Highly Rearranged Karyotypes and Multiple Sex Chromosome Systems in Armored Catfishes from the Genus Harttia (Teleostei, Siluriformes)

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.


Specimens
Three Harttia species not yet studied were investigated. Their collection sites, number, and sex of individuals are presented in Figure 1 and Table 2.

Chromosome Preparations and C-Banding
Mitotic chromosomes were obtained from cells of the anterior region of the kidney after in vivo colchicine treatment according to the protocol described in Bertollo et al. [26]. The experiments followed ethical and anesthesia procedures that were approved by the Ethics Committee on Animal Experimentation of the Universidade Federal de São Carlos (Process number CEUA 1853260315). The C-positive heterochromatin (C-banding) was identified according to Sumner [27] with some modifications according to Lui et al. [28].

Fluorescence In Situ Hybridization (FISH)
Two tandemly arrayed rDNA probes were obtained by PCR from the nuclear DNA of Harttia intermontana. The 5S rDNA probe included 120 base pairs (bp) of the 5S rRNA transcript region and 200 bp of a non-transcribed spacer (NTS), isolated according to Pendás et al. [29]. The 18S rDNA probe contained a 1400 bp segment of the 18S rRNA gene and was isolated following Cioffi et al. [30].

Chromosome Preparations and C-Banding
Mitotic chromosomes were obtained from cells of the anterior region of the kidney after in vivo colchicine treatment according to the protocol described in Bertollo et al. [26]. The experiments followed ethical and anesthesia procedures that were approved by the Ethics Committee on Animal Experimentation of the Universidade Federal de São Carlos (Process number CEUA 1853260315). The C-positive heterochromatin (C-banding) was identified according to Sumner [27] with some modifications according to Lui et al. [28].

Fluorescence In Situ Hybridization (FISH)
Two tandemly arrayed rDNA probes were obtained by PCR from the nuclear DNA of Harttia intermontana. The 5S rDNA probe included 120 base pairs (bp) of the 5S rRNA transcript region and 200 bp of a non-transcribed spacer (NTS), isolated according to Pendás et al. [29]. The 18S rDNA probe contained a 1400 bp segment of the 18S rRNA gene and was isolated following Cioffi et al. [30]. The probes were directly labeled with the Nick-Translation mix kit (Jena Bioscience, Jena, Germany) using ATTO550-dUTP for the 5S rDNA and AF488-dUTP for the 18S rDNA, according to the manufacturer's manual. Telomeric (TTAGGG)n sequences were also mapped using the DAKO Telomere PNA FISH Kit/FITC (DAKO, Glostrup, Denmark). FISH experiments followed the methodology described in Yano et al. [31].

Comparative Genomic Hybridization (CGH)
The total genomic DNA (gDNA) from male and female specimens of H. intermontana, Harttia sp. 1, and H. carvalhoi were extracted from liver tissues by the standard phenol-chloroform-isoamyl alcohol method [32]. The CGH experiments were focused on inter and intraspecific comparisons, with special emphasis on the XY 1 Y 2 sex chromosomes. In the first set of experiments (intraspecific genomic comparisons), the male-derived gDNA of H. intermontana and Harttia sp. 1was labeled by nick translation (Jena Bioscience) with ATTO550-dUTP, while female gDNA was labeled with Atto488-dUTP. Repetitive sequences were blocked in all experiments by using unlabeled C 0 t-1 DNA (i.e., a fraction of genomic DNA enriched for highly and moderately repetitive sequences), prepared according to Zwick et al. [33]. The final hybridization mixture was applied on each slide, which was composed of male-and female-derived gDNAs (500 ng each), plus 25 µg of female-derived C 0 t-1 DNA from the respective species. The probe was ethanol-precipitated, and the dry pellets were resuspended in a hybridization buffer containing 50% formamide, 2× SSC, 10% SDS, 10% dextran sulfate, and Denhardt's buffer, pH 7.0. In the second set of experiments (interspecific genomic comparisons), the gDNA samples of all-male specimens now analyzed (plus the gDNA of H. carvalhoi, another species harboring the same multiple XY 1 Y 2 sex system) were hybridized against metaphase chromosomes of H. intermontana. For this purpose, male-derived gDNA of H. intermontana was labeled with Atto550-dUTP by nick translation (Jena Bioscience), while the gDNA samples of the other two species were labeled with Atto488-dUTP (Harttia sp. 1) and Atto425-dUTP (H. carvalhoi) also by nick translation (Jena Bioscience). The three probes were hybridized simultaneously, and the final probe cocktail was composed of 500 ng of the male-derived gDNA of each H. intermontana, Harttia sp. 1, and H. carvalhoi species and 10 µg of the female-derived C 0 t-1 DNA of each species. The chosen ratio of probe vs. C 0 t-1 DNA amount was based on fish experiments previously performed in our laboratory [19,[34][35][36]. The CGH experiments followed the methodology described in Symonová et al. [37].

