A new genus of Anostomidae (Ostariophysi: Characiformes): Diversity, phylogeny and biogeography based on cytogenetic, molecular and morphological data

Accepted Manuscript A New Genus of Anostomidae (Ostariophysi: Characiformes): Diversity, Phylogeny and Biogeography Based on Cytogenetic, Molecular and Morphological Data Jorge L. Ramirez, José L.O. Birindelli, Pedro M. Galetti Jr. PII: DOI: Reference:

S1055-7903(16)30373-6 http://dx.doi.org/10.1016/j.ympev.2016.11.012 YMPEV 5671

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

24 June 2016 14 November 2016 24 November 2016

Please cite this article as: Ramirez, J.L., L.O. Birindelli, J., Galetti, P.M. Jr., A New Genus of Anostomidae (Ostariophysi: Characiformes): Diversity, Phylogeny and Biogeography Based on Cytogenetic, Molecular and Morphological Data, Molecular Phylogenetics and Evolution (2016), doi: http://dx.doi.org/10.1016/j.ympev. 2016.11.012

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A New Genus of Anostomidae (Ostariophysi: Characiformes): Diversity, Phylogeny and Biogeography Based on Cytogenetic, Molecular and Morphological Data

Jorge L. Ramireza*, José L. O. Birindellib, Pedro M. Galetti Jr.a

a

Laboratório de Biodiversidade Molecular e Conservação, Departamento de Genética e

Evolução, Universidade Federal de São Carlos, Rodovia Washington Luis Km 235, 13595-905, São Paulo, Brazil. E-mail: [email protected] b

Departamento de Biologia Animal e Vegetal, Universidade Estadual de Londrina, Caixa Postal

10.011, 8657-090, Londrina, PR, Brazil. E-mail: [email protected]

* Corresponding author: [email protected]

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Abstract A new genus of Anostomidae (Characiformes) is described to include ten valid extant species previously classified in Leporinus or Hypomasticus and distributed throughout most major river basins in South America: L. brinco, L. conirostris, L. elongatus, H. garmani, L. macrocephalus, L. muyscorum, L. obtusidens, L. piavussu, L. reinhardti, and L. trifasciatus. The monophyly of Megaleporinus is well-supported in a phylogenetic analysis based on two mitochondrial and three nuclear genes, as well as its sister group relationship to Abramites. Megaleporinus is diagnosed by having the exclusive combination of three unicuspid teeth on each premaxillary and dentary bone and a color pattern composed of one to four dark midlateral blotches. Additional distinguishing features and possible synapomorphies include a unique ZZ/ZW sex chromosome system confirmed for six congeners and a drumming apparatus wherein the first rib is elongated and associated with hypertrophied intercostal muscles, which was confirmed for three congeners as exclusive to mature males. Furthermore, our study identified at least four undescribed cryptic species, emphasizing the need for further taxonomic work and genetic analyses. A time-calibrated phylogenetic and biogeographical analysis of the new genus suggests that speciation in the proto-Amazon-Orinoco lineage was primarily driven by paleogeographic processes, such as the formation of the Orinoco and Tocantins basins. Dispersal and diversification of the genus in coastal basins draining the Eastern Brazilian Shield appears to have been facilitated by connections between paleo-basins during low sea level periods and headwater captures between coastal and inland watersheds. The present contribution demonstrates the importance of integrating data from morphology, DNA sequences and cytogenetics to advance the taxonomy and systematics of any complex species group.

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Keywords: Megaleporinus; cryptic species; ZW sex chromosomes; taxonomy; evolution; neotropical

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1. Introduction The neotropical freshwater ichthyofauna is perhaps the most diverse in the world but remains poorly known, as the total number of extant species is uncertain (Reis et al., 2016). In the last three decades, molecular data have significantly expanded our knowledge of neotropical fish diversity (Albert and Reis, 2011; Pereira et al., 2013). Analyses of mitochondrial and nuclear genes have added missing pages to the evolutionary history of many large and complex neotropical fish groups (e.g., Oliveira et al. 2011; Roxo et al. 2014; Thompson et al. 2014; Ramirez et al. 2016). In addition, DNA barcoding has disclosed taxonomic uncertainties and hidden diversity within the major groups of neotropical fishes (Pereira et al., 2013; Ramirez and Galetti Jr., 2015). The family Anostomidae is broadly distributed in South America and comprises approximately 150 described species distributed in 14 genera (Garavello and Britski, 2003; Sidlauskas and Vari, 2008). Leporinus is the most species-rich genus with approximately 90 valid species (Birindelli et al., 2013; Burns et al., 2014) showing wide morphological diversity, especially in snout shape, mouth position and dentition (Sidlauskas, 2007; Sidlauskas and Vari, 2008). The taxonomy of Anostomidae was substantially improved by Myers (1950), who defined eight genera (Leporellus, Gnathodolus, Synaptolaemus, Rhytiodus, Leporinus, Abramites, Schizodon, and Laemolyta) based on tooth morphology and mouth position to allocate the 75 species known at the time (Fowler, 1950). Myers (1950) considered previous attempts to split Leporinus to be inconsistent, as for example Myocharax (created for L. desmotes) and Hypomasticus (created for species with a distinctly ventral mouth such as H. mormyrops) (Borodin, 1929; Fowler, 1914). The only comprehensive taxonomic assessment of Leporinus was

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performed by Garavello (1979), who defined species groups within Leporinus based on coloration patterns. Garavello’s groups were adopted by some authors to aid in species-level identification (e.g., Sidlauskas and Vari, 2012), even though most of them proved to be artificial units (Sidlauskas and Vari, 2008). Only two comprehensive morphological studies have addressed the evolutionary history of Anostomidae, one focused on small-sized species with an upturned mouth, previously assigned to Anostominae (Winterbottom, 1980), and another focused on the familial relationships within the broader Anostomoidea (Sidlauskas and Vari, 2008). The latter study recovered Leporinus as paraphyletic, and possibly including Abramites. A study based on molecular data (Ramirez et al., 2016) obtained similar results with species of Leporinus interleaved among other genera (Abramites, Hypomasticus, Laemolyta, Rhytiodus, and Schizodon), revealing that it is feasible and imperative to split Leporinus. Cytogenetic studies on Leporinus have shown that most species lack sex chromosomes (Galetti Jr. et al., 1991, 1981). Six species, however, have a distinctive ZZ/ZW sex chromosome system (Galetti Jr. et al., 1995, 1981; Molina et al., 1998; Venere et al., 2004): Leporinus conirostris, L. macrocephalus, L. obtusidens [cited as L. elongatus (Paraná basin) and L. cf. elongatus (São Francisco basin)], L. piavussu (cited as L. obtusidens), L. reinhardti, and L. trifasciatus. In the ZZ/ZW system, the W chromosome is the largest one in the female karyotype and is absent from the male complement. Galetti Jr. et al. (1995) proposed the ZZ/ZW sex chromosome system as a synapomorphy for a putative monophyletic species group. However, the cytogenetics of most anostomid species remains unstudied, suggesting that potential ZW Leporinus might still be described. A recent phylogenetic analysis based on molecular data also grouped species bearing the ZW chromosome system as monophyletic (Clade C, Ramirez et al., 2016).

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The species group treated herein includes the largest body-sized species of the family (up to 600 mm standard length (SL)), which are exploited in commercial and subsistence fisheries throughout tropical South America (Garavello and Britski, 2003). Its members are mostly herbivorous (Alvim and Peret, 2004) and potentially good seed dispersers (Galetti et al., 2008). This fish is characterized by a dental formula of 3/3. This number of teeth is an important feature for Leporinus species diagnoses (Britski and Birindelli, 2008). As a monophyletic group of ecologically equivalent species (Galetti Jr. et al., 1995) occupying all major hydrographic basins in South America, the systematics of this group may serve as an excellent model for studies of biogeography and the geology underlying watershed history (Hubert et al., 2007). In the present study, two mitochondrial and three nuclear genes were used to reconstruct the evolutionary relationships of Anostomidae species bearing ZW sex chromosomes, as well as other potentially closely related species. We tested whether molecular and anatomical synapomorphies support the monophyly of the ZW clade, describing a new genus. We also inferred intrageneric relationships and the evolutionary processes involved in the diversification of this fish.

