Species delimitation reveals an underestimated diversity of Andean catfishes of the family Astroblepidae (Teleostei: Siluriformes)

Luz E. Ochoa Bruno F. Melo Jorge E. García-Melo Javier A. Maldonado-Ocampo Camila S. Souza Juan G. Albornoz-Garzón Cristhian C. Conde-Saldaña Francisco Villa-Navarro Armando Ortega-Lara Claudio Oliveira About the authors

ABSTRACT

Catfishes of the family Astroblepidae form a group composed by 82 valid species of the genus Astroblepus inhabiting high-gradient streams and rivers throughout tropical portions of the Andean Cordillera. Little has been advanced in the systematics and biodiversity of astroblepids other than an unpublished thesis, a single regional multilocus study and isolated species descriptions. Here, we examined 208 specimens of Astroblepus that apparently belong to 16 valid species from several piedmont rivers from northern Colombia to southern Peru. Using three single-locus approaches for species delimitation in combination with a species tree analysis estimated from three mitochondrial genes, we identified a total of 25 well-delimited lineages including eight valid and 17 potential undescribed species distributed in two monophyletic groups: the Central Andes Clade, which contains 14 lineages from piedmont rivers of the Peruvian Amazon, and the Northern Andes Clade with 11 lineages from trans- and cis-Andean rivers of Colombia and Ecuador, including the Orinoco, Amazon, and Magdalena-Cauca basins and Pacific coastal drainages. Results of species delimitation methods highlight several taxonomical incongruences in recently described species denoting potential synonymies.

Keywords:
Andes; Catfishes; Delimitation; Ostariophysi; Systematics; Taxonomy

RESUMEN

Los bagres de la familia Astroblepidae son un grupo compuesto por 82 especies válidas del género Astroblepus que habitan quebradas y ríos de alto gradiente a través de la porcion tropical en la Cordillera de los Andes. Poco se ha avanzado en la sistemática y biodiversidad de los astroblepidos aparte de una tesis no publicada, un único estudio multilocus regional y descripciones aisladas de especies. Aquí, examinamos 208 especímenes de Astroblepus que aparentemente pertenecen a 16 especies válidas provenientes de ríos de pie de monte de la cordillera de los Andes, desde el norte de Colombia hasta el sur de Perú. Utilizando tres metodologías de delimitación de especies para un único locus en combinación con análisis de un árbol de especies a partir de tres genes mitocondriales, identificamos un total de 25 linajes bien definidos que incluyen ocho especies válidas y 17 potenciales especies no descritas distribuidas en dos grupos monofiléticos: un clado de los andes centrales, que contiene 14 especies de los ríos de pie de monte de la Amazonía peruana y un clado de los andes del norte con 11 especies de los ríos trans y cisandinos de Colombia y Ecuador, incluyendo las cuencas del Orinoco, Amazonas y Magdalena-Cauca así como drenajes costeros del Pacífico. Los resultados de los métodos de delimitación de especies destacan varias incongruencias taxonómicas en especies recientemente descritas que denotan posibles sinonimias.

Palabras clave:
Andes; Bagres; Delimitación; Ostariophysi; Sistemática; Taxonomía

INTRODUCTION

The family Astroblepidae includes 82 valid species within the single genus Astroblepus Humboldt, 1805 (Fricke et al., 2020Fricke R, Eschmeyer WN, van der Laan R. Eschmeyer’s catalog of fishes: genera, species, references [Internet]. San Francisco; 2020. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp
http://researcharchive.calacademy.org/re...
) characterized by a specialized head and body morphology, with the presence of a sucker-shaped mouth with fleshy and expanded lips and a dorsal opening to the gill chamber, between the dorsal margin of the opercle and the ventral edge of the pterotic (Schaefer, 2003Schaefer SA. Family Astroblepidae. In: Reis RE, Kullander SO, Ferraris CJ Jr., editors. Check list of freshwater fishes of South and Central America. Porto Alegre: Edipucrs ; 2003. p.312-17.). Species of this genus have bodies without bony plates and with a modified pelvic musculature completely separate from the hypaxial muscles, forming a pair of strings-like muscles between the pectoral and pelvic fins (the protractor ischii muscles; Shelden, 1937Shelden FF. Osteology, myology, and probable evolution of the nematognath pelvic girdle. Ann N Y Acad Sci. 1937; 37(1):1-95. https://doi.org/10.1111/j.1749-6632.1937.tb55481.x
https://doi.org/10.1111/j.1749-6632.1937...
). Alternating contraction of the protractor and ischii muscles anterior of the pelvic girdle and the retractor ischii muscles posterior of the pelvic girdle, combined with adhesion to substrates using the oral disk results in a remarkable ability of Astroblepus to move forward against very strong currents and even scale vertical surfaces (Shelden, 1937; De Crop et al., 2013De Crop W, Pauwels E, Van Hoorebeke L, Geerinckx T. Functional morphology of the Andean climbing catfishes (Astroblepidae, Siluriformes): Alternative ways of respiration, adhesion, and locomotion using the mouth. J Morphol. 2013; 274(10):1164-79. https://doi.org/10.1002/jmor.20169
https://doi.org/10.1002/jmor.20169...
), thus giving this clade the name “climbing catfishes” (Schaefer, Arroyave, 2010).

Astroblepids inhabit moderate to high elevation streams on eastern and western flanks of the Andes Mountains in Panama, Colombia, Venezuela, Ecuador, Peru and Bolivia (Schaefer, 2003Schaefer SA. Family Astroblepidae. In: Reis RE, Kullander SO, Ferraris CJ Jr., editors. Check list of freshwater fishes of South and Central America. Porto Alegre: Edipucrs ; 2003. p.312-17.). Species of astroblepids have generally restricted geographic distributions, being limited to small portions of isolated drainages between 400 and 4000 m, above sea level, but less frequent above 3,000 m (Schaefer, 2003; Schaefer, Arroyave, 2010). Distribution and ecological niche modeling analyses have demonstrated that astroblepids are physiologically confined to drainage islands in the Andes, where elevation and temperature oscillation strongly influence their geographical distribution and endemism (Vélez-Espino, 2006Vélez-Espino LA. Distribution and habitat suitability index model for the Andean catfish Astroblepus ubidiai (Pisces: Siluriformes) in Ecuador. Rev Biol Trop. 2006; 54(2):623-38. Available from: https://www.scielo.sa.cr/scielo.php?pid=S0034-77442006000200035&script=sci_arttext
https://www.scielo.sa.cr/scielo.php?pid=...
; Schaefer, Arroyave, 2010). Ecological specializations and limited habitat distributions make Astroblepus vulnerable to many environmental impacts such as water pollution, deforestation, species introductions and urbanization (Anderson, Maldonado-Ocampo, 2011Anderson EP, Maldonado-Ocampo JA. A regional perspective on the diversity and conservation of tropical Andean fishes. Conserv Biol. 2011; 25(1):30-39. https://doi.org/10.1111/j.1523-1739.2010.01568.x
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), e.g., Astroblepus vaillanti (Regan, 1904Regan CT. A monograph of the fishes of the family Loricariidae. Trans Zool Soc London. 1904; 17(3):191-350.) in Ecuador (Alexiades, Encalada, 2017Alexiades AV, Encalada AC. Distribution and habitat suitability of andean climbing catfish in the Napo River Basin, Ecuador. Trop Conserv Sci. 2017; 10:1-7. https://doi.org/10.1177/1940082917709598
https://doi.org/10.1177/1940082917709598...
).