Microscopic Analyses and Image Processing
At least 30 metaphase spreads per individual was analyzed to confirm the 2n, karyotype structure, and CGH results. Images were captured using an Olympus BX50 light microscope (Olympus Corporation, Ishikawa, Japan), with CoolSNAP camera, and the images were processed using the Image-Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, USA). Chromosomes were classified as metacentric (m); submetacentric (sm); subtelocentric (st), or acrocentric (a) according to Levan et al. [38] and arranged according to decreasing size in the karyotypes. The fundamental number (FN), or number of chromosome arms, was achieved considering just acrocentrics as having a single chromosome arm.
A small amount of C-positive heterochromatin was found in the three species, mostly in the centromeric/pericentromeric regions of some chromosome pairs (Figure 2b,d,f), without specific accumulation in the sex chromosomes of H. intermontana and Harttia sp. 1 (Figure 2b,d).

Chromosomal Distribution of rDNAs and Telomeric Repeats
Differentiation in number and location of the 5S and 18S rDNA sequences was found among the three species. In H. intermontana and Harttia sp. 2, a single locus of 5S rDNA occurs, but in different chromosomes, i.e., in the submetacentric pairs 11 and 9, respectively. In Harttia sp. 1, there are two 5S rDNA loci, one of which is located in the submetacentric pair 12, and the other in the acrocentric pair 20, with a syntenic location with the 18S rDNA in the latter ( Figure 3).
The 18S rDNA probe was detected in a single locus in all species, but was also found in different chromosomal locations as follows:

Chromosomal Distribution of rDNAs and Telomeric Repeats
Differentiation in number and location of the 5S and 18S rDNA sequences was found among the three species. In H. intermontana and Harttia sp. 2, a single locus of 5S rDNA occurs, but in different chromosomes, i.e., in the submetacentric pairs 11 and 9, respectively. In Harttia sp. 1, there are two 5S rDNA loci, one of which is located in the submetacentric pair 12, and the other in the acrocentric pair 20, with a syntenic location with the 18S rDNA in the latter ( Figure 3).
The 18S rDNA probe was detected in a single locus in all species, but was also found in different chromosomal locations as follows: in the short arms of the second metacentric pair in H. intermontana; in the long arms of the acrocentric pair 20 in Harttia sp. 1, and in the long arms of the acrocentric pair 22 in Harttia sp. 2. No differences in the number and site positions of rDNA were detected between males and females ( Figure 3).
Hybridization with the (TTAGGG)n probe evidenced signals only in the telomeric regions of all chromosomes, without ITS in H. intermontana and Harttia sp. 1 (Figure 3b,d). However, in Harttia sp. 2, four ITS were located in the long arms of the chromosome pairs 1, 9, 16, and 22. A double-FISH using both telomeric and 18S rDNA probes revealed that these sequences present a syntenic location in the chromosome pair 22 (Figure 3f).

