Accessibility / Report Error

Molecular characterization of Astyanax species (Characiformes: Characidae) from the upper Paraguaçu River basin, a hydrographic system with high endemism

Abstract

Molecular tools have been employed to improve the knowledge about freshwater Neotropical fishes. Such approaches supporting studies of groups including species complexes such as Astyanax, one of the most diversified and taxonomically complex genus of the family Characidae. Here, we employed species delimitation analyses in four Astyanax species described for the upper Paraguaçu River basin, a drainage within Northeastern Mata Atlântica freshwater ecoregion with high endemism. We implemented single and multilocus approaches based on two mitochondrial and one nuclear markers. Cytochrome c Oxidase I sequences previously available for Astyanax species were also added to our dataset. The single locus analyses showed A. epiagos, A. rupestris, and A. aff. rupestris as different Molecular Operational Taxonomic Units (MOTUs), while A. brucutu and A. lorien were grouped. However, the multilocus approach distinguished these two species and showed congruence for the remaining single locus results. Astyanax aff. rupestris was separated into two MOTUs using both approaches, highlighting the need for an integrative taxonomic revision including A. aff. rupestris. These findings contribute to a better understanding of the diversity of this fish group in the upper Paraguaçu, identifying hidden diversity and reinforcing the relevance of this hydrographic system as a notable hotspot for ichthyofauna biodiversity endemism.

Keywords:
Biodiversity; Caatinga fishes; Freshwater fish; Hidden genetic diversity; Species delimitation

Resumo

Ferramentas moleculares têm sido empregadas para melhorar o conhecimento sobre os peixes Neotropicais. Tais abordagens apoiam estudos de grupos que incluem complexos de espécies, como Astyanax, um dos gêneros mais diversificados e taxonomicamente complexos dentro da família Characidae. Neste estudo, nós empregamos análises de delimitação de espécies em quatro espécies de Astyanax recentemente descritas da bacia do alto rio Paraguaçu, uma drenagem dentro da ecorregião Mata Atlântica Nordeste que apresenta alto endemismo. Nós realizamos abordagens de loco único e multilocos baseadas em dois marcadores mitocondriais e um nuclear. Sequências de Citocromo c Oxidase I anteriormente disponíveis para espécies de Astyanax foram adicionadas ao nosso conjunto de dados. As análises de loco único mostraram A. epiagos, A. rupestris e A. aff. rupestris como diferentes Unidades Taxonômicas Operacionais Moleculares (MOTUs), enquanto A. brucutu e A. lorien foram agrupadas. Entretanto, a abordagem multilocos distinguiu estas duas espécies e mostrou congruência com os demais resultados das análises de loco único. Astyanax aff. rupestris foi separada em duas MOTUs usando ambas as abordagens, sugerindo a necessidade de uma revisão taxonômica integrativa incluindo A. rupestris e ambas A. aff. rupestris. Esses achados contribuem para uma melhor compreensão da diversidade desse grupo de peixes na bacia do rio Paraguaçu, identificando diversidade oculta e reforçando a relevância desse sistema hidrográfico como um notável hotspot de endemismo da biodiversidade da ictiofauna.

Palavras-chave:
Biodiversidade; Delimitação de espécies; Diversidade genética oculta; Peixe de água doce; Peixes de Caatinga

INTRODUCTION

Astyanax Baird & Girard, 1854 is one of the most diversified and taxonomically complex genus within the Characidae family (Characiformes), including 125 valid species widespread throughout nearly the entire Neotropical region (Rossini et al., 2016Rossini BC, Oliveira CAM, Melo FAG, Bertaco VA, Astarloa JMD, Rosso JJ et al. Highlighting Astyanax species diversity through DNA barcoding. PLoS ONE. 2016; 11(12):e0167203. https://doi.org/10.1371/journal.pone.0167203
https://doi.org/10.1371/journal.pone.016...
; de Pinna et al., 2018de Pinna M, Abrahão V, Reis V, Zanata A. A new species of Copionodon representing a relictual occurrence of the Copionodontinae (Siluriformes: Trichomycteridae), with a CT-scan imaging survey of key subfamilial features. Neotrop Ichthyol. 2018; 16(4):e180049. https://doi.org/10.1590/1982-0224-20180049
https://doi.org/10.1590/1982-0224-201800...
; Fricke et al., 2022Fricke R, Eschmeyer WN, Van der Laan R. Eschmeyer’s catalog of fishes: Genera, species, references [Internet]. San Francisco: California Academy of Sciences; 2022. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp).
http://researcharchive.calacademy.org/re...
). This genus comprises small species of about 40 to 200 mm standard length (Garutti, 1998Garutti V. Descrição de uma espécie nova de Astyanax (Teleostei, Characidae) da bacia do Tocantins, Brasil. Iheringia, Sér Zool. 1998; 85:115–22.), occurring in a wide diversity of niches and aquatic environments within freshwater drainages from the southern United States to central Argentina (Eigenmann, 1921Eigenmann CH. The American Characidae. Part 3. Mem Mus Comp Zool. 1921; 43:209–310.; Bertaco, Garutti, 2007Bertaco VA, Garutti V. New Astyanax from the upper rio Tapajós drainage, Central Brazil (Characiformes: Characidae). Neotrop Ichthyol. 2007; 5(1):25–30. https://doi.org/10.1590/S1679-62252007000100003
https://doi.org/10.1590/S1679-6225200700...
).

Characterized by presenting high phenotypic plasticity and adaptation to distinct environmental conditions (Orsi et al., 2004Orsi ML, Carvalho ED, Foresti F. Biologia populacional de Astyanax altiparanae Garutti & Britski (Teleostei, Characidae) do médio Rio Paranapanema, Paraná, Brasil. Rev Bras Zool. 2004; 21(2):207–18. https://doi.org/10.1590/s0101-81752004000200008
https://doi.org/10.1590/s0101-8175200400...
), Astyanax species are among the most important components of the freshwater food web, with significant participation in the diet of large predator fishes (Prioli et al., 2002Prioli SMAP, Prioli AJ, Júlio Jr HF, Pavanelli CS, Oliveira AV, Carrer H et al. Identification of Astyanax altiparanae (Teleostei, Characidae) in the Iguaçu River, Brazil, based on mitochondrial DNA and a RAPD markers. Genet Mol Biol. 2002; 25(4):421–30. https://doi.org/10.1590/S1415-47572006000300011
https://doi.org/10.1590/S1415-4757200600...
), being usually dominant in headwaters and small tributaries (Bertaco, Lucena, 2010Bertaco VA, Lucena CAS. Redescription of Astyanax obscurus (Hensel, 1870) and A. laticeps (Cope, 1894) (Teleostei: Characidae): Two valid freshwater species originally described from rivers of Southern Brazil. Neotrop Ichthyol. 2010; 8(1):7–20. https://doi.org/10.1590/S1679-62252010000100002
https://doi.org/10.1590/S1679-6225201000...
). Within the Brazilian Shield, the genus is commonly found in large river systems (e.g., Amazon, La Plata, and São Francisco) and in the northeastern Brazilian coastal basins, including the Paraguaçu River basin.

The Paraguaçu River basin is considered one of the largest basins in northeastern Brazil (Higuchi et al., 1990Higuchi H, Britski HA, Garavello JC. Kalyptodoras bahiensis, a new genus and species of thorny catfish from northeastern Brazil (Siluriformes: Doradidae). Ichthyol Explor Freshw. 1990; 3:219–25.) and an extremely relevant drainage of the Northeastern Mata Atlântica freshwater ecoregion (NMAF, ecoregion 328, sensuAbell et al., 2008Abell R, Thieme ML, Revenga C, Bryer M, Kottelat M, Bogutskaya N et al. Freshwater ecoregions of the world: A new map of biogeographic units for freshwater biodiversity conservation. BioScience. 2008; 58(5):403–14. https://doi.org/10.1641/b580507
https://doi.org/10.1641/b580507...
; Camelier, Zanata, 2014aCamelier P, Zanata AM. Biogeography of freshwater fishes from the northeastern Mata Atlântica freshwater ecoregion: Distribution, endemism, and area relationships. Neotrop Ichthyol. 2014a; 12(4):683–98. https://doi.org/10.1590/1982-0224-20130228
https://doi.org/10.1590/1982-0224-201302...
). The fish fauna of the basin has been recognized by its high level of endemism (Buckup, 2011Buckup PA. The Eastern Brazilian Shield. In: Albert JS, Reis RE, editors. Historical biogeography of Neotropical freshwater fishes. Los Angeles: University of California Press; 2011. p.203–10. ; Camelier, Zanata, 2014aCamelier P, Zanata AM. Biogeography of freshwater fishes from the northeastern Mata Atlântica freshwater ecoregion: Distribution, endemism, and area relationships. Neotrop Ichthyol. 2014a; 12(4):683–98. https://doi.org/10.1590/1982-0224-20130228
https://doi.org/10.1590/1982-0224-201302...
; de Pinna et al., 2018de Pinna M, Abrahão V, Reis V, Zanata A. A new species of Copionodon representing a relictual occurrence of the Copionodontinae (Siluriformes: Trichomycteridae), with a CT-scan imaging survey of key subfamilial features. Neotrop Ichthyol. 2018; 16(4):e180049. https://doi.org/10.1590/1982-0224-20180049
https://doi.org/10.1590/1982-0224-201800...
). Recently, new species have been described for this basin, including different fish groups, such as Astyanax (e.g., Camelier, Zanata, 2014bCamelier P, Zanata AM. A new species of Astyanax Baird & Girard (Characiformes: Characidae) from the rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil, with comments on bony hooks on all fins. J Fish Biol. 2014b; 84(2):475–90. https://doi.org/10.1111/jfb.12295
https://doi.org/10.1111/jfb.12295...
; Zanata et al., 2017Zanata AM, Lima FC, Dario F, Garhard P. A new remarkable and Critically Endangered species of Astyanax Baird & Girard (Characiformes: Characidae) from Chapada Diamantina, Bahia, Brazil, with a discussion on durophagy in the Characiformes. Zootaxa. 2017; 4232(4):491–510. https://doi.org/10.11646/zootaxa.4232.4.2
https://doi.org/10.11646/zootaxa.4232.4....
, 2018Zanata AM, Burger R, Camelier P. Two new species of Astyanax Baird & Girard (Characiformes: Characidae) from the upper rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2018; 4438(3):471–490. https://doi.org/10.11646/zootaxa.4438.3.3
https://doi.org/10.11646/zootaxa.4438.3....
; Burger et al., 2019Burger R, Carvalho FR, Zanata AM. A new species of Astyanax Baird & Girard (Characiformes: Characidae) from western Chapada Diamantina, Bahia, Brazil. Zootaxa. 2019; 4604(2):369–80. https://doi.org/10.11646/zootaxa.4604.2.9
https://doi.org/10.11646/zootaxa.4604.2....
), Characidium Reinhardt, 1867 (e.g., Zanata, Camelier, 2015Zanata AM, Camelier P. Two new species of Characidium Reinhardt (Characiformes: Crenuchidae) from northeastern Brazilian coastal drainages. Neotrop Ichthyol. 2015; 13(3):487–98. https://doi.org/10.1590/1982-0224-20140106
https://doi.org/10.1590/1982-0224-201401...
; Melo, Espíndola, 2016Melo MRS, Espíndola VC. Description of a new species of Characidium Reinhardt, 1867 (Characiformes: Crenuchidae) from the Chapada Diamantina, Bahia, and redescription of Characidium bimaculatum Fowler, 1941. Zootaxa. 2016; 4196(4):552–68. https://doi.org/10.11646/zootaxa.4196.4.5
https://doi.org/10.11646/zootaxa.4196.4....
), Copionodon de Pinna, 1992 (e.g., de Pinna et al., 2018de Pinna M, Abrahão V, Reis V, Zanata A. A new species of Copionodon representing a relictual occurrence of the Copionodontinae (Siluriformes: Trichomycteridae), with a CT-scan imaging survey of key subfamilial features. Neotrop Ichthyol. 2018; 16(4):e180049. https://doi.org/10.1590/1982-0224-20180049
https://doi.org/10.1590/1982-0224-201800...
) Moenkhausia Eigenmann, 1903 (e.g., Benine et al., 2009Benine RC, Mariguela TC, Oliveira C. New species of Moenkhausia Eigenmann, 1903 (Characiformes: Characidae) with comments on the Moenkhausia oligolepis species complex. Neotrop Ichthyol. 2009; 7(2):161–68. https://doi.org/10.1590/S1679-62252009000200005
https://doi.org/10.1590/S1679-6225200900...
), and Rhamdiopsis Haseman, 1911 (e.g., Bockmann, Castro, 2010Bockmann FA, Castro RMCC. The blind catfish from the caves of Chapada Diamantina, Bahia, Brazil (Siluriformes: Heptapteridae): Description, anatomy, phylogenetic relationships, natural history, and biogeography. Neotrop Ichthyol. 2010; 8(4):673–706. https://doi.org/10.1590/S1679-62252010000400001
https://doi.org/10.1590/S1679-6225201000...
).

