Anais da Academia Brasileira de Ciências (2019) 91(2): e20180131 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201920180131 www.scielo.br/aabc | www.fb.com/aabcjournal An integrated approach clarifies the cryptic diversity inHypostomus Lacépède 1803 from the Lower La Plata Basin YAMILA P. CARDOSO1, FLORENCIA BRANCOLINI2, LUCILA PROTOGINO3, ARIEL PARACAMPO3, SERGIO BOGAN4, PAULA POSADAS1 and JUAN I. MONTOYA-BURGOS5 1Laboratorio de Sistemática y Biología Evolutiva (LASBE), Facultad de Ciencias Naturales y Museo, Paseo del Bosque, s/n, B1900FWA, La Plata, Buenos Aires, Argentina 2Instituto de Investigación e Ingeniería Ambiental, Universidad Nacional de San Martín, CONICET, Campus Miguelete, 25 de Mayo y Francia, 1650, San Martín, Buenos Aires, Argentina 3Instituto de Limnología “Dr. Raúl A. Ringuelet”, CONICET La Plata, Facultad de Ciencias Naturales y Museo, C.C. 712, 1900, La Plata, Buenos Aires, Argentina 4Fundación de Historia Natural “Félix de Azara”, Departamento de Ciencias Naturales y Antropología, Universidad Maimónides, Hidalgo 775, piso 7, 1405BDB, Buenos Aires, Argentina 5Département de Génétique et Évolution, Université de Genève, 30 quai Ernest Ansermet, 1211, Genève 4, Switzerland Manuscript received on February 8, 2018; accepted for publication on August 11, 2018 How to cite: CARDOSO YP, BRANCOLINI F, PROTOGINO L, PARACAMPO A, BOGAN S, POSADAS P AND MONTOYA-BURGOS JI. 2019. An integrated approach clarifies the cryptic diversity in Hypostomus Lacépède 1803 from the Lower La Plata Basin. An Acad Bras Cienc 91: e20180131. DOI 10.1590/0001-3765201920180131. Abstract: Hypostomus commersoni Valenciennes 1836, Hypostomus cordovae (Günther 1880) and Hypostomus laplatae (Eigenmann 1907) have been little studied since their original descriptions. This study shows a comprehensive review of these species from the Lower La Plata Basin, including their taxonomic history, distribution, color patterns, morphology, and ecological and molecular phylogenetic data. Morphological and phylogenetic analyses based on D-loop sequences suggested that H. commersoni can be separated into two subclades, or subgroups. Based on these results and on the non-overlapping distribution range of the two subclades, we conclude that they represent two distinct species, thereby revalidating H. spiniger. The results also suggest that H. paranensis should be considered as species inquirenda and H. cordovae as valid species. This integrated approach provides key information for assessing the conservation status and biogeographic aspects of the genus Hypostomus in the Lower La Plata Basin. Key words: Argentina, Brazil, freshwater fishes, molecular phylogenetics, Paraná Basin. INTRODUCTION 2017). The species are widely distributed in South America, from Costa Rica to the Salado River Within the family Loricariidae, Hypostomus Basin in Argentina (Ringuelet 1975). Two recent Lacépède 1803 is the most diverse genus, with more than 146 valid species (Eschmeyer et al. studies show the phylogenetic relationships of several species of Hypostomus in the southern part of its distribution, which comprises the Lower Correspondence to: Yamila Paula Cardoso E-mail: [email protected] Paraná and the Río de la Plata Basin (Cardoso et al. ORCID: https://orcid.org/0000-0003-3497-4359 2012, 2016). BIOLOGICAL SCIENCES An Acad Bras Cienc (2019) 91(2) YAMILA P. CARDOSO et al. CRYPTIC DIVERSITY IN Hypostomus Cardoso et al. (2012) raised the question of the sources of diversity in Hypostomus in the La Plata Basin, showing that the richness of species has been shaped by both inter- and intra-basin pressures. However, no further information on the biology, validated distribution, ecology, taxonomic status, or abundance of most of the species inhabiting the basin is available. More than 20 species of Hypostomus are recorded in the Lower La Plata Basin (Koerber and Weber 2014, Litz and Koerber 2014, Mirande and Koerber 2015), but this is likely to be underestimated. This gap in basic biological data makes obtaining thorough knowledge of the evolutionary and biogeographical events shaping this group of catfish a challenge. The present study contributes to a more complete assessment of species diversity and expands available biological data on Hypostomus Figure 1 - Map of collection localities of Hypostomus species inhabiting the Lower La Plata Basin. This commersoni (triangle), H. spiniger (star), H. cordovae (circles) and H. laplatae (squares). White symbols are type localities work focuses on H. commersoni Valenciennes and grey symbols are specimens used only in molecular 1836, H. cordovae (Günther 1880), and H. analyses. laplatae (Eigenmann 1907), species that have been little studied since their original