2. Materials and Methods 2.1 Sampling Specimens from multiple populations of all species of Leporinus confirmed as ZW were used in this study (Table 1). Other anostomid species with three teeth on the premaxilla and dentary were added: Leporinus brinco (Contas basin), L. elongatus (Jequitinhonha basin), and L. muyscorum (Magdalena and Orinoco basins) (Birindelli et al., 2013; Britski and Birindelli, 2008) (Table 1). The only excluded anostomid with three teeth on the premaxilla and dentary was 6

Leporinus amblyrhynchus, a small-sized species that has no sex chromosome (Galetti Jr. et al., 1991) and a dark midlateral stripe and dark transverse bars on the body, a coloration pattern similar to L. taeniatus, L. britskii and others (see Birindelli and Britski, 2013; Birindelli et al., 2013 for more information). These features suggest that L. amblyrhynchus is unrelated to ZW Leporinus; however, data, especially from molecular markers, are needed to test this hypothesis. Hypomasticus garmani (Jequitinhonha basin), on the other hand, was included because its coloration pattern is similar to L. conirostris (i.e., a body with numerous dark transverse bars and a single dark midlateral blotch on the caudal peduncle) (Fig. 1). Additionally, Abramites hypselonotus and Leporinus striatus, two closely related species (Sidlauskas and Vari, 2008) that group with the ZW species of Leporinus (Ramirez et al., 2016) were also included, although neither has a ZW sex chromosome system (Galetti Jr. et al., 1995; Martins et al., 2000). Finally, several anostomid species and four outgroups used in Ramirez et al. (2016) were included (Table 1). Our final dataset included 60 individual of 44 nominal anostomid species distributed in eight nominal genera. Most of the study specimens are deposited in ichthyology collections (Table 1). Specimens of all nominal species of Anostomidae were examined and morphologically compared to species of the new genus. The type specimens of most anostomid species were also examined, including those of all species in the new genus. The material examined is listed in Appendix A. Institutional abbreviations follow Sabaj Pérez (2014).

2.2 Morphological Analysis Meristic and morphometric data for morphological analysis were taken according to Birindelli et al. (2013). Lateral-line scale counts included the pored scales extending onto the base of the median caudal-fin rays; counts of longitudinal scale rows above the lateral line

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excluded the lateral-line scale row and the middorsal scale row; and counts of longitudinal scale rows below the lateral line excluded the lateral-line scale row. The Weberian apparatus was treated as four vertebrae in counts and the compound caudal centrum (i.e., preural 1 fused to ural 1) counted as a single element. All examined specimens were alcohol-preserved, except those indicated by CS, which were cleared and stained according to Taylor and Van Dyke (1985), or SK, which were dry skeletons prepared using methods outlined by Bemis et al. (2004). Scanning electron microscopy (SEM) images of the premaxilla, maxilla and dentary were taken from cleared and double stained specimens. The osteological nomenclature follows Weitzman (1962) with the following exceptions as per Fink and Fink (1981): anguloarticular (fusion of angular and articular bones) replaces separate articular and angular; anterior and posterior ceratohyal replaces ceratohyal and epihyal, respectively; basipterygium replaces pelvic bone; mesethmoid replaces ethmoid; vomer replaces prevomer; and pharyngobranchial replaces suspensory pharyngeal.

2.3 DNA Extraction, Amplification and Sequencing Total DNA was extracted from tissues (fins, muscle or liver) using the standard phenolchloroform method. Fragments of cytochrome oxidase subunit I (COI; 698 bp) and cytochrome b (Cytb; 1110 bp) were amplified through PCR using the primer pairs AnosCOIF-AnosCOIR and AnosCytBF-AnosCytBR, respectively (Ramirez and Galetti Jr., 2015). Myosin heavy chain 6 cardiac muscle alpha gene (Myh6, ~750 bp), recombination activating gene 1 (RAG1, ~1500 bp), and recombination activating gene 2 (RAG2, ~1100 bp) were amplified using nested-PCR according to Oliveira et al. (2011). PCR products were sequenced for both strands using an ABI 3730xl automatic sequencer (Applied Biosystems, Waltham, MA, USA). The contigs were

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assembled and edited using BioEdit (Hall, 1999). The sequences were deposited in GenBank (Table 1).

2.4 Phylogenetic Analysis Global alignment of each gene was carried out independently using Clustal X2 (Larkin et al., 2007). Default parameters were used, and there were no difficulties in alignment because of the lack of gaps in the data sets. At first, each gene was checked for incongruences using maximum parsimony (MP) in PAUP* 4.0b10 (Swofford, 2003) to detect potential sequencing errors, contamination or mislabeled vials (Fig. S1). All phylogenetic analyses were rooted using Anodus orinocensis (Hemiodontidae). All alignments were concatenated and phylogenetically analyzed using the MP optimality criterion as implemented in PAUP* 4.0b10 (Swofford, 2003). Initial heuristic searches were conducted using random stepwise addition and tree-bisectionreconnection branch swapping and a bootstrap with 1000 replicates. The maximum likelihood for concatenated sequences was also phylogenetically analyzed using a maximum likelihood (ML) method as implemented in RAxML for XSEDE (Stamatakis, 2006; Stamatakis et al., 2008) on the CIPRES Science Gateway web server (Miller et al., 2010). For the ML analysis we used a mixed partition model determined by PartitionFinder (Lanfear et al., 2012), including seven partitions (Table S1). The analysis was conducted under the GTR+G evolutionary model, which is the only model available that is compatible with RAxML (Stamatakis et al., 2008). All other parameters were set to default values. Support for nodes was estimated by 1000 bootstrap replicates. A multilocus Bayesian species tree was estimated using *BEAST (Star-BEAST) (Heled and Drummond, 2010). The nucleotide substitution model was selected based on Bayesian

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criteria, using JModeltest 2 (Darriba et al., 2012). The models used were GTR+I+G, HKY+I+G, K+I+G, K+G, and K+G for COI, Cytb, RAG1, RAG2, and Myh6, respectively. The two mitochondrial genes were linked to share a single tree, since they are physically linked. The mitochondrial genes were set to have an effective population size of one quarter that of nuclear genes. Initially, an uncorrelated log normal relaxed clock was used in all partitions. However, as all nuclear genes showed a coefficient of variation below 0.05, indicating clock-like data (Drummond and Bouckaert, 2015), a strict clock for these genes was implemented. The speciation model used was the Calibrate Yule algorithm. Additionally, we calibrated the species tree to estimate divergence times. The rise of Cordillera Oriental was used as a calibration point. This vicariant event isolated the Magdalena basin approximately 12 Ma ago (Albert et al., 2006; Lundberg et al., 1998), isolating Leporinus muyscorum. The calibration point was set using a normal distribution with a mean of 12 and a standard deviation of 1 Ma. Markov Chain Monte Carlo was implemented using three independent runs of 300 million generations each, sampling every 5000 runs and with a burn-in of 10%. All generations were sampled from the stationary phase. Convergence of analyses and adequate sample size (>200) were evaluated in Tracer v. 1.6 (Rambaut et al., 2014).

A parsimony ancestral state reconstruction was estimated as implemented in Mesquite (Maddison and Maddison, 2016), using the topology obtained in the Bayesian species tree analysis, allowing the evolution of the ZW sex chromosome system to be traced on the tree.

2.5 Historical Biogeography

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The historical biogeography was inferred by reconstructions of ancestral distributions on phylogenetic trees using Statistical Dispersal-Vicariance Analysis (S-DIVA) (Yu et al., 2010) estimated in RASP 3.02 (Yu et al., 2015). The analyses were implemented using a random sampling of 100 trees from the Bayesian species tree. Connections between basins were allowed according to South American paleogeography (Albert and Reis, 2011). Therefore, our configuration included connections between the Magdalena and Amazonas (proto-AmazonasOrinoco), Amazonas and Tocantins, Amazonas and Orinoco, Amazonas and Paraguay, Amazonas and Paraná, Amazonas and São Francisco, Paraná and São Francisco, Paraná and Paraguay, Paraná and Paraiba do Sul, São Francisco and Doce, São Francisco and Jequitinhonha; and between coastal basins (Doce, Itapicuru, Paraiba do Sul and Jequitinhonha).

3. RESULTS

3.1 Taxonomy Megaleporinus, new genus Figure 1 and Table 2 Type species. — Curimatus obtusidens Valenciennes, 1837: pl. 8, fig. 2 [type locality: Buenos Aires, Argentina]. Included species. — Megaleporinus brinco, M. conirostris, M. elongatus, M. garmani, M. macrocephalus, M. muyscorum, M. obtusidens, M. piavussu, M. reinhardti, M. trifasciatus (Fig. 1).

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Diagnosis. — Megaleporinus is diagnosed among Anostomidae by having the unique combination of: premaxilla with three unicuspid teeth (vs. four teeth with truncate cutting edge, or three teeth with two or more distinct cusps) (Fig. 2); dentary with three unicuspid teeth (vs. three or four teeth with two or more distinct cusps) (Fig. 2); and body with one to four dark midlateral blotches (Fig. 1; vs. body with dark transverse bars, or dark longitudinal stripes, or more than four dark blotches). The unique ZZ/ZW sex chromosome system is exclusive to the genus and confirmed for six of ten congeners. See the Discussion for additional information and comments on species diagnoses.

Remarks. — Megaleporinus garmani has four teeth on dentary (vs. diagnostic count of three) and is included in the genus based on premaxillary dentition, coloration and molecular evidence from three nuclear and two mitochondrial gene sequences. Leporinus wolfei is herein considered as a junior synonym of M. trifasciatus, following the hypothesis advanced by Garavello and Britski (2003). See the Discussion for additional comments.

Description. — Size medium to large relative to congeners, maximum size ranging from 250 to 600 mm SL (Table 2). Head and body elongate and moderately compressed. Greatest body depth at dorsal-fin origin. Mouth inferior (M. garmani), subterminal (e.g., M. elongatus) or terminal (e.g., M. macrocephalus) (Fig. 1). Premaxilla with three unicuspid incisiform teeth gradually decreasing in size from symphyseal tooth (Fig. 2). Dentary with three (most species) or four (M. garmani) unicuspid incisiform teeth gradually decreasing in size posterolaterally.

