UNIVERSIDADE ESTADUAL PAULISTA -UNESP INSTITUTO DE BIOCIÊNCIAS LUZ ENEIDA OCHOA ORREGO ANÁLISE DAS RELAÇÕES FILOGENÉTICAS E PADRÕES DE DIVERSIFICAÇÃO DE TRICHOMYCTERIDAE (TELEOSTEI, SILURIFORMES) UTILIZANDO SEQUÊNCIAS DE DNA DOUTORADO Botucatu 2018 UNIVERISDADE ESTADUAL PAULISTA INSTITUTO DE BIOCIÊNCIAS Luz Eneida Ochoa Orrego ANÁLISE DAS RELAÇÕES FILOGENÉTICAS E PADRÕES DE DIVERSIFICAÇÃO DE TRICHOMYCTERIDAE (TELEOSTEI, SILURIFORMES) UTILIZANDO SEQUÊNCIAS DE DNA Tese apresentada ao Instituto de Biociências, Câmpus de Botucatu, Universidade Estadual Paulista - UNESP, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas, Área de Concentração: Genética. Orientador: Dr. Claudio Oliveira Co-Orientador: Dr. Fábio Fernandéz Roxo Botucatu 2018 7 Palavras-chave: Biogeography; Evolution; Neotropical region; catfishes. Ochoa Orrego, Luz Eneida. Análise das relações filogenéticas e padrões de diversificação de Trichomycteridae (Teleostei, Siluriformes) utilizando sequências de DNA / Luz Eneida Ochoa Orrego. - Botucatu, 2018 Tese (doutorado) - Universidade Estadual Paulista "Júlio de Mesquita Filho", Instituto de Biociências de Botucatu Orientador: Claudio Oliveira Coorientador: Fábio Fernández Roxo Capes: 20204000 1. Bagre (Peixe). 2. Evolução. 3. Biogeografia. 4. Filogenia. 5. Análise de DNA. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSANGELA APARECIDA LOBO-CRB 8/7500 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. 8 AVISO Esta tese é parte dos requerimentos necessários à obtenção do título de Doutor em Ciências Biológicas, Genética, 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 referencias publicas 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 Genetics 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. A meu Esposo e família!!! “The only way to do great work is to love what do you do. If you haven’t found it yet, keep looking. Don’t settle. As with all matters of the heart, you’ll know when you find it “ Steve Jobs. Agradecimentos Incontáveis são as pessoas que contribuíram para a realização deste trabalho. Tenho muito que agradecer ao céu e a terra, ao Brasil inteiro e em particular: À Pós-Graduação de Ciências Biológicas (AC: Genética), da Universidade Estadual Paulista – UNESP, Campus de Botucatu, que possibilitou o desenvolvimento do Doutorado. Pelo auxílio financeiro concedido processo nº 2014/06853-8, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Ao professor Dr. Claudio Oliveira, que esteve presente em toda minha carreira científica, orientando com responsabilidade todas as fases do projeto e, principalmente agradeço pelas oportunidades fornecidas e confiança nesses anos. Ao meu co-orientador Dr. Fabio Roxo, pelos incontáveis ensinamentos, as sugestões e o apoio. Ao Dr. Fausto Foresti, pelo exemplo como pesquisador e dedicação aos alunos. À Dra. Cristiane Shimabukuro-Dias, pela grande ajuda e inumeráveis conselhos nos momentos difíceis dessa etapa, Aos colaboradores externos Alessio Datovo, Carlos DoNascimiento, Jorge Enrique Garcia, Mark Sabaj, pelas sugestões, ensinamentos e incondicional ajuda na etapa de analises e discussão. Aos amigos do Laboratorio de Biologia e Genética de Peixes, em especial a Natalia Mendez, Gabriel, Guilherme, Camila, Bruno, Nadayca, Maria Ligia, Fabileni, Najila Angélica, Yuldi, Samia e o Cristian; obrigada por me suportar durante tudo esse tempo, pelas risadas, por cada café e brincadeira. A minha família, em especial a minha mai, Maria Melba Orrego, exemplo de amor e fortaleza, a meu Pai, Pascual Ochoa pela força e amor, a meus irmãos Juan Felipe e Jose Luis por seu exemplo e apoio, a minhas irmãs e sobrinhos.... desculpem pela ausência nestes anos mais vocês foram parte da inspiração. A meu esposo Geysson e sua família, por sempre estar presentes e me incentivar. A minha sogra Dona Margarita Garcia agradecimento especial pelo indispensável amor e conselhos em todo momento. A meus amigos colombianos: Luz Mery Martinez, Cintia Moreno, Natalia Silva, Angela Jaramillo, Henry Agudelo, Ariel Bermudez, Frank Alvarez, Patricia Pelayo, por que ainda desde longe sempre estiveram presentes me apoiando e animando. A Raquel Turba, Camila Medeiros e Gabriela Pinho pela companhia durante meu estagio na UCLA. Muito obrigado!!!! RESUMO Trichomycteridae é uma das famílias mais diversa da superfamília Loricarioidea com aproximadamente 300 espécies válidas, incluídas em 41 gêneros e oito subfamílias, e amplamente distribuídas pelas drenagens da América do Sul e Central. Trichomycteridae é caracterizada morfologicamente pela presença de um sistema opercular altamente modificado, envolvendo os ossos operculares e pré-operculares, assim como pela variação no tamanho do corpo e padrões de coloração. Também apresentam uma ampla diversidade trófica incluindo espécies onívoras, insetívoras, lepidófagas e hematófagas. A monofilia da família e suas subfamílias são bem suportadas por caracteres morfológicos, exceto Trichomycterinae, a qual inclui Trichomycterus, um grupo não-monofilético, taxonomicamente complexo, com elevado número de espécies e desconhecida diversidade. Embora múltiplos estudos tenham focado nas relações supragenéricas com reduzida representatividade de espécies, ainda não existem estudos utilizando caracteres moleculares com ampla amostragem de Trichomycteridae. Neste contexto, o presente trabalho tem como objetivo principal estudar as relações filogenéticas de Trichomycteridae através da análise de sequências de DNA, usando duas aproximações: a análise multilocus, incluindo três genes mitocondriais e dois nucleares, e a implementação de análises filogenômicas usando 851 elementos ultraconservados do genoma (ultraconserved elements, UCEs). Com base na filogenia obtida, analisamos padrões de origem e diversificação, assim como sua correlação com a evolução do tamanho do corpo. Além disso, foram realizadas análises de biogeografia paramétrica para a reconstrução das áreas ancestrais. Os resultados obtidos pelas duas metodologias corroboram hipóteses morfológicas em relação à monofilia das subfamílias, exceto Glanapteryginae e Sarcoglanidinae, e revelam novas hipóteses de relacionamento dentro do clado Tridentinae-Stegophiliniae-Vandelliinae-Sarcoglanidinae- Glanapteryginae (TSVSG). As análises de divergência indicaram que a origem de Trichomyctreridae data do Cretaceo inferior com múltiplos eventos cladogenéticos ocorridos durante o final do Eoceno e inicio do Mioceno. A família apresenta uma alta heterogeneidade nas taxas de diversificação, com um shift evidente na origem da subfamília Trichomycterinae, o qual não está correlacionado com a evolução do tamanho do corpo. A reconstrução de áreas ancestrais indicou que o ancestral comum mais recente de Trichomycteridae esteve amplamente distribuído na região amazônica e drenagens costeiras do Atlântico Sul do Brasil. Diferentes processos geomorfológicos de dispersão e vicariância, principalmente associados com eventos de captura de cabeceira modelaram a distribuição atual dos membros de Trichomycteridae. ABSTRACT Trichomycteridae is one of the most specious families in the superfamily Loricarioidea with approximately 300 valid species including 41 genera and eight subfamilies, widely distributed through the rivers in South and Central America. Trichomycteridae is characterized morphologically by the presence of a highly modified opercular system, involving the opercular and pre-opercular bones, as well as by variation in body size and coloration patterns. Trichomycterids also present a wide trophic diversity including omnivorous, insectivorous, lepidophagous and hematophagous species. The monophyly of the family and its subfamilies are well supported by morphological characters except Trichomycterinae, which includes Trichomycterus, a taxonomically complex non-monophyletic group with a high number of species and unknown diversity. Although, multiple studies have focused on suprageneric relationships with reduced species representativity, there are no studies using molecular characters with a large sample of Trichomycteridae. In this context, the main objective of this research is to study the phylogenetic relationships of Trichomycteridae through the analysis of DNA sequences using two approaches: multilocus analysis, including three mitochondrial and two nuclear genes, and the implementation of phylogenetic analyzes using 851 ultraconserved elements of the genome (ultraconserved elements, UCEs). Based on the phylogeny obtained, we analyzed patterns of origin and diversification, as well as their correlation with the evolution of body size. In addition, analyzes of parametric biogeography were carried out for the reconstruction of the ancestral areas. The results obtained by the two methodologies corroborate morphological hypotheses supporting the monophyly of the subfamilies, except Glanapteryginae and Sarcoglanidinae, and reveal new hypotheses of relationship within the clade Tridentinae-Stegophiliniae-Vandelliinae-Sarcoglanidinae-Glanapteryginae (TSVSG). The analysis of divergence indicated that the origin of Trichomyctreridae dates from the lower Cretaceous with multiple cladogenetic events occurring during the late Eocene and early Miocene. The family shows a high heterogeneity in the rates of diversification, with an evident shift in the origin of the subfamily Trichomycterinae, which is not correlated with the evolution of body size. The reconstruction of ancestral areas indicated that the most recent common ancestor of Trichomycteridae was widely distributed in the Amazon region and coastal drains of the South Atlantic of Brazil. Different geomorphological processes of dispersal and vicariance mainly associated with river capture events modeled the current distribution of Trichomycteridae species. Table of Contents Apresentação .............................................................................................................................. 1 Introdução .................................................................................................................................. 2 Família Trichomycteridae .......................................................................................................... 3 Justificativa ................................................................................................................................ 8 Objetivo Geral .......................................................................................................................... 10 Objetivos específicos ............................................................................................................... 