Delimitação De Espécies E Padrões De Distribuição De Neoplecostomini (Regan, 1904) Na Bacia Do Alto Paraíba

Universidade Federal do Rio de Janeiro Instituto de Biologia Programa de Pós-Graduação em Biodiversidade e Biologia Evolutiva

Delimitação de espécies e padrões de distribuição de Neoplecostomini (Regan, 1904) na bacia do alto Paraíba

do Sul (Siluriformes: Loricariidae)

Beatrizz Oliveira Mesquita

Rio de Janeiro, 2020

Delimitação de espécies e padrões de distribuição de Neoplecostomini (Regan, 1904) na bacia do alto Paraíba do Sul (Siluriformes: Loricariidae)

Beatrizz Oliveira Mesquita

Tese a ser apresentada ao Programa de Pós-Graduação em Biodiversidade e Biologia Evolutiva, do Instituto de Biologia, da Universidade federal do Rio de Janeiro, como parte dos requisitos necessários à obtenção do título de Mestre em Biodiversidade e Biologia Evolutiva.

Orientador: Wilson José Eduardo Moreira da Costa

Co-orientador: José Leonardo de Oliveira Mattos

Banca examinadora:

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Prof. Dr. (Presidente)

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Prof. Dr.

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Prof. Dr.

Rio de Janeiro

VII/2020

Dissertação realizada no Laboratório de Sistemática e Evolução de Peixes Teleósteos, Instituto de

Biologia, Universidade Federal do Rio de

Janeiro

Orientador: Dr.

Wilson José Eduardo Moreira da Costa

(Laboratório de Sistemática e Evolução de

Peixes Teleósteos, Departamento de

Zoologia, Instituto de Biologia, Universidade

Federal do Rio de Janeiro)

“But where shall wisdom be found? And where is the place of understanding? Men knoweth not the price thereof; neither is it found in the land of the living... For the price of wisdom is above rubies” -Neil Gaiman

A Alex Schomaker Bastos

Agradecimentos

Agradeço ao Prof. Wilson Costa, pela orientação e oportunidade para a realização desse projeto. Pelas viagens, amizade, conversas e discussões sobre biologia e sobre a vida.

Ao programa de Pós-Graduação em Biodiversidade e Biologia Evolutiva e seus professores associados, pelo conhecimento passado, e pela oportunidade de realização do mestrado na instituição.

A José Leonardo Mattos, pela co-orientação, sempre muito disponível para todas as minhas dúvidas, com muita paciência e cuidado, além de todas as ajudas com análises. Ao Pedro Amorim, quase um segundo co-orientador, sempre muito companheiro e pró-ativo, a me ajudar de diversas formas com o projeto; de coletas a análises. Ao Axel Katz por me ajudar com dicas e fotos, especialmente na parte inicial do projeto. Ao Paulo Vilardo, por sempre me repassar as informações referentes à pós. Esse projeto não teria sido possível sem a ajuda e apoio dessas pessoas.

Ao Laboratório de Sistemática e Evolução de Peixes Teleósteos, pelo ambiente de trabalho e por ter sido uma segunda casa nos últimos anos. A todos os seus membros, pelo acolhimento, ajuda, boas risadas, discussões biológicas, carinho, companheirismo e por terem se tornado uma segunda família para mim: Wilson Costa, Maria Anais Barbosa, José Leonardo Mattos, Felipe Ottoni, Pedro Fasura, Pedro Bragança, Axel Katz, Filipe Rangel-Pereira, Elisabeth Henschel, Paulo Vilardo, Orlando da Conceição e Gilvan Joaquim.

À minha família, por todo carinho e suporte ao longo desses anos, sem os quais esse projeto não teria sido possível: Luciana Oliveira Mesquita, Valder Freire Mesquita, Bruna Oliveira Mesquita e Regina Oliveira e Lucílio Garcia.

A todos os meus amigos que alegraram os meus dias mesmo em dias difíceis, em especial Maria Isabela Caro, Gabriela Leonardo, Tainá Lima, Marcos Malagris, Paula Fragoso, Maísa Furtado, Marllon Barbosa, Hugo Bittencourt, Carolina Matsutani, Rodrigo Duarte, Maria Antônia Magalhães, Francyllia Santana, Carlos Valpaços, Vítor Maciel, Aline Carvalho, Mariana Szrabman, Vítor Santana, Brenda Rodrigues, Luiz Botelho, Matheus Ferradaz, Luisa Costa, Patrícia Ribeiro, Rafael Jordão, Julia São Thiago, Ana Galvão, Julio Duarte, Jhana Monteiro, Anna Salvatori, Everthon Felipe, Emanuel Neuhaus, Natasha Raadsack, Hugo Dolsa, Suellen Nunes, Renata Netto e Maurício Muniz.

À Monique Judice, por me ensinar a lidar melhor com a ansiedade.

Esse projeto foi financiado pelo Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) e pela Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Delimitação de espécies e padrões de distribuição de Neoplecostomini (Regan, 1904) na bacia do alto Paraíba do Sul (Siluriformes: Loricariidae)

Beatrizz Oliveira Mesquita

Resumo

Neoplecostomini é uma tribo de peixes de pequeno a médio porte, habitando rios montanhosos do sudeste e sul do Brasil, pertencendo à família Loricariidae, popularmente conhecidos como “cascudos”. Dos gêneros pertencentes à essa tribo, Neoplecostomus e Pareiorhina estão presentes na bacia do Alto Paraíba do Sul, uma das bacias de maior importância da região e que, apesar de ser fonte de abastecimento doméstico e industrial em uma área densamente populosa, também possui níveis altos de degradação. O objetivo desse estudo é estimar a diversidade de espécies de Neoplecostomi presentes na bacia, utilizando para tal métodos de coalescência, além de determinar a distribuição geográfica das espécies delimitadadas. Foram utilizados dois métodos para tal, o bPTP e o GMYC, a partir de sequências geradas do gene Citrocromo oxidase 1. Na análise de bPTP de Neoplecostomus, quatro espécies foram delimitadas para o Alto Paraíba do Sul: A espécie tipo, N. microps, presente em diversas drenagens, N. sp1, do médio curso do Rio Preto, N. sp2, do alto curso do Rio Preto e N. sp3, de Pindamonhangaba. Segundo a análise de GMYC, existiria apenas uma espécie na bacia do Alto Paraíba do Sul, N. microps. Nas análises de Pareiorhina, o gênero não foi recuperado como sendo monofilético, possuindo dois grupos distintos, um de espécies relacionadas à espécie tipo, P. rudolphi, e outras espécies relacionadas à Neoplecostomus, tratadas no presente estudo como ‘Pareiorhina’. A análise de bPTP recuperou quatro espécies de Pareiorhina, a própria espécie tipo, P. rudolphi, de Piquete, P. sp1, do Rio Salto, P. sp2, do Alto Rio Preto e P. sp3, de São José dos Campos; enquanto recuperou quatro espécies de ‘Pareiorhina’: ‘Pareiorhina’ hyptiorhachys, do Médio Rio Preto, ‘Pareiorhina’ brachyrhyncha, de Pindamonhangaba, ‘Pareiorhina’ sp4, de Itatiaia e ‘Pareiorhina’ sp5, de Resende. A análise de GMYC apresentou o mesmo resultado, exceto por ‘Pareiorhina’ sp4 e ‘Pareiorhina’ sp5, que apareceram como uma única espécie, assim como Pareiorhina rudolphi e P. sp1 apareceram também como uma única espécie. Os resultados corroboram um alto grau de endemismo para a bacia do Alto Paraíba do Sul, possuindo toda a diversidade de Pareiorhina e metade da diversidade de ‘Pareiorhina’. Os resultados de GMYC de Neoplecostomus associado aos baixos valores de suporte de bPTP

podem ser indício da ocorrência de apenas uma espécie para essa bacia, N. microps o que indicaria uma alta vagilidade desse gênero, o qual rios largos como o Paraíba do Sul não configurariam uma barreira geográfica, como é o caso dos outros dois gêneros analisados.

