The combination of DNA barcodes and geometric morphometrics is useful to discriminate taxonomically controversial species, providing more precise estimates of biodiversity. Therefore, our goal was to assess the genetic and morphometric diversity in Nematocharax, a controversial monotypic and sexually dimorphic genus of Neotropical fish, based on sequencing of cytochrome c oxidase subunit I (COI) and morphometric analyses in seven populations of N. venustus from coastal rivers in Brazil. The average pairwise intrapopulation divergence in COI ranged from 0 to 2.2%, while the average pairwise interpopulation divergence varied from 0 to 7.5%. The neighbour-joining (NJ) tree resulted in five genetic groups (bootstrap ≥ 97%), which correspond to the five clusters delimited by the BIN System, GMYC, and bPTP, indicating that there might be at least five species (or OTUs) within Nematocharax. Morphometric differences among these genetic lineages were also identified. Apparently, sexual selection, restricted dispersal, and geographic isolation might have acted synergistically to cause the evolutionary split of populations. These data challenge the current view that Nematocharax is a monotypic genus inasmuch as evolutionarily significant units or even distinguished species were identified. Therefore, we recommend that the highly impacted coastal basins in northeastern Brazil should be prioritized in conservation plans.

Understanding the structure of hybrid zones provides valuable insights about species boundaries and speciation, such as the evolution of barriers to gene flow and the strength of selection. In river networks, studying evolutionary processes in hybrid zones can be especially challenging, given the influence of past and current river properties along with biological species‐specific traits. Here, we suggest that a natural hybrid zone between two divergent lineages of the sexually dimorphic Neotropical fish Nematocharax venustus was probably established by secondary contact as a result of a river capture event between the Contas and Pardo river basins. This putative river capture is supported by hydrogeological evidence of elbows of capture, wind gaps and geological faults. The morphological (colour pattern) and genetic (mtDNA and RADseq) variation reveal a clinal transition between parental lineages along the main river, with predominance of F2 hybrids at the centre of the hybrid zone, absence of early generation backcrosses and different levels of hybridization in the tributaries. We highlight that different sources of information are crucial for understanding how the riverscape spatial history influences the connectivity between and within river systems and, consequently, the dynamics of gene flow between freshwater lineages/species.

Characiformes is the most cytogenetically studied group of freshwater Actinopterygii, but karyotypical data of several taxa remain unknown. This is the case of Nematocharax, regarded as a monotypic genus and characterized by marked sexual dimorphism. Therefore, we provide the first cytogenetic report of allopatric populations of Nematocharax venustus based on distinct methods of chromosomal banding and fluorescence in situ hybridization (FISH) with repetitive DNA probes (18S and 5S rDNA). The karyotype macrostructure was conserved in all specimens and populations, independently on sex, since they shared a diploid number (2n) of 50 chromosomes divided into 8m+26sm+14st+2a. The heterochromatin was mainly distributed at pericentromeric regions and base-specific fluorochrome staining revealed a single pair bearing GC-rich sites, coincident with nucleolar organizer regions (NORs). On the other hand, interpopulation variation in both number and position of repetitive sequences was observed, particularly in relation to 5S rDNA. Apparently, the short life cycles and restricted dispersal of small characins, such as N. venustus, might have favored the divergence of repetitive DNA among populations, indicating that this species might encompass populations with distinct evolutionary histories, which has important implications for conservation measures.Characiformes é o grupo de Actinopterygii de água doce mais estudado citogeneticamente, porém dados cariotípicos de vários taxa permanecem desconhecidos. Este é o caso de Nematocharax, considerado um gênero monotípico e caracterizado pelo acentuado dimorfismo sexual. Em vista disso, nós fornecemos a primeira descrição citogenética de populações alopátricas de Nematocharax venustus, baseada em métodos distintos de bandamento cromossômico e hibridação fluorescente in situ (FISH) com sondas de DNA repetitivo (DNAr 18S e 5S). A macroestrutura cariotípica mostrou-se conservada em todos os espécimes e populações, independentemente do sexo, uma vez que compartilharam um número diploide (2n) de 50 cromossomos dividido em 8m+26sm+14st+2a. A heterocromatina distribuiu-se principalmente nas regiões pericentroméricas e a coloração com fluorocromos base-específicos revelou um único par portador de sítios GC-ricos, coincidentes com as regiões organizadoras de nucléolo (RONs). Por outro lado, foi observada uma variação interpopulacional no número e na posição das sequências repetitivas, especialmente em relação ao DNAr 5S. Aparentemente, ciclos de vida curtos e dispersão restrita dos pequenos caracídeos, tal como N. venustus, podem ter favorecido a divergência do DNA repetitivo entre as populações, indicando que essa espécie pode englobar populações com distintas histórias evolutivas, o que tem implicações importantes para medidas de conservação.

An integrative approach based on morphological and multilocus genetic data was used to describe a new species of Nematocharax from the headwaters of the upper Contas River on the Diamantina Plateau, north‐eastern Brazil and to infer the relationships among evolutionary lineages within this fish genus. Multispecies coalescent inference using three mitochondrial and five nuclear loci strongly supports a basal split between Nematocharax venustus and the new species, whose distinctive morphological characters include absence of filamentous rays on pelvic fins of maturing and mature males, reduced anal‐fin lobe length and lower body depth. The unique morphological and genetic traits of the population from the upper Contas River were supported by previous reports based on cytogenetics, DNA barcode and geometric morphometrics, reinforcing the validation of the new species. The conservation status of this new species is discussed.

1. Drainage rearrangements, either headwater captures or coastal paleodrainages formed when sea level was low, are often invoked to explain connectivity and isolation among fish populations. Unravelling these events is crucial for understanding the evolutionary processes that have shaped the genetic diversity and