Much of the research on Astroblepus has been focused on either their ecology and life history (Vélez-Espino, 2003Vélez-Espino LA. Taxonomic revision, ecology and endangerment categorization of the Andean catfish Astroblepus ubidiai (Teleostei: Astroblepidae). Rev Fish Biol Fish. 2003; 13:367-78. https://doi.org/10.1007/s11160-004-1096-8
https://doi.org/10.1007/s11160-004-1096-...
; Briñez-Vásquez et al., 2005Briñez-Vásquez GN, Villa-Navarro FA, Ortega-Lara A, Reinoso-Flórez G, García-Melo JG. Distribución altitudinal y diversidad de la familia Astroblepidae (Pisces, Siluriformes), en la cuenca del río Coello, Tolima. Dahlia Rev Asoc Colomb Ictiol . 2005; 8:39-46.; Vélez-Espino, Fox, 2005Vélez-Espino LA, Fox MG. Demographic and environmental influences on life-history traits of isolated populations of the Andean catfish Astroblepus ubidiai. Environ Biol Fishes. 2005; 72(189):189-204. https://doi.org/10.1007/s10641-004-0775-6
https://doi.org/10.1007/s10641-004-0775-...
; Schaefer, Arroyave, 2010Schaefer SA, Arroyave J. Rivers as islands: Determinants of the distribution of Andean astroblepid catfishes. J Fish Biol. 2010; 77(10):2373-90. https://doi.org/10.1111/j.1095-8649.2010.02822.x
https://doi.org/10.1111/j.1095-8649.2010...
), cytogenetic characterization (Conde-Saldaña et al., 2019Conde-Saldaña CC, Cunha MS, Albornoz-Garzón JG, Barreto CAV, Ibagón N, Villa-Navarro FA, Dergam JA. Karyotipic divergence of two co-occurring species of Andean climbing catfishes (Siluriformes: Loricarioidei: Astroblepidae). Zebrafish. 2019; 16(1):106-14. https://doi.org/10.1089/zeb.2018.1659
https://doi.org/10.1089/zeb.2018.1659...
), functional morphology (De Crop et al., 2013De Crop W, Pauwels E, Van Hoorebeke L, Geerinckx T. Functional morphology of the Andean climbing catfishes (Astroblepidae, Siluriformes): Alternative ways of respiration, adhesion, and locomotion using the mouth. J Morphol. 2013; 274(10):1164-79. https://doi.org/10.1002/jmor.20169
https://doi.org/10.1002/jmor.20169...
), or their molecular and physiological evolution (Castiglione et al., 2017Castiglione GM, Hauser FE, Liao BS, Lujan NK, Van Nynatten A, Morrow JM et al. Evolution of nonspectral rhodopsin function at high altitudes. Proc Natl Acad Sci U S A . 2017; 114(28):7385-90. https://doi.org/10.1073/pnas.1705765114
https://doi.org/10.1073/pnas.1705765114...
). However, a few studies have focused on the species-level systematics and taxonomic diversity of astroblepids, such as the single taxonomic revision of the genus (Regan, 1904Regan CT. A monograph of the fishes of the family Loricariidae. Trans Zool Soc London. 1904; 17(3):191-350.), an unpublished dissertation describing the osteology of some Colombian species (Buitrago-Suárez, 1995Buitrago-Suárez UA. Sistemática de las especies colombianas del género Astroblepus Humboldt 1805 (Pisces: Siluroidei: Astroblepidae). [Master Dissertation]. Bogotá: Universidad Nacional de Colombia; 1995.), a molecular study of endemics from southern Peru and northern Bolivia (Schaefer et al., 2011), and various isolated species descriptions (Cardona, Guerao, 1994Cardona L, Guerao G. Astroblepus riberae, una nueva especie de siluriforme cavernicola del Perú (Osteichhtyes: Astroblepidae). Mem Biospeol. 1994; 21:21-24.; Ardila-Rodriguez, 2011aArdila-Rodriguez CA. Astroblepus cacharas: (Teleostei: Siluriformes: Astroblepidae), nueva especie del río Cáchira, cuenca del río Magdalena, Colombia. Dahlia Rev Asoc Colomb Ictiol. 2011a; 11:23-33.,bArdila-Rodriguez CA. Astroblepus itae, Astroblepus acostai. Dos nuevas especies del río Cáchira y el río Sinú, Colombia. Barranquilla (Atlántico). 2011b. 1-16., 2012Ardila-Rodriguez CA. Astroblepus ardilai sp. nov. Una nueva especie de pez del Municipio de Floridablanca, Departamento de Santander-Colombia. Peces del Departamento de Santander-Colombia. Barranquilla (Atlántico) . 2012; 5:1-21., 2013aArdila-Rodriguez CA. Astroblepus hidalgoi - A. floridaensis - A. huallegaensis y A. cajamarcaensis. Cuatro nuevas especies de los Andes del Perú. Barranquilla (Atlántico) . 2013a.1-22.,bArdila-Rodriguez CA. Astroblepus martinezi y Astroblepus jimenezae. Dos nuevas especies del río Sinú y río Atrato, Colombia. Barranquilla (Atlántico) . 2013b. 1-16., 2014Ardila-Rodriguez CA. Astroblepus tamboensis sp. nov. - A. chinchaoensis sp. nov. y A. mayoensis sp. nov. Tres nuevas especies de los Andes del Perú. Barranquilla (Atlántico) . 2014. 1-18., 2015aArdila-Rodriguez CA. Cinco nuevas especies de Astroblepidos para los Andes Colombianos. Rev Asoc Col Cienc Biol. 2015a; 27:124-35.,bArdila-Rodriguez CA. Astroblepus verai sp. nov- A. pradai, A. curitiensis y A. onzagaensis. Cuatro nuevas especies del departamento de Santander - Colombia. Barranquilla (Atlántico) . 2015b; 7:1-20.). However, taxonomic limits among species of Astroblepus are questionable and problematic. High levels of inter and intraspecific variation in body shape, fin sizes and body coloration further contribute to the taxonomic complexity of the group (Schaefer et al., 2011). Most of the early descriptions of Astroblepus species were brief on descriptive details (Regan, 1904; Eigenmann, 1917Eigenmann CH. Eighteen new species of fishes from northwestern South America. Proc Am Philos Soc. 1917. 56(7):673-89. Available from: https://www.jstor.org/stable/983888
https://www.jstor.org/stable/983888...
) and diagnoses in the last years were based in a single-character or a combination of distinct and labile pigmentation patterns. Morphological characters, such as meristic features, coloration patterns and presence or absence of adipose fin do not allow to distinguish astroblepids, and it has been assumed that future taxonomic revisions including additional characters will reduce the overestimated number of valid species recognized nowadays (Schaefer, 2003). Although specimens of this group are abundant in ichthyology collections in the Andean region, a large proportion of the individuals are misidentified, unidentified, or identified with taxonomic uncertainty (e.g., species cataloged as ‘sp.’, ‘aff.’ and ‘cf.’) (Hernández-Zapata et al., 2018Hernández-Zapata M, Ochoa-Orrego LE, Jiménez-Segura LF. Métodos de ADN Barcode revelan diversidad de especies de Astroblepus en la cuenca del río Porce, Colombia. Actual Biol. 2018; 40(108):59-71. https://doi.org/10.17533/udea.acbi.v40n108a06
https://doi.org/10.17533/udea.acbi.v40n1...
).