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Scales cycloid. Lateral line complete, extending from supracleithrum to base of median caudal-fin rays, with 36 to 44 scales (Table 2). Longitudinal scale rows between dorsal-fin origin and lateral line 4–7. Longitudinal scale rows between lateral line and pelvic-fin origin 4–7. Longitudinal scale rows around caudal peduncle 12 (M. brinco, M. elongatus, M. garmani) or 16 (other congeners), but never 14. Dorsal-fin rays ii, 10. Dorsal-fin origin slightly anterior to midpoint of standard length and to vertical through pelvic-fin origin. Adipose fin small, teardrop-shaped with origin approximately at vertical through base of last anal-fin rays. Pectoral-fin rays i, 13–16. Pelvic-fin rays i, 9. Anal-fin rays ii, 8 or ii, 9. Principal caudal-fin rays i, 8, 9, i; caudal-fin forked, unbranched and first two or three branched rays of each lobe elongated in most intact specimens, making fin lobes weakly falcate. Supraneurals four or five. Vertebrae 34 to 39, with ribs on vertebrae five through 20 to 24.

Sexual dimorphism. — Mature males of Megaleporinus elongatus (MZUSP 106810, 194.0 mm SL), M. macrocephalus (MZUSP 89501, 345.0 mm SL) and M. obtusidens (unpreserved specimens) show the first rib distinctly elongated, posteriorly bent, and slightly thicker than immature males and females (Fig. 3). The first rib of mature males of these three species is directly associated with hypertrophied intercostal muscles and indirectly to the gas bladder situated in the dorsal-most portions of the abdominal cavity. See the Discussion for additional comments.

Distribution and habitat. — Megaleporinus is widespread in South America, occurring in all major river basins, including the Amazonas, Orinoco, Paraná (including upper Paraná), Paraguay,

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Parnaíba, São Francisco, Tocantins, Uruguay, trans-Andean drainages such as the Atrato, Magdalena and Sinú in Colombia, and coastal drainages in eastern Brazil, including the Guaíba, Paraíba do Sul, Doce, Mucuri, São Mateus, Jucuruçu, Contas, Jequitinhonha, and Pardo (Fig. 4). Megaleporinus is absent in the coastal drainages of Guianas and northeastern Brazil. Most species are known to migrate short to long distances during their reproductive season, usually at the end of the dry and beginning of the wet seasons (Britski et al., 2012; Godoy, 1975; Mojica et al., 2012). Most species feed on plants but also on a variety of other items, including insects, crustaceans and mollusks (Godoy, 1975; Melo and Röpke, 2004).

Etymology. — From the Greek mega, meaning large or largest, plus Leporinus, the genus of Anostomidae in which most species were previously assigned; in reference to the large size of most congeners.

Key to identification. — Key to the identification of all extant species of Megaleporinus based on easily observed external features (Fig. 1, Table 1) with notes on distribution (Fig. 4) in parentheses. 1. Four teeth on dentary; mouth inferior … Megaleporinus garmani (Jequitinhonha and Pardo) 1’. Three teeth on dentary; mouth subterminal or terminal … 2 2. 12 scale rows around caudal peduncle; 36 to 38 lateral-line scales … 3 2’. 16 scale rows around caudal peduncle; 37 to 44 lateral-line scales (37 or 38 exclusive to M. reinhardti) … 4 3. Three dark midlateral blotches that distinctly increase in size posteriorly; dark longitudinal lines between scale rows of body … Megaleporinus brinco (Rio de Contas)

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3’. Three dark midlateral blotches that distinctly decrease in size posteriorly; dark longitudinal lines absent between scale rows of body … Megaleporinus elongatus (Jequitinhonha and Pardo) 4. Body with a single dark blotch on caudal peduncle … Megaleporinus conirostris (Paraíba do Sul, Doce, Mucuri, São Mateus, Jucuruçu) 4’. Body with two or three midlateral dark blotches … 5 5. Body with two or three vertically elongate dark blotches (in addition to a small round dark blotch on caudal peduncle) … 6 5’. Body with three rounded dark blotches (one of them on caudal peduncle)… 7 6. Body with vertically elongate dark blotch on anterior body, between verticals through dorsalfin origin and opercle; dark longitudinal lines absent between scale rows of posterior portion of body … Megaleporinus trifasciatus (Amazonas) 6’. Anterior portion of body without dark blotch; dark longitudinal lines between scale rows of body posterior to vertical through dorsal-fin origin … Megaleporinus macrocephalus (Paraguay) 7. Lateral-line scales 37 to 39; body with three dark blotches longitudinally elongate and sometimes forming dark midlateral stripe from vertical through dorsal-fin origin to base of caudal fin median rays … Megaleporinus reinhardti (São Francisco, Itapicuru) 7’. Lateral-line scales 39 to 44; body with three rounded dark blotches (never longitudinally elongated) … 8 8. Lateral-line scales 39 to 41 (rarely 41); mouth terminal (gape above horizontal through ventral orbital margin in specimens larger than 150 mm SL) … Megaleporinus piavussu (upper Paraná) 8’. Lateral-line scales 41 to 44 (rarely 41); mouth subterminal (gape at or slightly below horizontal through ventral orbital margin in specimens larger than 150 mm SL) … 9

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9. Body with dark transverse bars usually persistent in adults (specimens larger than 150 mm SL); 6 or 7 scale rows between lateral line and dorsal-fin origin … Megaleporinus obtusidens (Paraná, Paraguay, Uruguay, Jacuí, São Francisco, Parnaíba) 9’. Body with dark transverse bars usually faded in adults (specimens larger than 150 mm SL); 5 scale rows between lateral line and dorsal-fin origin … Megaleporinus muyscorum (Atrato, Magdalena, Sinú, Orinoco)

3.2 Phylogenetic Analysis Alignment of COI, Cytb, RAG1, RAG2, and Myh6 resulted in 627, 1005, 1469, 1023, and 754 characters, with 232, 420, 260, 186, and 102 parsimony informative sites, respectively. There were no gaps in the alignments. All phylogenetic trees were strongly supported and showed the same topology (Fig. 5, see Fig. S2 for individual trees). We found high genetic divergences between basins within four nominal species: M. conirostris, M. muyscorum, M. obtusidens, and M. trifasciatus (see Discussion). In sum, we found 16 lineages distributed among ten valid species. Megaleporinus muyscorum from the Magdalena Basin was sister to all congeners, with the remaining species allocated into two clades (Fig. 5). Figure 6 shows the calibrated tree and the ancestral distribution reconstruction for species of Megaleporinus with each cladogenetic event numbered for the Discussion section. Figure 7 shows the ancestral state reconstruction for the ZW sex chromosome system.

4. DISCUSSION

4.1 Monophyly

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The monophyly of extant Megaleporinus is well supported in phylogenetic analyses based on sequences of two mitochondrial (COI, and Cytb) and three nuclear genes (RAG1, RAG2, and Myh6). The ZW sex chromosome system has been confirmed for six species of Megaleporinus: M. conirostris, M. macrocephalus, M. obtusidens, M. piavussu, M. reinhardti, and M. trifasciatus (Galetti Jr. et al., 1995, 1981; Molina et al., 1998; Venere et al., 2004). Based on the ancestral state reconstruction (Fig. 7), the ZW sex chromosome system can be dated back to Node 2, or even to Node 1 (the analysis considers both states as equally optimal). Therefore, we hypothesize the ZW chromosome condition to be shared by cytogenetically unstudied species of Megaleporinus descended from Node 2 (Fig. 7). If confirmed for M. muyscorum from the Magdalena Basin, the ZW sex chromosome is a solid synapomorphy supporting the monophyly of Megaleporinus. A complementary cytogenetic study is therefore necessary to confirm the existence of these ZW sex chromosomes in those unstudied species. Similarly, at least three species of Megaleporinus (M. elongatus, M. macrocephalus, and M. obtusidens) exclusively have secondary dimorphic features regarding the development of the first rib and associated muscles. In mature males of the aforementioned Megaleporinus, the first rib is distinctly enlarged, elongated and associated with hypertrophied intercostal muscles (Fig. 3). This modification is herein described for the first time in Anostomidae but remains to be confirmed for all species of Megaleporinus. A similar modification is present in some species of Curimatidae (Dorn, 1972; Schaller, 1974) and Prochilodontidae (Castro and Vari, 2004; Schaller, 1971), in which mature males are known to produce sounds during the breeding season (Godoy, 1975). Curimatidae and Prochilodontidae are sister families closely related to Anostomidae plus Chilodontidae in the superfamily Anostomoidea (Dillman et al., 2016; Vari, 1983). The