More than ten species of Astyanax are currently reported as occurring in the Paraguaçu hydrographic system (Santos, Caramaschi, 2007Santos ACA, Caramaschi EP. Composition and seasonal variation of the ichthyofauna from upper Rio Paraguaçu (Chapada Diamantina, Bahia, Brazil). Braz Arch Biol Technol. 2007; 50:663–72. https://doi.org/10.1590/s1516-89132007000400012
https://doi.org/10.1590/s1516-8913200700...
, 2011Santos ACA, Caramaschi EP. Temporal variation in fish composition and abundance in a perennial tributary of the rio Paraguaçu, a little-known drainage in the Brazilian Semi-Arid region. Neotrop Ichthyol. 2011; 9(1):153–60. https://doi.org/10.1590/S1679-62252011005000007
https://doi.org/10.1590/S1679-6225201100...
). From this total, six species are endemic to the upper Paraguaçu course and have allopatric distribution, occurring in different tributaries: A. brucutu Zanata, Lima, Dario & Garhard, 2017, Pratinha River (Zanata et al., 2017Zanata AM, Lima FC, Dario F, Garhard P. A new remarkable and Critically Endangered species of Astyanax Baird & Girard (Characiformes: Characidae) from Chapada Diamantina, Bahia, Brazil, with a discussion on durophagy in the Characiformes. Zootaxa. 2017; 4232(4):491–510. https://doi.org/10.11646/zootaxa.4232.4.2
https://doi.org/10.11646/zootaxa.4232.4....
); A. epiagos Zanata & Camelier, 2008, Jacuípe River (Zanata, Camelier, 2008Zanata AM, Camelier P. Two new species of Astyanax (Characiformes: Characidae) from upper rio Paraguaçu and rio Itapicuru basins, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2008; 1908(1):28–40. https://doi.org/10.11646/zootaxa.1908.1.2
https://doi.org/10.11646/zootaxa.1908.1....
); A. hamatilis Camelier & Zanata, 2014, Utinga, Una, and São José rivers (Camelier, Zanata, 2014bCamelier P, Zanata AM. A new species of Astyanax Baird & Girard (Characiformes: Characidae) from the rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil, with comments on bony hooks on all fins. J Fish Biol. 2014b; 84(2):475–90. https://doi.org/10.1111/jfb.12295
https://doi.org/10.1111/jfb.12295...
); A. lorien Zanata, Burger & Camelier, 2018, Santo Antônio River; A. rupestris Zanata, Burger & Camelier, 2018, Coisa Boa and Cumbuca rivers (Zanata et al., 2018Zanata AM, Burger R, Camelier P. Two new species of Astyanax Baird & Girard (Characiformes: Characidae) from the upper rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2018; 4438(3):471–490. https://doi.org/10.11646/zootaxa.4438.3.3
https://doi.org/10.11646/zootaxa.4438.3....
); and A. sincora Burger, Carvalho & Zanata, 2019, Tremedal stream (Burger et al., 2019Burger R, Carvalho FR, Zanata AM. A new species of Astyanax Baird & Girard (Characiformes: Characidae) from western Chapada Diamantina, Bahia, Brazil. Zootaxa. 2019; 4604(2):369–80. https://doi.org/10.11646/zootaxa.4604.2.9
https://doi.org/10.11646/zootaxa.4604.2....
). Furthermore, according to Zanata et al., (2018)Zanata AM, Burger R, Camelier P. Two new species of Astyanax Baird & Girard (Characiformes: Characidae) from the upper rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2018; 4438(3):471–490. https://doi.org/10.11646/zootaxa.4438.3.3
https://doi.org/10.11646/zootaxa.4438.3....
, the Piabinha River shelters a morphotype tentatively identified by the authors as Astyanax aff. rupestris, due to divergences in some morphological characters when compared to A. rupestris. The high richness within Astyanax and the fact of being traditionally defined by a combination of non-exclusive characters (see Eigenmann, 1921Eigenmann CH. The American Characidae. Part 3. Mem Mus Comp Zool. 1921; 43:209–310.), added to its recognized phenotype plasticity (Orsi et al., 2004Orsi ML, Carvalho ED, Foresti F. Biologia populacional de Astyanax altiparanae Garutti & Britski (Teleostei, Characidae) do médio Rio Paranapanema, Paraná, Brasil. Rev Bras Zool. 2004; 21(2):207–18. https://doi.org/10.1590/s0101-81752004000200008
https://doi.org/10.1590/s0101-8175200400...
), occasionally hinders accurate species identification. Consequently, some taxa are frequently identified only at the generic level or into species complexes (e.g., Moreira-Filho, Bertollo, 1991Moreira-Filho O, Bertollo LAC. Astyanax scabripinnis (Pisces, Characidae): a species complex. Rev Bras Genet. 1991; 14:331–57.; Garutti, Britski, 2000Garutti V, Britski HA. Descrição de uma espécie nova de Astyanax (Teleostei: Characidae) da bacia do alto rio Paraná e considerações sobre as demais espécies do gênero na bacia. Comun Mus Ciênc Tecnol PUCRS, Sér Zool. 2000; 13:65–88.). A recent integrative phylogeny (Terán et al., 2020Terán GE, Benitez MF, Mirande JM. Opening the Trojan horse: phylogeny of Astyanax, two new genera and resurrection of Psalidodon (Teleostei: Characidae). Zool J Linn Soc. 2020; 190(4):1217–34. https://doi.org/10.1093/zoolinnean/zlaa019
https://doi.org/10.1093/zoolinnean/zlaa0...
) recovered species attributed to Astyanax in different subfamilies and genera, including the resurrected Psalidodon Eigenmann, 1911 and a new genus, Andromakhe Terán, Benitez & Mirande, 2020 (Terán et al., 2020Terán GE, Benitez MF, Mirande JM. Opening the Trojan horse: phylogeny of Astyanax, two new genera and resurrection of Psalidodon (Teleostei: Characidae). Zool J Linn Soc. 2020; 190(4):1217–34. https://doi.org/10.1093/zoolinnean/zlaa019
https://doi.org/10.1093/zoolinnean/zlaa0...
). Dagosta, Marinho, (2022)Dagosta FCP, Marinho MMF. New small-sized species of Astyanax (Characiformes: Characidae) from the upper rio Paraguai basin, Brazil, with discussion on its generic allocation. Neotrop Ichthyol. 2022; 20(1):e210127. https://doi.org/10.1590/1982-0224-2021-0127
https://doi.org/10.1590/1982-0224-2021-0...
argue that although this study has been efficient in recovering the polyphyletic nature of Astyanax, it failed in providing consistent diagnosis characters for the proposed clades. None of the species evaluated here were analyzed by Terán et al., (2020)Terán GE, Benitez MF, Mirande JM. Opening the Trojan horse: phylogeny of Astyanax, two new genera and resurrection of Psalidodon (Teleostei: Characidae). Zool J Linn Soc. 2020; 190(4):1217–34. https://doi.org/10.1093/zoolinnean/zlaa019
https://doi.org/10.1093/zoolinnean/zlaa0...
, except A. brucutu that, due to the lack of molecular data, was inserted as incertae sedis in Gymnocharacini. In view of that, the species is herein assigned to Astyanax.

It is well known that, given the remarkable richness and phenotypic plasticity observed in the Neotropical freshwater ichthyofauna (Wimberger, 1992Wimberger PH. Plasticity of fish body shape. The effects of diet, development, family and age in two species of Geophagus (Pisces: Cichlidae). Biol J Linn Soc. 1992; 45:197–218. https://doi.org/10.1111/j.1095-8312.1992.tb00640.x
https://doi.org/10.1111/j.1095-8312.1992...
; Reis et al., 2016Reis RE, Albert JS, Di Dario F, Mincarone MMM, Petry PL, Rocha LR. Fish biodiversity and conservation in South America. J Fish Biol. 2016. https://doi.org/10.1111/jfb.13016
https://doi.org/10.1111/jfb.13016...
), and its high number of cryptic species (Piggott et al., 2011Piggott MP, Chao NL, Beheregaray LB. Three fishes in one: Cryptic species in an Amazonian floodplain forest specialist. Biol J Linn Soc. 2011; 102(2):391–403. https://doi.org/10.1111/j.1095-8312.2010.01571.x
https://doi.org/10.1111/j.1095-8312.2010...
), the genetic analysis is a powerful tool for improving our knowledge on taxonomy and evolution of this group (Bellafronte et al., 2013Bellafronte E, Mariguela TC, Pereira LHG, Oliveira C, Moreira-Filho O. DNA barcode of Parodontidae species from the La Plata River basin applying new data to clarify taxonomic problems. Neotrop Ichthyol. 2013; 11(3):497–506. https://doi.org/10.1590/S1679-62252013000300003
https://doi.org/10.1590/S1679-6225201300...
; Costa-Silva et al., 2015Costa-Silva GJ, Rodriguez MS, Roxo FF, Foresti F, Oliveira C. Using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. PLoS ONE. 2015; 10(9):1–12. https://doi.org/10.1371/journal.pone.0135075
https://doi.org/10.1371/journal.pone.013...
; Anjos et al., 2020Anjos MS, Bitencourt JA, Nunes LA, Sarmento-Soares LM, Carvalho DC, Armbruster JW et al. Species delimitation based on integrative approach suggests reallocation of genus in Hypostomini catfish (Siluriformes, Loricariidae). Hydrobiologia. 2020; 847:563–78. https://doi.org/10.1007/s10750-019-04121-z
https://doi.org/10.1007/s10750-019-04121...
). Different DNA-based approaches, such as DNA barcode (Hebert et al., 2003Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Royal Soc B Biol Sci. 2003; 270(1512):313–21. https://doi.org/10.1098/rspb.2002.2218
https://doi.org/10.1098/rspb.2002.2218...
; Ward, 2009Ward RD. DNA barcode divergence among species and genera of birds and fishes. Mol Ecol Res. 2009; 9(4):1077–85. https://doi.org/10.1111/j.1755-0998.2009.02541.x
https://doi.org/10.1111/j.1755-0998.2009...
), molecular species delimitation (Pons et al., 2006Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S et al. Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Syst Biol. 2006; 55(4):595–609. https://doi.org/10.1080/10635150600852011
https://doi.org/10.1080/1063515060085201...
; Puillandre et al., 2012Puillandre N, Lambert A, Brouillet S, Achaz G. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology. 2012; 21(8):1864–77. https://doi.org/10.1111/j.1365-294X.2011.05239.x
https://doi.org/10.1111/j.1365-294X.2011...
; Ratnasingham, Hebert, 2013Ratnasingham S, Hebert PDNN. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS ONE. 2013; 8(7):e66213. https://doi.org/10.1371/journal.pone.0066213
https://doi.org/10.1371/journal.pone.006...
), and molecular phylogeny analyses (Edwards, 2009Edwards SV. Is a new and general theory of molecular systematics emerging? Evolution. 2009; 63(1):1–19. https://doi.org/10.1111/j.1558-5646.2008.00549.x
https://doi.org/10.1111/j.1558-5646.2008...
), have been successfully used for defining Molecular Operational Taxonomic Units (MOTUs) and characterizing hidden biodiversity within Neotropical freshwater fish (e.g., Ramirez, Galetti Jr., 2015Ramirez JL, Galetti PM. DNA barcode and evolutionary relationship within Laemolyta Cope 1872 (Characiformes: Anostomidae) through molecular analyses. Mol Phylogenet Evol. 2015; 93:77–82. https://doi.org/10.1016/j.ympev.2015.07.021
https://doi.org/10.1016/j.ympev.2015.07....
; Carvalho et al., 2011Carvalho DC, Oliveira DAA, Pompeu PS, Leal CG, Oliveira C, Hanner R. Deep barcode divergence in Brazilian freshwater fishes: the case of the São Francisco River basin. Mitochondrial DNA. 2011; 22:80–86. https://doi.org/10.3109/19401736.2011.588214
https://doi.org/10.3109/19401736.2011.58...
; Pereira et al., 2011Pereira LHG, Maia GMG, Hanner R, Foresti F, Oliveira C. DNA barcodes discriminate freshwater fishes from the Paraíba do Sul River Basin, São Paulo, Brazil. Mitochondrial DNA. 2011; 22:71–79. https://doi.org/10.3109/19401736.2010.532213
https://doi.org/10.3109/19401736.2010.53...
, 2013Pereira LH, Hanner R, Foresti F, Oliveira C. Can DNA barcoding accurately discriminate megadiverse Neotropical freshwater fish fauna? BMC Genetics. 2013; 14(1):20. https://doi.org/10.1186/1471-2156-14-20
https://doi.org/10.1186/1471-2156-14-20...
; Machado et al., 2016Machado CB, Ishizuka TK, Freitas PD, Galetti Jr PM. DNA barcoding reveals taxonomic uncertainty in Salminus (Characiformes). System Biodivers. 2016; 15(4):372–82. https://doi.org/10.1080/14772000.2016.1254390
https://doi.org/10.1080/14772000.2016.12...
; Ramirez et al., 2017Ramirez JL, Birindelli JL, Carvalho DC, Affonso PRAM, Venere PC, Ortega H et al. Revealing hidden diversity of the underestimated neotropical ichthyofauna: DNA barcoding in the recently described genus Megaleporinus (Characiformes: Anostomidae). Front Genet. 2017; 8:149. https://doi.org/10.3389/fgene.2017.00149
https://doi.org/10.3389/fgene.2017.00149...
; Silva-Santos et al., 2018Silva-Santos R, Ramirez JL, Galetti PM, Freitas PD. Molecular evidences of a hidden complex scenario in Leporinus cf. friderici. Front Genet. 2018; 9:47. https://doi.org/10.3389/fgene.2018.00047
https://doi.org/10.3389/fgene.2018.00047...
; Souza et al., 2018Souza CR, de Mello Affonso PRA, de Araújo Bitencourt J, Sampaio I, Carneiro PLS. Species validation and cryptic diversity in the Geophagus brasiliensis Quoy & Gaimard, 1824 complex (Teleostei, Cichlidae) from Brazilian coastal basins as revealed by DNA analyses. Hydrobiologia. 2018; 809(1):309–21. https://doi.org/10.1007/s10750-017-3482-y
https://doi.org/10.1007/s10750-017-3482-...
; Lopes et al., 2020Lopes U, Galetti Jr PM, Freitas PD. Hidden diversity in Prochilodus nigricans: A new genetic lineage within the Tapajós River basin. PLoS ONE. 2020; 15(8):e0237916. https://doi.org/10.1371/journal.pone.0237916
https://doi.org/10.1371/journal.pone.023...
).

