Zoosyst. Evol. 100 (1) 2024, 69-85 | DOI 10.3897/zse.100.112778 Zags SOMA 9 Se MON) > PENSUFT. pdb Population variation of Diapoma pampeana (Characiformes, Characidae, Stevardiinae) from an isolated coastal drainage in Uruguay, with new records: comparing morphological and molecular data James Anyelo Vanegas-Rios?, Wilson Sebastian Serra Alanis**, Maria de las Mercedes Azpelicueta’, Thomas Litz*, Luiz Roberto Malabarba® 1 Division Zoologia Vertebrados, Facultad de Ciencias Naturales y Museo, Unidades de Investigacién Anexo Museo, Gabinete 104, CONICET, UNLP, La Plata, Buenos Aires, Argentina Seccion Ictiologia, Departamento de Zoologia, Museo Nacional de Historia Natural, Montevideo, Uruguay Centro Universitario Regional del Este (CURE) Sede Rocha, Rocha, Uruguay Friedhofstr. 8, 88448 Attenweiler, Germany Laboratorio de Ictiologia, Departamento de Zoologia, Universidade Federal do Rio Grande do Sul (UF RGS), Av. Bento Gongalves, 9500, 91501-970 Porto Alegre, RS, Brazil oa FF W PY https://zoobank. org/BAC6CFC9-E0EC-459A-9A7B-18F0A5F71D68 Corresponding author: James Anyelo Vanegas-Rios (anyelovr@fcnym.unlp.edu.ar) Academic editor: Nicolas Hubert # Received 20 September 2023 Accepted 8 December 2023 @ Published 26 January 2024 Abstract Diapoma pampeana was recently described to occur in the upper Negro basin in Uruguay and Brazil. An isolated population ten- tatively identified as D. pampeana from the Pando stream, a perturbed coastal drainage in Uruguay, is studied and compared under the light of morphological and molecular data to test if there is evidence to consider it as a separate species. New geographical re- cords for the species are presented and included in the comparisons. The specimens analyzed were pooled into four groups: Pando, Santa Lucia, Middle Negro and Upper Negro. We analyzed 32 morphological characters using statistical procedures and recovered a COI-based phylogeny of different populations of D. pampeana to test if they may represent different species. Size-corrected PCA revealed that the Pando and Upper Negro groups are greatly diverging in both morphometric and meristic data along PC1 (mainly by the snout to dorsal-fin origin, dorsal to adipose-fin origins, number of longitudinal scales and predorsal scales). This deviating pattern was also obtained in a cluster analysis. The Santa Lucia and Middle Negro groups were found to be intermediate morpho- types. In contrast, molecular analyses revealed that the Pando and Upper Negro specimens resemble genetically and, thus, are placed together in the Neighbor-joining and Bayesian topologies, as part of a monophyletic Diapoma. We proposed that the Pando population, despite its deviating morphology observed, can be classified as D. pampeana. Therefore, this population constitutes a remarkable example of an isolated population that is morphologically divergent but genetically similar to the geographically most distant conspecific population. Key Words Body shape variation, integrative taxonomy, Neotropical fish, phylogeny, size-corrected PCA Introduction small-sized species (no more than 100 mm SL) (Thomaz et al. 2015; Mirande 2019; Ferreira et al. 2021; Ito et al. 2022; The Neotropical fish genus Diapoma Cope, 1894 isamem- Fricke et al. 2023). Diapoma is recognized as a monophy- ber of the tribe Diapomini, which is one of the largest mono- _letic group based on molecular data and combined evidence phyletic groups within the Stevardiinae with ~ 135 relatively (Thomaz et al. 2015; Mirande 2019; Ito et al. 2022). Copyright Vanegas-Rios, J.A. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 70 Vanegas-Rios, J.A. et al.: Population variation of Diapoma pampeana from Pando stream To date, this genus includes sixteen valid species that are distributed along different river drainages in Argen- tina, Brazil, Paraguay and Uruguay, mainly within the Rio de la Plata basin. Five species are known from the Parana-Paraguay system, D. guarani (Mahnert & Géry, 1987) (several streams flowing into the Parana basin in the border region between Alto Parana, Paraguay and Mi- siones, Argentina), D. obi (Casciotta, Almiron, Pialek & Rican, 2012) (some tributaries from the Paranay-Guazu drainage and the Moreno stream in Misiones, Argentina), D. nandi Vanegas-Rios, Azpelicueta & Malabarba, 2018 (the Piray-Mini stream in Misiones, Argentina), the re- cently described D. potamohadros Ito, Carvalho, Pava- nelli, Vanegas-Rios & Malabarba, 2022 (endemic from the Rio Iguazu basin in Argentina and Brazil) and D. ter- ofali (Géry, 1964) (the Rio Lujan and other streams flow- ing into the Rio de la Plata basin in Buenos Aires, Argen- tina) (Mahnert and Géry 1987; Menezes and Weitzman 2011; Casciotta et al. 2012; Vanegas-Rios et al. 2018; Ito et al. 2022). The Rio Uruguay basin is the water body that possesses the greater number of Diapoma species regis- tered so far (seven spp.), D. alegretense (Malabarba & Weitzman, 2003) (the Rio Ibicui system in Rio Grande do Sul, Brazil, and Laguna Redonda in Artigas, Uruguay), D. guarani (Barragem Sanchuri in Rio Grande do Sul, Brazil), D. lepiclastum (Malabarba, Weitzman & Casci- otta, 2003) (from the Rio Pelotas and Rio Canoas to the Lageado Uniao stream in Brazil, and in the eastern region of Misiones, Argentina), D. pampeana Ito, Carvalho, Pavanelli, Vanegas-Rios & Malabarba, 2022 (the upper Rio Negro basin in Brazil and Uruguay), D. pyrrhopteryx Menezes & Weitzman, 2011 (the Rio Pelotas basin in Rio Grande do Sul, Brazil, and the Pepiri Iguazu basin in Mi- siones, Argentina), D. terofali (streams flowing into the Rio Uruguay system in Rio Grande do Sul, Brazil, and in Artigas and Cerro Largo, Uruguay), and D. uruguayense (Messner, 1962) (tributaries of the Rio Uruguay in the border region between Argentina, Uruguay, and Brazil, and from the headwaters of the Rio Negro) (Malabarba and Weitzman 2003; Zarucki et al. 2010; Menezes and Weitzman 2011; Thomaz et al. 2015; Almiron et al. 2016). Four species have been recorded for the Laguna dos Patos basin and the coastal drainages of south Brazil, D. dicrop- otamicum (Malabarba & Weitzman, 2003) (northern trib- utaries of the Rio Jacui from the Serra Geral formation), D. itaimbe (Malabarba & Weitzman, 2003) (Tramandai, Mampituba, Ararangua river basins, southern coast of Brazil), D. speculiferum Cope, 1894, D. thauma Menez- es & Weitzman, 2011 (tributaries of the Rio Jacui basin, Rio Grande do Sul, Brazil) and D. tipiaia (Malabarba & Weitzman, 2003). Finally, D. alburnum (Hensel, 1870) is the most widely distributed species occurring in the Uruguay (e.g. the Rio Queguay basin), Parana (the Rio Gualeguay basin), Laguna dos Patos (e.g. the Rio Jacui system) basins and coastal drainages of southern Brazil (Malabarba 1983; Malabarba and Weitzman 2003; Proto- gino and Miquelarena 2012; Paullier et al. 2019). Diapoma pampeana, recently described from the upper Rio Negro basin, reaches 35 mm SL and can be zse.pensoft.net differentiated from all its congeners by a combination of characters, mainly from body pigmentation (Ito et al. 2022), including the presence of: a narrow and con- spicuous black line along the horizontal septum, never forming a wide lateral stripe; a longitudinal black stripe extending posteriorly on the middle caudal-fin rays; and a small black blotch, restricted to the base of the middle caudal-fin rays. We found specimens that potentially could be identified as D. pampeana from the Pando stream, a coastal drain- age flowing into the Rio de la Plata estuary in Uruguay, based on the resemblance of the humeral mark, midlateral stripe, and caudal-fin pigmentation. The possible pres- ence of this species in the Pando stream caught our atten- tion because the preliminary morphometric data obtained were somewhat incongruent with the data reported in the description by Ito et al. (2022), and because no conge- ner has been recorded in this area so far (Malabarba and Weitzman 2003; Menezes and Weitzman 2011; Gurdek and Acufia-Plavan 2017). This small drainage, which is located in the middle of urban and agricultural areas, has been greatly modified and affected by anthropogenic factors such as pollution, industrial activities, and urban waste originated from anthropogenic actions (Echevar- ria et al. 2011; Achkar et al. 2012; Gutiérrez et al. 2015; Muniz et al. 2019) and is considered of great importance, among other reasons, because its sub-estuarine mouth plays a role in the breeding and nursery for grounds of fish (Defeo et al. 2009; Acufia et al. 2017; Muniz et al. 2019). There are several studied cases in which species pre- viously considered as distributed in the Rio Uruguay drainage and Atlantic river coastal drainages have been separated in two different species [e.g. Parapimelodus nigribarbis (Boulenger, 1889) vs. P. valenciennis (Lut- ken, 1874), see Lucena et al. (1992); Pimelodus pintado Azpelicueta, Lundberg & Loureiro, 2008 vs. P. maculatus Lacepeéde, 1803, see Azpelicueta et al. (2008); Bunoceph- alus erondinae Cardoso, 2010 vs. B. doriae Boulenger, 1902, see Cardoso (2010); Pseudocorynopoma stanleyi Malabarba, Chuctaya, Hirschmann, Oliveira & Thomaz, 2020 vs. P. doriae Perugia, 1891, see Malabarba et al. (2021)]. So, the present study aims to carry out a mor- phological (mainly morphometric and meristic data) and COlI-based comparison between the population of D. pampeana from the Rio Negro basin, a tributary of the lower Rio Uruguay, and the population tentatively identi- fied as D. pampeana from the Pando stream, that empties directly in the Atlantic Ocean. We expect to test if there is evidence to consider