Intraspecific and Interspecific Comparative Genomic Hybridizations
Intraspecific genomic comparisons between males (Figure 4b

Numerical Chromosome Changes in Harttia Species
The Loricariidae family is an outstanding group to investigate chromosomal breaks and rearrangements that gave rise to extremely diverse karyotypes among its representatives [39][40][41][42]. These fishes are characterized by a sedentary lifestyle, with rare migratory events [43]. Their species occur in small and isolated populations [6] where the fixation of chromosomal rearrangements could occur at higher rates [44][45][46][47]. In fact, the Loricariinae subfamily shows extensive numerical chromosome variation (36 to 74), which is attributed to chromosomal rearrangements, mainly to Robersonian fusions (Rb fusion) and fissions [41,[48][49][50]. The Harttia genus, in which several cryptic and undescribed species are believed to occur, displays the second-largest chromosomal variation among the Loricariinae (52 to 62, Table 1, Figure 6). In addition, there is also strong evidence for evolutionary breakpoint regions (EBRs) promoting intrachromosomal remodeling, which are still being studied [51].
A putative ancestral karyotype, probably with 2n = 58 chromosomes, is attributed to the Harttia lineage, and this same 2n number occurs in its sister group Farlowella [52] and in basal species from Harttia phylogenetic relationships [7,8]. However, Harttia presents different pathways in relation to the 2n diversification, some species keeping 58 chromosomes, some others increasing this chromosome number by centric fissions (i.e., H. absaberi and Harttia sp. 2 now studied), with others decreasing this number due to Rb fusions (Table 1, Figure 6).
ITS generally reveal chromosomal rearrangements, such as Rb fusions or in tandem fusions [53]. In previous studies, ITS were identified in three Harttia species (H. loricariformis, 2n = 56; H. torrenticola, 2n = 56, and H. carvalhoi, 2n = 52♀/53♂), as vestiges of Robertsonian rearrangements [8,9]. It was proposed that fusion events were responsible for originating the largest metacentric pair found in H. torrenticola (pair 1) and H. carvalhoi (X chromosome), due to the presence of a proximal ITS on their short arms [8]. Harttia intermontana and Harttia sp. 1 also share a similar large metacentric X chromosome, but no ITS were detected. It is likely that this absence is due to the fact that not all chromosome fusions retain some telomeric DNA repeats at the fusion points [54]. Moreover, the occurrence of different chromosomal rearrangements and modifications of the non-functional telomeric arrays can be also considered [55]. In the last situation, a successive loss and degeneration of the non-functional telomeric repeats that were retained at the fusion sites leads to their gradual shortening, and, consequently, to an insufficient amount to be highlighted by FISH [53,56].
To date, the first largest metacentric pair of Harttia is shared by all species that have 2n = 56 chromosomes or a smaller number, except for H. loricariformis, and this could be considered as being derived from an Rb fusion chromosome. In Harttia sp. 2 the first chromosome pair is also a large metacentric-bearing ITS, however, this chromosome has a small size compared to the chromosome 01 of H. carvalhoi, H. intermontana, H. torrenticola, and Harttia sp. 1, thus indicating that additional rearrangements probably played a role on its origin. Noteworthy, Harttia sp. 2 presents four bi-armed chromosome pairs bearing ITS at the proximal regions of the long arms. According to the instability genomic proposal, ITS are hotspots for chromosomal breakage [57], and telomeric DNA damage can be irreparable, causing persistent activation in response to DNA damage [58] or remaining as EBRs on the genome [51,59]. This suggests that both ITS and terminal telomeric sequences are naturally prone to breakage, leading to chromosome plasticity [56,60,61]. Here, we propose that Harttia sp. 2 increased its chromosome number by centric fissions from an ancestral ITS bearing lineage, which acted as instable sites and promoted double strand breaks (DSBs) triggering further chromosomal rearrangements. This proposal is corroborated by the extensive FN modification among Harttia species (Table 1), since only Robertsonian rearrangements keep the FN unchanged throughout the karyotype evolution. It is known that chromosomal rearrangements might play an important role in speciation [47,62]. In this sense, the expressive rearranged karyotypes that are found among Harttia species may have acted as significant post-zygotic isolating mechanisms throughout the evolutionary history of this group.
Genes 2020, 11, x FOR PEER REVIEW 11 of 18 Figure 6. Representative idiograms of Harttia species from distinct Brazilian regions based on the distribution of rDNA sequences in their karyotypes, according to the present study, Blanco et al. [8], and Sassi et al. [11] data. The location of the 18S and 5S rDNA sites on the chromosomes are indicated in green and red, respectively. Inserts depict the male sex chromosomes. Figure 6. Representative idiograms of Harttia species from distinct Brazilian regions based on the distribution of rDNA sequences in their karyotypes, according to the present study, Blanco et al. [8], and Sassi et al. [11] data. The location of the 18S and 5S rDNA sites on the chromosomes are indicated in green and red, respectively. Inserts depict the male sex chromosomes.

