PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO GRANDE DO SUL FACULDADE DE BIOCIÊNCIAS PROGRAMA DE PÓS-GRADUAÇÃO EM ZOOLOGIA

FILOGENIA DE DUAS SUBFAMÍLIAS DE CASCUDOS (SILURIFORMES, ), USANDO DADOS NUCLEARES, MITOCONDRIAIS E MORFOLÓGICOS

Christian Andreas Cramer Orientador: Dr. Roberto E. Reis Co-orientador: Dr. Sandro L. Bonatto

TESE DE DOUTORADO PORTO ALEGRE – RS – BRASIL 2009 PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO GRANDE DO SUL FACULDADE DE BIOCIÊNCIAS PROGRAMA DE PÓS-GRADUAÇÃO EM ZOOLOGIA

FILOGENIA DE DUAS SUBFAMÍLIAS DE CASCUDOS (SILURIFORMES, LORICARIIDAE), USANDO DADOS NUCLEARES, MITOCONDRIAIS E MORFOLÓGICOS

Christian Andreas Cramer Orientador: Dr. Roberto E. Reis Co-orientador: Dr. Sandro Luis Bonatto

TESE DE DOUTORADO PORTO ALEGRE – RS – BRASIL 2009

AVISO

Esta tese é parte dos requisitos necessários para obtenção do título de doutor, área de 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). Dessa forma, quaisquer informações inéditas, opiniões e hipóteses, assim como nomes novos, não estão disponíveis na literatura zoológica. Pessoas interessadas devem estar cientes de que referências públicas ao conteúdo desse estudo, na sua presente forma, somente devem ser feitas com aprovação prévia do autor.

NOTICE

This thesis is a partial requirement for the PhD degree 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 are unavailable in the zoological literature. 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. SUMÁRIO

Agradecimentos ...... ii

Resumo ...... v

Abstract ...... vi

Apresentação ...... 1

Capítulo I - The phylogenetic relationships of the and (Siluriformes: Loricariidae) as inferred from mitochondrial cytochrome c oxidase I sequences ...... 5

Capítulo II - Molecular Phylogeny of the Neoplecostominae and Hypoptopomatinae (Siluriformes: Loricariidae) using Multiple Genes ...... 15

Capítulo III - A Total Evidence Phylogeny of the Neoplecostominae and Hypoptopomatinae (Siluriformes: Loricariidae) ...... 54

Conclusões Gerais ...... 98

Referências ...... 101

i Agradecimentos

Agradeço aos meus orientadores pela ajuda na conclusão da minha tese. Sou um dos sortudos alunos que pôde confiar em duas pessoas ‐ Roberto Reis como orientador e Sandro Bonatto como co‐orientador. Sem eles nunca teria chegado ao meu objetivo

O Roberto, responsável pelos assuntos ictiológicos, deu‐me total apoio, sugerindo a mudança inicial do projeto, organizando coletas para aumentar a amostragem de espécies. Sua paciência com minha ignorância inicial sobre a subfamília Neoplecostominae, a assistência a todos os problemas que surgiram nesse período e todas as discussões até a revisão da minha tese foram fundamentais para o desenvolvimento do meu trabalho.

A área do Sandro era a parte molecular. MUITAS coisas não funcionaram durante os 54 meses, como os PCR que não funcionaram ou que mais pareciam com marcadores moleculares, purificações que fizeram o DNA desaparecer e dúvidas na análise dos dados. A sua ajuda foi essencial e seu papel foi diferente, mas com certeza não foi mais fácil.

Lothar Beck (Universidade de Marburg) apoiou‐me à distância, inclusive com uns kits absurdamente caros por aqui.

Agradeço aos meus colegas do Genoma e da Ictiologia pela convivência. Muitos deles viraram amigos importantes, alguns de imediato, outros só com o tempo. A ajuda deles vem desde conversas sobre Deus e o mundo para esvaziar a cabeça a discussões sobre assuntos biológicos em geral, problemas específicos (que não faltaram neste tempo!), dicas, idéias, críticas e soluções práticas. Felizmente, descobri uma moeda forte para retribuir esses favores: meus bolos, tiramisus e outros doces sempre foram aceitos em troca.

Ainda merecem ser citadas algumas pessoas que foram muito importantes nessa jornada:

A Cristina e o Val, que além de me ajudarem nos assuntos da biologia, também foram ótimos parceiros para passeios e cinema. Espero poder visitá‐los novamente em Manaus!

A Mónica, mais uma integrante internacional do nosso laboratório multicultural, logo virou uma amiga próxima. Com a realização de uma viagem para o norte da , provamos que trabalho e lazer não se excluem.

Outros “espeixalistas” importantes que renderam muitas conversas, discussões e risadas e aos quais não posso deixas de referir, são: Bárbara, Edson, Tiago e Vivianne. A Vivi nem sabe quanta falta fez nos meses que passou nos EUA. Ainda há os outros ictiólogos, cascudólogos ou não, que,

ii de alguma forma, fizeram parte e deixaram marcas em minha vida e no meu trabalho: Alexandre Cardoso, Alexandre Charcansky, Aloísio, Fernanda, Fernando, Giovanni (peixes voadores), Héctor, Ignácio (peixes grandes e vivíparos), José, Juliana, Maria Laura, Mariangeles, Marta, Natália, Pablo, Vinicius e o casal Lucena. Em especial, ao Edson, ao Pablo e à Vivi, que tiveram um papel fundamental nas semanas de correria antes de terminar essa tese, meu sincero agradecimento.

No Genoma, já pela quantidade de pessoas, foi mais difícil conhecer bem todo mundo. Mas mesmo assim, muitos dos genômicos viraram meus amigos. Quem contribuiu significativamente para meu trabalho e evitou que eu pirasse totalmente, foram Cladinara, Fernanda Britto, André e Mirian. Mirian é uma das pessoas mais pacientes que conheço e a ajuda dela foi importantíssima para meu trabalho.

A Cacá virou uma amiga e parceira importante, sempre cheia de surpresas. Eu queria ter seu poder de superar obstáculos!

A Katu é uma pessoa extremamente querida que não quero imaginar longe da minha vida. Depois de Réveillon e carnaval espero ter mais possibilidades para passar tempo junto com ela e o Gabi.

A Déa sempre está bem humorada e garante boas risadas. Nem depois de 500 perguntas e pedidos de lotes da UFRGS ela perdeu a paciência comigo!

A famíla Richetti salvou meu Natal 2008 me adotando para passar esta festa em família. Só quem já passou Natal longe de casa sabe o significado desse gesto.

Não há espaço para entrar em detalhes sobre todos que, de alguma maneira, me animaram ou foram parceiros. Logo, se deixei de mencionar algum nome, isso não significa que ele ou ela não tenham sido relevantes para mim: Ana Lúcia, Ane Hahn, Cris, Fernanda Pedone, Henrique, Lari, Lisie, Luisa, Manoel, Marina, Paola, Taiana, Talita, Thomaz,...

Alexandros Stamatakis, Felipe, Pablo Goloboff, Ricardo e, especialmente, Taran pacientemente responderam todas as minhas perguntas filogenéticas e me deram várias dicas além.

Os seguintes colegas, amigos, instituições e empresas emprestaram ou doaram material: Jon W. Armbruster (AUM), Paulo Buckup (MNRJ), Raphael Covain (MNHG), Michael Hardman (INHS), Francisco Langeani (DZJRP), Nathan Lovejoy (UTSC), Margarete e Carlos (MCP), Gladys Monasterio de Gonzo (MCNI), Dirk Neumann (ZSM), Claudio Oliveira (LBP), Mark Sabaj (ANSP), Frank Schäfer/Aquarium Glaser (Rodgau, Germany), Ingo Seidel/Aqua Global (Seefeld, Germany), Oscar Shibatta (MZUEL), Andréa Thomaz (UFRGS), Thomas Weidner (Iffeldorf, Germany), André Werner/Transfish (Planegg, Germany) e Claudio Zawadski (NUP).

iii Um apoio elementar foi o financiamento do meu projeto pela Gesellschaft für Ichthyologie (Sociedade Alemã de Ictiologia; ajuda inicial), pelo DAAD (Serviço Alemão de Intercâmbio Académico; dois anos de bolsa) e pelo CNPq (bolsa de um ano).

Além da faculdade, outros amigos tiveram forte influência na minha vida aqui no Brasil.

A Sibele virou uma irmã para mim. As muitas horas das nossas conversas me ajudaram a superar muitas baixas e ela é uma companheira fiel para cinema, jantares, passeios. Nas últimas semanas ela me socorreu me emprestando seu laptop quando o meu decidiu fazer motim.

A Anna me acompanhou, animou e aguentou por quase dois anos. Foi um tempo muito importante para mim.

Outros amigos fiéis são o Luiz, a Luiza e a Thais e não posso deixar de lembrar Timóteo e Vinicius, meus parceiros de apartamento.

Quanto mais longe se passa dos amigos, melhor se percebe quais são as amizades mais firmes. Verdadeiras amizades não dependem de espaço ou de tempo. Lembro então dos amigos especiais que ficaram na Alemanha como o Dirk, a Mónica, a Thomais e a Verena. Pena que eu não tenho mais possibilidades de passar tempo com eles!

Last, but not least, quero agradecer à minha família. Sei que meus pais Ursula e Claus Cramer sofrem bastante com a distância, mas mesmo assim sempre me apoiaram e me motivaram, seja pessoalmente, por email, por telefone ou com remessas de chocolate nos momentos cruciais. E minha irmã Eva Cramer não é só uma irmã, mas também uma das minhas melhores amigas. Infelizmente ela também encontrou o seu paraíso longe da casa de nossos pais, de maneira que fica ainda mais complicado o nosso encontro que se dá somente uma vez por ano!!

Muito obrigado, muchas gracias, vielen Dank!!!

iv Resumo

Loricariidae é uma das mais diversas famílias de peixes, atualmente incluindo cerca de 800 espécies reconhecidas. Os loricarídeos são popularmente conhecidos como cascudos ou acaris e têm tamanho de poucos centímetros a mais que um metro. São encontrados somente em água doce, com ampla variação tanto na temperatura, como tipo de ambiente, ou seja, podem ser encontrados desde em córregos frios das montanhas até em lagos da área de inundação do rio Amazonas. A distribuição do grupo é ampla, abrangendo praticamente todas as bacias hidrográficas da América do Sul e parte da América Central, da Argentina até a Costa Rica. Apesar das primeiras espécies terem sido descritas por Linnaeus em 1758 e do trabalho realizado deste então, especialmente nos últimos 15 anos, as relações filogenéticas dos loricarídeos estão apenas parcialmente resolvidas. Os resultados das análises moleculares são conflitantes com os agrupamentos propostos pelas análises morfológicas e um consenso ainda não foi alcançado. Os grupos mais problemáticos são as subfamílias , Hypoptopomatinae e Neoplecostominae. Os estudos morfológicos e moleculares sugerem que Hypoptopomatinae e Neoplecostominae formam um grupo monofilético, porém a monofilia das mesmas ainda é incerta. Por esta razão, o principal objetivo da presente tese é testar a monofilia destas duas subfamílias atráves de uma análise de evidência total. Num primeiro estudo, sequências de um fragmento de 709 pares de bases da primeira subunidade do gene mitocondrial citocromo c oxidase (COI) foram usadas. As análises de Máxima Parcimônia (MP) e de Máxima Verossimilhança (ML) incluíram dados de 83 espécies, pertencentes a 29 gêneros, representando cinco subfamílias de Loricariidae. Adicionalmente, oito espécies de quatro famílias próximas foram usadas como grupo externo. Os resultados confirmaram Delturinae como a subfamília mais e mostraram a Hypoptopomatinae + Neoplecostominae como grupo irmão de Hypostominae. Corroborando estudos moleculares anteriores, Neoplecostominae é monofilética somente quando incluído o gênero Pseudotocinclus (Hypoptopomatinae). Neoplecostominae ficou inserida dentro de Hypoptopomatinae, que assim formou um grupo parafilético. Num segundo estudo, foram adicionados mais táxons e sequências parciais dos genes nucleares recombination activating gene 1 (RAG1) e 2 (RAG2) e F-Reticulon 4, resultando na análise de 136 espécies e 4678 pares de bases. As análises de MP, ML e Bayesianas confirmaram a maioria dos resultados moleculares anteriores, exceto pela polifilia de Pareiorhaphis, Neoplecostomus e Neoplecostominae. A análise final dos dados constituiu uma análise de evidência total, com o intuito de compreender melhor os conflitos entre as análises morfológicas e moleculares e encontrar, assim, uma solução mais robusta para o grupo. O novo conjunto de dados inclui 207 espécies e sequências concatenadas de COI, RAG1 e RAG2, bem como 472 caracteres morfológicos de estudos anteriores, resultando na maior filogenia de bagres já elaborada. A análise de MP confirmou a monofilia de Hypoptopomatinae e de Neoplecostominae + Pseudotocinclus. Dentro de Neoplecostominae, somente o gênero Pareiorhina ficou polifilético, provavelmente devido à falta de dados morfológicos. A filogenia do gênero Pareiorhaphis revelou um padrão biogeográfico de distribuição previamente desconhecido. Das duas tribos de Hypoptopomatinae, Hypoptopomatini foi recuperada como monofilética, mas Otothyrini se manteve parafilética. O gênero Parotocinclus ficou polifilético, formando três clados monofiléticos e parte das espécies espalhada na filogenia. O resultado obtido pela análise de evidência total conseguiu resolver vários conflitos entre as propostas filogenéticas anteriores para Loricariidae. Porém, os grupos não resolvidos foram os que apresentaram a menor quantidade de caracteres, mostrando que, para resolver as relações filogenéticas dentro desta família, é necessária a complementação dos caracteres, assim como a adição de novos táxons.

v Abstract

The Loricariidae, or armored , is one of the most diverse families, currently containing nearly 800 recognized . They are solely freshwater inhabitants, with a distribution from Uruguay and Argentina to Costa Rica. Loricariids occur in every kind of waters, from cool mountain streams to lakes in the Amazon floodplain and reach sizes from a few centimeters up to more than one meter. Though the first species have been described by Linnaeus in 1758 and much effort has been done, especially in the last 15 years, their phylogenetic relationships could be only partly resolved so far. In particular, molecular analyses showed some groupings that conflict with the results from morphological analyses. The most problematic groups are the subfamilies Hypostominae, Hypoptopomatinae, and Neoplecostominae. Both morphologic and molecular studies suggest that the latter two form a monophyletic group, although leaving doubts if they are monophyletic separately. Therefore, the Hypoptopomatinae and the Neoplecostominae were chosen as subjects for the present project. In a first study, sequences from a 709 basepair fragment of the first subunit of the mitochondrial cytochrome c oxidase gene (COI) were used. The maximum parsimony (MP) and maximum likelihood (ML) analyses included data from 83 loricariid species from 29 genera representing five loricariid subfamilies. Additionally, eight species from four closely related families were used as outgroup. The results confirmed the Delturinae as the most basal subfamily and showed the Hypoptopomatinae + Neoplecostominae as sister to the Hypostominae. Corroborating previous molecular studies, the Neoplecostominae was monophyletic only when including the hypoptopomatine Pseudotocinclus. The Hypoptopomatinae formed a paraphyletic group, embracing the Neoplecostominae. In a second study, more taxa as well as partial sequences from the nuclear recombination activating genes 1 (RAG1) and 2 (RAG2), and the F-Reticulon 4 gene were included, increasing the data set to a total of 136 species and 4678 basepairs. The MP, ML, and Bayesian analyses confirmed most previous molecular results, but some new polyphyletic taxa were found such as Pareiorhaphis, Neoplecostomus, and the Neoplecostominae. To try to better understand the conflicts between the morphological and molecular approaches and to reach a more complete solution, a total evidence analysis was undertaken, since this approach had already yielded good solutions for similar problems. A new data set with a total of 207 species and concatenated sequences from COI, RAG1, and RAG2, as well as 472 morphological characters from previous studies was analyzed using MP, resulting in the largest phylogeny done so far. The Hypoptopomatinae and the Neoplecostominae were recovered as monophyletic sister groups, the latter including the genus Pseudotocinclus. Inside the Neoplecostominae, only the genus Pareiorhina remained polyphyletic, probably because of the lack of morphological data. The phylogeny of the genus Pareiorhaphis showed a previously unknown structured biogeographic pattern. From the two hypoptopomatine tribes, Hypoptopomatini was recovered as monophyletic, but Otothyrini remained paraphyletic. Although three monophyletic clades were found for the genus Parotocinclus, part of its species remained scattered in the phylogeny. Summarizing, the total evidence analysis was able to resolve several of the previous uncertainties in the loricariid phylogeny, but a further complementation of characters and an expansion of the taxon sampling will be necessary to completely resolve the phylogenetic relationships of this group.

vi

People cannot discover new lands until they have the courage to lose sight of the shore…

André Gide

vii Apresentação

A ordem Siluriformes é distribuída em todos os continentes, com a exceção da Antártica. Com cerca de 3100 espécies em 36 famílias (Ferraris, 2007), bagres somam aproximadamente 10% de todas as espécies de peixes. Eles são principalmente habitantes de águas doces, com somente duas famílias marinhas, e são mais fortemente representados na América do Sul, com 14 famílias e 64% das espécies (Moyle e Cech, 2000; Rodiles- Hernández et al., 2005). Seis destas famílias formam a superfamília Loricarioidea, um grupo bem documentado com cerca de 1280 espécies ou 41% de todos os bagres: Astroblepidae, Callichthyidae, Loricariidae, Nematogenyidae, Scoloplacidae e Trichomycteridae (Schaefer, 1990). A família Loricariidae, ou cascudos, é uma das mais diversas famílias de peixes, atualmente compreendendo 785 espécies reconhecidas em cerca de 100 gêneros (Eschmeyer e Fricke, 2009). Diferente de outros peixes, cascudos têm a boca modificada em forma de uma ventosa e o corpo é coberto com placas ossificadas. Este grupo mega-diverso ocorre do Uruguai e norte da Argentina à Costa Rica e é encontrado em todos os tipos de águas, de riachos frios nas montanhas com correntezas fortes aos lagos da área de inundação do rio Amazonas, tendo um papel importante na biodiversidade. Ultimamente, loricariídeos foram descobertos introduzidos nos EUA e na Ásia (Chavez et al., 2006; Nico et al., 2009), em alguns lugares em quantidades surpreendentes com consequências ainda desconhecidas para a fauna local. Apesar de muito trabalho e centenas de publicações deste Linnaeus, ainda existem muitas espécies por serem descobertas e descritas e sua filogenia está somente parcialmente resolvida. A família Loricariidae foi descrita por Rafinesque em 1815 e em seguida, começando em 1831, oito subfamílias foram estabelecidas, das quais seis ainda são reconhecidas (Armbruster, 2004; Reis et al., 2006): Lithogeninae Eigenmann, 1909 (1 gênero, 3 espécies), Delturinae Reis et al., 2006 (2 gêneros, 7 espécies), Neoplecostominae Regan, 1904 (5 gêneros, 39 espécies), Hypoptopomatinae Eigenmann e Eigenmann, 1890 (18 gêneros, 103 espécies), Loricariinae Bonaparte, 1831 (~ 36 gêneros, 222 espécies) e Hypostominae, Kner, 1853 (~ 40 gêneros, 411 espécies). Esta divisão em subfamílias não tem sido estável e ao longo do tempo foram feitas várias mudanças. Ancistrinae e Hypostominae foram descritas em 1853 (Kner, 1853). Descrevendo a Neoplecostominae, Regan (1904) somente incluiu o gênero Neoplecostomus. Mais tarde, Gosline (1947) adicionou os gêneros Canthopomus, Corymbophanes, Delturus, Hemipsilichthys, Isbrueckerichthys (as espécies foram listadas como Pareiorhaphis por

1 que Isbrueckerichthys somente foi descrito em 1996), , Pareiorhaphis, Pareiorhina, , Pogonopomoides e Upsilodus. Isbrücker (1980) reconheceu seis subfamílias, mas listou Neoplecostomus como único gênero de Neoplecostominae e transferiu os outros gêneros para Hypostominae. A primeira filogenia de Loricariidae foi publicada por Howes (1983). Baseado em exames de osteologia e de músculos, ele descreveu a subfamília Chaetostomatinae para os gêneros Chaetostoma, Hemipsilichthys, Lasiancistrus e Lipopterichthys e pôs Ancistrinae na sinonímia de Hypostominae. Parecido a esta situação, as filogenias de Schaefer (1986, 1987, 1988) mostraram Hypostominae como parafilética por causa da separação da Ancistrinae; ele não reconheceu a Chaetostomatinae. Mesmo assim, Schaefer decidiu manter o status de subfamília para Ancistrinae, usando a classificação de Isbrücker (1980). O primeiro estudo filogenético com foco em Hypoptopomatinae foi feito por Schaefer (1991), mas somente 16 espécies foram incluídas. Baseado nos seus resultados, ele descreveu as tribos Hypoptopomatini e Otothyrini. Montoya-Burgos et al. (1997, 1998) foram os primeiros a fazer uma análise molecular, usando sequências de nucleotídeos dos genes do rRNA de 12S e 16S. Com exceção de Neoplecostominae, monotípica, Loricariinae foi a única subfamília a ser recuperada como monofilética. Duas descobertas importantes foram feitas neste estudo (Montoya-Burgos et al., 1998): pela primeira vez, Hemipsilichthys gobio foi mostrado como táxon basal para todos os outros loricariídeos, e Pseudotocinclus (Hypoptopomatinae) foi identificado como grupo irmão de Pareiorhina (Neoplecostominae), fazendo uma conexão entre as duas subfamílias. A primeira filogenia ampla para a Loricariidae foi publicada por Armbruster (2004). Ela foi focada em Hypostominae, mas incluiu mais que 120 espécies de todas as subfamílias, usando 215 caracteres morfológicos. Para reter a monofilia da Hypostominae, Armbruster sinonimizou Ancistrinae com Hypostominae e dividiu Hypostominae nas cinco tribos , Corymbophanini, Hypostomini, e Pterygoplichthini. Ademais, ele encontrou Otothyrini como parafilética e, mesmo sendo um táxon parafilético, devolveu os gêneros Isbrueckerichthys, Kronichthys, Pareiorhaphis e Pareiorhina para Neoplecostominae. Adicionalmente, Schaefer confirmou a descoberta de Montoya-Burgos et al. (1998) que Hemipsilichthys gobio, junto com Delturus angulicauda, forma o grupo-irmão de todos os outros loricariídeos, com exceção de Lithogenes. Baseado nestes resultados, Pereira (2005) ressuscitou o gênero Pareiorhaphis Miranda Ribeiro, 1918 para a maioria das espécies de Hemipsilichthys, deixando somente H. gobio, a espécie-tipo, H. papillatus e H. nimius neste último. No mesmo ano, a filogenia morfológica de Hypoptopomatinae de Gauger e

2 Buckup (2005), incluindo 31 espécies de quase todos os gêneros da subfamília, mostrou Otothyrini e o gênero Parotocinclus como grupos parafiléticos. No ano seguinte, Lehmann (2006) utilizou 169 caracteres morfológicos e um total de 114 espécies, na sua tese de doutorado sobre a filogenia de Hypoptopomatinae. As suas análises filogenéticas recuperaram o gênero Kronichthys como grupo mais basal de uma Hypoptopomatinae monofilética. De acordo com os seus resultados, Pseudotocinclus não tem nenhuma relação mais próxima com Neoplecostominae, e ele encontrou Otothyrini sendo parafilética. Focados em Hypoptopomatinae e incluindo sequências de DNA de apenas um gene e 44 espécies, Chiachio et al. (2008) encontraram Pseudotocinclus fazendo parte de Neoplecostominae, qual foi encontrada dentro de Hypoptopomatinae. A solução dos autores foi elevar Hypoptopomatini e Otothyrini ao nível de subfamília e redefinir Neoplecostominae, resultando em três grupos monofiléticos. Esta mudança nomenclatural deve ser considerada prematura por causa do baixo número de espécies incluídas, do uso de um único gene e das contradições com os estudos recentes, sejam moleculares ou morfológicos. Pouco depois, Pereira (2008) terminou sua tese de doutorado, dedicada à filogenia de Neoplecostominae. Representando a mais completa amostragem de táxons deste grupo e incluindo 303 caracteres morfológicos, este estudo recuperou Hypoptopomatinae e Neoplecostominae como grupos irmãos monofiléticos. Porém, os gêneros Pareiorhina e Kronichthys foram revelados como táxons irmãos na base de Hypoptopomatinae e, consequentemente, foram incluídos nesta subfamília. Infelizmente, Pseudotocinclus não foi incluído na análise de Pereira. A presente tese reúne os resultados da análise filogenética das duas subfamílias Hypoptopomatinae e Neoplecostominae, usando dados moleculares nucleares e mitocondriais e dados morfológicos. Como todos os estudos anteriores mostram que estas duas subfamílias formam um grupo monofilético, elas foram escolhidas como tópico do presente estudo. Considerando os diferentes resultados apresentados na literatura atual sobre as relações filogenéticas de Neoplecostominae e Hypoptopomatinae, este estudo teve como objetivo ampliar os dados sobre estes grupos, enfocando a relação entre os dados moleculares e as análises morfológicas, constituindo-se, assim, numa análise de evidência total. Foram elaborados três artigos. O primeiro, utilizando sequências parciais da primeira subunidade do gene mitocondrial citocromo c oxidase (COI) para 83 táxons foi publicado no Bulletin of Fish Biology (Revista da Sociedade Alemã de Ictiologia), em co-autoria com Ana M. R. Liedke, Sandro L. Bonatto e Roberto E. Reis. Os resultados deste estudo também recuperaram o gênero Pseudotocinclus como membro de Neoplecostominae e,

3 novamente, Neoplecostominae foi encontrada dentro de Hypoptopomatinae. Contrário a resultados anteriores, Hypoptopomatini e Otothyrini não formaram grupos monofiléticos. O segundo artigo contém os resultados de análises utilizando sequências parciais de COI e dos genes nucleares recombination activating gene 1 (RAG1) e 2 (RAG2) e F-Reticulon 4 para um total de 136 táxons, e será submetido para a revista Molecular Phylogenetics and Evolution, em co-autoria com Sandro L. Bonatto e Roberto E. Reis. Com o objetivo de testar os resultados prévios usando múltiplos genes, os métodos Máxima Parcimônia (MP), Máxima Verossimilhança (ML) e análise Bayesiana foram utilizados. Somente as subfamílias Delturinae e Loricariinae foram encontradas monofiléticas. Outra vez, o gênero Pseudotocinclus foi incluído em Neoplecostominae, mas Pareiorhaphis foi recuperado como polifilético, inclusive com uma espécie colocada fora de Neoplecostominae. As tribos Hypoptopomatini e Otothyrini se mantiveram não- monofiléticas. No terceiro artigo, objetivando resolver as contradições entre os dados morfológicos e moleculares, utilizou-se a análise de evidência total, unindo sequências de COI, RAG1 e RAG2 com dados morfológicos das teses de Pablo Lehmann e Edson H. L. Pereira, para 207 táxons. Este artigo será submetido para a revista Systematic Biology, junto com Pablo Lehmann, Edson H. L. Pereira, Sandro L. Bonatto e Roberto E. Reis. A análise de MP recuperou Hypoptopomatinae e Neoplecostominae como grupos irmãos monofiléticos. Quase todos os gêneros de Neoplecostominae resultaram em grupos monofiléticos, com a exceção de Pareiorhina, que permanece polifilético. A filogenia do gênero Pareiorhaphis mostrou um padrão biogeográfico antes desconhecido. Dentro de Hypoptopomatinae, no entanto, somente Hypoptopomatini foi encontrada como grupo natural, dentro de uma tribo Otothyrini parafilética, e os gêneros Hisonotus e Parotocinclus foram recuperados como polifiléticos.

4

Capítulo I

The phylogenetic relationships of the Hypoptopomatinae and

Neoplecostominae (Siluriformes: Loricariidae) as inferred from

mitochondrial cytochrome c oxidase I sequences

5 Bulletin of Fish Biology Volume 9 Nos. 1/2 15.12.2007 51-59

The phylogenetic relationships of the Hypoptopomatinae and Neoplecostominae (Siluriformes: Loricariidae) as inferred from mitochondrial cytochrome c oxidase I sequences

Die Phylogenie der Hypoptopomatinae und Neoplecostominae (Siluriformes: Loricariidae) auf Grundlage von Sequenzen des mitochondrialen Cytochrom-c- Oxidase-I-Gens

Christian A. Cramer 1,2, Ana M. R. Liedke2, Sandro L. Bonatto2 & Roberto E. Reis1

1Laboratório de Sistemática de Vertebrados, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90.619-900 Porto Alegre, RS, Brasil. 2Centro de Biologia Molecular e Genômica, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90.619-900 Porto Alegre, RS, Brasil; [email protected] (corresponding author)

Summary: The phylogenetic relationships of the loricariid subfamilies Neoplecostominae and Hypopto- pomatinae are assessed using sequences of the subunit 1 of the cytochrome c oxidase gene (COI), in order to test the contradictory results of previous, mostly morphologic analyses. We obtained an alignment of 709 contiguous nucleotides for 105 sequences of a fragment of COI for 83 species from 29 loricariid genera from representatives of five loricariid subfamilies and for eight outgroup species from four lorica- rioid families. Both, Maximum Likelihood and Parsimony analyses were conducted. Results show a mono- phyletic clade composed of Hypoptopomatinae + Neoplecostominae as sister to Hypostominae and this clade sister to Loricariinae. However, neither the Hypoptopomatinae nor the Neoplecostominae are mono- phyletic groups. Also, the genera Pareiorhaphis, Hisonotus and Parotocinclus turned out to be polypheletic.

Key words: phylogeny, Loricariidae, Hypoptopomatinae, Neoplecostominae, cytochrome c oxidase, armo- red catfish

Zusammenfassung: Die phylogenetischen Verwandtschaftsverhältnisse der Harnischwels-Unterfamilien Neo- plecostominae und Hypoptopomatinae werden mit Hilfe von Sequenzen der ersten Untereinheit der Cytochrom- c-Oxidase (COI) untersucht, um die widersprüchlichen Ergebnisse früherer, meist morphologischer Arbeiten zu prüfen. Wir erhielten ein Alignment von 709 zusammenhängenden Nukleotiden für 105 Sequenzen eines COI- Fragments für 83 Arten von 29 Gattungen aus fünf Unterfamilien der Harnischwelse sowie acht Außengruppen- Arten aus vier Familien der Loricarioidea. Es wurden Maximum-Parsimony- und Maximum-Likelihood-Analy- sen durchgeführt. Die Ergebnisse zeigen eine monophyletische Gruppe aus Hypoptopomatinae und Neopleco- stominae als Schwestergruppe der Hypostominae, und diese zusammen als Schwestergruppe der Loricariinae. Allerdings sind weder die Hypoptopomatinae noch die Neoplecostominae monophyletische Gruppen. Ebenso stellen sich die Gattungen Pareiorhaphis, Hisonotus und Parotocinclus als polyphyletisch heraus.