Palavras chave: Neoplecostomini, Paraíba do Sul, delimitação de espécies

Abstract

Neoplecostomini is a tribe of small to medium sized fishes, inhabiting mountainous river from South and Southeast of Brazil, belonging to the Loricariidae family, popularly known as armored catfishes. On the Neoplecostomini, the genera Neoplecostomus and Pareiorhina occur in the Upper Paraíba do Sul basin, one of the most important in the region, which is also highly degraded, although being the main source of industrial and domestic water usage. The goal of this study is to estimate the species diversity of Neoplecostomini with occurrence on this basin, utilizing coalescent methods for such, as well as determining the geographic distribution of the species found. BPTP and GMYC analyses were conducted, using the gene Cytochrome Oxidase 1. On the bTP Neoplecostomus analysis, four species were delimited for the Upper Paraíba do Sul basin: the type species, N. microps, occurring in multiple drainages, N. sp1 from the Medium Preto river, N. sp2, from the Upper Rio Preto river and N. sp3, from Pindamonhangaba. According to the GMYC analysis there is only one species from the Upper Paraíba do Sul basin, N. microps. The Pareiorhina analyses did not recover the genus as being monophyletic, with two distinct groups, one of species related to the type-species, P. rudolphi, and the other with species related to Neoplecostomus, referred to in this study as ‘Pareiorhina’. The bPTP analysis recovered four Pareiorhina species, the type species itself, P. rudolphi, from Piquete, P. sp1 from the Rio Salto river, P. sp2 from the Upper Rio Preto river and P. sp3, from São José dos Campos; while it recovered four ‘Pareiorhina’ species: ‘Pareiorhina’ hyptiorhachis, from the Medium Rio Preto river, ‘Pareiorhina’ brachyrhyncha, from Pindamonhangaba, ‘Pareiorhina’ sp4, from Itatiaia and ‘Pareiorhina’ sp5, from Resende. The GMYC analysis had the same the result, except for ‘Pareiorhina’ sp4 and ‘Pareiorhina’ sp5, which appeared as a single species, as did Pareiorhina rudolphi and P.sp1. The results corroborated a high degree of endemism for the Upper Paraíba do Sul basin, possessing all of the diversity of Pareiorhina and half of the diversity of ‘Pareiorhina’. The GMYC Neoplecostomus results associated with the low bPTP supports could indicate the occurrence of a single species to this basin, N. microps, which would indicate a high vagility

of this genus, of which wide rivers like Paraíba do Sul river would not be a geographic barrier, like is the case for the other two genera analyzed.

Key words: Neoplecostomini, Paraíba do Sul basin, species delimitation

Sumário

Agradecimentos...... 6

Resumo...... 8

Abstract...... 9

Lista de imagens...... 12

Lista de informações suplementares...... 13

Lista de abreviaturas e siglas...... 14

Capítulo 1- Species delimitation of Neoplecostomini (Siluriformes: Loricariidae) from Upper Paraíba do Sul basin using coalescent methods...... 15

Abstract...... 16

Introduction...... 16

Materials and Methods...... 24

Results...... 26

Discussion...... 29

Acknowledgements...... 32

References...... 32

Appendix 1...... 40

Lista de Imagens

Figura

1- Resultados da delimitação de espécies de Neoplecostomus por bPTP (barra rosa) e GMYC (barra azul). Descontinuidades nas barras representam espécies diferentes.As drenagens do grupo focal estão evidenciadas. Suportes dos nós indicam a probabilidade posterior das espécies delimitadas por bPTP.

2- Distribuição geográfica de espécies de Neoplecostomus delimitadas por bPTP. Cada símbolo representa uma espécie.

3- Resultados da delimitação de espécies de Pareiorhina lato sensu por bPTP (barra rosa) e GMYC (barra azul). Descontinuidades nas barras representam espécies diferentes.As drenagens do grupo focal estão evidenciadas. Suportes dos nós indicam a probabilidade posterior das espécies delimitadas por bPTP.

4- Distribuição geográfica de espécies de Pareiorhina lato sensu delimitadas por bPTP. Cada símbolo representa uma espécie.

Lista de informações suplementares

Apêndice 1- Çista de espécies e seus respectivos números de catálogo, localidades, coordenadas e o número de acesso das sequências de COI do Genbank

Lista de Abreviaturas e Siglas

12S- Gene 12S ribossomal RNA

16S- Gene 16S ribossomal RNA

BPTP- método de delimitação de espécies Bayesian Poisson Tree Process

Capes- Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

CNPQ- Conselho Nacional de Desenvolvimento Científico e Tecnológico

COI- Gene mitocondrial Citocromo Oxidase 1

Cyt b- Gene mitocondrial Citocromo b

DNA- Ácido desoxiribonucleico

GMYC- método de delimitação de espécies General Mixed Yule Process

MCMC- Cadeias de Markov

MS-222- tricaina metanosulfonada

MyH6- gene Myosin Heavy Chain 6

PCR: Reação em cadeia da polimerase

RAG1- gene Recombination activating gene 1

RAG2- gene Recombination activating gene 2

RNA- Ácido ribonucleico rRNA- RNA ribossomal

UFRJ- Universidade Federal do Rio de Janeiro

-Capítulo 1-

Species delimitation of Neoplecostomini (Siluriformes: Loricariidae) from Upper Paraíba do Sul basin using coalescent methods

Beatrizz Oliveira Mesquita

Species delimitation of Neoplecostomini (Siluriformes: Loricariidae) from Upper Paraíba do Sul basin using coalescent methods

Abstract

Neoplecostomini is a tribe of small to medium sized fishes, inhabiting mountainous river from South and Southeast of Brazil, belonging to the Loricariidae family. The genera Neoplecostomus and Pareiorhina occur in the Upper Paraíba do Sul basin, one of the most important in the region. The goal of this study is to estimate the species diversity of Neoplecostomini on this basin, as well as determining the geographic distribution of the species found. BPTP and GMYC analyses were conducted, using the gene Cytochrome Oxidase 1. On the bTP Neoplecostomus analysis, four species were delimited: N. microps, N. sp1, N. sp2 and N. sp3. According to the GMYC analysis there is only one species from the Upper Paraíba do Sul basin, N. microps. The Pareiorhina analyses did not recover the genus as being monophyletic, with two distinct groups, one of species related to the type-species, and the other with species related to Neoplecostomus, referred to in this study as ‘Pareiorhina’. The bPTP analysis recovered four Pareiorhina species: P. rudolphi, P. sp1, P. sp2 and P. sp3; while it recovered four ‘Pareiorhina’ species: ‘Pareiorhina’ hyptiorhachis, ‘Pareiorhina’ brachyrhyncha, ‘Pareiorhina’ sp4 and ‘Pareiorhina’ sp5, from Resende. The GMYC analysis had the same the result, except for ‘Pareiorhina’ sp4 and ‘Pareiorhina’ sp5, which appeared as a single species, as did Pareiorhina rudolphi and P.sp1. The results corroborated a high degree of endemism for the Upper Paraíba do Sul basin. The Neoplecostomus analyses results could indicate the occurrence of a single species to this basin, N. microps, which would indicate a high vagility of this genus.