10 Chapter 1 Phylogenomic analysis of trichomycterid catfishes (Teleostei:Siluriformes) inferred from Ultraconserved Elements. ............................................................................. 11 1.1 Introduction ...................................................................................................................... 13 1.2 Material and methods ...................................................................................................... 14 1.2.1 Taxon sampling ............................................................................................................... 14 1.2.2 UCE methods .................................................................................................................. 15 1.2.3 Phylogenetic inference .................................................................................................... 16 1.2.4 Time Calibrated tree in BEAST ...................................................................................... 17 1.2.5 Analyses of speciation/extinction and body size rates .................................................... 17 1.2.6 Ancestral reconstruction of feeding modes ..................................................................... 18 1.3 Results ............................................................................................................................... 19 1.3.1 Phylogenomic inferences for the Trichomycteridae family ............................................ 19 1.3.2 Speciation and extinction rates in Trichomycteridae ...................................................... 22 1.4 Discussion .......................................................................................................................... 24 1.4.1 Phylogenetics relationships in Trichomycteridae ........................................................... 24 1.4.2 Diversification pattern in Trichomycteridae ................................................................... 28 1.5 References ......................................................................................................................... 30 Chapter 2 A phylogenomic perspective on the historical biogeography of Trichomycteridae inferred from target enrichment of DNA ultraconserved elements. . 60 2.1 Introduction ...................................................................................................................... 62 2.2 Material and methods ...................................................................................................... 65 2.2.1 Taxon sampling and Species distribution ....................................................................... 65 2.2.2 Phylogeny construction ................................................................................................... 65 2.2.3 Divergence time estimates .............................................................................................. 67 2.2.4 Biogeographic methods: Ancestral range inference ....................................................... 68 2.3 Results ............................................................................................................................... 69 2.3.1 Phylogenomic inference of Trichomycteridae family ..................................................... 69 2.3.2 Diversification time of Trichomycteridae ....................................................................... 70 2.3.3 Ancestral range inference ................................................................................................ 71 2.4 Discussion .......................................................................................................................... 72 2.4.1 Phylogenetic relationships of Trichomycteridae ............................................................. 72 2.4.2 Biogeographical signature of river capture events .......................................................... 74 2.4.3 Influence of vicariance events in the biogeographical distribution of trans-Andean clades ........................................................................................................................................ 76 2.4.4 Ancestral reconstruction and its correspondence with biogeographic patterns in the eastern of Brazil ....................................................................................................................... 77 2.5 References ......................................................................................................................... 79 Supplement 1 Multilocus analysis of the catfish family Trichomycteridae (Teleostei: Ostariophysi: Siluriformes) supporting a monophyletic Trichomycterinae. .......................... 105 Supplement 2 New species of Trichomycterus (Siluriformes: Trichomycteridae) lacking pelvic fins from Paranapanema basin, southeastern Brazil. ................................................... 141 Referencias introdução ........................................................................................................ 160 Apresentação Dentro da ordem Siluriformes, a família Trichomycteridae corresponde a um dos maiores grupos monofiléticos de peixes Neotropicais, devido a sua excepcional riqueza de espécies, ampla gama de especializações morfológicas, fisiológicas e ecológicas. Estudos filogenéticos, principalmente baseados em caracteres morfológicos têm contribuído no conhecimento da história evolutiva do grupo, entanto inúmeros problemas sistemáticos e taxonômicos persistem. Dessa forma, visando contribuir para o conhecimento das relações filogenéticas dos peixes de água-doce na região Neotropical, o objetivo principal deste trabalho foi analisar as relações filogenéticas da família Trichomycteridae com base em caracteres moleculares. O manuscrito começa com uma introdução geral, destacando prévios estudos das relações filogenéticas da família, apresentando as principais hipóteses propostas até o momento; em seguida são apresentadas a justificativa e objetivos do trabalho. Os resultados foram organizados em dois capítulos inéditos. Cada um contendo um artigo em preparação. No primeiro capítulo são apresentados os resultados da análise genômica usando 851 Elementos Ultraconservados do genoma (UCE´s) para 150 espécies, que representam aproximadamente 41% da diversidade de espécies da família. Novos relacionamentos entre as subfamílias foram identificados assim como o reconhecimento de vários clados dentro do gênero Trichomycterus. Neste artigo é incluída também a analise macroevolutiva avaliando o tamanho do corpo e a evolução dos hábitos alimentares. No segundo capitulo, o manuscrito está focado a testar quais processos biogeográficos (dispersão, vicariância, evento fundador) tem modelado a distribuição da família na região tropical e sua relação com a extensa história geológica da América do Sul. Finalmente em anexo são apresentados dois manuscritos publicados durante o desenvolvimento do projeto. O primeiro, contem à primeira hipótese molecular para a família baseada numa análise multilocus, e o segundo corresponde a descrição de uma nova espécie do gênero Trichomycterus. Introdução A ictiofauna Neotropical de água-doce é bastante rica, incluindo mais de 7000 espécies válidas, representando aproximadamente 10% de todas as espécies conhecidas de vertebrados (Lundberg et al., 2000; Berra, 2001; Reis et al., 2003; Lévêque et al., 2005; Lévêque et al., 2008; Petry, 2008; Albert e Reis, 2011; Eschmeyer e Fong 2017). Os peixes das ordens Siluriformes e Characiformes juntas compreendem cerca de 74% das espécies de peixes Neotropicais e formam as duas ordens mais especiosas, com 3.700 espécies para os Siluriformes e 2.100 para Characiformes (Reis et al., 2016; Eschmeyer e Fong, 2017). Tal diversidade é também refletida na alta variação morfológica e ecológica destes grupos, constituindo um excelente modelo para investigar processos macroevolutivos. No contexto da biologia evolutiva, compreender os processos que modelaram a diversidade de espécies é desafiador (Ricklefs, 2007). A base para estes estudos está em conhecer os relacionamentos ancestrais-descendentes entre as espécies. Neste sentido, os estudos filogenéticos têm sido foco de importantes avanços na ultima década, principalmente relacionado ao progressivo avanço das técnicas de sequenciamento de DNA, o qual tem aumentado consideravelmente o número de caracteres usados nas inferências filogenéticas, e potenciam seu poder de resolução (Zou et al., 2012; McCormarck et al., 2012; Faircloth et al., 2013). Dentro da ordem Siluriformes, a superfamília Loricarioidea (sensu de Pinna, 1998), ou também chamada de subordem Loricarioidei (sensu Sullivan et al., 2006), representa o mais diverso e amplamente distribuído grupo de peixes neotropicais, encontrado em praticamente todos os hábitats de água-doce na região tropical (Reis et al., 2003; Nelson, 2006). Além de sua excepcional riqueza de espécies, esse grupo exibe uma ampla gama de especializações morfológicas, fisiológicas e ecológicas, ocupando muitos hábitats e níveis tróficos (Reis, 1998; de Pinna, 1998; Nelson, 1999; Brito, 2002). Com aproximadamente 1.420 espécies válidas (Eschmeyer e Fong, 2017), Loricarioidea é composto por seis famílias sendo as mais diversas Loricariidae e Trichomycteridae. A primeira tem sido foco de diversos estudos filogeneticos (e.g. Howes, 1983; Schaefer, 1987, 1991, 1998,2003; Amrbruster, 1998, 2004) e moleculares (e.g. Montoya Burgos et al., 1997, 1998, 2002; Zawadzki et al., 2005; Chiachio et al., 2008; Roxo et al., 2012a, b, 2014, 2017; Lujan et al., 2014; Covain et al., 2015). Estes estudos, com uma ampla 3 amostragem de táxons em grupos específicos dentro de Loricariidae, tem levantado questões biogeográficas e evolutivas importantes e têm auxiliado no esclarecimento dos mecanismos e processos geradores da ampla diversidade de espécies. As relações entre famílias de Loricarioidea encontram-se relativamente bem estabelecidas (Arratia, 1987; 1990; 1998; de Pinna, 1992), com base em dados morfológicos e moleculares (de Pinna, 1998; Britto, 2002; Sullivan et al., 2006; Lundberg et al., 2007). Por outro lado, as relações entre espécie, dentro de cada família ainda necessitam de estudos adicionais (e.g., Reis et al., 2003; Alexandrou et al., 2011; Roxo et al., 2012a, b). Trichomycteridae é a segunda família mais diversa em número de espécies dentro de Loricarioidea, foco de diversos estudos filogenéticos usando caracteres morfológicos (Baskin, 1973; de Pinna, 1988, 1989, 1992, 1998; de Pinna e Starnes, 1990; Costa e Bockman, 1993; Wosiacki, 2002; Datovo e Bockmann, 2010). Porém, poucos estudos têm sido realizados usando caracteres moleculares, além de se focar nas relações ao nível infrafamiliar (Fernandez e Schaefer, 2009) e na identificação genética de espécies (da Silva et al., 2010). Sete das oito subfamílias reconhecidas em Trichomycteridae (Copionodontinae, Trichogeninae, Sarcoglanidinae, Glanapteryginae, Tridentinae, Stegophilinae e Vandelliinae) são diagnosticadas por caracteres exclusivos; entretanto a subfamília Trichomycterinae e seu gênero mais diverso, Trichomycterus são considerados polifiléticos, basicamente diagnosticados pela ausência de especializações das outras subfamílias (Baskin, 1973; Costa e Bockmann, 1993; de Pinna, 1998; Datovo e Bockmann, 2010). Adicionalmente, o incompleto conhecimento da diversidade de espécies, hipóteses conflitantes ou incompletamente resolvidas para a família representa um dos maiores desafios na ictiologia neotropical. O acelerado descobrimento de novas espécies e a dificuldade em identificar caracteres sinapomorficos, requerem da aplicação de novas metodologias assim como o incremento na representatividade de espécies para tentar estabelecer as relações filogenéticas em Trichomycteridae. Família Trichomycteridae A família Trichomycteridae é um dos mais diversos grupos monofiléticos de peixes de água-doce (de Pinna, 1998), distribuído nas Américas Central e do Sul, desde a Costa Rica até a Patagônia (sul da Argentina e Chile), de ambos lados dos Andes, em águas turvas até os rios costeiros (Wosiacki, 2004). A família atualmente é representada por aproximadamente 300 espécies válidas (Eschmeyer e Fong, 2017), 41 gêneros e oito subfamílias (i.e. 4 Copionodontinae, Glanapteryginae, Sarcoglanidinae, Stegophylinae, Trichogeninae, Trichomycterinae, Tridentinae, Vandelliinae). Trichomycteridae é considerada a segunda família mais diversa de Loricarioidea e sua diversidade é ainda subestimada (de Pinna e Wosiack, 2003), com várias espécies identificadas, porém ainda não foram descritas formalmente (Reis et al., 2003). A família é também de especial interesse devido a sua posição chave na filogenia dos Siluriformes, que junto com Nematogenyidae, Callichthyidae, Scoloplacidae, Astroblepidae e Loricariidae constituem a primeira linhagen em diversificar (Datovo e Bockmann, 2010) da subordem Loricarioidei (sensu Sullivan et al., 2006). A monofilia de Trichomycteridae é suportada por um grande número de sinapomorfias, sendo a mais conspícua, o aparato opercular altamente modificado (Baskin, 1973; de Pinna, 1992a, 1998; Datovo e Bockmann, 2010), que inclui a presença de vários odontódeos sobre o opérculo e inter-opérculo; estrutura que em muitos membros, forma um complexo sistema músculo-esquelético que permite aos peixes ancorar-se ao substrato ou ao corpo de seus hospedeiros, no caso dos semiparásitos “candirus” (Datovo e Bockmann, 2010), nome popular relacionado com as espécies da subfamília Vandelliinae, mas algumas vezes usado para todas as espécies da família. Inicialmente a família Trichomycteridae foi alocada como subfamília “Trichomycteriformes” na família Siluroidei segundo Bleeker (1863). Posteriormente foi reconhecida como família por Gill (1872). Embora, Eigenmann e Eigenmann (1888, 1890) e Eigenmann (1918) usaram o nome Pygidiidae, não existe duvida que o nome adequado para a família é Trichomycteridae (Miranda-Ribeiro, 1922) como foi estabelecido por Tchernavin (1944) que revisou a questão nomenclatural dos membros da família e tratou Trichomycteridae como sinônimo junior de Pygiididae. Um dos primeiros e mais importantes estudos da família foi realizado por Eigenmann (1918) que revisou todas as espécies descritas até esse momento e subdividiu Pygiididae (=Trichomycteridae) em seis subfamílias: Nematogeninae, Pygiidinae, Pareiodontinae, Stegophilinae, Vandelliinae e Tridentinae. Entre 1944 e 1966 foram propostas duas subfamílias, a primeira para alocar dois novos gêneros e duas novas espécies, Pygidianops eigenmanni e Typhlobelus ternetzi, além de alocar a espécie Glanaptaeryx anguila previamente descrita por Myers (Myers, 1944, 1927). Em 1966 Myers e Weitzman descreveram a subfamília, Sarcoglanidinae, para alocar dois novos gêneros e duas novas espécies, Sarcoglanis simplex e Malacoglanis gelatinosus, baseados em três 5 espécimes, um único espécime de S. simplex do Alto rio Negro e dois espécimes de M. gelatinosus do rio Orteguaza, bacia do rio Caquetá na Colômbia. A análise mais detalhada das relações filogenéticas da subfamília Trichomycterinae e a primeira em proporcionar suporte cladístico para a monofilia de Trichomycteridae (de Pinna, 2016), foi realizada por Baskin em 1973, com seu trabalho “Structure and relationships of the Trichomycteridae”, recentemente publicada (de Pinna, 2016). O autor dividiu todos os trichomycteridos conhecidos em dois grupos monofileticos, o primeiro denominado “Trichomycterinae group” que incluiu as subfamílias Glanapteryginae, Sarcoglanidinae e Trichomycterinae; e o segundo “Vandelliinae group” composto pelas subfamílias Stegophilinae, Tridentinae e Vandelliinae. Baskin (1973) não achou evidências suficientes para suportar o monofiletismo da subfamília Trichomycterinae, hipóteses que posteriormente foi testada por de Pinna (1989), apontando para uma possível polifilia do grupo. Posteriormente de Pinna (1992) descreveu a subfamília Copionodontinae, diagnosticada pela posição anterior da nadadeira dorsal, a presença de uma nadadeira adiposa desenvolvida e a forma espatulada evidente dos dentes mandibulares. Essa subfamília, composta por dois gêneros e três espécies, foi suportada como monofilética e proposta como uma das linhagens irmãs dos Trichomycteridos devido a que apresenta as seguintes condições plesiomórficas: presença de ductus pneumaticus; separação do “Pterosfenotico”, “esfenotico” e “prootico”, a presença de “intercalarium”, canal infraorbital incompleto, presença de “interhyal” e a ampla abertura da capsula da bexiga natatoria; unicamente compartilhadas com Trichogenes (de Pinna, 1992). Novas espécies e gêneros foram descobertas nos anos seguintes –e.g. Ituglanis Costa e Bockmann (1993), que não foi assinalado a nenhuma subfamília definida, porém os autores sugeriram ser o grupo irmão do clado composto por Vandelliinae, Stegophilinae, Tridentinae, Glanapteryginae e Sarcoglanidinae (clado-VSTGS). Ituglanis é considerado essencial, para compreender o surgimento das adaptações morfológicas e ecológicas dos membros de Trichomycteridae, devido a sua posição filogenética intermediária entre as formas mais generalistas (Trichogeninae, Copionodontinae e Trichomycterinae), assim como das formas mais especializadas que compreendem o clado-TSVSG (Lima et al., 2013). Após o estudo de Baskin (1973), uma nova hipótese para a família Trichomycteridae foi apresentada e discutida por de Pinna (1998). Nessa hipótese os gêneros Scleronema e Ituglanis não foram assinalados para nenhuma subfamília conhecida, e as subfamílias 6 Copionodontinae e Trichogeninae formaram uma tricotomia com os demais membros da subfamília Trichomycterinae. Posteriormente, a maior parte dos estudos relacionados com a família concentrou-se na subfamília Trichomycterinae. O primeiro deles foi a descrição de Silvinichthys (Arratia 1998) e a proposta de quatro sinapomorfias para Trichomycterinae (Arratia 1990), sendo a subfamília composta pelos gêneros Eremophilus, Rhizosomichthys, Scleronema e Trichomycterus. Entretanto, a autora deixou claro que estabelecer os limites de Trichomycterinae e de Trichomycterus requereria uma análise completa de todas as espécies da subfamília. Wosiacki (2002) estudando 205 caracteres morfológicos de 73 espécies de Trichomycteridae incluindo 49 espécies válidas, assim como espécies não descritas, encontrou quatro árvores igualmente parcimoniosas. O cladograma de consenso estrito resultou na identificação de 70 clados. Para uma adequada organização das espécies, frente aos resultados obtidos, Wosiacki propôs 14 novos gêneros e 14 novas subfamílias. Além disso, propôs Scleronema como grupo irmão do clado-VSTGS e Ituglanis como grupo irmão de Scleronema+VSTGS. Wosiacki (2002) estudando as relações filogenéticas de Trichomycterinae também confirma a polifilia desta subfamilia, já apontado anteriormente por de Pinna (1998). Mais recentemente, análises moleculares empregando genes mitocondriais foram realizadas com o objetivo de testar as relações entre algumas espécies de Trichomycteridae (Fernandez e Schaefer, 2009). Os resultados obtidos com seis subfamílias, 17 gêneros e 21 espécies foram congruentes com hipóteses propostas previamente usando dados morfológicos (Eigenmann, 1918; Myers, 1944; Baskin, 1973; de Pinna, 1998) as quais estabeleceram que as subfamílias Stegophilinae e Vandelliinae conformam clados irmãos. O estudo mais recente incluindo 35 espécies representantes de todas as subfamílias de Trichomycteridae foi realizado por Datovo e Bockmann (2010). Estes autores propuseram uma hipótese de relacionamento baseada exclusivamente em caracteres morfológicos da musculatura da região dorsolateral da cabeça. Os resultados obtidos corroboram hipóteses previas de relacionamento entre os membros da família, além de invalidar muitos dos caracteres morfológicos inicialmente propostos para definir grupos dentro de Trichomycteridae. Os resultados indicaram que as subfamílias Copionodontinae e Trichogeninae formaram uma linhagem monofilética, correspondente ao grupo-irmão de todos os demais trichomycteridos, além de corroborar o monofiletismo do clado C ou clado -TSVSG (Tridentinae, Stegophilinae, Vandelliinae, Glanapteryginae). Entretanto, neste trabalho duas hipóteses são discordantes com relação às hipóteses anteriores para a família: a relação de 7 grupo irmão entre Tridentinae e Stegophilinae e o monofiletismo de Trichomycterinae lato sensu (Datovo e Bockmann, 2010), i.e., incluindo os gêneros Trichomycterus, Scleronema, Ituglanis, Bullockia e Hatcheria. Nesse contexto, considerando as hipóteses conflitantes para as relações de parentesco entre os membros de Trichomycteridae, o incremento acelerado na descrição de novas espécies, a dificuldade em identificar caracteres morfológicos únicos para os membros de Trichomycterus, a ausência de estudos baseados em caracteres moleculares, e a importância da família para o entendimento das relações evolutivas em Silurifomres, faz-se necessário a realização de uma filogenia molecular incluindo uma grande amostragem de espécies, como base fundamental para a realização de análises macroevolutivas e biogeográficas, que permitam identificar os mecanismos e processos evolutivos responsáveis pela origem e diversificação dos membros de Trichomycteridae. 