Schlüsselwörter: Phylogenie, Loricariidae, Hypoptopomatinae, Neoplecostominae, Cytochrome-c-Oxi- dase, Harnischwelse

1. Introduction the exception of two marine families, they are solely freshwater inhabitants. Catfish are most With around 2,600 species in 36 families (FER- strongly represented in South America with RARIS 2007), the order Siluriformes contains 14 families and 64% of the species (MOYLE & about 10% of all fish species worldwide. With CECH 2000). The armored catfishes (Lorica-

Bull. Fish Biol. 9 (1/2) 51

6 riidae), emphasized here, are endemic to South of molecular evolution that is about three and Central America. They are widespread and times higher than that of 12S or 16S rDNA play an important role in biodiversity, as they (KNOWLTON & WEIGT 1998). So, the evolu- are one of the most species-rich fish families. tion of this gene is rapid enough to allow Presently approximately 90 genera with about the separation of not only closely related spe- 700 species are recognized. Along with five cies, but also phylogeographic groups within closely related families, they form the super- a single species (COX & HEBERT 2001, WARES family Loricariodea (SCHAEFER & LAUDER 1986, & CUNNINGHAM 2001). Because changes in SCHAEFER 1990). its amino-acid sequence occur more slowly The family Loricariidae is divided into six than in any other mitochondrial genes (LYNCH subfamilies (ARMBRUSTER 2004, REIS et al. & JARRELL 1993), COI is more likely to pro- 2006). The subfamily Lithogeneinae with one vide deeper phylogenetic insights than alter- genus and only two species is the smallest natives such as cytochrome b (SIMMONS & and less known one. The Delturinae contains WELLER 2001), even if other mitochondrial two genera with seven species. Six genera with genes may equally resolve cases of recent di- 38 species comprise the subfamily Neople- vergence. costominae, and around 94 species in 18 genera are included in the subfamily Hypop- 2. Materials and methods topomatinae. The remaining taxa are dis- persed throughout the subfamilies Loricari- 2.1. Taxon sampling inae and Hypostominae. Although LINNAEUS (1758) described the first loricariid catfish The specimens and species used in this study and since then many scientists have been are listed in Appendix A. Our aim was to in- working with this family, their systematics is clude most of the species of the subfamilies still insufficiently resolved. So, the monophyly Hypoptopomatinae and Neoplecostominae to- of at least two subfamilies and two tribes gether with representatives of the other lorica- (Neoplecostominae and Hypostominae; An- riid subfamilies. Unfortunately there was no cistrini and Hypostomini) has been rejected fresh tissue from Lithogenes available. As out- by molecular studies (MONTOYA-BURGOS et al. group taxa we used representatives from the 1998, 2002, HARDMAN 2005), contrary to mor- other families of the Loricarioidea but Tricho- phologic results, and many genera are not mycteridae (SCHAEFER & LAUDER 1986, SCHAE- adequately defined. MONTOYA-BURGOS et al. FER 1990, DE PINNA 1998). (1998) and ARMBRUSTER (2004) found the Neoplecostominae and the Hypoptopomati- 2.2. DNA amplification and sequencing nae to cluster together. The most recent phy- logenetic study based on morphology is LEH- From total genomic DNA extracted from MANN (2006). He found the Neoplecostomi- fresh or ethanol-preserved tissue using the nae to be a paraphyletic group. The Hypop- QIAmp tissue kit (Qiagen, Hilden, Germany), topomatinae came out to be monophyletic. we amplified and sequenced a 709 basepair Therefore our aim is to test the findings on fragment of COI, using the primers these two subfamilies using molecular DNA LCO1490 and HCO2198 (HEBERT et al. sequence data. Our choice was the cytochro- 2003). Each PCR was carried out in 20 μl me c oxidase I gene (COI) because it shows reactions with the following concentrations: a greater range of phylogenetic signal than 1x Invitrogen PCR buffer (Invitrogen, São EBERT any other mitochondrial gene (H et al. Paulo), 1.5-2.5 mM MgCl2, 0.2% Triton, 2003). Like other protein-coding genes, its 200 μM of each dNTP, 0.025 U/μl taq poly- third-position nucleotides show a high inci- merase, 0.2 μM of each primer and up to dence of base substitutions, leading to a rate 2 μl of DNA solution.

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7 We used our lab’s standard protocol for this 3. Results primer pair with an initial denaturation step of 1 min at 96 °C followed by 40 cycles of 94 °C 3.1. Data analysis for 30 s, annealing at 50 °C for 20 s, 48 °C for 5 s, 46 °C for 5 s, 44 °C for 5 s, 42 °C for 5 s, 40 °C We obtained an alignment of 709 contiguous for 20 s and extension at 72 °C for 1 min. This nucleotides and 105 sequences of a fragment was followed by a final 3 min at 72 °C exten- of COI for 83 species from 29 ingroup genera sion step. Amplification success was evaluated and for eight outgroup species. No gaps were on GelRedTM (BioTium, São Paulo) or ethidium found. The character matrix contained 278 par- bromide-stained agarose gels (1%) in 0.5% TBE simony-informative characters. buffer (SAMBROOK et al. 1989). PCR products were purified using PEG8000, ExoSAP-IT® 3.2. Parsimony analysis (USB) or the ilustraTM GFX PCR and Gel Band Purification Kit (GE Healthcare, Bucking- The MP analysis with PAUP* resulted in 4,400 hamshire, UK). Sequencing was done using the equally parsimonious trees with a length of DYEnamicTM ET dye terminator kit (GE 2,827 steps. Their summarized strict consen- Healthcare, São Paulo) and a MegaBace1000 sus tree is shown in fig. 1. TNT found the same sequencer. consensus tree. Non-parametric bootstrap pro- Sequences were edited and combined using portions did not provide evidence of convinc- BioEdit 7.0.1 (HALL 1999). ing resolution for deeper nodes. The lack of support suggests weak signal overall and prob- 2.3 Analysis able inaccuracy among deeper nodes recovered by the analysis of these data. Sequences were aligned using Clustal X 1.83 (THOMPSON et al. 1997) using the standard 3.3. Maximum likelihood analysis settings. PAUP*4.0b10 (SWOFFORD 2001) was used to analyze the data with respect to the The best tree found with RAxML has a -ln L- parsimony criterion with the tree-bisection- score of -12410.368924 and is shown in fig. 2. reconnection (TBR) search algorithm with 1000 Parameter estimates for each of the codon-ba- replicates in which taxa were added randomly sed models are shown in table 1. to the starting tree. TNT using its ratchet algo- Both, the MP and the ML tree have little boot- rithm (GOLOBOFF et al. 1999) was also used and strap support for the deep nodes. As the result found the exact same results. All characters were from the ML analyses shows higher bootstrap treated as unordered and transformations were confidence intervals and fewer conflicts with the assigned equal weight. Nodal support was eva- morphologic phylogenies, we will not discuss the luated with 2000 nonparametric bootstrap pseu- MP analysis and will only focus on the ML result. doreplicates (FELSENSTEIN 1985) using the TBR search algorithm on a starting tree to which taxa 4. Discussion were added randomly. Multiple optimal topol- ogies were summarized through consensus ARMBRUSTER (2004) found Neoplecostominae methods. and Hypoptopomatinae to be a monophyletic For the maximum likelihood (ML) analysis we sister group to the Hypostominae + Loricari- used RAxML-HPC 7 (STAMATAKIS 2006) with 300 inae. In our results, they are sister to the Hypos- replicates under the GTRGAMMA model. Pa- tominae and the three subfamilies together are rameters were estimated for each of four sub- sister to the Loricariinae, corroborating the stitution categories over each of three codon findings of LEHMANN (2006). positions. Nodal support was evaluated with 1000 The topology of the Hypoptopomatinae nonparametric bootstrap pseudoreplicates. found here is nearly inverse to that of LEH-

Bull. Fish Biol. 9 (1/2) 53

8 Fig.1: Summarized strict consen- sus of 4,400 most parsimonious topologies each of 2,827 steps. Numbers above nodes are boot- strap values recovered by the ma- ximum likelihood analysis for those clades common to both parsimony and likelihood topol- ogies. Abb. 1: Zusammengefasster strikter Konsensus der 4.400 be- sten Bäume der Parsimonie-Ana- lyse mit jeweils 2.827 Schritten. Die Zahlen oberhalb der Äste sind Bootstrap-Werte der Likeli- hood-Analyse für die Gruppen, die sowohl in der Likelihood- als auch in der Parsimony-Analyse gefunden wurden.

54

9 Fig. 2: Best tree from the likelihood analysis (-ln L- 12100.250237). Branch lengths are means estimated by RAxML. Parameter estimates are provided in table 1. Abb. 2: Der beste Baum der Likelihood-Analyse (-ln L -12100,250237). Die Astlängen wurden von RAxML errechnet. Die Parameter-Werte sind in Ta- belle 1 gegeben.

Bull. Fish Biol. 9 (1/2) 55

10 Table 1: Model parameters estimated by RAxML minae, and should also include sequences from during likelihood analysis. nuclear genes. Additionally, both molecular and Tabelle 1: Von RAxML während der Likelihood- morphological data should be joined in a total Analyse berechnete Modell-Parameter evidence analysis. This procedure already hel- ped to resolve problems in similar cases (GA- TESY et al. 2003).

Acknowledgements

Thanks are due to our colleagues Edson H. L. PEREIRA and Tiago CARVALHO, who helped with identification of the samples, to Cladinara RO- BERTS SARTURI, Fernanda BRITTO and Mirian Tieko NUNES TSUCHIYA for support with the lab work. The following persons and companies donated specimens for this work: Thomas WEID- NER (Iffeldorf, Germany), Ingo SEIDEL, Fa. Aqua Global (Seefeld, Germany), André WERNER, Fa. Transfish (Planegg, Germany), and Frank SCHÄ- MANN (2006). In his study, Eurycheilichthys is the FER, Fa. Aquarium Glaser (Rodgau, Germany). most basal taxon of the Hypoptopomatinae and The following institutions provided tissue sam- the most derived one. Like LEHMANN ples for this work: ANSP, AUM, DZJRP, LBP, (2006), we found Acestridium, and MCP, MNHG, MNRJ, NUP, ZSM. as closely related. Corumbataia, Otothy- Financial support: DAAD (German Academ- ris and Schizolecis are closely related, as in LEH- ic Exchange Service), GfI (Gesellschaft für Ich- MANN (2006). The genus Pseudotocinclus was thyologie) and CNPq (Brazilian Council for found to be a sister taxon to Pareiorhina, inside Technologic and Scientific Development). the Neoplecostominae, with high bootstrap support. This finding is the same as in MON- Literature TOYA-BURGOS et al. (1998) where these two ge- nera and Hypoptopoma are closer related with ARMBRUSTER. J. W. 2004. Phylogenetic relationships the Neoplecostominae than with the rest of the of the suckermouth armoured catfishes (Lorica- Hypoptopomatinae. Pareiorhaphis, Parotocinclus riidae) with emphasis on the Hypostominae and and Hisonotus came out as polyphyletic taxa. the Ancistrinae. Zoologiucal Journal of the Lin- nean Society 141, 1-80. Parotocinclus has already been found to be poly- AZPELICUETA, M., & A. E. ALMIRÓN. 2007. Hisono- phyletic by LEHMANN (2006). No phylogenetic tus hungy sp. n. (Siluriformes, Loricariidae), a study on Hisonotus has been published so far, new species from arroyo Tirica, Misiones, Ar- but AZPELICUETA & ALMIRÓN (2007) and BRITS- gentina. Revue Suisse de Zoologie 114, 591- KI & GARAVELLO (2003, 2007) state that the ge- 598. nus is insufficiently defined and possibly poly- BRITSKI, H. A., & J. C. GARAVELLO. 2003. Hisonotus phyletic. insperatus: new species, from the upper Rio Paraná As the present results do not have high boot- basin (Pisces: Ostariophysi: Loricariidae). Copeia strap support, especially on the deeper nodes, 2003, 588-593. BRITSKI, H. A., & J. C. GARAVELLO. 2007. Description there is a need of additional work. To further of two new species of the genus Hisonotus EIGEN- clarify the relationships of the loricariid subfa- MANN & EIGENMANN, 1889, from upper Rio Ta- milies, a future work should sample additional pajós, Mato Grosso State, Brazil (Pisces: Os- species to include representatives of all genera tariophysi: Loricariidae). Brazilian Journal of Bio- of the Hypoptopomatinae and Neoplecosto- logy 67, 631-637.

56

11 COX, A. J., & P. D. N. H EBERT. 2001. Coloniza- cena & C.A.S. Lucena (eds). Phylogeny and Clas- tion, extinction and phylogeographic pattern- sification of Neotropical . EDIPUCRS, ing in a freshwater . Molecular Porto Alegre. Ecology 10, 371-386. MONTOYA-BURGOS, J.I., C. WEBER, & P.-Y. LE BAIL. ESCHMEYER, W. 2008. The Catalog of Fishes on-line. 2002. Phylogenetic relationships within Hyposto- http://research.calacademy.org/research/ich- mus (Siluriformes : Loricariidae) and related ge- thyology/catalog/fishcatsearch.html. Version nera based on mitochondrial D-loop sequences. from 29 of January 2008. Revue Suisse de Zoologie 109, 369-382. FELSENSTEIN, J. 1985. Confidence limits on phylo- MOYLE, P.B., & J.J. CECH. 2000. Fishes – An Intro- genies: an approach using the bootstrap. Evolu- duction to ichthyology. 4th edition. Prentice-Hall, tion 39, 783-791. Upper Saddle River. FERRARIS, C. J. 2007. Checklist of catfishes, recent and DE PINNA, M.C.C. 1998. Phylogenetic Relationships fossil (Osteichthyes: Siluriformes), and catalogue of Neotropical Siluriformes (Teleostei: Ostario- of siluriform primary types. Zootaxa 1418, 1-628. physi): Historical Overview and Synthesis of GATESY, J., G. A. AMATO, M. NORELL, R. DESALLE, & Hypotheses. Pp. 279-330. In: Malabarba, L.R., R.E. C. HAYASHI. 2003. Combined support for whole- Reis, R.P. Vari, Z.M.S. Lucena & C.A.S. Lucena sale taxic atavism in gavialine crocodylians. Sys- (eds). Phylogeny and Classification of Neotropi- tematic Biology 52: 403-422. cal Fishes. EDIPUCRS, Porto Alegre. GOLOBOFF, P.A., FARRIS, J.S., & K.C. NIXON. 1999. POSADA, D., & K.A. CRANDALL. 1998. Modeltest: test- TNT: Tree Analysis Using New Technology, ing the model of DNA substitution. Bioinformat- Available from www.cladistics.com. ics 14, 817–818. HALL, T.A. 1999. BioEdit: a user-friendly biological REIS, R.E., J. W. ARMBRUSTER, & E.H.L. PEREIRA. 2006. sequence alignment editor and analysis program Delturinae, a new loricariid catfish subfamily (Te- for Windows 95/98/NT. Nucleic Acids Sympo- leostei, Siluriformes), with a revision of Delturus. sium Series 41, 95-98. Zoological Journal of the Linnean Society 147, HARDMAN, M. 2005. The phylogenetic relationships 277-299. among non-diplomystid catfishes as inferred from SAMBROOK, J., E. F. FRITSCH, & T. MANIATIS. 1989. Mol- mitochondrial cytochrome b sequences; the ecular cloning: A laboratory manual. 2nd Edition. search for the ictalurid sister taxon (Otophysi: Si- Cold Spring Harbor Press, Cold Spring Harbor, luriformes). Molecular Phylogenetics and Evolu- New York tion 37, 700-720. SCHAEFER, S.A. 1990. Anatomy and relationship of the HEBERT, P. D. N., A. CYWINSKA, S. L. BALL, & J. R. DE scoloplacid catfishes. Proceedings of the Academy WAARD. 2003. Biological identifications through of Natural Sciences of Philadelphia 142, 167-210. DNA barcodes. Proceedings of the Royal Society SCHAEFER, S.A., & G.V. LAUDER. 1986. Historical trans- of London B 270, 313-321. formation of functional design: evolutionary KNOWLTON, N., & L. A. WEIGT. 1998. New dates and morphology of feeding mechanisms in loricario- new rates for divergence across the Isthmus of id catfishes. Systematic Zoology 35, 489-508. Panama. Proceedings of the Royal Society of SIMMONS, R.B., & S. J. WELLER. 2001. Utility and evo- London B 265, 2257-2263. lution of cytochrome b in insects. Molecular LEHMANN, P. 2006. Anatomia e relações filogenéticas Phylogenetics and Evolultion 20, 196-210. da família Loricariidae (Ostariophysi: Siluriformes) STAMATAKIS, A. 2006. RAxML-VI-HPC: Maximum com ênfase na subfamília Hypoptopomatinae. Likelihood-based Phylogenetic Analyses with Unpublished PhD thesis, PUCRS, Porto Alegre. Thousands of Taxa and Mixed Models. Bioinfor- LINNAEUS, C. von. 1758. Systema naturae. 10th editi- matics 22, 2688-2690. on. (Apud, Eschmeyer, W. 2008). SWOFORD, D. L. 2001. PAUP*. Phylogenetic Analysis LYNCH, M., & P. E. Jarrell. 1993. A method for calibra- Using Parsimony (*and other Methods). Version ting molecular clocks and its application to 4. Sinauer Associates, Sunderland, Massachusetts. mitochondrial DNA. Genetics 135, 1197-1208. THOMPSON, J. D., T. J. GIBSON, F. PLEWNIAK, F. JEAN- MONTOYA-BURGOS, J. I., S. MULLER, C. WEBER, & J. MOUGHIN, & D. G. HIGGINS. 1997. The PAWLOWSKI. 1998. Phylogenetic relationships of CLUSTAL_X windows interface: flexible strat- the Loricariidae (Siluriformes) based on mito- egies for multiple sequence alignment aided by chrondrial rRNA gene sequences, pp. 363-374. In: quality analysis tools. Nucleic Acids Research 25, Malabarba, L.R., R.E. Reis, R.P. Vari, Z.M.S. Lu- 4876-4882.

Bull. Fish Biol. 9 (1/2) 57

12 WARES, J. P., & C. W. CUNNINGHAM. 2001. Phylogeo- Received: 01.11.2007 graphy and historical ecology of the North At- Accepted: 01.12.2007 lantic intertidal. Evolution 12, 2455-2469.

Appendix: GenBank and depository information for species included in this study

Astroblepidae: Astroblepus sp. 1 ANSP 180581 (tag 4805) EU359404. Astroblepus sp. 2 ANSP 180605 (tag 4490) EU359405. Astroblepus sp. 3 ANSP 180613 (tag 4453) EU359406. Astroblepus sp. 4 ANSP 180616 (tag 4436) EU359407. Callichthyidae: Callichthys callichthys MCP 29384 EU359408. Hoplosternum littorale MCP 21196 EU359416. Loricariidae: Acestridium martini ANSP 182901 (tag V5276) EU359398. Acestridium martini ANSP 182901 (tag V5277) EU359399. Ace- stridium sp. 1 MCP 37783 EU359400. Acestridium sp. 2 MCP 37785 EU359401. Ancistrus brevipin- nis MCP 21246 EU359402. Apistoloricaria ommation ANSP 182331 (tag P6265) EU359403. Chae- tostoma sp. 1 ANSP 180446 (tag P4772) EU359409. Chaetostoma sp. 2 ANSP 180448 (tag P4814) EU359410. Corumbataia cuestae LBP 876 EU371019. Epactionotus bilineatus MCP 23679 EU371006. Epactionotus bilineatus MCP 26964 EU371008. Epactionotus bilineatus MCP 26964 EU371009. Epactionotus cf. gracilis MCP 35156 EU371007. Epactionotus gracilis MCP 23606 EU371005. Epac- tionotus itaimbezinho MCP 23683 EU371004. Eurycheilichthys limulus MCP 21270 EU370989. Eu- rycheilichthys limulus MCP 21270 EU370990. Eurycheilichthys pantherinus MCP 22373 EU371000. Eurycheilichthys sp.1 MCP 21207 EU370995. Eurycheilichthys sp. 1 MCP 21207 EU370998. Eu- rycheilichthys sp. 2 MCP 22374 EU370992. Eurycheilichthys sp. 2 MCP 22800 EU370994. Eurychei- lichthys sp. 3 MCP 35049 EU370999. Eurycheilichthys sp. 4 MCP 22199 EU370991. Eurycheilichthys sp. 5 MCP 22790 EU370993. Eurycheilichthys sp. 7 MCP 35071 EU370997. Eurycheilichthys sp. 7 MCP 35124 EU370996. Hemiancistrus subviridis AUM 42930 (tag P4648) EU359411. Hemipsilich- thys nimius MCP 30671 EU359412. Hisonotus cf. aky MCP 40029 EU359413. Hisonotus charrua MCP 21644 EU371013. Hisonotus francirochai DZJRP 7727 EU359415. Hisonotus laevior MCP 23005 EU371015. Hisonotus leucofrenatus MCP 31819 EU371001. Hisonotus leucofrenatus LBP 873 EU371002. Hisonotus sp. 1 MCP 23744 EU371010. Hisonotus sp. 2 MCP 25139 EU371014. Hisonotus sp. 3 MCP 25159 EU371012. Hisonotus sp. 4 LBP 810 EU371018. Hisonotus sp. 5 MCP 37682 EU359414. Hisonotus taimensis MCP 21375 EU371011. Hypoptoma bilobatum MHNG 2588.092 EU370986. Hypoptopoma cf. inexspectatum ANSP uncat. (tag 5089) EU359417. Hypopto- poma guianensis ANSP 180669 (tag T2215) EU359418. Hypoptopoma gulare ANSP 178340 (tag 1551) EU359419. Hypoptopoma inexspectatum ANSP uncat. (tag 5047) EU359420. Hypoptopoma steindachneri ANSP 182723 (tag P6233) EU359421. Hypostomus boulengeri NUP uncat. (Z49935) EU359422. Hypostomus commersonii MCP 22767 EU359423. Ixinandria montebelloi GEN 1713 EU359425. Ixinandria sp. MCNI uncat. EU359424. Ixinandria steinbachi MCNI 1222 EU359426. Kronichthys subteres MCP 31600 EU371021. Microlepidogaster perforatus MCP 41912 EU359427. Neoplecostomus microps MNR 24005 (#170) EU359429. Neoplecostomus sp. MCP 30672 EU359430. Otocinclus affinis DZJRP 7610 EU359431. Otocinclus cf. hoppei MHNG 2613.057 EU370985. Oto- cinclus cocama MCP 34842 EU359432. Otocinclus flexilis MCP 25234 EU370983. Otocinclus flexilis MCP 41907 EU370984. Otocinclus vittatus MCP 35848 EU359433. juquiae MCP unreg. EU359434. Otothyropsis sp. MHNG 2587.011 EU371003. Panaqolus changae ANSP 181097 (tag P6218) EU359435. Panaqolus sp. ZSM 32728 EU359436. Pareiorhaphis azygolechis MCP 41909 EU359437. Pareiorhaphis calmoni MCP 41275 EU359438. Pareiorhaphis eurycephalus MCP 41458 EU359439. Pareiorhaphis hypselurus MCP 21695 EU359440. Pareiorhaphis hystrix MCP 22787 EU359441. Pareiorhaphis nudula MCP 41906 EU359442. Pareiorhaphis parmula MCP 41747 EU359443. Pareiorhaphis sp. 1 MCP 22339 EU359444. Pareiorhaphis sp. 2 MCP 28683 EU359445.

58

13 Pareiorhaphis sp. 3 MCP 40111 EU359446. Pareiorhaphis sp. 4 MCP 41296 EU359447. Pareiorha- phis sp. 5 MCP 41457 EU359448. Pareiorhaphis splendens MCP 22330 EU359449. Pareiorhaphis splendens MCP 41263 EU359450. Pareiorhaphis steindachneri MCP 41289 EU359451. Pareiorhaphis stomias MCP 41910 EU359452. Pareiorhina sp. MNRJ 26518 (#281) EU359453. Parotocinclus cf. aripuanensis MCP unreg. EU359454. Parotocinclus eppleyi AUM 43947 (tag V5576) EU359455. Parotocinclus jumbo ZSM 32727 EU359456. Parotocinclus maculicauda MCP 41911 EU359457. Peckoltia vermiculata AUM 39245 (tag V060) EU359458. Peckoltia vittata AUM 39248 (tag V114) EU359459. Pseudacanthicus leopardus ANSP 179613 (tag 2450) EU359460. Pseudotocinclus juquiae LBP 616 EU370988. Pseudotocinclus tietensis LBP 696 EU370987. obtusa MCP 33330 EU371016. Pseudotothyris obtusa MCP 33330 EU371017. Rineloricaria sp. MCNI 1222 EU359461. Schizolecis guentheri MCP 31722 EU359462. Schizolecis guentheri MCP 31724 EU371020. Nematogenyidae: Nematogenys inermis ANSP 180477 (tag 1) EU359428. Scoloplacidae: Scoloplax distolothrix MCP 40282 EU359463.

Bull. Fish Biol. 9 (1/2) 59

14

Capítulo II

Molecular Phylogeny of the Neoplecostominae and Hypoptopomatinae

(Siluriformes: Loricariidae) using Multiple Genes

15 Molecular Phylogeny of the Neoplecostominae and Hypoptopomatinae (Siluriformes:

Loricariidae) using Multiple Genes.

Christian Andreas Cramera,b,*, Sandro Luis Bonattob, and Roberto E. Reisa

aLaboratório de Sistemática de Vertebrados, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil. bCentro de Biologia Molecular e Genômica, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil.

*Corresponding author. Tel.: +55 51 33534413. Fax: +55 51 33293903. E-mail address:

[email protected] (C. A. Cramer)

16 Abstract

A phylogenetic analysis of nearly all genera of the loricariid subfamilies Neoplecostominae and Hypoptopomatinae is provided based on fragments of the subunit 1 of the cytochrome c oxidase gene (COI), the recombination activating genes 1 (RAG1) & 2 (RAG2), and the

F-reticulon 4 gene in order to test the contradictory results of previous analyses. We obtained an alignment of 4678 contiguous nucleotides for 136 species from 50 loricariid genera from representatives of five loricariid subfamilies plus ten outgroup species from five loricarioid families, resulting in the largest phylogeny of the Loricariidae published so far. Our results from Maximum Likelihood, Maximum Parsimony, and Bayesian analyses show a monophyletic clade composed by the Hypoptopomatinae + Neoplecostominae as sister to the

Hypostominae and this clade sister to the Loricariinae. Delturinae is the sister-group of the above clade. However, neither the Hypoptopomatinae nor the Neoplecostominae were recovered as monophyletic groups. Previously hypothesized monophyly of the

Hypoptopomatini and Otothyrini could not be confirmed. Furthermore, the genera

Pareiorhaphis, Pareiorhina, Hisonotus and Parotocinclus were recovered as polyphyletic.

Keywords: Loricariidae, Neoplecostominae, Hypoptopomatinae, phylogeny, armored catfish,

COI, RAG1, RAG2

17 Introduction

With around 3100 species in 36 families (Ferraris, 2007), the order Siluriformes contains about 10% of all fish species worldwide. With the exception of two marine families, they are solely freshwater inhabitants. Catfish are most strongly represented in South America with 14 families and 64% of the species (Moyle and Cech, 2000; Rodiles-Hernández et al., 2005).

Loricariidae (armored catfishes) is the largest catfish family, with approximately 100 genera and 785 species currently recognized (Eschmeyer and Fricke, 2009). Armored catfishes are endemic to South and Central America, but recently there are more and more records of introduced species from North America and Asia. As loricariids inhabit all kinds of waters from small and cool mountain streams to large and warm rivers, they play an important role in biodiversity. Along with five closely related families, they form the superfamily Loricarioidea

(Schaefer and Lauder, 1986; Schaefer, 1990). The family Loricariidae is divided into six subfamilies (Armbruster, 2004; Reis et al., 2006): Lithogeninae (1 genus, 3 species),

Delturinae (2 genera, 7 species), Neoplecostominae (5 genera, 39 species), Hypoptopomatinae

(18 genera, 103 species), Loricariinae (~ 36 genera, 222 species), and Hypostominae (~ 40 genera, 411 species). However, the classification of genera in subfamilies has not been stable.

The Neoplecostominae was modified several times (for a good overview see Reis et al.,

2006). The monophyly of at least two subfamilies and two tribes (Neoplecostominae and

Hypoptopomatinae; Ancistrini and Hypostomini) has been rejected by molecular studies

(Montoya-Burgos et al., 1998, 2002; Hardman, 2005; Cramer et al., 2008), contrary to morphologic results.

Based on morphological characters, Schaefer (1997, 1998) considered the

Hypoptopomatinae to be monophyletic, consisting of the two monophyletic tribes

Hypoptopomatini and Otothyrini. Montoya-Burgos et al. (1998), using molecular data, were the first to find the Neoplecostominae and the Hypoptopomatinae closely connected, with 18 Pseudotocinclus being the sister group of Pareiorhina (Fig. 1). Cramer et al. (2008) came to similar results, with the Neoplecostominae as a paraphyletic group inside the

Hypoptopomatinae (Fig. 2). In contrast to this, based on morphology, Armbruster (2004) found the Hypoptopomatinae to be a monophyletic group inside the Neoplecostominae

(Fig. 3). Recently, Chiachio et al. (2008) made an attempt to solve these problems including

Pseudotocinclus in the Neoplecostominae and elevating the Otothyrini to subfamily level.

Unfortunately, however, this does not seem to be the ultimate solution as well. as it contradicts the most recent morphological analysis (Lehmann, 2006), our former results

(Cramer et al., 2008), and the findings presented here.

As there are strong contradictions between the morphological phylogenies and the molecular studies published so far (the most recent ones based on a single gene each; both with relatively few species), our aim is to join data from mitochondrial and nuclear sequences and to include the maximum number of species available. Despite the differences between previous results, both morphological and molecular studies found the Hypoptopomatinae +

Neoplecostominae to form a monophyletic group. Therefore these two subfamilies were chosen to be the main focus of our study presented here.

19

Fig. 1 Phylogeny from Montoya-Burgos et al. (1998: modified from Fig. 3) based on sequence data from

mitochondrial 12S and 16S.

20

Fig. 2 Phylogenetic interrelationships of the Fig. 3 Phylogenetic interrelationships of the

Loricariidae, modified from Cramer et al. Loricariidae modified from Armbruster

(2008) (2004)

2. Material and methods:

2.1. Taxon sampling and marker selection

The specimens and species used in this study are listed in Appendix A. Our aim was to include all genera and as many species as possible of the subfamilies Hypoptopomatinae and Neoplecostominae together with representatives of the other loricariid subfamilies.

Unfortunately there was no fresh tissue from Niobichthys or from Lithogenes available. As outgroup taxa we used representatives from the other five families of the Loricarioidea

(Schaefer and Lauder, 1986; Schaefer, 1990; de Pinna, 1998). 21 Four genes were used in these analyses.

The closely linked nuclear recombination activating genes (RAG1 and RAG2) are present in all jawed vertebrates and code for components of the recombinase involved in V (D) J recombination of T-receptor and immunoglobulin genes (Bartl et al., 1994; Bernstein et al.,

1996; Peixoto et al., 2000). Genes with immunological functions should provide an estimate of evolution largely uncorrelated with morphologic adaptations. These genes are usually highly conservative and underlay very little evolutionary pressure for adaptations on the environment (Hoofer et al., 2003). RAG1 and RAG2 have shown to be useful to reconstruct deep phylogenetic relationships in a series of studies (e.g. Sullivan et al., 2000,

2006; Lovejoy and Collette, 2001; Hardman and Page, 2003; Hardman, 2004; Lavoué and

Sullivan, 2004; López et al., 2004; Calcagnotto et al., 2005).

The cytochrome c oxidase I gene (COI) shows a greater range of phylogenetic signal than any other mitochondrial gene (Hebert et al., 2003). Like other protein-coding genes, its third-position nucleotides show a high incidence of base substitutions, leading to a rate of molecular evolution that is about three times higher than that of 12S or 16S rDNA

(Knowlton and Weigt, 1998). So, the evolution of this gene is rapid enough to allow the separation of not only closely related species, but also phylogeographic groups within a single species (Cox and Hebert, 2001; Wares and Cunningham, 2001).

Additional sequences from the nuclear F-Reticulon 4 gene were available for the group we are studying from Chiachio et al. (2008). Thirty of their 53 sequences matched taxa we used and were included in our analyses.

22 2.2. DNA amplification and sequencing

Total genomic DNA was extracted from ethanol-preserved tissue using the QIAmp tissue kit (Qiagen, Hilden, Germany). PCR were carried out in 20 µl reactions. Primers are shown in Table 1. If necessary, a nested PCR was done using internal primers we designed.

We amplified a 690 bp fragment of the subunit 1 of the cytochrome c oxidase using the following PCR conditions: 1x Invitrogen PCR buffer (Invitrogen, São Paulo), 1.5-2.5 mM

® MgCl2, 0.2% Triton X-100, 200 µM of each dNTP, 0.025 U/µl Platinum Taq polymerase

(Invitrogen, São Paulo), 0.2 µM of each primer, and up to 2 µl of DNA solution.

We used our lab’s standard protocol for this primer pair with an initial denaturation step of 1 min at 96 °C followed by 40 cycles of 94 °C for 30 s, annealing at 50 °C for 20 s,

48 °C for 5 s, 46 °C for 5 s, 44 °C for 5 s, 42 °C for 5 s, 40 °C for 20 s, and extension at

72 °C for 1 min. This was followed by a final 3 min at 72 °C extension step.

For the recombination activating gene 1 we amplified a 983 bp fragment using the

® PCR conditions as described above, but 1.5 mM MgCl2, 0.05 U/µl Platinum Taq polymerase (Invitrogen, São Paulo), and 0.5 µM of forward and reverse primer, usually without Triton X-100.

The following thermocycler conditions were used (S. Hoegg, personal communication): an initial denaturation step of 5 min at 94 °C followed by 10 cycles of

94 °C for 30 s, annealing at 52 to 57 °C (each +0.5 °C for the first 10 cycles) for 40 s, extension at 72 °C for 4 min, followed by 25 cycles at 94 °C for 30 s, 55 °C for 40 s, 72 °C for 4 min, and a final extension at 72 °C for 5 min.