Here, we performed species delimitation analyses in four recently described species of Astyanax plus the morphotype A. aff. rupestris, all endemic to the upper Paraguaçu River basin. We aimed to produce a DNA barcode reference library for the focal species and to investigate the existence of hidden diversity, contributing thus to a better knowledge of this relevant fish group and its diversification. Using mitochondrial and nuclear sequences, we combined single and multilocus-based methods to carry out genetic analyses. Our sequence data were compared to those that had already been published in Astyanax species studies.

MATERIAL AND METHODS

Biological sampling. Biological samples of four endemic species and one morphotype of Astyanax were collected from 76 specimens distributed along six tributaries in the upper Paraguaçu River basin, Bahia, Brazil (Fig. 1). The material analyzed included A. brucutu from the Pratinha River, Iraquara (n = 3); A. epiagos from the Ferro Doido River, Jacobina (n = 5); A. lorien from the Preto River, Palmeiras (n = 15); A. rupestris from the Coisa Boa River, Andaraí (n = 17) and Cumbuca River, Mucugê (n = 3); and the morphotype Astyanax aff. rupestris from the Piabinha River, Mucugê (n = 33). Fin fragments were sampled from each specimen, using tweezers and scissors, and then stored in ethanol (95%) in a freezer at 4oC. Vouchers were deposited in the ichthyological collection of the Museu de História Natural da Bahia, Salvador, Bahia, Brazil. All information related to the sampling localities, specimens, and vouchers is available in Tab. S1.

We complemented our biological sampling by downloading 1,792 COI sequences available in the BOLD system database (http://www.boldsystems.org/, accessed on March 31, 2020) for 66 nominal Astyanax species of several hydrographic basins from localities informed by the database depositors (Tab. S2). That dataset included A. hamatilis from São José River (n = 7); and unidentified specimens of Astyanax sp. from Coité (n = 2) and Piabinha (n = 2) rivers from the upper Paraguaçu basin. Altogether we analyzed five from the six Astyanax species endemic to the upper Paraguaçu River, except A. sincora, that was not collected and was not available in the BOLD system as well.

FIGURE 1 |
Map of the Paraguaçu River basin, Bahia, northeastern Brazil, showing collection sites of Astyanax species sampled in this study and for two Astyanax sp. available in the BOLD system database (*), with the exception of A. hamatilis. Astyanax rupestris from the Coisa Boa River (dark blue square) and Cumbuca River (dark blue circle), A. aff. rupestris from the Piabinha River (half yellow and blue circle), Astyanax sp. from the Piabinha River (orange circle*), A. lorien from the Preto River (pink circle), Astyanax sp. from Coité River (brown circle*), A. brucutu from the Pratinha River (red circle), and A. epiagos from the Ferro Doido River (green circle). The colors of the symbols on the map are in accordance with Fig. 2. Scale 1:1300723.

DNA isolation, amplification, purification, and sequencing. DNA extraction was performed using buffer saline protocol (Aljanabi, 1997Aljanabi S. Universal and rapid salt-extraction of high quality genomic DNA for PCR- based techniques. Nucleic Acids Res. 1997; 25(22):4692–93. https://doi.org/10.1093/nar/25.22.4692
https://doi.org/10.1093/nar/25.22.4692...
), and DNA was quantified using a Biophotometer (Eppendorf, Hamburg, Germany). Partial Cytochrome c Oxidase subunit I (COI), Cytochrome b (Cytb) and the first intron of the S7 ribosomal protein (S7) genes were amplified using the following oligonucleotides: COI FishF1 and COI FishR1 (Ward et al., 2005Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN. DNA barcoding Australia’s fish species. Philos Trans R Soc B Biol Sci. 2005; 360(1462):1847–57. https://doi.org/10.1098/rstb.2005.1716
https://doi.org/10.1098/rstb.2005.1716...
), AnosCytBF and AnosCytBR (Ramirez, Galetti, 2015Ramirez JL, Galetti PM. DNA barcode and evolutionary relationship within Laemolyta Cope 1872 (Characiformes: Anostomidae) through molecular analyses. Mol Phylogenet Evol. 2015; 93:77–82. https://doi.org/10.1016/j.ympev.2015.07.021
https://doi.org/10.1016/j.ympev.2015.07....
), and S7RPEX1F and S7RPEX2R (Chow, Hazama, 1998Chow S, Hazama K. Universal PCR primers for S7 ribosomal protein gene introns in fish. Mol Ecol. 1998; 7(9):1255–56. https://doi.org/10.1046/j.1365-294x.1998.00406.x
https://doi.org/10.1046/j.1365-294x.1998...
). Polymerase Chain Reactions (PCRs) were performed according to their respective authors.

The amplified products were checked on agarose gel 1% by electrophoresis, and then purified with a polyethyleneglycol (PEG) 20% protocol (Lis, 1980Lis JT. Fractionation of DNA fragments by polyethylene glycol induced precipitation. Meth Enzymol. 1980; 65:347–53. https://doi.org/10.1016/S0076-6879(80)65044-7
https://doi.org/10.1016/S0076-6879(80)65...
). Sequencing was run on an automated sequencer ABI3730XL (Applied Biosystems, Little Chalfont, UK), and all sequences were aligned and edited with the Geneious 6.1.6c software (Kearse et al., 2012Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28(12):1647–49. https://doi.org/10.1093/bioinformatics/bts199
https://doi.org/10.1093/bioinformatics/b...
). The plugin “find heterozygotes” of this software was used with a 0.80 threshold in order to identify heterozygous positions and assign ambiguity codes in the nuclear sequences, such as eventual NUMTS (nuclear mitochondrial DNA segments). The S7 haplotypes were estimated using the SEQPHASE web tool (Flot, 2010Flot JF. Seqphase: A web tool for interconverting phase input/output files and FASTA sequence alignments. Mol Ecol Res. 2010; 10(1):162–66. https://doi.org/10.1111/j.1755-0998.2009.02732.x
https://doi.org/10.1111/j.1755-0998.2009...
). COI sequences were deposited in the BOLD system under Project name ASTBA. Cytb and S7 sequences were deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/) under specific accession numbers as shown in the Tab. S1.

Single locus analyses. To obtain a general picture of genetic relationships within Astyanax, we first implemented a broad Bayesian inference analysis with BEAST 2.4.6 (Heled, Drummond, 2010Heled J, Drummond AJ. Bayesian inference of species trees from multilocus data. Mol Biol Evol. 2010; 27(3):570–80. https://doi.org/10.1093/molbev/msp274
https://doi.org/10.1093/molbev/msp274...
) using a large COI sequence dataset, representing the four species studied herein and other 66 nominal Astyanax species obtained from BOLD. Two independent runs were performed following the parameters: 100 million generations (Markov chain Monte Carlo, MCMC), sampling every 10,000, a strict lognormal clock for all partitions, and the Yule speciation model. The best-fitting model (GTR+I+G) was selected under the Bayesian Information Criterion (BIC) by jModeltest 2 (Darriba et al., 2012Darriba D, Taboada GL, Doallo RR, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8):772–72. https://doi.org/10.1038/nmeth.2109
https://doi.org/10.1038/nmeth.2109...
). A consensus tree was combined and resampled in LogCombiner with 30% burn-in, and then summarized in TreeAnnotator using BEAST 2.4.6 (Heled, Drummond, 2010Heled J, Drummond AJ. Bayesian inference of species trees from multilocus data. Mol Biol Evol. 2010; 27(3):570–80. https://doi.org/10.1093/molbev/msp274
https://doi.org/10.1093/molbev/msp274...
). An effective sample size (ESS) of 200 or higher was required for all parameters and checked in TRACER 1.6 (Rambaut et al., 2014Rambaut A, Suchard MA, Xie D, Drummond AJ. Tracer v1.6 2014. Available from: http://beast.bio.ed.ac.uk/Tracer.
http://beast.bio.ed.ac.uk/Tracer....
).

Based on the COI tree, only the species recovered in a single clade, which included the four targeted Astyanax species, were hereafter analyzed. For this new dataset, we implemented three species delimitation approaches using the COI sequences: Barcode Index Number (BIN, Ratnasingham, Hebert, 2013Ratnasingham S, Hebert PDNN. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS ONE. 2013; 8(7):e66213. https://doi.org/10.1371/journal.pone.0066213
https://doi.org/10.1371/journal.pone.006...
), Automatic Barcode Gap Discovery (ABGD, Puillandre et al., 2012Puillandre N, Lambert A, Brouillet S, Achaz G. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology. 2012; 21(8):1864–77. https://doi.org/10.1111/j.1365-294X.2011.05239.x
https://doi.org/10.1111/j.1365-294X.2011...
), and General Mixed Yule Coalescent (GMYC, Pons et al., 2006Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S et al. Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Syst Biol. 2006; 55(4):595–609. https://doi.org/10.1080/10635150600852011
https://doi.org/10.1080/1063515060085201...
). The BIN analysis was performed automatically in the BOLD system. Our sampling was assembled in a preexisting BIN database or assigned to a new BIN (Ratnasingham, Hebert, 2013Ratnasingham S, Hebert PDNN. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS ONE. 2013; 8(7):e66213. https://doi.org/10.1371/journal.pone.0066213
https://doi.org/10.1371/journal.pone.006...
). For the ABGD we used the K2P (Kimura-2-parameters) modified parameters (Pmin = 0.04, Pmax = 0.1, relative value gap X = 0.1), and 100 steps. The GMYC analysis was implemented in the SPLITS package for R statistical software (R Development Core Team, 2017R Development Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2017. Available from: https://www.r-project.org/
https://www.r-project.org/...
), using a single threshold under the standard parameters (interval = c(1,10)). This analysis uses an ultrametric tree to establish species limits based on the Yule (pure-birth) and Kingman models (coalescence), and to calculate the probability of splits in a lineage based on speciation rates (Ratnasingham, Hebert, 2013Ratnasingham S, Hebert PDNN. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS ONE. 2013; 8(7):e66213. https://doi.org/10.1371/journal.pone.0066213
https://doi.org/10.1371/journal.pone.006...
). As input, we used an ultrametric tree obtained with a lognormal relaxed clock, birth-death speciation model, HKY + G substitution model, 50 million MCMC sampling every 5,000 and burn-in of 10% in BEAST 2.4.6. Convergence was assessed by estimating the effective sampling size (ESS) using Tracer 1.7 software (Rambaut et al., 2014Rambaut A, Suchard MA, Xie D, Drummond AJ. Tracer v1.6 2014. Available from: http://beast.bio.ed.ac.uk/Tracer.
http://beast.bio.ed.ac.uk/Tracer....
) and accepting ESS values of 200 or more.

We calculated the genetic distances among the MOTUs obtained through the three species delimitation methods using the K2P model with MEGA 7.0.26 (Kumar et al., 2016Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7):1870–74. https://doi.org/10.1093/molbev/msw054
https://doi.org/10.1093/molbev/msw054...
). We used the K2P, since this model allows us to compare the values found here with those previously reported in other Astyanax studies (e.g., Carvalho et al., 2011Carvalho DC, Oliveira DAA, Pompeu PS, Leal CG, Oliveira C, Hanner R. Deep barcode divergence in Brazilian freshwater fishes: the case of the São Francisco River basin. Mitochondrial DNA. 2011; 22:80–86. https://doi.org/10.3109/19401736.2011.588214
https://doi.org/10.3109/19401736.2011.58...
; Pereira et al., 2011Pereira LHG, Maia GMG, Hanner R, Foresti F, Oliveira C. DNA barcodes discriminate freshwater fishes from the Paraíba do Sul River Basin, São Paulo, Brazil. Mitochondrial DNA. 2011; 22:71–79. https://doi.org/10.3109/19401736.2010.532213
https://doi.org/10.3109/19401736.2010.53...
; Rossini et al., 2018). Collins et al., (2012)Collins RA, Boykin LM, Cruickshank RH, Armstrong KF. Barcoding’s next top model: an evaluation of nucleotide substitution models for specimen identification. Methods Ecol Evol. 2012; 3:457–65. https://doi.org/10.1111/j.2041-210X.2011.00176.x
https://doi.org/10.1111/j.2041-210X.2011...
tested whether the K2P is a well-fitted model at the species level by comparing it to the other models (JC, F81, TrN, HKY, HKY+C and GTR+C) using data sets from different animal groups, including fish. The results indicate that the differences in distance between K2P and other models were usually minimal, and the identification success rates were largely unaffected by model choice, even when interspecific threshold values were reassessed.

Multilocus analyses. To obtain a multilocus Bayesian species tree (ST) we considered the nominal species recognized by morphological studies and the results generated by the GMYC analysis. This analysis was performed in BEAST (Star-BEAST) using 500 million MCMC, sampling every 10,000, relaxed clock and Yule models, and a burn-in of 20%. Nucleotide substitution models were selected based on BIC (Bayesian Information Criterion) using jModeltest 2 (Darriba et al., 2012Darriba D, Taboada GL, Doallo RR, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8):772–72. https://doi.org/10.1038/nmeth.2109
https://doi.org/10.1038/nmeth.2109...
). The best-fitting models were HKY for COI and Cytb, and F81 + G for S7. All generations were sampled from the stationary phase. The convergence of analyses and adequate ESS (>200) were evaluated in Tracer v1.7 (Rambaut et al., 2014Rambaut A, Suchard MA, Xie D, Drummond AJ. Tracer v1.6 2014. Available from: http://beast.bio.ed.ac.uk/Tracer.
http://beast.bio.ed.ac.uk/Tracer....
).

