The Neotropical region harbors most of the known fish diversity, with more than 5,200 valid species, however, there is additional evidence that many of them are yet to be described (Fricke et al., 2021). Several reasons are suggested to be related to this huge diversity, and among them, the evolution of reproductive barriers reducing gene flow plays a key role in creating reproductive incompatibilities between species (Mayr, 1963). The evolution of strong reproductive barriers is essential for coexistence of species, especially for sympatric ones (those whose geographical distribution overlaps), even if the isolation in sympatry has preceded the overlap of species that currently coexist in the same region, in a secondary contact (Mayr, 1999; Coyne and Orr, 2004).

Among animals, most cases of post-zygotic isolation are caused by genetic incompatibilities, among which chromosomal rearrangements including specific sex chromosome-systems are of fundamental importance (Bateson, 1909; Dobzhansky, 1933; Muller, 1942; Coyne and Orr, 2004). Such chromosomal rearrangements can prevent introgression and reduce gene flow by suppressing recombination (Machado et al., 2007; Yannic et al., 2009; McGaugh and Noor, 2012; Ostberg, et al., 2013; Presgraves, 2018; Potter, et al., 2021). However, the contribution of these processes for biodiversity in Neotropical fish species is still poorly understood.

The Lebiasinidae family is distributed throughout Central and South America, with 75 species divided into two subfamilies: Lebiasininae and Pyrrhulininae (Weitzman and Weitzman, 2003; Froese and Pauly, 2014; Fricke et al., 2021). The subfamily Pyrrhulininae is the most diverse, including the genera Derhamia, Nannostomus, Copella, Copeina, and Pyrrhulina (Fricke et al., 2021). Recent cytogenetic and molecular data have added significant contributions to understanding evolutionary relationships of these genera (Moraes et al., 2017, 2019, 2021; Sassi et al., 2019; Toma et al., 2019; Sember et al., 2020). Particularly, concerning Pyrrhulina, its diploid number (2n) ranges from 32 to 42 chromosomes, with Pyrrhulina aff. australis, P. australis (Eigenmann and Kennedy, 1903), Pyrrhulina aff. marilynae and Pyrrhulina sp. present 2n = 40 chromosomes, whereas P. brevis (Steindachner, 1876), P. obermulleri (Allen, Meyers and Myers, 1926), and Pyrrhulina cf. laeta have 2n = 42 chromosomes, both in males and females. Differently from the others, P. semifasciata (Steindachner, 1876) has divergent 2n among sexes (2n = ♀42 and ♂41) due to its exclusive multiple ♀X₁X₁X₂X₂/♂X₁X₂Y sex chromosome system (Moraes et al., 2017; Moraes et al., 2019). In addition, P. marilynae (Netto-Ferreira and Marinho, 2013) also differs from its congeneric species having 2n = 32 chromosomes, due to a series of chromosomal fusions (Moraes et al., 2021).

The development and improvement of large-scale genotyping-by-sequencing (GBS) procedures allowed high-resolution analysis using Single Nucleotide Polymorphisms (SNPs) for genetic diversity studies in non-model organisms (Steane et al., 2011; Yang et al., 2013; Sánchez-Sevilla et al., 2015; Alkimin et al., 2018; Souza et al., 2019; Oliveira et al., 2020; Sassi et al., 2021). Combining such genomic and cytogenetic data for pairs of species/populations in which there are variations related to geographic distribution, analysis of chromosomal rearrangements, and sex chromosome systems provide a critical tool to understanding the role of these events to generate biodiversity. The present study applies cytogenetic and genomic approaches in five Pyrrhulina species with distinct geographic distributions. Here, we aimed to assess the genetic diversity in five Pyrrhulina species by combining population genomic with previously obtained cytogenetic data. Our results highlighted that genomic diversity is consistent with the chromosomal features and provided hypothesis on the potential role of chromosomal rearrangements, especially those involving the sex chromosomes, in fostering genetic differentiation.

Here the first genomic evaluation of five Pyrrhulina species was performed. Our results highlight that genetic diversity among the analyzed Pyrrhulina species is consistent with their chromosomal features. In addition, evidence was found that the occurrence of chromosomal rearrangements including variation in sex chromosome systems might have contributed to increase differentiation.