The most recent phylogenetic study combined mitochondrial and nuclear loci to investigate nine morphospecies of Astroblepus from northern Bolivia and southern Peru (Schaefer et al., 2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
). Results supported seven species and the authors argued that allopatric species from distant drainages are more related to each other than to syntopic species or species from adjacent drainages (i.e., disjunct distribution of sister taxa). Despite relatively low species coverage (~11%), their study produced a valuable molecular dataset that can be used to incorporate additional species from other Andean regions. Similarly, a growing number of molecular studies have examined species-level diversity in Neotropical freshwater fishes (e.g., Pereira et al., 2013Pereira LHG, Hanner R, Foresti F, Oliveira C. Can DNA barcoding accurately discriminate megadiverse Neotropical freshwater fish fauna? BMC Genet. 2013; 14(20):1-14. https://doi.org/10.1186/1471-2156-14-20
https://doi.org/10.1186/1471-2156-14-20...
; 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):e0135075. https://doi.org/10.1371/journal.pone.0135075
https://doi.org/10.1371/journal.pone.013...
; 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):1-12. https://doi.org/10.1038/s41598-018-26550-x
https://doi.org/10.1038/s41598-018-26550...
; García-Melo et al., 2019García-Melo JE, Oliveira C, Costa-Silva GJ, Ochoa-Orrego LE, Pereira LHG, Maldonado-Ocampo JA. Species delimitation of neotropical Characins (Stevardiinae): Implications for taxonomy of complex groups. PLoS One . 2019; 14(6):1-22. https://doi.org/10.1371/journal.pone.0216786
https://doi.org/10.1371/journal.pone.021...
), however none have focused on astroblepids. Besides, various multilocus, coalescent-based approaches have yielded better resolution of species limits in morphologically cryptic radiations across different organisms (e.g., Barley et al., 2013Barley AJ, White J, Diesmos AC, Brown RM. The challenge of species delimitation at the extremes: Diversification without morphological change in philippine sun skinks. Evolution. 2013; 67(12):3556-72. https://doi.org/10.1111/evo.12219
https://doi.org/10.1111/evo.12219...
; Myers et al., 2013Myers EA, Rodríguez-Robles JA, DeNardo DF, Staub RE, Stropoli A, Ruane S et al. Multilocus phylogeographic assessment of the California Mountain Kingsnake (Lampropeltis zonata) suggests alternative patterns of diversification for the California Floristic Province. Mol Ecol. 2013; 22(21):5418-29. https://doi.org/10.1111/mec.12478
https://doi.org/10.1111/mec.12478...
; Satler et al., 2013Satler JD, Carstens BC, Hedin M. Multilocus species delimitation in a complex of morphologically conserved trapdoor spiders (Mygalomorphae, Antrodiaetidae, Aliatypus). Syst Biol . 2013; 62(6):805-23. https://doi.org/10.1093/sysbio/syt041
https://doi.org/10.1093/sysbio/syt041...
; Welton et al., 2013Welton LJ, Siler CD, Oaks JR, Diesmos AC, Brown RM. Multilocus phylogeny and Bayesian estimates of species boundaries reveal hidden evolutionary relationships and cryptic diversity in Southeast Asian monitor lizards. Mol Ecol . 2013; 22(13):3495-510. https://doi.org/10.1111/mec.12324
https://doi.org/10.1111/mec.12324...
) including freshwater fishes (Niemiller et al., 2012Niemiller ML, Near TJ, Fitzpatrick BM. Delimiting species using multilocus data: Diagnosing cryptic diversity in the southern cavefish, Typhlichthys subterraneus (Teleostei: Amblyopsidae). Evolution. 2012; 66(3):846-66. https://doi.org/10.1111/j.1558-5646.2011.01480.x
https://doi.org/10.1111/j.1558-5646.2011...
; Bagley et al., 2015Bagley JC, Alda F, Breitman MF, Bermingham E, Van Den Berghe EP, Johnson JB. Assessing species boundaries using Multilocus species delimitation in a morphologically conserved group of Neotropical freshwater fishes, the Poecilia sphenops species complex (Poeciliidae). PLoS One. 2015; 10(4):1-30. https://doi.org/10.1371/journal.pone.0121139
https://doi.org/10.1371/journal.pone.012...
).

Here, we combined newly generated mitochondrial sequences with those previously generated by Schaefer et al. (2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
) to study species diversity, delimit species boundaries within Astroblepus from central and northern Andes in Colombia, Ecuador and Peru, and to test Schaefer et al. (2011) hypothesis of the disjunct distribution of sister taxa along the Andean Cordillera.

MATERIAL AND METHODS

Taxon sampling. Sequences of a total of 172 specimens of Astroblepus were newly generated, in which 77 were previously identified in 16 valid species representing 20% of species diversity and, the remaining 95 specimens were identified at the genus level, as “aff.” or “cf.”. Additionally, we included 36 sequences available from GenBank for eight morphospecies identified with the letters A-I as delimited by the molecular study of Schaefer et al. (2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
). Four species of Loricariidae were included as outgroup taxa: Farlowella nattereri Steindachner, 1910, Lamontichthys stibaros Isbrücker & Nijssen, 1978, Loricaria simillimaRegan, 1904Regan CT. A monograph of the fishes of the family Loricariidae. Trans Zool Soc London. 1904; 17(3):191-350., and Pterygoplichthys multiradiatus (Hancock, 1828); specimens of Loricariidae were chosen due to previous phylogenies indicating the sister relationship with Astroblepidae (de Pinna 1998de Pinna MCC. Phylogenetic relationships of Neotropical Siluriformes (Teleostei: Ostariophysi): Historical Overview and Synthesis of Hypotheses. In: Malabarba LR, Reis RE, Vari R, Lucena ZMS, Lucena CAS, editors. Phylogeny and classification of Neotropical fishes . Porto Alegre: Edipucrs ; 1998. p.279-330.; Lujan et al., 2015Lujan NK, Meza-Vargas V, Astudillo-Clavijo V, Barriga-Salazar R, López-Fernández H. A multilocus molecular phylogeny for Chaetostoma Clade Genera and Species with a review of Chaetostoma (Siluriformes: Loricariidae) from the Central Andes. Copeia. 2015; 103(3):664-701. https://doi.org/10.1643/ci-14-194
https://doi.org/10.1643/ci-14-194...
; Roxo et al., 2019Roxo FF, Ochoa LE, Sabaj MH, Lujan NK, Covain R, Silva GSC et al. Phylogenomic reappraisal of the Neotropical catfish family Loricariidae (Teleostei: Siluriformes) using ultraconserved elements. Mol Phy Evol. 2019; 135:148-65. https://doi.org/10.1016/j.ympev.2019.02.017
https://doi.org/10.1016/j.ympev.2019.02....
). Specimens were either collected during the course of this study or available in museum collections. Voucher identifications were determined according to original descriptions and geographic distributions, with the exception of eight morphospecies proposed by Schaefer et al. (2011), whose specimens were unavailable for examination in this study. Unfortunately, samples from intervening or adjacent regions (e.g., Ecuador, Bolivia) were also limited or unavailable here. Vouchers are deposited in various ichthyology collections: the Academy of Natural Sciences of Drexel University, Philadelphia, USA (ANSP), Auburn University Museum of Natural History, Auburn, USA (AUM), Colección Zoológica de la Universidad del Tolima, Ibagué, Colombia (CZUT-IC), Instituto de Ciencias Naturales, Museo de Historia Natural, Universidad Nacional de Colombia, Bogotá, Colombia (ICNMHN), Instituto de Investigación Recursos Biológicos Alexander von Humboldt, Villa de Leyva, Colombia (IAvH-P), Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista, Botucatu, Brazil (LBP), Museo Javeriano de Historia Natural “Lorenzo Uribe”, Bogotá, Colombia (MPUJ) and Royal Ontario Museum, Toronto, Canada (ROM). Catalog numbers, localities and GenBank accession numbers of all terminals are summarized in S1. Photographs for representative species are included in the Fig. 1, and geographical distribution of Astroblepus species and samples included in this study are shown in the Fig. 2.

