Karyotype analysis of three species of Corydoras ( Siluriformes : Callichthyidae ) from southern Brazil : rearranged karyotypes and cytotaxonomy

The genus Corydoras comprises a diversity of species with different diploid numbers. We compared cytogenetic data among Corydoras species from different rivers of the Ponta Grossa Arch region in southern Brazil. Corydoras ehrhardti and C. aff. paleatus have a similar karyotype formula and the same diploid number (2n = 44). Corydoras lacrimostigmata has a higher diploid number, with 2n = 58 chromosomes. Fluorescence in situ hybridization using 5S and 18S ribosomal DNA probes suggests that these ribosomal DNA sequences are involved in chromosomal rearrangements in these Corydoras species. 5S rDNA is a chromosomal marker that is considered to be unique to the species analyzed in this study. Signals of interstitial telomeric sites are seen in a chromosome pair of C. lacrimostigmata, suggesting chromosomal rearrangements via fusions or translocations. This study revealed that C. ehrhardti and C. aff. paleatus have exclusive chromosomal markers associated with chromosome differentiation, which we speculate to prevent genetic introgression.


e160056[2]
To date, only six species of Corydoras, from disparate river basins of southern Brazil, have been investigated cytogenetically.Like the genus as a whole, these species show considerable variation in diploid number (2n = 44, 2n = 46, 2n = 58, and 2n = 66), chromosome structure, and karyotype formula (Oliveira et al., 1993;Artoni et al., 2006;Rocha et al., 2016).Although these chromosome variations make Corydoras a promising model for the study of karyotype evolution, molecular analyses of the genus are relatively lacking.Nucleolar organizer regions (NORs) sites have been mapped in all six southern Brazilian species for which karyotypes are available (Oliveira et al., 1993;Artoni et al., 2006).In situ localization of both 5S and 18S rDNA sequences has been performed in a single species, Corydoras carlae Nijssen & Isbrücker, 1983(Rocha et al., 2016).
Here, we set out to collect and compare cytogenetic and associated molecular data for several species of Corydoras from a geographical region near the Ponta Grossa Arch (Fig. 1).This region includes the headwater boundaries of the Tibagi River (Upper Paraná River), the Ribeira River (Atlantic drainage), the Iguaçu River (Lower Paraná River), and a sub-tributary of the Ivaí River (Upper Paraná River) located near the left bank of the Tibagi headwater.The Ponta Grossa Arch is characterized by elevation changes resulting from adjustments at faults along the eastern margin of the underlying platform.Reactivation of these ancient rifts has led to vertical movements between blocks, promoting the capture of adjacent upland drainages and faunal interchange (Ribeiro, 2006).This is reflected by the occurrence of species typical of lowland Atlantic coastal rivers in upland regions of the upper Paraná, such as Mimagoniates microlepis (Steindachner, 1877), Hyphessobrycon griemi Hoedeman, 1957 (Characidae) and Trichomycterus davisi (Haseman, 1911) (Trichomycteridae).
In this study, we focus on three southern Brazilian Corydoras species: C. ehrhardti Steindachner, 1910, C. lacrimostigmata Tencatt, Britto & Pavanelli, 2014, and C. aff. paleatus (Jenyns, 1842).Corydoras ehrhardti occurs in rivers of the Atlantic basins of the Paraná and Santa Catarina states, including the Iguaçu River and Tibagi River.The geographical range of Corydoras lacrimostigmata is limited to the Ivaí River basin (Tencatt et al., 2014).Corydoras aff.paleatus occurs throughout the Lower Paraná River, where the species was scientifically described, including the Iguaçu River.Corydoras aff.paleatus is also found in the Tibagi River, presumably as a result of former headwater river capture events (Artoni et al., 2006(Artoni et al., , 2009)).Comparison of karyotypes associated with chromosomal markers has become the method of choice for the analysis of structural chromosome reorganization (Oliveira et al., 2016).Here, we combine classical cytogenetic methods with molecular approaches to describe the cytogenetic characteristics of C. ehrhardti, C. lacrimostigmata, and C. aff.paleatus, and to seek evidence for chromosome rearrangements.Classical cytogenetic methods utilized herein include Giemsa staining, C-banding (which exposes the heterochromatic regions), and karyotyping techniques to describe the karyotype arrangement of the species.We also utilize Fluorescence in situ hybridization (FISH), which permits the mapping of molecular markers by localizing DNA probes with a specific sequence within a target sequence (Pinkel et al., 1986).FISH techniques are a powerful tool for the localization of chromosomal rearrangements, especially with site-specific probes that can identify rearranged regions when it is difficult to detect banding patterns in a karyotype (Pucci et al., 2014).