Heterochromatin and rDNA Sites Rearrangements in Harttia Species
The presence of small amounts of heterochromatin is probably an intrinsic characteristic of the Harttia species [8]. Indeed, H. intermontana and Harttia sp. 1 present the same pattern already described for other species of the genus, while in Harttia sp. 2, some more prominent pericentromeric bands are colocated with the ITS in the chromosome pairs 1, 9, 16, and 22. The epigenetic regulation of repetitive sequences, such as histone modifications and DNA methylation to form heterochromatin, is proposed to protect ITS from breakages and play important roles in regulation of gene expression [56,63]. In this way, the colocalization of the heterochromatin and ITS may be an expression of an epigenetic property of the Harttia sp. 2 genome. In addition, the rDNA loci colocalization with ITS (5S in pair 9 and 18S in pair 22) indicates that these multigene families are also probably associated with chromosomal rearrangements in Harttia sp. 2. In the same way, the wide differentiation of the chromosomes carrying the rDNA sequences among Harttia species demonstrates that these repetitive sequences may also be closely related to deep chromosomal changes that have occurred within the genus. In fact, in some groups of Loricariidae, the involvement of rDNA pseudogenes colocalized to ITS in chromosomal rearrangements have been demonstrated [40,41,50].
As a whole, three general conditions are found concerning the location of the rDNA genes among in which the first acrocentric carries the 18S rDNA site, while the 5S occurs in variable locations of different meta/submetacentric chromosomes (except for H. rondoni that has 18S rDNA site in the largest sm). In the third pattern, the 5S locus is found in a submetacentric pair, while the chromosome that carries the 45S rDNA is a large metacentric resulting from a fusion event, as found in H. intermontana and H. absaberi karyotypes (Figures 3 and 6).
EBRs are DNA clustered regions that are more prone to break and reorganize into genomes, and these specific regions have been described to be re-used during the evolution among related species [64][65][66][67]. According to the model, the evolutionary re-use of DSB regions and multiple locus repositioning among karyotypes corroborate to probable EBR occurrences adjacent to rDNA sites in the Harttia lineage, similar to those described in other loricariids, such as Ancistrus [40] and Rineloricaria [41].