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modification in Megaleporinus could also be related to male sound production during the breeding season. The most conspicuous morphological feature that diagnoses Megaleporinus is related to the reduction and the shape of the teeth on the jaw bones. All species of Megaleporinus have three unicuspid teeth on the premaxillary and dentary bones (except M. garmani, which has four dentary teeth). As previously mentioned, the number and morphology of jaw teeth are important features for diagnosing the genera of Anostomidae (Myers, 1950). Most species of the family have four teeth on the premaxillary and dentary bones, including Anostomoides, Anostomus, Laemolyta, Leporellus, Rhytiodus, Schizodon, and Synaptolaemus. Three teeth on the premaxilla are only found in Abramites, Megaleporinus and a few species of Hypomasticus and Leporinus (Britski and Birindelli, 2008; Sidlauskas and Vari, 2008). The only species that have only three teeth on the dentary are Abramites spp., Megaleporinus spp. (except M. garmani with four), Leporinus amblyrhynchus, L. desmotes, L. jatuncochi, L. venerei and Sartor spp. Note that L. venerei and Sartor spp. also have four teeth on the premaxilla. Therefore, the only species that share with Megaleporinus the presence of three teeth on each premaxillary and dentary bone are those of Abramites and L. amblyrhynchus, L. desmotes, and L. jatuncochi. The teeth in Abramites are distinct from all other anostomids by exhibiting a distinct notch on the cutting edge of the first two dentary teeth (Fig. 2). According to our phylogeny, the reduction from four to three premaxillary teeth is a synapomorphy for the clade, including Abramites, Megaleporinus and Leporinus striatus, whereas the reduction from four to three dentary teeth is a synapomorphy of Abramites plus Megaleporinus. Note that the last dentary tooth in Leporinus striatus is much smaller than the last tooth of most congeners and remote from the other dentary teeth (Birindelli

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and Britski, 2013), perhaps a transitional state in the loss of the last dentary tooth as in Abramites and Megaleporinus. Similarly, most anostomid genera have bicuspid or multicuspid teeth (Fig. 2), including Abramites, Anostomoides, Anostomus, Gnathodolus, Laemolyta, Pseudanos, Petulanos, Rhytiodus, Sartor, and Schizodon (Myers, 1950; Sidlauskas & Vari, 2008). The only genera that have unicuspid teeth are Hypomasticus, Leporellus, and Leporinus. Considering the phylogenetic position of Megaleporinus (Fig. 5), the presence of unicuspid teeth is a plesiomorphic feature of Anostomidae, excepting Anostominae (Anostomus and Pseudanos clade). Megaleporinus includes the largest members of the family, with at least four species (M. macrocephalus, M. obtusidens, M. piavussu, and M. trifasciatus) reaching more than 500 mm SL (Table 2). Other large anostomids with estimated maximum sizes up to 400 mm SL are Leporinus friderici, Rhytiodus microlepis, and Schizodon fasciatus (Britski et al., 2007; Santos et al., 2006; Santos and Feitosa, 2013). This is an additional interesting characteristic of Megaleporinus, although its evolutionary and ecological significance remains obscure. The most common color pattern among species of Anostomidae is the presence of one to four dark brown midlateral blotches in adults. Departures from this coloration pattern are the presence of dark brown transverse bars or one to six dark longitudinal stripes on the body in adults. Transverse bars are found in Abramites, some Leporinus (e.g., L. fasciatus), and Synaptolaemus. Longitudinal stripes occur in Anostomus, some Hypomasticus (e.g., H. despaxi), some Laemolyta (e.g., L. taeniata), Leporellus, some Leporinus (e.g., L. striatus), Pseudanos winterbottomi, and some Schizodon (e.g., S. scotorhabdotus). A color pattern composed of dark midlateral blotches (as in Megaleporinus) seems to have evolved or reversed independently

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many times in the evolution of Anostomidae and, therefore, does not provide clues for the phylogenetic position of Megaleporinus.

4.2 Diversity The molecular analysis presented herein corroborates previous results (Avelino et al., 2015; Ramirez et al., 2016; Ramirez and Galetti Jr., 2015) that considered the diversity of Anostomidae to be underestimated, and further highlights the need for taxonomic revisions and species descriptions. Moreover, is possible to detect new lineages within the nominal species if more multiple genetic samples from widespread species are included. Megaleporinus muyscorum was described by Steindachner (1900) based on a single holotype from the Río Magdalena, Colombia. The specimen, vouchered in Munich, was destroyed in WWII (Neumann, 2006). A neotype designation is unnecessary as the species was comprehensively described and illustrated by Steindachner (1900, 1902), and later redescribed in more detail by Garavello (2000). The latter author considered specimens of Leporinus muyscorum from the Orinoco Basin co-specific with trans-Andean specimens, expanding the known distribution of the species to cis-Andean South America. Based on molecular data (Fig. 5), however, the specimens from the Orinoco basin are more closely related to M. trifasciatus and M. macrocephalus, and characterize a different lineage (M. cf. muyscorum) and a potentially undescribed species. A detailed morphological comparison between trans-Andean and Orinoco specimens of M. muyscorum is therefore warranted. Megaleporinus trifasciatus was described by Steindachner (1876) based on a single specimen from Tefé in the Brazilian Amazon. The species is broadly distributed in the Amazon river basin, from Belém to at least the border between Brazil and Peru (Garavello and Britski,

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2003), and also occurs in the Tocantins-Araguaia drainage (Melo et al., 2005; Santos et al., 1984). The species is readily distinguished from all other Anostomidae based on its unique coloration (Fig. 1). Leporinus wolfei, described by Fowler (1939) based on two specimens from the Ucayali River in Peru, is a junior synonym of M. trifasciatus, as the type specimens have the same unique coloration and lie within the range of meristic features diagnostic of M. trifasciatus (Table 2; Garavello and Britski 2003). Our results revealed the existence of two lineages within M. trifasciatus, one from Upper Amazon (Ucayali and Madeira basins) and another from the Tocantins-Araguaia Basin. The Tocantins-Araguaia specimens, here designated as M. cf. trifasciatus, represent a lineage more closely related to M. macrocephalus, and denote possibly another undescribed species. Further studies based on morphological and molecular data are necessary to define the limits of M. trifasciatus and to resolve the status of the TocantinsAraguaia lineage. Steindachner (1875) described Megaleporinus conirostris based on many specimens from various Brazilian coastal drainages, including the Paraíba do Sul, Doce and Mucuri rivers. Our molecular results showed 4.1% sequence divergence in the COI gene between populations in the Doce and Paraíba do Sul, a high value compared to other anostomids (Ramirez and Galetti Jr., 2015) and other neotropical fishes (Pereira et al., 2013). Additional studies are needed to evaluate the distinctiveness of populations of M. conirostris inhabiting isolated drainages along the coast of eastern Brazil. The two obtained lineages were herein arbitrarily identified as M. conirostris (Paraiba do Sul river) and M. cf. conirostris (Doce river). The taxonomic history of Megaleporinus obtusidens is long and complex, dating back more than 150 years and including at least six other nominal species (L. aguapeiensis, L. bimaculatus, L. elongatus, L. pachyurus, L. piavussu, and L. silvestrii) (Britski et al., 2012).

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Megaleporinus obtusidens was recently redescribed based on type and additional specimens from the Paraná (lower and upper), Paraguay, Uruguay, São Francisco, Guaíba and Parnaíba basins (Britski et al., 2012). The molecular data suggest that M. obtusidens represents a complex with up to three distinct species (with a COI interspecific distances above 2.5%), herein identified as M. obtusidens (Paraná, Uruguay, and Jacuí basins), M. cf. obtusidens Paraguay, and M. cf. obtusidens São Francisco. This hypothesis differs from a recent analysis (Avelino et al., 2015) based on sequences of 16S rRNA, Cytb, COI, and α-tropomyosin that identified four putative lineages within this group. Further molecular analyses, including samples of numerous specimens from the entire distributional range of M. obtusidens, remain necessary for defining and diagnosing cryptic species within M. obtusidens.

4.3 Phylogeny The molecular data strongly support Megaleporinus as sister to Abramites, and that clade as sister to Leporinus striatus (Fig. 5; Ramirez et al., 2016). Sidlauskas and Vari (2008) considered Abramites and Leporinus striatus to be sister taxa, as supported by 10 morphological characters. Unfortunately, no species of Megaleporinus were included in that analysis and some of the characters used (Sidlauskas and Vari, 2008) vary within Megaleporinus. Leporinus scalabrinii, a fossil species previously classified as a mammal (Arrhinolemur scalabrinii) and recently recognized as a valid species of Leporinus (Bogan et al., 2012), has the overall morphology consistent with Megaleporinus. The fossil taxon is based on a single relatively well-preserved skull from the late Miocene Ituzaingó Formation of Argentina, which ranges from approximately 9 Ma to 6 Ma. According to Bogan et al. (2012), the overall morphology of the visible portions of the jaws, neurocranium, and suspensorium are comparable

