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Molecular studies have improved our knowledge on the neotropical ichthyofauna. DNA barcoding has successfully been used in fish species identification and in detecting cryptic diversity. Megaleporinus (Anostomidae) is a recently described freshwater fish genus within which taxonomic uncertainties remain. Here we assessed all nominal species of this genus using a DNA barcode approach (Cytochrome Oxidase subunit I) with a broad sampling to generate a reference library, characterize new molecular lineages, and test the hypothesis that some of the nominal species represent species complexes. The analyses identified 16 (ABGD and BIN) to 18 (ABGD, GMYC, and PTP) different molecular operational taxonomic units (MOTUs) within the 10 studied nominal species, indicating cryptic biodiversity and potential candidate species. Only Megaleporinus brinco, Megaleporinus garmani, and Megaleporinus elongatus showed correspondence between nominal species and MOTUs. Within six nominal species, a subdivision in two MOTUs was found, while Megaleporinus obtusidens was divided in three MOTUs, suggesting that DNA barcode is a very useful approach to identify the molecular lineages of Megaleporinus, even in the case of recent divergence (< 0.5 Ma). Our results thus provided molecular findings that can be used along with morphological traits to better define each species, including candidate new species. This is the most complete analysis of DNA barcode in this recently described genus, and considering its economic value, a precise species identification is quite desirable and fundamental for conservation of the whole biodiversity of this fish.

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Monophyly of the genus Leporinus (Characiformes: Anostomidae) was tested by sequencing and analysing a total of 4732 bp, including two mitochondrial [cytochrome oxidase subunit 1 (CO1) and cytochrome b (Cytb)] and three nuclear [myosin heavy chain 6 cardiac muscle alpha (Myh6), recombination activating gene 1 (RAG1) and recombination activating gene 2 (RAG2)] loci for 22 species of Leporinus, or c. 25% of all described species in the genus. Phylogenetic tree analyses (maximum parsimony, maximum likelihood and Bayesian species tree) indicate Leporinus to be paraphyletic, with monophyly being rejected by both Kishino-Hasegawa and Shimodaira-Hasegawa tests. The sequenced species of Leporinus are distributed across five clades that are interleaved among other anostomid genera. Several taxonomic changes are suggested as being necessary to restore monophyly for the group. The clade containing the type species, Leporinus fasciatus, should be considered Leporinus sensu stricto and at least three new genera should be described for other species currently considered part of Leporinus.

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The megadiversity of the neotropical ichthyofauna has been associated to recent diversification processes, reflecting in subtle or lacking morphological differentiation between species, challenging the classical taxonomic identification. Leporinus friderici occurs in several river basins of South America, and its nominal taxonomic validity has been questioned. Its wide distribution within the Brazilian Shield suggests that this species could be genetically structured among the hydrographic basins, despite a sharp morphological similarity. In this study, we performed phylogenetic analyses, based on three nuclear (recombination activating gene 1, RAG1, recombination activating gene 2, RAG2, and myosin heavy chain 6 cardiac muscle alpha gene, Myh6) and two mitochondrial (COI and Cytochrome b, Cytb) markers, in specimens morphologically similar to L. friderici and related species from different hydrographic basins in South America. Our phylogenetic tree identified four well-supported clades, which point out to the existence of taxonomic inconsistencies within this fish group. A clade named L. cf. friderici sensu stricto included eight Molecular Operational Taxonomic Units recently diversified in the Brazilian Shield basins. These results were also confirmed by a single-gene species delimitation analysis. It is suggested that this clade includes a species complex, characterizing taxonomic uncertainties. Another clade recovered only L. friderici from the Suriname rivers, validating this nominal species in its type locality. A third no-named clade, characterized by deeper species divergence, recovered five different nominal species interleaved with other undescribed forms previously also recognized as L. cf. friderici, indicating taxonomic errors. The fourth clade only included L. taeniatus. Our results showed a complex scenario involving the morphotype L. cf. friderici and allowed us to address aspects related to evolutionary diversification of this fish group and historical processes involved with, highlighting the importance of revealing hidden biodiversity for the taxonomy and conservationist action plans of these fish.