Key words: Neoplecostomini, Paraíba do Sul basin, species delimitation

Introduction

The Neotropical region has the most diverse fish fauna in the world, with over eight thousand species, representing a fourth of all fish species (Fricke et al., 2020; Reis, 2013; Reis et al., 2016). The order Siluriformes is among one of the most species-rich, popularly known as “catfishes”. Within this order, the superfamily Loricarioidea is a well-established monophyletic group, with a great importance in species number, containing the families Astroplepidae, Callichthydae, Loricariidae, Nematogenidae, Scoloplacidae and Trichomycteridae (de Pinna, 1992). Fishes of those families are characterized by having

odontodes, tooth-like structures outside of the oral cavity (de Pinna, 1998). The family Loricariidae, popularly known as “suckermouth armored catfishes”, is the most diverse, with 1004 valid species divided in six Subfamilies: Delturinae, Lithogeninae, Loricariinae, Hypoptopomatinae, Hypostominae and Rhinelepinae (Fricke et al., 2020). The subfamily Hypoptopomatinae sensu Roxo et al, 2019 is composed of three tribes, Hypoptopomatini, Otothyrini and Neoplecostomini. The Neoplecostomini are small to medium-sized fishes, inhabiting south-eastern and southern river basins of Brazil, being typically found in mountainous, swift-flowing streams (Armbruster, 2004; Langeani, 1990), being the focus of this study.

Eigenmann & Eigenmann (1890), in their work on South American catfishes, were the first to propose a subfamily division of the Loricariidae, in which they recognized three subfamilies: Loricariinae, Hypoptopomatinae and Plecostominae. Neoplecostomus, the only genus described then of Neoplecostomini, was allocated to the Plecostominae subfamily. Regan (1904) in his monograph on catfishes, first created the subfamily Neoplecostominae, allocating the single genus Neoplecostomus into it, as well as recognizing the other subfamilies Loricariinae, Hypoptopomatinae and Argiinae, the last currently belonging to a family of its own, Astroblepidae. Gosline (1945) conducted a revision of the Loricariidae, recognizing four subfamilies: Plecostominae, Hypoptopomatinae, Loricariinae and Neoplecostominae, with the last family remaining monogeneric and, including only Neoplecostomus. The author then recognized Astroblepidae (Regan’s 1904 Argiinae) as a family of its own. Later on, Gosline (1947) made an extensive revision of the Loricariidae, and separated the more than 400 described species into the previous recognized subfamilies Astroblepinae, which went back to being recognized as a Loricariidae subfamily, Hypoptopomatinae, Loricariinae, Plecostominae and Neoplecostominae; as well as describing a new subfamily, Lithogeninae, with a somewhat intermediate species between Astroblepinae and the other subfamilies of Loricariidae. He also proposed changes in the composition on the Neoplecostominae subfamily, of which besides the Neoplecostomus genus being included, the inclusion of the genera: Upsilodus, Hemipsilichthys, Pareiorhaphis, Kronichthys, Delturus, Corymbophanes, Canthopomus, Pogonopoma, Pogonopomoides, Rhinelepis and Pareiorhina, this last being described in this author’s study. Over thirty years after Gosline’s (1947) classification paper, Isbrücker (1980) allocated the approximately 600 species and 70 genera by then into 6 subfamilies: Lithogeneinae, Loricariinae, Hypostominae, Ancistrinae, Hypoptopomatinae and Neoplecostominae,

although the author did not provide characters to justify his classification. He also removed all the genera except Neoplecostomus from Neoplecostominae, retaining Regan’s (1904) concept of it being a monogeneric subfamily. Howes (1983) was the first author to apply phylogenetic methods to the Loricariidae taxonomic classification. Using cranial muscles as characters, he divided the family into 5 subfamilies: Chaetostominae, Loricariinae, Hypostominae, Hypoptopomatinae and Neoplecostominae. No specimens belonging to the previously recognized subfamily Lithogeninae were examined, so the author did not make any taxonomic decision about it. He also considered Neoplecostomus as being the sole genus of the Neoplecostominae, based on “plesiomorphic characters”, according to the author. Schaefer (1987) also employed phylogenetic methods in his analysis, using osteological information as characters. He recognized the subfamilies Lithogeninae, Neoplecostominae, Hypoptopomatinae, Loricariinae and Hypostominae plus Ancistrinae. Hypostominae was found not to be monophyletic without the Ancistrinae. Neoplecostominae was considered to be monogeneric, with the sole inclusion of Neoplecostomus, although he did not analyze all the genera assigned to the Neoplecostominae by Gosline (1947); only having been analyzed the genera Corymbophanes, Kronichthys, Pogonopomoides and Rhinelepis, all considered to be part of Hypostominae. Neoplecostominae was considered by Schaefer to be the sister group of all the remaining Loricariidae, with the exception of Lithogeninae. Montoya-Burgos et al. (1998) conducted the first molecular phylogeny study of the Loricariidae, using partial sequences of the 12S and 16S rRNA genes, analyzing 5 of the 6 families recognized by Isbrücker (1980), with the exception of the Lithogeninae. They found only the Loricariinae subfamily to be monophyletic, finding no support of monophyly of the other families. They also endorsed an enlarged concept of the Neoplecostominae proposed by Gosline (1947), except for the Hemipsilichthys gobio, referred to as Upsilodus victori by Miranda Ribeiro, 1924 (Pereira & Reis, 2017), which was considered non-monophyletic and the sister-group of all the other Loricariidae, and Pseudorinelepis agassizi, more related to what the author called “higher” Loricariidae. Armbruster (2004) in his morphology-based phylogeny recognized by then 646 species of Loricariidae, placing them in the subfamilies: Hypostominae, Loricariinae, Hypoptopomatinae, Neoplecostominae and a fifth family, composed by the genera Delturus and Upsilodus. Lithogenes grouped with the Astroblepidae in his analysis, based on two bone synapomorphies, so it was not considered as a Loricariidae subfamily. The hypostominae was not considered to be monophyletic without the Ancistrinae, so it became a tribe of the Hypostominae, alongside with Hypostomini, Corymbophanini, Pterygoplichthini and

Rhinelepini, the last three being created by the author. In the Neoplecostominae, the genera Hemipsilichthys (Pareiorhaphis, sensu Pereira & Reis, 2002), Isbrueckerichthys, Kronichthys, Neoplecostomus and Pareiorhina were allocated to it, although the author did not consider this subfamily to be monophyletic, being more related to Chaetostoma according to him. He also indicated a new subfamily, to contain the genera Delturus and Upsilodus (Hemipsilichthys gobio, sensu Miranda Ribeiro 1924) sister group to all the other Loricariidae, though the author did not suggest a new subfamily name. In 2006, Armbruster Pereira and Reis suggested then a new subfamily name, Delturinae, to allocate Delturus and Hemipsilichthys, formally considering Upsilodus victori a synonym to Hemipsilichthys gobio. In 2008, Chiachio et al. made a phylogenetic and biogeography study with the Hypoptopomatinae and Neoplecostominae, using a nuclear gene, F-reticulon4. They found the subfamily Hypoptopomatinae to be paraphyletic, the tribe Hypoptopomatini to be monophyletic and the sister-group to Neoplecostominae plus Ototyrini. The Otothyrini was also considered paraphyletic, for Pseudotocinclus tietensis was found to be related to Pareiorhina rudolphi, whereas Neoplecostominae could only be monophyletic with the inclusion of Pseudotocinclus tietensis. The authors elevated the tribes Hypoptopomatini and Othothyrini to subfamily levels. Also in 2008, Cramer et al. conducted a phylogenetic study about the relationships between the Hypoptopomatinae and the Neoplecostominae as well, though this time using as marker the Cytochrome c Oxidase I (COI). Similarly to Chiachio et al. (2008), Pseudotocinclus is more closely related to Pareiorhina, not being the Neoplecostominae monophyletic without the inclusion of this genus. The Hypoptopomatinae was also found to be non-monophyletic, though, with low bootstraps values, the authors decided not to take taxonomic decisions about this subfamily. In 2011 the same authors Cramer et al. published another molecular phylogenetic study on the Neoplecostominae and Hypoptopomatinae, though this time combining three nuclear genes and mitochondrial Cytochrome c Oxidase I (COI). The taxa Otothyrini, Hypoptopomatini and Neoplecostominae as described by the authors, were not recovered as monophyletic. Once again, Pseudotocinclus appeared as more closely related to Pareiorhina rudolphi, and Pareiorhina was not recovered as monophyletic. Neither was Neoplecostomus, Isbrueckerichthys and Pareiorhaphis. The authors once more chose not to make taxonomic decisions. Roxo et al., 2012a conducted a molecular phylogenetic study specifically on Neoplecostominae, using as markers the genes 12S and 16S rRNA, Cytochrome c Oxidase I