description. specimens were fixed in 4% formalin and deposited The descriptions are outdated and imprecise, in the ichthyology collection of the Fundación de and subsequent taxonomic work has often been Historia Natural Félix de Azara, Buenos Aires, undertaken without examination of the type material Argentina (CFA-IC). Specimens and photographs or was based on a small number of specimens or of type series were examined from the AMNH, a limited distribution range. Here, an integrated American Museum of Natural History, New York; approach, based on taxonomic history, geographic BMNH, British Natural History Museum, London; distribution, morphology, morphometrics, live color pattern, and ecological and molecular CAS, California Academy of Sciences, San phylogenetic data of the type-series, together with Francisco; MACN, Museo Argentino de Ciencias newly collected topotypes of these species, were Naturales “Bernardino Rivadavia,” Buenos Aires; used to re-evaluate their taxonomy. MCP, Museu de Ciências e Tecnologia da Pontifícia Universidade Católica do Rio Grande do Sul, Porto MATERIALS AND METHODS Alegre; MFA-ZV, Museo Provincial “Florentino Fishes were collected with gillnets, trammel nets, Ameghino,” Santa Fe; MHNG, Muséum d’Histoire hand nets, and cast nets. Locations sampled are Naturelle, Genève; MNHN, Museum National presented in Fig. 1. A tissue sample was taken from d’Histoire Naturelle, Paris; MLP, Museo de La each specimen for molecular analysis, preserved Plata, Buenos Aires, and ZMB, Zoological Museum in 96% ethanol, and stored at -20°C. Voucher of Berlin, Berlin (see Appendix). An Acad Bras Cienc (2019) 91(2) e20180131 2 | 25 YAMILA P. CARDOSO et al. CRYPTIC DIVERSITY IN Hypostomus Total DNA was obtained using the salt- continuous morphometric variables and 12 discrete extraction protocol (Aljanabi and Martinez 1997). meristic variables of 91 specimens (Tables I and The PCR amplification of the control region II). Missing data were estimated using the least- (D-loop) of the mitochondrial DNA was performed squares method with SL as explanatory variable. as in Cardoso et al. (2011). The PCR products were All measurements were standardized according purified and sequenced by Macrogen Inc. (Korea). to SL and log-transformed to control for size. New sequences were deposited in GenBank for: H. This transformation, equivalent to the additive commersoni (JF290450 to JF290458, MG457220 log ratio of Aitchinson (1986), controls for size to MG457223, MG457234 to MG457242), H. effect, preserves and linearizes allometric growth, cordovae (KX852401 to KX852410), H. laplatae and prevents spurious correlations of simple (KX852411 to KX852418), and H. spiniger ratios (Atchley et al. 1976, Atchley and Anderson (MG457224 to MG457233). Also, published data 1978, Hills 1978). The data were submitted to a for Hypostomus (Montoya-Burgos 2003, Cardoso between-class analysis (BCA), a particular case of et al. 2012, 2016) were used, so some specimens principal component analysis (PCA) with respect to were incorporated only in the molecular analyses instrumental variables in which there is only a single (see Fig. 1). In total, 109 sequences were edited factor as explanatory variable. In this case, species and manually aligned using BioEdit 7.0.1 (Hall was used as the explanatory factor. To explore the 1999). Two phylogenetic analyses were conducted, more closely related species, a second analysis, regardless of species assignment. Appropriate based on 30 specimens of H. commersoni, 31 of H. substitution models were selected with the Akaike spiniger, five H. affinis (Steindachner 1877), two information criterion (AIC) and the phylogeny H. interruptus (Miranda Ribeiro 1918) and three was inferred using maximum likelihood (ML) H. ancistroides (Ihering 1911) was submitted to implemented in MEGA.7 (Kumar et al. 2016). further BCA. Both analyses were performed with Confidence values for the limits of the ML tree the ade4 1.4-14 (Dray and Dufour 2007) package were computed with 1000 bootstrap replications in R 3.4.1 (R Core team 2014). (Felsenstein 1985). Subsequently, Bayesian Assessment of the conservation threat level inference (BI) analysis was conducted in MrBayes of the analyzed species was primarily based on 3.2.2 (Huelsenbeck and Ronquist 2001, Ronquist the application of the IUCN Red List Criterion et al. 2012) on CIPRES Science Gateway (Miller B (IUCN 2001). This criterion focuses on two et al. 2010). Four chains were run simultaneously spatial measures related to the distribution of the (three heated, one cold) for 30 million generations, species: area of occupancy (AOO) and extent of with sampling every 500 generations. Following occurrence (EOO) (IUCN 2017). Both measures graphic