Harttia comprises an armored catfish genus endemic to the Neotropical region, including 27 valid species with low dispersion rates that are restricted to small distribution areas. Cytogenetics data point to a wide chromosomal diversity in this genus due to changes that occurred in isolated populations, with chromosomal fusions and fissions explaining the 2n number variation. In addition, different multiple sex chromosome systems and rDNA loci location are also found in some species. However, several Harttia species and populations remain to be investigated. In this study, Harttia intermontana and two still undescribed species, morphologically identified as Harttia sp. 1 and Harttia sp. 2, were cytogenetically analyzed. Harttia intermontana has 2n = 52 and 2n = 53 chromosomes, while Harttia sp. 1 has 2n = 56 and 2n = 57 chromosomes in females and males, respectively, thus highlighting the occurrence of an XX/XY1Y2 multiple sex chromosome system in both species. Harttia sp. 2 presents 2n = 62 chromosomes for both females and males, with fission events explaining its karyotype diversification. Chromosomal locations of the rDNA sites were also quite different among species, reinforcing that extensive rearrangements had occurred in their karyotype evolution. Comparative genomic hybridization (CGH) experiments among some Harttia species evidenced a shared content of the XY1Y2 sex chromosomes in three of them, thus pointing towards their common origin. Therefore, the comparative analysis among all Harttia species cytogenetically studied thus far allowed us to provide an evolutionary scenario related to the speciation process of this fish group.

The armored Harttia catfishes present great species diversity and remarkable cytogenetic variation, including different sex chromosome systems. Here we analyzed three new species, H. duriventris, H. villasboas and H. rondoni, using both conventional and molecular cytogenetic techniques (Giemsa-staining and C-banding), including the mapping of repetitive DNAs using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) experiments. Both H. duriventris and H. villasboas have 2n = ♀56/♂55 chromosomes, and an X1X1X2X2 /X1X2Y sex chromosome system, while a proto or neo-XY system is proposed for H. rondoni (2n = 54♀♂). Single motifs of 5S and 18S rDNA occur in all three species, with the latter being also mapped in the sex chromosomes. The results confirm the general evolutionary trend that has been noticed for the genus: an extensive variation on their chromosome number, single sites of rDNA sequences and the occurrence of multiple sex chromosomes. Comparative genomic analyses with another congeneric species, H. punctata, reveal that the X1X2Y sex chromosomes of these species share the genomic contents, indicating a probable common origin. The remarkable karyotypic variation, including sex chromosomes systems, makes Harttia a suitable model for evolutionary studies focusing on karyotype differentiation and sex chromosome evolution among lower vertebrates.

Ancistrus is a specious genus of armored catfishes that has been extensively used for cytogenetic studies in the last 17 years. A comparison of the extensive karyotypic plasticity within this genus is presented with new cytogenetic analysis for Ancistrus cf. multispinis and Ancistrus aguaboensis. This study aims to improve our understanding of chromosomal evolution associated with changes in the diploid number (2n) and the dispersion of ribosomal DNAs (rDNAs) within Ancistrus. Ancistrus cf. multispinis and A. aguaboensis exhibit 2n of 52 and 50 chromosomes, respectively. Given that A. cf. multispinis shares a 2n = 52 also found in Pterygoplichthyini, the sister group for Ancistrini, a Robertsonian (Rb) fusion event is proposed for the 2n reduction in A. aguaboensis. 5S rDNAs pseudogenes sites have already been associated with Rb fusion in Ancistrus and our analysis suggests that the 2n reduction in A. aguaboensis was triggered by double strand breaks (DSBs) and chromosomal rearrangements at 5S rDNA sites. The presence of evolutionary breakpoint regions (EBRs) into rDNA cluster is proposed to explain part of the Rb fusion in Ancistrus. Cytogenetic data presented extends the diversity already documented in Ancistrus to further understand the role of chromosomal rearrangements in the diversification of Ancistrini.

Cytogenetic data showed the enrichment of repetitive DNAs in chromosomal rearrangement points between closely related species in armored catfishes. Still, few studies integrated cytogenetic and genomic data aiming to identify their prone-to-break DNA sites. Here, we aimed to obtain the repetitive fraction in Rineloricaria latirostris to recognize the microsatellite and homopolymers flanking the regions previously described as chromosomal fusion points. The results indicated that repetitive DNAs in R. latirostris are predominantly DNA transposons, and considering the microsatellite and homopolymers, A/T-rich expansions were the most abundant. The in situ localization demonstrated the A/T-rich repetitive sequences are scattered on the chromosomes, while A/G-rich microsatellites units were accumulated in some regions. The DNA transposon hAT, the 5S rDNA, and 45S rDNA (previously identified in Robertsonian fusion points in R. latirostris) are clusterized with some microsatellites, especially (CA)n, (GA)n, and poly-A, which also are enriched in regions of chromosomal fusions. Our findings demonstrated that repetitive sequences such as rDNAs, hAT transposon, and microsatellite units flank probable evolutionary breakpoint regions in R. latirostris. However, due to the sequence unit homologies in different chromosomal sites, these repeat DNAs only may have facilitated chromosome fusion events in R. latirostris rather than work as a double-strand breakpoint site.