8 Justificativa Compreender as relações filogenéticas entre organismos é um pré-requisito de todos estudos evolutivos. Até os anos 1970, a reconstrução filogenética foi baseada em análises de caracteres morfológicos ou ultraestruturais, contudo, esta abordagem é dificultada pelo número limitado de caracteres homólogos. No final de 1980, o acesso a sequências de DNA aumentou o número de caracteres que podem ser comparados de menos de 100 para mais de 1.000, melhorando consideravelmente o poder de resolução das inferências filogenéticas (Delsuc et al. 2005). As análises empregando dados moleculares são considerados uma ótima ferramenta na resolução de problemas taxonômicos e evolutivos (ancestrais-descendente). A utilização de caracteres moleculares nas análises filogenéticas em peixes tem crescido consideravelmente nas últimas décadas contribuindo significativamente na compreensão das relações entre grupos complexos (Bermingham e Avise 1986; Alves-Gomes et al. 1995; Ortí e Meyer 1997; Sivasundar et al. 2001; Shimabukuro-Dias et al. 2004; Calcagnotto et al. 2005; López- Fernandez et al. 2005; Chiachio et al. 2008; Javonillo et al. 2010; Lovejoy et al. 2010; Alexandrou et al., 2011; Oliveira et al., 2011; Carvalho-Costa et al. 2011; entre outros). No caso dos siluriformes a maioria dos estudos filogenéticos de nível superior têm-se centrado em grupos de poucas famílias, mas com amostragem de táxons mais completa internamente, por exemplo, loricarídeos (Baskin, 1973; Pinna, 1992; Schaefer, 1990); cetopsídeos (Vari e Pinna, 1995), pimelodídeos (Bockmann, 1998, Lundberg e McDade, 1986; Lundberg et al., 1991a, b; de Pinna, 1998); doradídeos e ariídeos (Ferraris, 1988, Lundberg, 1993; Royero, 1987). Contudo, não existem até o momento estudos em sistemática molecular abordando as relações da família Trichomycteridae com o uso de um grande número de táxons e representantes de todas subfamílias. A pesar dos múltiplos estudos desenvolvidos usando caracteres morfológicos para estabelecer o relacionamento evolutivo da família Trichomycteridae (Eigenmann, 1918; Peyer 1922; Berg 1940; Baskin 1973; de Pinna 1998; Datovo e Bockmann, 2010), existem conflitos ainda não resolvidos. Os únicos estudos que abordaram essas relações foram realizados com grupos abrangentes, suprafamíliares (de Pinna, 1998; Britto, 2002; Sullivan et al., 2006) ou intrafamíliares (Arratia, 1990; Costa e Bockmann, 1993, Wosiacki, 2002, Datovo 2010), porém, utilizando poucos táxons no grupo interno de Trichomycteridae. Após Datovo e 9 Bockmann (2010) nenhum estudo com o propósito de testar as relações filogenéticas de Trichomycteridae foi realizado. A pequena representatividade da diversidade de espécies da família em todos os estudos já realizados, o incremento no numero de espécies descritas e o limitado numero de caracteres morfológicos únicos para suportar relacionamentos dentro de Trichomycteridae, evidenciam a importância de um estudo aprofundado das relações filogenéticas utilizando uma amostragem mais ampla assim como o uso de caracteres moleculares e novas metodologias genéticas. A disponibilidade de uma hipótese filogenética robusta em Trichomycteridae pode fornecer suporte para a análise dos fatores que determinam a alta diversidade e padrões biogeográficos do grupo na região Neotropical. 10 Objetivo Geral Inferir hipóteses de relacionamento entre os táxons constituintes de Trichomycteridae usando caracteres moleculares. Objetivos específicos • Testar a hipótese de monofiletismo para as subfamílias de Trichomycteridae. • Estimar taxas de diversificação de acordo com a hipóteses filogenética e analizar sua correlação com a evolução morfológica do tamanho do corpo. • Identificar padrões biogeográficos relacionados com a diversificação das espécies através de análises de biogeografia paramétrica. 11 “The tree of life was always there. Evolution just fills in the gaps” Simon Conway Morris Chapter 1 Phylogenomic analysis of trichomycterid catfishes (Teleostei:Siluriformes) inferred from Ultraconserved Elements. Chapter 1 12 Phylogenomic analysis of trichomycterid catfishes (Teleostei:Siluriformes) inferred from Ultraconserved Elements. By Luz Eneida Ochoa Orrego Abstract The Trichomycteridae family is one of the most diverse groups of freshwater catfishes in South America; with approximately 290 valid species, eight subfamilies and 41 genera. Its members are widely distributed through out South America and the family is recognized by a high trophic diversity including generalized predators, algivores, carrion-feeders, scale and mucus eaters and the specialized parasites. Likewise, this group shows a high morphological variation and unevenly diversity distribution. Different studies using morphological characters and molecules have been addressed to understand the phylogenetic relationships within each subfamily corroborating their monophyletic status. Nevertheless, the increased knowledge of the taxonomic diversity of Glanapteryginae and Sarcoglanidinae has revealed a series of slightly differentiated taxa that have been difficult to confidently assign to one of these subfamilies. In order to assess the phylogenetic relationships of Trichomycteridae, we collected sequence data from ultraconserved elements (UCE) of the genome from 132 members of Trichomycteridae and 11 species of the outgroups. We used a concatenated matrix to infer the relationships by Bayesian (B) and Maximum Likelihood (ML) inferences. The results show a highly-resolved phylogeny with broad agreement between B and ML trees. The results provide overwhelming support for the monophyletic status of Trichomycterinae including Ituglanis and Scleronema. Previous hypotheses of relationships among subfamilies, as the sister relationship between Copionodontinae and Trichogeninae forming a sister clade to the remaining trichomycterids and the intrafamilial clade TSVSG are corroborated, while the monophyly of Glanapteryginae and Sarcoglanidinae is not recovered and unexpected novel relationships between members of both subfamilies are found with biogeographic correspondence. The macroevolutionary analysis revels heterogeneity in diversification rates and decouple body size evolution from speciation, suggesting that diversification processes in Trichomycteridae may be more related with ecological specialization. Chapter 1 13 1.1 Introduction Unraveling the relationships of major sections of the Tree of Life is one of the most daunting challenges of the evolutionary biology. Next-generation DNA sequencing (so-called massively parallel sequencing) (Crawford et al. 2012; Faircloth et al. 2012; Mccormack et al. 2012; Smith et al. 2014) is a promising tool that is helping to resolve the interrelationships of longstanding problematic taxa (Faircloth et al. 2013). One of the most common class of phylogenomic methods involves the sequence capture of nuclear regions flanking and including ultraconserved elements (UCEs) (Faircloth et al. 2012). Recent studies on ray-finned fishes (Faircloth et al. 2013) and flatfishes (Harrington et al. 2016), among other vertebrates groups (Mccormack et al. 2012; Crawford et al. 2015), have shown that UCE’s are ideal markers for phylogenetic studies because of their ubiquity among taxonomic groups (Siepel et al. 2005), low degrees of paralogy (Derti et al. 2006) and low saturation (Mccormack et al. 2012). Target enrichment of UCE loci has been used to investigate questions at deep timescales across diverse groups of taxa as consequence of nearly invariant core regions. Simultaneously, the more variable flanking UCE regions allow a better resolution of nodes across a range of evolutionary timescales in a given phylogeny (Faircloth et al. 2012). As variation in the flanks increases with distance from the core UCE, this combined approach display a balance between having a high enough substitution rate while minimizing saturation, thus providing information for estimating phylogenies at multiple evolutionary timescales (Faircloth et al. 2012; Mccormack et al. 2012). UCEs are rarely found in duplicated genomic regions (Derti et al. 2006), making the determination of orthology more straightforward than other markers (Mccormack et al. 2013). According to Gilbert et al. (2010), the phylogenetic informativeness of the combined flank and core regions of UCEs is superior to the ones derived from sets of protein-coding genes. Additionally, phylogenomic approaches are characterized by their potential to collect data from at least one order of magnitude more loci than the traditional protein-coding sequencing techniques. The present survey is the first to employ these recent advances in phylogenomics and high-throughput sequencing to address evolutionary relationships in the catfish family Trichomycteridae, which includes the so-called pencil and parasitic catfishes. The family contains approximately 290 species (Eschmeyer & Fong 2017) characterized by a highly modified opercular system, with opercular and interopercular bones usually armed with patches of sharp odontodes. Additionally, the family is also characterized by a high variation in body size with some species miniature due to a small body and paedomorphic features associated Chapter 1 14 with the degree of development of the laterosensory canal system and reduction of fin rays, as well the bones of the head (Weitzman and Vari 1988). Trichomycterids have one of the broadest ranges of trophic adaptations known within any single catfish family, including insectivores, omnivores, carnivores, necrophagous, mucophagous, lepidophagous, and hematophagous (Kelley & Atz 1964; Goulding 1979, 1980; Machado & Sazima 1983; Winemiller & Yan 1989; de Pinna 1998; Zuanon & Sazima 2004; Fernández & Schaefer 2009). The family has a wide distribution in the Neotropical freshwater drainages (de Pinna 1998) of Central and South America (Wosiacki 2004) from Costa Rica to Patagonia, occurring on both versants of the Andes, and even in a few insular freshwater environments (de Pinna & Wosiacki 2003; Fernández & Schaefer 2005). Eight trichomycterid subfamilies are currently recognized: Copionodontinae, Glanapteryginae, Sarcoglanidinae, Stegophilinae, Trichogeninae, Trichomycterinae, Tridentinae, and Vandelliinae. Only two publications with explicit cladistics analyses tested the interrelationships among all these subfamilies, being one based on morphological data (Datovo & Bockmann 2010) and another on combined nuclear and mitochondrial genes (Ochoa et al. 2017). The present phylogenomic analysis assembled a dataset of ultraconserved DNA elements (UCEs) and their flanking regions representing over 851 loci from 150 taxa including the outgroup (about and 41% of species diversity of the family). A new well-supported hypothesis of relationships for the Trichomycteridae emerged from this analysis, which serve to explore macroevolutionary dynamics across the tree, as well as to identify changes in speciation and extinction rates and its relationship with body size and trophic evolution. 