For the recombination activating gene 2 we amplified a 961 bp fragment using the

® PCR conditions as described for RAG1, but 3.0 mM MgCl2, 0.04 U/µl Platinum Taq

(Invitrogen, São Paulo), and 0.4 µM of forward and reverse primer. We used an initial denaturation step of 1min at 94 °C followed by 35 cycles of 94 °C for 30 s, annealing at 23 59 °C (51 °C for the nested PCR) for 30 s, and extension at 72 °C for 2 min. This was followed by a final 10 min at 72 °C extension step (Sullivan et al., 2006). Sometimes we used additionally 0.1 mg/µl BSA, 1M Betaine, 0.3% Trealose, and/or 0.2% Triton X-100.

Amplification success was evaluated on GelRedTM (BioTium, São Paulo) or ethidium bromide-stained agarose gels (Sambrook, 1989). PCR products were purified using

PEG8000, ExoSAP-IT® (USB) or the ilustraTM GFX PCR and Gel Band Purification Kit

(GE Healthcare, Buckinghamshire, UK). In case of multiple PCR products, an extraction from the gel was done using the ilustraTM GFX PCR and Gel Band Purification Kit.

Sequencing was done using the DYEnamicTM ET dye terminator kit (GE Healthcare, São

Paulo) read in a MegaBace1000 sequencer. Chromatograms were visualized, edited and assembled using BioEdit 7.0.1 (Hall, 1999). Sequence alignments for each partition were done using Clustal X 1.83 (Thompson et al., 1997) with the standard settings and concatenated in a single alignment.

24 Table 1 Primers used

Gene fragment Primer sequence Source

LCO1490 5‘-GGT CAA CAA ATC ATA AAG ATA TTG G-3‘ Hebert et al. (2003)

HCO2198 5‘-TAA ACT TCA GGG TGA CCA AAA AAT CA-3‘ Hebert et al. (2003)

RAG1-L3a 5’-GCR TTN CCA ATG TCA CAR TG-3’ Martin (1999)

RAG1-MFL1 5’-AGC TGC AGY CAR TAY CAY AAR ATG TA-3’ Martin (1999)

RAG2-MHF1 5’-TGy TAT CTC CCA CCT CTG CGy TAC C-3’ Sullivan et al. (2006)

RAG2-MHR1 5’- TCA TCC TCC TCA TCk TCC TCw TTG TA-3’ Sullivan et al. (2006)

Internal primers

COI-CMF 5‘-GCT AGC CTG TTA ATT CG-3‘ This study

COI-CMR 5‘-AAA GTG GTG TTH AAG TTT CG-3‘ This study

RAG1-CCF 5‘-TGG ACG TCG ATC TTT CAA CCC-3‘ This study

RAG1-CCR 5‘-CTA ATG TGG GCT GTG TCT CCA T-3‘ This study

RAG2-MCF 5‘-CCG TAC ACC CAA TGA-3‘ This study

RAG2-MCR 5‘-AAA TTC AGT AGA TTC TTG ACT GC-3‘ This study

2.3. Phylogenetic analyses

Maximum likelihood (ML) analyses were done using RAxML 7.0.3 (Stamatakis,

2006) with 1000 replicates under the GTRGAMMA model. The model parameters were estimated for the following seven partitions: each of the three codon positions for COI,

RAG1 and RAG2 each, the two introns, and the two exons of the F-Reticulon fragment.

Nodal support was evaluated with 2000 nonparametric bootstrap pseudoreplicates.

25 Bayesian analyses were done using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001;

Ronquist and Huelsenbeck, 2003). The same partitions as under ML were used.

Appropriate substitution models for each of the partitions were estimated with the Akaike

Information Criterion (AIC) as implemented in MrModeltest 2.3 (Nylander, 2004). Some of the Bayesian analyses were run on the Bioportal TITAN server (Oslo University).

Maximum Parsimony (MP) analyses were done using the new technology search as implemented in TNT 1.1 (Goloboff et al., 2008). We executed 1000 sequential ratchets of

200 iterations each, followed by sectorial searches and tree fusion. For nodal support, the

Bremer support (decay index) was calculated using the converse constraint method as implemented in TNT 1.1. 2000 nonparametric bootstrap pseudoreplicates were also calculated.

To test our topologies, we made six more MP analyses using constraints to enforce the monophyly of the subfamilies Hypostominae and Neoplecostominae, and the genera

Neoplecostomus, Pareiorhaphis, and Pareiorhina. The resulting alternative topologies were evaluated using the Kishino and Hasegawa test (KH) (Kishino and Hasegawa, 1989) as implemented in PAUP 4b10 (Swofford, 2001).

3. Results and Discussion

The present study is the largest loricariid phylogeny in number of species published so far. We included representatives from five of the six loricariid subfamilies. The

Lithogeninae could not be included because of the poor quality of the DNA extracted from formalin fixed tissue. The Hypoptopomatinae and the Neoplecostominae were included with taxa from all described genera but Niobichthys. The latter is a monotypic genus only known from one location in the Neblina Mountains in Venezuela. It was not possible to get tissue from this taxon. The Neoplecostominae is represented by most of its described

26 species plus some new taxa, lacking only four species of Pareiorhaphis (two of them without confirmed locations and only known from few specimens) and five of Neoplecostomus.

3.1. Sequence statistics

We were able to sequence fragments of up to 690 bp for COI, 983 bp for RAG1, and

961 bp for RAG2. Additional sequences were taken from GenBank (Cramer et al. (2008) and Rodriguez et al. (2008): COI; Chiachio et al. (2008): F-Reticulon 4). In our analysis we included sequences of 161 specimens from 146 species and 57 genera, most of them represented by sequences of the three genes we amplified (species and GenBank accession numbers in Appendix A). 136 species from 50 genera are loricariids, 103 species from 30 genera are from the subfamilies Neoplecostominae and Hypoptopomatinae. Nematogenys inermis is the most basal taxon of the super family Loricarioidea according to Schaefer and

Lauder (1986) and was used to root the trees.

Together with the F-Reticulon sequences, we obtained a total alignment length of

4678 bp. Out of these, 2258 (48.2%) were variable and 1588 (33.9%) were parsimony- informative. For the separate partitions these values were: COI: 1st codon position: 81

(35.2%) and 57 (24.7%), 2nd codon position: 25 (10.9%) and 8 (3.5%), 3rd codon position:

224 (97.4%) and 221 (96.1%), RAG2: 616 (64.1%) and 455 (47.3%), RAG1: 462 (47.0%) and 348 (35.4%), F-Reticulon: intronic positions: 724 (58.0%) and 424 (33.9%), and exonic positions: 134 (15.1%) and 80 (9.0%).

27 3.2. Phylogenetic analyses

The ML analyses resulted in one best tree (-ln L 51854.558340) that is shown in

Fig. 4. Most clades received high bootstrap values. To test our best tree, we ran another

ML analysis taking one of the most parsimonious trees as starting tree. This analysis resulted in the same topology as the previous best tree.

For the Bayesian analyses, the best models of sequence evolution for the data partitions found according to the AIC criterion were: SYM + G + I for the 1st codon position in COI, F81 + I for the 2nd codon position in COI, GTR + G + I for the 3rd codon position in COI and RAG2, HKY + G + I for RAG1 and the F-Reticulon exonic positions, and GTR + G for the F-Reticulon intronic positions. Two runs of four chains each (three heated, one cold) were run simultaneously with tree space sampled every 1000th generation. Convergence between chains occurred after 10 million generations (standard deviation of split frequency < 0.01). A graphical analysis of the evolution of the likelihood scores showed that the stationary phase was reached after 600,000 generations. Therefore, the first 2 million generations (20%) were discarded as burn-in. The remaining trees were used to calculate the consensus tree (Fig. 5; Appendix B). There are only few minor differences between the best tree from the ML analyses and the consensus from the

Bayesian analysis, with one exception, positions changed only one node. The only striking difference is that the Bayesian analysis did not resolve Lampiella gibbosa as sister to the genus Otocinclus, but as sister to Hisonotus leucofrenatus. To test this result, we performed additional MP and ML analyses (not shown here) for only the COI, RAG1, and

RAG2 sequences, without F-Reticulon. Analyzing each gene separately or the three together led to very similar topologies, always revealing Lampiella gibbosa as sister to

Hisonotus leucofrenatus. 28 The MP analyses resulted in 358 best trees with 9363 steps (CI: 0.36; HI: 0.64; RI:

0.69; RC: 0.25). Their strict consensus is shown in Fig. 6 (Appendix C).

Besides the lower resolution of the consensus of MP trees, there are only five minor differences between the results from the MP and the ML analyses, all with little support.

ML shows Delturus carinotus as sister taxon of the genus Hemipsilichthys, MP revealed it as sister taxon to Hemipsilichthys nimius. In the ML analysis, aspera is sister to all loricariids but the Delturinae, in the MP analyses the Delturinae and the

Loricariinae are basal to Rhinelepis. MP resolved Pareiorhaphis hypselurus, P. nudula, and P. stomias as sister group to Isbrueckerichthys, whereas ML puts them as sister group to the remaining Pareiorhaphis. ML shows Pareiorhaphis parmula as sister of P. azygolechis, P. sp. 1, and P. sp.4, MP resolved P. azygolechis as sister of the other named species. The only taxon whose position differed more than one node is Parotocinclus jumbo. MP showed it as sister to Lampiella gibbosa on the base of the genus Otocinclus, whereas ML placed it basal to the clade containing the majority of the Otothyrini.

29 Pareiorhaphis sp. 4 Pareiorhaphis sp. 1 Pareiorhaphis eurycephal us Pareiorhaphis parmula Pareiorhaphis sp. 5 100 Oxyropsis wrightiana Pareiorhaphis vestigipinni s Pareiorhaphis sp. 3 Pareiorhaphis hystrix 100 Pareiorhaphis sp. 6 Oxyropsis carinata Pareiorhaphis calmoni Pareiorhaphis steindachneri Pareiorhaphis azygolechi s 99 Kronichthys subteres Oxyropsis acutirostra Kronichthys subteres Kronichthys heylandi Kronichthys lacerta Pareiorhaphis splendens 100 Hypoptopoma bilobatum Pareiorhaphis splendens Pareiorhaphis hypselurus Pareiorhaphis stomias 100 Pareiorhaphis nudula Hypoptopoma guianense Isbrueckerichthys calvus Isbrueckerichthys saxicola Isbrueckerichthys epakmos Nannoptopoma spectabile Neoplecostomus ribeirensis Isbrueckerichthys alipionis Pareiorhaphis nasuta 89 Pareiorhaphis sp. 2 100 Hypoptopoma inexpectatum Hypoptopomatini Neoplecostom i na e nov. gen .1 Pareiorhaphis bahiana Neoplecostom i na e nov .g en. 3 Neoplecostom i na e nov. gen .2 100 Hypoptopoma cf. inexpectatum Pareiorhina brachyrhyncha Pareiorhina carrancas Neoplecostom u s franciscoensis Neoplecostomus paranensis 91 Hypoptopoma steindachneri Neoplecostomus espiritosante nsis Neoplecostomus microps Neoplecostomus sp. Pareiorhina sp. 100 Hypoptopoma gulare Pareiorhina sp. Pseudotocinclus juquiae Pseudotocin clu s tietensis Pareiorhaphis garbei -- 51 Otocinclus cf. hoppei Pareiorhaphis garbei Otothyris juquiae Schizolecis guentheri Schizolecis guentheri Otocinclus vestitus Hisonotus sp. 2 Hisonotus laevior 94 Hisonotus laevior Hisonotus armatus Otocinclus vittatus Hisonotus armatus Hisonotus taimensis 99 Hisonotus charrua a Hisonotus insperatus Otocinclus cocama Hisonotus charrua b Hisonotus leucofrenat us Hisonotus leucofrenat us Epactionotus gracilis 99 Otocinclus cf. mariae Epactionotu s itaimbezinho Epactionotus bilineatus Eurycheilichthys pantherinus Eurycheilichthys sp. 1 Otocinclus xakriaba Eurycheilichthys limulus 81 Eurycheilichthys limulus Hisonotus nigricauda Hisonotus ringueleti Otocinclus affinis Hisonotus iota 53 100 Hisonotus sp. 1 Pseudotothyris obtusa Pseudotothyris obtusa 62 Otocinclus flexilis Hisonotus francirochai Hisonotus sp. 4 M icrolepidogast er sp. 2 Otothyropsis sp. Otocinclus flexilis Parotocinclus bidentatus Parotocinclus maculicauda M icrolepidogast er sp. 1 Microlepidogast er perforatus Lampiella gibbosa Parotocinclus spilosoma Parotocinclus sp. Parotocinclus eppleyi Parotocinclus aripuanensi s 100 Acestridium martini Parotocinclus collinsae Parotocinclus cf aripuane nsis Parotocinclus britskii 89 Otothyris travassosi Acestridium martini Parotocinclus jumbo Gymnotocinclus anosteos Corumbataia cuestae 100 Hisonotus luteofrenatus Acestridium scutatum Hypoptopomati nae sp. Surinam Oxyropsis wrightiana Oxyropsis carinata Oxyropsis acutirostra Acestridium gymnogaster Hypoptopoma bilobatum Hypoptopoma guianense Nannoptopoma spectabile Hypoptopoma inexpectat um 92 Hypostomus boulengeri Hypoptopoma cf. inexpectatum Hypoptopoma steindachneri Hypoptopoma gulare -- Otocinclus cf. hoppei Hypostomus commersonii Otocinclus vestitus Otocinclus vittatus Otocinclus cocama 96 Otocinclus cf. mariae Hypostomus cochliodon Otocinclus xakriaba Otocinclus affinis Otocinclus flexilis 99 85 Otocinclus flexilis Hypostomus albopunctatus Lam piella gibbosa Acestridium martini Acestridium martini Acestridium scutatum 99 Hemiancistrus fuliginosus Acestridium gymnogaster Hypostomus boulengeri -- Hypostomus commersonii Hypostomus cochliodon Hemiancistrus punctulatus Hypostomus albopunct atu s Hemiancistrus fuliginosus Hemiancistrus punctulatus Liposarcus anisitsi 100 Liposarcus anisitsi Liposarcus pardalis Panaqolus sp. Panaqolus changa e Scobinancistrus pariolispos Liposarcus pardalis Ancistomus sabaji Peckoltia vermiculata -- Peckoltia vittata Hypancistrus contradens 90 Panaqolus sp. Hemiancistrus subviridis Panaque cf. nigrolineatus Panaque cf. cochliodon -- Hypostominae M egalancistrus paranan us Panaqolus changae Pseudacant hic us leopardus Corymbophane s kaiei Ancistrus brevipinnis Chaetostom a sp. 2 Scobinancistrus pariolispos Chaetostom a sp. 1 Rineloricaria sp. Ixinandria steinbachi 96 Apistoloricaria ommation 99 88 Ancistomus sabaji Harttia kronei Hem ipsilichtys papillatus 85 Hemipsilichthys gobio Peckoltia vermiculata Hemipsilichthys nimius Delturus carinotus Astroblepus sp. 1 -- Astroblepus sp. 3 Peckoltia vittata Astroblepus sp. 2 Astroblepus sp. 4 88 Scoloplax distolothrix Callichthys callichthys Hoplosternum littorale 58 Hypancistrus contradens ourastigma Trichomycterus sp. Nematogenys inermis Hemiancistrus subviridis 99 100 Panaque cf. nigrolineatus Panaque cf. cochliodon -- 91 parananus 96 Pseudacanthicus leopardus Corymbophanes kaiei 99 100 Ancistrus brevipinnis 100 Chaetostoma sp. 2

Chaetostoma sp. 1 Loricariinae 100 Rineloricaria sp. 100 Ixinandria steinbachi 99 98 Apistoloricaria ommation Harttia kronei Rhinelepis aspera * Delturinae 100 Hemipsilichtys papillatus 58 Hemipsilichthys gobio 96 100 Hemipsilichthys nimius * Delturus carinotus -- Astroblepus sp. 1 -- Astroblepus sp. 3 100 -- Astroblepus sp. 2 64 Outgroup Astroblepus sp. 4 Scoloplax distolothrix -- Callichthys callichthys 100 Hoplosternum littorale 56 Corydoras ourastigma Trichomycterus sp. Nematogenys inermis Fig. 4a Relationships of the taxa within the Delturinae, Loricariinae, Hypostominae and Hypoptopomatini based on the Maximum Likelihood analysis. This is part of the best tree (-lnL 51854.558340), remainder of the tree is in Fig. 4b and c. Likelihood bootstrap proportions shown on the branches (≥ 50). Branches marked with * differ in their position from the MP analysis; branches marked with S differ in their position from the Bayesian analysis.

30 Pareiorhaphis sp. 4 Pareiorhaphis sp. 1 Pareiorhaphis eurycephalus Pareiorhaphis parmula Pareiorhaphis sp. 5 Pareiorhaphis vestigipinni s Pareiorhaphis sp. 3 Pareiorhaphis hystrix Pareiorhaphis sp. 6 Pareiorhaphis calmoni Pareiorhaphis steindachneri Pareiorhaphis azygolechi s Kronichthys subteres Kronichthys subteres Kronichthys heylandi Kronichthys lacerta Pareiorhaphis splende ns Pareiorhaphis splende ns Pareiorhaphis hypselurus Pareiorhaphis stomias Pareiorhaphis nudula Isbrueckerichthys calvus Isbrueckerichthys saxicola Isbrueckerichthys epakmos Isbrueckerichthys duseni Neoplecostom u s ribeirensis Isbrueckerichthys alipionis Pareiorhaphis nasuta Pareiorhaphis sp. 2 Neoplecostom i na e nov. gen .1 Pareiorhaphis bahiana Neoplecostom i na e nov .g en. 3 Neoplecostom i na e nov. gen .2 Pareiorhina brachyrhynch a Pareiorhina carrancas Neoplecostomus franciscoensis Neoplecostom u s paranensis Neoplecostom u s espiritosante nsis Neoplecostomus microps Neoplecostomus sp. Pareiorhina sp. Pareiorhina sp. Pseudotocinclus juquiae Pseudotocin clu s tietensis Pareiorhaphis garbei Pareiorhaphis garbei Otothyris juquiae Schizolecis guentheri Schizolecis guentheri Hisonotus sp. 2 Hisonotus laevior Hisonotus laevior Hisonotus armatus Hisonotus armatus Hisonotus taimensis Hisonotus charrua a Hisonotus insperatus Hisonotus charrua b Hisonotus leucofrenat us Hisonotus leucofrenat us Epactionotus gracilis Epactionotu s itaim bezinho Epactionotus bilineatus Eurycheilichthys pantherinus Eurycheilichthys sp. 1 Eurycheilichthys limulus Eurycheilichthys limulus Hisonotus nigricauda Hisonotus ringueleti Hisonotus iota Hisonotus sp. 1 Pseudotothyris obtusa Pseudotothyris obtusa Hisonotus francirochai Hisonotus sp. 4 Microlepidogast er sp. 2 Otothyropsis sp. Parotocinclus bidentatus Parotocinclus m aculicauda Microlepidogast er sp. 1 Microlepidogast er perforatus Parotocinclus spilosoma Parotocinclus sp. Parotocinclus eppleyi Parotocinclus aripuanensi s Parotocinclus collinsae Parotocinclus cf aripuanensis 58 Parotocinclus britskii Hisonotus sp. 2 Otothyris travassosi Parotocinclus jumbo Gymnotocinclus anosteos 75 Corumbataia cuestae Hisonotus laevior Hisonotus luteofrenatus Hypoptopomati n ae sp. Surinam Oxyropsis wrightiana -- Oxyropsis carinata Hisonotus laevior Oxyropsis acutirostra Hypoptopoma bilobatum Hypoptopoma guianen se 74 Nannoptop oma spectabile 79 Hisonotus armatus Hypoptopoma inexpectat um Hypoptopoma cf. inexpectatum Hypoptopoma steindach n eri Hypoptopoma gulare Hisonotus armatus Otocinclus cf. hoppei Otocinclus vestitus 61 Otocinclus vittatus Otocinclus cocama Hisonotus taimensis Otocinclus cf. mariae Otocinclus xakriaba Otocinclus affinis Otocinclus flexilis 90 77 Hisonotus charrua a Otocinclus flexilis Lam piella gibbosa Acestridium martini Acestridium martini Hisonotus insperatus Acestridium scutatum Acestridium gymnogaster -- Hypostomus boulengeri Hypostomus commersonii Hisonotus charrua b Hypostomus cochliodon Hypostomus albopunct atu s Hemiancistrus fuliginosus Hemiancistrus punctulatus 95 Hisonotus leucofrenatus Liposarcus anisitsi Liposarcus pardalis Panaqolus sp. Panaqolus changa e Hisonotus leucofrenatus Scobinancistrus pariolispos 80 Ancistomus sabaji Peckoltia vermiculata Peckoltia vittata 95 Epactionotus gracilis Hypancistrus contradens Hemiancistrus subviridis Panaque cf. nigrolinea tus 99 Panaque cf. cochliodon Epactionotus itaimbezinho Megalancistrus parananus Pseudacant hic us leopardus Corymbophane s kaiei Ancistrus brevipinnis 87 Epactionotus bilineatus Chaetostom a sp. 2 Chaetostom a sp. 1 Rineloricaria sp. 50 Ixinandria steinbachi Eurycheilichthys pantherinus Apistoloricaria ommation 98 Harttia kronei 76 Rhinelepis aspera Hemipsilichtys papillatus Eurycheilichthys sp. 1 Hemipsilichthys gobio 99 Hemipsilichthys nimius Delturus carinotus Astroblepus sp. 1 99 Eurycheilichthys limulus Astroblepus sp. 3 Astroblepus sp. 2 Astroblepus sp. 4 Scoloplax distolothrix Eurycheilichthys limulus Callichthys callichthys Hoplosternum littorale Corydoras ourastigma Trichomycterus sp. 90 Hisonotus nigricauda Otothyrini Nematogenys inermis 93 Hisonotus ringueleti 53 77 Hisonotus iota Hisonotus sp. 1 100 Pseudotothyris obtusa -- Pseudotothyris obtusa 76 Hisonotus francirochai -- 80 Hisonotus sp. 4 64 Microlepidogaster sp. 2 Otothyropsis sp. 53 53 99 Parotocinclus bidentatus -- Parotocinclus maculicauda -- Microlepidogaster sp. 1 Microlepidogaster perforatus -- Parotocinclus spilosoma 98 Parotocinclus sp. 88 Parotocinclus eppleyi 52 63 Parotocinclus aripuanensis 58 Parotocinclus collinsae 51 93 Parotocinclus cf. aripuanensis Parotocinclus britskii Otothyris travassosi * Parotocinclus jumbo 100 -- Gymnotocinclus anosteos 99 Corumbataia cuestae 100 Hisonotus luteofrenatus Hypoptopomatinae sp. Surinam

Fig. 4b Relationships of the taxa within the Otothyrini (without Otothyris, Schizolecis, and Pseudotocinclus) based on the Maximum Likelihood analysis. This is part of the best tree (-lnL 51854.558340), remainder of the tree is in Fig. 4a and c. Likelihood bootstrap proportions shown on the branches (≥ 50). Branches marked with * differ in their position from the MP analysis; branches marked with S differ in their position from the Bayesian analysis.

31 Pareiorhaphis sp. 4 Pareiorhaphis sp. 1 Pareiorhaphis eurycephal us Pareiorhaphis parmula Pareiorhaphis sp. 5 Pareiorhaphis vestigipinni s Pareiorhaphis sp. 3 Pareiorhaphis hystrix Pareiorhaphis sp. 6 Pareiorhaphis calmoni Pareiorhaphis steindachneri Pareiorhaphis azygolechi s Kronichthys subteres Kronichthys subteres Kronichthys heylandi Kronichthys lacerta Pareiorhaphis splendens Pareiorhaphis splendens Pareiorhaphis hypselurus Pareiorhaphis stomias Pareiorhaphis nudula Isbrueckerichthys calvus Isbrueckerichthys saxicola Isbrueckerichthys epakmos Isbrueckerichthys duseni Neoplecostomus ribeirensis Isbrueckerichthys alipionis Pareiorhaphis nasuta Pareiorhaphis sp. 2 Neoplecostom i na e nov. gen .1 Pareiorhaphis bahiana Neoplecostomi nae nov .gen. 3 Neoplecostom i na e nov. gen .2 Pareiorhina brachyrhyncha Pareiorhina carrancas Neoplecostomus franciscoensis Neoplecostom u s paranensis Neoplecostomus espiritosantensis Neoplecostomus microps Neoplecostomus sp. Pareiorhina sp. Pareiorhina sp. Pseudotocin clu s juquiae Pseudotocin clu s tietensis Pareiorhaphis garbei Pareiorhaphis garbei Otothyris juquiae Schizolecis guentheri Schizolecis guentheri Hisonotus sp. 2 Hisonotus laevior Hisonotus laevior Hisonotus armatus Hisonotus armatus Hisonotus taimensis Hisonotus charrua a Hisonotus insperatus Hisonotus charrua b Hisonotus leucofrenatus Hisonotus leucofrenatus Epactionotus gracilis Epactionotu s itaimbezinho Epactionotu s bilineatus Eurycheilichthys pantherinus Eurycheilichthys sp. 1 Eurycheilichthys limulus Eurycheilichthys limulus Hisonotus nigricauda Hisonotus ringueleti Hisonotus iota Hisonotus sp. 1 Pseudotothyris obtusa Pseudotothyris obtusa 83 Pareiorhaphis sp. 4 Hisonotus francirochai Hisonotus sp. 4 -- Microlepidogast er sp. 2 Otothyropsis sp. Pareiorhaphis sp. 1 Parotocinclus bidentatus Parotocinclus maculicauda 63 Microlepidogast er sp. 1 Microlepidogast er perforatus Pareiorhaphis eurycephalus Parotocinclus spilosoma 91 * Parotocinclus sp. Parotocinclus eppleyi Parotocinclus aripuanensi s Pareiorhaphis parmula Parotocinclus collinsae Parotocinclus cf aripuane nsis 94 Parotocinclus britskii Otothyris travassosi Pareiorhaphis sp. 5 Parotocinclus jumbo Gymnotocinclus anosteos Corumbataia cuestae Hisonotus luteofrenatus 70 Pareiorhaphis vestigipinnis Hypoptopomati nae sp. Surinam Oxyropsis wrightiana Oxyropsis carinata Oxyropsis acutirostra 99 Pareiorhaphis sp. 3 Hypoptopoma bilobatum Hypoptopoma guianense 99 Nannoptopoma spectabile Hypoptopoma inexpectat um Pareiorhaphis hystrix Hypoptopoma cf. inexpectatum 94 Hypoptopoma steindachneri Hypoptopoma gulare Otocinclus cf. hoppei Pareiorhaphis sp. 6 Otocinclus vestitus Otocinclus vittatus Otocinclus cocama 99 Otocinclus cf. mariae Pareiorhaphis calmoni Otocinclus xakriaba Otocinclus affinis -- Otocinclus flexilis Otocinclus flexilis Pareiorhaphis steindachneri Lam piella gibbosa Acestridium martini Acestridium martini Acestridium scutatum 50 Pareiorhaphis azygolechis Acestridium gymnogaster Hypostomus boulengeri Hypostomus commersonii 99 Hypostomus cochliodon Kronichthys subteres Hypostomus albopunct atus Hemiancistrus fuliginosus 68 Hemiancistrus punctulatus Liposarcus anisitsi Kronichthys subteres Liposarcus pardalis Panaqolus sp. 99 Panaqolus changa e Scobinancistrus pariolispos Kronichthys heylandi Ancistomus sabaji Peckoltia vermiculata -- Peckoltia vittata 54 Hypancistrus contradens Kronichthys lacerta Hemiancistrus subviridis Panaque cf. nigrolineatus Panaque cf. cochliodon 99 Megalancistrus paranan us Pareiorhaphis splendens Pseudacant hic us leopardus Corymbophane s kaiei Ancistrus brevipinnis Chaetostom a sp. 2 Pareiorhaphis splendens Chaetostom a sp. 1 Rineloricaria sp.

Ixinandria steinbachi Neoplecostominae Apistoloricaria ommation 97 Pareiorhaphis hypselurus Harttia kronei Rhinelepis aspera Hem ipsilichtys papillatus 100 Hemipsilichthys gobio Pareiorhaphis stomias Hemipsilichthys nimius Delturus carinotus 98 Astroblepus sp. 1 * Astroblepus sp. 3 Pareiorhaphis nudula Astroblepus sp. 2 Astroblepus sp. 4 Scoloplax distolothrix Callichthys callichthys 60 Isbrueckerichthys calvus Hoplosternum littorale Corydoras ourastigma 57 Trichomycterus sp. Nematogenys inermis Isbrueckerichthys saxicola 92 Isbrueckerichthys epakmos 89 Isbrueckerichthys duseni 99 Neoplecostomus ribeirensis 97 Isbrueckerichthys alipionis 80 Pareiorhaphis nasuta 99 Pareiorhaphis sp. 2 -- Neoplecostominae nov. gen. 1 62 Pareiorhaphis bahiana 95 Neoplecostominae nov. gen. 3 77 Neoplecostominae nov. gen. 2 99 97 Pareiorhina brachyrhyncha Pareiorhina carrancas 66 -- Neoplecostomus franciscoensis -- Neoplecostomus paranensis 99 Neoplecostomus espiritosantensis 97 Neoplecostomus microps 56 Neoplecostomus sp. 100 Pareiorhina sp. 100 Pareiorhina sp. 100 Pseudotocinclus juquiae Pseudotocinclus tietensis 100 Pareiorhaphis garbei

97 Pareiorhaphis garbei Otothyrini 75 Otothyris juquiae 100 Schizolecis guentheri Schizolecis guentheri

Fig. 4c Relationships of the taxa within the Neoplecostominae plus Otothyris, Schizolecis, and Pseudotocinclus based on the Maximum Likelihood analysis. This is part of the best tree (-lnL 51854.558340), remainder of the tree is in Fig. 4a and b. Likelihood bootstrap proportions shown on the branches (≥ 50). Branches marked with * differ in their position from the MP analysis; branches marked with S differ in their position from the Bayesian analysis.

32 3.3. Phylogenetic relationships

The few differences between the results of MP, ML, and the Bayesian analyses are low support resolutions and are not unacceptable in morphological ground. We therefore will use the ML tree for further discussion because of its better resolution than the consensus from MP and its similarity to the consensus from the Bayesian analyses. Branch lengths from the ML tree are similar to the ones from the Bayesian analysis, so they are only shown once (Fig. 5).

Below we discuss the results of the ML analyses starting from the base of the tree and climbing upwards.

Inside the outgroup, an interesting finding is the long branch of Astroblepus sp. 4. The

COI and RAG1 sequences from the four Astroblepus are relatively similar compared with the other species from the genus (0.876-0.965 and 0.981 to 0.991, respectively). The same is true for the RAG2 sequences from species 1 to 3 (0.992 to 0.997), only the one from species 4 is significantly different from its congeners (0.716 to 0.722), but the translation to amino acids resulted in very similar sequences (0.956 to 0.972 vs. 0.984 to 1 between species 1 to 3 and 0.995/0.985 to 1 in COI/RAG1).

The Loricariidae forms a monophyletic group. Without the presence of taxa from the

Lithogeninae, the Delturinae is the most basal loricariid clade, as Reis et al. (2006) already suggested based on morphological results. A somewhat strange finding is that Rhinelepis is located clearly separated from the other hypostomine taxa. Most other studies placed members of the Rhinelepini as relatively basal taxa within the Hypostominae (e.g.

Schaefer, 1986; Armbruster, 2004), though Montoya-Burgos et al. (1998) revealed

Pseudorinelepis (Rhinelepini) as sister taxon of the Loricariinae. Rhinelepis differs from all other Hypostominae in some aspects. Besides its somewhat archaic appearance, they are 33 the only loricariids which do spawning migration and which produce millions of eggs (up to 180,000; vs. dozens or a few hundred in other loricariids) that are spread freely in the current (Suzuki et al., 2000). Rhinelepis are the only hypostomins without parental care of eggs or fries.

An alternative topology with an enforced monophyly of the Hypostominae resulted in seven additional steps and showed Rhinelepis as sister of the remaining hypostomine taxa.

This constrained topology could not be statistically refused by the KH test (p=0.6283).

Inside the Hypostominae, Chaetostoma was placed as the most basal taxon and not

Corymbophanes as in Armbruster (2004). Also, Panaqolus and Scobinancistrus are clearly separated from Panaque, suggesting that they are not synonyms or subgenera as proposed by Armbruster (2004). Neither the Ancistrini nor the Hypostomini seem to form monophyletic groups.