(COI), Cytochrome b and F-reticulon 4. The results once again indicated that Neoplecostominae could only be monophyletic with the inclusion of Pseudotocinclus, which one more time appeared to be associated with Pareiorhina rudolphi. The genera Pareiorhina and Neoplecostomus were found to be paraphyletic, in accordance with the findings of Cramer et al. (2011). Two years later, Roxo et al. (2014) conducted another phylogenetic study of the Neoplecostominae, but this time also encompassing the subfamilies Otothyrinae and Hypoptopomatinae, using the genes 16S rRNA, Cytochrome c Oxidase I (COI), Cytochrome b and F-reticulon 4. In accordance to Chiachio et al. (2008), Otothyrinae is the sister-group to Neoplecostominae, and together they are the sister-group to the Hypoptopomatinae. The three subfamilies were found to be monophyletic with high support, with Pseudotocinclus formally included as part of the Neoplecostominae. Only the genera Kronichthys and Isbrueckerichthys were recovered as monophyletic, while Pareiorhaphis, Pareiorhina and Neoplecostomus, were not. In the same year, Pereira et al. (2014) described a new genus from a coastal basin in Bahia state, Hirtella, which was ascribed to the Neoplecostominae by a combination of characters, although they did not determine the phylogenetic position of the genus. Lujan et al. (2015) made the most complete molecular phylogenetic study on the Loricariidae up until then, with representatives of all previously recognized subfamilies, using as markers the genes 16S, Cytochrome b, RAG1, RAG2 and MyH6. The results supported the existence of the previously recognized subfamilies Delturinae, Loricariinae, Hypoptopomatinae and Hypostominae. The genera Rhinelepis and Pseudorinelepis formed a clade, which made the author create the family Rhinelepinae, forming a polytomy with the incertae sedis ‘Pseudancistrus’ genisetiger and all the other Loricariidae, exclusive of Delturinae and Lithogeninae. Hypoptopomatinae was supported as monophyletic inclusive of Neoplecostominae, which was considered as a tribe, Neoplecostomini, sister group to Otothyrini. Both were found to be sister group to Hypoptopomatinae, which was considered paraphyletic. While Neoplecostomini appeared as monophyletic, only 9 species were analyzed in the study, which had a greater focus on the Hypostominae. Pseudotocinclus appeared as sister-group to all the other Neoplecostomini, not appearing as related to Pareiorhina rudolphi like the previous studies; even because this species was not analyzed. Pareiorhaphis and Neoplecostomus were not recovered as monophyletic. A year later, Silva et al. (2016) described a new genus from the Tocantins basin, Microplecostomus, designated according to molecular data to the Neoplecostominae, which

the authors considered to be a subfamily instead of a tribe. It was found to be sister group to all the other Neoplecostominae. In 2017 Pereira & Reis conducted a morphology-based phylogeny focused on Neoplecostominae, which the authors considered to be a subfamily instead of a tribe. Lithogeninae was once again found to be sister group to all remaining Loricariidae, as well as Delturinae was found to be sister group to all remaining Loricariidae with the exception of Lithogeninae. The subfamilies Loricariinae, Hypostominae and Hypoptopomatinae were considered to be monophyletic, this last recovered as sister group to Neoplecostominae. The genera Nannoplecostomus and Microplecostomus were considered to be incertae sedis. Neoplecostominae was recovered as monophyletic exclusive of Microplecostomus, a recently described genus appointed as a Neoplecostominae (Silva et al., 2016), and Pseudotocinclus, contradicting the previous molecular phylogenetic studies. All the remaining genera previously allocated to Neoplecostominae were considered to be monophyletic. Kronichthys was found to be sister-group of all remaining Neoplecostominae, and Euryochus, a new genus described by the authors from Espírito Santo, was sister group to all Neoplecostominae, with the exception of Kronichthys. It was ascribed to the Neoplecostominae by the presence of the synapomorphies described in the study. Like Hirtella, no DNA sequence belonging to Euryochus was made available. Hirtella was sister group to Pareiorhaphis, and both genera were considered to be sister group of Pareiorhina plus Isbrueckerichthys and Neoplecostomus. In 2019 Roxo et al conducted the first phylogenomic study of the Loricariidae, comprising 240 species and 75 genera, although they did not analyze any Lithogeninae, nor ‘Pseudancistrus’ genisetiger. The data supported Delturinae as the sister group to all remaining Loricariidae, followed by Rhinelepinae, sister group to all Loricaridae exclusive of Delturinae. According to their time-tree estimation, Loricariidae originated at about 65.4 Ma, during the Late Cretaceous/Paleogene boundary. The clade composed of Hypostominae plus Hypoptopomatinae originated at about 35.8 Ma, in accordance with previous studies proposed by Roxo et al. (2014). Their results corroborated the monophyly of four loricariid subfamilies: Delturinae, Loricariinae, Hypostominae and Hypoptopomatinae, although they also referred to “Rhinelepinae” as a subfamily. The clades previously treated as subfamilies, Otothyrinae and Neoplecostominae according to the authors could not be delimited as such, being therefore treated as tribes, Otothyrini and Neoplecostomini, within the Hypoptopomatinae subfamily. In accordance to Pereira & Reis (2017), the genus Microplecostomus did not fall into the Neoplecostomini. Like previous molecular studies, Pareiorhina and Neoplecostomus were not found to be monophyletic; the type-species of

Pareiorhina, P. rudolphi was sister to Pseudotocinclus tietensis, and sister to all the other Neoplecostomini, while Neoplecostomus ribeirensis was more related to species of Isbrueckerichthys, Kronichthys and Pareiorhaphis, than to other Neoplecostomus. The genus Plesioptopoma was sister to ‘Pareiorhina’ carrancas, opposing the the results found in Reis et al. (2012) and Pereira & Reis (2017), for Plesioptopoma lacks a Neoplecostomini synapomorphy, not even being considered as a Neoplecostomini previously. Pareiorhaphis plus Kronichthys was found sister group of Isbrueckerichthys plus Neoplecostomus ribeirensis.

At the current study, Neoplecostomini is considered as a tribe instead of a subfamily, in accordance to the latest Loricariidae phylogenomic study of Roxo et al. (2019), although since the genera Hirtella and Euryochus were not analyzed in such study, both are also being considered as Neoplecostomini, following the results found in Pereira & Reis (2017). Neoplecostomini is then composed by the genera Euryochus, Pereira & Reis, 2017; Hirtella Pereira, Zanata, Cetra & Reis, 2014; Isbrueckerichthys Derijst, 1996; Kronichthys Miranda Ribeiro, 1908; Neoplecostomus Eigenmann & Eigenmann, 1888; Pareiorhaphis Miranda Ribeiro, 1918; Pareiorhina Gosline, 1947; Plesioptopoma Reis, Pereira & Lehmann, 2012 and Pseudotocinclus Ihering, 1907. Neoplecostomus and Pareiorhina are the only Neoplecostomini genera to occur in the upper Paraíba do Sul basin, the focus area of this study.