1.2 Material and methods 1.2.1 Taxon sampling Tissues samples and voucher species used in this project were deposited in the collection of Laboratório de Biologia e Genética de Peixes UNESP, Botucatu, Brazil (LBP), Instituto de Pesquisas da Amazonia, Manaus, Brazil (INPA), and The Academy of Natural Sciences of Drexel University (ANSP). The table 1.1 synthesize pertinent data from all the samples belonging to ingroup and outgroup. Our analysis includes representatives of the all eight subfamilies and from 26 genera and 132 species of Trichomycteridae. The outgroup includes species of the: Nematogenyidae (Nematogenys inermis), Callichthyidae (Corydoras elegans, Corydoras gossei and Hoplosternum littorale), Scoloplacidae (Scoloplax dicra, Scoloplax distolothrix) Astroblepidae (Astroblepus grixalvii and Astroblepus sp.), Loricaiidae Chapter 1 15 (Lasiancistrus saetiger, Falorwella oxrryncha and Lamonichthys filamentosus) as representatives of the Loricarioidei and the resulting trees were rooted in the characiform Leporinus striatus. 1.2.2 UCE methods DNA extractions were done from approximately 25 mg of tissue using Qiagen DNeasy Tissue kits following the manufacturer’s protocols, and we ran all genomic DNA extractions on an agarose gel to assess quality. We quantified 2µl of each sample using fluorometry (Qubit, Life Technologies). The samples used in the library preparation presented a concentration between 10-40 ng/µl. To prepare the libraries initially we sheared 1-2µg of DNA to 400-600 bps in length using a Diagenode Bioruptor Standard (UCD 200) with 6-8 cycles of sonication (depending on DNA quality). The DNA libraries from 150 species were prepared using the Nextera (Epicentre Biotechnologies, Inc.) library preparation protocol for solution-based target enrichment following Faircloth et al. (2012) and increasing the number of PCR cycles following the tagmentation reaction to 20 as recommended by Faircloth et al (2013). We 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), then used the Epicentre Nextera kit to prepare transposase-mediated libraries with insert sizes averaging 100 bp (95% CI: 45 bp) following Adey et al. (2010). The libraries were enriches using a probe set developed for application to ostariophysan fishes to generate sequences data for approximately 2500 UCE loci (Faircloth et al. in prep). We converted the DNA 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 6 cycles using the manufacturer's recommended thermal profile and dual P5 and P7 indexed primers (see Kircher et al. 2012 (doi: 10.1093/nar/gkr771) for primer configuration). After purification with SPRI beads, libraries were quantified with the Quant-iT(TM) Picogreen(R) dsDNA Assay kit (ThermoFisher). We then enriched pools comprising 100 ng each of 8 libraries (800 ng total) using the MYbaits(R) Target Enrichment system (MYcroarray) following manual version 3.0. After capture cleanup, the bead-bound library was resuspended in the recommended solution and amplified for 10 cycles using a universal P5/P7 primer pair and KAPA HiFi reagents. After purification, each captured library pool was quantified with PicoGreen, and combined with all other pools in projected equimolar ratios prior to sequencing. Sequencing was performed across two Illumina HiSeq paired-end 100 bp lanes using v4 chemistry. Chapter 1 16 1.2.3 Phylogenetic inference We used the software Phyluce v1.5.0 (Faircloth 2016) to the analysis of UCE’s. The first step was to clean the data of adapter contamination, low quality bases and sequences containing ambiguous bases, using the program illumiprocessor, included in the Phyluce software. We assembled reads and generated consensus contigs for each species using ABySS (version 2.0.2) (Simpson et al. 2009) with a kmer value equal to 55. ABySS is the most accurate assembler which runs read-based error correction prior to assembly resulting in more accurate contigs. Following assembly, we proceeded to identify those contigs that were UCE loci and align species-specific contigs to the set probes/UCEs used for enrichment. These processes were realized with Python program (phyluce_assembly_match_contigs_to_probes.py) integrating LASTZ (Harris 2007) a pairwise aligner to match contigs and UCE loci. During the matching, the program creates a relational data base of matches to UCE loci by taxon. We removed reciprocal and non-reciprocal duplicates UCE loci and create a database of UCE loci recovered. The monolithic FASTA files were used to generate the alignments using MAFFT (version 7.130b) and we trimmed resulting alignments using the algorithm implemented by the seqcap_align.py script within phyluce. Every alignment was cleaned from the locus name using phyluce_align_remove_locus_name_from_nexus_lines and Gblocks. From the trimmed alignments, we created an incomplete matrix with 50, 75, and 85% completeness in order to evaluate the role of missing data in our matrices, tree topology and clade support values. For each matrix we prepared a concatenated alignment in PHYLIP format and every matrix was analyzed using maximum likelihood (ML) algorithm in RAxML v8.2.X (Stamatakis 2014) to compare the topologies with different levels of completeness. The best-fitting partitioning scheme was obtained using the Bayesian Information Criterion and hcluster search in Partitionfinder v1.1.1 (Lanfear et al. 2012) and the best scheme grouping together loci having the same substitution model was used in subsequent analyses. The phylogenetic analysis was performed using maximum likelihood inference in RAxML v. 7.2.6 (Stamatakis, 2010) with “GTRGAMMA” option. A posteriori bootstrapping analysis were conducted with RAxML’s autoMRE tool indicated that trees converged after 50 replicates, we reconciled the best fitting ML tree with the bootstrap replicates. Bayesian inference were performed in ExaBayes version 1.5 (Aberer, Kobert & Stamatakis 2014) with 1’000,000 iterations (2 chains; bur-in:25%) with parameters in default. Chapter 1 17 1.2.4 Time Calibrated tree in BEAST Divergence times estimates were performed in BEAST v 1.8.0 (Drummond et al. 2012) using a reduced matrix, of 90% completeness, with a total 66,845 pb and uncorrelated lognormal clock and birth and death speciation process. We ran two independent analyses of 50,000,000 generations each. To verify effective sampling of all parameters and to assess convergence of independent chains, we examined output log files in Tracer v.1.6 (Rambaut et al. 2014). After removing 25% of samples as burn-in independent runs were combined and a maximum clade-credibility (MCC) tree was constructed using TreeAnnotator v1.8.0 (Drummond et al. 2012). We offset the minimum ages of three nodes across the phylogeny using a combination of fossil and secondary priors. The fossil was described from the Monte Hermoso Formation in Argentina (Bogan & Agnolin 2009; Tomassini et al. 2013), it was placed under a log normal distribution with a mean of 4.5 Myr and standard deviation of 1.5, allowing for the origin of the subfamily Trichomycterinae. Secondary priors were placed under a normal distribution on the root, with the origin of Siluriforms reported by Betancur-R et al (2015) of 150 Myr and the search was conducted among the interval of 136.3-163.7 Myr using the lower and upper quantiles of 2.5%, respectively. The other point was in the origin of Trichomycteridae about 106 million years ago (Myr) as was estimated by Betancur-R et al. (2017). We implanted a normally distributed prior with mean of 106 and standard deviation of 7. The search was conducted among the interval of 92.28-119.7 Myr using the lower and upper quantiles of 2.5%, respectively. 1.2.5 Analyses of speciation/extinction and body size rates To estimate the number of distinct evolutionary regimes across our phylogenetic tree, we used the Bayesian Analysis of Macroevolutionary mixture (BAMM). This Bayesian approach uses reversible jump Markov chain Monte Carlo (RJMCMC) sampling to explore shifts in macro-evolutionary regimes assuming they occur across the branches of a phylogeny under a compound Poisson process, and explicitly accommodates diversification rate variation through time and among lineages. BAMM is both time sensitive and diversity-dependent, allowing rate shifts to occur anywhere on a branch based on the posterior tree density (Rabosky 2014). Speciation and extinction were inferred using the ‘speciation-extinction’ module, with correction for differential sampling across genera. The body size evolution was inferred using the continuous ‘trait’ module. The sampling fraction of each genus was determined by comparing the number sampled with the Chapter 1 18 number of species reported in data base as Fishbase (http://www.fishbase.org/search.php), Catalogue of Fishes (CAS) (http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp) and Siluriformes data (http://silurus.acnatsci.org/ACSI/taxa/Families.html). Data on standard length (LS) were obtained mainly from morphological descriptions and the reporters in the Check List of Freshwater Fishes of South and Central America (Reis, Kullander & Ferraris 2003), the specimens identified just to genus level were directly measure, all measurements were log-transformed. Priors for BAMM were generated using the R package BAMM-tools v.2.0.2 (Rabosky et al. 2014a) by providing the MCC tree from BEAST and total species numbers of the family. Two independent MCMC chains of 10’000,000 generations were run in BAMM and convergence was assessed by computing the effective sample sizes of log likelihoods, as well as the number of shift events present in each sample using the R package coda v. 0.16-1 (Plummer et al., 2006). After removing 10% of trees as burn-in, we analyzed the BAMM output using BAMMtools (Rabosky et al. 2014a) to estimate summary statistics, such as phylorate plots (showing rates and rate shifts in diversification), 95% credible sets of rate shifts configuration and average rates of speciation and extinction. 