But, as the Hypostominae is not the focus of this article, we included only few taxa and further studies have to analyze this subfamily with more details.

Concordant with former studies (e.g. Armbruster, 2004; Cramer et al., 2008), we found the Hypoptopomatinae + Neoplecostominae to form a monophyletic clade, but our data did not recover the Hypoptopomatini, Otothyrini (sensu Schaefer, 1991), or the

Neoplecostominae as natural groups. Some of the most parsimonious trees even show the

Hypoptopomatini as monophyletic like found by Lehmann (2006) and Chiachio et al.

(2008), but with low support.

The results from the complete dataset using ML and MP place Lampiella gibbosa as sister to the genus Otocinclus. Analyzing only the sequences from COI, RAG1 and RAG2, this species is positioned as sister to Hisonotus leucofrenatus, getting high bootstrap values. The Bayesian analyses of the complete dataset come to the same result. Therefore, 34 it still cannot be resolved if Lampiella really is a distinct genus or if it is a synonym of

Otocinclus as proposed by Schaefer (2003). Hypoptopoma forms a paraphyletic group including the genera Nannoptopoma and Oxyropsis. If Nannoptopoma (as proposed by

Chiachio et al. [2008]) and potentially Oxyropsis should be treated as synonyms of

Hypoptopoma, or if a new genus should be established for part of the Hypoptopoma species, should be investigated by a future study including more species and morphological data.

Corumbataia, Gymnotocinclus, Hisonotus luteofrenatus, and Hypoptopomatinae sp.

Surinam (probably the same species as shown in Le Bail et al. [2000 p. 262] and Schaefer

[1998: taxon 3]) form a group basal to the remaining Otothyrini + Neoplecostominae, with

Corumbataia being sister to Gymnotocinclus. As already predicted by Schaefer (Le Bail et al., 2000: p. 262), Hisonotus luteofrenatus (which belongs to the same undescribed genus, in preparation by Buckup, Britto, and Reis) and Hypoptopomatinae sp. Surinam also are sister taxa.

The next bigger clade is formed by the Neoplecostominae + Pseudotocinclus,

Schizolecis, and Otothyris juquiae. The two latter taxa, together with Pareiorhaphis garbei, form the basal group of this clade. Thus, Otothyris turns out to be a polyphyletic taxon. Lehmann (2006) also found Otothyris and Schizolecis to be sister taxa, but there is no morphological evidence for a closer relation between Pareiorhaphis garbei and these two genera (Pereira, 2008). Pareiorhaphis garbei shows characteristics that are exclusive for the Neoplecostominae (e.g. presence of an accessory process on the crest of the muscle levator arcus palatine and a branched canal on the canal plate) or for the genus

Pareiorhaphis (e.g. canal plate strongly articulated with the pre-operculum in mature males) (Pereira, 2008). Our additional constrained MP analyses enforced monophyly for 35 the genus Pareiorhaphis and the Neoplecostominae. The first analysis resulted in significantly worse trees (p=0.0001), by KH test, with 46 additional steps. The second analysis generated best trees with six additional steps, showing Pareiorhaphis garbei as sister to the remaining neoplecostomines. This result could not be rejected by the KH test

(p=0.4796).

Inside the Neoplecostominae, Pseudotocinclus and part of the genus Pareiorhina form the most basal group. That corroborates the results of Montoya-Burgos et al. (1998),

Cramer et al. (2008), and Chiachio et al. (2008), but this is the first time that Pareiorhina is recovered as polyphyletic.

An enforced monophyly for Pareiorhina caused 35 additional steps and showed all

Pareiorhina as sister of the genus Pseudotocinclus. The KH test refused the constrained topology as significantly worse (p=0.0012).

Our results place Neoplecostomus ribeirensis inside the genus Isbrueckerichthys.

There is no morphological corroboration for this finding as N. ribeirensis shares the morphological synapomorphies of the genus Neoplecostomus (dorsal profile of the unbranched pectoral spine with accentuated curvature; leteropterygium with small expansion in the distal portion; lower lip with papillae forming conspicuous series localized posterior of the dentary) and does not show the synapomorphy of the genus

Isbrueckerichthys (crest of the levator arcus palatine with dorsal direction) (Pereira, 2008).

To exclude the possibility of a misidentification or contamination, we reexamined the voucher specimen, repeated the DNA extraction and sequenced the three fragments again, with the very same result. The remaining species of Neoplecostomus form a well supported monophyletic group, as expected from morphologic data (Pereira, 2008). A constrained topology with an enforced monophyly for the genus Neoplecostomus resulted in significantly worse trees (p=0.0140) with 28 additional steps. 36 Contrary to the results of Pereira (2008), we found Pareiorhaphis to be polyphyletic.

Pareiorhaphis bahiana and the new genera Neoplecostominae nov. gen. 2 & 3 form a biogeographical clade. The three taxa are from coastal rivers in Bahia state.

Neoplecostominae nov. gen. 1, Pareiorhaphis nasuta, and P. sp. 2 form another geographical clade from the rio Doce basin. Being sister clades, the two groups form a third biogeographical clade from the rio São Francisco drainage. In agreement with

Pereira, (2008) we found P. hypselurus, P. nudula, and P. stomias as well as P. calmoni and P. steindachneri to form two groups with strong support. Our constraint forcing the genus Pareiorhaphis (without P. garbei) as monophyletic resulted in significantly worse trees (p=0.0222) with 28 additional steps.

The remaining taxa of the Otothyrini (sensu Schaefer, 1991) form a poorly supported group. Concordant with Gauger and Buckup (2005) and Lehmann (2006), Parotocinclus turned out to be polyphyletic. Parotocinclus aripuanensis, P. britskii, P. collinsae, P. eppleyi and an undescribed species form a geographical group from the Guyana shield.

Parotocinclus bidentatus and P. maculicauda are well supported sister taxa. Hisonotus is another genus that is highly polyphyletic, needing a morphological revision.

Striking in our results is that the “Hypoptopomatini” was recovered as the most basal hypoptopomatine taxon and Eurycheilichthys one of the most derived. This is the extreme opposite of what the morphologic data of Lehmann (2006) revealed.

At present, our molecular data suggest that Hypoptopomatini, Otothyrini, and

Neoplecostominae are not monophyletic. To further investigate this group with respect of the differences between molecular and morphologic evidences we suggest a combined analysis of these data, an approach that has been successful in similar cases 37 (e.g. Gatesy et al., 2003; Mattern and McLennan, 2004), together with a continuing expansion of the species sampling.

Acknowledgements

Thanks are due to our colleagues Edson H. L. Pereira, Tiago Pinto Carvalho, and

Pablo Lehmann, who helped with the identification of the samples, and to Cladinara

Roberts Sarturi, Fernanda Britto and Mirian Tieko Nunes Tsuchiya for support with the lab work. Bárbara Calegari, Alexandre Rodrigues Cardoso, Tiago Pinto Carvalho, and

Fernando C. Jerep helped collecting specimens. Taran Grant, Felipe Grazziotin, and

Alexandros Stamatakis helped us patiently with the phylogenetic analyses. The following persons and companies donated specimens for this work: Thomas Weidner (Iffeldorf,

Germany), Ingo Seidel/Aqua Global (Seefeld, Germany), André Werner/Transfish

(Planegg, Germany), and Frank Schäfer/Aquarium Glaser (Rodgau, Germany). The following colleagues and institutions provided tissue samples for this work: ANSP (Mark

Sabaj), AUM (Jon W. Armbruster), DZJRP (Francisco Langeani), INHS (Michael

Hardman), LBP (Claudio Oliveira), MCP (Margarete Lucena), MCNI (Gladys Monasterio de Gonzo), MNHG (Raphael Covain), MNRJ (Paulo Buckup), MZUEL (Oscar Shibatta),

NUP (Claudio Zawadski), UF (Nathan Lovejoy), UFRGS (Andréa Thomaz), and ZSM

(Dirk Neumann). Financial support: DAAD (German Academic Exchange Service) and

CNPq (Brazilian Council for Technologic and Scientific Development).

38 Appendix A: Species, depository information and GenBank accession numbers (COI, RAG2,

RAG1, F-Reticulon) included in this study. Sequences marked with * were taken from

GenBank; O: no sequence available.

Astroblepidae: Astroblepus sp. 1 ANSP 180581 (tag 4805) EU359404*, GQ225437, O, O.

Astroblepus sp. 2 ANSP 180605 (tag 4490) EU359405*, GQ225438, GQ214567, O. Astroblepus sp. 3 ANSP 180613 (tag 4453) EU359406*, GQ225439, GQ214568, O. Astroblepus sp. 4 ANSP

180616 (tag 4436) EU359407*, GQ225440, GQ214569, O. Callichthyidae: Callichthys callichthys MCP 29384 EU359408*, GQ225443, GQ214572, O. Corydoras ourastigma MCP

28799 GQ225387, GQ225447, GQ214575, O. Hoplosternum littorale MCP 21196 EU359416*,

GQ225475, GQ214599, O. Loricariidae: Acestridium martini ANSP 182901 (tag V5276)

EU359398*, GQ225430, GQ214560, O. Acestridium martini ANSP 182901 (tag V5277)

EU359399*, GQ225431, GQ214561, O. Acestridium sp. 1 MCP 37783 EU359400*, GQ225432,

GQ214562, O. Acestridium sp. 2 MCP 37785 EU359401*, GQ225433, GQ214563, O. Ancistomus sabaji AUM 35537 (tag T2031) O, GQ225434, GQ214564, O. Ancistrus brevispinnis MCP 21246

EU359402*, GQ225435, GQ214565, O. Apistoloricaria ommation ANSP 182331 (tag P6265)

EU359403*, GQ225436, GQ214566, O. Chaetostoma sp. 1 ANSP 180446 (tag P4772)

EU359409*, GQ225444, GQ214573, O. Chaetostoma sp. 2 ANSP 180448 (tag P4814)

EU359410*, GQ225445, GQ214574, O. Corumbataia cuestae LBP 876 EU371019*, GQ225446,

O, EU817521*. Corymbophanes kaiei AUM 28163 (tag MH217) O, GQ225448, GQ214576, O.

Delturus carinotus MCP no number (P 22) GQ225388, GQ225449, GQ214577, O. Epactionotus bilineatus MCP 26964 EU371008*, GQ225450, O, EU817553*. Epactionotus gracilis MCP 23606

EU371005*, GQ225451, GQ214578, O. Epactionotus itaimbezinho MCP 23683 EU371004*,

GQ225452, O, O. Eurycheilichthys limulus MCP 21270 EU370989*, GQ225453, GQ214579, O.

Eurycheilichthys limulus MCP 21270 EU370990*, O, O, O. Eurycheilichthys pantherinus MCP

22373 EU371000*, GQ225454, GQ214580, O. Eurycheilichthys sp. 1 MCP 21207 EU370995*, O,

O, EU817529*. Gymnotocinclus anosteos UFRGS no number O, GQ225456, O, O. Harttia kronei

MCP 31596 GQ225390, O, GQ214582, O. Hemiancistrus fuliginosus MCP 37566 O, GQ225457,

GQ214583, O. Hemiancistrus punctulatus MCP 21248 O, GQ225458, GQ214584, O. 39 Hemiancistrus subviridis AUM 42930 (tag P4648) EU359411*, GQ225459, GQ214585, O.

Hemipsilichthys gobio MCP 42452 GQ225391, GQ225460, GQ214586, EU817547*.

Hemipsilichthys nimius MCP 30671 EU359412*, GQ225461, GQ214587, O. Hemipsilichtys papillatus MCP 43954 GQ225392, GQ225462, GQ214588, O. Hisonotus armatus MCP 25159

EU371012*, O, O, O. Hisonotus armatus MCP 37682 EU359414*, GQ225463, GQ214589, O.

Hisonotus charrua a MCP 21644 EU371013*, O, O, EU817523*. Hisonotus charrua b UFRGS

7185 GQ225393, GQ225465, GQ214590, O. Hisonotus francirochai DZJRP 7727 EU359415*,

GQ225466, GQ214591, EU817518*. Hisonotus insperatus O, GQ225467, O, EU817524*.

Hisonotus iota MCP 40029 EU359413*, GQ225464, O, O. Hisonotus laevior MCP 23005

EU371015*, GQ225469, GQ214593, O. Hisonotus laevior MCP 37684 O, GQ225468, GQ214592,

O. Hisonotus leucofrenatus MCP 31819 EU371001*, GQ225470, GQ214594, EU817530*.

Hisonotus leucofrenatus LBP 873 EU371002*, GQ225471, GQ214595, O. Hisonotus luteofrenatus

MCP 32668 O, GQ225483, O, O. Hisonotus nigricauda UFRGS 9120 GQ225394, GQ225472,

GQ214596, EU817522*. Hisonotus ringueleti UFRGS 9119 GQ225395, GQ225473, GQ214597,

EU817531*. Hisonotus sp. 1 MCP 23744 EU371010*, O, O, O. Hisonotus sp. 2 MCP 25139

EU371014*, O, O, O. Hisonotus sp. 4 LBP 810 EU371018*, O, O, O. Hisonotus taimensis MCP

21375 EU371011*, GQ225474, GQ214598, O. Hypancistrus contradens AUM 39241 O,

GQ225476, GQ214600, O. Hypoptoma bilobatum MHNG 2588.092 EU370986*, GQ225477, O,

O. Hypoptopoma cf. inexspectatum ANSP uncat. (tag 5089) EU359417*, GQ225478, GQ214601,

O. Hypoptopoma guianensis ANSP 180669 (tag T2215) EU359418*, GQ225479, GQ214602, O.

Hypoptopoma gulare ANSP 178340 (tag 1551) EU359419*, GQ225480, GQ214603, EU817541*.

Hypoptopoma inexspectatum ANSP uncat. (tag 5047) EU359420*, GQ225481, GQ214604,

EU817555*. Hypoptopoma steindachneri ANSP 182723 (tag P6233) EU359421*, GQ225482,

GQ214605, O. Hypoptopomatinae sp. Surinam MCP 43953 GQ225396, GQ225484, O, O.

Hypostomus albopunctatus MCP 37990 O, GQ225485, GQ214606, O. Hypostomus boulengeri

NUP uncat. (Z49935) EU359422*, GQ225486, GQ214607, EU817560*. Hypostomus cochliodon

NUP uncat. Z64017 GQ225397, GQ225487, GQ214608, O. Hypostomus commersonii MCP 22767

EU359423*, GQ225488, GQ214609, O. Isbrueckerichthys alipionis MCP no number GQ225398,

40 GQ225489, GQ214610, EU817566*. Isbrueckerichthys calvus MZUEL 4949 GQ225399,

GQ225490, GQ214611, O. Isbrueckerichthys duseni MCP 42421 GQ225400, GQ225491,

GQ214612, EU817548*. Isbrueckerichthys epakmos MCP 42436 GQ225401, GQ225492,

GQ214613, O. Isbrueckerichthys saxicola MCP 43154 O, GQ225493, GQ214614, O. Ixinandria steinbachi EU359426*, GQ225494, GQ214615, O. Kronichthys heylandi MCP 31574 GQ225402,

GQ225495, GQ214616, O. Kronichthys lacerta MCP no number GQ225403, GQ225496,

GQ214617, O. Kronichthys subteres MCP 42443 GQ225404, GQ225497, GQ214618, O.

Kronichthys subteres MCP 31600 EU371021*, GQ225498, GQ214619, O. Lampiella gibbosa

MCP 43883 GQ225405, GQ225499, GQ214620, EU817545*. Liposarcus anisitsi MCP 37992 O,

GQ225500, O, O. Liposarcus pardalis ANSP 178396 tag: 1559 O, GQ225501, O, O.

Megalancistrus parananus MCP 37991 O, GQ225503, GQ214621, O. Microlepidogaster perforatus MNRJ 31886 O, GQ225505, GQ214623, O. Microlepidogaster sp. 1 MCP 41913

GQ225407, GQ225506, GQ214624, O. Microlepidogaster sp. 2 MCP 41912 EU359427*,

GQ225504, GQ214622, O. Nannoptopoma spectabile MCP 43952 GQ225408, GQ225507,

GQ214625, O. Neoplecostominae nov. gen. 1 MCP 42693 GQ225389, GQ225455, GQ214581, O.

Neoplecostominae nov. gen. 2 MCP 42460 GQ225386, GQ225441, GQ214570, O.

Neoplecostominae nov. gen. 3 MCP no number O, GQ225442, GQ214571, O. Neoplecostomus espiritosantensis MNRJ 22457 O, GQ225509, GQ214627, O. Neoplecostomus franciscoensis

MCP 42428 GQ225409, GQ225510, GQ214628, O. Neoplecostomus microps MNRJ 24005 (#170)

EU359429*, GQ225511, GQ214629, EU817568*. Neoplecostomus paranensis MNRJ 23974 O,

GQ225512, O, O. Neoplecostomus ribeirensis MCP 42480 GQ225410, GQ225513, GQ214630, O.

Neoplecostomus sp. MCP 30672 EU359430*, GQ225514, GQ214631, O. Otocinclus affinis

DZJRP 7610 EU359431*, GQ225515, GQ214632, O. Otocinclus cf. hoppei MHNG 2613.057

EU370985*, O, O, O. Otocinclus cf. mariae MHNG no number (SU07-350) GQ225411,

GQ225516, GQ214633, EU817558*. Otocinclus cocama MCP 34842 EU359432*, GQ225517,

GQ214634, O. Otocinclus flexilis MCP 25234 EU370983*, GQ225518, GQ214635, EU817546*.

Otocinclus flexilis MCP 41907 EU370984*, O, GQ214636, O. Otocinclus vestitus INHS 54583

GQ225412, GQ225519, GQ214637, O. Otocinclus vittatus MCP 35848 EU359433*, GQ225520,

41 GQ214638, EU817544*. Otocinclus xakriaba ANSP 180689 (tag: SAS93-13B no.4) GQ225413,

GQ225521, GQ214639, O. Otothyris juquiae MCP 43983 EU359434*, GQ225522, GQ214640, O.

Otothyris travassosi MNRJ 22947 (#645) O, GQ225523, O, EU817526*. Otothyropsis sp. MHNG

2587.011 EU371003*, GQ225524, GQ214641, O. Oxyropsis acutirostra ANSP 180816 (tag: 4015)

GQ225414, GQ225525, GQ214642, EU817542*. Oxyropsis carinata INHS 52488 GQ225415,

GQ225526, O, O. Oxyropsis wrightiana UF 126342 GQ225416, GQ225527, O, O. Panaqolus changae ANSP 181097 (tag P6218) EU359435*, GQ225528, GQ214643, O. Panaqolus sp. ZSM

32728 EU359436*, GQ225529, AY552031, O. Panaque cf. cochliodon O, GQ225530, AY552039,

O. Panaque cf. nigrolineatus GQ225417, GQ225531, AY896736, O. Pareiorhaphis azygolechis

MCP 41909 EU359437*, GQ225532, GQ214644, EU817564*. Pareiorhaphis bahiana MCP no number GQ225418, GQ225533, GQ214645, O. Pareiorhaphis calmoni MCP 41275 EU359438*,

GQ225534, GQ214646, O. Pareiorhaphis eurycephalus MCP 41458 EU359439*, GQ225536,

GQ214648, O. Pareiorhaphis garbei MCP 43598 O, GQ225537, GQ214649, O. Pareiorhaphis garbei MCP 43597 O, GQ225538, GQ214650, O. Pareiorhaphis hypselurus MCP 21695

EU359440*, GQ225539, GQ214651, O. Pareiorhaphis hystrix MCP 22787 EU359441*,

GQ225540, GQ214652, O. Pareiorhaphis nasuta MCP 37176 GQ225420, GQ225541, GQ214653,

O. Pareiorhaphis nudula MCP 41906 EU359442*, GQ225542, GQ214654, O. Pareiorhaphis parmula MCP 41747 EU359443*, GQ225543, GQ214655, O. Pareiorhaphis sp. 1 MCP 22339

EU359444*, GQ225546, GQ214658, O. Pareiorhaphis sp. 2 MCP 28683 EU359445*, GQ225547,

GQ214659, O. Pareiorhaphis sp. 3 MCP 40111 EU359446*, GQ225548, GQ214660, O.

Pareiorhaphis sp. 4 MCP 41296 EU359447*, GQ225544, GQ214656, O. Pareiorhaphis sp. 5

MCP 41457 EU359448*, GQ225545, GQ214657, O. Pareiorhaphis sp. 6 MCP 41460 GQ225419,

GQ225535, GQ214647, O. Pareiorhaphis splendens MCP 22330 EU359449*, GQ225549,

GQ214661, EU817565*. Pareiorhaphis splendens MCP 41263 EU359450*, GQ225550,

GQ214662, O. Pareiorhaphis steindachneri MCP 41289 EU359451*, GQ225551, GQ214663, O.

Pareiorhaphis stomias MCP 41910 EU359452*, GQ225552, GQ214664, O. Pareiorhaphis vestigipinnis MCP 43034 GQ225421, GQ225553, GQ214665, O. Pareiorhina brachyrhyncha

MCP 42434 GQ225422, GQ225554, GQ214666, O. Pareiorhina carrancas MCP 36915

42 GQ225423, GQ225555, GQ214667, O. Pareiorhina sp. MNRJ 26518 (#281) EU359453*,

GQ225556, GQ214668, O. Pareiorhina sp. MNRJ 26529 (#282) GQ225424, GQ225557,

GQ214669, O. Parotocinclus aripuanensis MCP 35884 O, GQ225558, GQ214670, O.

Parotocinclus bidentatus MCP 42430 GQ225425, GQ225559, GQ214671, O. Parotocinclus britskii ANSP 179131 (tag: 2112) GQ225426, O, O, O. Parotocinclus cf. aripuanensis MCP 43950

EU359454*, GQ225560, O, O. Parotocinclus collinsae ANSP 179140 (tag: 2268) O, GQ225561,

GQ214672, O. Parotocinclus eppleyi AUM 43947 (tag V5576) EU359455*, O, GQ214673,

EU817528*. Parotocinclus jumbo ZSM 32727 EU359456*, GQ225562, AY552048, O.

Parotocinclus maculicauda MCP 41911 EU359457*, GQ225563, GQ214674, EU817527*.

Parotocinclus sp. MCP 35875 O, GQ225564, GQ214675, O. Parotocinclus spilosoma MCP 43951

GQ225427, O, O, O. Peckoltia vermiculata AUM 39245 (tag V060) EU359458*, GQ225565,

GQ214676, O. Peckoltia vittata AUM 39248 (tag V114) EU359459*, GQ225566, GQ214677, O.

Pseudacanthicus leopardus ANSP 179613 (tag 2450) EU359460*, GQ225567, GQ214678, O.

Pseudotocinclus juquiae LBP 616 EU370988*, GQ225568, O, O. Pseudotocinclus tietensis LBP

696 EU370987*, GQ225569, GQ214679, EU817519*. Pseudotothyris obtusa MCP 33330

EU371016*, GQ225570, GQ214680, EU817525*. Pseudotothyris obtusa MCP 33330 EU371017*,

GQ225571, GQ214681, O. Rhinelepis aspera NUP uncat. (PR108) GQ225428, GQ225572,

GQ214682, O. Rineloriciaria sp. MCNI 1224 EU359461*, GQ225573, GQ214683, O. Schizolecis guentheri MCP 31722 EU359462*, GQ225574, O, EU817536*. Schizolecis guentheri MCP 31724

EU371020*, GQ225575, GQ214684, EU817539*. Scobinancistrus pariolispos ANSP 177883

GQ225429, GQ225576, GQ214685, O. Nematogenyidae: Nematogenys inermis ANSP 180477

(tag 1) EU359428*, GQ225508, GQ214626, O. Scoloplacidae: Scoloplax distolothrix MCP 40282

EU359463*, DQ492323*, GQ214686, O. Trichomycteridae: Trichomycterus sp. MCP 41292 O,

GQ225577, DQ492431*, O.

43 Appendix B: Majority rule consensus of the Bayesian analyses

1 Pareiorhaphis sp. 4 - Pareiorhaphis sp. 1 0,71 Pareiorhaphis eurycephal us 1 Pareiorhaphis parm ula 1 Pareiorhaphis sp. 5 1 Pareiorhaphis vestigipinnis 1 Pareiorhaphis hystrix - 1 Pareiorhaphis sp. 3 Pareiorhaphis sp. 6 1 1 Pareiorhaphis calmoni 0,74 Pareiorhaphis steindachneri Pareiorhaphis azygolechis 1 Pareiorhaphis splende ns 0,56 Pareiorhaphis splende ns 1 Kronichthys subteres 1 Kronichthys subteres 0,64 1 Kronichthys heylandi Kronichthys lacerta 1 Pareiorhaphis hypselurus 1 Pareiorhaphis stomias 1 Pareiorhaphis nudula 0,6 Isbrueckerichthys calvus 0,89 Isbrueckerichthys saxicola 0,99 Isbrueckerichthys epakmos 0,96 Isbrueckerichthys duseni 1 Neoplecostomu s ribeirensis Isbrueckerichthys alipionis 1 - Neoplecostomus franciscoensis - Neoplecostomus paranensis 1 Neoplecostom us espiritosante nsis 1 Neoplecostomu s microps 0,86 Neoplecostomus sp. 1 Pareiorhina brachyrhyncha 0,98 Pareiorhina carrancas 1 1 Neoplecostomina e nov. gen. 2 0,79 1 Neoplecostomin a e nov. gen. 3 Pareiorhaphis bahiana 0,99 Pareiorhaphis nasuta 1 Pareiorhaphis sp. 2 Neoplecostomina e nov. gen. 1 0,99 1 Pareiorhina sp. 1 Pareiorhina sp. 1 Pseudotocinclus juquiae Pseudotocin clus tietensis 1 Schizolecis guentheri 0,99 Schizolecis guentheri 1 Otothyris juquiae 1 Pareiorhaphis garbei Pareiorhaphis garbei - Hisonotus laevior 1 Hisonotus sp 2 - Hisonotus laevior - Hisonotus armatus 0,97 Hisonotus armatus 0,76 Hisonotus taimensis 1 0,83Hisonotus charrua a Hisonotus insperatus 0,96 Hisonotus charrua b 1 1 Hisonotus leucofrenatu s Hisonotus leucofrenatus Lampiella gibbosa 1 1 Eurycheilichthys limulus 1 Eurycheilichthys limulus 1 Eurycheilichthys sp. 1 1 1 Eurycheilichthys pantherinus 1 1 Epactionotus gracilis 1 Epactionotus itaim bezinho Epactionotus bilineatus Oxyropsis carinata 0,93Hisonotus sp. 1 1 1 Hisonotus iota 1 1 Hisonotus nigricauda Hisonotus ringueleti 1 0,67 Hisonotus francirochai Oxyropsis wrightiana 1 Hisonotus sp. 4 - 1 Pseudotothyris obtusa 1 Pseudotothyris obtusa 0,58 Microlepidogast er sp. 2 1 Oxyropsis acutirostra - Otothyropsis sp. 1 - M icrolepidogast er perforatus 1 Microlepidogast er sp. 1 1 Parotocinclus bidentatus Parotocinclus maculicauda - Hypoptopoma bilobatum Parotocinclus spilosoma 1 0,86 Parotocinclus eppleyi 1 1 0,96 Parotocinclus sp. 0,99 Parotocinclus aripuanensi s 0,84 Parotocinclus britskii Hypoptopoma guianense 1 Parotocinclus cf. aripuane nsis 0,89 Parotocinclus collinsae Otothyris travassosi 1 Parotocinclus jumbo 1 Corumbataia cuestae Hypoptopoma cf. inexpectatum 1 Gym notocinclus anosteos 1 Hisonotus luteofrenatus 1 Hypoptopomati nae sp. Surinam 1 Oxyropsis carinata Hypoptopoma inexpectatum 1 Oxyropsis wrightiana 1 1 Oxyropsis acutirostra - 1 Hypoptopoma bilobatum Hypoptopoma guianense Hypoptopoma gulare 1 Hypoptopoma cf. inexpectatum 1 0.96 1 Hypoptopoma inexpectat um 1 1 0,96 Hypoptopoma gulare Hypoptopoma steindachneri 1 Hypoptopoma steindachneri Nannoptop oma spectabile 0,96 - Otocinclus cf. hoppei 0,6 1 Otocinclus vestitus 1 Otocinclus vittatus 1 Nannoptopoma spectabile Otocinclus cocama Otocinclus cf. mariae 1 0,93 Otocinclus flexilis 0.96 1 Otocinclus flexilis 0,69 Otocinclus affinis - Otocinclus cf. hoppei Otocinclus xakriaba 1 Acestridium martini 0,99 Acestridium martini 0.6 1 Acestridium scutatum Otocinclus vestitus Acestridium gymnogaster 0,7 Hypancistrus contradens 1 - Scobinancistrus pariolispos Otocinclus vittatus 1 Panaqolus changae 1 1 Panaqolus sp. 0,95Ancistomus sabaji 1 - 1 Peckoltia verm iculata Peckoltia vittata Otocinclus cocama 1 Liposarcus anisitsi Liposarcus pardalis 1 1 Hypostomus boulengeri - Hypostomus com mersonii Otocinclus cf. mariae 1 Hypostomus cochliodo n 0,73 Hypostomus albopu nct atu s 1 0,98 1 Hemiancistrus fuliginosus 0,92 1 Hemiancistrus punctulat us 0.93 Otocinclus flexilis Hemiancistrus subviridis 1 1 Panaque cf. cochliodon Panaque cf. nigrolineat us 1 0,86 M egalancistrus paranan us 1 Otocinclus flexilis Pseudacant hic us leopardus 1 - Ancistrus brevipinnis 1 Corymbophanes kaiei 0.69 1 Chaetostoma sp. 1 Otocinclus affinis Chaetostoma sp. 2 1 Ixinandria steinbachi 1 Rineloricaria sp. 1 1 Apistoloricaria ommation Otocinclus xakriaba Harttia kronei Rhinelepis aspera 0,68 Delturus carinotus 1 Hemipsilichthys nim ius 1 1 Acestridium martini 1 Hem ipsilichthys gobio Hemipsilichtys papillatus 0,72 Astroblepus sp. 1 0,87 Astroblepus sp. 3 0.99 Acestridium martini 1 Astroblepus sp. 2 0,92 Astroblepus sp. 4 Scoloplax distolothrix 1 1 Callichtys callichthys Acestridium scutatum 1 Hoplosternum littorale 0,98 Corydoras ourastigma Trichom ycterus sp. Nem atogenys inermis Acestridium gymnogaster 0.7 Hypancistrus contradens - Scobinancistrus pariolispos 1 Panaqolus changae 1 1 Panaqolus sp. 0,95 Ancistomus sabaji - 1 Peckoltia vermiculata Peckoltia vittata 1 Liposarcus anisitsi Liposarcus pardalis 1 1 Hypostomus boulengeri - Hypostomus commersonii 1 Hypostomus cochliodon 0.73 Hypostomus albopunctatus 0.98 1 Hemiancistrus fuliginosus 0.92 1 Hemiancistrus punctulatus Hemiancistrus subviridis 1 1 Panaque cf. cochliodon Panaque cf. nigrolineatus 1 0.86 Megalancistrus parananus Pseudacanthicus leopardus 1 - Ancistrus brevipinnis 1 Corymbophanes kaiei 1 Chaetostoma sp. 1 Chaetostoma sp. 2 1 Ixinandria steinbachi 1 Rineloricaria sp. 1 1 Apistoloricaria ommation Harttia kronei Rhinelepis aspera 0.68 Delturus carinotus 1 Hemipsilichthys nimius 1 1 Hemipsilichthys gobio Hemipsilichtys papillatus 0.72 Astroblepus sp. 1 0.87 Astroblepus sp. 3 1 Astroblepus sp. 2 0.92 Astroblepus sp. 4 Scoloplax distolothrix 1 Callichtys callichthys 1 Hoplosternum littorale 0.98 Corydoras ourastigma Trichomycterus sp. Nematogenys inermis

0.2 substitution / site

Fig. 5a Majority rule consensus of the Bayesian analyses, remainder of the tree is in Fig. 5b. Branch lengths are drawn proportional to the amount of change. Branches marked with S differ in their position from the best ML tree. Bayesian posterior probabilities of clades marked on branches (≥ 0.50).