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to that typical for many members of Anostomidae, Abramites and Leporinus, in particular. As Megaleporinus, L. scalabrinii has only three teeth on the premaxillary bone and possibly three on the dentary bone, and a terminal mouth (vs. downturned in Hypomasticus, or upturned in Anostomoides, Anostomus, Gnathodolus, Laemolyta, Pseudanos, Petulanos, Sartor, Schizodon, and Synaptolaemus). Other anostomids with three premaxillary teeth are Abramites, Leporinus amblyrhynchus, L. desmotes, L. jatuncochi. Leporinus scalabrinii is distinguished from Abramites based on jaw tooth morphology, the former having unicuspid teeth with blunt cutting edge (vs. a symphyseal premaxillary tooth bicuspid, and the first two dentary teeth with a deep notch on the cutting edge). Leporinus scalabrinii is distinguished from L. amblyrhynchus by having a terminal mouth (vs. subinferior), and from L. desmotes and L. jatuncochi by having slightly larger symphyseal premaxillary and dentary teeth, less than twice the length of the lateral ones (vs. symphyseal premaxillary and dentary teeth much larger, at least two times longer than the lateral ones). On the other hand, L. scalabrinii has the frontal–parietal fontanel largely closed, a feature absent in all species of Megaleporinus, and present only in Abramites, Anostomus and some species of Pseudanos among anostomids (Sidlauskas and Vari, 2008). The phylogenetic position of L. scalabrinii is therefore dubious, although the morphological evidences support its relationship with the Abramites and Megaleporinus clade. Within Megaleporinus, M. muyscorum from the Magdalena Basin was sister to all congeners (see below for biogeographical implications). Except for M. muyscorum, Megaleporinus is divided into two clades, one with L. cf. muyscorum (Orinoco), L. trifasciatus (upper Amazon), L. cf. trifasciatus (Araguaia) and L. macrocephalus (Paraguay) (Fig. 5). This clade is also supported by the color pattern; its members are unique among congeners by having vertically elongated dark brown blotches on the body (vs. rounded midlateral blotches). The only

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other anostomid species that possess vertically elongated blotches belong to Anostomoides, Laemolyta, Rhytiodus and Schizodon. Those genera are closely related to each other and distant from Megaleporinus. Therefore, vertically elongated midlateral blotches are convergent in the two lineages. The other clade within Megaleporinus comprises M. conirostris (Paraíba do Sul) and M. cf. conirostris (Doce) as the sister group to M. brinco (Contas), M. elongatus (Jequitinhonha), M. garmani (Jequitinhonha), M. obtusidens (Paraná, Uruguay, and Jacuí), M. cf. obtusidens Paraguay (Paraguay), M. cf. obtusidens São Francisco (São Francisco), M. piavussu (Paraná), M. reinhardti (São Francisco), and M. cf. reinhardti (Itapicuru). Five taxa, Megaleporinus elongatus, M. obtusidens, M. cf. obtusidens Paraguay, M. cf. obtusidens São Francisco and M. piavussu, are more closely related to each other than to congeners (Fig. 5). Britski et al. (2012) considered M. obtusidens as very similar to L. piavussu, distinguishable only by minor differences in lateralline scale counts and snout shape. According to those authors, both species are highly similar to M. elongatus, a name that was long applied to specimens of M. obtusidens. Our results corroborated the close relationships between those three species and found M. obtusidens to be a complex composed of three cryptic species separable by drainage. Birindelli et al. (2013) considered M. brinco as closely related to either M. elongatus based on meristic data (Table 2) or M. conirostris, based on some meristic data and color pattern. Although not yet conclusive, our results suggest M. brinco is more closely related to M. elongatus than to M. conirostris (Fig. 5), and should be further investigated. Megaleporinus garmani has the downturned mouth typical of Hypomasticus and was included in that genus by Sidlauskas and Vari (2008). Our molecular data, however, place H. garmani firmly within Megaleporinus. Our analysis included Hypomasticus megalepis, H.

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mormyrops, and H. pachycheilus, and recovered Hypomasticus as a polyphyletic genus. Our results grouped L. copelandii and L. steindachneri with H. mormyrops (type species of the genus) and H. megalepis (Fig. 5) in a relatively basal clade. Hypomasticus pachycheilus, however, grouped with numerous species of Leporinus, including L. fasciatus (the type species of Leporinus). Further analyses, both molecular and morphological, are still needed to resolve the apparent polyphyly of Hypomasticus.

4.4 Historical Biogeography The most recent common ancestor of Megaleporinus was dated at 12 Ma (Fig. 6: Node 1), based on the rise of the Cordillera Oriental in the northern Andes, which isolated the Magdalena River from cis-Andean basins, corresponding to our calibration. The second cladogenetic event was estimated at 9.27 Ma (Fig. 6: Node 2) and corresponds to the divergence between lineages inhabiting the proto-Amazonas-Orinoco (including Tocantins) and the Eastern Brazilian Shield (Paraná, São Francisco and Coastal basins). This event was recovered by S-DIVA as a geodispersal (i.e., removal of a barrier, allowing range expansion (Ho et al., 2015) from protoAmazonas-Orinoco (including Tocantins) into the São Francisco). Geodispersal events from the Amazonas to basins of the Eastern Brazilian shield were previously reported (Hubert et al., 2007; Montoya-Burgos, 2003), and a biogeographic relationship between São Francisco and the upper Tocantins was already claimed to explain the presence of shared monophyletic groups (Lima and Caires, 2011). The most recent common ancestor for the proto-Amazonas-Orinoco lineages was dated at 5.97 Ma (Fig. 6: Node 3) and likely corresponds to separation of the Orinoco and Amazonas basins. Similar results were reported for Prochilodus (Prochilodontidae), where the Orinoco-

25

Amazon separation was estimated at 3.9 to 5.2 Ma (Sivasundar et al., 2001) or 3 Ma (Turner et al., 2004). The rise of Vaupés arch (Lujan and Armbruster, 2011), estimated between 10 Ma (Gregory-Wodzicki, 2000; Hoorn, 1994) and 8 Ma (Hoorn, 1993; Hoorn et al., 1995), is thought to have separated the Orinoco and Amazon basins. Biogeographical evidence suggests that the vicariance was more recent (Albert and Carvalho, 2011). The 4th cladogenetic event (Fig. 6) likely corresponds to the separation of the Tocantins from the Amazonas basin. The Tocantins-Araguaia basin has a complex geological history, its position changing according to tectonic reactivations (Rossetti and Valeriano, 2007). Vicariant events between the Tocantins and Amazonas are thought to be due to the emergence of the Gurupá arch, estimated at 5.66 Ma (Hubert et al., 2007), with the final establishment of the modern Tocantins estimated at 1.8 Ma (Rossetti and Valeriano, 2007). Our estimation of the separation between the Tocantins and Amazonas (3.98 Ma, Fig. 6) falls between the rise of the Gurupá arch and the origin of the modern Tocantins. The last speciation event of the proto-Amazon-Orinoco clade corresponds to the Paraguay-Amazonas separation (2.22 Ma, Fig. 6). This event was indicated by the S-DIVA analysis as primarily dispersal to the Paraguay basin and later speciation by vicariance. This pattern is expected due to hydrogeological events, such as a headwater capture of the Alto Amazonas by the Paraguay River. Those basins have a long shared history, over the last 40 Ma, with major capture events and formation of semipermeable barriers until the emergence of the Michicola arch, which finally separated the basins approximately 11.8 - 10 Ma (Lundberg et al., 1998). Other molecular analyses are quite variable for dating the Amazon and Paraguay vicariance, ranging from 11.4 to 10.5 Ma (Montoya-Burgos, 2003) to 4.1 to 2.3 Ma (Sivasundar

26

et al., 2001), indicating that connections between those basins are more complex than previously thought (Carvalho and Albert, 2011). Node 6 (7.67 Ma, Fig. 6) denotes the most recent common ancestor of the Eastern Brazilian Shield clade. This area includes two major river basins, the São Francisco and the upper Paraná, as well as several smaller coastal basins (Buckup, 2011). In this region, geological instability has produced several cases of headwater capture between major basins, as well as adjacent coastal basins (Buckup, 2011). The cladogenetic event marked by this node is probably a headwater capture that allowed Megaleporinus to disperse from the São Francisco River into the coastal basins. Subsequent and recurrent captures involving the coastal basins likely lead to the divergences represented by nodes 9 and 10. Node 7 (Fig. 6) marks the separation of the M. conirostris lineages (Doce vs. Paraiba do Sul), estimated at 1.8 Ma (Fig. 6). This geodispersal between two coastal basins appears related to fluctuations in sea level (Buckup, 2011) over the last million years (Albert and Reis, 2011; Miller et al., 2005). Dramatic rises in sea level isolate coastal basins via marine incursion, whereas periods of low sea level (marine regression) favor deltaic connections between adjacent basins. Evidence exists for paleo-basins along the coast of eastern Brazil and a complex river system that is currently submerged on the continental shelf (Abreu et al., 2005; Menezes et al., 2008). The process of range expansion into new basins during marine regressions and subsequent isolation by marine incursions likely contributed to differentiation among populations of M. conirostris.