Pareiorhina Gosline, 1947 was created to allocate Rhinelepis rudolphi (Miranda Ribeiro, 1911). Miranda Ribeiro (1911) proposed R. rudolphi as a substitute name for Hypostomus microps (Ihering, 1907), since this name was already assigned to H. microps (Steindachner, 1877), currently Neoplecostomus microps (Bockmann & Ribeiro, 2003; Langeani, 1990). It was a monotypic genus, with its single species Pareiorhina rudolphi, until Bockmann & Ribeiro described P. carrancas in 2003, from the Rio Grande basin. Since then, five more species were allocated to this genus: P. brachyrhyncha Chamon, Aranda & Buckup, 2005, from the Paraíba do Sul basin, P. cepta Roxo, Silva, Mehanna & Oliveira, 2012 from the São Francisco basin, P. hyptirhachis Silva, Roxo & Oliveira, 2013, also from the Paraíba do Sul basin, P. pelicicei Santos & Roxo, 2015, another species from Rio Grande basin, and more recently P. rosai Silva, Roxo & Oyakawa, 2016, from the São Francisco basin.

Neoplecostomus Eigenmann & Eigenmann, 1888 was created to allocate N. microps (Steindachner, 1876), previously described as Plecostomus microps (Steidachner, 1876), from the Rio Paraíba basin and N. granosus, previously described as Hypotomus granosus

(Valenciennes, 1840), from the Rio de Janeiro state and Caiena. In 1990 Langeani revised the genus, describing four additional species: N. espiritosantensis Langeani, 1990; N. franciscoensis Langeani, 1990; N. paranensis Langeani, 1990 and N. ribeirensis Langeani, 1990. Since then, thirteen additional species have been described: N. variipictus Bizerril, 1995 from the Paraíba do Sul basin; N. corumba Zawadzki, Pavanelli & Langeani, 2008; N. selenae Zawadzki, Pavanelli & Langeani, 2008; N. yapo Zawadzki, Pavanelli & Langeani, 2008, N. bandeirante Roxo, Oliveira & Zawadzki, 2012; N. botucatu Roxo, Oliveira & Zawadzki, 2012; N. langeanii Roxo, Oliveira & Zawadzki, 2012, the last six from the upper Paraná basin; N. docensis Roxo, Silva, Zawadzki & Oliveira, 2014, from the Rio Doce basin; N. jaguari Andrade & Langeani, 2014 from the upper Paraná basin; N. paraty Cherobim, Lazzarotto & Langeani, 2016, from the Perequê-Açú basin; N. canastra Roxo, Silva, Zawadzki & Oliveira, 2017; N. watersi Silva, Reia, Zawadzki & Roxo, 2018, the last two from the Upper Paraná basin and N. pirangaensis Oliveira & Oyakawa, 2019, from the Rio Doce basin.

The Paraíba do Sul river has an extension of about 1080 km, with a drainage area of about 57.000 km² on the states of São Paulo, Rio de Janeiro and Minas Gerais, in Brazil (Simões, 1977). It is formed by the junction of the rivers Paraibuna and Paraitinga, being one of the most relevant of the southeast region (Bizerril, 1999), the most densely populated in Brazil. Its origin dates back to the Triassic period, when a single, large, elongated basin in Gondwana arose as a result of the rifting process (Da Rosa & Garcia, 2000), that would culminate in the split of the supercontinent into the African and south American continents, at about 98 to 93 million years ago (Cretaceous period) (Cesero & Ponte, 1997). With the complete fission, independent, isolated South American Atlantic river basins arose, known as the Coastal Drainages of Eastern Brazil, which contains the Paraíba do Sul river basin (Ribeiro, 2006).

Despite its importance in providing water to domestic and industrial usage throughout São Paulo and Rio de Janeiro states, Paraíba do Sul river is highly degraded, due to inappropriate management of its waters (Silveira et al., 1979; Barroso, 1989). Even so, it has a high degree of endemism (Teixeira et al., 2004), which makes the correct assessment of its biodiversity urgent. On this matter, accurate species delimitation is fundamental in conservation biology, to correctly determine endangered species (Ziheng & Rannala, 2010) and define conservational policies.

Species are a fundamental biological unit, being as important as other lower level units, such as cells and organisms (Mayr, 1982). Species concept has also been a main theme in biology, and in the past century, many taxonomists have proposed different concepts (e.g. Mayr, 1942; Simpson, 1951; Hennig, 1966; Van Valen, 1976; Rosen, 1979, Nelson & Platnick, 1991; Davis & Nixon, 1992; Baum & Shaw, 1995). De Queiroz (2007) shed light to the matter of species delimitation, which has typically been confused with the concept of species. He proposed a new concept, which contained the similarities present in all previous ones: species are separately evolving metapopulations segments of lineages. The different properties that were used to define previous species concepts, were suggested by De Queiroz to be operational criteria or lines of evidence to the separation of lineages – in other words, to be the basis of species delimitation methods.

Traditionally, species have been delimited by morphologic data; however, evolutionary processes, such as morphological convergence, can conceal and underestimate the number of species, especially if they’re cryptic (Ziheng & Rannala, 2010). In this context, genetic data can help to clarify speciation processes and species delimitation (Hey, 2010). In the present study, we use two coalescence methods to delimitate species of two genera of armored catfish from the tribe Neoplecostomini, Pareiorhina and Neoplecostomus, from different populations from the upper Paraíba do Sul basin.

Materials and Methods

1.Taxon sampling

Field trips were conducted between the years of 2017 and 2020, and specimens collected with dip nets of different size. They were then euthanized right after collection by submergence in a buffered solution of tricaine methane sulphonate (MS-222) at a concentration of 250mg/L for a period of 10 minutes, or until completely ceasing opercular movement, following the guidelines of the Journal of the American Veterinary Medical Association (AVMA Guidelines) (Leary et al. 2013) and the European Comission DGXI consensus for fish euthanasia (Close et al. 1996, 1997). Afterwards, specimens were fixed and preserved in absolute ethanol, and were deposited at the fish collection of the Institute of Biology, Federal University of Rio de Janeiro (UFRJ). A list of the specimens and their Genbank access number is available on Appendix 1.

2.DNA extraction, amplification and sequencing

Total genomic DNA was extracted from the right side of the muscle tissue from the caudal peduncle, using DNeasy Blood and Tissue Kit (Qiagen), following the manufacturer’s instructions. The Cytochrome c oxidase subunit 1 (COI) mitochondrial gene was sampled, and target DNA fragments were amplified through the polymerase chain reaction (PCR) using the primers L5698-ASN and H7271-COIASN (5’- AGG CCT CGA TCC TAC AAA GKT TTA GTT AAC -3’) e H7271-COI (5’- GTG GTG GGC TCA TAC AAT AAA -3’) (Villa-Verde et al. 2012). Double-stranded PCR amplifications were performed in 60 μl with reagents at the following concentration: 5× GreenGoTaq Reaction Buffer (Promega), 3,7 mm MgCl2, 1 μm of each primer, 75ng of total genomic DNA, 0.2 mm of each dNTP and 1 U of standard Taq polymerase Promega. The thermocycling profile was as follows: initial denaturation for 4 min at 94°C; 35 cycles of denaturation for 50s at 92°C, annealing for 1 min at 45–60°C, extension for 90s at 72°C; and terminal extension for 4 min at 72°C. In all PCR reactions negative controls were made without DNA to check for contamination. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega). Sequencing reactions were made by Macrogen Inc. (South Korea) using the BigDye Terminator Cycle Sequencing Mix (Applied Biosystems).

3.Phylogenetic Analyses

Since there were two genera found in the upper Paraíba do Sul basin, there were conducted four independent analyses for species delimitation; one for Neoplecostomus and one for Pareiorhina for each species delimitation coalescent method, General Mixed Yule Coalescent (GMYC) and Bayesian Poisson Trees Process (bPTP). Both the Pareiorhina bPTP and GMYC matrixes had 618 characters; whilst the GMYC matrix had 16 in-group terminals, the bPTP had 23. Both had out-groups; each representing genera of the Neoplecostomini. Meanwhile, the Neoplecostomus bPTP matrix had 22 in-group terminals, from the upper Paraíba do Sul basin, three out-groups represented by Neoplecostomus outside of the area of interest, and one out-group represented by Pareiorhaphis, a Neoplecostomini genus. The GMYC matrix had 10 in-group terminals and 6 out-groups, also represented by Neoplecostomus outside Paraíba do Sul river basin. Both the matrixes had 661 characters. Most of the out-groups sequences were taken from Genbank. A list of specimens, their locality and respective GenBank accession number is represented in Appendix 1.