The Bayesian ST was used as guide tree to the Bayesian species delimitation approach using multilocus data (Yang, Rannala, 2010Yang Z, Rannala B. Bayesian species delimitation using multilocus sequence data. Proc Natl Acad Sci USA. 2010; 107(20):9264–69. https://doi.org/10.1073/pnas.0913022107
https://doi.org/10.1073/pnas.0913022107...
; Rannala, Yang, 2013Rannala B, Yang Z. Improved reversible jump algorithms for Bayesian species delimitation. Genetics. 2013; 194(1):245–53. https://doi.org/10.1534/genetics.112.149039
https://doi.org/10.1534/genetics.112.149...
) in the BP&P 3.3 software (Yang, 2015Yang Z. The BPP program for species tree estimation and species delimitation. Curr Zool. 2015; 61(5):854–65. https://doi.org/10.1093/czoolo/61.5.854
https://doi.org/10.1093/czoolo/61.5.854...
). This software uses a coalescent model, calculating the posterior probability of potential species considering the coalescent process of lineage sorting. The basic model used by BP&P involves two types of parameters: the population sizes on the species tree (θs), and the species divergence times (τs). To evaluate the impact of these parameters on species delimitation results and consider a range of speciation histories, we tested different gamma prior configurations and some default parameters in four distinct combinations. A first one assumed relatively large ancestral population sizes and deep divergences (θ ~ G(1,10) and τ0 ~ G(1, 10)) among species; a second combination considered small ancestral population sizes and shallow divergences among species (θ ~ G(2, 2000) and τ0 ~ G(2, 2000)); and the other two combinations assumed either large ancestral populations sizes (θ ~ G(1, 10)) and relatively shallow divergences among species (τ0 ~ G(2, 2000)), or small ancestral population sizes and deep divergences (θ ~ G(2, 2000), τ0 ~ G(1, 10)).

RESULTS

From the 76 individuals sampled, we obtained 75 COI sequences, comprised of 614 bp, without stop-codons, deletions, or insertions. Our primary Bayesian tree, obtained with a total of 1,867 COI sequences (75 generate herein and 1,792 downloaded from BOLD), grouped all Astyanax species belonging to the upper Paraguaçu River basin in a clade named hereafter Clade 1, with 0.95 probability posterior value (Fig. 2A), except A. hamatilis. This latter species was joined with species from other hydrographic basins (Astyanax taeniatus Jenyns, 1842 from the Ribeira da Terra Firme River; A. burgeraiZanata & Camelier, 2009Zanata AM, Camelier P. Astyanax vermilion and Astyanax burgerai: New characid fishes (Ostariophysi: Characiformes) from northeastern Bahia, Brazil. Neotrop Ichthyol. 2009; 7(2):175–84. https://doi.org/10.1590/S1679-62252009000200007
https://doi.org/10.1590/S1679-6225200900...
from the Almada River; Astyanax sp. from the Marcanaí River, and Astyanax sp. from the Macacuá River) in a distant clade from Clade 1.

The Clade 1 recovered 19 nominal Astyanax species from 17 hydrographic basins, which are part of the Brazilian crystalline shield and the Atlantic coast drainages (Figs. 2A, B), representing 390 sequences for A. bifasciatusGaravello & Sampaio, 2010Garavello JC, Sampaio FAA. Five new species of genus Astyanax Baird & Girard, 1854 from Rio Iguaçu, Paraná, Brazil (Ostariophysi, Characiformes, Characidae). Braz J Biol. 2010; 70(3):847–65. https://doi.org/10.1590/S1519-69842010000400016
https://doi.org/10.1590/S1519-6984201000...
, A. bockmanniVari & Castro, 2007Vari RP, Castro RMC. New species of Astyanax (Ostariophysi: Characiformes: Characidae) from the upper rio Paraná system, Brazil. Copeia. 2007; 2007(1):150–62. https://doi:10.1643/0045-8511(2007)7[150:NSOAOC]2.0.CO;2
https://doi:10.1643/0045-8511(2007)7[150...
, A. aff. bockmanni, A. dissimilisGaravello & Sampaio, 2010Garavello JC, Sampaio FAA. Five new species of genus Astyanax Baird & Girard, 1854 from Rio Iguaçu, Paraná, Brazil (Ostariophysi, Characiformes, Characidae). Braz J Biol. 2010; 70(3):847–65. https://doi.org/10.1590/S1519-69842010000400016
https://doi.org/10.1590/S1519-6984201000...
, A. fasciatus Cuvier, 1819, A. gymnodontus Eigenmann, 1911, A. gymnogenys Eigenmann, 1911, and A. minor Garavello & Sampaio, 2010 from Paraná River basin; A. paranae Eigenmann, 1914 (Paraná and Paraguay basins); A. intermedius Eigenmann, 1908 from Paraíba do Sul basin; A. aff. intermedius from Paraná and Paraíba do Sul basins; A. cf. fasciatus and A. rivularis Lutken, 1875 from Paraná and São Francisco basins; A. scabripinnis Jennys, 1842 from Paraná, Paraíba do Sul, Paraguay, São Francisco, and Doce basins; A. bimaculatus Linnaeus, 1758 from São Francisco basin; A. laticeps Cope, 1894 and A. obscurus Hensel, 1870 from Itapocu basin; A. aff. fasciatus and A. aff. jequitinhonhae Steindachner, 1877 from Jequitinhonha basin; A. xavanteGarutti & Venere, 2009Garutti V, Venere PC. Astyanaxxavante, a new species of characid from middle rio Araguaia in the cerrado region, Central Brazil (Characiformes: Characidae). Neotrop Ichthyol. 2009; 7(3):377–83. https://doi.org/10.1590/S1679-62252009000300004
https://doi.org/10.1590/S1679-6225200900...
from Araguaia basin; and A. brucutu, A. epiagos, A. lorien, A. rupestris, and A. aff. rupestris from upper Paraguaçu basin (Fig. 2B).

FIGURE 2 |
Bayesian tree showing phylogenetic relationships among Astyanax species, using 1,792 COI sequences available in the Bold system database, and 75 ones produced in this study for specimens of Astyanax endemic to the upper Paraguaçu River basin. A. Clusters (black) for the clade 1 formed by Astyanax species closely related to the specimens collected in the Paraguaçu River; and clusters (grey) for the remaining analyzed Astyanax species. B. Clade 1 in details, depicting the delimitation species results using BIN, ABGD, and GMYC approaches. Black rectangles represent the distinct number of MOTUs identified by the three analyses: BIN (MOTU 1-5); ABGD (MOTU 1-19); GMYC (MOTU 1-50). The numbers in the nodes correspond to the main clusters of species (Tab. S2). Nodes marked with an asterisk denote posterior probabilities greater than 0.9. Species of Astyanax from the upper Paraguaçu River basin are highlighted in colored rectangles. Astyanax sp. sequences were download from BOLD system database. Our studied species are in bold letters. The colors of the species name are highlighted in accordance with Fig. 1.

Specimens of Astyanax sp. were named following the indication of the collection site reported in the BOLD database. Among the specimens identified at the genus level only (i.e., Astyanax sp.), two sequences belong to individuals from the Coité River and two belong to individuals collected in the Piabinha River, both rivers from the upper Paraguaçu River basin. Details about the samples are available in the Tab. S2.

Single locus species delimitation analyses. Our single locus delimitation analyses for the species obtained in Clade 1, with COI sequences, showed different results among the three approaches used herein (BIN, ABGD, GMYC, Fig. 2B). The BIN approach recovered five distinct MOTUs. Focusing on the species from the Paraguaçu River basin, A. brucutu, A. epiagos, and A. lorien were grouped with Astyanax sp. Coité and thirteen nominal species in a single MOTU (MOTU 1, BIN AAC5910). Astyanax rupestris (MOTU 4, BIN ADI2769) was separated from A. aff. rupestris (MOTU 5, BIN ACR6356), while Astyanax sp. Piabinha was grouped with this latter. The mean divergence within BINs ranged from 0% (MOTU 2, BIN ABZ0055) to 1.7% (MOTU 1, BIN AAC5910), and the pairwise divergence between BINs ranged from 1.8% (MOTU 4, BIN ADI2769 and MOTU 5, BIN ACR6356) to 3.6% (MOTU 4, BIN ADI2769 and MOTU 3, BIN ABZ6219).

The ABGD analysis indicated the presence of 19 distinct MOTUs into Clade 1, with four singletons, i.e., four MOTUs represented only by a single individual. The average genetic distances within and between these MOTUs were 0.18% and 2.5%, respectively. We found A. brucutu, A. lorien, and Astyanax sp. Coité grouped into the MOTU 1 with 12 nominal species. The species A. epiagos and A. rupestris were separately recovered in the MOTU 4 and 17, respectively. The genetic distance between MOTU 1 and 4 was 1.8%. Differently from the BIN analysis, A. aff. rupestris was divided in two MOTUs named A. aff. rupestris 1 (MOTU 18) and A. aff. rupestris 2 (MOTU 19), with a genetic distance between them equal to 0.7%.

The GMYC results showed a total of 50 MOTUs, of which eight were singletons. The confidence limit for the estimated number of entities ranged from 49 to 60. The null model likelihood (L0 = 3877.946) was significantly (p < 0.01) lower than the GMYC model likelihood (L = 4039.65), indicating that there is probably more than one species in our sample. We observed A. brucutu and A. lorien grouped in the same MOTU (MOTU 26), while Astyanax sp. Coité River was clustered to the species A. bimaculatus, A. cf. fasciatus, and A. fasciatus from Miriri and São Francisco basins (MOTU 25). On the other hand, A. epiagos (MOTU 18), A. rupestris (MOTU 48), A. aff. rupestris 1 (MOTU 49), and A. aff. rupestris 2 (MOTU 50) were recovered as independent MOTUs. The average genetic distance values were 0.14% for intra- and 1.8% for inter-MOTUs.

The average genetic distance values calculated between Astyanax from the Paraguaçu River basin, defined by the BP&P analysis, were 0.0% (intra-MOTU) and 2.1% (inter-MOTU). The maximum intra-MOTU distance was 0.001% (A. rupestris), and the minimum inter-MOTU distance was 0.3% (A. brucutu and A. lorien). Astyanax aff. rupestris 1 and A. aff. rupestris 2 showed 0.7% inter-MOTU distance, while both diverged 1.8% from A. rupestris (see Tab. S3).

Multilocus species delimitation. After edition and alignment, the dataset for A. brucutu, A. epiagos, A. lorien, A. rupestris, and A. aff. rupestris consisted of 140 S7 sequences with 684 bp, and 75 COI and 73 Cytb sequences, comprising fragments with 614 bp and 1,032 bp, respectively. No stop-codons, deletions or insertions were observed.

The BEAST* analyses used to obtain a guide tree reached apparent convergence, with ESS of at least 300 for all parameters, showing convergence between runs. The BP&P results, using this prior information, separated A. brucutu, A. epiagos, A. lorien, and A. rupestris, and, similarly to the ABGD and GMYC results, suggested the existence of two genetic lineages within A. aff. rupestris (A. aff. rupestris 1 and A. aff. rupestris 2). The speciation probabilities assumed maximum values (1.0) on all nodes. Moreover, the species delimitation results were not affected by different prior settings, and we recovered maximum speciation probability values for all internal nodes in all tested combinations, indicating consistent results among runs (Fig. 3).

FIGURE 3 |
Species tree showing phylogenetic relationships for Astyanax’s MOTUs from the upper Paraguaçu River basin. The tree was generated using approximately 2,230 bp obtained for the COI, Cytb, and S7 sequences for the samples indicated in Tab. S1. The topology corresponds to the Bayesian tree. The numbers on the branches are bootstrap values for the posterior probability for Bayesian species tree and speciation probability values of BP&P species delimitation. The scale bar indicates nucleotide substitutions per site.