44 1 Pareiorhaphis sp. 4 - Pareiorhaphis sp. 1 0,71 Pareiorhaphis eurycephal us 1 Pareiorhaphis parmula 1 Pareiorhaphis sp. 5 1 Pareiorhaphis vestigipinni s 1 Pareiorhaphis hystrix - 1 Pareiorhaphis sp. 3 Pareiorhaphis sp. 6 1 1 Pareiorhaphis calmoni 0,74 Pareiorhaphis steindachneri Pareiorhaphis azygolechis 1 Pareiorhaphis splende ns 0,56 Pareiorhaphis splendens 1 Pareiorhaphis sp. 4 1 Kronichthys subteres 1 Kronichthys subteres 0,64 1 Kronichthys heylandi - Kronichthys lacerta Pareiorhaphis sp. 1 1 Pareiorhaphis hypselurus 1 Pareiorhaphis stomias 0.71 1 Pareiorhaphis nudula 0,6 Isbrueckerichthys calvus Pareiorhaphis eurycephalus 0,89 Isbrueckerichthys saxicola 0,99 Isbrueckerichthys epakmos 1 0,96 Isbrueckerichthys duseni 1 Neoplecostomu s ribeirensis Pareiorhaphis parmula Isbrueckerichthys alipionis 1 - Neoplecostomus franciscoensis 1 - Neoplecostomu s paranensis 1 Neoplecostomus espiritosantensis Pareiorhaphis sp. 5 1 Neoplecostomus microps 0,86 Neoplecostomus sp. 1 Pareiorhina brachyrhynch a 0,98 Pareiorhina carrancas Pareiorhaphis vestigipinnis 1 1 Neoplecostomin a e nov. gen. 2 1 0,79 1 Neoplecostomin ae nov. gen. 3 Pareiorhaphis bahiana 0,99 Pareiorhaphis nasuta 1 Pareiorhaphis hystrix 1 Pareiorhaphis sp. 2 Neoplecostomina e nov. gen. 1 0,99 1 Pareiorhina sp. 1 1 Pareiorhina sp. Pareiorhaphis sp. 3 1 Pseudotocin clus juquiae - Pseudotocinclus tietensis 1 Schizolecis guentheri 0,99 Schizolecis guentheri Pareiorhaphis sp. 6 1 Otothyris juquiae 1 Pareiorhaphis garbei Pareiorhaphis garbei 1 - Hisonotus laevior 1 Pareiorhaphis calmoni 1 Hisonotus sp 2 - Hisonotus laevior - Hisonotus armatus 0,97 Hisonotus armatus Pareiorhaphis steindachneri 0,76 Hisonotus taimensis 1 0,83Hisonotus charrua a 0.74 Hisonotus insperatus 0,96 Hisonotus charrua b Pareiorhaphis azygolechis 1 1 Hisonotus leucofrenatus Hisonotus leucofrenatu s Lampiella gibbosa 1 1 Eurycheilichthys limulus 1 1 Pareiorhaphis splendens Eurycheilichthys limulus 1 Eurycheilichthys sp. 1 1 1 Eurycheilichthys pantherinus 1 1 Epactionotus gracilis Pareiorhaphis splendens 1 Epactionotus itaimbezinho 0.56 Epactionotus bilineatus 1 0,93Hisonotus sp. 1 Hisonotus iota Kronichthys subteres 1 1 Hisonotus nigricauda 1 Hisonotus ringueleti 0,67 Hisonotus francirochai 1 Hisonotus sp. 4 1 Kronichthys subteres - 1 Pseudotothyris obtusa 1 Pseudotothyris obtusa 0,58 Microlepidogast er sp. 2 1 - Otothyropsis sp. - 0.64 Kronichthys heylandi 1 Microlepidogast er perforatus 1 Microlepidogast er sp. 1 1 Parotocinclus bidentatus Parotocinclus maculicauda Kronichthys lacerta 0,86 Parotocinclus spilosoma 1 Parotocinclus eppleyi 1 0,96 Parotocinclus sp. 0,99 Parotocinclus aripuanensi s 0,84 Parotocinclus britskii 1 Pareiorhaphis hypselurus Parotocinclus cf. aripuane nsis 1 0,89 Parotocinclus collinsae Otothyris travassosi 1 Parotocinclus jumbo Pareiorhaphis stomias 1 Corumbataia cuestae 1 Gymnotocinclus anosteos 1 Hisonotus luteofrenatus Hypoptopomati nae sp. Surinam Pareiorhaphis nudula 1 Oxyropsis carinata 1 1 Oxyropsis wrightiana 1 Oxyropsis acutirostra 0.6 Isbrueckerichthys calvus - 1 Hypoptopoma bilobatum Hypoptopoma guianen se 1 Hypoptopoma cf. inexpectatum 1 0.89 1 Hypoptopoma inexpectat um Isbrueckerichthys saxicola 1 0,96 Hypoptopoma gulare Hypoptopoma steindachneri 1 0.99 Nannoptop oma spectabile 0,96 Isbrueckerichthys epakmos - Otocinclus cf. hoppei 0,6 Otocinclus vestitus 1 Otocinclus vittatus 0.96 1 Otocinclus cocama Isbrueckerichthys duseni Otocinclus cf. mariae 1 0,93 Otocinclus flexilis 1 1 Otocinclus flexilis 0,69 Otocinclus affinis Neoplecostomus ribeirensis Otocinclus xakriaba 1 Acestridium martini 0,99 Acestridium martini Isbrueckerichthys alipionis 1 Acestridium scutatum Acestridium gymnogaster 0,7 Hypancistrus contradens 1 Neoplecostomus franciscoensis - Scobinancistrus pariolispos - 1 Panaqolus changae 1 1 Panaqolus sp. 0,95 Ancistomus sabaji - Neoplecostomus paranensis - 1 Peckoltia vermiculata Peckoltia vittata 1 Liposarcus anisitsi 1 Neoplecostomus espiritosantensis Liposarcus pardalis 1 1 Hypostomus boulen geri - Hypostomus commersonii 1 Hypostomus cochliodon Neoplecostomus microps 0,73 1 Hypostomus albopunct atu s Hemiancistrus fuliginosus 0.86 0,98 1 0,92 1 Hemiancistrus punctulat us Neoplecostomus sp. Hemiancistrus subviridis 1 1 Panaque cf. cochliodon Panaque cf. nigrolineat us 1 0,86 Megalancistrus paranan us 1 Pareiorhina brachyrhyncha Pseudacant hic us leopardus 1 - Ancistrus brevipinnis 1 Corymbophanes kaiei 0.98 Pareiorhina carrancas 1 Chaetostoma sp. 1 Chaetostoma sp. 2 1 Ixinandria steinbachi 1 Rineloricaria sp. 1 1 1 Neoplecostominae nov. gen. 2 1 Apistoloricaria ommation Harttia kronei 0.79 Rhinelepis aspera 1 Neoplecostominae nov. gen. 3 0,68 Delturus carinotus 1 Hemipsilichthys nimius 1 1 Hemipsilichthys gobio Hemipsilichtys papillatus Pareiorhaphis bahiana 0,72 Astroblepus sp. 1 0,87 Astroblepus sp. 3 0.99 1 Astroblepus sp. 2 0,92 Astroblepus sp. 4 Pareiorhaphis nasuta Scoloplax distolothrix 1 Callichtys callichthys 1 1 Hoplosternum littorale Pareiorhaphis sp. 2 0,98 Corydoras ourastigma Trichomycterus sp. Nematogenys inermis Neoplecostominae nov. gen. 1 0.99 1 Pareiorhina sp. 1 Pareiorhina sp. 1 Pseudotocinclus juquiae Pseudotocinclus tietensis 1 Schizolecis guentheri 0.99 Schizolecis guentheri 1 Otothyris juquiae 1 Pareiorhaphis garbei Pareiorhaphis garbei - Hisonotus laevior 1 Hisonotus sp 2 - Hisonotus laevior - Hisonotus armatus 0.97 Hisonotus armatus 0.76 Hisonotus taimensis 1 0.83Hisonotus charrua a Hisonotus insperatus 0.96 Hisonotus charrua b 1 1 Hisonotus leucofrenatus Hisonotus leucofrenatus Lampiella gibbosa 1 1 Eurycheilichthys limulus 1 Eurycheilichthys limulus 1 Eurycheilichthys sp. 1 1 1 Eurycheilichthys pantherinus 1 1 Epactionotus gracilis 1 Epactionotus itaimbezinho Epactionotus bilineatus 0.93 1 Hisonotus sp. 1 Hisonotus iota 1 1 Hisonotus nigricauda Hisonotus ringueleti 0.67 Hisonotus francirochai 1 Hisonotus sp. 4 - 1 Pseudotothyris obtusa 1 Pseudotothyris obtusa 0.58 Microlepidogaster sp. 2 - Otothyropsis sp. 1 - Microlepidogaster perforatus 1 Microlepidogaster sp. 1 1 Parotocinclus bidentatus Parotocinclus maculicauda 0.86 Parotocinclus spilosoma 1 Parotocinclus eppleyi 1 0.96 Parotocinclus sp. 0.99 Parotocinclus aripuanensis 0.84 Parotocinclus britskii Parotocinclus cf. aripuanensis 1 0.89 Parotocinclus collinsae Otothyris travassosi Parotocinclus jumbo 1 Corumbataia cuestae 1 Gymnotocinclus anosteos 0.2 substitution / site 1 Hisonotus luteofrenatus Hypoptopomatinae sp. Surinam

Fig. 5b Majority rule consensus of the Bayesian analyses, remainder of the tree is in Fig. 5a. Branch lengths are drawn proportional to the amount of change. Branches marked with S differ in their position from the best ML tree. Bayesian posterior probabilities of clades marked on branches (≥ 0.50). 45 Appendix C: Results of the MP analyses

Pareiorhaphis sp. 4 Pareiorhaphis sp. 1 Pareiorhaphis parmula Pareiorhaphis eurycephal us Pareiorhaphis sp. 5 Pareiorhaphis vestigipinni s Pareiorhaphis sp. 3 Pareiorhaphis hystrix Pareiorhaphis sp. 6 Pareiorhaphis azygolechi s Pareiorhaphis calmoni Pareiorhaphis steindach n eri Pareiorhaphis splende ns Pareiorhaphis splende ns Pareiorhaphis hypselurus Pareiorhaphis stomias Pareiorhaphis nudula Isbrueckerichthys alipionis Neoplecostomu s ribeirensis Isbrueckerichthys duseni Isbrueckerichthys epakmos Isbrueckerichthys calvus Isbrueckerichthys saxicola Kronichthys subteres Kronichthys subteres Kronichthys heylandi Kronichthys lacerta Pareiorhaphis nasuta Pareiorhaphis sp. 2 Neoplecostomi na e nov. gen. 1 Pareiorhina brachyrhyncha Pareiorhina carrancas Neoplecostomu s sp. Neoplecostomu s microps Neoplecostomu s franciscoensis Neoplecostomu s paranensis Neoplecostomu s espiritosantensis 100 Corumbataia cuestae Neoplecostomi na e nov. gen. 3 Neoplecostomi na e nov. gen. 2 Pareiorhaphis bahiana 80 10 Gymnotocinclus anosteos Pareiorhina sp. Pareiorhina sp. Pseudotocin clu s juquiae 5 100 Hisonotus luteofrenatus Pseudotocin clu s tietensis Hisonotus laevior Hisonotus sp. 2 8 Hypoptopomatinae sp. Surinam Hisonotus laevior Hisonotus armatus Hisonotus armatus Otocinclus cf. hoppei Hisonotus taimensis 95 Hisonotus insperatus Hisonotus charrua a 92 Otocinclus vestitus Hisonotus charrua b 19 Hisonotus leucofrenat us Hisonotus leucofrenat us 5 Epactionotu s itaimbezinho 96 Otocinclus vittatus Epactionotus gracilis Epactionotu s bilineatus Eurycheilichthys pantherin us 9 Otocinclus cocama Eurycheilichthys sp. 1 Eurycheilichthys limulus 100 Otocinclus cf. mariae Eurycheilichthys limulus Hisonotus ringueleti' 19 Hisonotus nigricauda Otocinclus xakriaba Hisonotus iota 77 Hisonotus sp. 1 Pseudotothyris obtusa Otocinclus affinis Pseudotothyris obtusa -- 4 100 Hisonotus francirochai Hisonotus sp. 4 -- Otocinclus flexilis Microlepidogast er sp. 2 4 21 Otothyropsis sp. Microlepidogast er perforatus 1 Parotocinclus maculicauda Otocinclus flexilis Parotocinclus bidentatus Microlepidogast er sp. 1 -- Parotocinclus spilosoma Lampiella gibbosa Otothyris travassosi Parotocinclus cf. aripuane nsis 4 Parotocinclus jumbo Parotocinclus britskii * Parotocinclus aripuanensi s Parotocinclus sp. 100 Parotocinclus eppleyi Acestridium martini Parotocinclus collinsae 80 Pareiorhaphis garbei 34 Pareiorhaphis garbei Acestridium martini Otothyris juquiae 100 5 Schizolecis guentheri Schizolecis guentheri Acestridium scutatum Corumbataia cuestae 45 Gymnotocinclus anosteos Hisonotus luteofrenatu s Acestridium gymnogaster Hypoptopomati n ae sp. Surinam Otocinclus cf .hoppei 100 Otocinclus vestitus Oxyropsis carinata Otocinclus vittatus 99 Otocinclus cocama 16 Oxyropsis wrightiana Otocinclus cf .mariae Otocinclus xakriaba 9 Otocinclus affinis 92 Oxyropsis acutirostra Otocinclus flexilis Otocinclus flexilis Lampiella gibbosa 5 100 Hypoptopoma bilobatum Parotocinclus jumbo Acestridium martini 100 Acestridium martini 20 Hypoptopoma guianense Acestridium scutatum Acestridium gymnogaster 37 Oxyropsis carinata Nannoptopoma spectabile Oxyropsis wrightiana Oxyropsis acutirostra 80 Hypoptopoma bilobatum 100 Hypoptopoma cf inexpectatum Hypoptopoma guianen se Nannoptopoma spectabile 4 Hypoptopoma cf inexpectatum 100 15 Hypoptopoma inexpectatum Hypoptopoma inexpectat um 73 Hypoptopoma gulare Hypoptopoma steindach n eri 19 64 Ancistomus sabaji 4 Hypoptopoma gulare Peckoltia vermiculata Peckoltia vittata 1 Panaqolus sp. Hypoptopoma steindachneri Panaqolus changae Scobinancistrus pariolispos 74 Hypancistrus contradens Ancistomus sabaji Hypostomus boulengeri 52 Hypostomus commersonii 1 Hypostomus albopunct atu s Peckoltia vermiculata Hypostomus cochliodon 1 Hemiancistrus fuliginosus Hemiancistrus punctulat us Peckoltia vittata Liposarcus pardalis Liposarcus anisitsi 88 Panaque cf. nigrolinea tus 97 Panaqolus sp. Panaque cf. cochliodon Hemiancistrus subviridis 4 Pseudacant hic us leopardus 2 Panaqolus changae Megalancistrus parananus Ancistrus brevipinnis Corymbophane s kaiei Scobinancistrus pariolispos Chaetostoma sp. 2 Chaetostoma sp. 1 Rhinelepis aspera Hypancistrus contradens Rineloricaria sp Ixinandria steinbachi 85 Apistoloricaria ommation Hypostomus boulengeri Harttia kronei Delturus carinotus 98 3 Hemipsilichthys nimius 87 Hypostomus commersonii Hemipsilichthys gobio Hemipsilichtys papillatus 6 Astroblepus sp. 3 3 Hypostomus albopunctatus Astroblepus sp. 1 65 Astroblepus sp. 2 Hypostomus cochliodon Astroblepus sp. 4 Scoloplax distolothrix 51 1 Trichomycterus sp. 100 Hemiancistrus fuliginosus Hoplosternum littorale Callichthys callichthys 3 Corydoras ourastigma 6 Hemiancistrus punctulatus Nematogenys inermis 80 100 Liposarcus pardalis 3 5 Liposarcus anisitsi 100 Panaque cf. nigrolineatus 14 Panaque cf. cochliodon 78 Hemiancistrus subviridis 2 67 98 Pseudacanthicus leopardus 1 20 98 Megalancistrus parananus 13 Ancistrus brevipinnis Corymbophanes kaiei 100 Chaetostoma sp. 2 21 Chaetostoma sp. 1 93 * Rhinelepis aspera 7 100 Rineloricaria sp. 98 30 Ixinandria steinbachi 91 11 Apistoloricaria ommation 11 Harttia kronei -- 51 * Delturus carinotus 0 100 2 Hemipsilichthys nimius 33 100 Hemipsilichthys gobio 22 Hemipsilichtys papillatus 61 Astroblepus sp. 3 100 2 Astroblepus sp. 1 100 100 23 Astroblepus sp. 2 0 10 Astroblepus sp. 4 Scoloplax distolothrix Trichomycterus sp. 97 Hoplosternum littorale 100 10 Callichthys callichthys 18 Corydoras ourastigma Nematogenys inermis

Fig. 6a Result of the MP analysis. Basal part of the strict consensus of 358 most parsimonious trees with 9363 steps, CI: 0.36, remainder of the tree is in Fig. 6b. Branches marked with * differ in their position from the best ML tree. Parsimony bootstrap proportions (≥ 50) are shown above branches, decay (Bremer) indices are shown below. 46 51 Pareiorhaphis sp. 4 -- Pareiorhaphis sp. 4 1 Pareiorhaphis sp. 1 Pareiorhaphis sp. 1 Pareiorhaphis parmula 64 1 Pareiorhaphis eurycephal us Pareiorhaphis parmula Pareiorhaphis sp. 5 Pareiorhaphis vestigipinnis 66 2 Pareiorhaphis sp. 3 Pareiorhaphis eurycephalus Pareiorhaphis hystrix Pareiorhaphis sp. 6 78 1 Pareiorhaphis azygolechi s Pareiorhaphis sp. 5 Pareiorhaphis calmoni Pareiorhaphis steindachneri 3 Pareiorhaphis splendens 75 Pareiorhaphis vestigipinnis Pareiorhaphis splendens Pareiorhaphis hypselurus Pareiorhaphis stomias 1 99 Pareiorhaphis sp. 3 Pareiorhaphis nudula Isbrueckerichthys alipionis 95 Neoplecostomus ribeirensis 5 Pareiorhaphis hystrix Isbrueckerichthys duseni Isbrueckerichthys epakmos 88 10 Isbrueckerichthys calvus Pareiorhaphis sp. 6 Isbrueckerichthys saxicola Kronichthys subteres 5 Kronichthys subteres Pareiorhaphis azygolechis Kronichthys heylandi Kronichthys lacerta Pareiorhaphis nasuta 99 Pareiorhaphis calmoni Pareiorhaphis sp. 2 Neoplecostomi nae nov. gen. 1 Pareiorhina brachyrhyncha 8 Pareiorhaphis steindachneri Pareiorhina carrancas Neoplecostomus sp. Neoplecostomus microps 100 Pareiorhaphis splendens Neoplecostomu s franciscoensis Neoplecostomus paranensis Neoplecostomus espiritosantensis 20 Pareiorhaphis splendens Neoplecostomi nae nov. gen. 3 Neoplecostomi nae nov. gen. 2 Pareiorhaphis bahiana 98 Pareiorhaphis hypselurus Pareiorhina sp. Pareiorhina sp. 100 Pseudotocinclu s juquiae 6 Pareiorhaphis stomias Pseudotocinclu s tietensis Hisonotus laevior * 16 Hisonotus sp. 2 98 Pareiorhaphis nudula Hisonotus laevior 72 Hisonotus armatus Hisonotus armatus 9 Isbrueckerichthys alipionis Hisonotus taimensis 2 Hisonotus insper atus 97 Hisonotus charrua a Neoplecostomus ribeirensis Hisonotus charrua b Hisonotus leucofrenat us 6 -- Hisonotus leucofrenat us Isbrueckerichthys duseni Epactionotus itaimbezinho Epactionotus gracilis 1 Epactionotus bilineatus 69 Isbrueckerichthys epakmos Eurycheilichthys pantherinus Eurycheilichthys sp. 1 Eurycheilichthys limulus 1 Isbrueckerichthys calvus Eurycheilichthys limulus Hisonotus ringueleti' Hisonotus nigricauda Isbrueckerichthys saxicola Hisonotus iota Hisonotus sp. 1 Pseudotothyris obtusa 100 Kronichthys subteres Pseudotothyris obtusa Hisonotus francirochai Hisonotus sp. 4 4 Microlepidogast er sp. 2 67 Kronichthys subteres Otothyropsis sp. Microlepidogast er perforatus Parotocinclus maculicauda 12 Kronichthys heylandi Parotocinclus bidentatus Microlepidogast er sp. 1 Kronichthys lacerta Parotocinclus spilosoma Otothyris travassosi 99 Parotocinclus cf. aripuanensis 87 Pareiorhaphis nasuta Parotocinclus britskii Parotocinclus aripuanensi s 4 93 Parotocinclus sp. 3 Pareiorhaphis sp. 2 Parotocinclus eppleyi Parotocinclus collinsae 6 Pareiorhaphis garbei Neoplecostominae nov. gen. 1 Pareiorhaphis garbei Otothyris juquiae Schizolecis guentheri 92 Pareiorhina brachyrhyncha Schizolecis guentheri Corumbataia cuestae Gymnotocinclus anosteos 6 Pareiorhina carrancas Hisonotus luteofrenatus Hypoptopomati nae sp. Surinam Otocinclus cf .hoppei 98 Neoplecostomus sp. Otocinclus vestitus Otocinclus vittatus Otocinclus cocama 2 Neoplecostomus microps Otocinclus cf .mariae Otocinclus xakriaba 98 Otocinclus affinis 99 Neoplecostomus franciscoensis Otocinclus flexilis Otocinclus flexilis 3 Lampiella gibbosa 12 Neoplecostomus paranensis Parotocinclus jumbo Acestridium martini Acestridium martini Neoplecostomus espiritosantensis Acestr idium scutatum Acestr idium gymnogaster Oxyropsis carinata 88 Neoplecostominae nov. gen. 3 Oxyropsis wrightiana Oxyropsis acutirostra 64 Hypoptopoma bilobatum 3 Neoplecostominae nov. gen. 2 Hypoptopoma guianense Nannoptopoma spectabile 3 Hypoptopoma cf inexpectatum Pareiorhaphis bahiana Hypoptopoma inexpectat um Hypoptopoma gulare Hypoptopoma steindachneri 100 Pareiorhina sp. Ancistomus sabaji Peckoltia vermiculata Peckoltia vittata 100 24 Pareiorhina sp. Panaqolus sp. Panaqolus changa e Scobinancistrus pariolispos 20 100 Pseudotocinclus juquiae Hypancistrus contradens Hypostomus boulengeri Hypostomus commersonii 23 Pseudotocinclus tietensis Hypostomus albopunct atus Hypostomus cochliodon Hemiancistrus fuliginosus Hisonotus laevior Hemiancistrus punctulat us Liposarcus pardalis Liposarcus anisitsi Hisonotus sp. 2 Panaque cf. nigrolineatus Panaque cf. cochliodon Hemiancistrus subviridis Hisonotus laevior Pseudacant hic us leopardus Megalancistrus parananus Ancistrus brevipinnis Hisonotus armatus Corymbophanes kaiei Chaetostoma sp. 2 88 Chaetostoma sp. 1 Hisonotus armatus Rhinelepis aspera 2 Rineloricaria sp Ixinandr ia steinbachi Hisonotus taimensis Apistoloricaria ommation Harttia kronei Delturus carinotus Hisonotus insperatus Hemipsilichthys nimius -- Hemipsilichthys gobio Hemipsilichtys papillatus Hisonotus charrua a Astroblepus sp. 3 3 Astroblepus sp. 1 Astroblepus sp. 2 Hisonotus charrua b Astroblepus sp. 4 Scoloplax distolothrix Trichomycterus sp. 99 Hisonotus leucofrenatus Hoplosternum littorale Callichthys callichthys 68 Corydoras ourastigma 4 Hisonotus leucofrenatus Nematogenys inermis 7 97 Epactionotus itaimbezinho 100 5 Epactionotus gracilis 77 9 Epactionotus bilineatus 4 62 99 Eurycheilichthys pantherinus -- 4 10 97 Eurycheilichthys sp. 1 98 2 4 Eurycheilichthys limulus 3 Eurycheilichthys limulus 99 Hisonotus ringueleti 83 7 Hisonotus nigricauda -- 3 Hisonotus iota 2 Hisonotus sp. 1 100 Pseudotothyris obtusa 94 5 Pseudotothyris obtusa 95 2 Hisonotus francirochai 4 83 Hisonotus sp. 4 -- 3 70 Microlepidogaster sp. 2 2 2 Otothyropsis sp. Microlepidogaster perforatus 96 Parotocinclus maculicauda 5 Parotocinclus bidentatus -- Microlepidogaster sp. 1 8 -- Parotocinclus spilosoma 4 Otothyris travassosi Parotocinclus cf. aripuanensis Parotocinclus britskii Parotocinclus aripuanensis Parotocinclus sp. Parotocinclus eppleyi Parotocinclus collinsae 100 Pareiorhaphis garbei 68 12 Pareiorhaphis garbei 2 74 Otothyris juquiae 3 100 Schizolecis guentheri 12 Schizolecis guentheri

Fig. 6b Result of the MP analysis. Upper part of the strict consensus of 358 most parsimonious trees with 9363 steps, CI: 0.36, remainder of the tree is in Fig. 6a. Branches marked with * differ in their position from the best ML tree. Parsimony bootstrap proportions (≥ 50) are shown above branches, decay (Bremer) indices are shown below.

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53

Capítulo III

A Total Evidence Phylogeny of the Neoplecostominae and

Hypoptopomatinae catfishes (Siluriformes: Loricariidae)

54 A Total Evidence Phylogeny of the Neoplecostominae and Hypoptopomatinae catfishes

(Siluriformes: Loricariidae).

Christian A. Cramera,b,*, Pablo Lehmanna, Edson H. L. Pereiraa, Sandro L. Bonattob &

Roberto E. Reisa

aLaboratório de Sistemática de Vertebrados, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil. bCentro de Biologia Molecular e Genômica, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil.

*Corresponding author. Tel.: +55 51 33534413. Fax: +55 51 33293903. E-mail address:

[email protected] (C. A. Cramer)

55 Abstract

A total evidence phylogeny of all genera of the loricariid subfamilies Neoplecostominae and

Hypoptopomatinae is presented combining 472 morphological characters and 2634 basepairs from fragments of three genes, both nuclear and mitochondrial. We obtained data for 207 species from 53 loricariid genera with representatives of five loricariid subfamilies and ten outgroup species from five loricarioid families, resulting in the largest catfish phylogeny published so far. Maximum Parsimony analyses were conducted in order to seek a solution for the contradictory results of previous solely morphological or molecular analyses. Our results recovered the Hypoptopomatinae and the Neoplecostominae as monophyletic sister groups and revealed a biogeographic distribution pattern for the genus Pareiorhaphis. Concordant with previous publications, the genus Pseudotocinclus was placed into the Neoplecostominae and the Delturinae was confirmed as the most basal loricariid subfamily except of Lithogenes.

Inside the Hypoptopomatinae, the tribe Hypoptopomatini formed a natural group, but the previously hypothesized monophyly of the Otothyrini could not be confirmed. Furthermore, the genera Hisonotus, Pareiorhina and Parotocinclus were recovered as polyphyletic, though three monophyletic groups could be separated for the latter.

Keywords: Loricariidae, Hypoptopomatinae, Neoplecostominae, phylogeny, total evidence,

COI, RAG1, RAG2

56 The order Siluriformes is distributed on all continents but the Antarctic. With around

3100 species in 36 families (Ferraris 2007), catfishes sum about 10% of all fish species. They are mainly freshwater inhabitants, with only two marine families, and are most strongly represented in South America with 14 families and 64% of the species (Moyle and Cech

2000; Rodiles-Hernández et al. 2005). Six of these families form the superfamily

Loricarioidea, a well documented clade with about 1280 species or 41% of all catfishes:

Astroblepidae, Callichthyidae, Loricariidae, Nematogenyidae, Scoloplacidae, and

Trichomycteridae (Schaefer and Lauder 1986; Schaefer 1990). The family Loricariidae is one of the most diverse fish families, currently containing 785 recognized species distributed in around 100 genera (Eschmeyer and Fricke 2009). Unlike most other catfishes, they possess a mouth that is modified into a sucking disk and are armor-plated, giving them the common name “armored catfishes”. This mega diverse group is spread from Uruguay and northern

Argentina to the Costa Rica and its species inhabit every kind of water, from cool fast running hill streams to the lakes in the Amazon floodplain. Ultimately they even have been found introduced in Asia and North America (Chavez et al. 2006; Page and Robins 2006; Nico et al.

2009), sometimes in astonishing quantities with yet unknown consequences for the native fauna. Loricariid size ranges from few centimeters (e.g. Nannoptopoma) up to more than one meter (e.g. Acanthicus) (Evers and Seidel 2005). In spite of much effort and hundreds of publications since Linnaeus, there still are many species to be discovered and described and their phylogeny is only partly resolved. The family Loricariidae was described by Rafinesque in 1815 and subsequently, beginning from 1831, eight subfamilies have been established, of which six are still recognized (Armbruster 2004; Reis et al. 2006): Lithogeninae Eigenmann,

1909 (1 genus, 3 species), Delturinae Reis et al., 2006 (2 genera, 7 species),

Neoplecostominae Regan, 1904 (5 genera, 39 species), Hypoptopomatinae Eigenmann and

Eigenmann, 1890 (18 genera, 103 species), Loricariinae Bonaparte, 1831 (~ 36 genera,

222 species), and Hypostominae, Kner, 1853 (~ 40 genera, 411 species). This division into 57 subfamilies has not been stable, and along the time, several changes have been made. The

Ancistrinae and the Hypostominae were described in 1853 (Kner 1853). When erecting the

Neoplecostominae, Regan (1904) only included the genus Neoplecostomus. Later, Gosline

(1947) added the genera Canthopomus, Corymbophanes, Delturus, Hemipsilichthys,

Isbrueckerichthys (the species were listed as Pareiorhaphis since Isbrueckerichthys was only described in 1996), Kronichthys, Pareiorhaphis, Pareiorhina, Pogonopoma,

Pogonopomoides, and Upsilodus. Isbrücker (1980) recognized six subfamilies and listed only the genus Neoplecostomus for the Neoplecostominae, transferring the other genera to the

Hypostominae.

The first phylogeny for the Loricariidae was published by Howes (1983). Based on his examinations of osteology and muscles he described the subfamily Chaetostomatinae for the genera Chaetostoma, Hemipsilichthys, Lasiancistrus, and Lipopterichthys and put the

Ancistrinae in the synonymy of the Hypostominae (Fig. 1). This division did not last a long time as Schaefer (1986, 1987, 1988) did not recognize the Chaetostomatinae. Though his phylogenies of show the Hypostominae as paraphyletic because of the separation of the

Ancistrinae (Fig. 2), Schaefer decided to maintain the subfamily status of the Ancistrinae, using Isbrücker’s (1980) classification. The first phylogenetic examination with focus on the

Hypoptopomatinae was done by Schaefer (1991), but only 16 species were included. Based on his results, he described the tribes Hypoptopomatini and Otothyrini. Montoya-Burgos et al.

(1997, 1998) made the first molecular analyses of the family, using 12S and 16S rRNA nucleotide sequences. Besides the monotypic Neoplecostominae, the Loricariinae was the only subfamily that was recovered as monophyletic. Two important discoveries were made in that study (Montoya-Burgos et al. 1998): for the first time, Hemipsilichthys gobio was shown as a taxon basal to all other loricariids and Pseudotocinclus (Hypoptopomatinae) was placed as sister of Pareiorhina (Neoplecostominae), making a connection between the two subfamilies (Fig. 3). The first ample loricariid phylogeny was published by Armbruster 58 (2004). It was focused on the Hypostominae, but included more than 120 species from all subfamilies using 215 morphological characters. In order to retain the monophyly of the

Hypostominae, Armbruster synonymized the Ancistrinae under the Hypostominae and split the Hypostominae into the five tribes Ancistrini, Corymbophanini, Hypostomini, Rhinelepini, and Pterygoplichthini. Furthermore, he found the Otothyrini as paraphyletic and, even being a paraphyletic taxon, he returned the genera Isbrueckerichthys, Kronichthys, Pareiorhaphis, and

Pareiorhina to the Neoplecostominae (Fig. 4). Additionally, he confirmed the findings of

Montoya-Burgos et al. (1998) that Hemipsilichthys gobio, together with Delturus angulicauda, is a sister group to all other loricariids except Lithogenes Eigenmann, 1909.