FIGURE 1
| Species of Astroblepus included in this study, A. A. ardiladuartei (LBP 26696 topotype live, 4.54 mm SL), B. A. cachara (LBP 26712 topotype live, 4.23 mm SL), C. A. caquetae (CZUT-IC 18464 topotype of museum, 7.84 mm SL), D. A. curitiensis (LBP 97118 topotype live, 5.92 mm SL), E. A. homodon (CZUT-IC 18390, 6.15 mm SL), F. A. gr. grixalvii (LBP24242 topotype live, 11.70 mm SL); F’. A. gr. grixalvii (CZUT-IC 18498 specimen of Magdalena basin 6,01 mm SL); F”. A. gr. grixalvii (CZUT-IC 18320 specimen of Cauca basin, 15.25 mm SL), G. A. itae (topotype live, 3.58 mm SL), H. A. latidens (topotype live, 13.40 mm SL), I. A. onzagaensis (topotype live, 7.82 mm SL), J. A. pradai (topotype live, 4.53 mm SL), K. A. trifasciatus (topotype of museum, 9.65 mm SL), K’. A. trifasciatus (topotype of museum, 9.01 mm SL), L. A. aff. trifasciatus (specimen of Magdalena basin, 7.94 mm SL), M. A. verai (topotype live, 3.51 mm SL).

FIGURE 2
| Map of northwestern South America showing the geographic distribution of samples used in this study and species distribution of Astroblepus reported in Global Biodiversity Information Facility (GBIF) and the California Academy of Sciences (CAS) databases.

DNA extraction and sequencing. DNA was extracted from tissues preserved in 95% EtOH using a silica-based method (Ivanova et al., 2006Ivanova NV, Dewaard JR, Hebert PDN. An inexpensive, automation-friendly protocol for recovering high-quality DNA. Mol Ecol Notes. 2006; 6(4):998-1002. https://doi.org/10.1111/j.1471-8286.2006.01428.x
https://doi.org/10.1111/j.1471-8286.2006...
). Partial sequences of three mitochondrial genes 16S rRNA (16S), cytochrome oxidase c subunit I (COI) and cytochrome b (Cytb) were amplified by polymerase chain reaction (PCR) with the primers described in S2. Amplifications were performed in a total volume of 12.5 μl with 1.25 μl of 10X buffer (10 mM Tris-HCl+15 mM MgCl2), 0.5 μl dNTPs (200 nM of each), 0.5 μl each 5 mM primer, 0.05 μl Platinum® Taq Polymerase (Invitrogen), 1 μl genomic DNA (10-50 ng), and 8.7 μl ddH2O. The thermo-cycler profile consisted of an initial denaturation (4 min at 95ºC) followed by 30 cycles of chain denaturation (30 s at 95ºC), primer hybridization (30-60 s at 52ºC for 16S and Cytb, 54ºC for COI), nucleotide extension (30-60 s at 72ºC), and a final extension (10 min at 72ºC). We observed heterozygosity in seven individuals in both forward and reverse sequences of COI indicating a putative co-amplification of a nuclear pseudogene. In this case, the primers L6252-Asn and H7271-COXI (Melo et al., 2011Melo BF, Benine RC, Mariguela TC, Oliveira C. A new species of Tetragonopterus Cuvier, 1816 (Characiformes: Characidae: Tetragonopterinae) from the rio Jari, Amapá, northern Brazil. Neotrop Ichthyol . 2011; 9(1):49-56. https://doi.org/10.1590/S1679-62252011000100002
https://doi.org/10.1590/S1679-6225201100...
) were used and increased annealing temperature (56ºC) in the PCR reactions.

All PCR products were first visually identified on 1% agarose gel and purified using ExoSap-IT® (USB Corporation) following manufacturer instructions. The purified PCR products were sequenced using the Big DyeTM Terminator v 3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems), purified again by ethanol precipitation and loaded onto an automatic sequencer 3130-Genetic Analyzer (Applied Biosystems) in the Instituto de Biociências, Universidade Estadual Paulista, Botucatu, Brazil.

Alignment and species delimitation analyses. Consensus sequences were assembled and edited in Geneious 7.1.4 (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...
) and aligned using the MUSCLE algorithm (Edgar, 2004Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32(5):1792-97. https://doi.org/10.1093/nar/gkh340
https://doi.org/10.1093/nar/gkh340...
). We estimated the index of substitution saturation (Iss) (Xia et al., 2003Xia X, Xie Z, Salemi M, Chen L, Wang Y. An index of substitution saturation and its application. Mol Phylogenet Evol. 2003; 26(1):1-7. https://doi.org/10.1016/S1055-7903(02)00326-3
https://doi.org/10.1016/S1055-7903(02)00...
; Xia, Lemey, 2009Xia X, Lemey P. Assesing substitution saturation with DAMBE. In: Lemey P, Salemi M, Vandamme AM, editors. The Phylogenetic Handbook: a pratical approach to phylogenetic analysis and hypothesis testing. Cambridge: Cambridge University Press; 2009. p.611-26.) in DAMBE v6 (Xia, 2017Xia X. DAMBE6: New tools for microbial genomics, phylogenetics, and molecular evolution. J Hered. 2017; 108(4):431-37. https://doi.org/10.1093/jhered/esx033
https://doi.org/10.1093/jhered/esx033...
). Three independent matrices were assembled for each gene and nucleotide substitution models were selected according to the Bayesian Information Criterion (BIC) in the test module of MEGA v7 (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...
).

