxons terminais; 5) a diversidade molecular de espécies não é conhecida. A Tabela 1 mostra o número de táxons analisados pelos principais estudos morfológicos e moleculares de Characidae (Mirande 2018), morfológico da subfamília Characinae (Mattox & Toledo-Piza 2012) e molecular de Characidae (Oliveira et al., 2011). Pode-se notar uma necessidade de maior amostragem de espécies de Characinae para geração de hipóteses filogenéticas mais robustas. Além disso, vários problemas taxonômicos ainda existentes em vários gêneros de Characinae podem ser esclarecidos com a identificação molecular de espécies. Finalmente, os recentes avanços na sistemática molecular com a utilização de supermatrizes em escala genômica tem proporcionado uma ótima oportunidade para obter filogenias mais robustas, e sua consequente utilização em estudos macroevolutivos. 22 Tabela 1. Número de espécies analisadas por gênero de Characinae em estudos anteriores e em nosso estudo. Gênero Espécies válidas Oliveira et al., 2011 Mattox & Toledo-Piza, 2012 Mirande, 2018 Este estudo Acanthocharax 1 0 1 0 1 Acestrocephalus 8 1 4 1 3 Charax 17 1 4 2 10 Cynopotamus 12 2 5 1 9 Galeocharax 3 1 3 1 3 Phenacogaster 23 1 4 3 13 Roeboides 21 1 6 2 18 Total 85 7 27 10 57 OBJETIVOS Com base nas hipóteses prévias de relacionamentos interespecíficos e intergenéricos da subfamília Characinae, e desta com outras subfamílias de Characidae, o presente projeto teve como objetivos: 1) Elaborar e testar as hipóteses de relações filogenéticas entre os gêneros da subfamília Characinae e desta com os demais membros de Characidae; 2) Elaborar e testar as hipóteses de relações filogenéticas entre as espécies de cada um dos gêneros de Characinae; 3) Realizar a reconstrução ancestral da dieta de Characinae e testar se as mudanças na dieta influenciaram a diversificação morfológica do tamanho e forma do corpo; 4) Gerar sequências utilizando a técnica de DNA barcode para permitir uma identificação molecular dos peixes da subfamília Characinae; 5) Identificar linhagens/ espécies descritas ou não descritas. 23 BIBLIOGRAFIA Alfaro, M. 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PLoS Biology 3, e7 29 Chapter 1 Phylogenomic analysis of the Neotropical fish subfamily Characinae using ultraconserved elements (Teleostei: Characidae) 30 Phylogenomic analysis of the Neotropical fish subfamily Characinae using ultraconserved elements (Teleostei: Characidae) Camila S. Souza Departamento de Biologia Estrutural e Funcional, Instituto de Biociências, Universidade Estadual Paulista, R. Prof. Dr. Antônio C. W. Zanin 250, 18618-689, Botucatu, SP, Brazil Abstract Characinae is one of the most species-rich subfamilies of Characidae and holds special taxonomic importance because it includes Charax, type-genus of Characidae and Characiformes. Currently, the monophyly and the hypotheses of intergeneric and interspecific relationships of Characinae are based on a few morphological and molecular studies but all with low species coverage. Given their diversity, taxonomic importance, and the lack of a taxon-dense phylogeny, we sought to buttress the systematic understanding of Characinae collecting DNA sequence data from ultraconserved elements (UCEs) of the genome from 98 specimens covering 57 species (67%) plus 17 characiforms as outgroups. We used maximum likelihood, Bayesian inference, and coalescent-based species tree approaches and the resulting phylogeny with 1,300 UCE loci (586,785 characters) reinforced the monophyly of the subfamily as well as of six genera: Acestrocephalus, Charax, Cynopotamus, Galeocharax, Phenacogaster, and Roeboides. The phylogeny reveals a novel hypothesis of intergeneric and interspecific relationships for the subfamily with Phenacogaster sister to all genera, and Acanthocharax sister to Cynopotamini (Cynopotamus (Acestrocephalus Galeocharax)) and Characini (Charax Roeboides). We propose a new tribe Acanthocharacini to allocate Acanthocharax, two subclades for Phenacogaster, two for Cynopotamus, three for Charax, and 31 reinforced the four subclades for Roeboides previously identified by morphological studies. Additionally, we generated a time-calibrated phylogeny for Characinae that suggested that the subfamily originated during the Miocene at around 19.4 million years ago. The results obtained here will contribute to the development of further research on the evolutionary processes modulating species diversification in Characinae. Keywords: biodiversity, Characiformes, freshwater fish, Neotropics, Ostariophysi, systematics. 1. Introduction The Neotropical ichthyofauna is mainly composed by non-cypriniform otophysan fishes (i.e., Characiformes, Siluriformes, and Gymnotiformes), which constitutes about 77% of the total species richness (Albert et al., 2011a, b). Among Neotropical otophysan groups, Characiformes represents one of the most ecomorphologically diverse orders of fishes with approximately 2,200 valid species (Fricke et al., 2022). Within the order, the family Characidae, as proposed by Oliveira et al. (2011), is the most species-rich with a total of 1,228 species (Fricke et al., 2022). Due to its great diversity, Characidae includes some of the greatest taxonomic and systematic challenges among Neotropical fishes (Oliveira et al., 2011; Mirande, 2019), with the general morphology and anatomy of the species being highly conservative, and most members attaining maximum body sizes of 10 cm standard length (SL) or less (Froese & Pauly, 2021). Characinae is the third most species-rich subfamily of Characidae and includes Charax Scopoli, 1777, type genus of Characiformes. The subfamily currently comprises 85 species (sensu