Material and Methods
This study is based on cytogenetic data from 68 specimens of the three species of Corydoras from the region of the Ponta Grossa Arch (Fig. 1).Chromosomes were extracted from kidney cells following air-drying procedures (Bertollo et al., 1978), applying the modifications of Blanco et al. (2012).C-banding was performed using the Sumner (1972) technique.We used three types of repetitive probes as chromosomal markers: (1) an 18S rDNA probe (approximately 1,800 bp), which was synthetized by polymerase chain reaction (PCR) according to Hatanaka, Galetti Júnior.( 2004); (2) a 5S rDNA probe synthetized by PCR according to Martins, Galetti Júnior. (1999); and (3) a probe targeting the universal vertebrate telomere sequence, the minisatellite (TTAGGG)n, synthetized by PCR according to Ijdo et al. (1991).The 18S rDNA probe was labeled with biotin-11-dUTP (Roche Applied Science), while the 5S rDNA and (TTAGGG)n probes were labeled with digoxigenin 11-dUTP by nick translation.
In Corydoras ehrhardti, FISH with 18S and 5S rDNA probes showed a syntenic location on the terminal and proximal positions of the long arm of the metacentric pair 3 (Fig. 3a).In C. aff.paleatus (Iguaçu basin), the 18S rDNA sites were located on the metacentric pair 5 and submetacentric pair 11, both on the terminal position of the long arm, while the 5S rDNA site was revealed to be at the proximal region of the short arm of the submetacentric pair 20 (Fig. 3b).In C. aff.paleatus (Ribeira basin), the 18S rDNA was found at the terminal end of the long arm of the metacentric pair 5, while the 5S rDNA was detected at the 4 e160056 [4] proximal region of the long arm of the submetacentric pair 12 (Fig. 3c).Finally, in C. lacrimostigmata, the 18S rDNA was at the terminal region of the short arm of submetacentric pairs 20 and 25, while the 5S rDNA was revealed to be at the terminal end of the long arm of metacentric pair 4 (Fig. 3d).
The telomeric probe (TTAGGG)n showed uniform signals on the telomeres of all chromosomes of all species analyzed here (data no shown).Additionally, in C. lacrimostigmata, the (TTAGGG)n probe showed small signals of interstitial telomeric sites (ITS) on the submetacentric pair 17, which matches the location of a heterochromatic block (Fig. 3, in detail).

Discussion
We analyzed the karyotype organization of three Corydoras species.We found that C. ehrhardti and both populations of C. aff.paleatus have identical diploid chromosome numbers and karyotype formulae; however, heterochromatin (Fig. 2) and 18S and 5S rDNA FISH analyses (Fig. 3) revealed divergences in the location of chromosome regions.Such divergence has also been observed in both allopatric and sympatric populations of other species (Tab. 1, Fig. 4).In the Tibagi population of C. ehrhardti, the constitutive heterochromatin blocks show a similar distribution to that found in a previous study of the Upper Tibagi (Artoni et al., 2006).Likewise, a similar pattern of heterochromatin has also been reported from an allopatric Atlantic drainage population from the coastal region of the Santa Catarina State, Brazil (Oliveira et al., 1993).Corydoras aff.paleatus populations of the Ribeira and Iguaçu river basins (this study) and populations from the Atlantic drainage of southern Brazil (Oliveira et al., 1993) and the Tibagi River basin (Artoni et al., 2006) present consistent differences in the number and localization of the heterochromatin blocks in their karyotypes.