1.2.6 Ancestral reconstruction of feeding modes For the ancestral character reconstruction analysis, we obtained information about the trophic level from species description and www.fishbase.org. We considered six trophic categories according to description, algivorous, which consume mainly periphyton and phytoplankton; lepidophagous, feeding mucus and scale; hematophagous, parasite species feeding blood; carrion feeders; insectivorous, consume aquatic insects and small arthropods; and omnivorous consume aquatic and terrestrial insects, plant and organic material. To reconstruct the ancestral feeding modes we estimated stochastic character mapping using the function “make.simmap” and the best fit discrete model of evolution equal rates (ER) , symetric rates (SYM), all rates different (ARD) to data as it is implemented in the R packages Phytools (Revell 2017) and Geiger (Harmon et al. 2008). Chapter 1 19 1.3 Results 1.3.1 Phylogenomic inferences for the Trichomycteridae family Sequencing produced a total of 176 million reads with a mean of 1’216,052 per sample from 143 taxa. Using ABySS, we assembled the DNA reads into a mean of 62,859 contigs (95CI, min = 1,813, max = 497,245) per sample, having an average length of 194 pb (Table 1.2). Contigs matching no UCEs and UCE loci matching multiple contigs were subsequently removed, thus an average of 1,290 unique contigs matching UCE loci from each species. The gene trees inferred from the individual locus alignments of the matrices with 50% (1383 loci), 75% (915 loci), and 85% (669 loci) complete alignments exhibit identical topologies and strong node support. Phylogenetic trees estimated from 50% and 85% matrices are shown in the sl Figures 1.3 and 1.4 respectively. The phylogeny in the figure 1 correspond with the results of maximum likelihood and Bayesian estimation using the 75% complete matrix composes by 851 loci having a mean length of 162 bp (2.71 CI) per alignment, and a total of 160,440 bp of aligned sequences. We recover a strong-supported phylogeny of Trichomycteridae that is identical across maximum likelihood and Bayesian analysis with a strong statistical support in most nodes (i.e., 100% bootstrap support or posterior probabilities of 1.0) and few cases of nodes receiving moderate or low support represent by gray circle in the Figure 1.1 (bootstrap support between 50 and 75%, posterior probabilities between 0.5 and 0.8). The phylogenetic hypothesis was partially congruent with previous morphological and molecular hypothesis of the relationships among trichomycterids (Eigenmann 1918; Myers 1944; Baskin 1973; Fernández & Schaefer 2009), with some differences in the relationships among subfamilies and genera. Monophyly of the Trichomycteridae and the subfamilies Copionodontinae (100% of the genera sampled), Stegophilinae (81.8% of the genera sampled), Vandelliinae (50% of the genera sampled), Tridentinae sensu stricto (Baskin, 1973; 75% of the genera sampled) and Trichomycterinae sensu Datovo & Bockmann (2010) and Ochoa et al. (2017) are supported by maximum values of bootstrap (100%) and posterior probabilities (p=1). Our analysis of UCE data provide strong evidence for the monophyly of the clade B, composed by Copionodontinae and Trichogeninae (clade B, by Datovo & Bockmann 2010) as the sister group to all remaining Trichomycterids (100% bootstrap support and posterior probabilities of 1.0). Copionodontinae Chapter 1 20 and the genus Copionodon (C. pecten, C. orthiocarinathus and C. sp. n) are both recovered as monophyletic. Clade C is recovered with a basal dichotomy between the Trichomycterinae and TSVSG clade. Our phylogenomic data provide strong evidence for the monophyly of Trichomycterinae (bootstrap=100%, posterior probabilities p=1) as defined by the morphological study of Datovo & Bockmann (2010) and the recent multilocus analysis of Ochoa et al (2017). This definition of the subfamily includes Ituglanis and Scleronema and excludes Potamoglanis. The latter genus was recently erected (Henschel et al. 2017) to include miniature species group previously allocated in Trichomycterus and often referred to as the Trichomycterus hasemani group. Our study confirms the non-monophyly of Trichomycterus, the largest genus of the Trichomycteridae that concentrates 72% of the family diversity. In our topology, an undescribed trichomycterine (currently in analysis by DoNascimiento) from the coast in Venezuela is placed as sister to all remaining trichomycterines, which are grouped into two major lineages. The first lineage is further subdivided into six main clades. Four of these clades appear as successive sister taxa: the first clade in diversify includes Trichomycterus cachirensis, T. sandovali, and the monotypic Eremophilus mutisii, all from the Magdalena basin (Clade D1’); the second joins T. guianensis and T. cf guianensis from the Essequibo basin (Clade D2); the third, T. trasandianus and T. aff. spilosoma from Magdalena basin and Dos bocas river in Ecuador repectively (Clade D2’); and the fourth T. striatus, T. ruitoquensis, T. banneaui, and T. sp. 1 from the Magdalena basin (clade D2’’). A group of five undescribed species of Trichomycterus from Paraná-Paraguay basin forms a monophyletic group that is sister to Chilean Clade E, composed by Bullockia maldonadoi, T. aerolatus, and T. chiltoni. Finally, the monophyletic Ituglanis is located at the apical portion of this major trichomycterine lineage. The sampled species of the genus are grouped into four main clades: one containing the species from the Tocantins basin (I. goya, I. ramiroi, and I. sp. 1); other with species from coastal Atlantic drainages (I. boitata, I. parahybae, and I. sp. Ribeira) and an undescribed species from the Amazon basin (I. sp. 2); the third and fourth clades combine species from the Amazon and La Plata system, being Ituglanis cf amazonicus, I. eichhorniarum plus two undescribed species (I. sp. 3 and I. sp. 4) in one clade and I. amazonicus, I. parkoi, I. herberti and an undescribed species (I. sp. 5) in the other. The second major trichomycterine lineage includes two successive basal clades, being one composed by T. punctulatus (Central Andean Pacific slopes) and T. cf. knerii (Orinoco), and another by T. cf. oroyae and T. quechuorum (both from Amazonas High Andes) (Clade D1). Chapter 1 21 Remaining taxa are grouped into a large clade that includes Scleronema and all species of Trichomycterus from the La Plata, Northeastern Atlantic, and Southeastern Atlantic provinces. Clade D4 clusters Scleronema (S. minutum) and several species of Trichomycterus from southeastern Brazil (T. inhering, T. balios, T. poikilos, T. perkos, T. davisi, T. zonatus, T. stawiarski and T. cubataonis). Clade D5 contains three main subclades: the first includes T. nigroauratus, T. pradensis, T. albinotatus, T. alternatus, T. cf. auroguttatus, T. mimosensis, and T. immaculatus; the second T. brasiliensis, T. cf. brasiliensis, T. pirabitira, T. candidus, plus four undescribed species (T. sp. n. Grande, T. sp. SF, T. sp. 6, and T. sp. 7); the third T. reinhardti, T. pauciradiatus, T. piratymbara, T. septemradiatus, T. cf. septemradiatus, and one undescribed species (T. sp. 8). The TSVSG clade includes the subfamilies Tridentinae, Stegophilinae, Vandelliinae, Sarcoglanidinae and Glanapteryginae. In our analysis, we included five representatives of the three currently recognized glanapterygine genera, including the most generalized Listrura (L. camposi and L. picinguabae) and the highly derived psammophilic Typhlobelus (T. guacamaya) and Pygidianops (P. slender, and P. sp); only Glanapteryx could not be sampled. For Sarcoglanidinae we included half of the genera (Sarcoglanis simplex, Stauroglanis gouldingi and Microcambeva barbata), lacking Stenolicmus, Malacoglanis and Ammoglanis. Additionally, a new undescribed genus seemingly belonging to the Glanaperyginae (Trichomycteridae n. gen.; de Pinna & Datovo; pers. comm.) was incorporated to our study. The resulting hypothesis recovers the monophyly of Tridentinae, Stegophilinae and Vandelliinae with exception of Glanapteryginae and Sarcoglanidinae. Representatives of both subfamilies from coastal Atlantic drainages (Southeastern South America) are grouped in one clade and those from the Amazon and Orinoco into another group along with Potamoglanis from Amazon and Paraguay (Northwestern South America). The Southeastern lineage is at the base of the TSVSG clade and includes Trichomycteridae n. gen., Microcambeva barbata (Sarcoglanidinae) and the two species of Listrura (Glanapteryginae). The Northwestern clade clusters Potamoglanis hasemani, Stauroglanis gouldingi, Sarcoglanis simplex, Typhlobelus guacamaya and the two Pygidianops species. This clade is the sister to the so-called Vandelliinae-group, a node with strong support that includes the Tridentinae, Stegophilinae, and Vandelliinae, with the last two subfamilies appearing as sister taxa. Our results not support the recently allocation of Potamoglanis hasemani in the Tridentinae contra Henschel et al (2017) and resolve Tridens sp. as sister to the clade formed by Tridensimilis brevis and Tridentopsis pearsoni. Chapter 1 22 With a representation of nine among the 11 genera of the Stegophilinae (only Schultzichthys and Apomatoceros are missing), the internal relationships of the subfamily is well resolved and mostly in agreement with a recent morphological phylogeny of the group (DoNascimiento 2015). Our hypothesis divides the Stegophilinae in two major groups. The largest group contains Homodiaetus (Ho. passarellii and Ho. anisitsi) at the base and two subclades: one composed by monotypic Megalocentor echthrus and Henonemus (He. intermedius, He. punctatus, and He. sp. 1) and the second by Pareiodon as sister of Pseudostegophilus + Acanthopoma. The latter genus is monotypic and included into a non-monophyletic Pseudostegophilus (P. haemomyzon, P. nemurus, P. paulensis, and P. sp.). The second major stegophiline group clusters Ochmacanthus (O. reinhardti, O. sp. 1, O. sp. 2, and O. sp. 3) as sister group of the clade composed by Stegophilus panzeri and the monotypic Haemomaster venezuelae. Two of the four vandelliine genera were included in our analysis, Paracanthopoma and Vandellia. Monophyly of both genera and the whole subfamily are strongly supported, but species-level interrelationships showed low support. 