This study is primarily directed to the most poorly known species of the genus Trichomycterus, comprising five nominal species (T. florensis, T. immaculatus, T. nigricans, T. paquequerensis and T. santaeritae) endemic to south-eastern Brazil. One of them, T. nigricans, is the type species of the genus, involved in taxonomic problems for over 150 years. A detailed historical review, accompanied by examination of type specimens and recent collections, revealed that the correct type locality of T. nigricans is in the vicinity of Rio de Janeiro, not Santa Catarina as commonly appears in the literature; specimens previously misidentified as T. nigricans from Santa Catarina belong to a possibly undescribed species of the genus Cambeva; T. paquequerensis is a synonym of T. immaculatus, and T. florensis is a synonym of T. santaeritae; and the hypothesis that T. santaeritae is closely related to the Amazon Sarcoglanidinae is refuted. The three valid species are redescribed. These species are members of a clade also including T. caipora that is highly supported by molecular data, diagnosed by a pronounced posterior maxillary process and caudal fin emarginate at least in larger specimens. A subclade comprising T. caipora, T. nigricans and T. santaeritae is diagnosed by a long maxilla and a bifid anterior extremity of hypobranchial 3.

The Jenynsia lineata species complex comprises J. lineata from Montevideo, Uruguay and Jenynsia multidentata, from coastal basins of Brazil, Uruguay and Argentina up to 1200 m a.s.l. Taxonomic divisions within this group were tested using three different species delimitation methods, which found the two existing names to be synonyms and revealed a new cryptic species. Jenynsia darwini sp. nov. is distinguished from all congeners by having a unique combination of character states, including the shape of the dorsal postcleithrum (three times higher than wide v. less than two times higher than wide) and female colour pattern in the half of the caudal peduncle with rows of chromatophores segmented in unaligned spots (v. aligned spots forming lines). The new species also differs from J. lineata by having 26 nucleotide substitutions in the mitochondrial gene cytochrome c oxidase I (coI). Phylogenetic analysis of the genus based on morphological characters proposed by previous studies corroborates monophyly of the subgenera Plesiojenynsia and Jenynsia, with the new species being allocated to the subgenus Jenynsia as the sister group of J. lineata.

The high diversity of Neotropical fishes has been attributed to major South American palaeogeographic events, such as Andean uplift, rise of the Isthmus of Panama and marine transgressions. However, the unavailability of temporal information about evolution and diversification of some fish groups prevents the establishment of robust hypotheses about correlations between species diversification and proposed palaeogeographical events. One example is the Anablepidae, a family of teleost fishes found mostly in coastal habitats of Central and South America, but also in some inner river basins of South America. Historical aspects of the distribution patterns of the Anablepidae were never analysed and no accurate estimation of time of its origin and diversification is presently available. A multi-gene analysis was performed to estimate Anablepidae phylogenetic position, age and biogeography, comprising seven nuclear genes. The suborder Cyprinodontoidei was recovered in three major clades, one comprising all the Old World Cyprinodontoidei and two comprising New World lineages. Anablepidae was recovered as the sister group of the New World Poeciliidae, with the Amazonian genus Fluviphylax as their sister group. The ages found for the origin and diversification of Cyprinodontiformes were congruent with the pattern recorded for other vertebrate groups, with an origin anterior to the Cretaceous-Paleogene (K-Pg) transition and diversification during the Paleogene. The age estimated for the split between the Atlantic and Pacific lineages of Anableps was congruent with the rise of Panamanian Isthmus. The results suggest Miocene marine transgressions as determinant to the current distribution of Jenynsia.

Psammocambeva, a subgenus of Trichomycterus s.s., includes a clade endemic to south-eastern Brazil, the Psammocambeva alpha-clade (PAC), containing species with similar colour pattern and fin morphology, making difficult their identification without accurate examination. The greatest diversity of PAC species occurs in the Rio Paraíba do Sul basin area (RPSA), situated within the Atlantic Forest, one of the most important and endangered biodiversity centres in the world. Herein, we: perform a multigene phylogeny focusing on species of PAC; revise morphological characters diagnosing species of PAC from the RPSA, with special attention to those equivocally synonymised in a recent study; describe two new species, and provide a key for species identification. Molecular and morphological evidence supported the recognition of eight valid species belonging to four species complexes. Data indicated that T. auroguttatus, T. travassosi, and T. longibarbatus are valid species. Finally, we discuss the negative impacts of underestimating species diversity in regions under the intense process of natural habitat loss, concluding that integrative approaches are important tools to estimate species diversity, but they should include a range of morphological characters informative to delineate and diagnose groups and their respective species, in association with phylogenies generated by robust molecular datasets.