Corydoras lacrimostigmata (Ivaí River basin) is morphologically similar (albeit with some diagnostic differences) to its congeneric species Corydoras flaveolus Ihering, 1911 (Tencatt et al., 2014).Its chromosomal heterochromatin blocks and the 18S rDNA sites on two chromosomal pairs are similar to those visualized with Ag-NORs in a previous study of C. flaveolus (Oliveira et al., 1992).From a geological viewpoint, C. lacrimostigmata of the Ivaí basin is unlikely to have been involved in the headwater capture events of Ponta Grossa Arch.It might, e160056[5] Tab. 1. Cytogenetic data for Corydoras species from southern Brazil.2n= diploid number; PR = Paraná State; SC = Santa Catarina State; RS = Rio Grande do Sul State; M = metacentric; SM = submetacentric; ST = subtelocentric; A = acrocentric; 18S = number of 18S rDNA sites; 5S = number of 5S rDNA sites; NOR = number of 18S rDNA sites detected by Ag-NOR staining.* The river was not identified in the original paper.however, have differentiated in the headwaters of the Ivaí River, a sub-basin of the Upper Paraná River.In line with this, the karyotype organization of C. lacrimostigmata found here is similar to that described for C. flaveolus, a species located in another sub-basin of the upper Paraná River far from the Ponta Grossa Arch (Oliveira et al., 1992), albeit with some differences in the karyotype formula.
A comparison of the karyotypes of C. ehrhardti, C. aff.paleatus, and C. lacrimostigmata shows some variation in the localization of heterochromatin.These are likely due to population-specific repetitive DNA accumulation in the absence of gene flow, a process previously suggested for other fish taxa (e.g., Vicari et al., 2010;Pucci et al., 2014).FISH analyses revealed a differentiated pattern of both location and number of 18S and 5S rDNA sites among the populations studied here.Corydoras ehrhardti was the only species in which the 18S and 5S ribosomal sites were syntenic.Previous studies of C. ehrhardti from the upper Tibagi River found a single chromosome pair carrying 45S rDNA (Artoni et al., 2006).However, the location of Ag-NORs in allopatric populations of C. ehrhardti from the Atlantic basin (Tab. 1) suggests additional chromosomal pairs bearing 45S rDNA (Oliveira et al., 1993), consistent with the occurrence of rearranged karyotypes.Chromosomal rearrangements, such as inversions, translocations, and transposon-mediated transpositions (Symonová et al., 2013), could explain the diversification of the major rDNA clusters in Corydoras.
A comparison of our observations in C. aff.paleatus with previous studies indicates some differences in the number of 18S rDNA sites.The occurrence of two chromosome pairs bearing 18S rDNA in the Iguaçu population of C. aff.paleatus is similar to what has been found in other populations from the Lower Paraná River (Tab.1), while the Ribeira population of C. aff.paleatus only has a single 18S rDNA site.An analysis of C. aff.paleatus specimens from Lagoa Dourada (Upper Tibagi River) using an 18S rDNA probe revealed signals on three chromosomes (Artoni et al., 2006).Based on the premise of a historical interchange of the ichthyofauna in adjacent basins on the Ponta Grossa Arch, the specimen analyzed by Artoni et al. (2006) may represent an intraspecific hybrid between Tibagi and Ribeira populations of C. aff.paleatus.
In contrast, the 5S rDNA is a chromosomal marker that is specific to the species analyzed in this study.The localization of the 5S rDNA sites is divergent among C. ehrhardti, C. aff.paleatus, and C. lacrimostigmata.In C. ehrhardti, the in situ analysis of the 5S and 18S rDNA sequences revealed a syntenic localization of both rDNA sites on chromosome pair 3, albeit in independent clusters.In C. aff.paleatus (Upper Iguaçu and Upper Ribeira rivers) and in C. lacrimostigmata, 5S and 18S rDNA sequences were found to be non-syntenic and on different chromosomes, giving rise to heterokaryotypes due to chromosome rearrangements, such as translocations, inversions, and transpositions.According to Symonová et al. (2013), there is evidence to suggest that ribosomal DNA spreading is involved in chromosome rearrangements, thereby affecting recombination rates in both genomes and ultimately leading to a rapid genome divergence.Thus, the detection of the rearranged rDNA chromosome sites is an important source of evidence for mechanisms that could prevent genetic introgression.