5. Conclusions

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Megaleporinus has a complex evolutionary history. Speciation in the proto-AmazonOrinoco lineage was likely facilitated by paleogeographic processes, such as the isolation of the Orinoco and Tocantins basins from the paleo-Amazonas. The diversification process in the Eastern Brazilian Shield lineage, on the other hand, seems to be predominantly hydrogeological, with dispersals facilitated by headwater captures and connections between paleo-basins during marine regressions. The present study allows us to predict the ZW chromosome system for species that are completely unstudied cytogenetically. Similarly, the presence of an internal secondary dimorphic feature is likely to occur in most, if not all, species of Megaleporinus. Both hypotheses make excellent goals for future investigations. Furthermore, our results identified at least four potential cryptic and undescribed species, emphasizing the need for further taxonomic revisions and genetic analyses. Considering the ecological and economic significance of the species of Megaleporinus, answers to those open questions are imperative for their conservation. The diversity of Megaleporinus appears to be underestimated, putting undescribed species at risk. Recently, a major dam failure killed millions of fish along much of the Doce river (Escobar, 2015), threatening populations of M. cf. conirostris, a potentially undescribed species. The present contribution demonstrates the need to integrate data from disparate sources (in our case, cytogenetics, molecular sequences and morphology) if the final goal is to improve the phylogeny and classification of a complex species group. Separately, these data have distinct limitations that prevent them alone from determining Megaleporinus with precision. However, combining these data makes it possible to robustly infer the composition, diagnosis, phylogeny

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and biogeography of Megaleporinus. Given the incredible diversity and complexity of neotropical fish fauna, such integrative studies are strongly encouraged.

Acknowledgments This work was supported by the Conselho Nacional de Desenvolvimento Cientifíco e Tecnológico (Universal 473474/2011-5 to P.M.G.J.; Universal 47890/2013-9 to J.L.O.B.), the SISBIOTA-Brazil Program (CNPq, 563299/2010-0; FAPESP, 10/52315-7), and the South American Characiformes Inventory Project (FAPESP 2011/50282-7). The authors received fellowship grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (2011/21836-4, J.L.R.; 10/51250-9, J.L.O.B.). The authors received productivity research grants from Conselho Nacional de Desenvolvimento Cientifíco e Tecnológico (304440/2009-4 to P.M.G.J.) and Fundação Araucária (641/2014 to J.L.O.B.). We are grateful to C. Cramer, C. Doria, D. Carvalho, H. Ortega, J.C. Riofrio, J. Rodriguez-Pulido, M. Carrillo, P. R. Afonso, P. Venere and W. Troy for help obtaining part of the tissue samples and ICMBIO/MMA for sampling fish authorization (32215-1). For loans and assistance during collection visits we thank Mark Sabaj and John Lundberg (ANSP), James Maclaine, Oliver Crimmen, Patrick Campbell and Ralf Britz (BMNH), Tomio Iwamoto and Dave Catania (CAS), Kevin Swagel and Susan Mochel (FMNH), Lucia Rapp Py-Daniel and Renildo Oliveira (INPA), Carlos Alberto Lucena and Roberto E. Reis (MCP), Karsten E. Hartel, Karel F. Liem and Andrew Williston (MCZ), Patrice Pruvost (MNHN), Paulo Buckup and Marcelo Britto (MNRJ), Wolmar Wosiacki, Alberto Akama and André NettoFerreira (MPEG), Max Hidalgo and Hernan Ortega (MUSM), Heraldo Britski, Náercio Menezes, Mario de Pinna, Aléssio Datovo and Osvaldo Oyakawa (MZUSP), Helmut Wellendorf and Christian Pollman (NMW), and Larry Page (UF). Comments and suggestions on the MS were

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provided by Mark Sabaj, supported by the iXingu Project (NSF DEB-1257813). The authors thank Brian Sidlauskas, Nathan K. Lujan and a third anonymous reviewer for suggestions and comments that improved the revised manuscript.

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Table 1. Voucher data, GenBank accession numbers and cytogenetic knowledge for specimens included in the analyses. Species

River

Basin

Abramites hypselonotus

Madeira

Madeira

Sex chromosomes No

Abramites hypselonotus

Yarinacocha

Amazonas

Anostomus ternetzi

Arinos

Hypomasticus megalepis Hypomasticus mormyrops

Museum voucher

COI

Cytb

RAG1

RAG2

Myh6

UFRO-I 8234*

KF568968

KF569011

KF569054

KF569097

KF569140

No

MUSM 47356

KU134848 KU134866

KU134884

KU134902 KU134920

Tapajós

No

MZUSP 113996

KF568970

KF569013

KF569056

KF569099

Pitinga

Uatuma

Unknown

MZUEL 10200

KX020571 KX020574

KX020580

KX020583 KX020577

Bananal

Paraíba do Sul

No

MZUEL 08022

KX020572 KX020575

KX020581

KX020584 KX020578

Madeira

Unknown

UFRO-I 2156*

KF568973

KF569016

KF569059

KF569102

KF569145

Hypomasticus pachycheilus

KF569142

Laemolyta fernandezi

Araguaia

Tocantins

Unknown

GEPEMA 5598

KF568974

KF569017

KF569060

KF569103

KF569146

Leporellus vittatus

Sepotuba

Paraguay

No

MZUSP 113987

KF568980

KF569023

KF569066

KF569109

KF569152

Leporinus affinis

Tocantins

Tocantins

Unknown

-

KF568975

KF569018

KF569061

KF569104

KF569147

Leporinus bleheri

Guaporé

Paraguay

Unknown

MZUSP – 113988

KF568976

KF569019

KF569062

KF569105

KF569148

Leporinus cf. parae

Madeira

Madeira

Unknown

UFRO-I 9367*

KF568999

KF569042

KF569085

KF569128

KF569171

Leporinus copelandii

Manhuaçu

Doce

No

MCNIP 0459

KF568978

KF569021

KF569064

KF569107

KF569150

Leporinus desmotes

Araguaia

Tocantins

No

GEPEMA 5553

KF568979

KF569022

KF569065

KF569108

KF569151

Leporinus fasciatus

Madeira

Madeira

No

UFRO-I 3343

KF568981

KF569024

KF569067

KF569110

KF569153

Leporinus friderici Paraná

Turvo

Paraná

No

MZUSP 113983

KF568982

KF569025

KF569068

KF569111

KF569154

Leporinus friderici Paraguay

Sepotuba

Paraguay

No

MZUSP – 113985

KF568983

KF569026

KF569069

KF569112

KF569155

Leporinus geminis

Araguaia

Tocantins

ZZ/ZW1

GEPEMA 5530

KF568984

KF569027

KF569070

KF569113

KF569156

Leporinus jatuncochi

Candeias

Madeira

Unknown

UFRO-I 10845

KX020573 KX020576

KX020582

KX020585 KX020579

Leporinus lacustris Paraguay

Cuiaba

Paraguay

No

MZUSP – 113991

KF568985

KF569028

KF569071

KF569114

KF569157

Leporinus lacustris Paraná

Tiete

Paraná

No

MZUSP – 113994

KF568986

KF569029

KF569072

KF569115

KF569158

Leporinus octomaculatus Paraguay

Bandeirantes

Paraguay

Unknown

MZUSP – 113984

KF568988

KF569031

KF569074

KF569117

KF569160

Leporinus octomaculatus Paraguay

Sumidouro

Amazonas

Unknown

MZUSP – 113992

KF568989

KF569032

KF569075

KF569118

KF569161

Leporinus piau

Urucuia

São Francisco

No

-

KF568990

KF569033

KF569076

KF569119

KF569162

Leporinus sp.