Overall, the genetic diversity indexes (π; θ _W ) (Table 2) were low, being the highest ones observed in P. obermulleri and P. semifasciata and the lowest in P. marilynae followed by P. brevis. Low diversity values are somewhat expected due to the nature of our sequencing technique (see methods) and similar results were found in populations from Hoplias malabaricus under the same sequencing technique (Souza et al. submitted). According to Dxy and Da values (Figure 2), P. semifasciata and P. marilynae are very distinct from each other, presenting the highest observed values (0.01770 and 0.0158). They are also distinct morphologically (Netto-Ferreira and Marinho, 2013) and present non-overlapping distributions in the Amazon basin. Pyrrhulina semifasciata occur in the Purus, Juruá, Negro, Branco rivers, Amazonas main channel (Dagosta and De Pinna, 2021), and Madeira River while P. marilynae is restricted to the upper portions of Tapajós and Xingu (Netto-Ferreira & Marinho, 2013) (Figure 1). Similarly, values of Dxy and Da between P. semifasciata and P. australis are also very high (0.0171 and 0.0144), indicating their genetic distinctiveness. Furthermore, their distribution does not overlap (Figure 1) and they are also quite distinct morphologically and probably distantly related species, according to a morphology-based phylogenetic analysis (Marinho, 2014). This results are also in agreement with the species tree, as P. marilynae and P. australis were recovered as sister species in a distinct clade (Figure 1). However, it is important to emphasize that a more comprehensive species-level sampling is still necessary before claiming sister species relationships, especially in taxa that are apparently divergent such as P. brevis and P. obermulleri.

Pyrrhulina semifasciata, the only species in the family up to now analyzed with heteromorphic sex chromosomes, presents high genetic differentiation values when compared to all other species. This could suggest that the presence of such multiple sex system promoted genetic differentiation in the species. The association of sex chromosomes and a high genetic differentiation was also evidenced in previous studies for some populations of other species such as the wolf fishes Hoplias malabaricus (Souza et al., submitted) and Erythrinus erythrinus (Souza et al., 2022). In addition, other studies suggest that an event of sex chromosome turnover in plants could have promoted the reduction of gene flow between groups of Rumex hastatulus, which could have contributed to further accumulation of genetic differences (Beaudry et al., 2019). Furthermore, the emergence of a neo-sex chromosome system is suggested as a factor that may have driven the speciation process in sticklebacks (Kitano et al., 2009). Therefore, the emergence of sex neo-chromosomes could result in a rapid evolutionary mechanism fixing genetic variation in different species (Beaundry et al., 2019). Indeed, our results suggest that P. semifasciata, which was collected in syntopy with P. brevis and presents a different chromosomal structure with a multiple sex chromosome system, is also genetically highly divergent from that latter species.

Therefore, we present three main hypotheses for the observed scenario (I) P. brevis and P. semifasciata evolved in allopatry. Later, their distribution overlapped and the current genetic differences are the result of this previous allopatric period and not related to the sex chromosomes emergence (II) these species evolved in allopatry and the sex chromosomes present in P. semifasciata may have acted as an additional barrier to introgression, fostering their genetic differentiation after they became sympatric, and (III) the emergence of a multiple sex chromosome system of the X₁X₂Y-type in P. semifasciata have acted as key factor to reduction of the gene flow and contributed to the speciation process and the significant genetic differentiation observed for this species.

The PCoA result also suggested that P. semifasciata stayed isolated from all other species, while P. brevis clustered with P. obermulleri (Figure 3). Many works highlight the important role of neo-sex chromosomes in preventing introgression, which is agreement with the lack of introgression between sympatric species detected here (Table 4), due to pairing problems associated with meiotic segregation of heterozygous rearrangements (e.g., McKee et al., 1998; Kitano and Peichel, 2012; Beaudry et al., 2019). Similarly, sex chromosomes might promote speciation, as some genes that play a role in reproductive isolation between populations can be accumulated in neo-sex chromosomes (Reinhold, 1998; Lindholm and Breden, 2002; Kitano and Peichel, 2012). Haldane’s rule postulates that hybrids of the heterogametic sex from closely related species are often sterile, thus hybrid incompatibility also may act as a reproductive isolating barrier and contribute to prevent introgression (Haldane, 1922; Coyne and Orr, 2004). In both scenarios, the neo-sex chromosomes system could have contributed to genetic differentiation or speciation. Hence, it may be possible that the chromosomal rearrangements present in P. semifasciata may have had an impact on the diversity of Pyrrhulina species.