Sequences were edited using the software Mega v. 7 (Kumar et al. 2016) and aligned using the algorithm Clustal W (Chenna et al. 2003), with default parameters for gap opening and extension. Gaps were considered as informative characters. The best- fitting models of

molecular evolution were performed under the Akaike criteria with small sample correction in the software PartitionFinder (Lanfear, Calcott, Ho & Guindon, 2012). The selection was determined to each codon position. Bayesian Inference (BI) analyses were conducted with BEAST v.2.3 (Drummond & Rambaut, 2007). Two independent Markov Chain Monte Carlo (MCMC) were performed for each analysis. The Neoplecostomus bPTP MCMC runs were performed with 100 million generations each, the Neoplelcostomus GMYC MCMC runs with 10 million generations each, the Pereiorhina bPTP MCMC runs with 50 million generations each, and the Pareiorhina GMYC MCMC runs with also 50 million generations. All the analyses had a tree sampling frequency of every 1 thousand generations. All analyses were performed under an uncorrelated relaxed clock model. The convergence of the independent MCMC chains and the proper burn-in values were assessed by evaluating the stationary phase of the chains using Tracer v.1.6 (Rambaut, Suchard, Xie, Drummond, 2014). The BI final consensus tree and its Bayesian posterior probabilities were generated with the remaining trees after removing the first 25% of the samples as burn-in.

4.Species Delimitation

Two coalescent species delimitation methods were applied: General Mixed Yule Coalescent (GMYC) (Monaghan et al., 2009; Pons et al., 2006) and Bayesian Poisson-Tree Process (bPTP) (Zhang et al., 2013). Both GMYC and bPTP analyses were performed online, using as input the trees calculated with BEAST; the bPTP analysis on the bPTP web server (http://species.h-its.org), using 50 million MCMC generations, with a thinning value of 1000 and a burn-in of 0.25; and the GMYC analysis on the GMYC web server (http://species.h- its.org/gmyc/), using multiple-threshold (Monaghan et al., 2009; Zhang et al., 2013).

Results

36 sequences were generated from specimen of Neoplecostomus and Pareiorhina lato sensu, generating a 687 character matrix in Pareiorhina and a 661 one in Neoplecostomus. The analysis of bPTP in Neoplecostomus from the upper Paraíba do Sul resulted in 4 clusters, whilst the GMYC analysis resulted in only 1. The Pareiorhina bPTP analysis resulted in 4 clusters, and the GMYC analysis, 3.

1.Neoplecostomus

Fig.1: bPTP (pink bar) and GMYC (blue bar) species delimitation topology results of Neoplecostomus. Each bar discontinuity represents a different species. The drainages on which the focal group occur are highlighted. Node supports indicate the posterior probability of the species delimited by bPTP.

The results from the bPTP and GMYC Neoplecostomus delimitation analyses are represented in Fig. 1. The focal group of the present study are the Neoplecostomus populations from numerous drainages of the upper Paraíba do Sul basin, represented by UFRJ 12405; UFRJ 12900; UFRJ 11941; UFRJ 11943; UFRJ 11948; UFRJ 11947; UFRJ 11946; UFRJ 11949; FJ434531; UFRJ 11928; UFRJ 11934; EU359429; KU608295; UFRJ 11937; KU608294; UFRJ 12640.2; UFRJ 12354; UFRJ 12640.1; FJ434531, as shown in Fig. 2. On the bPTP analysis, four species were recognized from the upper Paraíba do Sul basin, the type species, N. microps, from various drainages across the Paraiba do Sul basin, two from Rio Preto, a Paraíba do Sul tributary containing its headwaters on the Itatiaia massif and flowing into the Paraibuna river: one species from the middle course of the Rio Preto river, N. sp1 (UFRJ 12405), and one from the upper course, N. sp2 (UFRJ 12900; UFRJ 11941; UFRJ 11943; UFRJ 11948; UFRJ 11947; UFRJ 11946; UFRJ 11949). These two species from the Rio Preto river appear as sister group from the rest of Neoplecostomus from the Paraíba do Sul basin. The first species, N. sp3 (FJ434531), from Pindamonhangaba, is sister from the species here determined as N. microps, (UFRJ 11928; UFRJ 11934; EU359429; KU608295; UFRJ 11937; KU608294; UFRJ 12640.2; UFRJ 12354; UFRJ 12640.1). On the other hand, the GMYC analysis identified only one species from the upper Paraíba do Sul basin, N. microps

(UFRJ 12405; UFRJ 12900; UFRJ 11941; UFRJ 11943; UFRJ 11948; UFRJ 11947; UFRJ 11946; UFRJ 11949; FJ434531; UFRJ 11928; UFRJ 11934; EU359429; KU608295; UFRJ 11937; KU608294; UFRJ 12640.2; UFRJ 12354; UFRJ 12640.1; FJ434531). The geographic distribution of the Neoplecostomus species found by the bPTP results is represented in Fig. 2.

Fig 2.: Geographic distribution of the Neoplecostomus species found by bPTP analysis. Each symbol corresponds to a different species. According to the GMYC analysis, all the haplotypes belong to a single species, N. microps.

2.Pareiorhina lato sensu

Fig.3: bPTP (pink bar) and GMYC (blue bar) species delimitation topology results of Pareiorhina lato sensu. Each bar discontinuity represents a different species. The drainages on which the focal group occur are highlighted. Node supports indicate the posterior probability of the species delimited by bPTP.

The results from the bPTP and GMYC Pareiorhina delimitation analyses are represented in Fig. 3. The focal group of the present study are the Pareiorhina lato sensu populations from numerous drainages of the upper Paraíba do Sul basin, represented by UFRJ 12406; GU702272; UFRJ 11927; UFRJ 11950.1; UFRJ 11950.2; GU702275; JN89800; GU702273; GU702274; GU702278; UFRJ 12353; UFRJ 11959; UFRJ 11933; UFRJ 11980; UFRJ 12026; UFRJ 11962; UFRJ 11986; UFRJ 11979; UFRJ 10479; UFRJ 11985; UFRJ 12024; UFRJ 11983; UFRJ 12402.1; UFRJ 12402.2; UFRJ 12355.1; UFRJ 12355.2; UFRJ 12355.3, as shown in Fig. 3. Pareiorhina was not recovered as a monophyletic genus. Pareiorhina rudolphi, type-species of the genus, appears as being more related to Pseudotocinclus tietensis, whilst the other ‘Pareiorhina’ appear with high support as being more relaated to Neoplecostomus paraty. The bPTP analysis resulted in a group containing ‘Pareiorhina’ hyptiorhachis (UFRJ 12406), from the middle course of Rio Preto river; ‘Pareiorhina’ brachyrhyncha (GU702272), from Pindamonhangaba; ‘Pareiorhina’ sp4 (UFRJ 11927), from Itatiaia and ‘Pareiorhina’ sp5 (UFRJ 11950.1; UFRJ 11950.2), from Resende; all three last species from tributaries of the Paraíba do Sul river. This group is sister to a group containing species of Pseudotocinclus and of Pareiorhina related to the type species: the type species itself, Pareiorhina rudolphi (GU702275; JN89800; GU702273; GU702274;

GU702278; UFRJ 12353), from Piquete (type-locality), from a tributary of the Paraíba do Sul river; P. sp2 (UFRJ 11959; UFRJ 11933; UFRJ 11980; UFRJ 12026; UFRJ 11962; UFRJ 11986; UFRJ 11970; UFRJ 10479; UFRJ 11985; UFRJ 12024; UFRJ 11683) from Resende and Itatiaia area, also containing haplotypes from the upper Rio Preto river; P. sp3 (UFRJ 12402.1; UFRJ 12402.2), from São José dos Campos, on a Paraíba do Sul tributary and P. sp1 (UFRJ 12355.1; UFRJ 12355.2; UFRJ 12355.3), also from Resende, but from a different drainage from P. sp2, on a Paraíba do Sul tributary. The GMYC analysis had the same result, except for ‘Pareiorhina’ sp4 and sp5, which appeared as a single species; also, Pareiorhina rudolphi and P. sp1 also appeared as a single species. The geographic distribution of the Pareiorhina species found by the bPTP results is represented in Fig. 4.