these isolated populations as sepa- rate species or to treat them as a single species, describing any intraspecific variation between them. Additionally, a recent examination of specimens of Diapoma at Museo Nacional de Historia Natural, Montevideo (MHNM), revealed two lots of individuals similar to D. pampea- na in the body shape and meristic data from the Yi and Santa Lucia river basins (as fixed in 10% formalin, DNA extraction was unavailable). Consequently, they were in- cluded in the morphological analyses to enhance the com- parisons and are also presented as new records. Zoosyst. Evol. 100 (1) 2024, 69-85 Materials and methods Specimens were collected in the Pando stream (permis- sion No. 202/717/04, DINARA, Uruguay) between 2003 and 2004. They were fixed in formalin 10% and preserved in alcohol 70%. Some of them, which were preserved originally in ethanol 96%, were rehydrated before being preserved and catalogued as the others. Additional stud- ied specimens of D. pampeana and comparative species are deposited in the following institutions: MACN-ict, MLP, MHNG, MHNM, UFRGS, and UNMDP (abbrevi- ations according to Sabaj 2020). Morphological analysis Measurements and other counts were taken following Fink and Weitzman (1974), with the modifications pre- sented by Ito et al. (2022). Twenty-two measurements were taken point to point with a digital caliper under a stereomicroscope and are expressed as percentages of standard length (SL) or head length (HL) for units of the head. Specimens were cleared and stained (c&s) follow- ing Taylor and Dyke (1985). The total number of ver- tebrae was counted in c&s specimens. Those counts in- cluded the first preural centrum plus the first ural centrum (PU1 + U1) counted as one element and all four vertebrae of the Weberian apparatus. The specimens from the Pando stream were compared with type specimens of D. pampeana from the Rio Negro basin under different statistical procedures. Additionally, the specimens presumably belonging to D. pampeana from the Yi and Santa Lucia river basins in Uruguay were only processed in the morphological analyses because they were not suitable for DNA extraction. To facilitate comparisons, a morphometric data matrix that included all of these specimens was pooled into groups (based on geographic drainages) as follows: Pando (n = 17), Santa Lucia (Canelon Grande, n=2), Middle Negro (Y1, n= 15), and Upper Negro (several localities, n = 35). Nearly all the specimens analyzed in all groups were adults, except for a few immature specimens that were also added in the comparisons (excluding the bone hooks, no morphomet- ric or meristic differences were observed between them and the respective adults). This dataset was analyzed using the “allometric vs. standard” procedure (Elliott et al. 1995), under which the allometric coefficients are cal- culated concerning a standard (reference, such as over- all length) measurement (each variable is regressed onto this after log-transformation). The size-corrected mor- phometric dataset was analyzed using a principal com- ponent analysis (PCA), based on the covariance matrix. For the PCA, the number of significant principal compo- nents (PCs) was decided by two criteria: the broken-stick model (Frontier 1976) and the scree plot method (Cattel 1966). To compare the dissimilarity between the groups associated with the size-corrected morphometric data, a hierarchical cluster analysis was performed using Ward’s method (Ward 1963) and Euclidean distances, under 1000 71 bootstrap replicates. Missing values in measurements (e.g. some fin rays were broken in a few individuals) were imputed from predictor values obtained under maximum likelihood from the EM algorithm (Dempster et al. 1977; Pigott 2001) using 500 iterations. For the morphometric data, confidence intervals of 95% were calculated using 9000 bootstraps. Meristic data showing slightly different patterns be- tween the Pando group and the other groups were analyzed using a PCA on the root-squared transformed values and the correlation matrix (Quinn and Keough 2002) (outli- ers that could not be reexamined in the specimens were omitted and the mean was imputed for missing data). To illustrate the distinctive patterns within the Pando group, we used Tukey box plots for the variables, which provide a clearer representation of the observed variability. When it comes to counts, both mean and mode values are re- ported, and they are separated by a slash. For those analyses, normality was tested using a Sha- piro—Wilk statistic (W) in each case (a < 0.05) and data were log-transformed when needed to better approximate to a multivariate normality. Statistical procedures were carried out in PAST 4.12 (Hammer et al. 2001), IBM SPSS Statistics 26.0 (IBM 2019), and GraphPad Prism 9.4.1 (GraphPad Software, San Diego, CA, USA). Molecular analysis The mitochondrial cytochrome c oxidase subunit I gene (COI) was obtained from two specimens from the Pando stream. DNA extraction and polymerase chain reaction (PCR) were carried out following the standard COI proto- cols (Ivanova et al. 2006; Rosso et al. 2012), under differ- ent sets of primer cocktails for fishes (Ivanova et al. 2007). In total each amplification reaction produced a vol- ume of 12.375 uwL from 2 wl of DNA template, 6.25 wL of 10% trehalose, 2 uL of molecular biology grade wa- ter, 1.25 wL of 10x reaction buffer, 0.625 wL of MgCl2 (50 uM), 0.0625 wL of dNTP (10 mM), 0.0625 wL of each primer (10 uM) and 0.0625 uL of Invitrogen’s Platinum Taq. polymerase (5 U wL-1). The amplification condi- tions consisted of 2 min at 95 °C, followed by 35 cycles at 94 °C for 30 s, at 52 °C for 40 s and at 72 °C for 1 min, and ended at 72 °C for 10 min. E-Gels (Invitrogen) were used to check the amplification success. The COI gene was sequenced in Macrogen (Korea) and IGEVET-UNLP (Argentina). Sequence chromatograms were edited using BioEdit 7.2.5 (Hall 1999). For comparative purposes, in addition to the newly generated sequences, 72 COI sequences were selected from representative specimens of the valid species of Dia- poma (except D. nandi) (Ito et al. 2022) and genera close- ly related to it (detailed later herein) that are available in GenBank and Barcode of Life database (BOLD, available at http://www.boldsystems.org) (accession numbers for all sequences analyzed are provided in Suppl. material 1). The COI sequences were aligned with MUSCLE (1000 iterations) (Edgar 2004) and generated as a data matrix zse.pensoft.net 72 Vanegas-Rios, J.A. et al.: Population variation of Diapoma pampeana from Pando stream partitioned by the first three codon positions in MEGA 11.0.13 (Tamura et al. 2021). The COI dataset was upload- ed to Zenodo (https://do1.org/10.5281/zenodo.8361520). To analyze the phylogenetic placement of the specimens from the Pando stream through different methods, the COI data matrix was analyzed by the phylogenetic procedures and conditions described hereafter. Modeltest-NG (Darri- ba et al. 2019) was used for selecting the best-fit nucleo- tide substitution model available for each procedure (and computational package) based on the partitioned align- ment when necessary. The AIC and BIC statistical criteria were explored, but the latter was used to choose the best model among the candidate models. The neighbor-joining (NJ) tree (10000 bootstrap) was constructed based on the Tajima+Nei model with rates gamma-distributed as im- plemented in Mega. Bayesian analyses were conducted in MRBAYES 3.2.2 (Ronquist et al. 2012) using two runs, each with four Markov chains, which ran for 60 million generations (25% discarded as burn-in, sampling a tree every 3000 generations) under the models SYM+I (posi- tion 1), F81 (position 2) and HK Y+G (position 3). Tracer 1.7.2 (Rambaut et al. 2018) was used to evaluate the re- sults of the MRBAYES analyses from each run (ESS val- ues and InL plots). The trees were visualized and prepared with FIGTREE 1.4.4 (Rambaut 2018). The CIPRES por- tal (Miller et al. 2010) was used to run the following com- putational programs: MODELTEST-NG and MRBAYES 3.2.2. The outgroup was composed of species from Bry- conamericus Eigenmann, 1907, Hypobrycon Malabarba & Malabarba, 1994, Nantis Mirande, Aguilera & Azpeli- cueta, 2006, Odontostoechus Gomes, 1947, Piabarchus Myers, 1928, and Piabina Reinhardt, 1867, genera that have been considered in preceding studies (Ferreira et al. 2011; Mirande 2019; Ito et al. 2022) to be closely related to Diapoma (trees were rooted in Piabina species when required). Additionally, to compare the interspecific and within-species variability, the uncorrected pairwise ge- netic distances (gamma distributed and pairwise deletion) were calculated in MEGA 11.0.13 (Tamura et al. 2021). To examine the potential variability associated with polymorphism between the Pando and Upper Negro specimens, despite the limited number of samples avail- able, the specimens of D. pampeana in the COI data matrix were pooled into the two respective groups using DNASP 6.12.03 software (Rozas et al. 2017) to calculate the polymorphism sites, nucleotide diversity (7) (Nei and Li 1979), net (Da) and absolute (Dxy) divergences (Nei 1987), and to generate the haplotypes set including in- variant sites and gaps. POPART (Leigh and Bryant 2015) was used to construct a haplotype network using a medi- an-joining algorithm and default settings. Results Based on the comparisons carried out (detailed below), we confirmed that the examined specimens from the Pan- do stream (Figs 1, 2A, B), Santa Lucia system (Fig. 2C), and middle Rio Negro basin (Fig. 2E) correspond to new zse.pensoft.net records of D. pampeana, which extend its distribution to the southwest from the upper Negro basin (in straight-line distances: ~ 200 km to Middle Negro, ~ 280 km to Santa Lucia, and ~ 290 km to Pando) (Fig. 3). The list of exam- ined specimens of D. pampeana is presented in Table 1. Morphological comparisons The measurements of the examined specimens are sum- marized in Table 2. Comparing the morphometric data between the Pando group and the other groups, discrete differences between the ranges obtained were not detect- ed. Some tendencies based on the mean in some measure- ments, with partially overlapping ranges, were observed. The distance between the snout and dorsal-fin origin tend- ed to be slightly longer in the Pando, Santa Lucia and Mid- dle Negro groups than in the Upper Negro group (53.5- 58.2% SL, mean = 55.7%+1.4 in Pando; 54.2—55.7% SL, mean 54.9%+1.1 in Santa Lucia; 54.2-57.6% SL, mean = 55.8%+1.0 in Middle Negro vs. 49.0-56.2% SL, mean = 52.7%+1.7 in Upper Negro). The Pando group present- ed a slightly smaller distance between the dorsal- and adipose-fin origins compared to the Upper Negro group (30.4-34.8% SL, mean = 33.0%+1.1 vs. 33.0-39.9% SL, mean = 37.1%+1.4), but almost similar to the other groups (33.6—-34.4 & SL, mean = 34.0+0.6 in Santa Lucia; 32.0-36.1% SL, mean = 33.9%+1.0 in Middle Negro, re- spectively). In other measurements, as the caudal pedun- cle length and snout length, the Pando group tended to show greater mean values as follow (Pando, Santa Lucia, Middle Negro and Upper Negro, respectively): for cau- dal peduncle length 12.1-14.6% SL, mean = 13.6%+0.7; 12.1-13.4% SL, mean = 12.8+0.9; 12.5—15.0% SL, mean = 13.4%+0.6; 8.9-13.4% SL, mean = 11.3%+1.0; and for snout length 19.3—22.3% HL, mean = 21.0%+0.8; 21.5— 21.6% HL, mean = 21.5%+0.1; 20.1—22.1% HL, mean = 20.8%+0.6; 16.3—22.1% HL, mean = 19.1%+1.6. Based on the consensus between the scree plot method and broken-stick model (Suppl. material 2), to ensure that did not discard biologically pertinent data, four eigenvec- tor elements were selected in the PCA on the size-correct- ed data, which accounted for 70.7% of the total variance (Suppl. material 3; Fig. 4A, B). Along the first axis 1n the PC1 vs. PC2 plot (Fig. 4A: explained 53.2% of the total variance), the Pando group was almost fully differentiat- ed from the Upper Negro group, but overlapped with the majority of the specimens of the Santa Lucia and Middle Negro groups. In the PC3 vs. PC4 plot (Fig. 4B explained 17.6% of total variance), the groups appeared to overlap, and there was no clear distinction between them. PC1 was most heavily loaded by the following measurements (Fig. 4A, Table 3): negatively by the snout to dorsal-fin origin (-0.5), snout to anal-fin origin (-0.3), caudal pe- duncle length (-0.3), snout to pelvic-fin origin (-0.2), and caudal peduncle depth (-0.2); and positively by the dor- sal- to adipose-fin origins (0.5) dorsal fin to caudal-fin base (0.2), dorsal-fin length (0.2), and anal-fin base length (0.2). PC2 was most influenced by positive variables such Zoosyst. Evol. 100 (1) 2024, 69-85 73 Figure 1. Coloration in life of D. pampeana (A, B) from the Pando stream, Canelones Uruguay. Photo by J. Pfleiderer. Table 1. New records and material examined of D. pampeana. n= number of examined specimens. Group names corresponds with those described in the text. Accession numbers: OR533516* and OR533515**. Group Santa Lucia Santa Lucia Pando Pando Pando Pando Pando Middle Negro Upper Negro Upper Negro Upper Negro Upper Negro Upper Negro Upper Negro Upper Negro Upper Negro n 10 SL (mm) IB) 23:9 32.0=33.9 30.6 22:2=2019 25.3-34.8 252 19.6-29.8 29:9=33.6 27.4-28.7 24.3 27.2-32.0 27.3-29.1 255 25:9-33.6 29.6 Catalog number MHNM 1125 MHNM 1189 MHNM 812 MLP 14443 MLP 11444* MLP 11445 UNMDP 5219** MHNM 4018 UFRGS 8119 UFRGS 8120 UFRGS 8121 UFRGS 8122 UFRGS 8123 UFRGS 8429 UFRGS 8464 UFRGS 28705 Country Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Brazil Brazil Brazil Locality Canelones, Rio Santa Lucia basin, Canelon Grande stream Canelones, Rio Santa Lucia basin, Canelon Grande stream Canelones, Canada de Ramos, Pando, Pando stream Canelones, Canada de Ramos, Pando, Pando stream Canelones, Canada de Ramos, Pando, Pando stream Canelones, Canada de Ramos, Pando, Pando stream Canelones, Canada de Ramos, Pando, Pando stream Durazno, marginal lagoon to Rio Yi, Estancias del Lago Cerro Largo, small stream at Route 26, ca. 59 km from Melo, between Sauce creek and Fraile Muerto creek Tacuarembé, Rio Tacuaremb6, at Route 26, Villa Ansina Rivera, Mazangano Bridge at Route 44 Rivera, lateral puddles and Corrales creek, affluent of Rio Tacuaremb6, Route 27 Tacuarembé, Caraguata creek, tributary to Rio Tacuarembé, Route 26, Las Toscas Rio Grande do Sul, Bagé, road between Acegua and Bagé, Rio Negro Rio Grande do Sul, Bagé, road between Acegua and Bagé, BR-153, Cinco Saltos creek, affluent of Rio Negro Rio Grande do Sul, Bagé, road between Acegua and Bagé, BR-153, Cinco Saltos creek, affluent of Rio Negro Latitude/Longitude 34°29'14.00'S, 56°20'33.61"W 34°29'14.70'S, 56°20'34.54"W 34°43'39.01"S, 55°56'39"W 34°44'19.2'S, 55°56'27"W 34°42'12'S, 55°56'42.6'W 34°44'19.2'S, 55°56'27"W 34°42'12'S, 55°56'42.6'W 33°21'47.16'S, 56°35'23.43'W 32°17'39'S, 54°44'59"W JIE DO.33,5,.00- Lolo w, 32°06'33'S, 54°40'08.6"W o1723. 20-9, 00 VOL4a W D2n09 29-3; 00-0 27a, 31°28'37'S, 54°08'20"W 31°36'53'S, 54°08'42"W 31°36'53'S, 54°08'42"W Remarks 1 c&s: 28.7 mm SL 15 fully measured 3 c&s: 30.4— 31.5 mm SL holotype zse.pensoft.net 74 Vanegas-Rios, J.A. et al.: Population variation of Diapoma pampeana from Pando stream Figure 2. Extern morphology of studied specimens of Diapoma pampeana. A. MLP 11443, male, 30.6 mm SL, Uruguay, Pando Stream; B. MLP 11445, female, 35.1 mm SL, Uruguay, Pando Stream; C. MHNM 1125, female, 31.3 mm SL, Uruguay, Canelon Grande Stream; D. MHNM 4018, male, 29.8 mm SL, Uruguay, marginal lagoon to Rio Yi; E. MHNM 4018, female, 29.3 mm SL, Uru- guay, marginal lagoon to Rio Yi. Photographs of the specimens from the Upper Negro are available in Ito et a/. (2022). Scale bar: 1 mm. zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 69-85 hs 9°0’ jr iS a SSN . -7°0" -23°0' Loic i. -39°0' 710 ~~! = 0 N O O ». PC1 (27.0 %) PC3 (13.2 %) Figure 4. Most discriminant axes obtained from the PCA analyses performed using morphometric and meristic data of studied spec- imens of Diapoma pampeana (in each plot, the loadings are scaled to 90% of the PC scores). Size-corrected measurements: A. PC1 vs. PC2 plot; B. PC3 vs. PC4 plot. Meristic data; C. PC1 vs. PC2 plot; D. PC3 vs. PC4 plot. Only these variables that most loaded the components are indicated as follows: E- depth at dorsal-fin origin; F- snout to dorsal-fin origin; G- snout to pelvic-fin origin; H- Snout to anal-fin origin; I- distance between dorsal- and adipose-fin origins; J- dorsal fin to caudal-fin base; K- anal-fin base length; L- caudal peduncle length; M- longitudinal scales; N- lateral-line scales; P- scales between lateral line-dorsal origin; Q- scales between lateral line-pelvic origin; R- circumpeduncular scales; S- predorsal scales; T- number of branched anal-fin rays; U- gill rakers on up- per limb of branchial arch; V- gill rakers on lower limb of branchial arch; W- number of maxillary teeth; X- number of dentary teeth. as the snout to anal-fin origin (0.3), depth at dorsal-fin origin (0.2), snout to pelvic-fin origin (0.2), and distance between the dorsal- and adipose-fin origins (0.2). PC3 was most affected by the dorsal fin to caudal-fin base (-0.3), whereas PC4 was most strongly loaded by the anal-fin base (0.3). The cluster analysis showed that the four groups ana- lyzed were distributed into two large clusters (most boot- strap values were below 50). All the specimens of the Up- per Negro group (except three) were almost completely separated from the Pando, Santa Lucia, and Middle Ne- gro groups. In contrast, the specimens of the Pando group were not clustered separately, but were instead mixed mainly with the specimens of the Santa Lucia and Middle Negro groups (Suppl. material 4). The comparative results obtained in the meristic data are presented in Table 4. The first four components (ex- plained 64.4% of the total variance, Suppl. material 3) zse.pensoft.net were chosen as significant to analyze the variation in the meristic data, following the same criteria used for the morphometric data (Fig. 4C, D; Suppl. material 2). The Pando group was slightly differentiated from the Upper Negro group along the horizontal axis in the PC1 vs. PC2 plot (accounted for 41.3% of the total variance, Fig. 4C), but overlapped almost completely with the other groups. In the PC3 vs. PC4 plot (explained 23.0% of the total variance, Fig. 4D), the Pando group was not separately distributed from the other groups along the axes. PC1 was most strongly influenced by the number of longitudinal scales (0.7) and predorsal scales (0.7) (Fig. 4C, Table 3). PC2 was strongly loaded by the number of dentary (0.7) and maxillary (0.5) teeth, and number of gill rakers on the upper limb of the first branchial arch (0.5) (Table 3). PC3 was mainly influenced by the number of pored lat- eral-line scales (-0.7), whereas PC4 was greatly affected by the number of branched anal-fin rays (-0.7). Tukey Zoosyst. Evol. 100 (1) 2024, 69-85 Table 3. Loadings obtained from the PCA analyses using mor- phometric and meristic data. Percentages of variance are reported. Variables Components 1 2 3 4 Morphometric data: 41.5% 11.6% 9.2% 8.4% Depth