The Rare XX/XY 1 Y 2 System in Fish Species
Based on an overview of available fish karyotype data [68], only about 5% of the analyzed species possess heteromorphic sex chromosomes, including approximately 47 cases of multiple sex chromosomes [69]. Among them, some different systems, such as ♀X 1 X 1 X 2 X 2 /♂X 1 X 2 Y; ♀XX/♂XY 1 Y 2 ; ♀X 1 X 1 X 2 X 2 /♂X 1 Y 1 X 2 Y 2 ; ♂ZZ/♀ZW 1 W 2 , and ♂Z 1 Z 1 Z 2 Z 2 /♀Z 1 W 1 Z 2 W 2 , were already identified as scattered on the fish phylogeny and independently evolved in many lineages and, sometimes, even within a same genus or species [70].
In the Harttia genus, two multiple sex chromosome systems were previously described, the X 1 X 1 X 2 X 2 /X 1 X 2 Y one in H. punctata, H. duriventris, and H. villasboas and the XX/XY 1 Y 2 system in H. carvalhoi [8,11]. While the first one is well-represented among a variety of fish families [18], the XX/XY 1 Y 2 system is found in only a few fish species (Table 3). Here, like in H. carvalhoi [8], two additional cases were identified in H. intermontana and Harttia sp. 1.
Multiple XX/XY 1 Y 2 sex chromosome systems are proposed to have originated by one bi-armed chromosome fission leading to Y 1 and Y 2 formation [71][72][73] or by X-autosome fusion forming a large bi-armed X chromosome and subsequent centric fission in the origination of the Y 1 and Y 2 chromosomes [74][75][76][77]. In Harttia species, the large metacentric 1 observed in H. torrenticola is comparable to X chromosome in H. carvalhoi, H. intermontana, and Harttia sp. 2 and was proposed to be originated from an Rb fusion [8].
To date, Harttia lineages from the south/southeast Brazilian drainages have no proto-sex or XY chromosomes identified, which would corroborate the proposal of an X-autosome fusion acting in the origin of the XY 1 Y 2 system. However, the occurrence of H. torrenticola (without differentiated sex chromosomes) and H. carvalhoi (XY 1 Y 2 ) in the same branch of the phylogenetic relationship [7] and the same CGH pattern among H. carvalhoi, H. intermontana, and Harttia sp. 1 concerning sex chromosomes, point to an Rb fusion leading to their large metacentric X-chromosome, as well as to the similar large metacentric pair 1 of H. torrenticola.
Although H. intermontana and H. carvalhoi possess the same 2n and sex chromosome system (XX/XY 1 Y 2 ), significant differences occur between the karyotype structure of these two species. The absence of several large submetacentric pairs in H. intermontana as well as the occurrence of its large second metacentric pair carrying 18S rDNA cistrons are remarkable. Besides that, the morphology of their Y 2 chromosome also differs, corresponding to a subtelocentric in H. intermontana and to an acrocentric chromosome in H. carvalhoi. By comparing the chromosomal morphology and the distribution of the ribosomal sites, it is possible to infer that some additional rearrangements, such as Rb fusion and/or reciprocal translocation, pericentric inversion, and loss or gain of 5S sequences, took place in the chromosome evolution of these species. All data corroborate EBRs occurrence in adjacent regions to rDNA loci and in the pericentromeric region of the largest metacentric pair in the chromosomal diversification of the Harttia species inhabiting south and southeast Brazilian drainages. Ancistrus dubius 38♀, 39♂ X-A tandem fusion and further neo-Y chromosome fission [75,77] Harttia carvalhoi 52♀, 53♂ Y-chromosome fission [8,9,79,80] Harttia intermontana 52♀, 53♂ X-A tandem fusion and further neo-Y chromosome fission Present study Harttia sp. 1 56♀, 57♂ X-A tandem fusion and further neo-Y chromosome fission Present study

Conclusions
Our study provided additional evidence on the evolutionary pathways followed by fish species of the genus Harttia, highlighting both shared and specific chromosomal features that have emerged throughout their life story. We were also able to identify two new cases of the rare XX/XY 1 Y 2 multiple sex chromosomes systems among fishes, displaying a significant particular incidence in the Harttia lineages from south/southeast Brazil. The species in this branch, which include the H. intermontana, Harttia sp. 1, and Harttia sp. 2 here studied, experienced different ways of chromosome diversification, such as 2n reduction and increase by Rb fusions and centric fissions, respectively, and the emergence of a XX/XY1Y2 sex chromosome system in different species, in contrast to what occurred with the lineages from north Brazilian regions where the X 1 X 1 X 2 X 2 /X 1 X 2 Y system stands out. The occurrence of deeply reorganized karyotypes in the species here studied are in accordance with EBRs present in the Harttia genome, which could be reused for chromosome speciation in this group. As a whole, the present study highlights the importance of cytogenetics as a tool for evolutionary studies and, particularly in the present case, detaching the highly differentiated patterns followed by the Harttia lineages from two main Brazilian geographic regions. Funding: This research was founded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (302449/2018-3) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (2018/22033-1). FMCS was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (2020/02681-9). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Finance Code 001.