Fig 4.: Geographic distribution of Pareiorhina lato sensu species found by bPTP analysis. Each symbol corresponds to a different species. According to the GMYC analysis, ’Pareiorhina’ sp4 and ’Pareiorhina’ sp5 correspond to a single species, as does Pareiorhina sp1 and Pareiorhina rudolphi.

Discussion

When describing the second species of Pareiorhina in 2003, Bockmann & Ribeiro allocated ‘Pareiorhina’ carrancas to the Pareiorhina genus for the fact that by comparison with other

small loricariids, it was the one which shared more common traits with the new species. Since there were no proposed synapomorphies assigned to Pareiorhina, the authors already admitted the possibility that the new taxon could belong to another genus, but preferred not to describe a new one. Since then, the non-monophyly of the genus has been appointed in several studies (Crammer et al., 2011; Roxo et al., 2012a; Roxo et al., 2014; Roxo et al 2019), which is corroborated by the present study. The results show the occurrence of three groups of species from the upper Paraiba do Sul basin, including two genera, Neoplecostomus and Pareiorhina, and a third one that hasn’t been described yet, presented on this study as ‘Pareiorhina’.

The bPTP analysis, with four species delimited, and the GMYC analysis, with three species, show a high ‘Pareiorhina’ diversity to this basin. Besides the already described species, ‘Pareiorhina’brachyrhyncha and ‘Pareiorhina’ hyptiorhachis, there’s at least a third species, occurring in Resende and Itatiaia (‘Pareiorhina’ sp4 delimited on the GMYC analysis), corresponding to half of the diversity known for this new genus.

The genus Pareiorhina stricto sensu, containing species associated to the type species, was believed to be monotypic, with its type-species P. rudolphi. However, according to this study there are in fact four possible species, three from which were recovered for both bPTP and GMYC delimitation methods. Hence, this genus would be endemic to the upper Paraíba do Sul basin, containing all Pareiorhina stricto sensu species. These results suggest that wider river on their middle course, like the Paraíba do Sul river, could be a geographic barrier for the four species delimited by bPTP, all of which occurring on the upper courses of its tributaries. The Itatiaia massif could also be a geographic barrier between P. sp2, from upper Rio Preto river, and P. sp1, from Rio Salto river, for although being on the same Resende municipality, they occur on different drainages of the massif.

Neoplecostomus microps has been commonly treated as the only species of the genus found on the Paraíba do Sul basin, as N.granosus and N.variipictus are only known by their type- series, and by the fact that the type locality of N. granosus is probably incorrect (Langeani, 1990). However, the bPTP analysis here presented demonstrates the occurrence of four possible species, including the type-species, although the GMYC analysis has identified only one. Despite the high diversity of the genus is found on the upper Paraná basin, this result would triplicate the occurrence of the Neoplecostomus on the upper Paraíba do Sul basin. The difference of results between the two analyses could be because of the fact that bPTP is often more sensitive to close-related species delimitation (Zhang, 2013). The GMYC analysis on

this case could underestimate the number of species. On the other hand, the low support values of bPTP species delimitation associated with the GMYC result could also indicate that there’s in fact only one species of Neoplecostomus on this basin. On this case, the results could suggest a higher vagility when compared to Pareiorhina lato sensu, of which wider and less-oxygenated middle course rivers and mountain chains would not be a geographic barrier to Neoplecostomus. Future studies with more genetic markers associated with morphological analyses are required to clarify this matter.

Acknowledgments

Thanks are due to Axel Katz and Paulo Vilardo for field assistance and for the photos taken. Special thanks to Claudia Bove for the hospitality during the field trips. This study was supported by CAPES (Coordenação de Aperfeicoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico - Ministério de Ciência e Tecnologia).

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Appendix

Appendix 1: A list with the specimens’s voucher number, Genbank acces number, Locality, geographic position and References.

Genbank Accessio Geographic Referenc Voucher Species Locality n Number position e (COI)

Order Siluriformes - - -

Family Loricariidae - - -

Neoplecostomina Subfamily - - - e Ribeirão da Jacutinga river,under the Neoplecostomus bridge. Paraíba do 22°06'56.6"S Present UFRJ 12405 - sp1 Sul basin, Santa 44°04'49"W paper Rita do Jacutinga municipality, MG state -Brazil Prata waterfall Rio da Prata river. Rio Preto river Neoplecostomus 22°14'47.8"S Present UFRJ 12670 - drainage, Bocaina sp2 44°31'18.7"W paper de Minas municipality, MG state- Brazil Rio Alcantilado river, near the farm's main Neoplecostomus house. Rio Preto 22°17'47.5" S Present UFRJ 11941 - sp2 river drainage, 44°33'19.3" W paper Bocaina de Minas municipality, MG state- Brazil Muralha waterfall, Rio Alcantilado river. Neoplecostomus Rio Preto river 22°17'38.4"S Present UFRJ 11943 - sp2 draingae, Bocaina 44°33'26" W paper de Minas municipality, MG state - Brazil Neoplecostomus Rio Preto river, 22°19'33"S Present UFRJ 11948 - sp2 2km from 44°36'11.4"W paper

Maromba village. Rio Preto river drainage, Bocaina de Minas municipality, MG Rio das flores river, Rio Preto Neoplecostomus river drainage. 22°16'10.4" S Present UFRJ 11947 - sp2 Bocaina de Minas 44°30'21.1" W paper municipality, MG state - Brazil Prata waterfall Rio da Prata river. Rio Preto river Neoplecostomus 22°14'47.8"S Present UFRJ 11946 - drainage, Bocaina sp2 44°31'18.7"W paper de Minas municipality, MG state- Brazil Cachorros bridge, Rio Preto river. Rio Preto river Neoplecostomus 22°18'23.6"S Present UFRJ 11949 - drainage, Bocaina sp2 44°30'28.6" W paper de Minas municipality, MG state - Brazil Ribeirão Cajarana river.Paraíba do Neoplecostomus Sul basin, 22°46'00”S Roxo et LBP645 FJ434531 sp3 Pindamonhangab 45°27'00” W al., 2012 a municipality, SP state- Brazil Cherobim Neoplecostomus Guapi-Açu river NMG01 KU608296 Undetermined et al., sp4 basin 2016 Cherobim Neoplecostomus NMM4 KU608298 Macaé river basin Undetermined et al., sp5 2016 Cherobim Neoplecostomus NMM1 KU608297 Macaé river basin Undetermined et al., sp5 2016 Rio Jucu river. Oriental coastal Neoplecostomus basin, Domingos 20°24'30”S Roxo et LBP2551 FJ434530 espiritosantensis Martins 40°54'55”W al., 2012 municipality, ES state- Brazil Rio das Velhas Neoplecostomus 20°18'43”S Roxo et LBP6489 GQ214804 river. Rio São franciscoensis 43°34'02”W al., 2012 Francisco river