at dorsal-fin origin -0.1 0.2 0.2 0.2 Snout to dorsalfin origin 0.5 0.1 0.1 0.0 Snout to pectoral-fin origin -0.1 0.1 0.0 0.0 Snout to pelvic-fin origin 0.2 0.2 0.0 0.0 Snout to analfin origin -0.3 0.3 0.1 0.1 Distance between dorsal- and adipose-fin origins 0.5 0.2 0.1 0.2 Dorsal fin to caudal-fin base 0.2 0.1 0.3 0.2 Dorsalfin length 0.2 0.0 0.1 0.1 Dorsal-fin base length 0.1 0.0 0.1 0.0 Pectoral-fin length 0.1 0.1 0.0 0.0 Pelvic-fin length 0.1 0.1 0.1 0.1 Analfin base length 0.2 0.1 0.0 0.3 Caudal peduncle depth 0.2 0.0 -0.1 0.0 Caudal peduncle length 0.3 0.0 0.2 0.0 Head length -~0.1 0.1 0.0 0.0 Snout length -~0.1 0.0 0.0 0.0 Horizontal eye length 0.0 0.0 0.0 0.0 Postorbital head length 0.0 0.1 0.0 0.0 Least interorbital width 0.1 0.0 0.0 0.0 Upper jaw length 0.0 0.1 0.0 0.0 Lower jaw length 0.1 0.1 0.0 0.0 Meristic data: 27.0% 14.4% 13.2% 9.8% Longitudinal scales 0.7 -0.4 0.1 0.1 Lateralline scales 0.3 0.0 0.7 0.0 Scales between lateral line-dorsal origin -0.6 -0.4 0.2 -0.4 Scales between lateral line-pelvic origin -0.6 0.2 0.2 0.5 Circumpeduncular scales 0.5 0.3 0.6 0.4 Predorsal scales 0.7 0.3 0.1 0.0 Branched anal-fin rays 0.2 0.2 -0.4 0.7 Gill rakers upper limb of branchial arch 0.5 0.5 -0.3 0.0 Gill rakers lower limb of branchial arch 0.6 0.1 0.1 -0.2 Maxillary teeth -0.4 0.5 -~0.1 -0.1 Dentary teeth 0.0 0.7 0.5 0.1 box plots of counts that most affected PCA and were most distinctive for the Pando group are presented in Suppl. material 5. The multivariate analyses performed on the morphometric and meristic data converged in coincident results that, despite having overlap between some indi- viduals, showed the population from the Pando stream to be somewhat distinctive morphologically from the speci- mens from the upper Rio Negro basin. The number of ver- tebrae of the Pando group was observed within the range of variation of the Upper Negro group (34 vs. 34-35). 7/ The pigmentation pattern observed in the Pando group (Figs 1, 2A, B) was similar to that found in the Upper Ne- gro groups (Ito et al. 2022: figs 1-3), mainly character- ized by the vertically enlarged humeral spot, the narrow and conspicuous black line along the horizontal septum of body (in some specimens of the Pando group, it was observed to be somewhat silvery), the longitudinal black stripe extending posteriorly on the middle caudal-fin rays, and the presence of a small black blotch, restricted to the base of the middle caudal-fin rays. The specimens of the Middle Negro group (Fig. 2D, E) were observed to be sim- ilarly pigmented as the other groups, except for the cau- dal-fin blotch, which was not completely extended along the middle rays in some specimens. The examined speci- mens of the Santa Lucia group were found slightly faded (Fig. 2C), but some pigmentation characters of D. pam- peana as those aforementioned were found to be present. Molecular comparisons The genetic variation seen, based on the Tajima-Nei dis- tance between the Pando specimens and the specimens from the upper Negro basin of D. pampeana were found to be very low or nearly zero (< 0.002), even when boot- strapped (10000). In the NJ topology (Fig. 5), the two specimens of the Pando stream analyzed were placed to- gether with the remaining specimens of D. pampeana from the upper Negro basin and, particularly, as closely related to one specimen of that basin than between each other (Fig. 5). The Bayesian topology showed that D. pampeana was more related to D. guarani and D. obi within a com- mon clade with D. potamohadros and D. tipiaia (Fig. 6). In both the Bayesian and NJ trees, the analyzed specimens of Diapoma from the Pando stream were strongly placed together in the same clade with the specimens of D. pam- peana. The general pattern of interrelationships among the Diapoma species was found to be almost similar between both methods. The uncorrected pairwise mean distance obtained for D. pampeana ranged from 3.5 to 7.6% (Sup- pl. material 6: the lowest value with D. obi and the high- est value with D. itaimbe). The intraspecific variation of D. pampeana was observed to be varying from 0 to 0.2%. Only one specimen of the Upper Negro group (UFRGS Table 4. Comparative meristic data obtained for the studied specimens of D. pampeana. SD = standard deviation; n = number of examined specimens. Mean and mode values are reported. Group names corresponds with those described in the text. Pando Range Mean/ n Range Mo Mode+SD Longitudinal scales 35-38 =36.9/3740.9 17 35-37 LateraHine scales 7-9 7.8/7+0.8 17 5-8 Scales between lateral line-dorsal origin 5-5 5.0/5+0.0 17 5-5 Scales between lateral line-pelvic origin 4-5 4.1/4+0.2 17 5-5 Circumpeduncular scales 14-15 14.1/14+0.3 15 15 Predorsal scales 12-15 13.6/1340.8 17 14-15 Branched anal-fin rays 21-26 8=23.4/2341.4 17 22-26 Gill rakers upper limb of branchial arch 7-10 8.3/8+0.8 14 7 Gill rakers lower limb of branchial arch 14-18 14.9/1441.2 14 15 Maxillary teeth 1-3 1.6/2+0.6 15 2 Dentary teeth 7-11 8.8/9+1.1 15 12 Santa Lucia Middle Negro Upper Negro Mean/ n Range Mean/ n Range Mean/ n Mode+SD Mode+SD Mode+SD 36.0/N/A#1.4 2 35-39 37.0/3641.1 15 32-37 35.1/3641.2 35 6.5/N/A#2.1 2 5-8 6.7/7+1.0 15 5-9 7.3/8+1.0 35 5.0/N/At0.0 2 5-5 5.0/5+0.0 15 56 5.6/6+0.5 35 5.0/N/A40.0 2 4-5 4.1/4+0.4 15 45 4.5/4+0.5 34 N/A 1 14-15 14.3/1440.5 15 11-15 13.0/134+0.9 35 14.5/N/A+0.7. 2 11-13 12.3/12+0.6 15 10-14 12.3/12+0.8 35 24.0/N/A#2.8 2 21-25 22.8/2341.1 15 21-25 9 22.8/22+1.1 35 N/A 1 7-8 7.5/7+0.5 15 6-9 7.1/7+0.9 34 N/A 1 14-15 14.3/1440.5 15 13-15 13.9/14+0.8 35 N/A 1 1-2 1.3/1+0.5 15 1-4 2.1/3+0.9 34 N/A 1 7-11 9.0/9+1.0 15 6-14 9.0/9+1.3 35 zse.pensoft.net 78 Vanegas-Rios, J.A. et al.: Population variation of Diapoma pampeana from Pando stream 74 pF Diapoma pampeana PANDO UNMDP5219 22 | Diapoma pampeana UPPER NEGRO UFRGS12642_TEC1377b Diapoma pampeana PANDO MLP11444_2 Diapoma pampeana UPPER NEGRO UFRGS12642_TEC1377a rl | Diapoma pampeana UPPER NEGRO UFRGS12642_TEC1377d Diapoma pampeana UPPER NEGRO UFRGS$12642_TEC1377c Diapoma pampeana UPPER NEGRO UFRGS12642_TEC1377e 79 Diapoma guarani UFRGSTEC1379c Diapoma guarani UFRGSTEC1379d 93 Diapoma obi CY5 76 82 o9 I Diapoma obi CY6 49 y Diapoma potamohadros UFRGS27622_TEC1827d Diapoma potamohadros UFRGS27622_TEC1827q | y Diapoma potamohadros UFRGS27622_TEC1827a Diapoma potamohadros UFRGS27622_TEC1827b Diapoma potamohadros UFRGS27622_TEC1827e Diapoma tipiaia UFRGS15015_TEC1796b m= Diapoma speculiferum UFRGS12889_TEC1069 By o9 | Diapoma speculiferum UFRGS12890_TEC1084 Diapoma speculiferum UFRGS12388_TEC692 = Diapoma speculiferum UFRGS10004_TEC709 99 63 Diapoma pyrrhopteryx MCP44377 Diapoma terofali UFRGS12891_TEC339 82 Diapoma terofali UFRGS12892_TEC392 65 ss y Diapoma thauma UFRGS20044_TEC5420a 10 = Diapoma thauma UFRGS20044_TEC5420c | Diapoma thauma UFRGS20044_TEC5420b 90 Diapoma alegretense UFRGS10008_TEC714 Diapoma alegretense UFRGS10008_TEC8a = Diapoma lepiclastum CY4 Diapoma uruguayense UFRGS10962_TEC393 Diapoma uruguayense UFRGS11644_TEC457 1 Diapoma uruguayense UFRGS12401_TEC592 Diapoma dicropotamicum UFRGS12727_TEC1465a o4 Diapoma dicropotamicum UFRGS12727_TEC1465d Diapoma dicropotamicum UFRGS12727_TEC1465e bid Diapoma dicropotamicum UFRGS12727_TEC1465c Diapoma alburnum UFRGS12471_TEC1279 Diapoma alburnum MCP21291 Diapoma alburnum UFRGS15029_TEC1810c Diapoma alburnum UFRGS10753_TEC1390 9 Diapoma alburnum UFRGS12593_TEC1269 Diapoma itaimbe UFRGS12627_TEC1362h Diapoma itaimbe UFRGS16567_TEC2899b Diapoma itaimbe UFRGS12625_TEC1360h at | Diapoma itaimbe UFRGS12084_TEC1231h 86 ! Diapoma itaimbe UFRGS12634_TEC1369e Diapoma itaimbe UFRGS12635_TEC1370G 86 Diapoma itaimbe UFRGS12717_TEC1453 m Diapoma itaimbe UFRGS12603_TEC1303 Diapoma itaimbe UFRGS12587_TEC1263h Diapoma itaimbe isolate UFRGS12534_TEC1236e Diapoma itaimbe UFRGS12585_TEC1261h Diapoma itaimbe UFRGS12600_TEC1300e 48 | Diapoma itaimbe UFRGS12620_TEC1355g Diapoma itaimbe UFRGS12651_TEC1383 Diapoma itaimbe UFRGS16517_TEC2845c a7 40 93 92 a | Outgroups 60 er | 0.01 Figure 5. Neighbor-Joining topology of analyzed Diapoma specimens based on Tamura-Nei model and COI sequence data. Boot- strap values (10000 replicates) are shown below the branches. SBL = 0.783. zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 69-85 0.7569 —_—_— 0.9343 0.7476 0.9958 0.9995 a exw Outgroups 0.02 i Diapoma pampeana PANDO UNMDP5219 Diapoma pampeana UPPER NEGRO UFRGS12642 TEC1377a Diapoma pampeana UPPER NEGRO UFRGS12642_TEC1377b Diapoma pampeana UPPER NEGRO UFRGS12642_TEC1377c Diapoma pampeana UPPER NEGRO UFRGS12642_TEC1377d Diapoma pampeana UPPER NEGRO UFRGS12642_TEC1377e rere Diapoma pampeana PANDO MLP11444_2 Diapoma guarani UFRGS_TEC1379c Diapoma guarani UFRGS_TEC1379d g.soa4 Diapoma obi CY5 Diapoma obi CY6 0.8509 Diapoma potamohadros UFRGS27622_TEC1827d Diapoma potamohadros UFRGS27622_TEC1827g 4 Diapoma potamohadros UFRGS27622_TEC1827a Diapoma potamohadros UFRGS27622_TEC1827b Diapoma potamohadros UFRGS27622_TEC1827e Diapoma tipiaia UFRGS15015_TEC1796b _ Diapoma alegretense UFRGS10008_TEC714 none Diapoma alegretense UFRGS10008_TEC8a Diapoma lepiclastum CY4 0.9045 Diapoma uruguayense UFRGS10962_TEC393 okeas Diapoma uruguayense UFRGS11644_TEC457 0.9076 : Diapoma uruguayense UFRGS12401_TEC592 Diapoma thauma UFRGS20044_TEC5420a Diapoma thauma UFRGS20044_TEC5420b Diapoma thauma UFRGS20044_TEC5420c Diapoma speculiferum UFRGS10004_TEC709 1 Diapoma speculiferum UFRGS12388_TEC692 Diapoma speculiferum UFRGS12889_TEC1069 1 Diapoma speculiferum UFRGS12890_TEC1084 0.9543 Diapoma terofali UFRGS12891_TEC339 uae Diapoma terofali UFRGS12892_TEC392 Diapoma pyrrhopteryx MCP44377 Diapoma itaimbe UFRGS12587_TEC1263h 0.9785 Diapoma itaimbe UFRGS12603_TEC1303 Diapoma itaimbe UFRGS12635_TEC1370G Diapoma itaimbe UFRGS12717_TEC1453 0.8834 Diapoma itaimbe UFRGS12084_TEC1231h Diapoma itaimbe UFRGS12634_TEC1369e Diapoma itaimbe isolate UFRGS12534_TEC1236e 4 Diapoma itainmbe UFRGS12585_TEC1261h Diapoma itaimbe UFRGS12600_TEC1300e Diapoma itaimbe UFRGS12620_TEC1355g 0.9996 Diapoma itaimbe UFRGS12651_TEC1383 Diapoma itaimbe UFRGS16517_TEC2845c Diapoma itaimbe UFRGS12625_TEC1360h 09282'__ Diapoma itaimbe UFRGS12627_TEC1362h Diapoma itaimbe UFRGS16567_TEC2899b Diapoma dicropotamicum UFRGS12727_TEC1465a 0.999 Diapoma dicropotamicum UFRGS12727_TEC1465c Diapoma dicropotamicum UFRGS12727_TEC1465d 0.9597 1 0.9483 0.9626] Diapoma dicropotamicum UFRGS12727_TEC1465e Diapoma alburnum MCP21291 areas Diapoma alburnum UFRGS12471_TEC1279 7 Diapoma alburnum UFRGS10753_TEC1390 07718 Diapoma alburnum UFRGS12593_TEC1269 Diapoma alburnum UFRGS15029_TEC1810c Figure 6. Bayesian topology of phylogenetic relationships among the analyzed Diapoma species (comparing specimens of D. pampeana from the Pando stream and Upper Negro basin) based on COI sequence data. Numbers at nodes correspond to poste- rior probabilities. 