Sumidouro

Unknown

KF568993

KF569036

KF569079

KF569122

KF569165

Leporinus steindachneri

Jequitinhonha

Jequitinhonha

Unknown

KF568994

KF569037

KF569080

KF569123

KF569166

Leporinus striatus Paraguay

Sepotuba

Paraguay

No

MZUSP – 117733 MCNI-PUCMG0379 MZUSP 113986

KF568995

KF569038

KF569081

KF569124

KF569167

Leporinus striatus Uruguay

Uruguay

Uruguay

No

MZUSP 118669

KU134863 KU134881

KU134899

KU134917 KU134935

42

Leporinus taeniatus

Urucuia

São Francisco

No

MCP – 44097

KF568996

KF569039

KF569082

KF569125

KF569168

Leporinus tigrinus

Araguaia

Tocantins

Unknown

GEPEMA – 5514

KF568997

KF569040

KF569083

KF569126

KF569169

Leporinus unitaeniatus

Araguaia

Tocantins

Unknown

GEPEMA - 5549

KF569000

KF569043

KF569086

KF569129

KF569172

Leporinus venerei

Araguaia

Tocantins

Unknown

GEPEMA - 5549

KF569000

KF569043

KF569086

KF569129

KF569172

Contas

Unknown

MZUSP 118670

KU134850 KU134868

KU134886

KU134904 KU134922

Megaleporinus brinco Megaleporinus cf. conirostris

Doce

Doce

Unknown

MCNIP 0186

KF568977

KF569020

KF569063

KF569106

KF569149

Megaleporinus cf. muyscorum

Meta

Orinoco

Unknown

-

KU134851 KU134869

KU134887

KU134905 KU134923

Megaleporinus cf. obtusidens Paraguay Megaleporinus cf. obtusidens São Francisco

Cuiaba

Paraguay

ZZ/ZW

MZUSP 118668

KU134861 KU134879

KU134897

KU134915 KU134933

Pandeiros

São Francisco

ZZ/ZW

MCNIP 44805

KU134862 KU134880

KU134898

KU134916 KU134934

Megaleporinus cf. reinhardti

Itapicuru

Unknown

-

KU134849 KU134867

KU134885

KU134903 KU134921

Megaleporinus cf. trifasciatus

Araguaia

Itapicurúmirim Tocantins

ZZ/ZW

GEPEMA 5095

KF568998

KF569041

KF569084

KF569127

Megaleporinus conirostris

Paraibuna

Costa

ZZ/ZW

-

KU134852 KU134870

KU134888

KU134906 KU134924

Megaleporinus elongatus

Itacambiruçu

Jequitinhonha

Unknown

MCNIP 0375

KU134853 KU134871

KU134889

KU134907 KU134925

Megaleporinus elongatus

Jequitinhonha

Jequitinhonha

Unknown

-

KU134854 KU134872

KU134890

KU134908 KU134926

Megaleporinus garmani

Itacambiruçu

Jequitinhonha

Unknown

MCNIP 0021

KU134855 KU134873

KU134891

KU134909 KU134927

Megaleporinus macrocephalus

Cuiaba

Paraguay

ZZ/ZW

MZUSP 118667

KU134856 KU134874

KU134892

KU134910 KU134928

Megaleporinus muyscorum

Magdalena

Magdalena

Unknown

ICNMHN 19074

KU134857 KU134875

KU134893

KU134911 KU134929

Megaleporinus obtusidens

Turvo

Paraná

ZZ/ZW

MZUSP 113982

KU134858 KU134876

KU134894

KU134912 KU134930

Megaleporinus obtusidens

Turvo

Paraná

ZZ/ZW

-

KF568987

KF569030

KF569073

KF569116

Megaleporinus obtusidens

Jacuí

Jacuí

Unknown

MCP 25476

KU134859 KU134877

KU134895

KU134913 KU134931

Megaleporinus obtusidens

Ibicui

Uruguay

ZZ/ZW

MCP 28917

KU134860 KU134878

KU134896

KU134914 KU134932

Megaleporinus piavussu

Turvo

Paraná

ZZ/ZW

MZUSP 113981

KF568991

KF569034

KF569077

KF569120

KF569163

Megaleporinus reinhardti

Curimataí

São Francisco

ZZ/ZW

MCP 44776

KF568992

KF569035

KF569078

KF569121

KF569164

Megaleporinus trifasciatus

Madeira

Amazonas

ZZ/ZW

UFRO-I 4902

KU134864 KU134882

KU134900

KU134918 KU134936

Megaleporinus trifasciatus

Ucayali

Amazonas

ZZ/ZW

MUSM 47351

KU134865 KU134883

KU134901

KU134919 KU134937

Pseudanos trimaculatus

Cautário

Madeira

No

UFRO-I 14970

KF569003

KF569046

KF569089

KF569132

KF569175

Rhytiodus microlepis

Madeira

Madeira

No

UFRO-I 18647

KF569004

KF569047

KF569090

KF569133

KF569176

Rhytiodus lauzannei

Madeira

Madeira

Unknown

UFRO-I 6840

KF569005

KF569048

KF569091

KF569134

KF569177

Schizodon borellii

Cuiaba

Paraguay

No

MZUSP – 113990

KF569006

KF569049

KF569092

KF569135

KF569178

Schizodon fasciatus

Guaporé

Madeira

No

MZUSP 113989

KF569007

KF569050

KF569093

KF569136

KF569179

KF569170

KF569159

43

Schizodon intermedius

Tiete

Paraná

No

MZUSP – 113995

KF569008

KF569051

KF569094

KF569137

KF569180

Schizodon knerii

Urucuia

São Francisco

No

-

KF569009

KF569052

KF569095

KF569138

KF569181

Schizodon vittatus

Tocantins

Tocantins

No

-

KF569010

KF569053

KF569096

KF569139

KF569182

Caenotropus labyrinthicus

Araguaia

Tocantins

No

GEPEMA – 5230

KF568971

KF569014

KF569057

KF569100

KF569143

Curimata cyprinoides

Araguaia

Tocantins

No

GEPEMA – 4759

KF568972

KF569015

KF569058

KF569101

KF569144

Prochilodus nigricans

Araguaia

Tocantins

No

GEPEMA – 4679

KF569002

KF569045

KF569088

KF569131

KF569174

Anodus orinocensis

Araguaia

Tocantins

No

GEPEMA – 5216

KF568969

KF569012

KF569055

KF569098

KF569141

GEPEMA: Grupo de Estudos de Peixes do Médio Araguaia, UFMT. MCNIP: Museu de Ciências Naturais - PUC Minas. MCP: Museu de Ciências e Tecnologia da PUCRS. MZUSP: Museu de Zoologia da USP. UFRO-I: Universidade Federal de Rondonia. MZUEL: Museu de Zoologia da Universidade Estadual de Londrina. ICNMHN: Instituto de Ciencias Naturales, Museo de Historia Natural, Facultad de Ciencias, Universidad Nacional de Colombia. 1A different ZZ/ZW sex chromosome system described by Venere et al. (2004).

44

Table 2. Meristic and morphometric data for all extant species of Megaleporinus with comments on known distribution (DIS). EMS = estimated maximum size (reference), DET = number of dentary teeth (n), LLS = number of lateral-line scales (n), REF = main reference, PMT = number of premaxillary teeth (n), RMS = recorded maximum size (voucher), SAL= number of scale rows from lateral line to dorsal-fin origin (n), SBL = number of scale rows from lateral line to pelvic-fin origin (n), SCP = number of scale rows around caudal peduncle (n), SPN = number of supraneural (n), VER = vertebral count (n). RMS

EMS

PMT

DET

LLS

SAL

SBL

SCP

Megaleporinus brinco

187.2 mm SL (UFBA 4843)

300 mm SL

3 (20)

3 (20)

36 to 38 (29)

4 (29)

4 (29)

Megaleporinus conirostris

375 mm SL (MZUSP 500 mm SL 47891)

3 (100) 3 (100)

39 to 41 (132)

5 or 6 (132)

Megaleporinus elongatus

384 mm SL (MCZ 20422) 207.9 mm SL (NUP 15579)

500 mm SL

3 (20)

3 (20)

250 mm SL

3 (20)

4 (20)

Megaleporinus macrocephalus

400 mm SL (MZUSP 600 mm SL 3 (15) 14838) (Britski et al., 2007)

Megaleporinus muyscorum

260 mm SL (BMNH 1947.7.1.133-135)

400 mm SL (Mojica, 2012)

Megaleporinus obtusidens

Megaleporinus piavussu

Megaleporinus garmani

Megaleporinus reinhardti

SPN

VER

DIS

REF

12 (29) 5 (2)

35 or 36 (3)

Contas

Birindelli et al. (2013)

4 or 5 (132)

16 (132)

37 (1)

36 or 37 4 or 5 (31) (31) 36 to 38 5 (20) (20)

4 (31)

12 (31)

4 or 5 (20)

12 (20) 4 (1)

Paraíba, Doce, Mucuri, São Mateus, Jucuruçu Jequitinhonha, Pardo Jequitinhonha, Pardo

3 (15)

42 or 43 6 (15) (15)

5 or 6 (15)

16 (15) 4 (3)

38 or 39 (3)

Paraguay

Garavello & Britski (1988)

3 (14)

3 (14)

41 to 43 (16)

5 (16)

5 or 6 (16)

16 (16) 5 (1)

39 (2)

Magdalena, Sinú, Atrato, Orinoco

Garavello (2000)

510 mm SL (MZUSP 600 mm SL 43090)

3 (50)

3 (50)

41 to 44 (194)

6 or 7 (199)

5 to 7 (199)

16 (36) 5 (3)

39 (3)

Britski et al. (2012)

380.2 mm SL (MZUSP 48779) 242.5 mm SL (MZUSP 39375)

500 mm SL

3 (20)

3 (20) 3 (20)

5 or 6 (36) 5 or 6 (20)

39 (2)

3 (20)

5 to 7 (36) 5 or 6 (20)

16 (36) 5 (3)

300 mm SL

39 to 41 (35) 37 to 39 (20)

Paraná, Paraguay, Uruguay, Jacuí, São Francisco, Parnaíba upper Paraná

16 (20) 4 (2)

35 (3)

5 (1)

34 or 35 (2) 35 (1)

São Francisco, Itapicuru

Britski et al. (2012)

Britski et al. (2012) Britski et al. (1988)

45

Megaleporinus trifasciatus

260 mm SL (CAS 70569)

500 mm SL (Santos & Feitosa, 2013)

3 (20)

3 (20)

41 to 43 (20)

5 or 6 (20)

5 or 6 (20)

16 (20) 4 (3)

38 or 39 (5)

Amazonas

46

Figure captions Figure 1. Alcohol-preserved specimens of all extant species of Megaleporinus. M. brinco, UFBA 4843, 174.9 mm SL, paratype, de Contas river; M. conirostris, NMW 68697, 171.8 mm SL, syntype, Paraíba do Sul river; M. elongatus, MZUSP 87883, 150.5 mm SL, Pardo river; M. garmani, MZUSP 93768, 94.5 mm SL, Jequitinhonha river; M. macrocephalus, MZUSP 14513, 156.0 mm SL, paratype, Paraguay basin; M. muyscorum, CAS 70622, 125.2 mm SL, Atrato river; M. obtusidens, MZUSP 48820, 278.2 mm SL, Paraguay basin; M. piavussu, MZUSP 48779, 243.0 mm SL, paratype, upper Paraná basin; M. reinhardti, MCP 17152, 175.0 mm SL, São Francisco river; M. trifasciatus, MZUSP 6702, 182.0 mm SL, Negro river, Amazon basin. Scale bars equal 1 cm.