1.3.2 Speciation and extinction rates in Trichomycteridae Divergence times were very similar with the reported by Ochoa et al (2017), dating the diversification of Trichomycteridae family during the Lower Cretaceous about 107.52 Myr (93.9-120.61, 95% HPD) (Figure 1.5) around the time of the continental separation between Africa and South America. The oldest split within Trichomycteridae was estimated at 66.63 Myr (43.76-90.5, 95% HPD) and established the clade Copionodontinae+Trichogeninae subfamilies. Subsequently, the clade C composes by the remaining trichomycterids diversified in two big clades, the first group including Tridentinae, Stegophilinae and Vandelliinae, as well as, the representative species from Sarcoglanidinae and Glanapteryginae; and the second group represented by Trichomycterinae subfamily, which diversified during the Paleocene (41.97 Myr, 28.62-55.02, 95% HPD). BAMM analyses strongly supported a diversity-dependent speciation process across Trichomycteridae with a net diversification rate of 0.131 species/Myr (0.105-0.164, 95% HPD) and extinction rate of 0.041 species/Myr (0.009-0.088, 95%HPD). The highest speciation rates are seen in Trichomycterinae node (0.214 species/Myr) and the sister group Copionodontinae+Trichogeninae (0.087species/Myr). These two clades also show the highest extinction rates (0.045 species/Myr) (Table 1.3). The TSVSG clade exhibits the lowest Chapter 1 23 speciation and extinction rates within the family. The changes in speciation rates (cool colors=slow, warm=fast) along each branch of the Trichomycteridae phylogeny can be observed in the figure 1.2A. The 95% credible set of rate shift configurations sampled with BAMM included eight distinct shifts of which the configuration with the highest probability included two shifts. The figure 1.2B shows the shifts both along the stem of Trichomycterinae with a frequency f=0.94 and f=0.058 respectively. Although the specific placements are at different times, both rate shifts occur during Oligocene and Miocene. Rate-through-time plots (Figure 1.2C) revealed a constant speciation rate until ~14Mya, at which point speciation and net diversification rate began to increase with the highest average rate (~0.065 species/My) seen at present. The figure 1.2D representing the branch-specific marginal shift probabilities, the length of each branch represents the percentage of samples from the posterior that contain a rate shift; in this case, the large branch of Trichomycterinae corroborates the probability of rate shift in this clade. Rates of body size (SL) evolution exhibited a single background rate characterized by a low diversification for all subfamilies with exception of Trichomycterinae, where the T. aerolatus clade composes by T. aerolatus, T. chiltoni and Bullockia maldonadoi showed an increase in diversification rates (Figure 1.6). The results show nine shift configurations that account for more than 95% of samples and, all configurations show slowdowns in the family with subsequently increase in the T. aerolatus clade. The best shifts configurations are showed in gray, with shifts in Stegophilinae, Vandelliinae and three different shifts in the stem and recently diversified clades in Trichomycterinae. In the ancestral reconstruction of feeding modes, we evaluated three different models for discrete comparative data and the Equal Rates (ER) model showed the best fit to data with AIC=114.3731 (Table 1.4). The ER model assume a single parameter governs all transition rates and below this model we simulated 100 stochastic character maps. The aggregate map (Figure 1.7) suggests that the ancestral feeding mode should be considered as insectivorous. Subsequently, this was followed by the acquisition of the most specialized modes as lepidophagous and hematophagous, with an increase in the omnivorous, one of the most generalized trophic habits. Chapter 1 24 1.4 Discussion 1.4.1 Phylogenetics relationships in Trichomycteridae The present phylogenomic analysis recovered almost fully resolved trees with two different methods of phylogenetic inference (ML and B). The topology obtained is congruent with previous hypothesis of trichomycterid relationships based on morphology (Eigenmann 1918; Myers 1944; Baskin 1973; de Pinna 1992, 1998; Datovo & Bockmann 2010) and molecular datasets (Fernández & Schaefer 2009; Ochoa et al. 2017). The monophyly of the Trichomycteridae is supported by maximum values of bootstrap (BS=100%) and posterior probabilities (P=1). This result is congruent with all morphological studies, which provide a high number of unequivocal synapomorphies for the family (Eigenmann 1918; Myers 1944; Baskin 1973; de Pinna 1992, 1998; Datovo & Bockmann 2010). Our dataset support the monophyly of all subfamilies with the exception of Glanapteryginae and Sarcoglanidinae, and shows a perfect correspondence of the relative position of the early diverging branches with the most recent morphological and molecular hypothesis of the family (Datovo & Bockmann 2010; Ochoa et al. 2017), where the clade composed by Copionodontinae and Trichogeninae is the sister group of the clade C (Datovo & Bockmann 2010) clustering the remaining trichomycterids, represented by the TSVSG group (Tridentinae, Stegophilinae, Vandelliinae, Sarcoglanidinae, Glanapteryginae) (Costa & Bockmann 1994) and Trichomycterinae. The Copionodontinae and Trichogeninae have been considered basal lineages within the Trichomycteridae, and members of these subfamilies exhibit several morphological conditions that are intermediate between those present in remaining trichomycterids and other loricarioids (Eigenmann 1918; Myers 1944; Baskin 1973; de Pinna 1992, 1998; Datovo & Bockmann 2010). Clade C gathers all members of the Trichomycteridae except the basal Copionodontinae and Trichogeninae. This clade is basally divided into two lineages: Trichomycterinae (sensu Datovo & Bockmann 2010) and TSVSG clade. Strong morphological and molecular evidence support the monophyly of the clade, which has been unanimously recovered in all analyses of the Trichomycteridae including the present study. The longstanding controversy in the relationships in Trichomycterinae (Baskin 1973; de Pinna 1989, 1998), began to be elucidated with the support of its monophyletic status by morphological characters (Datovo & Bockmann 2010), as well as, the molecular evidence with the inclusion of genera Scleronema and Ituglanis in this subfamily, and the exclusion of Chapter 1 25 Potamoglanis (previously referred to as Trichomycterus hasemani group).This three trichomycterine subgroups were more explicitly proposed as aligned with the clade TSVSG, however, the relationship of Scleronema and Ituglanis with the clade TSVSG was rejected by Datovo & Bockmann (2010) who provided morphological evidence for the grouping of these genera with the remaining trichomycterines. In contrast, the inclusion of Potamoglanis within the TSVSG clade is supported by morphological (de Pinna 1989, 1998; Datovo & Bockmann 2010; DoNascimiento 2015) and molecular hypothesis (Henschel et al. 2017; Ochoa et al. 2017). On the other hand, the relationship within Trichomycterinae has never been extensively surveyed by any publication based on morphological data, notwithstanding some suggestion of small putative subgroups with restrict geographic distribution have been proposed (e.g.; (de Pinna 1989; Costa 1992; Wosiacki 2002; Fernández & Osinaga 2006; Wosiacki & De Pinna 2008; Barbosa & Costa 2010; Datovo, Carvalho & Ferrer 2012). In recent molecular analysis by Ochoa et al (2017) including a substantial taxonomic sampling of the subfamily, with a total of 70 trichomycterine terminals, were identified two major lineages and six main subclades (D1, D2, D3, D4, D5, and E). The genomic analysis expands this previous sampling of trichomycterines in about 15% and the resulting subfamily tree exhibits only three most significant divergences relative to that of Ochoa et al. (2017): the inclusion of an undescribed trichomycterine from the Caribbean coast in Venezuela (previously unsampled) at the base of the whole subfamily and the non-monophyly of Clades D1 and D2. Our dataset recovers the two major clades identified in Ochoa et al (2017) with a strong node support, the first including Eremophilus, Bullockia, Ituglanis, and most northern species of Trichomycterus; and the second, composed by Scleronema and all remaining species of Trichomycterus (predominantly from southeastern South America). Previous morphological analysis (Costa & Bockmann 1994; de Pinna 1998; Datovo & Bockmann 2010; DoNascimiento 2015) and molecular studies (Fernández & Schaefer 2009; Henschel et al. 2017; Ochoa et al. 2017) have supported the monophyly of the TSVSG clade, a lineage composed by Tridentinae, Stegophilinae, Vandelliinae, Sarcoglanidinae and Glanagapteryginae, our phylogenomic hypothesis strongly support this group as well. One of the most important changes in the relationship of TSVSG clade was the incorporation of the clade Potamoglanis (formely referred to as the Trichomycterus hasemani- group) as sister clade of some sarcoglanidines in molecular analysis (Ochoa et al. 2017). Chapter 1 26 Previous hypothesis about the relationship of this clade with TSVSG group were proposed initially by De Pinna (1989), who suggested the inclusion of Potamoglanis in Tridentinae, based in the following characters shared for both clades: the small size and the presence of a single enormous cranial fontanela. Subsequently, DoNascimiento (2015) in a morphological analysis including 520 characters and 49 terminal taxa, allocated Potamoglanis at the base of the entire TSVSG clade, refuting de Pinna’s (1989) hypothesis. Most recently, molecular studies by Henschel et al. (2017) recovers Potamoglanis as sister to tridentines and ignoring previous molecular results formally erected the genus Potamoglanis, as a subgroup of Tridentinae. Nevertheless, this phylogenomic hypothesis support a result similar to that of Ochoa et al. (2017) in grouping Potamoglanis with the sarcoglanidines and glanapterygines from Amazon and Orinoco. In light of these conflicting hypotheses of relationships for Potamoglanis, we consider the assignment of this group to any trichomycterid subfamily premature. In this way, for the sake of nomenclatural stability, the present paper follows the traditional concept of the Tridentinae, that is, not including Potamoglanis. Within the TSVSG clade morphological studies traditionally have proposed the sister relationship between Glanapteryginae and Sarcoglanidinae, recognized as Glanapteryginae- group (Baskin 1973; Costa & Bockmann 1994; de Pinna 1998; Datovo & Bockmann 2010). However, the most recent morphological analysis (DoNascimiento 2015) and molecular phylogenies of the Trichomycteridae (Henschel et al. 2017; Ochoa et al. 2017) refuted the monophyly of the Glanapteryginae-group. DoNascimiento (2015) resolved the Sarcoglanidinae and Glanapteryginae are successive sister taxa to the Vandelliinae-group. In the topology of Henschel et al. (2017), tridentines and Potamoglanis are intercalated between glanapterygines (at the base of the TSVSG clade) and sarcoglanidines. The Glanapteryginae is also placed at the base of the TSVSG clade in the hypothesis of Ochoa et al. (2017). In this scheme; however, the Sarcoglanidinae is not monophyletic, with Stauroglanis appearing closer to vandelliines and Sarcoglanis to Potamoglanis. The present analysis, which has the largest taxonomic sampling of both subfamilies to date, obtained an even more striking result in which neither Sarcoglanidinae nor Glanapteryginae are monophyletic. Members of both subfamilies are clustered into two clades that are successive