Based on molecular data and variation in the diploid number, the genus Corydoras has been divided into five groups (Oliveira et al., 1992).Corydoras ehrhardti and C. paleatus have been classified as part of the same group (group 4), the origin of which Oliveira et al. (1992) attribute to polyploidization and chromosome number reduction.It has been suggested that in separate species maintaining the same diploid number and similar karyotypes, genetic introgression is limited by geographical barriers (Oliveira et al., 1992).However, C. ehrhardti and C. aff.paleatus are found in sympatry in the Tibagi River basin (Artoni et al., 2006).The occurrence of different Corydoras species in sympatry could be a result of the historical headwater capture events and ichthyofaunal interchange of the Ponta Grossa Arch (Ribeiro, 2006).Corydoras aff.paleatus has not previously been reported to occur in the Ribeira River basin; its distribution across the Ponta Grossa Arch might be the recent result of headwater capture events between the Iguaçu and Ribeira basins.
Chromosome segregation failure and the ensuing production of unviable gametes due to the accumulation of chromosomal rearrangements might play an important role in speciation (Navarro, Barton, 2003;Faria, Navarro, 2010).In the same way, the genetic differences accumulated in the two divergent populations of C. aff.paleatus studied here could act as a meiotic barrier: A lack of synteny within the rearranged chromosome regions of heterokaryotypes inhibits crossover during meiosis, leading to irregular meiotic segregation and the production of genetically unbalanced germ cells.This leads to a reduced gene flow even after restoring contact between the two species.
It has previously been suggested that C. ehrhardti and C. paleatus have originated through polyploidization, inversion, and chromosome fusion events (Oliveira et al., 1992).Use of a telomeric probe failed to detect any vestiges of ITS in the chromosomes of C. ehrhardti and C. aff.paleatus, although it is possible that telomeric sequences have deteriorated or even been completely lost at the points of chromosomal rearrangements (Slijepcevic, 1998;Rosa et al., 2011;Primo et al., 2016).Corydoras lacrimostigmata has a diploid chromosome number of 58 (m/sm) and shows a weak ITS signal on a submetacentric pair, corroborating the occurrence of chromosomal rearrangements in Corydoras (Oliveira et al., 1992).The ITS site is localized on pair 17 and corresponds to a heterochromatic block at the same position.It can be classified as heterochromatic-ITS (het-ITS), meaning that it might represent possible fission points due to rearrangements, in which the telomeric 7 e160056 [7] repeats were retained after the reorganization.Furthermore, these het-ITS are considered sites of spontaneous and induced chromosome breakage, with the capacity to lead to further chromosome reorganization (Ruiz-Herrera et al., 2008;Barros et al., 2017).
In conclusion, we observed specific differences in chromosomal markers among C. ehrhardti, C. aff.paleatus, and C. lacrimostigmata with respect to heterochromatin distribution, chromosome mapping of 18S and 5S rDNA, and the distribution of telomeric sequences and presence of ITS.Our cytogenetic assessments revealed the accumulation of differences between populations of C. aff.paleatus from the Ribeira, Upper Paraná, and Lower Paraná River basins, which we hypothesize to be associated with rearrangements under conditions of reduced gene flow.To determine if these differences act as a mechanism of reproductive isolation, further analyses are needed.These could include tests for reduced gene flow and recombination within chromosomal rearrangements, in particular near the breakpoints, or a test for signatures of selection within chromosome rearrangements where adaptation drives the process.The mapping of genes in rearranged regions remains a challenge, especially in unknown genomes.