Based on this result, Pereira (2005) resurrected the genus Pareiorhaphis Miranda Ribeiro,

1918 for most of the species of Hemipsilichthys, remaining only H. gobio, H. papillatus, and

H. nimius in the latter. In the same year, a morphologic phylogeny of the Hypoptopomatinae

(Gauger and Buckup 2005), including 31 species from nearly all genera of this subfamily, showed the Otothyrini and the genus Parotocinclus as paraphyletic groups. In the following year, Lehmann (2006) used 169 characters and a total of 114 species, mainly hypoptopomatines, in his doctoral thesis. His phylogenetic analyses recovered the genus

Kronichthys as the most basal of a monophyletic Hypoptopomatinae. According to his results,

Pseudotocinclus did not have any closer relation to the Neoplecostominae, and again, he found the Otothyrini to be paraphyletic (Fig. 5). Within a few months, three studies dedicated to the phylogeny of the Hypoptopomatinae and the Neoplecostominae appeared in 2008. Two of them (Chiachio et al. 2008; Cramer et al. 2008) were based on sequences of a single gene

(F-Reticulon 4 and COI respectively). Focused on the Hypoptopomatinae and including only

44 species, Chiachio et al. (2008) found Pseudotocinclus to be part of the Neoplecostominae and the latter was found inside the Hypoptopomatinae. Their solution was to elevate the

Hypoptopomatini and the Otothyrini to subfamily rank and to redefine the Neoplecostominae, resulting in three monophyletic groups (Fig. 6). This act must be considered somewhat 59 premature because of the very low number of species included, the use of sequences of a single gene, and the contradictions to the most recent studies, both molecular and morphologic. Their results also conflict with the findings of Cramer et al. (2008) based on sequences for 83 loricariid species with focus on the Hypoptopomatinae and the

Neoplecostominae. These also found the genus Pseudotocinclus as a member of the

Neoplecostominae and the latter amidst the Hypoptopomatinae, but neither the

Hypoptopomatini nor the Otothyrini could be resolved as monophyletic (Fig. 7). The third study concluded that year was a doctoral thesis dedicated to the Neoplecostominae (Pereira

2008). Representing the most complete taxon sampling of this group and using 303 morphological characters, it revealed the Neoplecostominae and the Hypoptopomatinae as monophyletic sister groups. Pareiorhina and Kronichthys were resolved as sister taxa on the base of the Hypoptopomatinae and thus were included in the latter (Fig. 8). Unfortunately, no

Pseudotocinclus was included in that thesis. The most recent study treating the phylogeny of loricariid catfishes (Cramer et al. this volume) used sequences from four genes, both nuclear and mitochondrial, for 136 ingroup species, 103 of them representing 30 genera of the

Hypoptopomatinae and Neoplecostominae. Solely the Delturinae and the Loricariinae were found as monophyletic. Pseudotocinclus was again recovered inside the Neoplecostominae, and Pareiorhaphis ended up as polyphyletic, with one species even being placed outside the

Neoplecostominae. Once more, the Neoplecostominae caused a paraphyly of the

Hypoptopomatinae and the Otothyrini, and not even the Hypoptopomatini could be resolved as monophyletic (Fig. 9).

In summary, much effort has been done, but neither morphological nor molecular data alone have been able to resolve the phylogenetic relationships inside the Loricariidae, rather resulting in contradictory topologies. In similar cases, a total evidence analysis has been successful (e.g. Eernisse and Kluge 1993; Gatesy et al. 2003; Mattern and McLennan 2004;

Pretti et al. 2009). Therefore, the aim of the present study was to try this approach to find a 60 well supported solution for the Loricariidae, taking advantage of the large quantity of data already available. That way, we resolved the Neoplecostominae, the Hypoptopomatinae, and the Hypoptopomatini as monophyletic groups, but the Otothyrini and several genera were found to be non-monophyletic.

Panaque Chaetostoma Pseudacanthicus Dolichancistrus Leptoancistrus Acanthicus Lipopterichthys

Pseudancistrus Hypostominae Lasiancistrus Ancistrus

Ancistrus Ancistrinae Lithoxus Exastilithoxus Peckoltia Lithoxus Hemiancistrus Hemiancistrus Cochliodon Pseudacanthicus Hypostomus Pseudancistrus Oligancistrus Pterygoplichthys Chaetostomatinae Panaque Lasiancistrus Peckoltia Chaetostoma Cochliodon Hypostominae Lipopterichthys Hypostomus Hemipsilichthys Hypostomus Hypoptopomatinae Isorineloricaria Loricariinae Pterygoplichthys Pterygoplichthys Neoplecostominae punctatus Kronichthys Schizolecis Fig. 1 Phylogeny from Howes (1983) based on (Hypoptopomatinae) Loricariinae osteology and myology. Hypoptopomatinae Fig. 2 Phylogeny from Schaefer (1986) based on

osteology. Some names have been changed

to update taxonomy and to correct

misidentifications following Armbruster

(2004).

61

Fig. 3 Phylogeny from Montoya-Burgos et al. (1998: modified from Fig. 3) based on sequence data from

mitochondrial 12S and 16S.

62 Epactionotus Hisonotus Eurycheilichthys Hisonotus Hisonotus Parotocinclus Pseudotothyris new new Otothyris Microlepidogaster Otothyrinae Pseudotothyris Schizolecis Hisonotus Corumbataia Pareiorhaphis Isbrueckerichthys Isbueckerichthys

Neoplecostomus new Pareiorhina

Pseudotocinclus Neoplecostominae Otocinclus Lampiella Macrotocinclus Hypoptopoma

Nannoptopoma new Hypoptopoma

Oxyropsis Hypoptopomatinae Acestridium Hypostominae Loricariinae Delturinae

Fig. 6 Phylogeny from Chiachio et al. (2008) based on sequence data from nuclear F-Reticulon 4 (modified from Fig. 2). Fig. 4 Phylogenetic interrelationships of the Loricariidae based on osteology modified from Armbruster (2004) .

Hypoptopoma gen. nov. B Nannoptopoma Oxyropsis Acestridium Otocinclus Niobichthys Corumbataia Schizolecis Pseudotothyris Otothyris Otothyropsis Hypoptopomatinae Microlepidogaster Pseudotocinclus Lampiella Hisonotus notatus New taxon TT Hisonotus insperatus Parotocinclus Parotocinclus Epactionotus Parotocinclus Parotocinclus Parotocinclus Parotocinclus Eurycheilichthys gen. nov. A Kronichthys Neoplecostominae Pareiorhina gen.nov."Taxon12“ Pareiorhaphis Isbrueckerichthys Neoplecostomus Fig. 7 Phylogenetic interrelationships of the Hypostominae Loricariinae Loricariidae based on sequence data from Delturinae Astroblepidae mitochondrial COI, modified from Cramer Fig. 5 Phylogeny from Lehmann (2006) based et al. (2008). on morphology (simplified from Fig. 91).

63 Otocinclus Pareiorhaphis Kronichthys Hisonotus Pareiorhaphis Pareiorhaphis

Isbrueckerichthys Neoplecostominae

Parotocinclus maculicauda Hypoptopomatinae Neoplecostomus Epactionotus Isbrueckerichtys Nov. gen. 1 Eurycheilichthys Pareiorhaphis Pareiorhaphis Gen. nov. P Nov. gen. 2 Nov. gen. 3 Gen. nov. T Neoplecostomus Pareiorhina Parotocinclus jumbo Pareiorhina Pseudotocinclus Pareiorhina Pareiorhaphis garbei Schizolecis

Kronichthys Neoplecostominae Otothyris Hisonotus Gen. nov. B Epactionotus Eurycheilichthys Pareiorhaphis Hisonotus Pseudotothyris Isbrueckerichthys Hisonotus Hisonotus Otothyrini Neoplecostomus Microlepidogaster Otothyropsis Hypostominae Microlepidogaster Parotocinclus Loricariidae Microlepidogaster Delturinae Parotocinclus Guyana Shield Parotocinclus Astroblepidae Otothyris Parotocinclus Gymnotocinclus Fig. 8 Phylogeny from Pereira (2008) based on Corumbataia Hisonotus Hypoptopomatinae sp. morphologic characters (simplified from Oxyropsis Hypoptopomatinae Hypoptopoma Hypoptopoma Fig. 82). Nannoptopoma Otocinclus Lampiella Acestridium Hypostominae Loricariinae Rhinelepis aspera Delturinae Outgroup

Fig. 9 Phylogenetic interrelationships of the

Loricariidae based on sequence data from

mitochondrial COI and nuclear RAG1 and

RAG2. Simplified from Cramer et al. (this

volume: Fig. 4).

64 MATERIAL AND METHODS:

The nucleotide alignment from Cramer et al. (this volume) with all sequences for the genes cytochrome c oxidase I (COI), recombination activating gene 1 (RAG1), and recombination activating gene 2 (RAG2) was taken. Additional sequences for the COI fragment were used from Cramer et al. (2008) and are listed in the Appendix 1.

Morphological data are mostly osteological and were taken from Lehmann (2006: 169 characters for 114 species; focused on the Hypoptopomatinae) and Pereira (2008: 303 characters for 71 species; focused on the Neoplecostominae). These data were concatenated in one matrix. Collection numbers of vouchers and GenBank accession numbers are listed in the cited studies. Taxa that received different denominations by the different authors are listed in

Table 1.

Whenever possible, only data from the same species were joined. Five exceptions were made as there were no data available from the same species (see Table 2). If molecular data were available for more than one specimen from the same species and there were no doubts about the validity and the identification of the species, the same morphological data were assigned for both individuals. The kind of data available for each terminal is specified in

Fig. 10 and Table 3. Our resulting matrix consisted of a total of 3106 characters (2634 molecular and 472 morphological; 1561 of them informative for parsimony) for 235 terminals.

65

Table 1 Taxa where denomination differed in earlier studies

Denomination previously used by Cramer et al. (2008)1, Cramer Denomination used here et al. (this volume) 2, Lehmann (2006)3, or Pereira (2008)4 Acestridium sp. 11,3; Acestridium scutatum2 Acestridium scutatum Acestridium sp. 21,3; Acestridium gymnogaster2 Acestridium gymnogaster Eurycheilichthys sp. 11; Eurycheilichthys sp. n. „Pin“3 Eurycheilichthys sp. 1 Eurycheilichthys sp. 21; Eurycheilichthys sp. n. „Neo“3 Eurycheilichthys sp. 2 Eurycheilichthys sp. 31; Eurycheilichthys sp. n. „Lis”3 Eurycheilichthys sp. 3 Eurycheilichthys sp. 41; Eurycheilichthys sp. n. „Taq“3 Eurycheilichthys sp. 4 Eurycheilichthys sp. 71; Eurycheilichthys sp. n. „Pir“3 Eurycheilichthys sp. 7 Gymnotocinclus anosteus2; Gen. nov. T4 Gymnotocinclus anosteus Hisonotus cf. aky1 Hisonotus iota Hisonotus sp. 31; Hisonotus armatus2 Hisonotus armatus Hisonotus sp. 51; Hisonotus armatus2; Hisonotus sp. n.4 Hisonotus armatus Neoplecostominae nov. gen. 12; Gen. nov. A3; Gen. nov. P4 Neoplecostominae nov. gen. 1 Neoplecostominae nov. gen. 22; Gen. nov. unnamed “Taxon 12”3; Neoplecostominae nov. gen. 2 Gen. nov. B4 Neoplecostomus sp.1,2; Neoplecostomus P sp. n.4 Neoplecostomus sp. P New taxon TT sp. “Torp. BDX”3; Hisonotus luteofrenatus2 Hisonotus luteofrenatus New taxon TT sp. “Torp. QPT”3 Hisonotus chromodontus New taxon TT sp. “Torp. QPX”3 Hisonotus chromodontus Pareiorhaphis nasuta2; Pareiorhaphis M sp. n.4 Pareiorhaphis nasuta Pareiorhaphis sp. 21,2; Pareiorhaphis P sp. n.4 Pareiorhaphis sp. 2 Pareiorhaphis sp. 51,2; Pareiorhaphis T sp. n.4 Pareiorhaphis sp. 5 Pareiorhaphis sp. 62; Pareiorhaphis Ca sp. n.4 Pareiorhaphis sp. 6 Pareiorhina sp. n.3; Pareiorhina sp. B4 Pareiorhina sp. B Parotocinclus sp.2; Parotocinclus sp. 153 Parotocinclus sp. 15

66

Table 2 Cases where data from different species where combined

Species of the molecular Species of the morphological data Denomination used in the data text and figures Ancistrus brevipinnis Ancistrus reisi Ancistrus Astroblepus sp. 1 Astroblepus sp. “transandino” = Astroblepus 1 Astroblepus sp. 2 Astroblepus sp. 2 Astroblepus sp. “cisandino” = Astroblepus 2 Astroblepus sp. 1 Chaetostoma sp. 1 Chaetostoma leucomelas Chaetostoma Corymbophanes kaiei Corymbophanes andersoni Corymbophanes Harttia kronei Harttia loricariformis Harttia Rineloricaria sp. Rineloricaria strigilata Rineloricaria

Phylogenetic Analyses

Maximum Parsimony (MP) analyses were performed using the new technologies as implemented in TNT 1.1 (Goloboff et al. 2008). We executed the driven search option that chooses the parameters for the sectorial search, the ratched, and the tree fusion search algorithms, followed by tree bisection and reconnection (TBR). For nodal support, the Bremer support (decay index; Bremer 1994) was calculated using negative constraints as implemented in TNT 1.1. Also, 2000 nonparametric bootstrap pseudoreplicates were calculated. All characters were run unweighted and unordered.

As the strict consensus of all MP trees contained large polytomies, using the

“prunn =4” command in TNT, eight taxa were detected that jump along different positions in alternative trees, causing multiple nodal collapses in the consensus (see Table 3). These taxa were excluded from the strict consensus.

67 To test our topology, we made another maximum parsimony analysis, using constraints to enforce the monophyly of the genus Pareiorhina. The Kishino Hasegawa test (Kishino and

Hasegawa 1989) as implemented in PAUP* 4b10 (Swofford 2001) was used to evaluate the alterative constrained topologies.

To see if it was possible to resolve the relationships inside the genus Pareiorhaphis including the species P. cerosus and P. regani that have been excluded from the strict consensus in Figure 10, we made an additional analysis including all Pareiorhaphis plus four more species from the Neoplecostominae.

RESULTS AND DISCUSSION

The MP analyses resulted in 150 shortest trees of 11135 steps (CI: 0.28, HI: 0.72, RI:

0.71, and RC: 0.20). The strict consensus of these trees contained large polytomies. TNT detected eight taxa that caused multiple nodal collapses in the consensus (see Table 3). The clades where the polytomies were caused were numbered and are also given in Table 3. As the strict consensus where these taxa were excluded shows a significantly better resolution, it is used here for our further discussion (Fig. 10).

The separate MP analysis of the genus Pareiorhaphis resulted in three best trees with

1628 steps (CI: 0.50, HI: 0.50, RI: 0.51 and RC: 0.26; 426 characters were informative for parsimony). Their strict consensus contains only two trichotomies and is shown in Figure 11.

68

Table 3 Taxa that jump along different positions in the trees and that were excluded from the consensus.

Taxon Clade where the Data available for this taxon polytomy was caused Hisonotus notatus VI Morphological from Lehmann (2006) and Pereira (2008) Hypoptopomatinae sp. Surinam III Molecular (COI, RAG2) Microlepidogaster sp. 1 V Molecular (COI, RAG1, RAG2) New taxon 22 "Microhypostomus" I Morphological from Lehmann (2006) Parotocinclus bidentatus V Molecular (COI, RAG1, RAG2) Parotocinclus cristatus IV Morphological from Lehmann (2006) Pareiorhaphis cerosus II Morphological from Pereira (2008) Pareiorhaphis regani II Morphological from Pereira (2008)

Here we present the largest catfish phylogeny in number of species published so far and simultaneously the first phylogeny for the Loricariidae using total evidence. Our results include representatives from all loricarioid families and 207 species from the Loricariidae. Inside the Loricariidae, we were able to include species from five of the six subfamilies. Solely Lithogenes was not available because of its geographical restriction and the resulting rarity in zoological collections. The 58 species (34 described + 24 undescribed) of the Neoplecostominae include all known genera and nearly all described species, only five species of Neoplecostomus lacking. The 111 species (71 described + 40 undescribed) of the Hypoptopomatinae cover all described genera and most species of this subfamily.

69 99 Eurycheilichthys sp. 2 -- 9 Eurycheilichthys sp. 2 50 1 Eurycheilichthys sp. 4 82 -- 1 Eurycheilichthys sp. 1 3 1 Eurycheilichthys sp. 3 65 99 Eurycheilichthys limulus 99 4 7 Eurycheilichthys limulus a) 15 Eurycheilichthys sp. 7 82 Eurycheilichthys pantherinus 13 93 Epactionotus itaimbezinho 99 7 Epactionotus gracilis 16 Epactionotus bilineatus Hisonotus armatus -- Hisonotus armatus -- Hisonotus taimensis 15 * Hisonotus laevior -- Hisonotus laevior -- * Hisonotus sp. 2 * Hisonotus charrua -- 52 Hisonotus charrua 13 5 93 Hisonotus leucofrenat us 75 * Hisonotus leucofrenat us * Lam piella gibbosa 98 Hisonotus ringueleti -- 83 18 Hisonotus nigricauda 8 4 Hisonotus iota Hisonotus sp. 1 99 Pseudotothyris obtusa 5 Pseudotothyris obtusa Otothyropsis sp. -- -- Microlepidogast er sp. 2 1 3 Hisonotus francirochai Otothyropsis sp. „Can“ Hisonotus sp. 4 -- Pseudotocin clu s sp. „PP“ 1 Microlepidogast er perforatus Microlepidogast er sp. „AP“ -- Parotocinclus minutus 1 Parotocinclus sp. „Vitoria” Parotocinclus sp. 2 Parotocinclus sp. „jimi-cristatus” C Parotocinclus planicauda Parotocinclus jimi 63 Parotocinclus sp. 8 „Doce“ Rhinelepis aspera 56 * Parotocinclus doceanus -- -- * Parotocinclus sp. 1 „Intermedia“ C -- Parotocinclus maculicauda „Cubatã o“ 27 1 -- * Parotocinclus maculicauda * Parotocinclus sp. 7 62 Peckoltia vermiculata Parotocinclus sp. 3 87 61 Parotocinclus spilosoma 13 Parotocinclus cearensis C 1 Parotocinclus sp. 14 -- -- Parotocinclus sp. 10 * Ancistomus sabaji 9 Parotocinclus haroldoi Parotocinclus cesarpintoi Parotocinclus spilurus C Hisonotus insperatus * Parotocinclus sp. 11 Hypoptopom a inexpectat um Peckoltia vittata Hypoptopom a cf. inexpectatum -- Hypoptopom a gulare C 1 Hypoptopom a steindach n eri 52 Gen. nov. sp. „MAD“ 84 5 Gen. nov. sp. „LET“ Scobinancistrus pariolispos 56 Nannoptop om a sternoptych um 86 1 Nannoptop om a spectabile 4 97 Hypoptopom a guianen se C 81 20 Hypoptopom a bilobatum 98 * 4 Oxyropsis carinata 98 56 95 Hypancistrus contradens 17 Oxyropsis wrightiana 4 7 Oxyropsis acutirostra 74 Acestridium martini C -- Acestridium martini 72 4 20 2 100 4 Acestridium discus 83 Acestridium scutatum Panaqolus sp. 16 Acestridium gymnogaster Niobichthys ferrarisi --

C Hypostominae Otocinclus cocama 78 -- -- 4 12 -- Otocinclus batmani 6 1 Otocinclus vestitus * -- Panaqolus changae 1 Otocinclus vittatus 68 2 Otocinclus cf. hoppei 5 Otocinclus cf. mariae C -- 99 Otocinclus flexilis 14 99 2 Otocinclus affinis -- 4 99 Hemiancistrus punctulatus Otocinclus flexilis 5 Otocinclus xakriaba 99 Schizolecis guentheri C 84 13 Schizolecis guentheri 10 95 Otothyris travassosi -- * 11 Otothyris juquiae Hemiancistrus fuliginosus -- Parotocinclus sp. 18 1 Parotocinclus sp. 19 -- C Parotocinclus longirostris -- -- 1 Parotocinclus sp. 12 4 * 82 -- Parotocinclus sp. 13 Hypostomus commersonii Parotocinclus eppleyi 2 -- -- Parotocinclus polyochrus Parotocinclus sp. 15 C 7 1 * -- Parotocinclus sp. 16 1 Parotocinclus aripuanensi s * 70 Hypostomus boulengeri Parotocinclus sp. 6 6 -- Parotocinclus collinsae Parotocinclus cf. aripuane nsis C Parotocinclus britskii Parotocinclus sp. 17 -- Hisonotus chrom odontus Hypostomus albopunctatus -- * Hisonotus chrom odontus * -- * Hisonotus luteofrenatus -- 58 * New taxon TT sp. „Torp. BDT“ C -- * 54 New taxon TT sp. „Tromp. SF“ -- 1 * New taxon TT sp. „Tromp. AP“ Hypostomus cochliodon New taxon TT sp. „P. 4“ 5 69 Gymnotocinclus anosteos * 7 Corumbataia cuestae C Parotocinclus prata Parotocinclus sp. 5 98 98 Liposarcus pardalis Parotocinclus jumbo 11 98Parotocinclus sp. 9 50 Pareiorhaphis sp. 1 -- 11 C 1 -- Pareiorhaphis sp. 4 1 Pareiorhaphis parmula -- 1 -- * Pareiorhaphis vestigipinni s Liposarcus anisitsi 1 -- Pareiorhaphis sp. 5 61 1 Pareiorhaphis eurycephal us 92 C 1 Pareiorhaphis hystrix -- -- 4 Pareiorhaphis sp. 3 1 * 98 * Pareiorhaphis sp. 6 Panaque cf. nigrolineatus 54 Pareiorhaphis calmoni -- * Pareiorhaphis steindach n eri -- * Pareiorhaphis azygolechi s 92 C 99 Pareiorhaphis hypselurus * 99 17 Pareiorhaphis stomias -- * 12 Panaque cf. cochliodon Pareiorhaphis nudula 99 99 Pareiorhaphis splende ns 9 8 Pareiorhaphis splende ns 11 C -- Pareiorhaphis mutuca 4 Pareiorhaphis sp. C * 97 Pareiorhaphis garbei Hemiancistrus subviridis 5 Pareiorhaphis garbei -- Pareiorhaphis stephan us C * Pareiorhaphis bahiana 98 Pareiorhaphis nasuta 77 17 61 -- Pareiorhaphis sp. 2 Pseudancistrus leopardus * Pareiorhaphis sp. Z 1 Isbrueckerichthys epakmos 76 Isbrueckerichthys duseni C 81 2 Isbrueckerichthys calvus 24 Isbrueckerichthys saxicola * Megalancistrus parananus Isbrueckerichthys alipionis Neoplecostomu s m icrops 96 60 60 Neoplecostomu s sp. P 9 -- 3 I C L P 1 Neoplecostomu s sp. I 88 11 -- Neoplecostomus espiritosantensis 96 84 6 7 Neoplecostomus paranensis Corymbophanes 5 Neoplecostomu s franciscoensis Neoplecostomu s ribeirensis Neoplecostomi na e g en. nov. 2 sp. S3 3 C 70 Neoplecostomi na e g en. nov. 2 sp. S1 3 87 Neoplecostomi na e g en. nov. 2 sp. F 83 Chaetostoma sp. 2 6 Neoplecostomi na e g en. nov. 2 sp. CV Neoplecostomi na e g en. nov. 2 sp. D Neoplecostomn ia e g en. nov. 3 C L 97 Pareiorhina brachyrhyncha 79 3 9 Pareiorhina sp. D 92 2 -- Chaetostoma 77 Pareiorhina carrancas 85 12 8 Pareiorhina rudolphi 3 Pareiorhina sp. B L 100 Kronichthys subteres 90 12 Kronichthys subteres 1 -- 98 7 Kronichthys lacerta -- Lasiancistrus caucanus 62 -- 6 Kronichthys sp. P 20 3 99 1 61 15 Kronichthys sp. M C L Kronichthys heylandi 8 Neoplecostomi na e g en. nov. 1 1 94 Pseudotocin clu s tietensis Ancistrus 79 5 Pseudotocinclus juquiae 7 97 Pareiorhina sp. 29 Pareiorhina sp. C L P Rhinelepis aspera Loricariinae 62 Peckoltia vermiculata -- 100 Rineloricaria * Ancistomus sabaji * Peckoltia vittata 84 Scobinancistrus pariolispos C L P * Hypancistrus contradens 97 83 Panaqolus sp. 30 * Panaqolus changae Ixinandria steinbachi 99 Hemiancistrus punctulat us -- * -- Hemiancistrus fuliginosus C * 82 Hypostomus com mersonii 98 12 * -- * Hypostomus boulengeri 92 100 -- * Hypostomus albopunct atu s Apistoloricaria ommation 11 * Hypostomus cochliodon 98 Liposarcus pardalis 16 -- * Liposarcus anisitsi C L P * 98 Panaque cf. nigrolinea tus 3 -- * Panaque cf. cochliodon Harttia * Hemiancistrus subviridis 61 Pseudancistrus leopardus 96 * Megalancistrus parananus C L P 3 Corymbophane s 83 Chaetostoma sp. 2 -- 3 Chaetostoma 100 Hemipsilichthys gobio 1 -- Lasiancistrus caucanus 1 Ancistrus 100 C P Rineloricaria L 97 30 Ixinandria steinbachi 30 100 98 12 Apistoloricaria ommation Hemipsilichthys papillatus 3 16 Harttia 100 Hemipsilichthys gobio 30 Hemipsilichthys papillatus C L P Delturinae Delturus parahyba e 99 Delturus angulica u da 11 Hemipsilichthys nimius Delturus brevis Delturus carinotus Hemipsilichthys nimius L P Scoloplax distolothrix 99 Trichomycterus sp. Delturus angulicauda 100 Astroblepus 2 100 24 Astroblepus 1 Astroblepus sp. 3 49 11 L P Astroblepus sp. 4 86 Hoplosternum littorale 99 9 Callichthys callichthys Delturus brevis 42 Corydoras ourastigma Nematogenys inermis C Delturus carinotus L P Delturus parahybae C Scoloplax distolothrix C Trichomycterus sp. C L P Astroblepus 2 100 C L P

Astroblepus 1 Outgroup 100 24 C Astroblepus sp. 3 49 C Astroblepus sp. 4 C 86 Hoplosternum littorale C 99 9 Callichthys callichthys 42 C Corydoras ourastigma C Nematogenys inermis Fig. 10a First part of the strict consensus of the 150 most parsimonious trees (11135 steps; CI: 0.28) with the jumping taxa (Table 3) excluded. Numbers above branches are values from 2000 bootstrap replicates, numbers below are Bremer support. Remainder of the tree in Fig. 10b-d. Roman numbers name clades. C, L, and P specify the kind of data available for each terminal: C = molecular, L = morphological from Lehmann 2006, and P = morphological from Pereira 2008.