DISCUSSION

Our major phylogenetic analysis recovered all endemic species from the upper Paraguaçu River studied here in a single and large clade (Clade 1). It is noteworthy that although we have included four of the six endemic species, we did not include all Astyanax species described for the Paraguaçu River basin (Santos, Caramaschi, 2007Santos ACA, Caramaschi EP. Composition and seasonal variation of the ichthyofauna from upper Rio Paraguaçu (Chapada Diamantina, Bahia, Brazil). Braz Arch Biol Technol. 2007; 50:663–72. https://doi.org/10.1590/s1516-89132007000400012
https://doi.org/10.1590/s1516-8913200700...
, 2011Santos ACA, Caramaschi EP. Temporal variation in fish composition and abundance in a perennial tributary of the rio Paraguaçu, a little-known drainage in the Brazilian Semi-Arid region. Neotrop Ichthyol. 2011; 9(1):153–60. https://doi.org/10.1590/S1679-62252011005000007
https://doi.org/10.1590/S1679-6225201100...
), therefore, Clade 1 must be incomplete. Despite this, our findings suggest that fishes of this basin share an evolutionary history that can result in its high level of endemism (Buckup, 2011Buckup PA. The Eastern Brazilian Shield. In: Albert JS, Reis RE, editors. Historical biogeography of Neotropical freshwater fishes. Los Angeles: University of California Press; 2011. p.203–10. ; Camelier, Zanata, 2014aCamelier P, Zanata AM. Biogeography of freshwater fishes from the northeastern Mata Atlântica freshwater ecoregion: Distribution, endemism, and area relationships. Neotrop Ichthyol. 2014a; 12(4):683–98. https://doi.org/10.1590/1982-0224-20130228
https://doi.org/10.1590/1982-0224-201302...
; de Pinna et al., 2018de Pinna M, Abrahão V, Reis V, Zanata A. A new species of Copionodon representing a relictual occurrence of the Copionodontinae (Siluriformes: Trichomycteridae), with a CT-scan imaging survey of key subfamilial features. Neotrop Ichthyol. 2018; 16(4):e180049. https://doi.org/10.1590/1982-0224-20180049
https://doi.org/10.1590/1982-0224-201800...
). Interestingly, we observed a pattern of genetic proximity between species of Clade 1 and species from distinct hydrographic basins, such as the Brazilian crystalline shield and the Atlantic coastal drainages (i.e., São Francisco, Paraná, and Paraíba do Sul basins). According to Ribeiro, (2006)Ribeiro AC. Tectonic history and the biogeography of the freshwater fishes from the coastal drainages of eastern Brazil: An example of faunal evolution associated with a divergent continental margin. Neotrop Ichthyol. 2006; 4(2):225–46. https://doi.org/10.1590/S1679-62252006000200009
https://doi.org/10.1590/S1679-6225200600...
, geological events between upland crystalline drainages and Atlantic tributaries occurred at different times, causing seemingly distant basins to share species or even species complex. The Paraguaçu River has an extensive system of branching headwaters that are adjacent to the eastern streams of the São Francisco basin (Buckup, 2011Buckup PA. The Eastern Brazilian Shield. In: Albert JS, Reis RE, editors. Historical biogeography of Neotropical freshwater fishes. Los Angeles: University of California Press; 2011. p.203–10. ). In fact, several sister taxa or genetically related species between São Francisco River and NMAF ecoregion, presently separated by the Espinhaço Mountains, have been already reported (e.g., Camelier, Zanata, 2014aCamelier P, Zanata AM. Biogeography of freshwater fishes from the northeastern Mata Atlântica freshwater ecoregion: Distribution, endemism, and area relationships. Neotrop Ichthyol. 2014a; 12(4):683–98. https://doi.org/10.1590/1982-0224-20130228
https://doi.org/10.1590/1982-0224-201302...
; Sarmento-Soares et al., 2016Sarmento-Soares LM, Britski HA, Anjos M, Zanata AM, Martins-Pinheiro RF, Barretto M. First record of genus Imparfinis from a northeastern coastal Brazilian River Basin: I. borodini Mees & Cala, 1989 in Rio de Contas, Bahia. Check List. 2016; 12:1832–48. http://dx.doi.org/10.15560/12.1.1832
http://dx.doi.org/10.15560/12.1.1832...
; Ramirez et al., 2017Ramirez JL, Birindelli JL, Carvalho DC, Affonso PRAM, Venere PC, Ortega H et al. Revealing hidden diversity of the underestimated neotropical ichthyofauna: DNA barcoding in the recently described genus Megaleporinus (Characiformes: Anostomidae). Front Genet. 2017; 8:149. https://doi.org/10.3389/fgene.2017.00149
https://doi.org/10.3389/fgene.2017.00149...
; Anjos et al., 2020Anjos MS, Bitencourt JA, Nunes LA, Sarmento-Soares LM, Carvalho DC, Armbruster JW et al. Species delimitation based on integrative approach suggests reallocation of genus in Hypostomini catfish (Siluriformes, Loricariidae). Hydrobiologia. 2020; 847:563–78. https://doi.org/10.1007/s10750-019-04121-z
https://doi.org/10.1007/s10750-019-04121...
).

In turn, although the species delimitation methods can be delimiting lineages, but not necessarily species (Carstens et al., 2013Carstens BC, Pelletier TA, Reid NM, Satler JD. How to fail at species delimitation. Mol Ecol. 2013; 22:4369–83. https://doi.org/10.1111/mec.12413
https://doi.org/10.1111/mec.12413...
; Sukumaran, Knowles, 2017Sukumaran J, Knowles LL. Multispecies coalescent delimits structure, not species. Proc Natl Acad Sci USA. 2017; 114:1607–12. https://doi.org/10.1073/pnas.1607921114
https://doi.org/10.1073/pnas.1607921114...
), our results are in accordance with the taxa considered valid to the upper Paraguaçu basin (A. brucutu, A. epiagos, A. lorien, A. rupestris) and revealed the existence of two genetic lineages within A. aff. rupestris. However, the number of identified MOTUs was method-dependent, as previously reported in similar studies (e.g., Costa-Silva et al., 2015Costa-Silva GJ, Rodriguez MS, Roxo FF, Foresti F, Oliveira C. Using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. PLoS ONE. 2015; 10(9):1–12. https://doi.org/10.1371/journal.pone.0135075
https://doi.org/10.1371/journal.pone.013...
; Rossini et al., 2016Rossini BC, Oliveira CAM, Melo FAG, Bertaco VA, Astarloa JMD, Rosso JJ et al. Highlighting Astyanax species diversity through DNA barcoding. PLoS ONE. 2016; 11(12):e0167203. https://doi.org/10.1371/journal.pone.0167203
https://doi.org/10.1371/journal.pone.016...
; Machado et al., 2018Machado VN, Collins RA, Ota RP, Andrade MC, Farias IP, Hrbek T. One thousand DNA barcodes of piranhas and pacus reveal geographic structure and unrecognised diversity in the Amazon. Sci Rep. 2018; 8:8387. https://doi.org/10.1038/s41598-018-26550-x
https://doi.org/10.1038/s41598-018-26550...
). These discrepancies may likely be due to analytical differences inherent to each method. The BIN and ABGD methods are based on genetic distances. The former is a result of the refined single linkage (RESL), which associates COI sequences with an identifier (BIN) based on a distance value automatically delineated (Ratnasingham, Hebert, 2013Ratnasingham S, Hebert PDNN. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS ONE. 2013; 8(7):e66213. https://doi.org/10.1371/journal.pone.0066213
https://doi.org/10.1371/journal.pone.006...
), while ABGD requires a priori specification of an intraspecific distance threshold (Puillandre et al., 2012Puillandre N, Lambert A, Brouillet S, Achaz G. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology. 2012; 21(8):1864–77. https://doi.org/10.1111/j.1365-294X.2011.05239.x
https://doi.org/10.1111/j.1365-294X.2011...
). Contrastingly, GMYC uses coalescence approaches, and it requires an ultrametric gene tree in which branches are assigned to one lineage per species or multiple lineages per species (Pons et al., 2006Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S et al. Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Syst Biol. 2006; 55(4):595–609. https://doi.org/10.1080/10635150600852011
https://doi.org/10.1080/1063515060085201...
). In addition, inconsistencies between the species delimitation methods may be biased by the molecular markers used (Hebert et al., 2003Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Royal Soc B Biol Sci. 2003; 270(1512):313–21. https://doi.org/10.1098/rspb.2002.2218
https://doi.org/10.1098/rspb.2002.2218...
) and the limited sample sizes (Carstens et al., 2013Carstens BC, Pelletier TA, Reid NM, Satler JD. How to fail at species delimitation. Mol Ecol. 2013; 22:4369–83. https://doi.org/10.1111/mec.12413
https://doi.org/10.1111/mec.12413...
) common to many investigations. It has been suggested that since distance methods rely heavily on the disparity between intra- and interspecific variation, an incomplete taxonomic sampling could influence the accuracy of the method (Frézal, Leblois, 2008Frézal L, Leblois R. Four years of DNA barcoding: current advances and prospects. Infect Genet Evol. 2008, 8:727–36. https://doi.org/10.1016/j.meegid.2008.05.005
https://doi.org/10.1016/j.meegid.2008.05...
).

The BIN analysis showed more conservative results, grouping the nominal species A. brucutu, A. epiagos, and A. lorien, plus Astyanax sp. from Coité River, in a single MOTU; and separating A. rupestris from the A. aff. rupestris and Astyanax sp. Piabinha. The BIN method uses 2.2% threshold, splitting species in new BINs when this value is at least twice higher (e.g., 4.4%) (Ratnasingham, Hebert, 2013Ratnasingham S, Hebert PDNN. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS ONE. 2013; 8(7):e66213. https://doi.org/10.1371/journal.pone.0066213
https://doi.org/10.1371/journal.pone.006...
). Although genetic distances equal or higher than 2% are commonly used to separate MOTUs (Hebert et al., 2004Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM. Identification of birds through DNA Barcodes. PLoS Biology. 2004; 2(10):e312. https://doi.org/10.1371/journal.pbio.0020312
https://doi.org/10.1371/journal.pbio.002...
; Ward, 2009Ward RD. DNA barcode divergence among species and genera of birds and fishes. Mol Ecol Res. 2009; 9(4):1077–85. https://doi.org/10.1111/j.1755-0998.2009.02541.x
https://doi.org/10.1111/j.1755-0998.2009...
), this threshold can underestimate the number of species when applied to complex groups such as Astyanax. For Neotropical fishes, smaller genetic divergence values have been reported for congeneric species with recent divergence (e.g., Carvalho et al., 2011Carvalho DC, Oliveira DAA, Pompeu PS, Leal CG, Oliveira C, Hanner R. Deep barcode divergence in Brazilian freshwater fishes: the case of the São Francisco River basin. Mitochondrial DNA. 2011; 22:80–86. https://doi.org/10.3109/19401736.2011.588214
https://doi.org/10.3109/19401736.2011.58...
; Pereira et al., 2011Pereira LHG, Maia GMG, Hanner R, Foresti F, Oliveira C. DNA barcodes discriminate freshwater fishes from the Paraíba do Sul River Basin, São Paulo, Brazil. Mitochondrial DNA. 2011; 22:71–79. https://doi.org/10.3109/19401736.2010.532213
https://doi.org/10.3109/19401736.2010.53...
; Ramirez, Galetti, 2015Ramirez JL, Galetti PM. DNA barcode and evolutionary relationship within Laemolyta Cope 1872 (Characiformes: Anostomidae) through molecular analyses. Mol Phylogenet Evol. 2015; 93:77–82. https://doi.org/10.1016/j.ympev.2015.07.021
https://doi.org/10.1016/j.ympev.2015.07....
; Machado et al., 2016Machado CB, Ishizuka TK, Freitas PD, Galetti Jr PM. DNA barcoding reveals taxonomic uncertainty in Salminus (Characiformes). System Biodivers. 2016; 15(4):372–82. https://doi.org/10.1080/14772000.2016.1254390
https://doi.org/10.1080/14772000.2016.12...
; Ramirez et al., 2017Ramirez JL, Birindelli JL, Carvalho DC, Affonso PRAM, Venere PC, Ortega H et al. Revealing hidden diversity of the underestimated neotropical ichthyofauna: DNA barcoding in the recently described genus Megaleporinus (Characiformes: Anostomidae). Front Genet. 2017; 8:149. https://doi.org/10.3389/fgene.2017.00149
https://doi.org/10.3389/fgene.2017.00149...
; Ribolli et al., 2021Ribolli J, Zabonini Filho E, Scaranto BMS, Shibatta OA, Machado CB. Cryptic diversity and diversification processes in three cis-Andean Rhamdia species (Siluriformes: Heptapteridae) revealed by DNA barcoding. Genet Mol Biol. 2021; 44(3):e20200470. https://doi.org/10.1590/1678-4685-GMB-2020-0470
https://doi.org/10.1590/1678-4685-GMB-20...
), and 1% has been acclaimed as the optimal threshold for those belonging to species complexes (Hubert et al., 2008Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, Burridge M et al. Identifying canadian freshwater fishes through DNA barcodes. PLoS ONE. 2008; 3(6):e2490. https://doi.org/10.1371/journal.pone.0002490
https://doi.org/10.1371/journal.pone.000...
; Pereira et al., 2011Pereira LHG, Maia GMG, Hanner R, Foresti F, Oliveira C. DNA barcodes discriminate freshwater fishes from the Paraíba do Sul River Basin, São Paulo, Brazil. Mitochondrial DNA. 2011; 22:71–79. https://doi.org/10.3109/19401736.2010.532213
https://doi.org/10.3109/19401736.2010.53...
). Therefore, the nature of the algorithms used by the BIN method may suffer interference when employed in hyper-diverse groups. For these latter groups, GMYC analyses have been considered more efficient than other methods (Ratnasingham, Hebert, 2013Ratnasingham S, Hebert PDNN. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS ONE. 2013; 8(7):e66213. https://doi.org/10.1371/journal.pone.0066213
https://doi.org/10.1371/journal.pone.006...
; Costa-Silva et al., 2015Costa-Silva GJ, Rodriguez MS, Roxo FF, Foresti F, Oliveira C. Using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. PLoS ONE. 2015; 10(9):1–12. https://doi.org/10.1371/journal.pone.0135075
https://doi.org/10.1371/journal.pone.013...
; Ribolli et al., 2021Ribolli J, Zabonini Filho E, Scaranto BMS, Shibatta OA, Machado CB. Cryptic diversity and diversification processes in three cis-Andean Rhamdia species (Siluriformes: Heptapteridae) revealed by DNA barcoding. Genet Mol Biol. 2021; 44(3):e20200470. https://doi.org/10.1590/1678-4685-GMB-2020-0470
https://doi.org/10.1590/1678-4685-GMB-20...
), consisting in one of the most accepted approaches for species delimitation based on a single locus analysis (Costa-Silva et al., 2015Costa-Silva GJ, Rodriguez MS, Roxo FF, Foresti F, Oliveira C. Using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. PLoS ONE. 2015; 10(9):1–12. https://doi.org/10.1371/journal.pone.0135075
https://doi.org/10.1371/journal.pone.013...
). In Astyanax, this method was already chosen as the most appropriate for species delimitation (Rossini et al., 2016Rossini BC, Oliveira CAM, Melo FAG, Bertaco VA, Astarloa JMD, Rosso JJ et al. Highlighting Astyanax species diversity through DNA barcoding. PLoS ONE. 2016; 11(12):e0167203. https://doi.org/10.1371/journal.pone.0167203
https://doi.org/10.1371/journal.pone.016...
).