In contrast, some species showed low differentiation, indicating that they are more similar genetically. The Dxy and Da values between P. marilynae and P. australis were the lowest ones (0.008 and 0.0066 respectively) and these two species were recovered as sister species in our phylogenetic analysis (Figure 1). Also, both species share with P. vittata a series of unique morphological characters which indicate that they form a monophyletic clade (Netto-Ferreira and Marinho, 2013). Pyrrhulina brevis and P. obermulleri presented low Dxy and Da values as well (0.0121 and 0.0090) in addition to be close to each other in the PCoA analysis (Figure 3) and be recovered in the same clade in the species tree, although not as sister species (Figure 1). All Tajima’s D values were negative except for P. marilynae. Negative results may suggest a recent event of population expansion, or that purifying selection is acting mainly in P. semifasciata and P. brevis. The small positive value presented by P. marilynae could indicate a bottleneck event, but this seems unlikely due to the low value.

The results of PCoA indicated a clear clustering of species sampled in the same geographical region, except for P. semifasciata, which was collected in sympatry with P. brevis and did not cluster with any other species (Figure 3). In fastStructure K = 4 result, only P. australis and P. marilynae were grouped. In both fastStructure and PCoA results, P. australis and P. marilynae were closely related, and such a connection is also presented in the speciestree (Figure 1) and supported by the species tree presented by Marinho (2014).

In addition, AMOVA results suggest that grouping individuals by species maximize the variation between groups, although the groups generated by fastStructure and PCoA have also shown informative values (Table 3). Therefore, AMOVA results show that the clustering of individuals by species best explains the complexity of the data and therefore suggests that the species are well separated. The relationships between river reorganizations and biodiversity are a complex issue, as previously isolated habitats and populations can be merged, increasing dispersal and local diversity, and reducing speciation and extinction rates. They can also separate previously connected environments, leading to increased genetic isolation and speciation, and extinction rates (Tagliacollo et al., 2015; Albert et al., 2018). Therefore, the complex biogeographic history of the region complicates the understanding of Pyrrhulina genetic diversity considering that the current geographic distribution of species may not be the same as in the past and a comprehensive species-level phylogeny is still necessary to infer past geographic distributions.

It is widely known that chromosomal rearrangements might have a role in speciation (Rieseberg, 2001; Navarro and Barton, 2003; Basset et al., 2008). Chromosomal rearrangements have the potential to limit introgression, thus facilitating the origin and maintenance of reproductive isolation through recombination suppression (Faria and Navarro, 2010; Sichová et al., 2015). Here, P. australis and P. marilynae were recovered as sister species in our phylogenetic analysis. This suggests a low genetic difference despite their different chromosomal formulae. Conversely, the species P. semifasciata, the only with a multiple sex chromosome system, showed high differentiation to all other species. This furnishes additional evidence for the role of chromosomal rearrangements, especially those associated with sex chromosomes, as potential important reproductive barriers between these species.

Our results highlighted the distribution of the genetic diversity among the analyzed Pyrrhulina species and contributed to our understanding of their evolutionary history. However, the scarcity of phylogenetic data involving all Pyrrhulina species does not permit us to do a cause-and-effect correlation. Besides, the occurrence of chromosomal rearrangements leading to the presence of the X₁X₂Y sex chromosome system in P. semifasciata could have led to an intensification of the genetic differentiation of this species by preventing introgression with the other species. Future work with a better population and species-level sampling is still needed to bring new insights to 1) species delimitation, 2) infer demographic trends, 2) infer past distributional ranges, and 4) study the tempo and mode of speciation, including chromosomal rearrangements as a mechanism for isolation.