A remarkable morphological diversity and karyotype variability can be observed in the Neotropical armored catfish genus Harttia. These fishes offer a useful model to explore both the evolution of karyotypes and sex chromosomes, since many species possess male-heterogametic sex chromosome systems and a high rate of karyotype repatterning. Based on the karyotype organization, the chromosomal distribution of several repetitive DNA classes, and the rough estimates of genomic divergences at the intraspecific and interspecific levels via Comparative Genomic Hybridization, we identified shared diploid chromosome numbers (2n = 54) but different karyotype compositions in H. dissidens (20m + 26sm + 8a) and Harttia sp. 3 (16m + 18sm + 14st + 6a), and different 2n in H. guianensis (2n = 58; 20m + 26sm + 2st + 10a). All species further displayed similar patterns of chromosomal distribution concerning constitutive heterochromatin, 18S ribosomal DNA (rDNA) sites, and most of the surveyed microsatellite motifs. Furthermore, differences in the distribution of 5S rDNA sites and a subset of microsatellite sequences were identified. Heteromorphic sex chromosomes were lacking in H. dissidens and H. guianensis at the scale of our analysis. However, one single chromosome pair in Harttia sp. 3 males presented a remarkable accumulation of male genome-derived probe after CGH, pointing to a tentative region of early sex chromosome differentiation. Thus, our data support already previously outlined evidence that Harttia is a vital model for the investigation of teleost karyotype and sex chromosome dynamics.

The Neotropical genus Harttia comprises species with extensive chromosomal remodeling and distinct sex chromosome systems (SCSs). So far, three different SCSs with male heterogamety have been characterized in the group. In some species, the presence of the XX/XY1Y2 SCS is associated with a decrease in diploid numbers and several chromosomal rearrangements, although a direct relation to sex chromosome differentiation has not been shown yet. Here, we aimed to investigate the differentiation processes that have led to the establishment of the rare XX/XY1Y2 SCS and track its evolutionary history among other Harttia species. For that, four whole chromosome painting probes derived from chromosome 1 of H. torrenticola (HTO-1), chromosomes 9 and X of H. carvalhoi (HCA-9 and HCA-X), and chromosome X from H. intermontana (HIN-X) were applied in nine Harttia species. Homeologous chromosome blocks were located in Harttia species and demonstrated that Robertsonian (Rb) fusions originated HTO-1, HCA-9, and HCA-X chromosomes, while Rb fissions explain Y1 and Y2 sex chromosomes. Specifically, in H. intermontana, HCA-X, HCA-9, and the NOR-bearing chromosome demonstrated that homeologous blocks were used in the HIN-X and metacentric pair 2 origins. Consequently, diploid numbers changed between the studied species. Overall, the data also reinforce the existence of unstable genomic sites promoting chromosomal differentiation and remodeling within the genus Harttia.

Miniature fishes have always been a challenge for cytogenetic studies due to the difficulty in obtaining chromosomal preparations, making them virtually unexplored. An example of this scenario relies on members of the family Lebiasinidae which include miniature to medium-sized, poorly known species, until very recently. The present study is part of undergoing major cytogenetic advances seeking to elucidate the evolutionary history of lebiasinids. Aiming to examine the karyotype diversification more deeply in Pyrrhulina, here we combined classical and molecular cytogenetic analyses, including Giemsa staining, C-banding, repetitive DNA mapping, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) to perform the first analyses in five Pyrrhulina species (Pyrrhulina aff. marilynae, Pyrrhulina sp., P. obermulleri, P. marilynae and Pyrrhulina cf. laeta). The diploid number (2n) ranged from 40 to 42 chromosomes among all analyzed species, but P. marilynae is strikingly differentiated by having 2n = 32 chromosomes and a karyotype composed of large meta/submetacentric chromosomes, whose plesiomorphic status is discussed. The distribution of microsatellites does not markedly differ among species, but the number and position of the rDNA sites underwent significant changes among them. Interspecific comparative genome hybridization (CGH) found a moderate divergence in the repetitive DNA content among the species’ genomes. Noteworthy, the WCP reinforced our previous hypothesis on the origin of the X1X2Y multiple sex chromosome system in P. semifasciata. In summary, our data suggest that the karyotype differentiation in Pyrrhulina has been driven by major structural rearrangements, accompanied by high dynamics of repetitive DNAs.

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