Because the morphology-based identification of Astroblepus species is uncertain and difficult, we identified genetic lineages using first a single locus COI applying three species delimitation approaches. The first method correspond with a Bayesian implementation of the Poisson Tree Process model (bPTP, Zhang et al., 2013Zhang J, Kapli P, Pavlidis P, Stamatakis A. A general species delimitation method with applications to phylogenetic placements. Bioinformatics. 2013; 29(22):2869-76. https://doi.org/10.1093/bioinformatics/btt499
https://doi.org/10.1093/bioinformatics/b...
), using the best maximum likelihood (ML) tree as input file in the PTP web server (http://species.h-its.org) with default settings. The best ML tree was estimated with RAxML PTHREADS-SSE3 implemented in RAxML v8.019 (Stamatakis, 2014Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9):1312-13. https://doi.org/10.1093/bioinformatics/btu033
https://doi.org/10.1093/bioinformatics/b...
), which executed five searches with five randomized maximum-parsimony topologies using the GTRGAMMA model and 1,000 bootstrap pseudoreplicates by the autoMRE bootstopping criterion (see Pattengale et al., 2010Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME, Stamatakis A. How many Bootstrap Replicates are necessary?. J Comput Biol. 2010; 17(3):337-54. https://doi.org/10.1089/cmb.2009.0179
https://doi.org/10.1089/cmb.2009.0179...
).

The Generalized Mixed Yule-Coalescent model (GMYC) was the second method implemented (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...
; Fujisawa, Barraclough, 2013Fujisawa T, Barraclough TG. Delimiting species using single-locus data and the generalized mixed yule coalescent approach: A revised method and evaluation on simulated data sets. Syst Biol. 2013; 62(5):707-24. https://doi.org/10.1093/sysbio/syt033
https://doi.org/10.1093/sysbio/syt033...
) that aims to discern stochastic birth-death processes between species, from neutral coalescent processes within species, analyzing time intervals between branching events in a single time-calibrated gene tree (Pons et al., 2006). For this analysis, we estimated a calibrated tree using the lognormal relaxed molecular clock model, which assumes that the rates of molecular evolution are uncorrelated, but log-normally distributed among lineages as implemented in BEAST v1.8.2 (Drummond, Rambaut, 2007Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol. 2007; 7(214):1-8. https://doi.org/10.1186/1471-2148-7-214
https://doi.org/10.1186/1471-2148-7-214...
). We obtained an ultrametric gene tree with two independent runs of 30 million generations with one tree sampled every 1000th generation. BEAST log files were examined in Tracer v1.6 (Rambaut et al., 2018Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst Biol . 2018; 67(5):901-04. https://doi.org/10.1093/sysbio/syy032
https://doi.org/10.1093/sysbio/syy032...
) to access both stationary phase and parameter convergence. A maximum clade credibility tree was estimated in TreeAnnotator v1.8.1 based on the sampled trees after discarding the first 25% generations as a burn-in procedure. All likelihood and Bayesian analyses were performed on 2x 40 CPU 128GB Brycon server at LBP/UNESP. The ultrametric gene tree was used in the GMYC analysis conducted with standard parameters (interval = c (0,10)) using a single threshold via the splits package in R v1.3.1056 (http://r-forge.r-project.org/projects/splits) on a single gene tree.

The third method correspond with the Automatic Barcode Gap Discovery (ABGD; Puillandre et al., 2012Puillandre N, Modica MV, Zhang Y, Sirovich L, Boisselier MC, Cruaud C, Holford M, Samadi S. Large-scale species delimitation method for hyperdiverse groups. Mol Ecol . 2012; 21(11):2671-91. https://doi.org/10.1111/j.1365-294X.2012.05559.x
https://doi.org/10.1111/j.1365-294X.2012...
) that estimates divergence gaps in the interspecific comparison relative to the amount of intraspecific variation. We used a Pmax at 0.15 and a relative gap width of 1.5, with the Jukes-Cantor model (JC69) and other parameters at default in the ABGD webserver (wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html). In summary, the COI lineages identified by these methods were used in downstream concatenated analyses.

Using a concatenated matrix, we estimated a maximum likelihood tree in RAxML v8.019 (Stamatakis, 2014Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9):1312-13. https://doi.org/10.1093/bioinformatics/btu033
https://doi.org/10.1093/bioinformatics/b...
) with GTRGAMMA model and 1,000 bootstrap pseudoreplicates using the autoMRE bootstopping criterion (Pattengale et al., 2010Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME, Stamatakis A. How many Bootstrap Replicates are necessary?. J Comput Biol. 2010; 17(3):337-54. https://doi.org/10.1089/cmb.2009.0179
https://doi.org/10.1089/cmb.2009.0179...
). Afterward, we assigned COI lineages to species groups and estimated a species tree using the multispecies coalescent method *BEAST implemented in BEAST2 (Bouckaert et al., 2014Bouckaert R, Heled J, Kühnert D, Vaughan T, Wu CH, Xie D et al. BEAST 2: A software platform for Bayesian Evolutionary Analysis. PLoS Comput Biol. 2014; 10(4):1-6. https://doi.org/10.1371/journal.pcbi.1003537
https://doi.org/10.1371/journal.pcbi.100...
). We performed two BEAST2 runs of 350 million generations each, sampling every 100,000 generations and with other parameters at default. Log files from each run were checked for convergence and mixing and ensured that ESS scores were >200 in Tracer v1.4. Then, we implemented a species delimitation analysis using a concatenated matrix (COI, 16S and Cytb) to test this groupings on a multispecies coalescent model in the program Bayesian Phylogenetics and Phylogeography (BP&P) v3.2 (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...
; Yang, Rannala, 2010Yang Z, Rannala B. Bayesian species delimitation using multilocus sequence data. Proc Natl Acad Sci . 2010; 107(20):9264-69. https://doi.org/10.1073/pnas.0913022107
https://doi.org/10.1073/pnas.0913022107...
, 2014Yang Z, Rannala B. Unguided species delimitation using DNA sequence data from multiple loci. Mol Biol Evol . 2014; 31(12):3125-35. https://doi.org/10.1093/molbev/msu279
https://doi.org/10.1093/molbev/msu279...
; 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...
). The BP&P method incorporates both the coalescent theory and the phylogenetic uncertainty, accounts for gene tree variance and incomplete lineage sorting (ILS), and calculates mutation-scaled population size (θ) and divergence time (τ) estimates; it also assumes that no gene flow occurs following speciation, an analogy to the biological species concept (Mayr, 1942Mayr E. Systematic and the origin of species, from the viewpoint of a Zoologist. New York: Columbia University Press; 1942.). The algorithm explores different species delimitation models and different species phylogenies with fixed specimen assignments to populations. We ran the A10 analysis of BP&P, a species delimitation using a user-specified guide tree (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...
), based on the concatenated dataset partitioned by gene, used the BEAST2 species tree as a guide tree, and specifying a Dirichlet distribution (α = 2) to account for variation in mutation rates among loci. We conducted runs using three different combinations of gamma-distributed priors for ancestral θ with a default prior θ ~ G(3, 0.004), a diffuse prior θ ~ G(3,0.002) and an estimated prior from sequences θ ~ G(3, 0.003) and the same values were used for (τ0). The program implements two different rjMCMC algorithms tested for each species delimitation hypothesis, and the program auto-adjusted the fine-tuning parameters. We conservatively accepted daughter lineages from nodes with speciation probabilities >0.95 across all three priors as strongly supported species.

