BackgroundThe megadiverse Neotropical freshwater ichthyofauna is the richest in the world with approximately 6,000 recognized species. Interestingly, they are distributed among only 17 orders, and almost 80% of them belong to only three orders: Characiformes, Siluriformes and Perciformes. Moreover, evidence based on molecular data has shown that most of the diversification of the Neotropical ichthyofauna occurred recently. These characteristics make the taxonomy and identification of this fauna a great challenge, even when using molecular approaches. In this context, the present study aimed to test the effectiveness of the barcoding methodology (COI gene) to identify the mega diverse freshwater fish fauna from the Neotropical region. For this purpose, 254 species of fishes were analyzed from the Upper Parana River basin, an area representative of the larger Neotropical region.ResultsOf the 254 species analyzed, 252 were correctly identified by their barcode sequences (99.2%). The main K2P intra- and inter-specific genetic divergence values (0.3% and 6.8%, respectively) were relatively low compared with similar values reported in the literature, reflecting the higher number of closely related species belonging to a few higher taxa and their recent radiation. Moreover, for 84 pairs of species that showed low levels of genetic divergence (<2%), application of a complementary character-based nucleotide diagnostic approach proved useful in discriminating them. Additionally, 14 species displayed high intra-specific genetic divergence (>2%), pointing to at least 23 strong candidates for new species.ConclusionsOur study is the first to examine a large number of freshwater fish species from the Neotropical area, including a large number of closely related species. The results confirmed the efficacy of the barcoding methodology to identify a recently radiated, megadiverse fauna, discriminating 99.2% of the analyzed species. The power of the barcode sequences to identify species, even with low interspecific divergence, gives us an idea of the distribution of inter-specific genetic divergence in these megadiverse fauna. The results also revealed hidden genetic divergences suggestive of reproductive isolation and putative cryptic speciation in some species (23 candidates for new species). Finally, our study constituted an important contribution to the international Barcoding of Life (iBOL.org) project, providing barcode sequences for use in identification of these species by experts and non-experts, and allowing them to be available for use in other applications.

Background and aims: Considering the promising use of DNA barcoding for species identification, the importance of the freshwater fish fauna of the Paraíba do Sul River Basin, and its advanced stage of degradation, the present study evaluated the effectiveness of DNA barcoding to identify the fish species in this basin. Materials and methods: A total of 295 specimens representing 58 species belonging to 40 genera, 17 families, and 5 orders were sequenced. Results: The DNA barcodes discriminated all species analyzed without ambiguity. The results showed a pronounced difference between conspecific and congeneric pair-wise sequence comparisons, demonstrating the existence of a "barcode gap" for the species analyzed. The nearest-neighbor distance analysis showed only three cases with Kimura two-parameter values lower than a 2% divergence threshold. However, the patterns of divergence observed in each case remained sufficient to discriminate each species, revealing the accuracy of DNA barcoding even cases with relatively low genetic divergence. At the other extreme, three species displayed high genetic sequence divergence among conspecifics. For two cases, Characidium alipioi and Geophagus proximus, barcoding proved effective at flagging possible new species. For another case, Astyanax bimaculatus, the use of DNA barcoding of the comparison of shared freshwater fish fauna between different basins revealed itself as highly useful in disclosing that the previously identified A. bimaculatus "cluster A" probably represents the species Astyanax altiparanae. Conclusion: The present study is among the first to assess the efficiency of barcoding for the Brazilian freshwater fishes. The results demonstrate the utility of barcoding to identify the fauna from this basin, contribute to an enhanced understanding of the differentiation among species, and to help flag the presence of overlooked species.

–  Pseudoplatystoma corruscans is distributed widely in the La Plata Basin in Brazil, where it is a very important species because of its high commercial value and ecological role as a voracious predator. This species undertakes long migrations during the dry and rainy seasons to feed and reproduce, respectively. In this study, we analysed seven microsatellite loci to test the hypotheses of the existence of a single panmictic population of P. corruscans in the La Plata Basin. All microsatellites analysed were highly polymorphic with the number of alleles per locus ranging from seven (Pcor28) to 30 (Pcor10). Across all samples, 107 alleles were detected. Thirty alleles were private, i.e., found in only one sample. Highly significant genetic differentiation was observed among samples in three of the four analyses performed: FST (from 0.03435 to 0.16349, P < 0.05), Analysis of molecular variance (amova)FST = 8.25% (P < 0.0001) and amovaRST = 9.72% (P < 0.0001). A microsatellite multilocus Bayesian assignment test with the programme structure confirmed the division of the fishes into six groups largely concurrent with main branches on a population neighbour‐joining tree. The obtained results rejected the panmixia hypothesis. On the other hand, the documented long‐distance movements of P. corruscans and the geographical scale of genetic differentiation found in this study indicate a strong tendency for fish of this species to utilise their natal nursery regions for reproduction, which represents the first demonstrated example of homing in a freshwater catfish in South America.