Following our single locus analysis and GMYC results, this study showed five MOTUs among the focused species in the Paraguaçu basin, recognizing A. epiagos (MOTU 28) and A. rupestris (MOTU 48) as distinct species, but joining the two nominal species A. lorien and A. brucutu in a single clade (MOTU 26). Of note, the multilocus species delimitation approach used here was able to separate the latter species into different MOTUs. Moreover, the description of both species was based on strong diagnostic morphological characters and distinct habitats (Zanata et al., 2017Zanata AM, Lima FC, Dario F, Garhard P. A new remarkable and Critically Endangered species of Astyanax Baird & Girard (Characiformes: Characidae) from Chapada Diamantina, Bahia, Brazil, with a discussion on durophagy in the Characiformes. Zootaxa. 2017; 4232(4):491–510. https://doi.org/10.11646/zootaxa.4232.4.2
https://doi.org/10.11646/zootaxa.4232.4....
, 2018Zanata AM, Burger R, Camelier P. Two new species of Astyanax Baird & Girard (Characiformes: Characidae) from the upper rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2018; 4438(3):471–490. https://doi.org/10.11646/zootaxa.4438.3.3
https://doi.org/10.11646/zootaxa.4438.3....
; Vita et al., 2020Vita G, Zanata AM, Datovo A. Anatomy and ontogenetic changes of the facial and gular musculature of the tetra Astyanax brucutu: A remarkable case of adaptation to durophagy. J Anat. 2020; 237(6):1136–50. https://doi.org/10.1111/joa.13280
https://doi.org/10.1111/joa.13280...
). According to these authors, Astyanax brucutu presents a unique mandibular morphology similarly found only in specimens of Creagrutus Günther, 1864 and Piabina Reinhardt, 1867. Furthermore, A. brucutu inhabits a geographical region characterized by a distinctive combination of environmental attributes, such as high transparent water, elevated levels of dissolved oxygen, patches of gastropod shells on the bottom and coarse substrate partially covered by aquatic macrophytes, which are not observed elsewhere in the basin or adjacent drainages (Zanata et al., 2017Zanata AM, Lima FC, Dario F, Garhard P. A new remarkable and Critically Endangered species of Astyanax Baird & Girard (Characiformes: Characidae) from Chapada Diamantina, Bahia, Brazil, with a discussion on durophagy in the Characiformes. Zootaxa. 2017; 4232(4):491–510. https://doi.org/10.11646/zootaxa.4232.4.2
https://doi.org/10.11646/zootaxa.4232.4....
). Additionally, these nominal species could be easily identified by the monophyly criterion and their allopatric distribution.

Although it is parsimonious to consider A. lorien and A. brucutu as valid species, an alternative hypothesis is to assume that the morphological differences among them are possibly related to local adaptations, since the presence of barriers to gene flow can promote such phenotypic differences in distinct populations or lineages (Zamudio et al., 2016Zamudio KR, Bell RC, Mason NA. Phenotypes in phylogeography: Species’ traits, environmental variation, and vertebrate diversification. Proc Natl Acad Sci USA. 2016; 113(29):8041–48. https://doi.org/10.1073/pnas.1602237113
https://doi.org/10.1073/pnas.1602237113...
). Rossini et al., (2016)Rossini BC, Oliveira CAM, Melo FAG, Bertaco VA, Astarloa JMD, Rosso JJ et al. Highlighting Astyanax species diversity through DNA barcoding. PLoS ONE. 2016; 11(12):e0167203. https://doi.org/10.1371/journal.pone.0167203
https://doi.org/10.1371/journal.pone.016...
argued that Astyanax local populations described as new species due only to their restricted geographical distribution or local adaptations could be synonymized in the future. Therefore, further phylogeographic studies incorporating a larger sampling, genomic data, and considering population size and divergence time as relevant parameters should be performed to reassess the genetic relationships between A. lorien and A. brucutu.

Meantime, low genetic distances among species have been often associated with recent divergences, in which the time to accumulate genetic differences is quite short (Ornelas-García et al., 2008Ornelas-García CP, Domínguez-Domínguez O, Doadrio I. Evolutionary history of the fish genus Astyanax Baird & Girard (1854) (Actinopterygii, Characidae) in Mesoamerica reveals multiple morphological homoplasies. BMC Evol Biol. 2008; 8:340. https://doi.org/10.1186/1471-2148-8-340
https://doi.org/10.1186/1471-2148-8-340...
). Previous studies using the COI gene have already detected low genetic distances among species of Astyanax, reporting 0.93% between A. cf. fasciatus and A. rivularis (e.g., Carvalho et al., 2011Carvalho DC, Oliveira DAA, Pompeu PS, Leal CG, Oliveira C, Hanner R. Deep barcode divergence in Brazilian freshwater fishes: the case of the São Francisco River basin. Mitochondrial DNA. 2011; 22:80–86. https://doi.org/10.3109/19401736.2011.588214
https://doi.org/10.3109/19401736.2011.58...
). Low interspecific genetic distance values related to recent divergence have also been described within other fish genera, such as Parodon Valenciennes, 1849 (0.4%; Bellafronte et al., 2013Bellafronte E, Mariguela TC, Pereira LHG, Oliveira C, Moreira-Filho O. DNA barcode of Parodontidae species from the La Plata River basin applying new data to clarify taxonomic problems. Neotrop Ichthyol. 2013; 11(3):497–506. https://doi.org/10.1590/S1679-62252013000300003
https://doi.org/10.1590/S1679-6225201300...
), Zungaro Bleeker, 1858 (0.4%; Pires et al., 2017Pires AA, Ramirez JL, Galetti Jr P, Troy WP, Freitas PD. Molecular analysis reveals hidden diversity in Zungaro (Siluriformes: Pimelodidade): a genus of giant South American catfish. Genetica. 2017; 145(3):335–40. https://doi.org/10.1007/s10709-017-9968-8
https://doi.org/10.1007/s10709-017-9968-...
), Megaleporinus Ramirez, Birindelli & Galetti Jr., 2017 (0.67%; Ramirez et al., 2017Ramirez JL, Birindelli JL, Carvalho DC, Affonso PRAM, Venere PC, Ortega H et al. Revealing hidden diversity of the underestimated neotropical ichthyofauna: DNA barcoding in the recently described genus Megaleporinus (Characiformes: Anostomidae). Front Genet. 2017; 8:149. https://doi.org/10.3389/fgene.2017.00149
https://doi.org/10.3389/fgene.2017.00149...
), Leporinus Agassiz, 1829 (0.7%; Silva-Santos et al., 2018Silva-Santos R, Ramirez JL, Galetti PM, Freitas PD. Molecular evidences of a hidden complex scenario in Leporinus cf. friderici. Front Genet. 2018; 9:47. https://doi.org/10.3389/fgene.2018.00047
https://doi.org/10.3389/fgene.2018.00047...
) Rineloricaria Bleeker, 1862 (0.8%; Costa-Silva et al., 2015Costa-Silva GJ, Rodriguez MS, Roxo FF, Foresti F, Oliveira C. Using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. PLoS ONE. 2015; 10(9):1–12. https://doi.org/10.1371/journal.pone.0135075
https://doi.org/10.1371/journal.pone.013...
), Apareiodon (0.9%; Bellafronte et al., 2013Bellafronte E, Mariguela TC, Pereira LHG, Oliveira C, Moreira-Filho O. DNA barcode of Parodontidae species from the La Plata River basin applying new data to clarify taxonomic problems. Neotrop Ichthyol. 2013; 11(3):497–506. https://doi.org/10.1590/S1679-62252013000300003
https://doi.org/10.1590/S1679-6225201300...
), and Laemolyta Cope, 1872 (0.9%; Ramirez, Galetti Jr., 2015Ramirez JL, Galetti PM. DNA barcode and evolutionary relationship within Laemolyta Cope 1872 (Characiformes: Anostomidae) through molecular analyses. Mol Phylogenet Evol. 2015; 93:77–82. https://doi.org/10.1016/j.ympev.2015.07.021
https://doi.org/10.1016/j.ympev.2015.07....
). The small body size of specimens of Astyanax and the fact of some species are geographically isolated in headwaters may enable the occurrence of vicariance events and speciation by geographic isolation (Castro, 1999Castro RMC. Evolução da ictiofauna de riachos sul-americanos: padrões gerais e possíveis processos causais. In: Caramaschi EP, Mazzoni R, Peres-Neto PR, editors. Ecologia de peixes de riachos. Rio de Janeiro: Série Oecologia Brasiliensis; 1999. p.139–55. https://doi.org/10.4257/oeco.1999.0601.04
https://doi.org/10.4257/oeco.1999.0601.0...
). Low genetic divergence between isolated species in distinct tributaries in the same basin may indicate that they had been through a recent vicariance followed by a fast morphological differentiation, without reaching a reciprocal monophyly (Costa-Silva et al., 2015Costa-Silva GJ, Rodriguez MS, Roxo FF, Foresti F, Oliveira C. Using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. PLoS ONE. 2015; 10(9):1–12. https://doi.org/10.1371/journal.pone.0135075
https://doi.org/10.1371/journal.pone.013...
). That might explain the low genetic divergence (0.3%) between A. lorien and A. brucutu herein observed.

The multilocus analysis, separating A. lorien from the remaining Astyanax species studied, including A. brucutu (Fig. 3), reinforces the taxonomic validity of these species. On the other hand, A. rupestris and A. aff. rupestris showed higher values of genetic distances (1.8%; see Tab. S3), suggesting that A. aff. rupestris may be indeed considered distinct from A. rupestris. This result is in accordance with the difficulties pointed by Zanata et al., (2018)Zanata AM, Burger R, Camelier P. Two new species of Astyanax Baird & Girard (Characiformes: Characidae) from the upper rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2018; 4438(3):471–490. https://doi.org/10.11646/zootaxa.4438.3.3
https://doi.org/10.11646/zootaxa.4438.3....
in the description of A. rupestris, in which the authors decided not to include the population of the Piabinha River within A. rupestris. According to the authors, specimens of A. aff. rupestris are very similar morphologically to A. rupestris but possess variations in some meristic characters that were not observed in the former. The Piabinha population also presents high frequency of specimens with variable lateral-line perforation, four premaxillary teeth in the inner row, and reduction in the number of branched dorsal- and pelvic-fin rays (Zanata et al., 2018Zanata AM, Burger R, Camelier P. Two new species of Astyanax Baird & Girard (Characiformes: Characidae) from the upper rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2018; 4438(3):471–490. https://doi.org/10.11646/zootaxa.4438.3.3
https://doi.org/10.11646/zootaxa.4438.3....
). Astyanax aff. rupestris is apparently restrict to the Piabinha River, a Cumbuca’s tributary, while A. rupestris is known to occurs in a somewhat broader distribution throughout both Cumbuca and Piaba River sub-basins (Zanata et al., 2018Zanata AM, Burger R, Camelier P. Two new species of Astyanax Baird & Girard (Characiformes: Characidae) from the upper rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2018; 4438(3):471–490. https://doi.org/10.11646/zootaxa.4438.3.3
https://doi.org/10.11646/zootaxa.4438.3....
).

Furthermore, the GMYC approach also pointed A. aff. rupestris divided in two MOTUs (A. aff. rupestris 1 and A. aff. rupestris 2), evidencing hidden genetic diversity and showing MOTUs in sympatry and reciprocal monophyly (see Fig. 3), though with less than 1% of genetic distance between them (0.7%). It appears that some Astyanax lineages from the upper Paraguaçu are in a gray zone (sensude Queiroz, 2007de Queiroz K. Species concepts and species delimitation. Syst Biol. 2007; 56(6):879–86. https://doi: 10.1080/10635150701701083
https://doi:...
), in which speciation is in process, and the boundaries among species are hardly identified (Costa-Silva et al., 2015Costa-Silva GJ, Rodriguez MS, Roxo FF, Foresti F, Oliveira C. Using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. PLoS ONE. 2015; 10(9):1–12. https://doi.org/10.1371/journal.pone.0135075
https://doi.org/10.1371/journal.pone.013...
; Anjos et al., 2020Anjos MS, Bitencourt JA, Nunes LA, Sarmento-Soares LM, Carvalho DC, Armbruster JW et al. Species delimitation based on integrative approach suggests reallocation of genus in Hypostomini catfish (Siluriformes, Loricariidae). Hydrobiologia. 2020; 847:563–78. https://doi.org/10.1007/s10750-019-04121-z
https://doi.org/10.1007/s10750-019-04121...
). In this sense, we agree that further population studies of A. rupestris, A. aff. rupestris 1, and A. aff. rupestris 2, using methods of integrative taxonomy, including molecular and morphological data, are necessary to clarify the taxonomic status of the A. rupestris putative species complex.

Our study was useful in confirming A. rupestris from the Piaba and Cumbuca sub-basins as a single molecular unit distinct from A. aff. rupestris from the Piabinha River. In addition, the data supported the existence of two genetic lineages within the A. aff. rupestris morphotype. The multilocus analysis was more efficient in identifying species with recent divergence when compared to the single locus analysis using COI sequence only. Altogether, we characterized six distinct MOTUs: Astyanax epiagos, A. brucutu, A. lorien, A. rupestris, A. aff. rupestris 1, and A. aff. rupestris 2. Regarding the two Astyanax sp. previously reported for the Paraguaçu River basin by Rossini et al., (2016)Rossini BC, Oliveira CAM, Melo FAG, Bertaco VA, Astarloa JMD, Rosso JJ et al. Highlighting Astyanax species diversity through DNA barcoding. PLoS ONE. 2016; 11(12):e0167203. https://doi.org/10.1371/journal.pone.0167203
https://doi.org/10.1371/journal.pone.016...
, the results indicated that Astyanax sp. from the Piabinha River and A. aff. rupestris 2 share the same COI haplotype, and, consequently, belonging to the same taxon (MOTU). On the other hand, Astyanax sp. from the Coité tributary needs to be taxonomically assessed, since it clustered to nominal species from São Francisco and Miriri basins, showing no genetic similarity to the endemic Astyanax species from the Paraguaçu River studied here. Overall, these findings contribute to a better understanding of the diversity of this fish group in the upper Paraguaçu River basin, pointing out hidden diversity and reinforcing the relevance of this hydrographic system for the biodiversity ichthyofauna.