Mattox & Toledo-Piza, 2012) distributed in rivers of the Neotropical region, including both sides of the Andes. In the east of the Andes, they occur from the northern coastal drainages of Venezuela to tributaries of the lower La Plata basin (Lucena & Menezes, 2003). Most members of the group are easily 32 recognized by their deep anterior bodies with a characteristic gibbosity (Figure 1), although this is absent in Phenacogaster Eigenmann, 1907 and Acestrocephalus Eigenmann, 1910 (Lucena & Menezes, 2003; Mattox & Toledo-Piza, 2012). They have adopted three distinct feeding strategies: carnivory, omnivory, and lepidophagy, although most species are carnivorous and feed mainly on insects and other fishes (Géry, 1977; Lucena & Menezes, 2003). Roeboides represents the only genus of Characinae with lepidophagous habits and possesses morphological specializations that aid in this unusual feeding style, such as external mammiliform teeth used to pluck scales from their prey (Sazima & Machado, 1982; Sazima, 1984). The taxonomic history of Characinae has changed significantly during the two last decades, with its present composition defined by Lucena (1998), Lucena & Menezes (2003), Mirande (2009, 2010) and Mattox & Toledo-Piza (2012). The definition of Characinae proposed by Lucena & Menezes (2003) included 12 genera (Acanthocharax Eigenmann, 1912; Acestrocephalus; Charax; Cynopotamus Valenciennes, 1850; Galeocharax Fowler, 1910; Gnathocharax Fowler, 1913; Heterocharax Eigenmann, 1912; Hoplocharax Géry, 1966; Lonchogenys Myers, 1927; Phenacogaster; Priocharax Weitzman & Vari, 1987; Roeboides) based on the relatively deep body shape, presence of more than 20 conical teeth along the maxilla, presence of a conspicuous pseudotympanum anterior to the first pleural rib, and the retention of larval pectoral fin in specimens up to 41 mm SL. Conversely, the hypothesis proposed by Mirande (2009, 2010) based on morphological features included eight of the 12 genera (sensu Lucena & Menezes, 2003): Acanthocharax, Acestrocephalus, Charax, Cynopotamus, Galeocharax, Phenacogaster, Priocharax, and Roeboides, with the tentative inclusion of Priocharax based on Lucena & Menezes (2003) (Mirande, 2010: 492). He also proposed three additional genera, Bryconexodon Géry, 1980, Exodon Müller & Troschel, 1844, and Roeboexodon Géry, 1959 as belonging to Characinae forming a clade sister to all remaining genera of the subfamily. This three-genera group is composed of strictly lepidophagous characids and some of them had been previously proposed as closely related 33 to Roeboides in a pre-cladistic context (e.g., Géry, 1964; Roberts, 1970). Later, with a broader set of taxa and characters, Mirande (2019:296) reinterpreted these results and assigned Bryconexodon, Exodon and Roeboexodon to their own subfamily, the Exodontinae. Figure 1. Representative specimens of Characinae. (A) Phenacogaster sp.; (B) Phenacogaster eurytaenia; (C) Acanthocharax microlepis; (D) Cynopotamus atratoensis; (E) Roeboides descalvadensis; (F) Galeocharax humeralis; (G) Charax condei; (H) Acestrocephalus sardina. Photographs by Martin Taylor (A, B), Johnny Jensen (C), Frank Schäefer (D, E), Frank Teigler (G), Pablo Giorgis (F), and Ralf Britz (H). 34 In parallel to these studies, Mattox & Toledo-Piza (2012) carried out a morphological study focusing on the subfamily Characinae to test its monophyly and the subunits proposed by Lucena (1998) and Lucena & Menezes (2003). The morphology-based results (Figure 2B) supported the monophyly of the seven genera in Characinae based on ten non-ambiguous synapomorphies. The authors arranged the genera in three tribes: Phenacogasterini as sister to Characini and Cynopotamini, with the intergeneric relationships (Phenacogaster ((Charax Roeboides) (Acanthocharax (Cynopotamus (Acestrocephalus Galeocharax))))). Acanthocharax was previously considered the sister-group to Charax and Roeboides (Lucena, 1998) but the former genus was found as sister to all other Cynopotamini more recently (Mattox & Toledo-Piza, 2012). Among the outgroups, Tetragonopterus has been consistently found as the sister group to Characinae based on both morphological and molecular data (Buckup, 1998; Javonillo et al., 2010; Oliveira et al., 2011; Mattox & Toledo-Piza, 2012; Melo et al., 2016; Betancur-R et al., 2019). Weitzman & Vari (1987) described the enigmatic genus Priocharax and suggested that its morphology was similar to members of Characinae and Cynopotaminae. Lucena (1998) was the first to include Priocharax in a phylogenetic context, which resulted as the most basal lineage in the Characinae, leading to the subsequent classification of this genus in Characinae (Lucena & Menezes, 2003; Mirande, 2010). Mattox & Toledo-Piza (2012) were the first to propose that Priocharax was more related to the Heterocharacinae, a subset of former members of the Characinae given their own subfamily rank (Mirande, 2010; Mattox & Toledo-Piza, 2012). Oliveira et al. (2011) did not analyze Priocharax but showed that Heterocharacinae is nested within Acestrorhynchidae rather than to Characinae and therefore transferred Heterocharacinae into Acestrorhynchidae. Mirande (2019), based on literature information, suggested the placement of Priocharax in the subfamily Characinae. Betancur et al. (2019) found Priocharax as sister to Roeboides but, after tissue extraction and publication, an additional specimen of the same lot was examined by us and the identification as Priocharax was not confirmed. Additionally, the voucher specimen used by 35 Betancur et al. (2019) is no longer available for reidentification (LBP 17836; C. Souza, personal