12642: TEC1377e) showed the greater p-distance (0.2%) in each comparison with the other specimens analyzed of D. pampeana (Suppl. material 6). In the polymorphism analysis comparing the Pando and Upper Negro specimens, 728 sites were analyzed (381: invariable; 347: with gaps or missing data), resulting in one polymorphic site (singleton) and two haplotypes (Hd = 0.286; variance = 0.034; standard deviation = 0.196). In general, the nucleotide diversity was extremely low (1 = 0.00095; 0 = 0.00107; k = 0.286) for the samples ana- lyzed. No variation was found within the Pando samples (x = 0.00000; k = 0.000). For the Upper Negro samples, only one polymorphic site (monomorphic in the Pando samples) was found and, thus, their diversity was slightly greater (1 = 0.00105; k = 0.400). There were no observed shared mutations between these two populations. Regard- ing the divergence between the populations compared, the values obtained were low (Dxy = 0.00052; Da = 0.00000). The haplotype network showed a simple structure of two groups without well-defined geographic structure and in which one of them was mixed (Suppl. material 7). Comparative examined material Diapomaalburnum:. UFRGS 13309, 11, 33.4-56.0 mm SL. Diapoma guarani: MHNG 2366.99, holotype, 31.7 mm SL. Diapoma lepiclastum: MACN-ict 9682, 47, 29.3— 42.0 mm SL. Diapoma obi: MLP 11312, 3, 29.5—35.6 mm SL. MACN-ict 9560, holotype, 52.6 mm SL. Diapoma uruguayense, MACN-ict 9681, 7, 31.6—-34.6 mm SL. Discussion The stevardiine species D. pampeana was recently de- scribed from several localities along the Rio Negro basin in Brazil and Uruguay (Ito et al. 2022). The freshwater fish fauna from the Pando stream is mainly known from studies focused on estuarine-influenced coastal waters (Plavan et al. 2010; Gurdek and Acufia-Plavan 2017). Al- though the body coloration was similar between the spec- imens of the Pando and Upper Negro groups, the former showed a striking morphometric and meristic divergence from the latter. Additionally, the intraspecific variation between the Pando and Upper Negro populations be- came much more subtle when compared with specimens from geographically intermediate areas such as the San- ta Lucia and Middle Negro basins (i.e. the specimens of these groups were slightly more similar to each other in the morphometric and meristic data than to the specimens of the Upper Negro). Frequently, in morphological com- parisons using multivariate methods, the populations ex- hibiting major differences in body shape correspond with those morphotypes that are located at the farthest distance geographically from the others (Lazzarotto et al. 2017; Vanegas-Rios et al. 2019; Rodrigues-Oliveira et al. 2023). In consequence, the analyzed specimens of D. pampea- na may be responding to a gradual pattern of divergence associated with spatial segregation, often observed in widespread species (Lazzarotto et al. 2017; Arroyave et al. 2019; Vanegas-Rios et al. 2019; Rodrigues-Oliveira et al. 2023). zse.pensoft.net 80 Vanegas-Rios, J.A. et al.: Population variation of Diapoma pampeana from Pando stream In the morphological comparisons performed herein, the Pando group was greatly differentiated in the size-cor- rected PCA from the Upper Negro group along PC1, which was mainly influenced by the following distances: snout to dorsal-fin origin, snout to anal-fin origin, dor- sal- and adipose-fin origins, and caudal peduncle length (Fig. 4A). Some measurements such as the snout to dor- sal-fin origin, snout to anal-fin origin, snout to pelvic-fin origin, distance between the dorsal- and pectoral-fin or- igins, head length, snout length, and eye diameter have been found to be taxonomically informative to discrimi- nate among Diapoma species (Malabarba and Weitzman 2003; Vanegas-Rios et al. 2018; Ito et al. 2022). Based on the meristic data, these groups were also found to be slightly divergent from each other along PC1. For this component, some main counts defining the variability were the number of longitudinal scales and predorsal scales (Table 3, Fig. 4C). Usually, the range is used as the main indicator to define the limits of the morpholog- ical variation among species in measurements or counts (Garavello et al. 1992; Aguirre et al. 2016; Lazzarotto et al. 2017; Arroyave et al. 2019; Vanegas-Rios et al. 2019; Malabarba et al. 2021). The main dilemma for defining these limits appears when data have varied degrees of overlapping between populations of study. Further statistical procedures are used as a complement to test if diverging tendencies in morphometric data are, or are not, significant (Lazzarotto et al. 2017; Arroyave et al. 2019; Vanegas-Rios et al. 2019; Rodrigues-Oliveira et al. 2023). For instance, it is frequently found that subtle or moderate intraspecific differences in body shape result in being statistically significant, but it may be also associat- ed with the degree of sensitivity involved in pairwise tests (e.g. means). Recently, the statistical potential behind morphometrically divergent patterns has been used to propose subspecies within gymnotids (Craig et al. 2017). Such criterion is not commonly used in modern ichthy- ology, so that the erection of infraspecific categories can be considered unnecessary to understand the intraspecific variation (e.g. clines) (Kottelat 1998; Kullander 1999). In the case of the Pando group, it seems unjustified to propose a new infraspecific category under the light of the current evidence described from morphological data, even more when it is geographically isolated and its phe- notypic variation is described and contextualized in the present contribution. In a similar case, geographically iso- lated coastal river populations of D. itaimbe that showed a Statistically significant difference in overlapping ranges of anal-fin ray counts were treated as structured isolated populations instead of separate species (Malabarba and Weitzman 2003; Hirschmann et al. 2015). When the discriminative tendencies are striking, as oc- curred here between the Pando and Upper Negro groups, including additional independent evidence, such as DNA data, can help to support the conclusion. The COI marker has played an important role in resolving taxonomic ques- tions in freshwater fishes (Pereira et al. 2013). In general, comparative studies using COI and morphology are deal- ing with cryptic species, species complexes or populations zse.pensoft.net with slight morphological variations (under a context of geographic isolation) (Serrano et al. 2019; Garavello et al. 2021; Guimaraes et al. 2021; Malabarba et al. 2021; Aguil- era et al. 2022). The phylogenetic signal of the COI marker within Diapoma has been studied and used to propose in- traspecific and interspecific limits, as well as new species (Casciotta et al. 2012; Hirschmann et al. 2015; Ito et al. 2022). However, the use of this marker by itself for recog- nizing species (e.g. as single locus without morphological or cytogenetic support) has its own methodological lim- its, and it is especially important to have this into account when dealing with complex groups of species or popu- lations (Castro Paz et al. 2014; Garcia-Melo et al. 2019; Klimov et al. 2019; Silva-Santos et al. 2023). This may imply that in some cases there is no guarantee of complete interspecific delimitation (e.g. Castro Paz et al. 2014). The phylogenetic comparison performed using the COI marker of all known species of Diapoma (except D. nandi from the Parana basin) recovered the Pando group as part of D. pampeana. It also demonstrates that the recognition of the Pando group as separate would make D. pampeana paraphyletic (Fig. 5: NJ topology). The variability of the p-distances calculated for the Pando group were observed to be lower than the average congeneric values (0O-0.2% vs. 1.3-8.0%), as it has been often reported in other characids (Pereira et al. 2011b; Garcia-Melo et al. 2019; Silva-Santos et al. 2023). Furthermore, the mean genetic distances were 3.5% and 4.3% between D. pampeana and its closest related species (D. obi and D. guarani, respec- tively). The intraspecific values obtained for D. pampea- na are within the conspecific variation reported in spe- cies of other characid groups such as Astvanax (from the Rio Paraguac¢u basin, mean = 0-1.7%); Hyphessobrycon Durbin, 1908 (from the Amazon basin, mean = 0-8.9%), and some stevardiines (Bryconamericus, Eretmobrycon Fink, 1976, Hemibrycon Gunther, 1864, Knodus Eigen- mann, 1911, and Piabina: 0-1.9%) (Pereira et al. 2011b; Castro Paz et al. 2014; Garcia-Melo et al. 2019; Sil- va-Santos et al. 2023). In barcoding studies, 2% threshold limit (at least 10 times the average conspecific values) has been used as the cutoff divergence value for delimiting species or molecular operational taxonomic units (Hebert et al. 2004; Ward 2009). However, an alternative thresh- old value of 1% has been considered for studying species complexes (Hubert et al. 2008; Pereira et al. 2011a; Sil- va-Santos et al. 2023). Therefore, these threshold values should be cautiously evaluated for each case in particular. The results obtained from the exploratory analysis using haplotype, allowed us to detect the potential presence of two haplotypes that were separated by a single mutational change. Additionally, the Pando group presented the same haplotype as the Upper Negro group, which again reinforc- es the great resemblance between both groups. Additional- ly, no well-defined lineages were detected in the molecular comparisons. However, this needs to be further investigat- ed so that within Diapoma, for instance, it has been found that D. itaimbe forms populations with well-defined struc- tural lineages associated with a coastal biographic pattern (Hirschmann et al. 2015). The recognition of species as Zoosyst. Evol. 100 (1) 2024, 69-85 separately evolving metapopulation lineages is a unifying concept in defining species (De Queiroz 2007), and so far, we have no support from molecular data to separate the Pando group from D. pampeana. Freshwater fishes have limited their ability to disperse across brackish, marine or terrestrial barriers, being bio- logically restricted to water bodies after their formation, and thus, the disjunct geographic range associated with Species and populations across several basins might be ex- plained by river captures or dispersal favored by temporary connections (Albert and Reis 2011; Thomaz et al. 2017; Camelier et al. 2018; Cassemiro et al. 2023). These poten- tial explanations would be plausible for testing in D. pam- peana if its distribution was really widespread along most coastal drainages in Uruguay, as our findings suggest. Conclusion We concluded that the specimens from the Pando stream, despite the morphological divergence observed, can be classified as D. pampeana. We supported our decision based on the following arguments: 1) the deviations found on the morphometric and meristic data (e.g. PCA) are not enough to erect a new species and, as consequence, the intraspecific variability is increased; 2) the specimens of the Pando group were similarly pigmented as the speci- mens of the Upper Negro group (sharing the same diag- nostic pattern on the humeral mark, midlateral stripe, and caudal-fin pigmentation); 3) the COI-based phylogenetic procedures supported the placement of the Pando group within the genetic variation of the Upper Negro group (typical distribution of D. pampeana), and 4) based on the genetic distances, the Pando group was found to be genetically similar to the Upper Negro group, with p-dis- tances (O—0.2%) being lower than the mean distances ob- tained between each congener (1.3—8.0%). Additionally, the present work also confirmed the presence of D. pam- peana in the Yi (Middle Negro basin) and Santa Lucia river basins, based on morphological evidence. Although it was not possible to separate species, our results provide new information that can be further appreciated. For in- stance, it has been proposed that diverging populations can represent separate evolutionarily significant units, which should be conserved (Moritz 1994; de J. May-Itza et al. 2012; Stockwell et al. 2013; Berger et al. 2018). The geographic range of D. pampeana seems to be incom- pletely understood and might be more widely represented along the Rio Negro basin and other coastal drainages in Uruguay (Fig. 3, Suppl. material 8). Future studies may bring new insights into the population variation of the species and other phylogeographic patterns if more specimens and new localities are analyzed. The Pando population of D. pampeana constitutes a remarkable ex- ample of an isolated population that is morphologically divergent (in the morphometric and meristic data) from the geographically most distant conspecific population (upper Rio Negro basin), but that shares a high degree of genetic resemblance with it. 81 Acknowledgments We thank the following institutions and museums for their assistance and support: G. Chiaramonte (MACN-ict); So- nia Fisch-Muller and Rafael Covain (MHNG); D. Nad- alin, Jorge R. Casciotta and Adriana E. Almiron (MLP); C. Lucena (MCP), Priscila M. Ito and Juliana M. Wingert (UFRGS). The authors are grateful for the financial sup- port provided by FONCyT (BID-PICT 2019-02419 and PIBBA 0654CO to JAVR). We are indebted to Juan José Rosso, Matias Delpiane, and Juan Martin Diaz de Astarloa (IMyC-UNMDP), and C. Bruno and G. Giovambattista (IGEVET-UNLP) for their assistance with the DNA pro- cedures. Nicolas Tizio (Fundacion Unidos por Naturale- za), J. Pfleiderer, and F. M. Frias helped with photographs. This paper benefited from valuable suggestions and com- ments of anonymous reviewers, W. Costa, and F. Araujo. References Achkar M, Dominguez A, Pesce F (2012) Cuenca del Rio Santa Lucia-Uruguay. Aportes para la discusi6n ciudadana. Montevideo. Acufia A, Mufioz N, Gurdek R, Machado I, Severi V (2017) Inter-es- tuarine and temporal patterns of the fish assemblage of subtropical subestuaries along the Rio de la Plata coast (Uruguay). Brazilian Journal of Oceanography 65(2): 173-186. https://doi.org/10.1590/ $1679-87592017131106502 Aguilera G, Teran GE, Mirande JM, Alonso F, Chumacero GM, Cardo- so Y, Bogan S, Faustino-Fuster DR (2022) An integrative approach method reveals the presence of a previously unreported species of Imparfinis Eigenmann and Norris 1900 (Siluriformes: Heptapte- ridae) in Argentina. Journal of Fish Biology 101(5): 1248-1261. https://doi.org/10.1111/)fb.15197 Aguirre WE, Navarrete R, Malato G, Calle P, Loh MK, Vital WF, Vala- dez G, Vu V, Shervette VR, Granda JC (2016) Body shape variation and population genetic structure of Rhoadsia altipinna (Characidae: Rhoadsiinae) in southwestern Ecuador. Copeia 104(2): 554-569. https://doi.org/10.1643/CG-15-289 Albert JS, Reis RE (2011) Historical Biogeography of Neotropical Freshwater Fishes. University of California Press, California, 388 pp. https://doi.org/10.1525/9780520948501 Almiron A, Casciotta J, Ricanova S, Dragova K, Pialek L, Rigéan R (2016) First record of Diapoma pyrrhopteryx Menezes & Weitzman, 2011 (Characiformes: Characidae) from freshwaters of Argentina. Ichthyological Contributions of Peces Criollos 40: 1-3. Arroyave J, Martinez CM, Stiassny MLJ (2019) DNA barcoding un- covers extensive cryptic diversity in the African long-fin tetra Bry- conalestes longipinnis (Alestidae: Characiformes). Journal of Fish Biology 95: 379-392. https://doi.org/10.1111/jfb.13987 Azpelicueta MM, Lundberg JG, Loureiro M (2008) Pimelodus pintado (Siluriformes: Pimelodidae), a new species of catfish from affluent rivers of Laguna Merin, Uruguay, South America. Proceedings of the Academy of Natural Sciences of Philadelphia 157(1): 149-162. https://do1.org/10.1635/0097-3157(2008)157[149:PPSPAN ]2.0.CO;2 Berger C, Stambuk A, Maguire I, Weiss S, Fiireder L (2018) Integrating genetics and morphometrics in species conservation—A case study on the stone crayfish, Austropotamobius torrentium. Limnologica 69: 28-38. https://doi.org/10.1016/j.limno.2017.11.002 zse.pensoft.net 82 Vanegas-Rios, J.A. et al.: Population variation of Diapoma pampeana from Pando stream Camelier P, Menezes NA, Costa-Silva GJ, Oliveira C (2018) Molec- ular and morphological data of the freshwater fish Glandulocauda melanopleura (Characiformes: Characidae) provide evidences of river captures and local differentiation in the Brazilian Atlantic For- est. PLOS ONE 13(3): e0194247. https://doi.org/10.1371/journal. pone.0194247 Cardoso AR (2010) Bunocephalus erondinae, a new species of banjo catfish from southern Brazil (Siluriformes: Aspredinidae). Neo- tropical Ichthyology 8(3): 607-613. https://doi.org/10.1590/S1679- 62252010000300005 Casciotta J, Almiron A, Pidlek L, Ri¢an O (2012) Cyanocharax obi, a new species (Characiformes: Characidae) and the first record of the genus from tributaries of the rio Parana basin, Argentina. Zootaxa 3391(1): 39-51. https://doi.org/10.11646/zootaxa.3391.1.3 Cassemiro FAS, Albert JS, Antonelli A, Menegotto A, Wtest RO, Cerezer F, Coelho MTP, Reis RE, Tan M, Tagliacollo V, Bailly D, da Silva VFB, Frota A, da Graga WJ, Ré R, Ramos T, Oliveira AG, Dias MS, Colwell RK, Rangel TF, Graham CH (2023) Landscape dynamics and diversification of the megadiverse South American freshwater fish fauna. Proceedings of the National Academy of Sci- ences of the United States of America 120(2): e2211974120. https:// doi.org/10.1073/pnas.2211974120 Castro Paz FP, Batista Jd S, Porto JIR (2014) DNA Barcodes of Rosy Tetras and Allied Species (Characiformes: Characidae: Hyphes- sobrycon) from the Brazilian Amazon Basin. PLOS ONE 9(5): e98603. https://doi.org/10.1371/journal.pone.0098603 Cattel R (1966) The scree test for the number of factors. Multivar- iate Behavioral Research 1: 245-276. https://doi.org/10.1207/ $15327906mbr0102_ 10 Craig JM, Crampton WGR, Albert JS (2017) Revision of the polytypic electric fish Gymnotus carapo (Gymnotiformes, Teleostei), with de- scriptions of seven subspecies. Zootaxa 