sister taxa to the Vandelliinae-group. Interestingly, sarcoglanidines and glanapterygines from the Atlantic coastal drainages (Microcambeva, Listrura, and trichomycterid n. gen.) are grouped into one clade, whereas the second clade gathers together members of both subfamilies from the Amazon and Orinoco (Sarcoglanis, Stauroglanis, Typhlobelus, and Pygidianops) along with Potamoglanis (Amazon Chapter 1 27 and Paraguay). The sarcoglanidines and glanapterygines grouped in the latter clade curiously share several reductive features, such as extreme reductions in the number of opercular odontodes, interopercular odontodes, premaxillary teeth, and pigmentation (Costa, 1994; de Pinna, 1989). The dismantling of the Glanapteryginae-group is a drastic change that obviously demands further investigation, but this result is not so surprising at all. A critical appraisal of the osteological characters listed to support various nodes of the Glanapteryginae-group tree demonstrates that several putative synapomorphies are of difficult polarization and known to exhibit some degree of homoplasy (de Pinna 1989, 1998; Costa & Bockmann 1994; de Pinna & Winemiller 2000b). Moreover, diagnoses and interrelationships among the putative basal most genera of the Glanapteryginae and Sarcoglanidinae are particularly unstable and the limits between each subfamily is increasingly blurry. For instance, new data suggest that Ammoglanis pulex is actually a glanapterygine, rather than a sarcoglanidine as originally described (de Pinna, 2016). Allocation of newly discovered taxa (e.g., trichomycterid n. gen.) into one or another subfamily is often difficult and possibly arbitrary (pers. obs.; de Pinna, 2016). In spite of the new relationships showed in this study are strongly supported, decisions about taxonomic boundaries are often less than ideal due to the absence of the keys taxa in both subfamilies (Glanapteryx, Ammoglanis, Malacoglanis and Stenolicmus). However, this results insight the importance of a comprehensive taxonomic examination of Glanapteryginae and Sarcoglanidinae. In contrast with the myological evidence presented by Datovo & Bockmann (2010) and the molecular support for the hypothesis of a tridentine-stegophiline group (Ochoa et al. 2017), our data recovered with a strong support the monophyly of the Vandelliinae-group as proposed by Baskin (1973) including Tridentinae as sister group of the candiru subfamilies and reinforce the molecular evidence presented by Fernandez & Schaefer (2009) to support the monophyly of Stegophilinae and Vandelliinae, a clade sharing derived conditions of the mesethmoid conua, maxillary and rictal babrbels, restricted gill openings, branchiostegal membrane lacking a free edge (Baskin 1973) and the presence of a median premaxilla (de Pinna 1998). Even though subfamilial analysis is beyond the scope of this study, our results show interesting results in the vandelliinae-group. Our study included three of the four genera of Tridentinae; only Miuroglanis was unavailable. According to Baskin (1973) derived characters as, a greater number of anal rays, the origin of the anal fin anterior to the dorsal origin, the ventral exposure and distinctly larger eyes support the monophyletic Tridens-group clustering Tridens, Tridensimilis and Tridentopsis; in so far as Miuroglanis shows primitive conditions in each of Chapter 1 28 these characters. As well as the following characters: fewer opercular teeth, rictal babel not visible externally, eyes face more ventrally than dorsally, Weberian capsule with an elongate neck and anal fin origin three or more vertebrae anterior to dorsal origin support the relationships within Tridens-group with Tridensimilis more closely related to Tridens than to Tridentopsis. Our results not support the morphological hypothesis of relationships among Tridens-group, with Tridensimilis more related to Tridentopsis than to Tridens involving a strong node support for this scheme of relationships. Regarding Vandelliinae, we included only two genera of the four, missing Plectrochilus and Paravandellia. Morphological hypothesis proposed by Schmidt (1993) support the sister- group between Paracanthopoma and Pletrochilus plus Vandellia based on the loss of median premaxillary teeth, proximal end of premaxilla and ethmoid cornua both forked, reduced numbers of dentary teeth, and interopercular odontodes directed posterior. Our hypothesis of interrelationships among stegophilines is almost identical to of the comprehensive revision of the subfamily published by DoNascimiento (2015). In both analyses, the Stegophiline has a basal dichotomy with a clade clustering Haemomaster, Ochmacanthus, and Stegophilus and another grouping all remaining genera. The only difference between the two topologies is the placement of the monotypic Acanthopoma among the species of Pseudostegophilus in our analysis. By contrast, DoNascimiento’s (2015) tree allocate Acanthopoma in a basal polytomy with Pareiodon and a monophyletic Pseudostegophilus. 1.4.2 Diversification pattern in Trichomycteridae Patterns of species diversity have been shaped by both speciation and extinction throughout the history of life, and one of the key questions in evolutionary biology is to understand the temporal and spatial dynamics of these processes (Nee, Mooers & Harvey 1992; Sanderson & Donoghue 1996; Barraclough & Nee 2001; Jablonski et al. 2003; Ricklefs 2007). Additionally, the fossil record and molecular phylogenetic data of extant lineages can provide valuable information on the process of diversification in form of branch length and the distribution of divergence times throughout the evolutionary history of a clade (Silvestro, Schnitzler & Zizka 2011). Relevant issues in detecting significant rate shifts include incorporating extinction, phylogenetic uncertainty, phylogenetic scale, sampling density, correlation and/or causality of biotic or niche attributes driving the rate shifts (Barraclough & Nee 2001). The program BAMM, as now implemented, can address a number of these issues. Chapter 1 29 Trichomycteridae is an ideal candidate for comparative evolutionary studies considering its relatively old origins in the Lower Cretaceous, a widespread biogeographical distribution, occupation of diverse habitats, considerable morphological diversity, the presence of both species-rich and depauperate clades, and now a well-supported phylogeny. The analysis of diversification rates in Trichomycteridae shows a high clade heterogeneity. The rates-throughout-time analysis exhibited long term speciation rates decline in the TSVSG and Copionodontinae+Trichogeninae clades with an extinction rate constant throughout its entire 66 Myr diversification until the present; in comparison, it was identified an increase in the speciation rate and evolutionary shift in Trichomycterinae, a clade is characterized by its species richness. The average rate of net diversification for Trichomycteridae is not striking (0.131 lineage/ Myr). This rate is similar to those estimated by Rabosky et al (2013) in an extensive analysis correlating diversification rates across approximately ~30.000 living species of ray-finned fishes. In a macroevolutionary context, shift in diversification rates in a phylogeny should be positively associated with phenotypic evolution (Eldredge & Gould 1972; Pennell, Harmon & Uyeda 2013). Regardless of the underlying causal mechanisms, a growing body of empirical research suggests that species diversification and morphological evolution are frequently coupled in nature (Rabosky et al. 2014b). Recent studies demonstrated that rates of species diversification are highly correlated with the rate of body size evolution across the 30,000 living species of ray-finned fishes that comprise the majority of vertebrate biological diversity (Rabosky et al. 2013). Despite the coupling in species diversification rates and body size evolution seem to be a general feature, our phylogeny not support the correlation of different regimen in diversification rate with the evolution of body size in Trichomycteridae. Researches in mammals, cetaceans, squamates and salamanders have shown similar results, decoupling morphological diversification from speciation and suggest that the processes that give rise to the morphological diversity of a class of animals are far more free to vary than previously considered (Adams et al. 2009; Venditti, Meade & Pagel 2011; Rabosky & Adams 2012; Burbrink et al. 2012). Evolutionary hypotheses related to the evolution of miniature species have been proposed for Trichomycteridae (Weitzman & Vari 1988; de Pinna 1998); however, little is known about the relationship between morphological evolution and speciation rates in this group. Chapter 1 30 The diversification rates in Trichomycterinae are probably influenced by ecological traits as its wide geographical distribution, occupying different types of environments and generalist trophic habit. According to Rundell & Price (2009) geographic range expansions is a critical part of “successful” speciation, but it may require that recently separated species are undergo ecological or morphological divergence, before sympatry is possible (Podrabsky, Garrett & Kohl 2010). This way, one interpretation of the higher speciation rates in Trichomycterinae can be related with the progressive colonization of a new regions and generalist trophic habit. According to ancestral reconstruction of feeding modes, Trichomycterinae exhibits a more generalist trophic habit with insectivorous and omnivorous species, that in combination with its wide geographical distribution and ecological diversity could have contributed in the increase of diversification rates. In comparison, clades with more specialized feeding (algivorous, hematophagous, lepidophagous and carrion feeders) have major constraint in its response to environmental changes. Most ecological studies indicate that specialists are more sensitive to extinction under environmental changes, as consequence of small size of local populations, an often restricted geographic range, and a limited utilization of resources and habitats (Colles, Liow & Prinzing 2009). In summary, our analysis showed that the diversification rates in Trichomycteridae are heterogeneous and diversity dependent, as it was evidenced in the increasing of diversification rates in Trichomycterinae. Our results do not show a direct association between speciation rates and body size evolution, probably that Trichomycteridae was predisposed to diversify in ways that do not involve adaptive divergence as might result from ecological specialization and allopatric speciation. 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