RESULTS

The molecular dataset included 208 sequences of COI (534 bp), 201 sequences of 16S (495 pb) and 177 sequences of Cytb (489 pb). All matrices did not present insertions, deletions, stop codons or sequencing errors due to cross-contamination. The Iss.c values were greater than Iss values, indicating the absence of saturation in all three datasets. The concatenated dataset included a total of 215 terminals and 1,517 pb.

The single-locus approaches revealed a combined result of 42 COI lineages of which 11 belong to valid species, eight correspond to the morphospecies reported by Schaefer et al. (2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
) and the 23 remaining represent unidentified lineages (Fig. 3). The ABGD analysis delimited 42 lineages (genetic distances 0.010-0.220), the tree-based bPTP analysis delimited 36 lineages (25% of species >0.95 bootstrap), and the GMYC analysis delimited 88 lineages (L=1988.87, a value not significantly better than the null model of coalescent branching rates L=1977.87). The later method delimited some entities represented by one or two individuals with very low genetic distances (0.000). Fourteen clusters were coincident among all methods, nine were coincident only between ABGD and PTP, 13 between ABGD and GMYC and two clusters between bPTP and GMYC (Fig. 3). According to these results, the preliminary species delimitation included 14 clusters coincident between three methods and 38 clusters recovered in at least two methods that, when compared to alternative groupings in the third approach, continue with interspecific genetic distance equal to 0. The overall mean genetic distance between the putative species was 0.108±0.009 and interspecific genetic distance values ranged from 0.009 ± 0.004 between Astroblepus sp. 28 and A. micrescens Eigenmann, 1918 to 0.187±0.022 between Astroblepus sp. A and A. trifasciatus (Eigenmann, 1912). The final number of 42 COI lineages of Astroblepus was used as an input prior for the multigene analysis.

FIGURE 3
| Results of single-locus approaches using cytochrome oxidase c subunit I (COI) for developing preliminary species delimitation hypothesis with 42 lineages. Results are represented on the ultrametric gene tree with collapsed nodes. All nodal support values were PP>0.95. Blocks at right of the tree represent hypothesized species groups and the values in the middle indicate the number of clusters identified by ABGD, bPTP and GMYC analyses for every collapsed node. COL: Colombia, ECU: Ecuador, PER: Peru.

Maximum likelihood tree of the concatenated matrix with nodal support is shown in S3 and the species tree (Fig. 4) presented highest posterior probability (PP=1) (ln L = -19303.083, ESS=211) for 73% of the COI lineages with 17 showing low support. The BP&P analysis supported only 59.5% of these lineages with a total of 25 species (PP = 1) recovering seven of 11 valid species and 18 from 23 unidentified lineages delimited in the singles-locus analysis (Fig. 4). The lineages supported were consistent in every set of priors used (S4, S5 and S6). The topology clearly shows two strongly-supported monophyletic groups that are well-defined biogeographically: the herein named “Northern Andes clade” with 23 lineages predominantly from Colombia, and the “Central Andes clade” with 19 lineages from Peru and Ecuador identified in the COI analysis (Fig. 4).

FIGURE 4
| Species tree inferred from the concatenated dataset of mitochondrial genes (COI, Cytb, and 16S). Nodal support values are Bayesian posterior probabilities. Non-significant speciation probabilities identified in BP&P analysis algorithm A10 (PP:<0.95) are indicate by black circles and species supported with asterisk.

In the Northern Andes Clade, the BP&P analysis supported 11 lineages from multiple trans and cis-Andean rivers, involving Orinoco, Amazon, and Magdalena-Cauca basins and Pacific coastal drainages of Colombia and Ecuador. These results also indicated poor support values for the delimitation of A. itae Ardila-Rodriguez, 2011, A. micrescens, A. onzagaensis Ardila-Rodriguez, 2015, A. latidens Eigenmann, 1918, A. aff. trifasciatus, Astroblepus sp. 22, 23, 25, 26, 27, 28, and 29, potential new species (Fig. 4, black circles). In the Central Andes Clade, the analysis supported 14 lineages and reveal low support for morphospecies A and I, as well as Astroblepus sp. 9, 12, and 13 from piedmont rivers of the Peruvian Amazon.

Northern and Central Andes clades. The Northern Andes clade includes 11 species from Colombia, except Astroblepus sp. 20 from the Río Almendro in the northern Peru and Astroblepus cf. regani from the Río Wiñacay in Ecuador. This clade also includes one species from piedmont rivers of the Orinoco basin (Astroblepus sp. 21), one species from the Caquetá river, upper Amazon basin (A. caquetae Fowler, 1943), one species from the Río Dagua, a coastal Pacific river (A. trifasciatus), and six species from the Magdalena-Cauca basin (A. grixalvii Humboldt, 1805, A. cacharas Ardila-Rodriguez, 2011, A. curitiensis Ardila-Rodriguez, 2015, A. homodon (Regan, 1904Regan CT. A monograph of the fishes of the family Loricariidae. Trans Zool Soc London. 1904; 17(3):191-350.), A. nicefori Myers, 1932, and Astroblepus sp. 24). The A. grixalvii group includes topotypes of the type species of the genus, A. grixalvii from Río Palace (upper Río Cauca) and specimens from several Colombian drainages such as upper Magdalena basin (Ríos Guarapas, Saldaña, Coello, Opia, Totare, and Sumapáz), middle Magdalena basin (Río Samaná), upper Cauca basin (Río Quindío), coastal Pacific rivers (Río San Juan and Río Patía), and the cis-Andean piedmont rivers of the Orinoco basin (Río Negro). The A. pradai Ardila-Rodriguez, 2015 lineage contained three nominal species (A. pradai, A. curitiensis, and A. floridablancaensis Ardila-Rodriguez, 2016) from tributaries of Río Sogamoso and Río Lebrija (middle Magdalena basin); the A. cacharas lineage includes two nominal species (A. cacharas and A. verai Ardila-Rodriguez, 2015) from tributaries of Río Lebrija and Río Sogamoso (middle Magdalena basin), and the A. itae lineage includes A. itae and A. ardiladuartei Ardila-Rodriguez, 2015 from tributaries of Río Lebrija (middle Magdalena basin). The species tree recovered A. trifasciatus (Río Dagua, Pacific drainage) as sister to the remaining taxa, and then A. caquetae and Astroblepus sp. 20 from the upper Amazonas (Río Caqueta and Río Almendro, respectively), with this clade being sister-group to all other remaining species of the Northern Andes clade (Fig. 4).

The Central Andes clade includes 14 species: two from the Madre de Dios drainage, a tributary of the Madeira basin (morphospecies B and C), 10 species from the Marañon-Ucayali system, including the ríos Apurimac, Urubamba, Chorobamba, and Pozuzo (morphospecies D, E, F-G, H and Astroblepus sp. 10, 11, 14, 15, 16, and 17), and two species from Río Wiñacay, a Pacific coastal drainage of Ecuador (Astroblepus sp. 18 and 19). The nine morphospecies (A-I) of Schaefer et al. (2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
) represented eight delimited species (i.e., their morphospecies F and G were delimited as a single species). Astroblepus cf. regani and Astroblepus sp. 20 (Northern Andes clade) and Astroblepus sp. 13, 17, 18, and 19 (Central Andes clade) occur either in southern Ecuador or northern Peru, representing the boundaries of the two major clades, and likely an intersection region for the diversification of Astroblepus (Fig. 4).