4318(3): 401-438. https:// doi.org/10.11646/zootaxa.4318.3.1 Darriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T (2019) ModelTest-NG: A new and scalable tool for the selection of DNA and protein evolutionary models. Molecular Biology and Evolution 37(1): 291—294. https://doi.org/10.1093/molbev/msz1 89 May-Itza W de J, Quezada-Euan JJG, Ayala R, De La Rua P ( (2012) Morphometric and genetic analyses differentiate Mesoamerican populations of the endangered stingless bee Melipona beecheii (Hymenoptera: Meliponidae) and support their conservation as two separate units. Journal of Insect Conservation 16: 723-731. https:// doi.org/10.1007/s10841-012-9457-4 De Queiroz K (2007) Species concepts and species delimi- tation. Systematic Biology 56(6): 879-886. _ https://doi. org/10.1080/10635150701701083 Defeo O, Horta S, Carranza A, Lercari D, de Alava A, Gomez J, Martinez G, Lozoya JP, Celentano E (2009) Hacia un Manejo Eco- sistémico de Pesquerias. Areas Marinas Protegidas en Uruguay. Fac- ultad de Ciencias-UNDECIMAR, Montevideo, 122 pp. Dempster AP, Laird NM, Rubin DB (1977) Maximum likelihood from incomplete data via the EM algorithm. Journal of the Royal Sta- tistical Society, Series B, Methodological 39(1): 1-38. https://doi. org/10.1111/j.2517-6161.1977.tb01600.x Echevarria L, Gomez A, Lale M, Lopez R, Nieto P, Pereyra G (2011) Plan de manejo costero integrado del tramo de costa A° Solis Chico —A° Solis Grande. In: Conde D (Ed) Manejo Costero Integrado en Uru- guay: ocho ensayos interdisciplinarios Centro Interdisciplinario para el Manejo Costero Integrado del Cono Sur, Montevideo, 123-152. zse.pensoft.net Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32(5): 1792— 1797. https://do1.org/10.1093/nar/gkh340 Elliott NG, Haskard K, Koslow JA (1995) Morphometric analysis of orange roughy (Hoplostethus atlanticus) off the continental slope of southern Australia. Journal of Fish Biology 46: 202-220. https://doi. org/10.1111/j.1095-8649.1995.tb05962.x Ferreira KM, Menezes NA, Quagio-Grassioto I (2011) A new genus and two new species of Stevardiinae (Characiformes: Characidae) with a hypothesis on their relationships based on morphological and his- tological data. Neotropical Ichthyology 9(2): 281-298. https://doi. org/10.1590/S1679-62252011000200005 Ferreira KM, Mirande JM, Quagio-Grassiotto I, Santana JCO, Baice- re-Silva CM, Menezes NA (2021) Testing the phylogenetic hy- potheses of Stevardiinae Gill, 1858 in light of new phenotypic data (Teleostei: Characidae). Journal of Zoological Systematics and Evolutionary Research 59(8): 2060-2085. https://doi.org/10.1111/ qz8.12517 Fink WL, Weitzman SH (1974) The so-called Cheirodontin fishes of Central America with descriptions of two new species (Pisces: Characidae) Smithsonian Contributions to Zoology 172: 1-45. https://doi.org/10.5479/s1.00810282.172. Fricke R, Eschmeyer WN, Van der Laan R (2023) Eschmeyer’s Cat- alog of Fishes: genera, species, references. http://researcharchive. calacademy. org/research/ichthyology/catalog/fishcatmain.asp [accessed 1 December.2023] Frontier S (1976) Etude de la décroissance des valeurs propres dans une analyse en composantes principales: comparaison avec le modele du baton brisé. Journal of Experimental Marine Biology and Ecology 25: 67-75. https://doi.org/10.1016/0022-0981(76)90076-9 Garavello JC, Dos Reis SF, Strauss RE (1992) Geographic variation in Leporinus friderici (Bloch) (Pisces: Ostariophysi: Anostomidae) from the Parana-Paraguay and Amazon River basins. Zoologica Scripta 21(2): 197-200. https://doi.org/10.1111/j.1463-6409.1992. tb00320.x Garavello JC, Ramirez JL, de Oliveira AK, Britski HA, Birindelli JLO, Galetti Jr PM (2021) Integrative taxonomy reveals a new species of Neotropical headstanding fish in genus Schizodon (Characiformes: Anostomidae). Neotropical Ichthyology 19(4): e210016. https://doi. org/10.1590/1982-0224-2021-0016 Garcia-Melo JE, Oliveira C, Da Costa Silva GJ, Ochoa-Orrego LE, Gar- cia Pereira LH, Maldonado-Ocampo JA (2019) Species delimitation of neotropical Characins (Stevardiinae): Implications for taxono- my of complex groups. PLOS ONE 14(6): e0216786. https://doi. org/10.1371/journal.pone.0216786 Guimaraes KL, Rosso JJ, Souza MF, Diaz de Astarloa JM, Rodrigues LR (2021) Integrative taxonomy reveals disjunct distribution and first record of Hoplias misionera (Characiformes: Erythrinidae) in the Amazon River basin: morphological, DNA barcoding and cy- togenetic considerations. Neotropical Ichthyology 19(2): e200110. https://doi.org/10.1590/1982-0224-2020-0110 Gurdek R, Acufia-Plavan A (2017) Temporal dynamics of a fish commu- nity in the lower portion of a tidal creek, Pando sub-estuarine sys- tem, Uruguay. Iheringia. Série Zoologia 107(0): e2017003. https:// doi.org/10.1590/1678-4766e2017003 Gutiérrez JM, Villar S, Acufia Plavan A (2015) Micronucleus test in fishes as indicators of environmental quality in subestuaries of the Rio de la Plata (Uruguay). Marine Pollution Bulletin 91(2): 518— 523. https://doi.org/10.1016/j.marpolbul.2014.10.027 Zoosyst. Evol. 100 (1) 2024, 69-85 Hall TA (1999) BioEdit: A User-Friendly Biological Sequence Align- ment Editor and Analysis Program for Windows 95/98/NT. Nucleic Acids Symposium Series. Oxford University Press, 95-98. Hammer Q, Harper DAT, Ryan PD (2001) PAST: Paleontological statis- tics software package for education and data analysis. Palaeontolo- gia Electronica 4: 1-9. Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identifica- tion of Birds through DNA Barcodes. PLOS Biology 2(10): e312. https://doi.org/10.1371/journal.pbio.00203 12 Hirschmann A, Malabarba LR, Thomaz AT, Fagundes NJR (2015) Riv- erine habitat specificity constrains dispersion in a Neotropical fish (Characidae) along Southern Brazilian drainages. Zoologica Scripta 44(4): 374-382. https://doi.org/10.1111/zsc.12106 Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, Burridge M, Watkin- son D, Dumont P, Curry A, Bentzen P, Zhang J, April J; Bernatchez L (2008) Identifying Canadian Freshwater Fishes through DNA Barcodes. PLOS ONE 3(6): e2490. https://doi.org/10.1371/journal.pone.0002490 IBM (2019) IBM SPSS Statistics for Windows, Version 26.0. IBM Corp, Armonk, NY. Ito PMM, Carvalho TP, Pavanelli CS, Vanegas-Rios JA, Malabarba LR (2022) Phylogenetic relationships and description of two new species of Diapoma (Characidae: Stevardiinae) from the La Plata River basin. Neotropical Ichthyology 20(1): e210115. https://doi. org/10.1590/1982-0224-2021-0115 Ivanova NV, Dewaard JR, Hebert PDN (2006) An inexpensive, auto- mation-friendly protocol for recovering high-quality DNA. Molec- ular Ecology Notes 6: 998-1002. https://doi.org/10.1111/j.1471- 8286.2006.01428.x Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN (2007) Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes 7: 544-548. https://doi.org/10.1111/).1471-8286.2007.01748.x Klimov PB, Skoracki M, Bochkov AV (2019) Cox1 barcoding versus multilocus species delimitation: validation of two mite species with contrasting effective population sizes. Parasites & Vectors 12: 1-8. https://doi.org/10.1186/s13071-018-3242-5 Kottelat M (1998) Systematics, species concepts and the conservation of freshwater fish diversity in Europe. The Italian Journal of Zool- ogy 65(sup1): 65—72. https://do1.org/10.1080/11250009809386798 Kullander SO (1999) Fish species — how and why. Reviews in Fish Biology and Fisheries 9(4): 325-352. https://doi. org/10.1023/A: 1008959313491 Lazzarotto H, Barros T, Louvise J, Caramaschi EP (2017) Morpho- logical variation among populations of Hemigrammus coeruleus (Characiformes: Characidae) in a Negro River tributary, Brazil- ian Amazon. Neotropical Ichthyology 15: e160152. https://doi. org/10.1590/1982-0224-20160152 Leigh JW, Bryant D (2015) popart: full-feature software for haplotype network construction. Methods in Ecology and Evolution 6: 1110— 1116. https://do1.org/doi.org/10.1111/2041-210X.12410 Lucena CA, Malabarba LR, Reis RE (1992) Resurrection of the neo- tropical pimelodid catfish Parapimelodus nigribarbis (Boulenger), with a phylogenetic diagnosis of the genus Parapimelodus (Teleoastei: Siluriformes). Copeia 1992(1): 138-146. https://doi. org/10.2307/1446545 Mahnert V, Géry J (1987) Deux nouvelles especes du genre Hyphesso- brycon (Pisces, Ostariophysi, Characidae) du Paraguay: H. guarani n. sp. et H. procerus n. sp. Bonner Zoologische Beitrage 38: 307-314. Malabarba LR (1983) Redescri¢ao e discussao da posi¢aéo taxondmica de Astyanax hasemani Eigenmann, 1914 (Teleostei, Characidae). 83 Comunica¢des do Museu de Ciéncias da PUCRS. Serie Zoologica 14: 177-199. Malabarba LR, Weitzman SH (2003) Description of new genus with six new species from southern Brazil, Uruguay and Argentina, with a discussion of a putative characid clade (Teleostei: Characiformes: Characidae). Comunicacgées do Museu de Ciéncias e Tecnologia da PUCRS. Série Zoologia 16: 67-151. Malabarba LR, Chuctaya J, Hirschmann A, de Oliveira EB, Thomaz AT (2021) Hidden or unnoticed? Multiple lines of evidence support the recognition of a new species of Pseudocorynopoma (Characidae: Corynopomini). Journal of Fish Biology 98(1): 219-236. https://doi. org/10.1111/jfb.14572 Menezes NA, Weitzman SH (2011) A systematic review of Diapoma (Teleostei: Characiformes: Characidae: Stevardiinae: Diapomini) with descriptions of two new species from southern Brazil. Papéis Avulsos de Zoologia 51(5): 59-82. https://doi.org/10.1590/S0031- 10492011000500001 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. 