ACKNOWLEDGEMENTS

The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2010/52315–7, 2016/19075–9, and 2017/09321–5). RSS and CBM thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES Financial Code 001). AMZ and PC thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (AMZ: CNPq 476449/2007–3, 562335/2010–2, 476495/2010–5; 563299/2010–0, 304477/2018-4; PC: 423760/2018-1). PDF and PMGJ thank CNPq (317345/2021-4 and 303524/2019-7, respectively) for the financial support. The authors also thank Rafael Burger for helping with the sampling collecting and Ueslei Lopes for contributing to improve this manuscript. The authors thank three anonymous reviewers for constructive comments that improved the manuscript.

REFERENCES

  • Abell R, Thieme ML, Revenga C, Bryer M, Kottelat M, Bogutskaya N et al Freshwater ecoregions of the world: A new map of biogeographic units for freshwater biodiversity conservation. BioScience. 2008; 58(5):403–14. https://doi.org/10.1641/b580507
    » https://doi.org/10.1641/b580507
  • Aljanabi S. Universal and rapid salt-extraction of high quality genomic DNA for PCR- based techniques. Nucleic Acids Res. 1997; 25(22):4692–93. https://doi.org/10.1093/nar/25.22.4692
    » https://doi.org/10.1093/nar/25.22.4692
  • Anjos MS, Bitencourt JA, Nunes LA, Sarmento-Soares LM, Carvalho DC, Armbruster JW et al Species delimitation based on integrative approach suggests reallocation of genus in Hypostomini catfish (Siluriformes, Loricariidae). Hydrobiologia. 2020; 847:563–78. https://doi.org/10.1007/s10750-019-04121-z
    » https://doi.org/10.1007/s10750-019-04121-z
  • Bellafronte E, Mariguela TC, Pereira LHG, Oliveira C, Moreira-Filho O. DNA barcode of Parodontidae species from the La Plata River basin applying new data to clarify taxonomic problems. Neotrop Ichthyol. 2013; 11(3):497–506. https://doi.org/10.1590/S1679-62252013000300003
    » https://doi.org/10.1590/S1679-62252013000300003
  • Benine RC, Mariguela TC, Oliveira C. New species of Moenkhausia Eigenmann, 1903 (Characiformes: Characidae) with comments on the Moenkhausia oligolepis species complex. Neotrop Ichthyol. 2009; 7(2):161–68. https://doi.org/10.1590/S1679-62252009000200005
    » https://doi.org/10.1590/S1679-62252009000200005
  • Bertaco VA, Garutti V. New Astyanax from the upper rio Tapajós drainage, Central Brazil (Characiformes: Characidae). Neotrop Ichthyol. 2007; 5(1):25–30. https://doi.org/10.1590/S1679-62252007000100003
    » https://doi.org/10.1590/S1679-62252007000100003
  • Bertaco VA, Lucena CAS. Redescription of Astyanax obscurus (Hensel, 1870) and A. laticeps (Cope, 1894) (Teleostei: Characidae): Two valid freshwater species originally described from rivers of Southern Brazil. Neotrop Ichthyol. 2010; 8(1):7–20. https://doi.org/10.1590/S1679-62252010000100002
    » https://doi.org/10.1590/S1679-62252010000100002
  • Bockmann FA, Castro RMCC. The blind catfish from the caves of Chapada Diamantina, Bahia, Brazil (Siluriformes: Heptapteridae): Description, anatomy, phylogenetic relationships, natural history, and biogeography. Neotrop Ichthyol. 2010; 8(4):673–706. https://doi.org/10.1590/S1679-62252010000400001
    » https://doi.org/10.1590/S1679-62252010000400001
  • Buckup PA. The Eastern Brazilian Shield. In: Albert JS, Reis RE, editors. Historical biogeography of Neotropical freshwater fishes. Los Angeles: University of California Press; 2011. p.203–10.
  • Burger R, Carvalho FR, Zanata AM. A new species of Astyanax Baird & Girard (Characiformes: Characidae) from western Chapada Diamantina, Bahia, Brazil. Zootaxa. 2019; 4604(2):369–80. https://doi.org/10.11646/zootaxa.4604.2.9
    » https://doi.org/10.11646/zootaxa.4604.2.9
  • Camelier P, Zanata AM. Biogeography of freshwater fishes from the northeastern Mata Atlântica freshwater ecoregion: Distribution, endemism, and area relationships. Neotrop Ichthyol. 2014a; 12(4):683–98. https://doi.org/10.1590/1982-0224-20130228
    » https://doi.org/10.1590/1982-0224-20130228
  • Camelier P, Zanata AM. A new species of Astyanax Baird & Girard (Characiformes: Characidae) from the rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil, with comments on bony hooks on all fins. J Fish Biol. 2014b; 84(2):475–90. https://doi.org/10.1111/jfb.12295
    » https://doi.org/10.1111/jfb.12295
  • Carstens BC, Pelletier TA, Reid NM, Satler JD. How to fail at species delimitation. Mol Ecol. 2013; 22:4369–83. https://doi.org/10.1111/mec.12413
    » https://doi.org/10.1111/mec.12413
  • Carvalho DC, Oliveira DAA, Pompeu PS, Leal CG, Oliveira C, Hanner R. Deep barcode divergence in Brazilian freshwater fishes: the case of the São Francisco River basin. Mitochondrial DNA. 2011; 22:80–86. https://doi.org/10.3109/19401736.2011.588214
    » https://doi.org/10.3109/19401736.2011.588214
  • Castro RMC. Evolução da ictiofauna de riachos sul-americanos: padrões gerais e possíveis processos causais. In: Caramaschi EP, Mazzoni R, Peres-Neto PR, editors. Ecologia de peixes de riachos. Rio de Janeiro: Série Oecologia Brasiliensis; 1999. p.139–55. https://doi.org/10.4257/oeco.1999.0601.04
    » https://doi.org/10.4257/oeco.1999.0601.04
  • Chow S, Hazama K. Universal PCR primers for S7 ribosomal protein gene introns in fish. Mol Ecol. 1998; 7(9):1255–56. https://doi.org/10.1046/j.1365-294x.1998.00406.x
    » https://doi.org/10.1046/j.1365-294x.1998.00406.x
  • Collins RA, Boykin LM, Cruickshank RH, Armstrong KF. Barcoding’s next top model: an evaluation of nucleotide substitution models for specimen identification. Methods Ecol Evol. 2012; 3:457–65. https://doi.org/10.1111/j.2041-210X.2011.00176.x
    » https://doi.org/10.1111/j.2041-210X.2011.00176.x
  • Costa-Silva GJ, Rodriguez MS, Roxo FF, Foresti F, Oliveira C. Using different methods to access the difficult task of delimiting species in a complex neotropical hyperdiverse group. PLoS ONE. 2015; 10(9):1–12. https://doi.org/10.1371/journal.pone.0135075
    » https://doi.org/10.1371/journal.pone.0135075
  • Dagosta FCP, Marinho MMF. New small-sized species of Astyanax (Characiformes: Characidae) from the upper rio Paraguai basin, Brazil, with discussion on its generic allocation. Neotrop Ichthyol. 2022; 20(1):e210127. https://doi.org/10.1590/1982-0224-2021-0127
    » https://doi.org/10.1590/1982-0224-2021-0127
  • Darriba D, Taboada GL, Doallo RR, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8):772–72. https://doi.org/10.1038/nmeth.2109
    » https://doi.org/10.1038/nmeth.2109
  • Edwards SV. Is a new and general theory of molecular systematics emerging? Evolution. 2009; 63(1):1–19. https://doi.org/10.1111/j.1558-5646.2008.00549.x
    » https://doi.org/10.1111/j.1558-5646.2008.00549.x
  • Eigenmann CH. The American Characidae. Part 3. Mem Mus Comp Zool. 1921; 43:209–310.
  • Flot JF. Seqphase: A web tool for interconverting phase input/output files and FASTA sequence alignments. Mol Ecol Res. 2010; 10(1):162–66. https://doi.org/10.1111/j.1755-0998.2009.02732.x
    » https://doi.org/10.1111/j.1755-0998.2009.02732.x
  • Frézal L, Leblois R. Four years of DNA barcoding: current advances and prospects. Infect Genet Evol. 2008, 8:727–36. https://doi.org/10.1016/j.meegid.2008.05.005
    » https://doi.org/10.1016/j.meegid.2008.05.005
  • Fricke R, Eschmeyer WN, Van der Laan R. Eschmeyer’s catalog of fishes: Genera, species, references [Internet]. San Francisco: California Academy of Sciences; 2022. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp).
    » http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp).
  • Garavello JC, Sampaio FAA. Five new species of genus Astyanax Baird & Girard, 1854 from Rio Iguaçu, Paraná, Brazil (Ostariophysi, Characiformes, Characidae). Braz J Biol. 2010; 70(3):847–65. https://doi.org/10.1590/S1519-69842010000400016
    » https://doi.org/10.1590/S1519-69842010000400016
  • Garutti V. Descrição de uma espécie nova de Astyanax (Teleostei, Characidae) da bacia do Tocantins, Brasil. Iheringia, Sér Zool. 1998; 85:115–22.
  • Garutti V, Britski HA. Descrição de uma espécie nova de Astyanax (Teleostei: Characidae) da bacia do alto rio Paraná e considerações sobre as demais espécies do gênero na bacia. Comun Mus Ciênc Tecnol PUCRS, Sér Zool. 2000; 13:65–88.
  • Garutti V, Venere PC Astyanaxxavante, a new species of characid from middle rio Araguaia in the cerrado region, Central Brazil (Characiformes: Characidae). Neotrop Ichthyol. 2009; 7(3):377–83. https://doi.org/10.1590/S1679-62252009000300004
    » https://doi.org/10.1590/S1679-62252009000300004
  • Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM. Identification of birds through DNA Barcodes. PLoS Biology. 2004; 2(10):e312. https://doi.org/10.1371/journal.pbio.0020312
    » https://doi.org/10.1371/journal.pbio.0020312
  • Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Royal Soc B Biol Sci. 2003; 270(1512):313–21. https://doi.org/10.1098/rspb.2002.2218
    » https://doi.org/10.1098/rspb.2002.2218
  • Heled J, Drummond AJ. Bayesian inference of species trees from multilocus data. Mol Biol Evol. 2010; 27(3):570–80. https://doi.org/10.1093/molbev/msp274
    » https://doi.org/10.1093/molbev/msp274
  • Higuchi H, Britski HA, Garavello JC Kalyptodoras bahiensis, a new genus and species of thorny catfish from northeastern Brazil (Siluriformes: Doradidae). Ichthyol Explor Freshw. 1990; 3:219–25.
  • Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, Burridge M et al Identifying canadian freshwater fishes through DNA barcodes. PLoS ONE. 2008; 3(6):e2490. https://doi.org/10.1371/journal.pone.0002490
    » https://doi.org/10.1371/journal.pone.0002490
  • Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S et al Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28(12):1647–49. https://doi.org/10.1093/bioinformatics/bts199
    » https://doi.org/10.1093/bioinformatics/bts199
  • Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7):1870–74. https://doi.org/10.1093/molbev/msw054
    » https://doi.org/10.1093/molbev/msw054
  • Lis JT. Fractionation of DNA fragments by polyethylene glycol induced precipitation. Meth Enzymol. 1980; 65:347–53. https://doi.org/10.1016/S0076-6879(80)65044-7
    » https://doi.org/10.1016/S0076-6879(80)65044-7
  • Lopes U, Galetti Jr PM, Freitas PD. Hidden diversity in Prochilodus nigricans: A new genetic lineage within the Tapajós River basin. PLoS ONE. 2020; 15(8):e0237916. https://doi.org/10.1371/journal.pone.0237916
    » https://doi.org/10.1371/journal.pone.0237916
  • Machado CB, Ishizuka TK, Freitas PD, Galetti Jr PM. DNA barcoding reveals taxonomic uncertainty in Salminus (Characiformes). System Biodivers. 2016; 15(4):372–82. https://doi.org/10.1080/14772000.2016.1254390
    » https://doi.org/10.1080/14772000.2016.1254390
  • Machado VN, Collins RA, Ota RP, Andrade MC, Farias IP, Hrbek T. One thousand DNA barcodes of piranhas and pacus reveal geographic structure and unrecognised diversity in the Amazon. Sci Rep. 2018; 8:8387. https://doi.org/10.1038/s41598-018-26550-x
    » https://doi.org/10.1038/s41598-018-26550-x
  • Melo MRS, Espíndola VC. Description of a new species of Characidium Reinhardt, 1867 (Characiformes: Crenuchidae) from the Chapada Diamantina, Bahia, and redescription of Characidium bimaculatum Fowler, 1941. Zootaxa. 2016; 4196(4):552–68. https://doi.org/10.11646/zootaxa.4196.4.5
    » https://doi.org/10.11646/zootaxa.4196.4.5
  • Moreira-Filho O, Bertollo LAC Astyanax scabripinnis (Pisces, Characidae): a species complex. Rev Bras Genet. 1991; 14:331–57.
  • Ornelas-García CP, Domínguez-Domínguez O, Doadrio I. Evolutionary history of the fish genus Astyanax Baird & Girard (1854) (Actinopterygii, Characidae) in Mesoamerica reveals multiple morphological homoplasies. BMC Evol Biol. 2008; 8:340. https://doi.org/10.1186/1471-2148-8-340
    » https://doi.org/10.1186/1471-2148-8-340