observation). Thus, the position of Priocharax in the phylogeny of Characidae remains unclear. Other characiform studies also included representatives of Characinae using both molecular (Javonillo et al., 2010; Oliveira et al., 2011; Tagliacollo et al., 2012; Melo et al., 2016; Betancur-R et al., 2019; Melo et al., 2021), and total evidence analysis (Mirande, 2019). These studies agree about the relationships between Characinae and Tetragonopterinae (i.e., Tetragonopterus) (Javonillo et al., 2010; Oliveira et al., 2011; Tagliacollo et al., 2012; Melo et al., 2016; Mirande, 2019; Betancur-R. et al., 2019); however, the molecular studies did not corroborate the monophyly of Cynopotamus (Oliveira et al., 2011; Tagliacollo et al., 2012; Melo et al., 2016). In addition, Acanthocharax, hypothesized as the sister group of either clades (Charax Roeboides) or (Cynopotamus (Acestrocephalus Galeocharax)) in morphological reconstructions (Lucena, 1998; Mattox & Toledo-Piza, 2012), had not been included in any molecular analysis. The hypotheses of interspecific relationships among genera of the Characinae are also limited to morphological analyses and/or arrangements proposals based on taxonomic revisions and species descriptions (Lucena, 1998; Lucena, 2003, 2007; Menezes, 2006; Lucena & Malabarba, 2010; Menezes & Lucena, 2014; Giovannetti et al., 2017). These relationships remain unclear also due to the low number of taxa used in phylogenetic analyses (Oliveira et al., 2011; Mattox & Toledo-Piza, 2012; Mirande, 2019) (Figure 2). So far, the morphological phylogeny that covered interspecific relationships with the highest number of species of Characinae (i.e., 30%) is that of Mattox & Toledo-Piza (2012), although their study focused on testing the monophyly of the subfamily and the intergeneric relationships. In the field of molecular phylogenetics, ultraconserved elements (UCEs; Faircloth et al., 2012) have been used to explore the relationships within various animal groups, including fishes (Harrington et al., 2016; Chakrabarty et al., 2017; Alfaro et al., 2018; Roxo et al., 2019; Ochoa et al., 2020; Mateussi et al., 2020; Melo et al., 2021). They constitute excellent markers 36 Figure 2. Intergeneric hypotheses of Characinae based on morphological (A- Lucena, 1998; B- Mattox & Toledo- Piza, 2012), total-evidence analysis (C- Mirande, 2019), and phylogenomic study (D- This study). Figures modified from original publications. for phylogenetic studies due to their presence among a wide range of taxonomic groups, low degrees of ambiguity, and low saturation (Siepel et al., 2005; Derti et al., 2006; McCormack et al., 2012; Alda et al., 2021), despite well-known problems with incomplete lineage sorting (Alda et al., 2019; Alda et al., 2021). Here, we performed a phylogenomic study of Characinae using 57 out of 85 species (67%), including representatives of the seven genera currently assigned to the subfamily based on an UCE dataset to (i) test the monophyly of the genera, (ii) test previous phylogenetic hypotheses of intergeneric and interspecific relationships, and (iii) to better understand the evolution and biogeography of this subfamily. 2. Materials and Methods 2.1. Taxon sampling Our study included 98 specimens from 57 species of Characinae (67%) with representatives of all seven genera. Numbers in parentheses represent sample size relative to the number of valid species: 37 Acanthocharax (1/1), Acestrocephalus (3/8), Charax (10/17), Cynopotamus (9/12), Galeocharax (3/3), Phenacogaster (13/23), and Roeboides (18/21). The outgroup included 16 species: one species of Acestrorhynchidae (Acestrorhynchus microlepis (Jardine, 1841)), one Cheirodontinae (Protocheirodon pi (Vari, 1978)), one Aphyocharacinae (Aphyocharax pusillus Günther, 1868), three Exodontinae (Bryconexodon juruenae Géry, 1980, Exodon paradoxus Müller & Troschel, 1844 and Roeboexodon guyanensis (Puyo, 1948)), two Spintherobolinae (Amazonspinther dalmata Bührnheim, Carvalho, Malabarba & Weitzman, 2008 and Spintherobolus papilliferus Eigenmann, 1911), two Stevardiinae (Markiana nigripinnis (Perugia, 1891) and Planaltina myersi (Böhlke, 1954), three Stethaprioninae (Astyanax jordani (Hubbs & Innes, 1936), Paracheirodon axelrodi (Schultz, 1956) and Hyphessobrycon compressus (Meek, 1904)), and three Tetragonopterinae (Tetragonopterus georgiae (Géry, 1965), T. argenteus Cuvier 1816, and T. chalceus Spix & Agassiz, 1829). Acestrorhynchus microlepis (Acestrorhynchidae) was used to root the trees. Separately, we used a 95% complete edge-trimmed matrix to include three species of Priocharax and only one specimen by species (85 taxa) to estimate a maximum likelihood (ML) tree and to time-calibrate the phylogeny to test the position and relationships of Priocharax. Voucher samples used in this study are deposited in the Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista, Botucatu, Brazil (LBP), Coleção dos Recursos Genéticos, Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil (INPA), Royal Ontario Museum, Toronto, Canada (ROM), Laboratório de Ictiologia, Museu de Ciências e Tecnologia, Porto Alegre, Brazil (MCP), Laboratório de Ictiologia e Pesca, Universidade Federal de Rondônia, Porto Velho, Brazil (UNIR), Laboratório de Genética e Biologia Molecular, Universidade Federal do Maranhão, São Luís, Brazil (LABGEN/UFMA), Coleção Ictiológica, Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil (MZUSP), and Smithsonian Tropical Research Institute, Panama City, Panama (STRI). The samples collected in this study are in agreement with Brazilian laws through SISBIO/MMA permit n. 3245 and procedures for collection, maintenance and analyses followed 38 the international guidelines for animal experiments through CEEAA IBB/UNESP protocol n. 304. The Supplementary Table 1 includes data from all ingroup and outgroup samples. 