Figure 2. Jaws of various Anostomidae species showing the distinct morphology and arrangement of teeth and jaw bones. Images of Megaleporinus elongatus and M. trifasciatus obtained as photographs; other images obtained through SEM. Scale bars equal 1 mm.

Figure 3. Photograph of a dry-skeleton specimen of Megaleporinus macrocephalus, MZUSP 89501, 345.0 mm SL, showing the elongated first rib of a mature male (arrow). Jaws, pectoral girdle, suspensorium, infraorbital series, and opercular series of left side removed. Scale bar equals 1 cm.

Figure 4. Map of South America showing the approximate distribution of all valid species of Megaleporinus.

47

Figure 5. Bayesian species tree showing phylogenetic relationships among species of Megaleporinus (bold branches) and related anostomids. Trees were generated using five genes (approx. 4900 bp). The numbers on the nodes are bootstrap values for separate maximum parsimony and maximum likelihood analyses, and posterior probability for the Bayesian species tree. The scale bar indicates nucleotide substitutions per site. Only values for nodes supported by at least two analyses are shown.

Figure 6. Biogeographical reconstruction of ancestral areas of Megaleporinus species using Statistical Dispersal-Vicariance Analysis (S-DIVA) and divergence times estimated in BEAST. The circled numbers represent the cladogenetic event used in the Discussion. Node values are divergence times in millions of years. Only divergences in supported nodes are shown. The bars represent confidence intervals of 95%. SF = São Francisco.

Figure 7. Ancestral state reconstruction using maximum parsimony for presence and absence of the ZW sex chromosome system.

48

77/96/0.99

53/82/0.99

Megaleporinus reinhardti (São Francisco) 100/100/1 M. cf. reinhardti (Itapicuru) M. garmani (Jequitinhonha) M. brinco (Contas) M. elongatus (Jequitinhonha) M. cf. obtusidens São Francisco (São Francisco) 100/100/1 M. obtusidens (Paraná, Uruguay, Jacuí) 69/78/ M. cf. obtusidens Paraguay (Paraguay) 0.93 M. piavussu (upper Paraná) M. conirostris (Paraíba do Sul) 100/100/1 96/81/0.99 M. cf. conirostris (Doce) M. cf. muyscorum (Orinoco) 100/100/1 M. macrocephalus (Paraguay) 97/100/1 99/100/1 M. trifasciatus (upper Amazon) 99/100/1 91/ M. cf. trifasciatus (Tocantins) 100/1 M. muyscorum (Magdalena) Abramites hypselonotus 92/100/1 L. striatus Uruguay 100/100/1 52/85/0.97 L. striatus Paraguay L. geminis Hypomasticus pachycheilus L. affinis 100/100/1 L. fasciatus 97/98/0.99 76/95/0.99 L. bleheri L. tigrinus 100/100/1 82/84/0.84 L. octomaculatus Paraguay 69/94/1 100/100/1 L. octomaculatus Amazonas 93/97/1 L. unitaeniatus 91/100/1 L. desmotes 100/100/1 L. jatuncochi L. cf. parae 68/100/1 100/100/1 L. venerei 100/100/1 L. lacustris Paraná 100/100/1 86/97/0.99L. lacustris Paraguay Leporinus taeniatus 100/100/1 Leporinus frideri Paraguay 100/95/0.97 Leporinus frideri Paraná 100/100/1 100/100/1 Leporinus piau Laemolyta fernandezi Rhytiodus lauzannei 99/100/1 100/100/1 R. microlepis S. borelli 100/100/0.99 100/100/1 S. intermedius 100/100/1 S. fasciatus S. vittatus 100/100/1 100/100/1 S. knerii Hypomasticus megalepis Hypomasticus mormyrops 98/100/1 100/100/1 Leporinus copelandii 100/100/1 Leporinus steindachneri 100/100/1 Leporinus sp. Anostomus ternetzi 100/100/1 Pseudanos trimaculatus 94/100/0.99 Leporellus vittatus Caenotropus labyrinticus Curimata cyprinoides Prochilodus nigricans Anodus orinocensis 8.0

M. cf. reinhardti (Itapicuru)

1. Magdalena/Proto-Amazonas (1.00) 2. Proto-Amazonas/São Francisco (0.82) 3. Orinoco/Amazonas (1.00) 4. Tocantins/Amazonas (1.00) 5. Amazonas/Paraguai (1.00) 6. São Francisco/Doce (0.73) 7. Doce/Paraíba do Sul (0.92) 8. São Francisco (0.6) 9. São Francisco/Paraná (0,35) São Francisco/Paraná/Jequitinhonha (0.31) 10. São Francisco/Itapicuru (1.00)

10 0.28

M. reinhardti (São Francisco)

4.48

M. garmani (Jequitinhonha)

6.01

M. elongatus (Jequitinhonha)

6.56

8

2.49 M. cf. obtusidens São Francisco (SF) 9 2.83 6

M. obtusidens (Paraná)

1.85

7.67

2.23

Eastern Brazilian Shield

M. brinco (Contas)

M. cf. obtsidens Paraguay (Paraguay) M. piavussu (Paraná) M. cf. conirostris (Doce)

2 9.27

7 1.8

M. conirostris (Paraíba do Sul)

1

M. macrocephalus (Paraguay)

3 5.97

12

5 4 3.98

2.22 M. trifasciatus (Upper Amazonas) M. cf. trifasciatus (Tocantins) M. muyscorum (Magdalena)

10

0 x106 Ma

proto-Amazonas-Orinoco

M. cf. muyscorum (Orinoco)

Node 2

Node 1

ZW Sex Chromosome system No Sex Chromosome No-related ZW Sex Chromosomes (Venere et al., 2004) Cytogenically unknown

Abramites hypselonotus Megaleporinus reinhardti M. cf. reinhardti M. garmani M. brinco M. elongatus M. cf. obtusindes São Francisco M. obtusidens M. cf. obtusidens Paraguay M. piavussu M. conirostris M. cf. conirostris M. cf. muyscorum M. macrocephalus M. trifasciatus M. cf. trifasciatus M. muyscorum L. striatus Uruguay L. striatus Paguay Leporinus geminis Hypomasticus pachycheilus Leporinus affinis L. fasciatus L. bleheri L. tigrinus L. octomaculatus Amazonas L. octomaculatus Paraguay L. unitaeniatus L. desmotes L. jatuncochi L. cf. parae L. venerei L. lacustris Paraná L. lacustris Paraguay L. taeniatus L. friderici Paraná L. friderici Paraguay L. piau Laemolyta fernandezi Rhytiodus lauzannei R. microlepis Schizodon borelli S. intermedius S. fasciatus S. vittatus S. knerii Hypomasticus megalepis H. mormyrops Leporinus copelandii L. steindachneri Leporinus sp. Anostomus ternetzi Pseudanos trimaculatus Leporellus vittatus Caenotropus labyrinticus Curimata cyprinoides Prochilodus nigricans Anodus orinocensis

Graphical Abstract

Megaleporinus

M. cf. reinhardti (Itapicuru) 10 0.28

M. reinhardti (São Francisco)

4.48

M. garmani (Jequitinhonha)

6.01

M. elongatus (Jequitinhonha)

6.56

8 Abramites hypselonotus L. striatus Uruguay L. striatus Paraguay L. geminis Hypomasticus pachycheilus

2.49 M. cf. obtusidens São Francisco (SF) 9 2.83 6

M. obtusidens (Paraná)

1.85

7.67

2.23

Eastern Brazilian Shield

M. brinco (Contas)

M. cf. obtsidens Paraguay (Paraguay)

Leporinus

M. piavussu (Paraná) M. cf. conirostris (Doce) 2 9.27

7 1.8

M. conirostris (Paraíba do Sul)

Rhytiodus Schizodon Hypomasticus megalepis Hypomasticus mormyrops Leporinus copelandii Leporinus steindachneri Leporinus sp. Anostomus ternetzi Pseudanos trimaculatus Leporellus vittatus Caenotropus labyrinticus Curimata cyprinoides Prochilodus nigricans Anodus orinocensis

1

M. macrocephalus (Paraguay)

3 5.97

12

5 4 3.98

2.22 M. trifasciatus (Upper Amazonas) M. cf. trifasciatus (Tocantins) M. muyscorum (Magdalena)

10

0 x106 Ma

proto-Amazonas-Orinoco

M. cf. muyscorum (Orinoco)

Laemolyta fernandezi

Highlights     

Megaleporinus is described to include ten valid extant species. Megaleporinus is supported by morphological, cytogenetical and molecular data. At least four undescribed, cryptic species, were identified. Synapomorphies include a ZW sex chromosome system. Paleogeographic and hydrogeological events have likely driven Megaleporinus speciation.

49