Background and aims. We analyzed a small and wide geographically distributed Neotropical freshwater fish, the Piabina argentea from the Upper Paraná Basin, to check the hypothesis that this species is composed of more than one biological unit, since it has a limited dispersion, through the DNA barcode technique. Materials and methods. Partial mitochondrial COI and CytB gene sequences were obtained for 58 specimens drawn from 13 localities. Results. Phylogenetic analysis revealed six major clusters of P. argentea. Kimura-two-parameter (K2P) genetic divergences among these six P. argentea clusters ranged from 2 to 5.6% and from 2.3 to 5.4% for COI and CytB genes, respectively, and these values were on average approximately nine times greater than intra-cluster K2P divergences. The fixation index (F ST ) among clusters showed very high values and the haplotype network analysis displayed seven unconnected units. Conclusion. These results reinforce the hypothesis that the widely distributed P. argentea species concept as currently conceived actually represents more than one species (possibly six). These results demonstrate the efficacy of DNA barcoding for the discovery of hidden diversity in Neotropical freshwater fishes, and we conclude that barcoding is a useful tool for alpha taxonomy.

Brachyplatystoma platynemum is a catfish species widely distributed in the Amazon basin. Despite being considered of little commercial interest, the decline in other fish populations has contributed to the increase in the catches of this species. The structure, population genetic variability, and evolutionary process that have driven the diversification of this species are presently unknown. Considering that, in order to better understand the genetic structure of this species, we analyzed individuals from seven locations of the Amazon basin using eight molecular markers: control region and cytochrome b mtDNA sequences, and a set of six nuclear microsatellite loci. The results show high levels of haplotype diversity and point to the occurrence of two structured populations (Amazon River and the Madeira River) with high values for FST. Divergence time estimates based on mtDNA indicated that these populations diverged about 1.0 Mya (0.2–2.5 Mya 95% HPD) using cytochrome b and 1.4 Mya (0.2–2.7 Mya 95% HPD) using control region. During that time, the influence of climate changes and hydrological events such as sea level oscillations and drainage isolation as a result of geological processes in the Pleistocene may have contributed to the current structure of B. platynemum populations, as well as of differences in water chemistry in Madeira River. The strong genetic structure and the time of genetic divergence estimated for the groups may indicate the existence of strong structure populations of B. platynemum in the Amazon basin.

Anthropogenic impacts are an increasing threat to the diversity of fishes, especially in areas around large urban centres, and many effective conservation actions depend on accurate species identification. Considering the utility of DNA barcoding as a global system for species identification and discovery, this study aims to assemble a DNA barcode reference sequence library for marine fishes from the coastal region of São Paulo State, Brazil. The standard 652 bp 'barcode' fragment of the cytochrome c oxidase subunit I (COI) gene was PCR amplified and bidirectionally sequenced from 678 individuals belonging to 135 species. A neighbour-joining analysis revealed that this approach can unambiguously discriminate 97% of the species surveyed. Most species exhibited low intraspecific genetic distances (0.31%), about 43-fold less than the distance among species within a genus. Four species showed higher intraspecific divergences ranging from 2.2% to 7.6%, suggesting overlooked diversity. Notably, just one species-pair exhibited barcode divergences of <1%. This library is a first step to better know the molecular diversity of marine fish species from São Paulo, providing a basis for further studies of this fauna - extending the ability to identify these species from all life stages and even fragmentary remains, setting the stage for a better understanding of interactions among species, calibrating the estimations about species composition and richness in an ecosystem, and providing tools for authenticating bioproducts and monitoring illegal species exploitation.

A total of five polymorphic microsatellites loci from Pseudoplatystoma corruscans were isolated and characterized. A population survey involving 43 specimens resolved a large number of alleles (range seven to eight among loci) and high observed heterozygosity (0.500–0.615), indicating its usefulness in population genetics studies. Cross‐species amplification was successful in four other Pimelodidae species.

Accurate species delimitation is crucial for studies of phylogeny, phylogeography, ecology, conservation and biogeography. The limits of species and genera in the Characidae family are controversial due to its uncertain phylogenetic relationships, high level of morphological homoplasy and the use of ambiguous morphological characters for descriptions. Here we establish species boundaries for Bryconamericus , Hemibrycon , Knodus and Eretmobrycon (Stevardiinae: Characidae), previously diagnosed with morphology, using three different barcoding approaches (GMYC, PTP, ABGD). Results revealed that species delimitation was successful by the use of a single-gene approach and by following a workflow in the context of integrative taxonomy, making evident problems and mistakes in the cataloging of Characidae species. Hence, it was possible to infer boundaries at genus level for clusters in the trees (GMYC and PTP) and automatic partitions (ABGD) which were consistent with some of recent taxonomic changes proposed in Characidae. We found that discordance cases between methods were linked to limitations of the methods and associated to putative species cluster closely related, some historically problematic in their diagnosis and identification. Furthermore, we suggested taxonomic changes and possibly new species, revealing a high degree of hidden diversity. Finally, we propose a workflow as a fast, accurate and objective way to delimit species from mitochondrial DNA sequences and to help clarify the classification of this group.