Additionally, most specimens that could not be allocated into valid species (“sp.”) or those showing morphological similarity with valid species identified in the categories as “aff.” and “cf.” were successfully grouped with valid species and potential species with high support values for the clusters and low genetic divergences (S3). From a total of 208 specimens included in this study, the species tree and species validation in a coalescent multilocus analysis allowed the identification of 97 individuals assigned to the categories “sp.”, “aff.” and “cf.”, as well as supported the eight morphotypes identified previously by Schaefer et al. (2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
).

DISCUSSION

The present study identified 25 well delimited lineages of Astroblepus, eight of which being allocated to valid species (9.7% of species diversity) and the other 17 corresponding to distinct genetic lineages and potential undescribed species. These results indicate an underestimated molecular diversity and represent a first step to improve the taxonomic, phylogenetic and biogeographic understanding of astroblepids. Results represent an expansion of the taxon sampling relative to the previous multilocus analysis of Astroblepus (e.g., Schaefer et al., 2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
), which proposed eight morphotypes of Astroblepus from the piedmont of the upper Río Madre de Dios and tributaries of the upper Marañon-Ucayali system, all major tributaries of the Amazon basin in Peru. Our results agree with the relationships among those morphotypes.

Although astroblepids are morphologically unmistakable regarding to other catfishes by having specialized mouth morphology and pelvic-fin musculature, the species identification is very complex. Regan (1904Regan CT. A monograph of the fishes of the family Loricariidae. Trans Zool Soc London. 1904; 17(3):191-350.) already highlighted morphological differences in the adipose fin due to size, individual variation and preservation of specimens. Morphological characters currently used to distinguish species of Astroblepus, such as tooth type, presence/absence of adipose spine, body shape, fin size and pigmentation pattern have extreme intraspecific variation (Schaefer et al., 2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
). Most described species in the last decade are supported by single-character diagnoses or color pattern. These ambiguous diagnoses with excessive intraspecific variation have generated confusion due to the fact that species were described without a comprehensive taxonomic or phylogenetic support (Ardila-Rodriguez, 2011aArdila-Rodriguez CA. Astroblepus cacharas: (Teleostei: Siluriformes: Astroblepidae), nueva especie del río Cáchira, cuenca del río Magdalena, Colombia. Dahlia Rev Asoc Colomb Ictiol. 2011a; 11:23-33.,b, 2012, 2013a,b, 2014, 2015a,b).

Several of the species diagnosed by those labile characters appeared as a single genetic lineage in our study. For example, our analysis delimited as a single species, topotypes representatives of Astroblepus curitiensi, A. pradai and A. floridablancaensis, described from tributaries of the same hydrographic basin (Río Sogamoso, middle Magdalena basin) with similar pigmentation patterns of body and fins, barbel length and oral disc diameter but diagnosed by variations in the morphological structure of the adipose fin (a denticulate imbedded spine in A. pradai vs. a well-developed adipose fin without spine in A. curitiensis vs. an adipose fin composed of spine posteriorly attached by a membrane to the caudal peduncle in A. floridablancaensis) (Ardila-Rodriguez, 2015bArdila-Rodriguez CA. Astroblepus verai sp. nov- A. pradai, A. curitiensis y A. onzagaensis. Cuatro nuevas especies del departamento de Santander - Colombia. Barranquilla (Atlántico) . 2015b; 7:1-20., 2016Ardila-Rodriguez CA. Astroblepus floridablancaensis sp. nov. una uneva especie de Jabonero (Siluriformes: Astroblepidae) del Municipio de Floridablanca, Departamento de Santander - Colombia. Peces del Departamento de Santander - Colombia. Barranquilla (Atlántico) . 2016; 8:1-20.). Likewise, our molecular results found a single species including A. cacharas from Río Cáchira and A. verai from Quebrada del Medio, a tributary of Río Chucuri (Ardila-Rodriguez, 2011a, 2015b). These species were described by a combination of features with high intraspecific variation, such as configuration of the adipose fin, pigmentation pattern and meristic data. While some differences can be detected between specimens of these species, such as length of the maxilary barbel (extending beyond the oral disc in A. cacharas vs. short maxillary barbel not surpassing the oral disc in A. verai) and shape of the outer row of premaxillary teeth (10-11 conic and one-two bicuspid in A. cacharas vs. 7-8 conic and one bicuspid in A. verai), the coloration pattern and size of oral disc clearly overlap.

In addition to taxonomic issues detected in recently described species discussed above, our results strongly support the recognition of two biogeographically structured clades of Astroblepus. The Northern Andes clade contains species mostly from Magdalena-Cauca, Orinoco and Caquetá (upper Amazon) basins in Colombia, including the type species of the genus, Astroblepus grixalvii, along with specimens from several Colombian regions (upper and middle Magdalena basin, Patía basin and piedmont Orinoco). This clade composes the large A. grixalvii species complex, as better delimited herein. Morphological studies of A. grixalvii are limited to an unpublished dissertation (Briñez-Vásquez, 2011Briñez-Vásquez GN. Redescripción de Astroblepus grixalvii Humboldt, 1805 (Siluriformes:Astroblepidae) y comparación de sus poblaciones trasandinas en la cuenca del rio Magdalena (Colombia). [Master Dissertation]. Ibagué: Universidad del Tolima; 2011.) in which the author identified morphological variation among isolated populations of the species in the Magdalena and Cauca rivers associated with discrepancies in habitat conditions. This result is supported by the presence of various lineages within the complex (S3). As one of the most widespread species of Astroblepus, it is expected that more species within the A. grixalvii complex will be recognized and described afterwards.

Although a detailed biogeographic analysis is beyond the scope of this paper, the structured pattern of Central and Northern Andes clades is an interesting result. Lujan et al. (2015Lujan NK, Meza-Vargas V, Astudillo-Clavijo V, Barriga-Salazar R, López-Fernández H. A multilocus molecular phylogeny for Chaetostoma Clade Genera and Species with a review of Chaetostoma (Siluriformes: Loricariidae) from the Central Andes. Copeia. 2015; 103(3):664-701. https://doi.org/10.1643/ci-14-194
https://doi.org/10.1643/ci-14-194...
) found a similar biogeographic pattern in loricariids of the Chaetostoma clade, that includes two northern Andean clades as sister to a group widely distributed in the central-southern Andes. Similarly, a biogeographic analysis using species distribution models for endemic birds in the tropical region, identified different events of isolation and diversification associated with geographical barriers, being one of the most important the Marañon River valley in the northen Peru. This barrier was responsible to isolate the Central and Northen Andean taxa since the Miocene splitting the areas in four bioregions: the Central Cordillera of Peru, the Eastern Side of the Central Andes, the Northen Central Cordillera and the Tumbesian Mountains (Hazzi et al., 2018Hazzi NA, Moreno JS, Ortiz-Movliav C, Palacio RD. Biogeographic regions and events of isolation and diversification of the endemic biota of the tropical Andes. Proc Natl Acad Sci U S A . 2018; 115(31):7985-90. https://doi.org/10.1073/pnas.1803908115
https://doi.org/10.1073/pnas.1803908115...
). According to those authors, one of the primary drivers of the biogeographic distribution was the formation of sizeable warm river valleys during periods of high Andean uplift, isolating species in the highland ecosystems (Hazzi et al., 2018). A central-northern pattern was also observed in Neotropical plants of the family Rubiaceae (Antonelli et al., 2009Antonelli A, Nylander JAA, Persson C, Sanmartín I. Tracing the impact of the Andean uplift on Neotropical plant evolution. Proc Natl Acad Sci U S A. 2009; 106(24):9749-54. https://doi.org/10.1073/pnas.0811421106
https://doi.org/10.1073/pnas.0811421106...
). Therefore, astroblepids represent an excellent model to further study how these processes influenced the distribution of freshwater fishes in the Andean region.