2.2. DNA extraction and sequencing DNA was extracted from muscle, gills, or fin tissues with a DNeasy Tissue kit (Qiagen Inc.) following manufacturer’s instructions. Then, 2 µl of each genomic DNA was quantified using fluorometry (Qubit, Life Technologies) to prepare the libraries using a concentration between 10– 40 ng/µl. DNA libraries and sequencing were performed at Arbor Biosciences (AB; arborbiosci.com; Ann Arbor, MI, USA). Whole genomic DNA was first sheared with a QSonica Q800R instrument and selected to modal lengths of approximately 500 nt using a dual-step SPRI bead cleanup. DNA libraries were prepared for the 117 specimens (98 ingroup taxa and 19 outgroup taxa) by modifying the Nextera (Epicentre Biotechnologies) library preparation protocol for solution-based target enrichment following Faircloth et al. (2012) and the number of PCR cycles following the recommendation of Faircloth et al. (2013). AB staff used the Nextera library preparation protocol of in vitro transposition followed by PCR to prune the DNA and attach sequencing adapters (Adey et al., 2010). The Epicentre Nextera kit was used subsequentially to prepare transposase-mediated libraries with insert sizes averaging 100 bp (95% CI: 45 bp) following Adey et al. (2010). The libraries were enriched using a new probeset developed for ostariophysan fishes that captures sequence data for 2,708 UCE loci (Faircloth et al., 2020). Then, the DNA was converted to Illumina sequencing libraries with a slightly modified version of the NEBNext(R) Ultra (TM) DNA Library Prep Kit for Illumina(R). After ligation of sequencing primers, libraries were amplified using KAPA HiFi HotStart ReadyMix (Kapa Biosystems) for six cycles using the manufacturer’s recommended thermal profile and dual P5 and P7 indexed primers (Kircher et al., 2012). After purification with 39 SPRI beads, libraries were quantified with the Quant-iT(TM) Picogreen(R) dsDNA Assay kit (ThermoFisher). AB staff then enriched pools comprising 100 ng each of eight libraries (800 ng total) using the MYbaits(R) Target Enrichment system (MYcroarray) following manual version 3.0. Sequencing was performed across two Illumina HiSeq paired-end 100 bp lanes using v4 chemistry. 2.3. Sequence data processing After sequencing, adapter contamination, low quality bases, and sequences containing ambiguous base calls were trimmed using the Illumiprocessor software pipeline (Faircloth, 2013; https://github.com/faircloth-lab/illumiprocessor). After trimming, the assembled Illumina were read into contigs on a species-by-species basis using Velvet (Zerbino & Birney, 2008) on VelvetOptimiser (https://github.com/VictorianBioinformatics-Consortium/VelvetOptimiser). Following assembly, a custom Python program (match_contigs_to_probes.py), available in PHYLUCE (Faircloth, 2016) was used, integrating LASTZ (Harris, 2007) to align species specific contigs to our probe-UCE set. During the alignment, the latter program creates a relational data base of matches to UCE loci by taxon. We then used the get_match_counts.py program (also in PHYLUCE) to query the database and generate FASTA files for UCE loci that were identified across all taxa. A custom Python program (seqcap_align_2.py) was then used to align contigs using the MUSCLE alignment algorithm (Edgar, 2004) and to perform edge trimming. 2.4. Phylogenetic analyses We performed phylogenetic analyses with 75% and 90% complementary matrices to test the effect of missing data: the 75% complete matrix contains loci present in at least 75% of taxa (i.e. 85 terminals or more), and the 90% complete matrix contains loci present in at least 90% of taxa (i.e. 40 103 terminals or more). We concatenated the matrices and performed analyses by ML in RAxML v8.019 (Stamatakis, 2014), and Bayesian inference (BI) in ExaBayes v1.4 (Aberer et al., 2014), and the coalescent-based species tree approach (AS) in ASTRAL-III (Zhang et al., 2018) using a 2×10 CPU, 256 GB Zungaro server at LBP-UNESP. For ML and BI analyses, the data was partitioned to account for variation in rates and patterns of molecular evolution among sites using PartitionUCE (Tagliacollo & Lanfear, 2018) and the substitution models estimated with hcluster in PartitionFinder v2 (Lanfear et al., 2014; 2016) through RAxML v8.0 (Stamatakis, 2006). The ML analysis was performed using GTRCAT (Stamatakis, 2006) with 20 alternative runs of distinct parsimony starting trees in RAxML v8.019 (Stamatakis, 2014). The posteriori bootstrapping analysis was conducted using the autoMRE function in RAxML using the bootstopping criteria (Stamatakis, 2014; Pattengale et al., 2010). Bayesian inference was performed in ExaBayes v 1.597 (Aberer et al., 2014), with two independent runs, each with four chains (one cold and three heated) with 50,000,000 iterations and other parameters as default. Tree space was sampled every 100 generations to yield a total of 10,001 trees. We assessed convergence of the posterior distribution examining the ESS > 200 (effective sample size) and evaluating posterior trace distribution in Tracer v1.6.1 (Rambaut et al., 2014). We obtained the most likely set of trees from the posterior distribution of possible topologies using the consensus algorithm of ExaBayes with 10% burn-in. The coalescent-based species tree approach (AS) was inferred from individual gene trees using ASTRAL-III (Zhang et al., 2018). The individual gene trees used as input to ASTRAL- III were estimated by a ML analysis using a RAxML bootstrapped using the parameter –N = 2 and the GTRGAMMA model. Then, ASTRAL-III was used to infer species trees from the best gene trees, and to reconstruct the majority-rule consensus tree of the results. 