70 99 Eurycheilichthys sp. 2 -- 9 Eurycheilichthys sp. 2 50 1 Eurycheilichthys sp. 4 -- 1 82 Eurycheilichthys sp. 1 1 3 Eurycheilichthys sp. 3 65 99 Eurycheilichthys limulus C P 99 4 7 Eurycheilichthys limulus 15 Eurycheilichthys sp. 7 82 Eurycheilichthys pantherinus 50 Pareiorhaphis sp. 1 13 93 Epactionotus itaimbezinho 99 b) 7 Epactionotus gracilis 16 C Epactionotus bilineatus Hisonotus armatus -- 1 -- Hisonotus armatus -- Pareiorhaphis sp. 4 Hisonotus taimensis 15 * Hisonotus laevior -- Hisonotus laevior -- 1 C P -- * Hisonotus sp. 2 * Hisonotus charrua -- 52 Hisonotus charrua Pareiorhaphis parmula 13 5 93 Hisonotus leucofrenat us 75 * Hisonotus leucofrenat us 1 C L P * Lampiella gibbosa 98 Hisonotus ringueleti -- -- 83 18 Hisonotus nigricauda Pareiorhaphis vestigipinnis 8 4 Hisonotus iota Hisonotus sp. 1 C P 99 Pseudotothyris obtusa 1 5 Pseudotothyris obtusa Otothyropsis sp. ------Pareiorhaphis sp. 5 Microlepidogast er sp. 2 1 3 Hisonotus francirochai P Otothyropsis sp. „Can“ C Hisonotus sp. 4 61 1 -- Pseudotocinclu s sp. „PP“ Pareiorhaphis eurycephalus Microlepidogast er perfor atus 1 Microlepidogast er sp. „AP“ -- Parotocinclus minutus 1 C L P 1 Parotocinclus sp. „Vitoria” Parotocinclus sp. 2 92 Pareiorhaphis hystrix Parotocinclus sp. „jimi-cristatus” Parotocinclus planicauda Parotocinclus jimi C 63 Parotocinclus sp. 8 „Doce“ 56 -- * Parotocinclus doceanus 4 -- -- * Parotocinclus sp. 1 „Intermedia“ Pareiorhaphis sp. 3 -- Parotocinclus maculicauda „Cubatão“ -- 27 1 -- * Parotocinclus maculicauda C P * Parotocinclus sp. 7 1 Parotocinclus sp. 3 87 Parotocinclus spilosoma Pareiorhaphis sp. 6 61 13 Parotocinclus cearensis * 1 Parotocinclus sp. 14 C L P -- Parotocinclus sp. 10 9 Par otocinclus har oldoi Parotocinclus cesarpintoi 54 Pareiorhaphis calmoni Parotocinclus spilurus Hisonotus insperatus Parotocinclus sp. 11 C P Hypoptopoma inexpectat um -- Hypoptopoma cf. inexpectatum -- * Pareiorhaphis steindachneri Hypoptopoma gulare 1 Hypoptopoma steindachneri 52 Gen. nov. sp. „ MAD“ C P 5 Gen. nov. sp. „ LET“ * 56 86 Nannoptopoma sternoptychum 1 Nannoptopoma spectabile -- Pareiorhaphis azygolechis 4 97 Hypoptopoma guianense 81 20 Hypoptopoma bilobatum C P 98 4 95 Oxyropsis carinata 56 17 Oxyropsis wrightiana 7 99 4 Oxyropsis acutirostra * Pareiorhaphis hypselurus 74 Acestridium martini -- Acestridium martini 72 4 C P 2 100 4 Acestridium discus Acestridium scutatum 99 16 17 Acestridium gymnogaster Pareiorhaphis stomias Niobichthys ferrarisi 78 -- Otocinclus cocama -- -- C P 12 -- 4 Otocinclus batmani 12 1 6 -- Otocinclus vestitus 1 Otocinclus vittatus 68 2 Pareiorhaphis nudula Otocinclus cf. hoppei 5 -- 99 Otocinclus cf. mariae Otocinclus flexilis C P 14 99 2 Otocinclus affinis -- 4 Otocinclus flexilis 5 99 Pareiorhaphis splendens Otocinclus xakriaba 99 Schizolecis guentheri 84 13 Schizolecis guentheri C P 10 95 Otothyris travassosi 11 Otothyris juquiae 8 -- Parotocinclus sp. 18 Pareiorhaphis splendens 1 Parotocinclus sp. 19 -- -- Parotocinclus longirostris P 1 Parotocinclus sp. 12 4 -- Parotocinclus sp. 13 Parotocinclus eppleyi Pareiorhaphis mutuca 2 -- -- Parotocinclus polyochrus II 1 Parotocinclus sp. 15 -- 7 -- P Parotocinclus sp. 16 1 Parotocinclus aripuanensi s 70 Parotocinclus sp. 6 Pareiorhaphis sp. C 6 Parotocinclus collinsae Parotocinclus cf. aripuanensis 4 Parotocinclus britskii C P Parotocinclus sp. 17 -- Hisonotus chromodontus -- 97 Pareiorhaphis garbei -- * Hisonotus chromodontus * Hisonotus luteofrenatus 58 * New taxon TT sp. „Torp. BDT“ C P -- * 54 New taxon TT sp. „Tromp. SF“ -- 1 * New taxon TT sp. „Tromp. AP“ 5 New taxon TT sp. „P. 4“ Pareiorhaphis garbei 5 69 Gymnotocinclus anosteos 7 Corumbataia cuestae P Parotocinclus prata Parotocinclus sp. 5 98 Parotocinclus jumbo -- Pareiorhaphis stephanus 11 98Parotocinclus sp. 9 50 -- 11Pareiorhaphis sp. 1 C P -- 1 Pareiorhaphis sp. 4 1 Pareiorhaphis parmula -- 1 Pareiorhaphis vestigipinni s * Pareiorhaphis bahiana 1 -- Pareiorhaphis sp. 5 61 1 Pareiorhaphis eurycephal us 1 92 C P -- Pareiorhaphis hystrix -- 4 Pareiorhaphis sp. 3 1 * Pareiorhaphis sp. 6 98 54 Pareiorhaphis nasuta Pareiorhaphis calmoni -- * Pareiorhaphis steindachneri -- * Pareiorhaphis azygolechis C P 99 Pareiorhaphis hypselurus 77 * 99 17 Pareiorhaphis stomias 17 12 Pareiorhaphis nudula Pareiorhaphis sp. 2 99 99 Pareiorhaphis splendens 9 8 Pareiorhaphis splendens -- P -- Pareiorhaphis mutuca * 4 Pareiorhaphis sp. C Neoplecostominae 97 Pareiorhaphis garbei Pareiorhaphis sp. Z 5 Pareiorhaphis garbei -- Pareiorhaphis stephanus 1 C P * Pareiorhaphis bahiana 99 98 Pareiorhaphis nasuta 77 -- 17 Pareiorhaphis sp. 2 Isbrueckerichthys epakmos * Pareiorhaphis sp. Z 1 Isbrueckerichthys epakmos 76 Isbrueckerichthys duseni 9 C L P 81 2 Isbrueckerichthys calvus 24 Isbrueckerichthys saxicola 76 Isbrueckerichthys duseni Isbrueckerichthys alipionis 96 60 Neoplecostomus microps 60 Neoplecostomus sp. P C 9 -- 3 1 Neoplecostomus sp. I 88 11 -- Neoplecostomus espiritosantensis 2 7 Isbrueckerichthys calvus 84 6 Neoplecostomus paranensis 81 5 Neoplecostomus franciscoensis Neoplecostomus ribeirensis C Neoplecostomi na e g en. nov. 2 sp. S3 70 Neoplecostomi na e g en. nov. 2 sp. S1 87 3 Neoplecostomi na e g en. nov. 2 sp. F 24 Isbrueckerichthys saxicola 6 Neoplecostomi na e g en. nov. 2 sp. CV Neoplecostomi na e g en. nov. 2 sp. D C P Neoplecostomniae gen. nov. 3 97 79 Pareiorhina brachyrhyncha 9 Pareiorhina sp. D Isbrueckerichthys alipionis 92 77 2 Pareiorhina carrancas 85 12 8 Pareiorhina rudolphi C L P 3 Pareiorhina sp. B 100 90 Kronichthys subteres -- 12 Kronichthys subteres Neoplecostomus microps 7 98 62 -- Kronichthys lacerta 6 Kronichthys sp. P 20 99 1 3 Kronichthys sp. M 60 C P 61 15 Kronichthys heylandi 8 96 Neoplecostomi nae gen. nov. 1 94 Neoplecostomus sp. P Pseudotocinclu s tietensis 79 5 Pseudotocinclu s juquiae 60 3 7 97 Pareiorhina sp. P 29 Pareiorhina sp. 9 -- Rhinelepis aspera 62 Peckoltia vermiculata Neoplecostomus sp. I -- 1 * Ancistomus sabaji * Peckoltia vittata C P 84 Scobinancistrus pariolispos 11 * Hypancistrus contradens 83 -- Panaqolus sp. 88 Neoplecostomus espiritosantensis * Panaqolus changae 99 Hemiancistrus punctulatus -- C P -- * Hemiancistrus fuliginosus * 82 Hypostomus commersonii 7 * -- * Hypostomus boulengeri 6 Neoplecostomus paranensis 92 -- * Hypostomus albopunct atus 11 Hypostomus cochliodon 84 * 98 C P Liposarcus pardalis -- * Liposarcus anisitsi * 98 Panaque cf. nigr olinea tus -- Neoplecostomus franciscoensis * Panaque cf. cochliodon 5 * Hemiancistrus subviridis 61 Pseudancistrus leopardus C L P 96 * Megalancistrus parananus 3 Corymbophane s 83 Neoplecostomus ribeirensis Chaetostoma sp. 2 -- 3 Chaetostoma 1 -- Lasiancistrus caucanus P 1 Ancistrus 100 Rineloricaria 97 30 Ixinandria steinbachi Neoplecostominae gen. nov. 2 sp. S3 100 98 12 Apistoloricaria ommation 3 16 Harttia L P 100 Hemipsilichthys gobio 30 Hemipsilichthys papillatus Delturus parahybae Neoplecostominae gen. nov. 2 sp. S1 99 70 Delturus angulicauda 11 Delturus brevis P Delturus carinotus Hemipsilichthys nimius Scoloplax distolothrix 3 87 Neoplecostominae gen. nov. 2 sp. F Trichomycterus sp. 100 Astroblepus 2 P 100 24 Astroblepus 1 Astroblepus sp. 3 49 6 Astroblepus sp. 4 86 Neoplecostominae gen. nov. 2 sp. CV 99 Hoplosternum littorale 9 Callichthys callichthys 42 Corydoras ourastigma C P Nematogenys inermis Neoplecostominae gen. nov. 2 sp. D C Neoplecostomniae gen. nov. 3 C P 97 Pareiorhina brachyrhyncha P 79 9 Pareiorhina sp. D C P 92 2 77 Pareiorhina carrancas 8 L P 12 85 Pareiorhina rudolphi L P 3 Pareiorhina sp. B C L P 100 Kronichthys subteres C L P 90 12 Kronichthys subteres -- 7 C P Kronichthys lacerta 62 -- 6 C P Kronichthys sp. P 3 99 1 P Kronichthys sp. M 61 15 C L P Kronichthys heylandi 8 C L P Neoplecostominae gen. nov. 1 C L 94 Pseudotocinclus tietensis C 79 5 Pseudotocinclus juquiae C 7 97 Pareiorhina sp. C 29 Pareiorhina sp. Fig. 10b Second part of the strict consensus of the 150 most parsimonious trees (11135 steps; CI: 0.28) with the jumping taxa (Table 3) excluded. Numbers above branches are values from 2000 bootstrap replicates, numbers below are Bremer support. Remainder of the tree in Fig. 10a, c, and d. Roman numbers name clades. C, L, and P specify the kind of data available for each terminal: C = molecular, L = morphological from Lehmann 2006, and P = morphological from Pereira 2008.

71 99 Eurycheilichthys sp. 2 -- 9 Eurycheilichthys sp. 2 50 1 Eurycheilichthys sp. 4 C L -- 1 82 Eurycheilichthys sp. 1 1 3 Eurycheilichthys sp. 3 65 99 Eurycheilichthys limulus Hypoptopoma inexpectatum 99 4 7 Eurycheilichthys limulus 15 Eurycheilichthys sp. 7 C c) 82 Eurycheilichthys pantherinus 13 93 99 Epactionotus itaimbezinho 7 Epactionotus gracilis Hypoptopoma cf. inexpectatum 16 Epactionotus bilineatus Hisonotus armatus C -- Hisonotus armatus -- Hisonotus taimensis 15 * Hisonotus laevior -- Hypoptopoma gulare -- Hisonotus laevior -- * Hisonotus sp. 2 C * Hisonotus charrua -- 52 Hisonotus charrua 1 13 5 93 Hisonotus leucofrenat us 75 Hypoptopoma steindachneri * Hisonotus leucofrenat us * Lampiella gibbosa 98 L Hisonotus ringueleti -- 83 18 Hisonotus nigricauda 8 4 Hisonotus iota 52 Gen. nov. sp. „MAD“ Hisonotus sp. 1 99 Pseudotothyris obtusa 5 Pseudotothyris obtusa L Otothyropsis sp. -- -- Microlepidogast er sp. 2 5 1 3 Gen. nov. sp. „LET“ Hisonotus francirochai Otothyropsis sp. „Can“ Hisonotus sp. 4 L -- Pseudotocinclu s sp. „PP“ 1 Microlepidogast er perforatus 56 Microlepidogast er sp. „AP“ Nannoptopoma sternoptychum -- Parotocinclus minutus 1 Parotocinclus sp. „Vitoria” C L Parotocinclus sp. 2 86 Parotocinclus sp. „jimi-cristatus” 1 Parotocinclus planicauda Nannoptopoma spectabile Parotocinclus jimi 63 Parotocinclus sp. 8 „Doce“ 56 4 C * Parotocinclus doceanus -- -- * Parotocinclus sp. 1 „Intermedia“ 27 1 -- Parotocinclus maculicauda „Cubatã o“ 97 Hypoptopoma guianense -- * Parotocinclus maculicauda * Parotocinclus sp. 7 C Parotocinclus sp. 3 87 61 Parotocinclus spilosoma 20 13 Parotocinclus cearensis Hypoptopoma bilobatum 1 Parotocinclus sp. 14 -- Parotocinclus sp. 10 81 C 9 Parotocinclus haroldoi Parotocinclus cesarpintoi Parotocinclus spilurus 98 Oxyropsis carinata Hisonotus insperatus 4 Parotocinclus sp. 11 Hypoptopoma inexpectat um C L Hypoptopoma cf. inexpectatum 95 -- 17 Hypoptopoma gulare Oxyropsis wrightiana 1 Hypoptopoma steindach neri 56 52 Gen. nov. sp. „MAD“ 5 Gen. nov. sp. „LET“ C 56 7 86 Nannoptop oma sternoptychum 1 Nannoptop oma spectabile 4 4 97 Hypoptopoma guianense Oxyropsis acutirostra Hypoptopomatini 81 20 Hypoptopoma bilobatum 98 4 95 Oxyropsis carinata C L 56 17 Oxyropsis wrightiana 4 7 Oxyropsis acutirostra 74 Acestridium martini Acestridium martini -- Acestridium martini 72 4 2 100 4 Acestridium discus 74 C L Acestridium scutatum 16 Acestridium gymnogaster Niobichthys ferrarisi Acestridium martini -- 78 -- Otocinclus cocama -- -- 4 4 12 -- Otocinclus batmani 72 1 L 6 -- Otocinclus vestitus 1 Otocinclus vittatus 68 2 Otocinclus cf. hoppei 5 Acestridium discus Otocinclus cf. mariae 2 -- 99 4 99 Otocinclus flexilis 2 14 100 C L Otocinclus affinis -- 4 Otocinclus flexilis 5 Otocinclus xakriaba 99 Acestridium scutatum Schizolecis guentheri 84 13 Schizolecis guentheri 16 10 95 Otothyris travassosi C L 11 Otothyris juquiae -- Parotocinclus sp. 18 Acestridium gymnogaster 1 Parotocinclus sp. 19 -- -- Parotocinclus longirostris 1 Parotocinclus sp. 12 L 4 -- Parotocinclus sp. 13 Parotocinclus eppleyi 2 -- Parotocinclus polyochrus Niobichthys ferrarisi -- 7 1 Parotocinclus sp. 15 -- Parotocinclus sp. 16 C L 1 Parotocinclus aripuanensi s 70 Parotocinclus sp. 6 6 Parotocinclus collinsae -- Otocinclus cocama Parotocinclus cf. aripuanensis Parotocinclus britskii 78 L Parotocinclus sp. 17 -- -- Hisonotus chromodontus -- -- 4 -- * Hisonotus chromodontus Otocinclus batmani * Hisonotus luteofrenatus 12 58 * New taxon TT sp. „Torp. BDT“ -- * 54 New taxon TT sp. „Tromp. SF“ -- 1 C L -- 1 * New taxon TT sp. „Tromp. AP“ 6 New taxon TT sp. „P. 4“ 5 Otocinclus vestitus 69 Gymnotocinclus anosteos 7 Corumbataia cuestae Parotocinclus prata -- 1 C Parotocinclus sp. 5 98 Parotocinclus jumbo Otocinclus vittatus 11 98Parotocinclus sp. 9 50 -- 11Pareiorhaphis sp. 1 2 1 Pareiorhaphis sp. 4 68 C -- 1 Pareiorhaphis parmula -- 1 Pareiorhaphis vestigipinni s 1 -- Otocinclus cf. hoppei Pareiorhaphis sp. 5 61 1 Pareiorhaphis eurycephal us 1 92 5 -- Pareiorhaphis hystrix C -- 4 Pareiorhaphis sp. 3 1 * Pareiorhaphis sp. 6 54 Pareiorhaphis calmoni Otocinclus cf. mariae -- * Pareiorhaphis steindach neri -- * Pareiorhaphis azygolechi s 99 C L P 99 Pareiorhaphis hypselurus * 99 -- 17 Pareiorhaphis stomias 12 Pareiorhaphis nudula Otocinclus flexilis 99 99 Pareiorhaphis splendens 9 8 Pareiorhaphis splendens 14 99 C L P -- Pareiorhaphis mutuca 2 4 Pareiorhaphis sp. C 97 Pareiorhaphis garbei Otocinclus flexilis 5 Pareiorhaphis garbei -- Pareiorhaphis stephanus -- 4 C * Pareiorhaphis bahiana 98 Pareiorhaphis nasuta 77 -- 17 Pareiorhaphis sp. 2 Otocinclus affinis * Pareiorhaphis sp. Z 1 Isbrueckerichthys epakmos 5 76 Isbrueckerichthys duseni C L P 81 2 Isbrueckerichthys calvus 24 Isbrueckerichthys saxicola Otocinclus xakriaba Isbrueckerichthys alipionis 96 60 Neoplecostomus microps 60 Neoplecostomus sp. P C L 9 -- 3 1 Neoplecostomus sp. I 88 11 -- Neoplecostomus espiritosante nsis 99

84 7 Neoplecostomus paranensis Schizolecis guentheri Hypoptopomatinae 6 5 Neoplecostomus franciscoensis Neoplecostomus ribeirensis C L Neoplecostomi nae gen. nov. 2 sp. S3 70 Neoplecostomi nae gen. nov. 2 sp. S1 13 87 3 Neoplecostomi nae gen. nov. 2 sp. F 84 Schizolecis guentheri 6 Neoplecostomi nae gen. nov. 2 sp. CV Neoplecostomi nae gen. nov. 2 sp. D C L Neoplecostomniae gen. nov. 3 97 79 Pareiorhina brachyrhyncha 10 9 Pareiorhina sp. D 95 Otothyris travassosi 92 77 2 Pareiorhina carrancas 85 12 8 Pareiorhina rudolphi C 3 Pareiorhina sp. B 100 90 Kronichthys subteres 11 -- 12 Kronichthys subteres Otothyris juquiae 98 7 Kronichthys lacerta 62 -- 6 99 Kronichthys sp. P 20 3 1 L Kronichthys sp. M 61 15 Kronichthys heylandi 8 -- Neoplecostomi nae gen. nov. 1 Parotocinclus sp. 18 94 Pseudotocinclu s tietensis 79 5 Pseudotocinclu s juquiae 7 97 Pareiorhina sp. L 29 Pareiorhina sp. Rhinelepis aspera 1 62 Parotocinclus sp. 19 Peckoltia vermiculata -- * Ancistomus sabaji * Peckoltia vittata 84 Scobinancistrus pariolispos * Hypancistrus contradens -- 83 Panaqolus sp. Parotocinclus longirostris * Panaqolus changa e 99 Hemiancistrus punctulat us L ------* Hemiancistrus fuliginosus * 82 Hypostomus commersonii * 1 -- * Hypostomus boulengeri Parotocinclus sp. 12 92 -- * Hypostomus albopunct atus 11 Hypostomus cochliodon * L 98 Liposarcus pardalis 4 -- * Liposarcus anisitsi * 98 Panaque cf. nigrolinea tus Parotocinclus sp. 13 -- * Panaque cf. cochliodon * Hemiancistrus subviridis -- C L 61 Pseudancistrus leopardus 96 * Megalancistrus parananus 3 Corymbophane s Parotocinclus eppleyi 83 Chaetostoma sp. 2 -- 3 Chaetostoma 1 -- Lasiancistrus caucanus 2 L 1 Ancistrus 100 Rineloricaria 97 Parotocinclus polyochrus 30 Ixinandria steinbachi -- 100 98 12 Apistoloricaria ommation -- 3 16 Harttia C L 100 Hemipsilichthys gobio 30 Hemipsilichthys papillatus Parotocinclus sp. 15 Delturus parahyba e 99 1 Delturus angulicau da 7 11 Delturus brevis L Delturus carinotus Hemipsilichthys nimius -- Scoloplax distolothrix Parotocinclus sp. 16 Trichomycterus sp. 100 Astroblepus 2 C L 100 24 Astroblepus 1 Astroblepus sp. 3 49 1 Astroblepus sp. 4 Parotocinclus aripuanensis 86 99 Hoplosternum littorale 9 Callichthys callichthys C 42 Corydoras ourastigma 70 Nematogenys inermis Parotocinclus cf. aripuanensis C L 6 Parotocinclus collinsae L Parotocinclus sp. 6 C L Parotocinclus britskii L Parotocinclus sp. 17 L -- Hisonotus chromodontus L -- * Hisonotus chromodontus -- C L * Hisonotus luteofrenatus III * L 58 New taxon TT sp. „Torp. BDT“ L -- * 54 New taxon TT sp. „Tromp. SF“ L 1 * New taxon TT sp. „Tromp. AP“ -- L New taxon TT sp. „P. 4“ 5 C L P 69 Gymnotocinclus anosteos C L 7 Corumbataia cuestae L Parotocinclus prata L Parotocinclus sp. 5 C L 98 Parotocinclus jumbo L 11 Parotocinclus sp. 9 Fig. 10c Third part of the strict consensus of the 150 most parsimonious trees (11135 steps; CI: 0.28) with the jumping taxa (Table 3) excluded. Numbers above branches are values from 2000 bootstrap replicates, numbers below are Bremer support. Remainder of the tree in Fig. 10a, b, and d. Roman numbers name clades. C, L, and P specify the kind of data available for each terminal: C = molecular, L = morphological from Lehmann 2006, and P = morphological from Pereira 2008.

72 99 Eurycheilichthys sp. 2 -- 9 Eurycheilichthys sp. 2 50 1 C L Eurycheilichthys sp. 4 -- 1 82 Eurycheilichthys sp. 1 1 3 Eurycheilichthys sp. 3 99 Eurycheilichthys sp. 2 65 99 Eurycheilichthys limulus 99 4 7 Eurycheilichthys limulus 15 Eurycheilichthys sp. 7 C L 82 Eurycheilichthys pantherinus -- 13 93 Epactionotus itaimbezinho 9 d) 99 Eurycheilichthys sp. 2 7 Epactionotus gracilis 16 Epactionotus bilineatus Hisonotus armatus 1 C L -- Hisonotus armatus -- Hisonotus taimensis 15 50 * Hisonotus laevior Eurycheilichthys sp. 4 -- Hisonotus laevior -- * Hisonotus sp. 2 C L * Hisonotus charrua -- 52 Hisonotus charrua 1 13 5 93 Hisonotus leucofrenat us 82 75 Eurycheilichthys sp. 3 * Hisonotus leucofrenat us * Lampiella gibbosa -- 98 Hisonotus ringueleti C L -- 83 18 Hisonotus nigricauda 3 8 4 Hisonotus iota Eurycheilichthys sp. 1 Hisonotus sp. 1 99 Pseudotothyris obtusa 1 5 Pseudotothyris obtusa C L P Otothyropsis sp. -- -- Microlepidogast er sp. 2 1 3 Hisonotus francirochai 65 99 Eurycheilichthys limulus Otothyropsis sp. „Can“ Hisonotus sp. 4 C L P -- Pseudotocinclus sp. „PP“ Microlepidogast er perforatus 4 1 7 Microlepidogast er sp. „AP“ Eurycheilichthys limulus -- Parotocinclus minutus 99 1 Parotocinclus sp. „Vitoria” Parotocinclus sp. 2 C L Parotocinclus sp. „jimi-cristatus” Parotocinclus planicauda Eurycheilichthys sp. 7 Parotocinclus jimi 15 63 Parotocinclus sp. 8 „Doce“ 56 * Parotocinclus doceanus C L P -- -- * Parotocinclus sp. 1 „Intermedia“ -- Parotocinclus maculicauda „Cubatão“ 27 1 -- 82 * Parotocinclus maculicauda Eurycheilichthys pantherinus * Parotocinclus sp. 7 Parotocinclus sp. 3 C 87 L 61 Parotocinclus spilosoma 13 Parotocinclus cearensis 13 1 Parotocinclus sp. 14 93 Epactionotus itaimbezinho -- Parotocinclus sp. 10 9 Parotocinclus haroldoi Parotocinclus cesarpintoi C L Parotocinclus spilurus 99 7 Hisonotus insperatus Epactionotus gracilis Parotocinclus sp. 11 Hypoptopoma inexpectat um Hypoptopoma cf. inexpectatum C L P -- 16 Hypoptopoma gulare 1 Hypoptopoma steindach neri 52 Gen. nov. sp. „ MAD“ Epactionotus bilineatus 5 Gen. nov. sp. „ LET“ 56 86 Nannoptop oma sternoptychum C P 1 Nannoptop oma spectabile 4 97 Hypoptopoma guianense 81 20 Hypoptopoma bilobatum Hisonotus armatus 98 4 95 Oxyropsis carinata 56 17 Oxyropsis wrightiana 7 C P 4 Oxyr opsis acutir ostra 74 Acestridium martini -- Acestridium martini 72 4 Hisonotus armatus 2 100 4 Acestridium discus Acestridium scutatum 16 Acestridium gymnogaster -- C Niobichthys ferr ar isi -- 78 -- Otocinclus cocama -- -- 4 Otocinclus batmani Hisonotus taimensis 12 -- 1 6 -- Otocinclus vestitus 15 1 Otocinclus vittatus 68 2 C Otocinclus cf. hoppei 5 -- 99 Otocinclus cf. mariae Otocinclus flexilis * 99 Hisonotus laevior 14 2 Otocinclus affinis -- 4 Otocinclus flexilis 5 C Otocinclus xakr iaba 99 -- Schizolecis guentheri 84 13 Schizolecis guentheri Hisonotus laevior 10 95 Otothyris travassosi 11 Otothyris juquiae -- Parotocinclus sp. 18 C 1 Parotocinclus sp. 19 * -- -- Parotocinclus longirostris Hisonotus sp. 2 1 Parotocinclus sp. 12 -- 4 -- Parotocinclus sp. 13 Parotocinclus eppleyi C 2 -- -- Parotocinclus polyochrus 7 1 Parotocinclus sp. 15 -- Parotocinclus sp. 16 * Hisonotus charrua 1 Parotocinclus aripuanensi s 70 Parotocinclus sp. 6 C 6 Parotocinclus collinsae VI Parotocinclus cf. aripuanensis -- 52 Parotocinclus britskii Hisonotus charrua Parotocinclus sp. 17 -- Hisonotus chromodontus -- C -- * Hisonotus chromodontus * Hisonotus luteofrenatus 13 5 58 * New taxon TT sp. „Torp. BDT“ 93 54 Hisonotus leucofrenatus -- * New taxon TT sp. „Tromp. SF“ -- 1 * New taxon TT sp. „Tromp. AP“ New taxon TT sp. „P. 4“ 5 C 69 Gymnotocinclus anosteos 75 7 Corumbataia cuestae Parotocinclus prata * Hisonotus leucofrenatus Parotocinclus sp. 5 98 Parotocinclus jumbo C L 11 98Parotocinclus sp. 9 50 Pareiorhaphis sp. 1 * -- 11 -- 1 Pareiorhaphis sp. 4 Lampiella gibbosa 1 Pareiorhaphis parmula -- 1 Pareiorhaphis vestigipinni s 1 -- Pareiorhaphis sp. 5 C 61 1 Pareiorhaphis eurycephal us 1 92 -- Pareiorhaphis hystrix 98 Hisonotus ringueleti -- 4 Pareiorhaphis sp. 3 1 * Pareiorhaphis sp. 6 54 Pareiorhaphis calmoni C -- * Pareiorhaphis steindach neri -- * Pareiorhaphis azygolechi s 18 99 Pareiorhaphis hypselurus Hisonotus nigricauda * 99 17 Pareiorhaphis stomias -- 83 12 Pareiorhaphis nudula C 99 99 Pareiorhaphis splendens 9 8 Pareiorhaphis splendens -- Pareiorhaphis mutuca 8 4 Hisonotus iota Hypoptopomatinae 4 Pareiorhaphis sp. C 97 Pareiorhaphis garbei C 5 Pareiorhaphis garbei -- Pareiorhaphis stephanus * Pareiorhaphis bahiana 98 Hisonotus sp. 1 Pareiorhaphis nasuta 77 -- 17 Pareiorhaphis sp. 2 * Pareiorhaphis sp. Z C L 1 Isbrueckerichthys epakmos 76 Isbrueckerichthys duseni 99 81 2 Isbrueckerichthys calvus Pseudotothyris obtusa 24 Isbrueckerichthys saxicola Isbrueckerichthys alipionis C L 96 60 Neoplecostomus microps 60 Neoplecostomus sp. P 5 9 -- 3 1 Neoplecostomus sp. I Pseudotothyris obtusa 88 11 -- Neoplecostomus espiritosantensis 84 6 7 Neoplecostomus paranensis C 5 Neoplecostomus franciscoensis Neoplecostomus ribeirensis Neoplecostomi nae gen. nov. 2 sp. S3 Otothyropsis sp. 70 Neoplecostomi nae gen. nov. 2 sp. S1 87 3 Neoplecostomi nae gen. nov. 2 sp. F -- 6 Neoplecostomi nae gen. nov. 2 sp. CV C Neoplecostomi nae gen. nov. 2 sp. D -- Neoplecostomniae gen. nov. 3 97 Microlepidogaster sp. 2 79 Pareiorhina brachyrhyncha 9 Pareiorhina sp. D 1 92 77 2 Pareiorhina carrancas C 8 85 3 12 Pareiorhina rudolphi 3 Pareiorhina sp. B 100 Kronichthys subteres Hisonotus francirochai 90 -- 12 Kronichthys subteres 98 7 Kronichthys lacerta 62 -- 6 L Kr onichthys sp. P 20 99 1 3 Kr onichthys sp. M 61 15 Kronichthys heylandi 8 Otothyropsis sp. „Can“ Neoplecostomi nae gen. nov. 1 94 Pseudotocinclus tietensis 79 5 Pseudotocinclus juquiae C 7 97 Pareiorhina sp. 29 Pareiorhina sp. Hisonotus sp. 4 Rhinelepis aspera 62 Peckoltia vermiculata -- * Ancistomus sabaji L * Peckoltia vittata 84 Scobinancistrus pariolispos * Hypancistrus contradens Pseudotocinclus sp. „PP“ 83 Panaqolus sp. * Panaqolus changa e -- C L 99 Hemiancistrus punctulat us -- -- * Hemiancistrus fuliginosus * 82 Hypostomus commersonii Microlepidogaster perforatus * -- * Hypostomus boulengeri 92 -- * Hypostomus albopunct atus 1 11 L * Hypostomus cochliodon 98 Liposarcus pardalis -- * Liposarcus anisitsi 98 Microlepidogaster sp. „AP“ * Panaque cf. nigrolineatus -- * Panaque cf. cochliodon * Hemiancistrus subviridis L 61 Pseudancistrus leopardus 96 * Megalancistrus parananus 3 Corymbophanes Parotocinclus minutus 83 Chaetostoma sp. 2 -- -- 3 Chaetostoma L 1 -- Lasiancistrus caucanus 1 Ancistrus 100 Rineloricaria 97 Parotocinclus sp. Vitoria” 30 „ Ixinandria steinbachi 1 100 98 12 Apistoloricaria ommation 3 16 Harttia L 100 Hemipsilichthys gobio 30 Hemipsilichthys papillatus Parotocinclus sp. 2 Delturus parahyba e 99 Delturus angulicau da 11 Delturus brevis L Delturus carinotus Hemipsilichthys nimius Scoloplax distolothrix Parotocinclus sp. „jimi-cristatus” Tr ichomycter us sp. 100 Astroblepus 2 L 100 24 Astroblepus 1 Astroblepus sp. 3 49 Astroblepus sp. 4 Parotocinclus planicauda 86 99 Hoplosternum littorale 9 Callichthys callichthys 42 L Corydoras ourastigma Nematogenys inermis Parotocinclus jimi L 63 Parotocinclus sp. 8 „Doce“ L 56 * Parotocinclus doceanus L IV V * Parotocinclus sp. 1 „Intermedia“ -- -- L 27 1 -- Parotocinclus maculicauda „Cubatão“ C L P -- * Parotocinclus maculicauda L * Parotocinclus sp. 7 L Parotocinclus sp. 3 C L 87 Parotocinclus spilosoma L 61 13 Parotocinclus cearensis 1 L Parotocinclus sp. 14 L -- Parotocinclus sp. 10 L 9 Parotocinclus haroldoi L Parotocinclus cesarpintoi L Parotocinclus spilurus C L Hisonotus insperatus L Parotocinclus sp. 11

Fig. 10d Fourth part of the strict consensus of the 150 most parsimonious trees (11135 steps; CI: 0.28) with the jumping taxa (Table 3) excluded. Numbers above branches are values from 2000 bootstrap replicates, numbers below are Bremer support. Remainder of the tree in Fig. 10a-c. Roman numbers name clades. C, L, and P specify the kind of data available for each terminal: C = molecular, L = morphological from Lehmann 2006, and P = morphological from Pereira 2008.

73 Our initial strict consensus contained some large polytomies. We detected eight

“jumping taxa” (see Table 3), species whose phylogenetic position were very unstable, causing large polytomies. After their exclusion from the consensus, most of these polytomies could be resolved. We suspect that most of the uncertain placements were due to lack of compatible data: Hypoptopomatinae sp. Surinam, Microlepidogaster sp. 1, and Parotocinclus bidentatus were only represented by molecular data, being placed in a larger group of taxa where only morphological data were available; the contrary is the case for Hisonotus notatus and New taxon 22 "Microhypostomus”, which were only represented by morphological data among species with molecular data only. Pareiorhaphis cerosus is only known from two type specimens and P. regani by the holotype only. Because of these circumstances, only characters from external morphology could be included for these two species. Nevertheless, we see no risk of biased groupings caused by the presence of only one kind of data as most clades have been found in similar ways by previous studies.

Below we will discuss our results beginning on the bottom of the tree, climbing upwards.

Delturinae

The monophyletic Delturinae is represented by all described species and was resolved as the most basal loricariid subfamily, corroborating earlier publications (e.g. Lehmann 2006;

Cramer et al. 2008; Pereira 2008; Cramer et al. this volume). This well supported group can easily be recognized by the combination of a high preadipose keel, formed by the azygous preadipose plates and almost symmetrically bifid jaw teeth (Reis et al. 2006). The lack of resolution within this clade might be due to an incomplete character sampling. Delturus carinotus is the only species where fresh tissue for molecular analyses was available, but at the same time, it is the only species where no specimens for morphological examinations

74 could be obtained. Morphologically, both genera can be easily distinguished: Delturus have a strong and massive body (vs. slender and elongate) and the dorsal-fin membrane is extended posteriorly, contacting the first preadipose plate (vs. not or slightly extended and never in contact with first preadipose plate) (Reis et al. 2006).

Loricariinae

Our results show the Loricariinae as a strongly supported monophyletic group. Since this subfamily was recovered as a natural group by all anterior studies (e.g. Schaefer 1987;

Rapp Py-Daniel 1997; Fichberg 2008), we included only a few species as representatives.

Members from this subfamily can be easily recognized by their long and flattened caudal peduncle and the absence of an adipose fin (Covain and Fisch-Muller 2007). Another particularity of this group is the diversity in lips structure, which can be strongly papillose, filamentous or smooth (Covain and Fisch-Muller 2007).

Hypostominae

Concordant with Cramer et al. (this volume), the Hypostominae is monophyletic, with the exception of Rhinelepis aspera being reallocated outside the subfamily, though no morphological data for Rhinelepis have been available. In spite of a high bootstrap value, the clade gets only low Bremer support. Hypostomines are typically bulkier than other loricariids and generally have thicker plates than neoplecostomines. They can be distinguished from other loricariids by the development of the spinelet that is large and V-shaped and clearly slides under the nuchal plate, whereas it is square or absent in most other loricariids and, when present, does not slide under the nuchal plate. The few hypoptopomatines that have a triangular spinelet can be distinguished from the Hypostominae by a completely or nearly completely exposed pectoral girdle (vs. at most some odontodes supported by the coracoid

75 strut) and an adductor fossa of the pectoral girdle covered by bone (vs. wholly exposed)

(Armbruster 2004).