The present study also supports the hypothesis that most astroblepids are endemic to single or adjacent rivers of the Andes and that their distribution generally does not cross major headwater divides (Schaefer et al., 2011Schaefer SA, Chakrabarty P, Geneva AJ, Sabaj-Pérez MH. Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae). Zool J Linn Soc. 2011; 162(1):90-102. https://doi.org/10.1111/j.1096-3642.2010.00673.x
https://doi.org/10.1111/j.1096-3642.2010...
), despite broader distributions of some lineages such as Astroblepus sp. D, E, and F and within the A. grixalvii species complex. Our results also corroborate the hypothesis of disjunct distribution of sister taxa of Astroblepidae (Schaefer et al., 2011). This can be clearly observed in species from the Pacific region of Ecuador and Colombia that are phylogenetically closer to species from Magdalena-Cauca, Orinoco, and western Amazon systems, forming a reticulate pattern of diversification, even separated by three mountain chains of the Andes: Western, Central and Eastern Cordilleras. Such pattern in the Andes was reported for trans-Andean (Géry, 1962Géry J. Notes on the ichthyology of Surinam and other Guianas.10. The distribution pattern of the genus Hemibrycon, with a description of a new species from Surinam and an incursion into ecotaxonomy. Bull Aquat Biol. 1962; 3(28):65-80.) and Amazonian freshwater fishes (Hubert, Renno, 2006Hubert N, Renno JF. Historical biogeography of South American freshwater fishes. J Biogeogr. 2006; 33(8):1414-36. https://doi.org/10.1111/j.1365-2699.2006.01518.x
https://doi.org/10.1111/j.1365-2699.2006...
; Lima, Ribeiro, 2011Lima FCT, Ribeiro AC. Continental-scale tectonic controls of biogeography and ecology. In: Albert JS, Reis RE, editors. Historical biogeography of neotropical freshwater fishes . Berkeley: University of California Press ; 2011. p.145-64.; Ribeiro et al., 2013Ribeiro AC, Jacob RM, Silva RRSR, Lima FCT, Ferreira DC, Ferreira KM et al. Distributions and phylogeographic data of rheophilic freshwater fishes provide evidences on the geographic extension of a central-Brazilian amazonian palaeoplateau in the area of the present day Pantanal Wetland. Neotrop Ichthyol . 2013; 11(2):319-26. https://doi.org/10.1590/S1679-62252013000200010
https://doi.org/10.1590/S1679-6225201300...
; Dagosta, de Pinna, 2017Dagosta FCP, de Pinna M. Biogeography of Amazonian fishes: deconstructing river basins as biogeographic units. Neotrop Ichthyol. 2017; 15(3):e170034. https://doi.org/10.1590/1982-0224-20170034
https://doi.org/10.1590/1982-0224-201700...
), and further genetic/morphological studies with genera having such distribution (e.g., Creagrutus Günther, 1864, Trichomycterus Valenciennes, 1832) might test this hypothesis. In addition, the apical position of the Pacific lineages in both clades and their non-monophyletic pattern show the complex association between phylogeographic connections with the hydrographic history in the Andean region. In this case mediated probably by the formation of the arc of Western Cordillera (Lundberg et al., 1998Lundberg JG, Marshall LG, Guerrero J, Horton B, Malabarba MCSL, Wesselingh F. The stage for Neotropical fish diversification: A history of tropical South American rivers. In: Malabarba LR, Reis RE, Vari RP, Lucena ZM, Lucena CAS, editors. Phylogeny and classification of Neotropical fishes. Porto Alegre: Edipucrs; 1998. p.13-48.; Albert, Reis, 2011Albert JS, Reis RE. Introduction to neotropical freshwaters. In: Albert JS, Reis RE, editors. Historical biogeography of neotropical freshwater fishes. Berkeley: University of California Press; 2011. p.3-19.) and the subduction of Caribbean and South America plates that drove the uplift of the Central Cordillera separating the Pacific drainages from the remaining systems in South America (Rincon-Sandoval et al., 2019Rincon-Sandoval M, Betancur-R R, Maldonado-Ocampo JA. Comparative phylogeography of trans-Andean freshwater fishes based on genome-wide nuclear and mitochondrial markers. Mol Ecol . 2019; 28(5):1096-115. https://doi.org/10.1111/mec.15036
https://doi.org/10.1111/mec.15036...
).

Overall, our results represent an important step aiming to better delimit and recognize the underestimated species diversity in Astroblepidae and it opens a window for future systematic research on these now well-defined clades. Future directions to investigate the species diversity in this family involve (1) inclusion of unsampled species from Panama and other regions, (2) the formal description of species based on robust and unambiguous morphological diagnoses improving the samples size and ontogenetic variation, (3) the phylogenetic inference based on a time-calibrated genome-scale analysis, and (4) further test our hypothesis of biogeographically-structured species and whether ancestral astroblepids diversified before or after the uplift of the Andean Cordillera.

ACKNOWLEDGMENTS

We are grateful to Jorge Enrique García Díaz who provided logistic support and to José Luis Lozano who kindly helped us in the ANDESCOL2 expedition to Colombian Andes. We also thank the CaVFish Colombia project-Catálogo Visual de Peces de Agua Dulce de Colombia for providing all pictures. This study is dedicated to our colleague Javier A. Maldonado-Ocampo (in memoriam), who was always interested in discovering and protecting the Andean freshwater fishes and for providing a large amount of material of this study collected along many years. The ANDESCOL expeditions and Brycon server used for analyses were funded by FAPESP 14/26508-3 (CO). Authors were funded by grants CNPq 409551/2017-1, FAPESP 14/06853-8, FAPESP 18/23883-9, FAPESP 2016/19075-9 (LEO), FAPESP 16/11313-8 (BFM), FAPESP 17/06551-0 (CSS), 18/09767-6 (CCCS), and FAPESP 14/26508-3 (CO).

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  • HOW TO CITE THIS ARTICLE

    Ochoa LE, Melo BF, García-Melo JE, Maldonado-Ocampo JA, Souza CS, Albornoz-Garzón JG, Conde-Saldaña CC, Villa-Navarro F, Ortega-Lara A, Oliveira C. Species delimitation reveals an underestimated diversity of Andean catfishes of the family Astroblepidae (Teleostei: Siluriformes). Neotrop Ichthyol. 2020; 18(4):e200048. https://doi.org/10.1590/1982-0224-2020-0048

Publication Dates

  • Publication in this collection
    04 Dec 2020
  • Date of issue
    2020

History

  • Received
    16 June 2020
  • Accepted
    09 Oct 2020
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