41 2.5. Divergence time estimation We estimated a time-calibrated phylogeny using an uncorrelated relaxed molecular clock (lognormal) using the BEAST v2 (Bouckaert et al., 2014). We used the 95% complete edge-trimmed matrix (91 UCE loci with 38,888bp) to estimate both the topology and node ages. We included a constraint on the root and two fossils as calibration points. First, a calibration point was assigned to the root of the tree (i.e., all taxa) to calibrate the node splitting Acestrorhynchidae and members of Characidae that, based on the recent time reconstruction of the entire order Characiformes (Melo et al., 2021), was estimated to have occurred in the Late Cretaceous at around 72 (83–60) millions of years ago (Ma) (normal distribution; offset = 72 Ma; mean = 0; standard deviation = 7.0). The second calibration point was based on the fossil †Paleotetra entrecorregos, UNG 2T-149, an articulated skeleton from the Entre-Córregos Formation in Minas Gerais, Brazil, dated to the Eocene-Oligocene boundary (Weiss et al., 2012). The fossil was hypothesized to be a stem Stevardiinae (Weiss et al., 2012) or a stem Characidae (Mirande, 2019). Given the natural uncertainty of the fossil and pending additional analysis with less missing data showing higher support for early nodes of Characidae, and considering that the origin of Characidae was estimated at around 65 Ma (Melo et al., 2021), we assigned it to the node stem of Stevardiinae following the original description and phylogenetic placement (Weiss et al., 2012) (log-normal distribution; offset = 33.9 Ma; mean = 5.0 Ma; standard deviation = 1.0). The third calibration point was †Megacheirodon unicus, MCP 3086-PV, an articulated skeleton from Oligocene-Miocene deposits of the Tremembé Formation in São Paulo, Brazil (Bührnheim et al., 2008). †Megacheirodon was hypothesized to be the sister clade to Spintherobolus and Amazonspinther (Bührnheim et al. 2008), thus we implemented it as a calibration of the node with both genera and other members of Characidae (log-normal distribution; offset = 23.8 Ma; mean = 1.0 Ma; standard deviation = 1.25). We also included a constraint prior for the monophyly of Characidae. The BEAST analyses were 42 conducted under a birth-death model for prior distributions and ran for 50 million generations sampling frequency at every 10,000th generation. We checked stationarity and sufficient mixing of parameters (ESS > 200) using Tracer v1.6 (Rambaut et al., 2014). A consensus tree was built using TreeAnnotator v1.8.2. All clade-age estimates are presented as the mean plus 95% highest posterior density (HPD) values. 3. Results and Discussion 3.1. Overall patterns and monophyly of Characinae Sequencing and data filtering yielded an initial edge-trimmed aligned matrix comprising 2,527 UCE loci with a total of 990,049 base pairs (bp) for 114 specimens (98 Characinae and 16 outgroups) (Supplementary Table 2). We used two UCE matrices with 114 taxa to infer phylogenetic relationships: the 75% complete matrix was composed of 1,300 UCE loci and 586,785 bp, each of which contained sequence data for at least 85 taxa and the 90% complete matrix with 297 UCE loci and 137,232 bp each of which contained sequence data for at least 103 taxa. Phylogenies were resolved with high statistical support for most nodes regardless of matrix completeness (75% or 90%), or method of phylogenetic inference (ML, BI, or ASTRAL-III) (Figures 3–4; S1–S5). The results of the ML trees and BI analyses of the edge-trimmed, 75% complete, partitioned matrices show identical topologies (Figures 3–4; S3). Overall, all phylogenetic inferences show the same topology to the intergeneric relationship, however ASTRAL-III presented the highest number of differences in interspecific relationships compared to the ML and the BI analyses. Details of the differences among each analysis can be observed in the resulted topologies (Figures 3–4; S1–S5). Discrepancies in interspecific relationships presented by ASTRAL-III included Galeocharax gulo and species of Phenacogaster and Roeboides (Figures S4-S5) that show rapid and/or recent 43 diversification (Figure 5). Disagreements among trees could be caused by different factors such as incomplete lineage sorting (Pollard et al., 2006; Carstens & Knowles, 2007), mutation and recombination rate (Pollard et al., 2006, Rosser et al., 2017), selective pressures (Malinsky et al., 2015, Sun et al., 2015), hybridization and introgression (Toews & Brelsford, 2012, Denton et al., 2014). Nevertheless, a significant explanation for different relationships found in ASTRAL-III can be attributable to deep coalescence processes, where multiples lineages tend to persist into the deeper portion of the species tree, that is common in species with rapid and/or recent diversification (Degnan & Rosenberg, 2006, 2009). Despite the few discordances among topologies, we constructed our discussion based on the results of the ML tree of the edge-trimmed, 75% complete, partitioned matrix with 100% bootstrap for 76.6% of the nodes (Figures 3-4) and the topology that best concord with the morphology of the subfamily. The UCE phylogeny supports the monophyly of Characinae and of all non-monotypic characin genera: Acestrocephalus, Galeocharax, Cynopotamus, Charax, Phenacogaster, and Roeboides (Figures 3-4; ML = 100). As our taxon sampling contained only one specimen of Acanthocharax, we could not test the monophyly of the genus. The