Like already stated elsewhere (Ferraris 2007; Cramer et al. this volume), there seems to be no closer connection between Panaque and the genera Panaqolus and Scobinancistrus, contradicting the hypothesis of these genera being synonyms or subgenera (Armbruster 2004).

Several genera appeared as non-monophyletic, such as Hemiancistrus. But since the

Hypostominae is not the main focus of the present study, only few taxa were included and further relationships inside this subfamily should be analyzed in a future study with a more complete taxon sampling.

Our strict consensus resulted in a polytomy for the Loricariinae, the Hypostominae,

Rhinelepis, and the Neoplecostominae + Hypoptopomatinae. That way, we could not resolve the relationships between these groups.

Neoplecostominae

The Neoplecostominae and the Hypoptopomatinae were recovered as monophyletic sister groups. The Neoplecostominae, however, was found to be monophyletic only when including the hypoptopomatine genus Pseudotocinclus (Fig. 10b), getting weak support but corroborating the results of Cramer et al. (2008) and Chiachio et al. (2008). In the last years there has been some confusion about the definition and the composition of the

Neoplecostominae. Based on morphological data, Armbruster (2004) recovered the

Neoplecostominae as monophyletic enclosing the Hypoptopomatinae, Lehmann (2006) revealed this subfamily as paraphyletic, and Pereira (2008) showed it to be monophyletic, but excluding the genera Kronichthys and Pareiorhina that were recovered as one clade sister taxon to the Hypoptopomatinae; though the latter study did not include Pseudotocinclus.

Based on DNA sequence data, Chiachio et al. (2008) and Cramer et al. (2008) found a monophyletic Neoplecostominae, including Pseudotocinclus, but these studies did not include 76 any representatives of or Kronichthys or Isbrueckerichthys. Cramer et al. (this volume), including all genera, came to a similar result, but unexpectedly excluding the species

Pareiorhaphis garbei from the subfamily.

To justify the inclusion of the genus Pseudotocinclus in a monophyletic

Neoplecostominae, Chiachio et al. (2008) found three non-exclusive morphological characters for this clade (dorsally positioned eyes, exposed preopercle, and incomplete fusion of the anterior abdominal dermal bony plates). Until a more complete morphological study (in preparation by EHLP) is available, we adopt these diagnostic characters here.

Inside the Neoplecostominae, as the most basal clade, we found Pseudotocinclus to be a sister taxon of part of Pareiorhina, though the latter was revealed as polyphyletic (see also

Cramer et al. this volume). Such polyphyly might be reverted by the inclusion of morphologic data of Pareiorhina sp. (in preparation by EHLP), not available so far. An alternative topology with an enforced monophyly of Pareiorhina resulted in 49 additional steps and maintained Pareiorhina and Pseudotocinclus as sister taxa. This constrained topology was statistically refused by the KH test (p=0. 0012).

To discard the possibility of a misidentification, we examined the voucher and other available specimens of Pareiorhina sp. and found them to share the non-exclusive synapomorphies (Pereira 2008) of Pareiorhina. The remaining species of Pareiorhina were recovered as a strongly supported monophyletic group. Future analyses (in preparation by

EHLP) will bring more light into this question.

The major clade of the Neoplecostominae, containing all taxa but Pseudotocinclus and

Pareiorhina sp., got high Bremer support.

Kronichthys was recovered as monophyletic, being sister to the new genus 1. Lehmann

(2006) already found them to be related in some proximity, but not as sister clades.

77 The new genera 2 and 3 form a monophyletic group consisting of one large polytomy.

Probably, that was caused by the character sampling as molecular data were only available for one species of the new genus 2 and no morphological data were available for the new genus 3.

Concordant with Pereira (2008), the genera Isbrueckerichthys and Neoplecostomus are sister taxa. Both were recovered as well supported monophyletic groups, contrary to Cramer et al. (this volume), who found Neoplecostomus ribeirensis inside the genus

Isbrueckerichthys, but without any morphological evidence for this finding.

Forming the most derived clade, the genus Pareiorhaphis was recovered as monophyletic. Cramer et al. (this volume) were the only ones who revealed it as polyphyletic, contrary to other studies that included more than only a few species (Cramer et al. 2008;

Pereira 2008). As described above, only few characters could be included for the two species

Pareiorhaphis cerosus and P. regani. For this reason, their placement inside the genus could not be resolved in the complete analyses, causing large polytomies. Therefore we decided to exclude these two species from the strict consensus in order to gain a better resolution

(Fig. 10b) and to discuss the results of the separate analyses of the genus including all species

(see Fig. 11).

78 55 Pareiorhaphis sp. 4 Rio Uruguay, rio Jacui Uruguay, rio Rio -- 1 Pareiorhaphis sp. 1 1 -- Pareiorhaphis parmula Iguaçu & rio 1 -- Pareiorhaphis vestigipinnis 1 -- Pareiorhaphis eurycephalus 66 1 Pareiorhaphis sp. 5 1 -- Pareiorhaphis sp. 6 1 93 Pareiorhaphis hystrix -- 1 Pareiorhaphis sp. 3 2

Pareiorhaphis azygolechis rivers coastal Southern R,S,suhr PR) southern SC, (RS, Pareiorhaphis cameronii -- -- Pareiorhaphis regani 1 2 Pareiorhaphis steindachneri 100 Pareiorhaphis splendens 5 -- Pareiorhaphis splendens -- 2 Pareiorhaphis nudula 99 1 10 99 Pareiorhaphis hypselurus

13 Pareiorhaphis stomias Northern coastal rivers(RJ,MG, ES, BA) & rio São Francisco São rio & BA) ES, -- Pareiorhaphis bahiana 1 -- Pareiorhaphis stephanus -- 1 Pareiorhaphis sp. C -- 2 Pareiorhaphis mutuca 1 -- Pareiorhaphis sp. Z 2 73 Pareiorhaphis nasuta -- 3 98 2 6 Pareiorhaphis sp. 2 97 Pareiorhaphis garbei -- 3 Pareiorhaphis garbei 2 Pareiorhaphis cerosus 94 Isbrueckerichthys alipionis 9 Neoplecostomus microps Kronichthys heylandi Pseudotocinclus tietensis

Fig. 11 Strict consensus of the three best trees (1628 steps; CI: 0.50) from the separate analysis of the

genus Pareiorhaphis, including P. cerosus and P. regani. Numbers above branches are values from

1000 bootstrap replicates, numbers below are Bremer support.

Pareiorhaphis cerosus came out as the most basal taxon. However its status is highly uncertain as this species is only known from the two type specimens without a type locality and only few external morphological characters could be obtained.

An interesting finding is that the remaining species of Pareiorhaphis clustered into about three geographically delimited clades (Fig. 11 and Fig. 12). The first group is from the northern coastal rivers (states Rio de Janeiro, Espirito Santo, and Bahia) and the headwaters of the rio São Francisco (Minas Gerais). Pareiorhaphis garbei is the most basal species with 79 known origin. It occurs in the rio Macaé and rio Macacu (Rio de Janeiro) and thus is also geographically separated from its congeners, even being more closely related to the monophyletic northern clade.

The second non-monophyletic group occurs in the southern coastal rivers (states Rio

Grande do Sul, Santa Catarina, and Paraná). Inside this group, the three species from the rivers Maquiné, Três Forquilhas, Mampituba, and Araranguá (Pareiorhaphis hypselurus, P. nudula, and P. stomias) form a clade. These rivers are considered as a region with high degree of endemism (Malabarba and Isaia 1992; Reis and Schaefer 1998), and recently were recognized as a distinct freshwater ecoregion in the WWF/TNC mapping project (Abell et al.

2008). Pareiorhaphis regani was described from the rio Curicuriari (rio Negro basin), but as the description is based on one single specimen and there are no other Neoplecostominae known from the whole Amazon region, this locality is highly doubtful. In spite of having longer odontodes than known from P. steindachneri, these two species might be synonyms.

But only the examination of the holotype may resolve this question.

The third geographical clade is formed by the species distributed in the basins that drain the southern Brazilian highlands westwards. Pareiorhaphis parmula occurs in the upper rio

Iguaçu, P. sp. 4 in the upper rio Jacui and P. hystrix is known from the upper reaches of both the rio Jacui and rio Uruguay. The remaining species are distributed in the upper rio Uruguay basin.

80 4

7

6

5

3

2 1

Northern coastal rivers & rio São Francisco Pareiorhaphis garbei Southern coastal rivers Rios Uruguay, Jacui & Iguaçu

Fig. 12 Map showing the distribution of the species from the genus Pareiorhaphis (without the species P.

cerosus and P. regani which do not have confirmed localities). 1 = Rio Jacui, 2 = rio Uruguai, 3 = rio

Iguaçu, 4 = rio São Francisco, 5 = rio Doce, 5 = rio Jequitinhonha, 6 = rio Contas.

Hypoptopomatinae

All morphological phylogenies of members of the Hypoptopomatinae (Schaefer 1991;

Schaefer 1997; Schaefer 1998; Armbruster 2004; Lehmann 2006; Pereira 2008) have recovered the subfamily as monophyletic. Contrastingly, none of the solely molecular analyses so far conducted (Montoya-Burgos et al. 1998; Chiachio et al. 2008; Cramer et al.

2008, this volume) was able to uncover a monophyletic Hypoptopomatinae. The molecular

81 analyses consistently find the Neoplecostominae amidst the Hypoptopomatinae (Figs. 6, 7, and 9). Our total evidence analysis, however, recovered the Hypoptopomatinae as a monophyletic group with moderately high Bremer support. A characteristic of this subfamily is the ventral surface of the pectoral fin skeleton covered by thin skin and usually bearing numerous odontodes, such that the bone appears to be exposed on the ventral surface

(Schaefer 2003).

Schaefer (1991) divided the subfamily into two purportedly monophyletic clades, the tribes Hypoptopomatini and Otothyrini. Our results confirm his findings for the former, concordant with some previous studies (based on morphology: Schaefer 1997, 1998;

Armbruster 2004; Lehmann 2006; based on molecular data: Chiachio et al. 2008). But we could not corroborate the monophyly of the Otothyrini, agreeing with Gauger and Buckup

(2005), Cramer et al. (2008), Lehmann (2006), Pereira (2008), and Cramer et al. (this volume) but contrary to Chiachio et al. (2008) and Schaefer (1997, 1998). Even with the Otothyrini being polyphyletic, here we continue using the denomination Hypoptopomatini to refer to this monophyletic clade.

Like already demonstrated by other studies (Gauger and Buckup 2005; Lehmann 2006;

Cramer et al. 2008; Cramer et al. this volume), we found Parotocinclus to be a highly paraphyletic group, albeit some monophyletic clades were recovered. For the strongly supported Parotocinclus jumbo and P. sp. 9 clade, a new genus is being described (PL and

RER, in preparation). The species from northeastern Brazil, with the exclusion of

Parotocinclus jumbo that is clearly different from this group, compose a well supported clade

(P. cearensis, P. cesarpintoi, P. haroldoi, P. spilosoma, P. spilurus, P. sp. 10, and P. sp. 14), likely representing a second new genus. Lehmann (2006) already suspected such a relationship but could not resolve them as monophyletic. Another geographical group is formed by the species from the Guyana Shield (Parotocinclus aripuanensis, P. britskii, P. collinsae, P. eppleyi, P. longirostris, P. polyochrus, and the undescribed species 6, 12, 13, and 82 15 – 19), as also found by Lehmann (2006) and Cramer et al. (this volume). These species differ clearly from its congeners in their smaller size, body shape, and coloration and likely comprise a third new genus.

The type species, Parotocinclus maculicauda, clustered with P. bidentatus, P. doceanus, the new species 1, 7, and 8, and P. maculicauda “Cubatão” (clade V, Fig. 10d), getting only low support. The latter probably is identical with the type species and was only separated because of some minor morphological differences. Unfortunately, we could not resolve which other species make part of this Parotocinclus sensu strictu, as there was a large polytomy on the base of this clade, containing P. jimi, P. minutus, P. planicauda, and some additional undescribed species. The inclusion of DNA sequence data for more species might resolve this problem.

Most unexpectedly, the “jumping” taxon Microlepidogaster sp. 1 was also recovered inside the clade V. This species was preliminary identified as a Microlepidogaster, but it is presently being described as a new genus (in preparation by Martins and Langeani, personal communication). The position of Parotocinclus cristatus (clade IV, Fig. 10d) could not be resolved as it is one of the jumping species that have been excluded from the consensus.

Another well supported clade is formed by Corumbataia cuestae and Gymnotocinclus, which were recovered as sister to a new genus, here entitled New taxon TT. This genus (under description by Buckup, Britto, and Reis) currently comprises six species. Four of them are new and two have recently been described as Hisonotus, albeit the authors already stated that there are uncertainties about this placement and that the genus Hisonotus is in need of a taxonomic revision (Britski and Garavello 2007). A seventh species, Hypoptopomatinae sp.

Surinam, probably the same species as shown in Le Bail et al. (2000: p. 262) and Schaefer

(1998: taxon 3), was found to belong to this group, but was excluded from the strict consensus. A lack of compatible data frustrated a precise grouping.

83 The next clade is formed by Otothyris and Schizolecis, getting high Bremer support, and being placed as sister to the Hypoptopomatini. This weakly supported position differs from all previous studies, although Lehmann (2006) came to a similar result, but including also the genera Corumbataia and Pseudotothyris.

Hypoptopomatini

Schaefer (1991) found this clade as monophyletic and described it as a new tribe, a result further corroborated by the present and most previous studies. The few studies that found a non-monophyletic Hypoptopomatini (Cramer et al. 2008; Cramer et al. this volume), obtained weak support for alternative resolutions. Schaefer (1991) gives the following characteristics for this tribe: Derived absence of a levator arcus palatini crest on the hyomandibula, reduced levator muscle, and presence of a few, relatively large plates at the anterior snout margin.

Our topology is very similar to Schaefer’s (1991), recovering Otocinclus and

Acestridium as monophyletic genera and revealing Niobichthys as sister to all hypoptopomatin genera but Otocinclus. On the other hand, we recovered the genus Hypoptopoma as polyphyletic, concordant with Chiachio et al. (2008) and Cramer et al. (this volume). Since the results of Chiachio et al. (2008) placed Oxyropsis as a separate clade, those authors tried to resolve the paraphyly of Hypoptopoma treating Nannoptopoma as a synonym of

Hypoptopoma, suggesting that the former is a neotenic form of the latter. However, as they included only few species of both genera in their analysis, it is no surprise that our inclusion of more taxa resulted in different topologies, an effect already described by Schaefer (1998), coincidently using hypoptopomatine taxa as examples. In their revision of Oxyropsis, Aquino and Schaefer (2002) considered the genus as valid and well defined. As we could not include the type species Hypoptopoma thoracatum, we prefer to maintain Nannoptopoma and

Oxyropsis as distinct monophyletic genera and to postpone the decision whether to split 84 Hypoptopoma or to synonymize the other two genera till a more complete dataset is available.

The taxonomic status of the possible new genus also could not be clarified as its two species were placed into a polytomy with part of the species of Hypoptopoma.

Isbrücker and Seidel (in Isbrücker et al. 2001) described the genus Macrotocinclus for the species Otocinclus affinis and O. flexilis, repeating basically Schaefer’s definition of the

Otocinclus affinis complex (Schaefer 1997). Based on our results (Lehmann 2006; Lehmann et al. submitted; Cramer et al. this volume; this study), Otocinclus is a strongly supported clade and there is no need for a split in two genera. Moreover, the recognition of

Macrotocinclus as defined by its authors would turn Otocinclus paraphyletic, as O. xakriaba,

O. mimulus, and O. arnoldi are more closely related to O. affinis and O. flexilis than to the remaining species of Otocinclus (Lehmann et al in press). Therefore, we consider

Macrotocinclus as a synonym of Otocinclus.

Going on with the remaining taxa of the Otothyrini (sensu Schaefer 1991), Hisonotus insperatus is placed in a larger polytomy, far from other congeners. This is not unexpected, because, even considering the necessity of a taxonomic revision of Hisonotus (Britski and

Garavello 2007), this species can be clearly distinguished from the remaining species (e.g. having one internasal plate [vs. three] that does not contact the rostral plate [vs. in contact]; predorsal plates fused and unique [vs. not fused and paired] [Lehmann 2006]) and is more akin to Parotocinclus.

Our clade IV received very high Bremer support and might serve as a base for a future description of a new subfamily. Inside clade IV, besides the bulk of species of Parotocinclus, treated above, the next monophyletic group comprises Microlepidogaster perforatus, M. sp.

“AP”, and Pseudotocinclus sp. “PP”. The latter was preliminary assigned to the genus

Pseudotocinclus by Lehmann (2006), because his results showed it as sister to 85 Pseudotocinclus tietensis. At present, the genus Microlepidogaster is being revised (in preparation by B. Calegari, personal communication) and the species “PP” is likely to make part of it (Calegari, personal communication). Respecting the ongoing revision and the low support of the clade, we do not make changes in this species generic assignment.

A heterogeneously composed clade is formed by Pseudotothyris obtusa, two undescribed species of Otothyropsis, Microlepidogaster sp. 2, Hisonotus francirochai, and

Hisonotus sp. 4. As already postulated by its authors (Ribeiro et al. 2005), Otothyropsis, which is currently being revised (in preparation by B. Calegari, personal communication), and

Pseudotothyris were found as closely related. Based on this grouping, we reexamined the voucher specimens of Microlepidogaster sp. 2 and Hisonotus francirochai. Both species appear to be more closely related to Otothyropsis than to the assigned genera (Calegari, personal communication). For example they share a crest with odontodes on the tip of the supraoccipital plate. Unfortunately, the voucher of Hisonotus sp. 4 was not available, but from its collecting locality in the rio Alambari in the rio Tietê basin, it might be a Otothyropsis, as the type species was described from this basin.

Concordant with earlier studies (Britski and Garavello 2007; Cramer et al. 2008;

Cramer et al. this volume), the genus Hisonotus was revealed as polyphyletic, comprising at least two clades, each with moderate support. However, we could not find any characters to distinguish the monophyletic groups. Even the species from the rio Uruguai basin (Carvalho and Reis 2009) could not be joined in one group (Hisonotus sp. 1 occurs in the rio Jacui basin;

Hisonotus charrua is distributed in the rio Uruguai basin, but was placed in the other clade, separated with high decay index). Additionally, Lampiella gibbosa was found as sister to

Hisonotus leucofrenatus. Currently, the status of Lampiella is uncertain. The species was first described as Otocinclus, and Chiachio et al. (2008) found it to be sister to that genus.

Lehmann (2006) showed it as more proximally related to Hisonotus than to Otocinclus, and our earlier molecular results resolved it as sister to either Otocinclus or Hisonotus 86 leucofrenatus (Cramer et al. this volume). Based on the lack of strong evidence for a taxonomic change, we maintain its generic status as separated from Otocinclus and Hisonotus.

The last clade of our consensus tree is composed by Epactionotus and Eurycheilichthys as sister taxa in a highly supported group. Both genera are clearly monophyletic, concordant with Lehmann (2006) and Cramer et al. (2008, this volume). Remarkably, based purely on morphological evidence, Lehmann (2006) recovered Eurycheilichthys as the most basal taxon in the Hypoptopomatinae, and not closely related to Epactionotus, contrary to the results of

Chiachio et al. (2008), Cramer et al. (2008; this volume) and the ones presented here. These two genera occur in small rivers draining the southern portion of the Brazilian Shield. A common pattern for the distribution of fishes is that basal taxa are distributed on the highlands, such as the Brazilian and the Guyana shields, and the more derived taxa are spread in the lowlands, such as the Amazon and the Orinoco basins. This pattern is caused by the older age of the shields and repeated incursions of marine water in the lower areas till the late

Miocene (Lundberg et al. 1998; Hulka et al. 2006), inhibiting the life of strict freshwater inhabitants. That way, diversification began on the shields and only later, the lowlands were invaded. Among others, Ribeiro et al. (2005) showed this for the genera Creagrutus and

Piabina, and Menezes et al. (2008) for the Glandulocaudinae, but there are several examples for loricariids, as well. The two most basal subfamilies, Lithogeninae and Delturinae, are solely distributed on the Guyana and the Brazilian shield, respectively (Reis et al. 2006;

Schaefer and Provenzano 2008). Inside the Loricariinae, Harttia is one of the most basal taxa

(Rapp Py-Daniel 1997), with its species occurring mostly on the Guyana and the Brazilian shields (Covain et al. 2006), whereas Pseudohemiodon as one of the most derived loricariine taxa is mainly distributed in the Amazon lowlands. The same is true for the Hypostominae, with the members from the most basal tribe Corymbophanini (Armbruster 2004) exclusively dispersed on the Guyana shield and the most derived tribe Ancistrini having its highest diversity in the Amazon region. 87 Further on this biogeographic evidence, morphology within the Hypoptopomatinae is highly variable and is also in disagreement with the phylogenetic findings. For instance,

Eurycheilichthys and Epactionotus are very generalized in their morphology, being hardly distinguishable from neoplecostomines or basal hypostomines. On the other hand, genera like

Hypoptopoma, Nannoptopoma, Oxyropsis, and especially Acestridium are highly modified morphologically from the general pattern of basal loricariids with some bizarre body shapes and other specializations.

The entire Neoplecostominae is restricted to the Brazilian shield. Therefore it is a somewhat unexpected finding that the Hypoptopomatini with species exclusively from the lowlands was recovered as one of the most basal clades inside the Hypoptopomatinae, and

Epactionotus + Eurycheilichthys, both from the highlands, were recovered as the most derived taxa.

Comparing the results from solely molecular or morphologic analyses with the ones obtained from the total evidence approach, several advantages of the latter were found. Other studies on the phylogeny of fishes already have come to the same conclusion, but only used few species (e.g. Betancur-R et al 2007; Pretti et al. 2009). With the present study probably being the largest total evidence analysis conducted for fishes so far, we would like to highlight the importance and the benefits of this method.

Results from solely molecular or morphological data have not been able to fully resolve the loricariid phylogeny, and different studies have come to conflicting results, even using the same type of data, whereas our total evidence phylogeny seems to be the best approach. Our results combine resolutions from previous analyses, resolving most suprageneric groups as monophyletic. The Neoplecostominae and the Hypoptopomatinae are groups where molecular and morphological studies came to highly controversial results. Our total evidence solution shows both as separate monophyletic clades, including Pareiorhina and Kronichthys in the

88 Neoplecostominae, as already proposed by Armbruster (2004), but with the addition of the genus Pseudotocinclus.

Doubts about the influence of the different quantities of molecular and morphological characters apparently are without fundament, as our matrix comprised above two times more parsimony informative molecular characters than morphologic ones (1089 vs. 472).

Nevertheless, the comparison of the different results shows that morphological characters have a strong influence on the result of the total evidence analysis. Solely molecular data did not recover the monophyly of the Hypoptopomatinae or the genera Neoplecostomus,

Isbrueckerichthys, and Pareiorhaphis, whereas they become monophyletic using the combined data set. Another finding that only the jointed data revealed, is the biogeographic pattern found for the genus Pareiorhaphis. Even groups with relatively scarce molecular data, such as the Parotocinclus from northeastern Brazil, benefited from the total evidence approach as morphology alone did not recover its monophyly.

Summarizing, we show that a total evidence approach is the most adequate method to explore the loricariid phylogeny since our combined dataset was able to resolve several of the known problems for the subfamilies Hypoptopomatinae and Neoplecostominae. The

Neoplecostominae was resolved as monophyletic, including Pseudotocinclus, as well as

Kronichthys and Pareiorhina that had been considered as hypoptopomatines by Pereira

(2008). Most of its genera were found to be monophyletic, except for Pareiorhina that requires additional morphological data. Both, the Hypoptopomatinae and the

Hypoptopomatini were also recovered as monophyletic. Conversely, the Otothyrini was found to be a polyphyletic taxon, corroborating other recent studies (Armbruster 2004; Lehmann

2006; Cramer et al. 2008; Pereira 2008; Cramer et al. this volume). Because of a larger polytomy in our consensus tree, we still cannot offer an approach for a split into well defined

89 monophyletic groups, though our clade IV is a strong candidate for a future suprageneric taxon.

The polyphyly of Parotocinclus could be partly resolved as we found three monophyletic groups that represent undescribed genera. Nevertheless, the species around P. maculicauda (the Parotocinclus sensu strictu) remain unresolved.

The status of the “Otothyrini” and the genera Hisonotus, Hypoptopoma, and

Parotocinclus should be resolved by a further improvement of the character as well as the taxon sampling. Our results suggest that filling the gaps in our matrix (especially for the jumping taxa from Table 3), as well as the inclusion of additional (e.g. currently still only half of the species of Hisonotus are represented) and especially some critical taxa (e.g.

Hypoptopoma thoracatum) will amend our understanding of the phylogeny of the remaining polyphyletic taxa.

ACKNOWLEDGMENTS:

Thanks are due do to Taran Grant and Pablo Goloboff who helped us patiently with questions about phylogenetic methods. Bárbara Calegari helped us with the re-identification of some hypoptopomatine taxa and Vivianne Sant’Anna gave several valuable technical advices. Financial support: Brazilian National Counsel of Technological and Scientific

Development (CNPq) and German Academic Exchange Service (DAAD).

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96

Appendix 1 Species, depository information and GenBank accession numbers for the additional COI

sequences from Cramer et al. (2008) used in this study.

Eurycheilichthys sp. 2 MCP 22374 EU370992. Eurycheilichthys sp. 2 MCP 22800

EU370994. Eurycheilichthys sp. 3 MCP 35049 EU370999. Eurycheilichthys sp. 4 MCP

22199 EU370991. Eurycheilichthys sp. 7 MCP 35071 EU370997.

97 Conclusões Gerais

No presente estudo foram elaboradas filogenias baseadas em sequências de DNA e de evidência total, incluindo dados morfológicos de estudos prévios. A análise de evidência total resultou na maior filogenia de bagres feita até o momento. Estes resultados foram comparados com as filogenias baseadas exclusivamente em dados morfológicos. Usando somente dados moleculares, de um ou de múltiplos genes, vários grupos foram encontrados como não-monofiléticos, tais como a subfamília Hypoptopomatinae e os gêneros Pareiorhina, Pareiorhaphis, Neoplecostomus, Hisonotus e Parotocinclus. Ao contrário dos estudos morfológicos, o gênero Pseudotocinclus sempre foi revelado como membro da subfamília Neoplecostominae, e não de Hypoptopomatinae, corroborando publicações anteriores. Além disto, os gêneros Kronichthys e Pareiorhina são encontrados dentro de Neoplecostominae, confirmando a classificação de Armbruster (2004), mas contrariando Lehmann (2006) e Pereira (2008). Com o objetivo de resolver estes conflitos entre os resultados de análises usando somente dados moleculares ou morfológicos, optou-se por fazer uma análise de evidência total. Os resultados deste conjunto de dados, incluindo 472 caracteres morfológicos e 2634 pares de bases de três genes para 207 espécies, conseguiram resolver vários dos conflitos conhecidos. As subfamílias Hypoptopomatinae e Neoplecostominae se apresentaram como monofiléticas, assim como a tribo Hypoptopomatini e todos os gêneros de Neoplecostominae, exceto Pareiorhina. Oito espécies foram excluídas do consenso estrito, porque elas agruparam com várias espécies diferentes em árvores diferentes, resultando em grandes politomias. A escassez de dados disponíveis para estes táxons provavelmente foi responsável por isto. Depois de terem sido retirados do consenso, a maioria destas politomias foi resolvida. Sem a possibilidade de incluir membros de Lithogeninae, a subfamília Delturinae foi encontrada como o clado mais basal da Loricariidae, corroborando Armbruster (2004) e Reis et al. (2006). Loricariinae foi encontrada monofilética, ficando em uma politomia junto com Hypostominae e Neoplecostominae + Hypoptopomatinae. Nas análises moleculares do capítulo II, Rhinelepis aspera foi encontrada fora de Hypostominae, contrariando Armbruster (2004). Neoplecostominae e Hypoptopomatinae foram recuperadas como grupos-irmãos, a primeira incluindo o gênero Pseudotocinclus. Dentro de Neoplecostominae, todos os gêneros exceto Pareiorhina formam grupos naturais. Provavelmente, a polifilia de

98 Pareiorhina é causada por falta de dados morfológicos para uma espécie. O resultado de uma análise forçando Pareiorhina como monofilético foi refutado como sendo estatisticamente pior. Concordando com todas as análises moleculares, Pseudotocinclus é mais proximamente relacionado à Pareiorhina. A análise de evidência total encontrou uma filogenia com um padrão biogeográfico antes desconhecido para o gênero Pareiorhaphis. Foram formados três grupos geográficos: dos rios costeiros do sul (RS, SC e PR), das cabeceiras dos rios drenando para o oeste (rios Uruguai, Jacui e Iguaçu) e dos rios costeiros no norte (RJ, ES, BA) + rio São Francisco (MG). Contrariando estudos exclusivamente moleculares, mas confirmando resultados morfológicos, a subfamília Hypoptopomatinae foi recuperada como monofilética. Entretanto, das duas tribos descritas por Schaefer (1991), somente Hypoptopomatini forma um grupo natural, deixando Otothyrini polifilética. Os gêneros Nannoptopoma e Oxyropsis dividem as espécies do gênero Hypoptopoma em dois grupos. Chiachio et al. (2008) encontraram uma situação parecida e sinonimizaram Nannoptopoma com Hypoptopoma, alegando neotenia. À base dos presentes resultados, seria necessária a sinonimização adicional de Oxyropsis para tornar Hypoptopoma monofilético. Como não foi possível incluir a espécie tipo, Hypoptopoma thoracatum, optou-se pela manutenção de Nannoptopoma até que uma análise mais completa possa oferecer uma solução mais bem suportada. Confirmando outros estudos (Gauger e Buckup, 2005; Lehmann, 2006; Britski e Garavello, 2007), os gêneros Hisonotus e Parotocinclus foram encontrados como polifiléticos. Porém, três grupos monofiléticos puderam ser encontrados no último: um clado com duas espécies do sul e do nordeste do Brasil, um clado da Amazônia e um clado do nordeste brasileiro. Mas, mesmo assim, as espécies em volta da espécie tipo permaneceram em uma politomia, provavelmente causada por falta de dados moleculares. Além das espécies que claramente não pertencem ao gênero Hisonotus, por exemplo, H. francirochai e H. insperatus, as espécies formam dois grupos, portanto, poucos dados morfológicos foram disponíveis para estas espécies. Epactionotus e Eurycheilichthys formam os táxons mais derivados de Hypoptopomatinae, confirmando outros estudos exclusivamente moleculares, mas contrariando os resultados de Lehmann (2006). Isto é surpreendente porque contradiz um padrão geral na distribuição de peixes. Exemplos para vários grupos mostram que, geralmente, os táxons mais basais têm a sua distribuição nos escudos e os grupos mais

99 derivados são encontrados nas áreas baixas do continente, como a bacia do Amazonas ou do Orinoco. Mesmo dentro da Loricariidae este padrão é comum: as duas subfamílias mais basais, Lithogeninae e Delturinae, bem como os táxons mais basais de Loricariinae e de Hypostominae, são dos escudos; portanto, os táxons mais derivados das duas últimas têm sua maior diversidade nas áreas baixas. Desta forma, com Hypoptopomatini distribuída principalmente na Amazônia, mas sendo um táxon basal dentro da subfamília, e Epactionotus + Eurycheilichthys sendo recuperados como mais derivados, mas ocorrendo no escudo brasileiro, Hypoptopomatinae não se enquadra neste padrão mais geral. O uso da evidência total resolveu vários conflitos das filogenias prévias e parece ser a melhor estratégia para acessar as relações dentro de Loricariidae, mesmo não tendo todos os tipos de dados para todos os táxons. Mesmo usando quase quatro vezes mais dados moleculares, a morfologia tem forte influência na topologia. Os dois tipos de dados contribuíram nas resoluções encontradas e, juntos, revelaram grupos antes não resolvidos (por exemplo, os Parotocinclus do nordeste) e o padrão biogeográfico do gênero Pareiorhaphis. Um futuro estudo deveria usar este método, incluindo as informações ausentes para os grupos que ainda não foram bem resolvidos, especialmente para estes onde principalmente apenas um tipo de dados está disponível (por exemplo, Hypostominae, Hisonotus e Parotocinclus). Além disto, algumas espécies importantes deverão ser acrescentadas. Por enquanto, por exemplo, não foi possível incluir a espécie- tipo de Hypoptopoma, o que impediu uma decisão sobre o futuro deste gênero, e somente metade das espécies de Hisonotus estavam disponíveis, impossibilitando uma conclusão final sobre este grupo parafilético.

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