basin, São Bartolomeu municipality, MG state- Brazil. Rio Campo Belo river, at Estrada do Parque road Neoplecostomus 1610. Campo 22°28'13.9"S Present UFRJ 11928 - microps Belo river 44°35'00.2"W paper drainage, Itatiaia municipality, RJ state- Brazil Rio Alambari river, under the bridge on the RJ- 22°17'36.23"S Neoplecostomus 163 road. Present UFRJ 11934 - 44°33'39.61" microps Alambari river paper W drainage, Resende municipality, RJ state - Brazil Neoplecostomus Cramer et Undetermined EU359429 Undetermined Undetermined microps al.,2007 Cherobim DZSJRP1391 Neoplecostomus 23°09'24.0"S KU608295 Cunha et al., 3 microps 44°51'20.0"W 2016 Rio Pirapetinga river, under the bridge on the RJ- Cherobim Neoplecostomus 163 road. 22°21'53.7"S UFRJ 11937 - et al., microps Pirapetinga river 44°30'06.5"W 2016 drainage, Resende municipality, RJ state - Brazil Cherobim DZSJRP1390 Neoplecostomus 22°39'15.0"S KU608294 Silveiras et al., 2 microps 44°46'01.0"W 2016 Neoplecostomus 23°09'25.3"S Present UFRJ 12640.2 - Cunha microps 44°51'22.0"W paper Ribeirão Benfica river, behind Águas Claras Neoplecostomus 22°35'31.3"S Present UFRJ 12354 - hotel. Paraíba do microps 45°12'13.8"W paper Sul basin, Piquete municipality, SP state - Brazil Neoplecostomus 23°09'25.3"S Present UFRJ 12640.1 - Cunha microps 44°51'22.0"W paper DZSJRP Neoplecostomus Stream on the 23°12'53.7”S Cherobim KU550707 13914 paraty road Parati- 44°47'28.6”W et al.,

Cunha. Perequê- 2016 Açu river basin, Parati minicipality, RJ state- Brazil Pareiorhaphis Rio Matipó river, Cramer et MCP 37176 GQ225420 Undetermined nasuta MG state, Brazil al., 2011 Rio Salto river, under the bridge at BR-354 road. 22°25'30.4"S Present UFRJ 12355 - Pareiorhina sp1 Paraíba do Sul 44°44'30.9"W paper basin, Resende municipality, RJ state - Brazil Rio Salto river, under the bridge at BR-354 road. 22°25'30.4"S Present UFRJ 12355.2 - Pareiorhina sp1 Paraíba do Sul 44°44'30.9"W paper basin, Resende municipality, RJ state - Brazil Rio Salto river, under the bridge at BR-354 road. 22°25'30.4"S Present UFRJ 12355.3 - Pareiorhina sp1 Paraíba do Sul 44°44'30.9"W paper basin, Resende municipality, RJ state - Brazil Rio Pirapetinga river tributary, on the RJ-163 road. 22°21'1.1"S Present UFRJ 11959 - Pareiorhina sp2 Pirapetinga 44°29'59"W paper drainage, Resende municipality, RJ state - Brazil Rio Alambari river, under the bridge on the RJ- 22°17'36.23"S 163 road. Present UFRJ 11933 - Pareiorhina sp2 44°33'39.61" Alambari river paper W drainage, Resende municipality, RJ state - Brazil Rio das flores river, Rio Preto river drainage. 22°16'10.4" S Present UFRJ 11980 - Pareiorhina sp2 Bocaina de Minas 44°30'21.1" W paper municipality, MG state - Brazil

Antas waterfall, Córrego das Antas river. Rio 22°16'52.5"S Present UFRJ 12026 - Pareiorhina sp2 Preto drainage, 44°32'02.4"W paper Bocaina de Minas municipality, MG state - Brazil Rio Alcantilado river, near the farm's main house. Rio Preto 22°17'47.5" S Present UFRJ 11962 - Pareiorhina sp2 river drainage, 44°33'19.3" W paper Bocaina de Minas municipality, MG state- Brazil Cachorros bridge, Rio Preto river. Rio Preto river 22°18'23.6"S Present UFRJ 11986 - Pareiorhina sp2 drainage, Bocaina 44°30'28.6" W paper de Minas municipality, MG state - Brazil Prata waterfall Rio da Prata river. Rio Preto river 22°14'47.8"S Present UFRJ 11979 - Pareiorhina sp2 drainage, Bocaina 44°31'18.7"W paper de Minas municipality, MG state- Brazil Rio Marimbondo river, near RJ 151 22° 20'5.13"S Present UFRJ 10479 - Pareiorhina sp2 road. Resende 44°32'32,14" paper municipality, MG W state - Brazil Poço do Marimbondo, at Rio Marimbondo 22°21'40.7"S Present UFRJ 11985 - Pareiorhina sp2 river. Rio Preto 44°35'14.5" W paper drainage, Itatiaia municipality, RJ state - Brazil Rio Preto river tributary, 1,5km from Fumaça waterfall. Rio 22°15'32.2"S Present UFRJ 12024 - Pareiorhina sp2 Preto drainage, 44°20'49.9"W paper Resende municipality, RJ state - Brazil

Escorrega waterfall, Rio Preto river at 22°19'53.85"S Maromba village. Present UFRJ 11683 - Pareiorhina sp2 44°36'56.69" Rio Preto paper W drainage, Itatiaia municipality, RJ state - Brazil Córrego do Futuro river tributary. Paraíba 22°57'16.44"S Present UFRJ 12402.2 - Pareiorhina sp3 do Sul basin, São 46°0'44.1"W paper José dos Campos municipality, SP state - Brazil Córrego do Futuro river tributary. Paraíba 22°57'16.44"S Present UFRJ 12402 - Pareiorhina sp3 do Sul basin, São 46°0'44.1"W paper José dos Campos municipality, SP state - Brazil Rio Campo Belo river, at Estrada do Parque road 1610. Campo 22°28'13.9"S Present UFRJ 11927 - "Pareiorhina" sp4 Belo river 44°35'00.2"W paper drainage, Itatiaia municipality, RJ state- Brazil Rio Pirapetinga river, under the bridge on the RJ- 163 road. 22°21'53.7"S Present UFRJ 11950.2 - "Pareiorhina" sp5 Pirapetinga river 44°30'06.5"W paper drainage, Resende municipality, RJ state - Brazil Rio Pirapetinga river, under the bridge on the RJ- 163 road. 22°21'53.7"S Present UFRJ 11950.1 - "Pareiorhina" sp5 Pirapetinga river 44°30'06.5"W paper drainage, Resende municipality, RJ state - Brazil Paraíba do Sul "Pareiorhina" 22°47'09.6"S Pereira et LBP-29871 GU702272 river basin, São brachyrhyncha 45°27'21.6"W al., 2011 Paulo state-

Brazil

Ribeirão Santana river, tributary of Rio Preto river. "Pareiorhina" Rio Preto river 22°01'02.1"S Present UFRJ 12406 - hyptiorhachis drainage, Rio 43°53'30.3"W paper Preto municipality, MG state - Brazil Ribeirão Benfica river, behind Águas Claras Pareiorhina 22°35'31.3"S Present UFRJ 12353 - hotel. Paraíba do rudolphi 45°12'13.8"W paper Sul basin, Piquete municipality, SP state - Brazil Pareiorhina 22°34'48.0"S Pereira et LBP-37495 GU702296 Undetermined rudolphi 45°10'12.0"W al., 2011 Pareiorhina 22°34'48.0"S Pereira et LBP-37775 GU702274 Undetermined rudolphi 45°10'12.0"W al., 2011 Pareiorhina 22°34'48.0"S Pereira et LBP-37774 GU702273 Undetermined rudolphi 45°10'12.0"W al., 2011 Ribeirão Piquete. Paraíba do Sul river basin, Pareiorhina 22°35'00"S Roxo et LBP8044 JN089800 Piquete rudolphi 45°10'08"W al., 2012 municipality, São Paulo state- Brazil Pareiorhina 22°34'48.0"S Pereira et LBP-37776 GU702275 Undetermined rudolphi 45°10'12.0"W al., 2011 Pseudotocinclus Cramer et LBP696 EU370987 Undetermined Undetermined tietensis al., 2008