intergeneric relationships of Characinae and the interspecific relationships in Cynopotamus and Galeocharax are strongly supported in most of the nodes (Figures 3-4; S1-S5). Our reconstruction also shows the closer relationship between Characinae and Tetragonopterus, with Exodontinae as the immediate sister clade. This reconstruction matches recent molecular phylogenies of related taxa (e.g. Oliveira et al., 2011; Melo et al., 2016, 2021). Furthermore, our results found Priocharax inside Stethaprioninae and the relationship between Priocharax and Hyphessobrycon and other characids rather than with Characinae (Figures 5 and S6). The morphology-based phylogeny (Mattox & Toledo-Piza, 2012) supported the monophyly of Characinae based on ten non-ambiguous synapomorphies (details in Supplementary data), and the authors proposed three tribes in Characinae: Phenacogasterini (Phenacogaster), Characini (Charax 44 Roeboides) and Cynopotamini ((Acanthocharax (Cynopotamus (Acestrocephalus Galeocharax)))). Herein, our phylogeny shows a slightly distinct arrangement: Phenacogasterini (Phenacogaster), Acanthocharacini new tribe (Acanthocharax), sister to Characini (Charax Roeboides) and Cynopotamini (Cynopotamus (Acestrocephalus Galeocharax)) (Figures 3-4). Given the new phylogenetic arrangement, Acanthocharacini Souza, Melo, Mattox & Oliveira, 2021 (ZooBank Nomenclatural Act: urn:lsid:zoobank.org:act:E32EDF2D-978C-4DB9-AD2C-8EAB9E1E5FE1), is herein proposed as a new tribe of Characinae. 3.1.1. Acanthocharacini Souza, Melo, Mattox & Oliveira, 2022 urn:lsid:zoobank.org:act:E32EDF2D-978C-4DB9-AD2C-8EAB9E1E5FE1 Included genus: Acanthocharax Eigenmann, 1912 Diagnosis: As for the genus (e.g., Eigenmann, 1912). Acanthocharacini is distinguished from all other tribes of Characinae by the presence of a spiniform projection on the preopercle (Eigenmann, 1912; Mattox & Toledo-Piza, 2012). The absence of predorsal scales were used previously to characterize the genus being useful to diagnose the tribe, albeit predorsal scales were independently lost in Charax condei and C. stenopterus. 45 Figure 3. Phylogenetic relationships of Characinae based on a maximum likelihood analysis of the 75% complete matrix of ultraconserved elements (1,300 loci; 586,785 bp), highlighting the phylogenetic relationships in Phenacogasterini, Cynopotamini, and the position of Acanthocharacini. All nodes have bootstrap values ≥90% except where indicated. 46 Figure 4. Phylogenetic relationships of Characinae based on a maximum likelihood analysis of the 75% complete matrix of ultraconserved elements (1,300 loci; 586,785 bp), highlighting the phylogenetic relationships in Characini. All nodes have bootstrap values ≥90% except where indicated. 47 3.2. Monophyly of genera and intergeneric relationships Monophyly of Phenacogaster, the sole genus in the tribe Phenacogasterini, was supported herein, including 14 species (13 valid and one undescribed species) out of the 23 currently recognized in the genus (Fricke et al., 2022). Morphologically, Phenacogaster has been diagnosed mainly by the presence of two longitudinal series of large, narrow, and elongate preventral scales and by the gap in the external tooth series of the premaxilla (Malabarba & Lucena, 1995; Lucena & Malabarba, 2010). The former character was interpreted as one of the five synapomorphies of Phenacogaster (Mattox & Toledo-Piza, 2012). Monophyly of the clade comprising the other six genera (i.e., Acanthocharax, Acestrocephalus, Charax, Cynopotamus, Galeocharax and Roeboides) was supported herein with 100% support, as in previous hypotheses based on morphology (Figure 2; Lucena, 1998; Mattox & Toledo-Piza, 2012). This clade was strongly supported by the latter authors based on nine non-ambiguous synapomorphies, with one synapomorphy exclusive for this clade: lateral wings of mesethmoid poorly developed (Mattox & Toledo Piza, 2012). Our phylogenetic hypothesis shows Acanthocharax as sister to the clade ((Charax Roeboides) (Cynopotamus (Acestrocephalus Galeocharax)) (Figure 3; ML = 100), which is congruent with the hypothesis based mainly on comparative myology (Howes, 1976). This result contrasts, however, to Lucena (1998), who proposed a closer relationship between Acanthocharax and the clade formed by Charax and Roeboides based on two synapomorphies. The more recent morphological phylogeny of the subfamily suggested the inclusion of Acanthocharax in the Cynopotamini based on five synapomorphies (Mattox & Toledo-Piza, 2012), four of which highly homoplastic in their analyses. The fifth synapomorphy is the shape of the lower pharyngeal toothplate which is more elongated posteriorly in Acanthocharax, Cynopotamus, Acestrocephalus and Galeocharax, but should be 48 reinterpreted either as convergent in the former genus and the Cynopotamini or as lost (i.e., reversed to the shorter, more basal shape) in Charax and Roeboides based on our results. The UCE phylogeny indicates Cynopotamus as sister to the clade with Acestrocephalus and Galeocharax, a relationship previously proposed by morphological studies (Menezes, 1976; Lucena, 1998; Mattox & Toledo-Piza, 2012). However, the relationships recovered herein do not support the total-evidence hypothesis that found Acestrocephalus as sister to a clade formed by Cynopotamus and Galeocharax (Mirande, 2019). Furthermore, Acestrocephalus, Cynopotamus and Galeocharax were individually corroborated as monophyletic assemblages (Figure 3; ML = 100), as shown in previous morphological studies (e.g., Menezes, 1976; 2006; Mattox & Toledo-Piza, 2012; Giovanetti et al., 2017). In the tribe Characini, Charax and Roeboides are resolved as sister genera in our molecular study and both are supported as monophyletic (Figure 4; ML = 100), a result also consistent with morphological studies (Lucena, 1998; 2000; Mirande, 2009; 2010; Mattox & Toledo-Piza, 2012). 