  • Orsi ML, Carvalho ED, Foresti F. Biologia populacional de Astyanax altiparanae Garutti & Britski (Teleostei, Characidae) do médio Rio Paranapanema, Paraná, Brasil. Rev Bras Zool. 2004; 21(2):207–18. https://doi.org/10.1590/s0101-81752004000200008
    » https://doi.org/10.1590/s0101-81752004000200008
  • Pereira LHG, Maia GMG, Hanner R, Foresti F, Oliveira C. DNA barcodes discriminate freshwater fishes from the Paraíba do Sul River Basin, São Paulo, Brazil. Mitochondrial DNA. 2011; 22:71–79. https://doi.org/10.3109/19401736.2010.532213
    » https://doi.org/10.3109/19401736.2010.532213
  • Pereira LH, Hanner R, Foresti F, Oliveira C. Can DNA barcoding accurately discriminate megadiverse Neotropical freshwater fish fauna? BMC Genetics. 2013; 14(1):20. https://doi.org/10.1186/1471-2156-14-20
    » https://doi.org/10.1186/1471-2156-14-20
  • Piggott MP, Chao NL, Beheregaray LB. Three fishes in one: Cryptic species in an Amazonian floodplain forest specialist. Biol J Linn Soc. 2011; 102(2):391–403. https://doi.org/10.1111/j.1095-8312.2010.01571.x
    » https://doi.org/10.1111/j.1095-8312.2010.01571.x
  • de Pinna M, Abrahão V, Reis V, Zanata A. A new species of Copionodon representing a relictual occurrence of the Copionodontinae (Siluriformes: Trichomycteridae), with a CT-scan imaging survey of key subfamilial features. Neotrop Ichthyol. 2018; 16(4):e180049. https://doi.org/10.1590/1982-0224-20180049
    » https://doi.org/10.1590/1982-0224-20180049
  • Pires AA, Ramirez JL, Galetti Jr P, Troy WP, Freitas PD. Molecular analysis reveals hidden diversity in Zungaro (Siluriformes: Pimelodidade): a genus of giant South American catfish. Genetica. 2017; 145(3):335–40. https://doi.org/10.1007/s10709-017-9968-8
    » https://doi.org/10.1007/s10709-017-9968-8
  • Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S et al Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Syst Biol. 2006; 55(4):595–609. https://doi.org/10.1080/10635150600852011
    » https://doi.org/10.1080/10635150600852011
  • Prioli SMAP, Prioli AJ, Júlio Jr HF, Pavanelli CS, Oliveira AV, Carrer H et al Identification of Astyanax altiparanae (Teleostei, Characidae) in the Iguaçu River, Brazil, based on mitochondrial DNA and a RAPD markers. Genet Mol Biol. 2002; 25(4):421–30. https://doi.org/10.1590/S1415-47572006000300011
    » https://doi.org/10.1590/S1415-47572006000300011
  • Puillandre N, Lambert A, Brouillet S, Achaz G. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology. 2012; 21(8):1864–77. https://doi.org/10.1111/j.1365-294X.2011.05239.x
    » https://doi.org/10.1111/j.1365-294X.2011.05239.x
  • de Queiroz K. Species concepts and species delimitation. Syst Biol. 2007; 56(6):879–86. https://doi: 10.1080/10635150701701083
    » https://doi:
  • R Development Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2017. Available from: https://www.r-project.org/
    » https://www.r-project.org/
  • Rambaut A, Suchard MA, Xie D, Drummond AJ. Tracer v1.6 2014. Available from: http://beast.bio.ed.ac.uk/Tracer.
    » http://beast.bio.ed.ac.uk/Tracer.
  • Ramirez JL, Birindelli JL, Carvalho DC, Affonso PRAM, Venere PC, Ortega H et al Revealing hidden diversity of the underestimated neotropical ichthyofauna: DNA barcoding in the recently described genus Megaleporinus (Characiformes: Anostomidae). Front Genet. 2017; 8:149. https://doi.org/10.3389/fgene.2017.00149
    » https://doi.org/10.3389/fgene.2017.00149
  • Ramirez JL, Galetti PM. DNA barcode and evolutionary relationship within Laemolyta Cope 1872 (Characiformes: Anostomidae) through molecular analyses. Mol Phylogenet Evol. 2015; 93:77–82. https://doi.org/10.1016/j.ympev.2015.07.021
    » https://doi.org/10.1016/j.ympev.2015.07.021
  • Rannala B, Yang Z. Improved reversible jump algorithms for Bayesian species delimitation. Genetics. 2013; 194(1):245–53. https://doi.org/10.1534/genetics.112.149039
    » https://doi.org/10.1534/genetics.112.149039
  • Ratnasingham S, Hebert PDNN. A DNA-based registry for all animal species: The Barcode Index Number (BIN) system. PLoS ONE. 2013; 8(7):e66213. https://doi.org/10.1371/journal.pone.0066213
    » https://doi.org/10.1371/journal.pone.0066213
  • Reis RE, Albert JS, Di Dario F, Mincarone MMM, Petry PL, Rocha LR. Fish biodiversity and conservation in South America. J Fish Biol. 2016. https://doi.org/10.1111/jfb.13016
    » https://doi.org/10.1111/jfb.13016
  • Ribeiro AC. Tectonic history and the biogeography of the freshwater fishes from the coastal drainages of eastern Brazil: An example of faunal evolution associated with a divergent continental margin. Neotrop Ichthyol. 2006; 4(2):225–46. https://doi.org/10.1590/S1679-62252006000200009
    » https://doi.org/10.1590/S1679-62252006000200009
  • Ribolli J, Zabonini Filho E, Scaranto BMS, Shibatta OA, Machado CB. Cryptic diversity and diversification processes in three cis-Andean Rhamdia species (Siluriformes: Heptapteridae) revealed by DNA barcoding. Genet Mol Biol. 2021; 44(3):e20200470. https://doi.org/10.1590/1678-4685-GMB-2020-0470
    » https://doi.org/10.1590/1678-4685-GMB-2020-0470
  • Rossini BC, Oliveira CAM, Melo FAG, Bertaco VA, Astarloa JMD, Rosso JJ et al Highlighting Astyanax species diversity through DNA barcoding. PLoS ONE. 2016; 11(12):e0167203. https://doi.org/10.1371/journal.pone.0167203
    » https://doi.org/10.1371/journal.pone.0167203
  • Santos ACA, Caramaschi EP. Temporal variation in fish composition and abundance in a perennial tributary of the rio Paraguaçu, a little-known drainage in the Brazilian Semi-Arid region. Neotrop Ichthyol. 2011; 9(1):153–60. https://doi.org/10.1590/S1679-62252011005000007
    » https://doi.org/10.1590/S1679-62252011005000007
  • Santos ACA, Caramaschi EP. Composition and seasonal variation of the ichthyofauna from upper Rio Paraguaçu (Chapada Diamantina, Bahia, Brazil). Braz Arch Biol Technol. 2007; 50:663–72. https://doi.org/10.1590/s1516-89132007000400012
    » https://doi.org/10.1590/s1516-89132007000400012
  • Sarmento-Soares LM, Britski HA, Anjos M, Zanata AM, Martins-Pinheiro RF, Barretto M. First record of genus Imparfinis from a northeastern coastal Brazilian River Basin: I. borodini Mees & Cala, 1989 in Rio de Contas, Bahia. Check List. 2016; 12:1832–48. http://dx.doi.org/10.15560/12.1.1832
    » http://dx.doi.org/10.15560/12.1.1832
  • Silva-Santos R, Ramirez JL, Galetti PM, Freitas PD. Molecular evidences of a hidden complex scenario in Leporinus cf. friderici Front Genet. 2018; 9:47. https://doi.org/10.3389/fgene.2018.00047
    » https://doi.org/10.3389/fgene.2018.00047
  • Souza CR, de Mello Affonso PRA, de Araújo Bitencourt J, Sampaio I, Carneiro PLS. Species validation and cryptic diversity in the Geophagus brasiliensis Quoy & Gaimard, 1824 complex (Teleostei, Cichlidae) from Brazilian coastal basins as revealed by DNA analyses. Hydrobiologia. 2018; 809(1):309–21. https://doi.org/10.1007/s10750-017-3482-y
    » https://doi.org/10.1007/s10750-017-3482-y
  • Sukumaran J, Knowles LL. Multispecies coalescent delimits structure, not species. Proc Natl Acad Sci USA. 2017; 114:1607–12. https://doi.org/10.1073/pnas.1607921114
    » https://doi.org/10.1073/pnas.1607921114
  • Terán GE, Benitez MF, Mirande JM. Opening the Trojan horse: phylogeny of Astyanax, two new genera and resurrection of Psalidodon (Teleostei: Characidae). Zool J Linn Soc. 2020; 190(4):1217–34. https://doi.org/10.1093/zoolinnean/zlaa019
    » https://doi.org/10.1093/zoolinnean/zlaa019
  • Vari RP, Castro RMC New species of Astyanax (Ostariophysi: Characiformes: Characidae) from the upper rio Paraná system, Brazil. Copeia. 2007; 2007(1):150–62. https://doi:10.1643/0045-8511(2007)7[150:NSOAOC]2.0.CO;2
    » https://doi:10.1643/0045-8511(2007)7[150:NSOAOC]2.0.CO;2
  • Vita G, Zanata AM, Datovo A. Anatomy and ontogenetic changes of the facial and gular musculature of the tetra Astyanax brucutu: A remarkable case of adaptation to durophagy. J Anat. 2020; 237(6):1136–50. https://doi.org/10.1111/joa.13280
    » https://doi.org/10.1111/joa.13280
  • Ward RD. DNA barcode divergence among species and genera of birds and fishes. Mol Ecol Res. 2009; 9(4):1077–85. https://doi.org/10.1111/j.1755-0998.2009.02541.x
    » https://doi.org/10.1111/j.1755-0998.2009.02541.x
  • Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN. DNA barcoding Australia’s fish species. Philos Trans R Soc B Biol Sci. 2005; 360(1462):1847–57. https://doi.org/10.1098/rstb.2005.1716
    » https://doi.org/10.1098/rstb.2005.1716
  • Wimberger PH. Plasticity of fish body shape. The effects of diet, development, family and age in two species of Geophagus (Pisces: Cichlidae). Biol J Linn Soc. 1992; 45:197–218. https://doi.org/10.1111/j.1095-8312.1992.tb00640.x
    » https://doi.org/10.1111/j.1095-8312.1992.tb00640.x
  • Yang Z. The BPP program for species tree estimation and species delimitation. Curr Zool. 2015; 61(5):854–65. https://doi.org/10.1093/czoolo/61.5.854
    » https://doi.org/10.1093/czoolo/61.5.854
  • Yang Z, Rannala B. Bayesian species delimitation using multilocus sequence data. Proc Natl Acad Sci USA. 2010; 107(20):9264–69. https://doi.org/10.1073/pnas.0913022107
    » https://doi.org/10.1073/pnas.0913022107
  • Zamudio KR, Bell RC, Mason NA. Phenotypes in phylogeography: Species’ traits, environmental variation, and vertebrate diversification. Proc Natl Acad Sci USA. 2016; 113(29):8041–48. https://doi.org/10.1073/pnas.1602237113
    » https://doi.org/10.1073/pnas.1602237113
  • Zanata AM, Burger R, Camelier P. Two new species of Astyanax Baird & Girard (Characiformes: Characidae) from the upper rio Paraguaçu basin, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2018; 4438(3):471–490. https://doi.org/10.11646/zootaxa.4438.3.3
    » https://doi.org/10.11646/zootaxa.4438.3.3
  • Zanata AM, Camelier P Astyanax vermilion and Astyanax burgerai: New characid fishes (Ostariophysi: Characiformes) from northeastern Bahia, Brazil. Neotrop Ichthyol. 2009; 7(2):175–84. https://doi.org/10.1590/S1679-62252009000200007
    » https://doi.org/10.1590/S1679-62252009000200007
  • Zanata AM, Lima FC, Dario F, Garhard P. A new remarkable and Critically Endangered species of Astyanax Baird & Girard (Characiformes: Characidae) from Chapada Diamantina, Bahia, Brazil, with a discussion on durophagy in the Characiformes. Zootaxa. 2017; 4232(4):491–510. https://doi.org/10.11646/zootaxa.4232.4.2
    » https://doi.org/10.11646/zootaxa.4232.4.2
  • Zanata AM, Camelier P. Two new species of Characidium Reinhardt (Characiformes: Crenuchidae) from northeastern Brazilian coastal drainages. Neotrop Ichthyol. 2015; 13(3):487–98. https://doi.org/10.1590/1982-0224-20140106
    » https://doi.org/10.1590/1982-0224-20140106
  • Zanata AM, Camelier P. Two new species of Astyanax (Characiformes: Characidae) from upper rio Paraguaçu and rio Itapicuru basins, Chapada Diamantina, Bahia, Brazil. Zootaxa. 2008; 1908(1):28–40. https://doi.org/10.11646/zootaxa.1908.1.2
    » https://doi.org/10.11646/zootaxa.1908.1.2

ADDITIONAL NOTES

  • HOW TO CITE THIS ARTICLE

    Silva-Santos R, Machado CB, Zanata AM, Camelier P, Galetti Jr. PM, Freitas PD. Molecular characterization of Astyanax species (Characiformes: Characidae) from the upper Paraguaçu River basin, a hydrographic system with high endemism. Neotrop Ichthyol. 2023; 21(2):e230032. https://doi.org/10.1590/1982-0224-2023-0032

Edited-by

Guillermo Ortí

Publication Dates

  • Publication in this collection
    12 May 2023
  • Date of issue
    2023

History

  • Received
    18 Jan 2022
  • Accepted
    09 Apr 2023
Sociedade Brasileira de Ictiologia Neotropical Ichthyology, Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura, Universidade Estadual de Maringá., Av. Colombo, 5790, 87020-900, Phone number: +55 44-3011-4632 - Maringá - PR - Brazil
E-mail: neoichth@nupelia.uem.br