2010 Gateway Computing Environments Workshop (GCE), 8 pp. https:// doi.org/10.1109/GCE.2010.5676129 Mirande JM (2019) Morphology, molecules and the phylogeny of Characidae (Teleostei, Characiformes). Cladistics 35(3): 282-300. https://do1.org/10.1111/cla.12345 Moritz C (1994) Defining ‘Evolutionarily Significant Units’ for conser- vation. Trends in Ecology & Evolution 9: 373-375. https://doi.org/ https://doi.org/10.1016/0169-5347(94)90057-4 Muniz P, Venturini N, Brugnoli E, Gutiérrez JM, Acufia A (2019) Chap- ter 30 — Rio de la Plata: Uruguay. In: Sheppard C (Ed.) World Seas: An Environmental Evaluation (2" edn.). Academic Press, 703-724. https://doi.org/10.1016/B978-0-12-805068-2.00036-X Nei M (1987) Molecular Evolutionary Genetics. Columbia University Press, New York, 514 pp. https://doi.org/10.7312/nei-92038 Nei M, Li WH (1979) Mathematical model for studying genetic vari- ation in terms of restriction endonucleases. Proceedings of the Na- tional Academy of Sciences of the United States of America 76(10): 5269-5273. https://do1.org/10.1073/pnas.76.10.5269 Paullier S, Bessonart J, Brum E, Loureiro M (2019) Lista de especies de peces de la cuenca del Rio Queguay, Rio Uruguay bajo. Boletin de la Sociedad Zooldgica del Uruguay 28: 66-78. https://doi. org/10.26462/28.2.3 Pereira LHG, Maia GMG, Hanner R, Foresti F, Oliveira C (2011a) DNA barcodes discriminate freshwater fishes from the Paraiba do Sul Riv- er Basin, SAo Paulo, Brazil. Mitochondrial DNA 22(sup1): 71-79. https://doi.org/10.3109/19401736.2010.532213 Pereira LHG, Pazian MF, Hanner R, Foresti F, Oliveira C (2011b) DNA barcoding reveals hidden diversity in the Neotropical freshwater fish Piabina argentea (Characiformes: Characidae) from the Up- per Parana Basin of Brazil. Mitochondrial DNA 22(sup1): 87-96. https://doi.org/10.3109/19401736.2011.588213 Pereira LHG, Hanner R, Foresti F, Oliveira C (2013) Can DNA barcod- ing accurately discriminate megadiverse Neotropical freshwater fish fauna? BMC Genetics 14(1): 1-20. https://doi.org/10.1186/1471- 2156-14-20 Pigott TD (2001) A review of methods for missing data. Educational Research and Evaluation 7(4): 353-383. https://doi.org/10.1076/ edre.7.4.353.8937 Plavan AA, Passadore C, Gimenez LJ (2010) Fish assemblage in a temperate estuary on the uruguayan coast: Seasonal variation and zse.pensoft.net 84 Vanegas-Rios, J.A. et al.: Population variation of Diapoma pampeana from Pando stream environmental influence. Brazilian Journal of Oceanography 58(4): 299-314. https://doi.org/10.1590/S 1679-875920 10000400005 Protogino LC, Miquelarena AM (2012) Cyanocharax alburnus (Hensel, 1870) (Characiformes: Characidae): First distribution record in Ar- gentina. Check List 8(3): 581-583. https://doi.org/10.15560/8.3.581 Quinn GP, Keough MJ (2002) Experimental desing and data analysis for biologist. Cambrigde University Press, Cambrigde. Rambaut A (2018) FigTree: Tree figure drawning tool. 1.4.4 ed, Institute of Evolutionary Biology, University of Edinburgh. Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posteri- or summarization in bayesian phylogenetics using Tracer 1.7. System- atic Biology 67(5): 901-904. https://doi.org/10.1093/sysbio/syy032 Rodrigues-Oliveira IH, Kavalco KF, Pasa R (2023) Body shape varia- tion in the Characid Psalidodon rivularis from Sao Francisco river, Southeast Brazil (Characiformes: Stethaprioninae). Acta Zoologica 104(3): 345-354. https://doi.org/10.1111/azo.12415 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 Rosso JJ, Mabragafia E, Gonzalez Castro M, Diaz de Astarloa JM (2012) DNA barcoding Neotropical fishes: Recent advances from the Pampa Plain, Argentina. Molecular Ecology Resources 12(6): 999-1011. https://doi.org/10.1111/1755-0998. 12010 Rozas J, Ferrer-Mata A, Sanchez-DelBarrio JC, Guirao-Rico S, Libra- do P, Ramos-Onsins SE, Sanchez-Gracia A (2017) DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Molecular Biology and Evolution 34(12): 3299-3302. https://doi.org/10.1093/ molbev/msx248 Sabaj MH (2020) Codes for natural history collections in ichthyology and herpeteology. Copeia 108(3): 593-669. https://doi.org/10.1643/ ASIHCODONS2020 Serrano EA, Melo BF, Freitas-Souza D, Oliveira MLM, Utsunomia R, Oliveira C, Foresti F (2019) Species delimitation in Neotropical fishes of the genus Characidium (Teleostei, Characiformes). Zoo- logica Scripta 48(1): 69-80. https://doi.org/10.1111/zsc.12318 Silva-Santos R, de Barros Machado C, Zanata AM, Camelier P, Galetti Jr PM, Domingues de Freitas P (2023) Molecular characteriza- tion of Astyanax species (Characiformes: Characidae) from the upper Paraguacgu River basin, a hydrographic system with high endemism. Neotropical Ichthyology 21(2): e230032. https://doi. org/10.1590/1982-0224-2023-0032 Stockwell CA, Heilveil JS, Purcell K (2013) Estimating divergence time for two evolutionarily significant units of a protected fish species. Conservation Genetics 14(1): 215-222. https://doi.org/10.1007/ $10592-013-0447-1 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolution- ary Genetics Analysis Version 11. Molecular Biology and Evolution 38(7): €30223027. https://doi.org/10.1093/molbev/msab120 Taylor WR, Dyke GCV (1985) Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. Cybium 9: 107-119. Thomaz AT, Arcila D, Orti G, Malabarba LR (2015) Molecular phy- logeny of the subfamily Stevardiinae Gill, 1858 (Characiformes: Characidae): classification and the evolution of reproductive traits. BMC Evolutionary Biology 15(1): e146. https://doi.org/10.1186/ $12862-015-0403-4 zse.pensoft.net Thomaz AT, Malabarba LR, Knowles LL (2017) Genomic signatures of paleodrainages in a freshwater fish along the southeastern coast of Brazil: Genetic structure reflects past riverine properties. Heredity 119(4): 287-294. https://doi.org/10.1038/hdy.2017.46 Vanegas-Rios JA, Azpelicueta MM, Malabarba LR (2018) A new species of Diapoma (Characiformes, Characidae, Stevardiinae) from the Rio Parana basin, with an identification key to the species of the genus. Jour- nal of Fish Biology 93(5): 830-841. https://doi.org/10.1111/jfb.13786 Vanegas-Rios JA, Britzke R, Mirande JM (2019) Geographic variation of Moenkhausia bonita (Characiformes: Characidae) in the rio de la Plata basin, with distributional comments on M. intermedia. Neotropical Ich- thyology 17(1): e170123. https://doi.org/10.1590/1982-0224-20170123 Ward Jr JH (1963) Hierarchical grouping to optimize an objective func- tion. Journal of the American Statistical Association 58(301): 236— 244. https://doi.org/10.1080/01621459.1963.10500845 Ward RD (2009) DNA barcode divergence among species and genera of birds and fishes. Molecular Ecology Resources 9(4): 1077-1085. https://doi.org/10.1111/j.1755-0998 .2009.02541.x Zarucki M, Gonzalez-Bergonzoni I, Teixeira-de-Mello F, Duarte A, Ser- ra S, Quintans F, Loureiro M (2010) New records of freshwater fish for Uruguay. Check List 6(2): 1-4. https://doi.org/10.15560/6.2.191 Supplementary material | All COI sequences analyzed in the present work Authors: James Anyelo Vanegas-Rios, Wilson Sebastian Serra Alanis, Maria de las Mercedes Azpelicueta, Thomas Litz, Luiz Roberto Malabarba Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.100.112778.suppl1 Supplementary material 2 Scree plots obtained from the morphometric and meristic data analyzed Authors: James Anyelo Vanegas-Rios, Wilson Sebastian Serra Alanis, Maria de las Mercedes Azpelicueta, Thomas Litz, Luiz Roberto Malabarba Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.100.112778 .suppl2 Zoosyst. Evol. 100 (1) 2024, 69-85 Supplementary material 3 Total variance accounted for the PCA performed for the morphometric and meristic data Authors: James Anyelo Vanegas-Rios, Wilson Sebastian Serra Alanis, Maria de las Mercedes Azpelicueta, Thomas Litz, Luiz Roberto Malabarba Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.100.112778.suppl3 Supplementary material 4 Cluster analysis (Ward’s method) of size-corrected morphometric data of analyzed specimens of Diapoma pampeana Authors: James Anyelo Vanegas-Rios, Wilson Sebastian Serra Alanis, Maria de las Mercedes Azpelicueta, Thomas Litz, Luiz Roberto Malabarba Data type: tif Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.100.112778.suppl4 Supplementary material 5 Tukey box plot of most distinctive meristic data observed in analyzed specimens of Diapoma pampeana Authors: James Anyelo Vanegas-Rios, Wilson Sebastian Serra Alanis, Maria de las Mercedes Azpelicueta, Thomas Litz, Luiz Roberto Malabarba Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.100.112778.suppl5 85 Supplementary material 6 Uncorrected pairwise genetic distances using the COI data matrix Authors: James Anyelo Vanegas-Rios, Wilson Sebastian Serra Alanis, Maria de las Mercedes Azpelicueta, Thomas Litz, Luiz Roberto Malabarba Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.100.112778.suppl6 Supplementary material 7 Haplotype network of the COI data analyzed of D. pampeana Authors: James Anyelo Vanegas-Rios, Wilson Sebastian Serra Alanis, Maria de las Mercedes Azpelicueta, Thomas Litz, Luiz Roberto Malabarba Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacom- mons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for oth- ers, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.100.112778.suppl7 Supplementary material 8 Table of coordinates used Authors: James Anyelo Vanegas-Rios, Wilson Sebastian Serra Alanis, Maria de las Mercedes Azpelicueta, Thomas Litz, Luiz Roberto Malabarba Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacom- mons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for oth- ers, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.100.112778.suppl8 zse.pensoft.net