3.3. Interspecific relationships Hypotheses of interspecific relationships in Characinae were limited to morphological analyses or arrangements based on taxonomic revisions and species descriptions (Lucena, 2000, 2003, 2007; Menezes, 2006, 2007; Lucena & Malabarba, 2010; Menezes & Lucena, 2014; Giovannetti et al. 2017). Phenacogaster, the most species-rich genus, lacks formal descriptions for many species as well as taxonomic definitions of subgroups (Lucena & Malabarba, 2010). Based on 56% of its species diversity, our phylogeny consistently supports the presence of two main Phenacogaster clades (ML=100; Figure 4). The first is the "Phenacogaster pectinata clade" (Figure 3), widely distributed across the Amazon and Paraguay basins with relatively strong node support (ML>86%). Species from the 49 Amazon (P. beni Eigenmann, 1911, P. capitulata Lucena & Malabarba, 2010, P. megalostictus Eigenmann, 1909, P. pectinata (Cope, 1870), P. prolata Lucena & Malabarba, 2010, P. aff. pectinata and P. aff. suborbitalis) are sister group to P. tegata (Eigenmann, 1911) from the Paraguay basin. The second is the "P. franciscoensis clade", that includes P. maculoblonga Lucena & Malabarba, 2010, from the Orinoco basin sister to P. carteri (Norman, 1934) from Mazaruni River, with this subclade as sister to the remaining species in the P. franciscoensis clade. Within the latter group, P. wayana Le Bail & Lucena, 2010 from eastern Guiana Shield (Amapá and Jari rivers) is sister to two subclades: one with P. aff. retropinna Lucena & Malabarba, 2010 (Tapajós) sister to an undescribed species from the Xingu River, and another with successive less inclusive clades containing P. eurytaenia Antonetti, Lucena & Lucena, 2018 (Tocantins) sister to P. naevata Antonetti, Lucena & Lucena, 2018 (Tocantins) which is sister to a clade of P. calverti (Fowler, 1941) (Parnaíba) and P. franciscoensis Eigenmann, 1911 (São Francisco). Interestingly, the species from Tocantins, Parnaíba and São Francisco basins are related to each other forming a geographically-structured subclade. This result is similar to studies with other fish groups distributed throughout those basins such as Otocinclus hasemani and O. xakriaba (Schaefer, 1997), Hisonotus sp. 1, Hisonotus sp. 2 and Parotocinclus aff. spilurus (Roxo et al., 2014), and Prochilodus lacustris and P. brevis (Melo et al., 2018). The time-calibrated phylogeny suggests that "Phenacogaster pectinata clade" and "P. franciscoensis clade" diverged during the Miocene, at approximately 8.4 Ma (10.4–6.4 Ma 95% HPD; Figure 5). Acanthocharax is the only monotypic genera of Characinae and restricted to its type species A. microlepis Eigenmann, 1912 with distribution through the Essequibo River and adjacent drainages of Guyana. In our analysis, it was represented by a single specimen from Kurupung River in Guyana and the phylogeny placed it as sister to the major clade containing Characini and Cynopotamini. Our phylogenetic hypothesis supported the monophyly of Cynopotamus based on nine out of 50 Figure 5. Time-calibrated phylogeny for Characinae based on a BEAST analysis of 91 UCE loci present for at least 95% of 85 specimens (66 Characinae and 19 outgroups). Node bars show the 95% highest posterior distribution of ages. 51 12 (75%) species of the genus. The species were grouped into two clades with high support (ML=100). The first is the "C. magdalenae clade" (Figure 3) that represents the first evidence of a close relationship among species from west of the Andes, with strong support for C. venezuelae (Schultz, 1944) from Santa Rosa River of Venezuela as sister to C. magdalenae (Steindachner, 1879) from Samaná River/Magdalena basin of Colombia. The second is the "Cynopotamus bipunctatus clade" formed by the following species from east of the Andes: C. bipunctatus Pellegrin, 1909, C. essequibensis Eigenmann, 1912, C. gouldingi Menezes, 1987, C. juruenae Menezes 1987, C. kincaidi (Schultz, 1950), C. tocantinensis Menezes, 1987, and C. xinguano Menezes 2007. According to timing estimates, the MRCA of Cynopotamus originated during the Miocene at approximately 10.0 Ma (12.5–8.0 Ma 95% HPD; Figure 5). The C. magdalenae clade (west of Andes) and C. bipunctatus clade (east of Andes) diverged between 9.0–5.5 Ma (95% HPD) which is coincident with the rise of the Eastern Cordillera (∼12 Ma) and the rise of the Merida Andes (∼8 Ma) with the isolation of the modern Maracaibo and Orinoco basins (Albert et al., 2006). Acestrocephalus is well supported as monophyletic in our analysis. Our phylogeny showed A. acutus Menezes, 2006 from the Tocantins River as sister to a clade formed by A. nigrifasciatus Menezes 2006 from Jamanxim/Tapajós and A. sardina (Fowler, 1913) from Sipapo and Negro rivers (Figure 3). Because our study included <50% of the species of Acestrocephalus, future studies with a greater number of species are necessary to better understand the relationships within the genus. Galeocharax was recently revised with only three valid species [G. goeldii (Fowler, 1913), G. gulo (Cope, 1870) and G. humeralis (Valenciennes, 1834)] with G. knerii Steindachner, 1879 from the Upper Paraná river basin considered a junior synonym of G. gulo (type locality: Pebas, Peru, and widely distributed throughout Amazonas, Orinoco, Tocantins, and upper Paraná Rivers) (Giovannetti et al., 2017). Here, we analyzed five samples of G. gulo from distinct drainages (Araguaia, Purus, Solimões, Amazon, and Paraná) and results did not support its monophyly (Figure 3; Figures S1-S5; ML=100). The topology found that the specimen from Araguaia River is sister to 52 the other two clades: one with G. gulo (Paraná; formerly G. knerii) and G. humeralis (Paraguay) and another with G. goeldii (Madeira) and G. gulo (Amazon drainages). Hence, our phylogeny does not support the proposal of synonymy of G. knerii and highlight that G. gulo from the Araguaia River might represent an undescribed species. Indeed, Giovannetti et al. (2017) discussed the morphological variation in populations from the Amazon, Paraná and Tocantins rivers, which would be interesting to be reinvestigated in light of our genomic data. In Charax, molecular results strongly supported C. stenopterus from the coastal drainages of southern Brazil, the "Charax stenopterus clade", as a sister group of a large clade composed by species from the Amazon, Orinoco, Paraguay and Mearim river basins (Figures 4, S1-S5; ML=100). Within the large clade, two subclades emerge: "C. tectifer clade" and "C. gibbosus clade". We did not find monophyly for C. condei and C. niger (Figure 4; Figures S1-S5; ML=100). Charax condei (Géry & Knöppel, 1976) (Trombetas) and C. aff. tectifer (Amazon) were closer to a clade with C. metae Eigenmann 1922 (Orinoco) and C. tectifer (Cope, 1870) (Ucayali). This is a clade restricted to the proto-Orinoco-Amazonas, a previous fluvial configuration in northwestern South America, with species shared between the Orinoco and Amazon basin (Hoorn et al., 2010; Albert et al., 2018). Within the Charax gibbosus clade, C. condei (Amazon and Negro) were related to a successive less inclusive clade with C. leticiae Lucena, 1987 (Paraguay), C. gibbosus (Linnaeus, 1758) (Rupununi), C. awa Guimarães, Brito, Ferreira & Ottoni, 2018 (Mearim), C. niger Lucena, 1989 (Oiapoque), C. niger (Amapá), C. pauciradiatus (Günther, 1864) (Tapajós) and C. michaeli Lucena, 1989 (Amazon). This biogeographic pattern approximates with the timing of the channelization of the Amazon River at approximately 10–5 Ma, and the subsequent connection and diversification of the fish faunas from western and eastern Amazon basin (Albert et al., 2018; 2021). In Roeboides, Lucena (1998) used osteological and meristic traits and proposed the division of Roeboides in four groups: R. dispar group, R. guatemalensis group, R. microlepis group, and the R. affinis group. In addition, Lucena (2000) considered R. guatemalensis and R. microlepis groups as 53 sister clades, with the R. affinis group as sister of the clade formed by these groups, and R. dispar as the sister to all other groups. Our results partially corroborated the composition of the four groups (herein termed clades) but did not support R. guatemalensis clade as sister to R. microlepis clade (Figure 4; Figures S1-S5). Our phylogenetic hypothesis showed the R. guatemalensis clade from west of the Andes as sister to a large monophyletic assemblage with R. dispar clade sister to R. microlepis and R. affinis clades (Figure 4). However, the relationship between the R. microlepis and R. affinis clades presented lower bootstrap support (ML=48%). Furthermore, our study did not support the monophyly of R. affinis, with one lineage of R. affinis (Araguaia) sister to a clade with R. affinis (Solimões and Juruá), R. sazimai (Mearim), R. biserialis (Amazon) and R. descalvadensis (Paraguay and Paraná). The time-calibrated tree estimated that the MRCA of R. guatemalensis clade (west-Andes) had its origin in the Pliocene (2.6 Ma, 3.3–2.0 Ma 95% HPD) and the last speciation event of this clade corresponds to species from Panama (0.7 Ma, 1.0–0.4 Ma 95% HPD) and Costa Rica (0.8 Ma, 1.1–0.5 Ma 95% HPD). This result suggests that the distribution and diversification of this clade into Central America are directly associated with the closure of the Isthmus of Panama at approximately 4–2.8 Ma (Bermingham & Martin, 1998; Coates & Stallard , 2013; McGirr et al., 2021). Overall, this study presents the most taxon-dense phylogeny for Characinae to date covering all genera and provides novel hypotheses for intergeneric and interspecific relationships. Limitations of our study include the relatively lower species coverage of Acestrocephalus (~38%), Charax (~58%) and Phenacogaster (~61%), the low support for the relationship between Phenacogaster aff. retropinna and P. sp. Xingu with others from the P. franciscoensis clade (48%), the low support for the relationship between the Roeboides microlepis clade and the R. affinis clade (48%), and the moderate support for the placement of R. affinis (56%) and R. sazimai (70%) within the R. affinis clade (Figs. 2–3). Finally, our results indicate various instances of relationships that are similar to previous morphological phylogenies (e.g. Lucena, 1998; Mattox & Toledo-Piza, 2012) and provide 54 an interesting framework for future investigation on the evolutionary processes modulating species diversification. Revisionary studies of species diversity and additional molecular data are also crucial to better understand the species diversity and evolution in Characinae. Acknowledgements We are grateful to Mary Burridge (ROM), María Fernando Castillo (STRI), Carlos Lucena (MCP), Carolina Doria (UFRO-ICT), Camila C. Ribas (INPA), Erick C. Guimarães (UFMA), and Michel D. Gianeti (MZUSP) for curatorial assistance and/or loan of tissues and vouchers. We are also thankful to LBP colleagues Luz E. Ochoa, Nadayca T. B. Mateussi, Fabio F. Roxo, Cristhian C. Conde-Saldaña and Eric. V. Ywamoto for advices and support with analyses. 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