Chromosomal Rearrangements and Origin of the Multiple XX/XY1Y2 Sex Chromosome System in Harttia Species (Siluriformes: Loricariidae)

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.


INTRODUCTION
Although sex determination can be environmentally determined in some species, it is usually genetically regulated, and often associated with the presence of sex chromosomes (Furman et al., 2020). According to a widely accepted model, sex chromosomes arise from an autosomal pair due to the emergence of a sex-specific locus in one of the homologous (Bull, 1983;Charl-esworth, 2002). Over time, the ancestral homologous pair undergoes divergences in its genetic composition, recombination rate, and morphology, leading to sex chromosomes differentiation (Charlesworth duriventris,and H. villasboas,and (2) a proto/neo-XX/XY system in H. rondoni, both belonging to clade II, and (3) the XX/XY 1 Y 2 system in H. carvalhoi, H. intermontana, and Harttia sp.1, species which belong to clade III (Centofante et al., 2006;Blanco et al., 2017;Sassi et al., 2020;Sassi et al., 2021;Deon et al., submitted). Chromosomal data compared to a phylogenetic framework indicate that ancestral karyotype with 2n = 58 chromosomes and without a differentiated SCS could represent a plesiomorphic condition for clade III (Deon et al., submitted). Belonging to the same clade III, the species H. torrenticola has a karyotype composed by 2n = 56, undifferentiated sex chromosomes (Blanco et al., 2013), and a large metacentric pair being morphologically similar to the X chromosome of H. carvalhoi. WCP-FISH experiments using X 1 and X 2 probes derived from H. punctata, confirmed that chromosomes that gave rise to the X 1 X 2 Y and the XY 1 Y 2 systems are evolutionary independent (Deon et al., submitted).
Here, we aimed to investigate the differentiation processes that have led to the establishment of the rare XX/XY 1 Y 2 SCS and to track its evolutionary history among other Harttia species. For that, we performed a WCP-FISH investigation using four distinct sex chromosome-specific probes hybridized in several species. The results allowed us to identify the main rearrangements involved in the origin of this unique SCS. Besides, the data provide new insights into the origin and evolution of such a rare XY-derived SCS, consequently increasing our knowledge about the evolution of vertebrate sex chromosomes.

Individuals and Chromosome Preparation
Representatives of Harttia species analyzed in this study are summarized in Table 1 Mitotic chromosomes were obtained from kidney cells, according to Bertollo et al. (2015). All procedures agreed with the Ethics Committee of Animal Usage of the Universidade Federal de São Carlos (Process number CEUA 1853260315), Brazil.

Chromosome Microdissection, Probe Preparation, and Labeling
Fifteen copies of each target chromosome were isolated by glassneedle-based microdissection, and obtained DNA was amplified by oligonucleotide primed-polymerase chain reaction (DOP-PCR) as described in . Chromosomes were chosen based on their morphology -bi-armed chromosomes that were suspected to be originated from Robertsonian fusions were targeted: the largest metacentric (HCA-X), and the largest submetacentric (HCA-9) from H. carvalhoi; the largest metacentric (HIN-X) from H. intermontana, and the largest metacentric (HTO-1) from H. torrenticola ( Figure 1). Probes were labeled with Spectrum Orange-dUTP or Spectrum Green-dUTP (Vysis, Downers Grove, United States) in a secondary DOP-PCR, using 1 μL of the primarily amplified product as a template DNA (Yang and Graphodatsky, 2009).

Fluorescence in situ Hybridization
Slides were prepared and pre-treated according to  and denatured in 70% formamide/2x SSC for 3 min at 72°C. For each slide, 12 μL of hybridization solution (containing 0.2 μg of each labeled probe, 50% formamide, 2×SSC, 10% dextran sulfate, and 5 μg of salmon sperm DNA) was denatured for 10 min at 75°C and incubated to pre-hybridize for 1 h at 37°C. To block the hybridization of high-copy repeat sequences, 20 μg of C 0 t-1 DNA, directly prepared from H. carvalhoi, H. torrenticola, and H. intermontana male genomes were used, according to Zwick et al. (1997). Hybridization took place for 48 h at 37°C in a moist chamber. Post-hybridization washes were performed in 1×SSC for 5 min at 65°C, and 5 min in 4×SSC/Tween at room temperature. Finally, the slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium (Vector, Burlingame, CA, United States).

Image Analyses and Processing
Metaphase plates were captured using an Olympus BX50 light microscope (Olympus Corporation, Ishikawa, Japan) coupled with a CoolSNAP camera. The images were processed using Image-Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, United States). The figures were edited and organized using Adobe Photoshop CC 2020 (San Jose, CA, United States) software.

RESULTS
Results obtained by HCA-X and HCA-9 probes are summarized in Figure 2 and Table 2. In H. carvalhoi (52\/53_ -XX/XY 1 Y 2 ), the HCA-X probe successfully identified their X chromosomes in females and the X, Y 1, and Y 2 chromosomes in males. Small centromeric signals in both acrocentric pairs 23 and 24 were also evidenced. In agreement, the HCA-9 probe correctly recognized the submetacentric pair 9 (Figures 2A,B). Similarly, in Harttia sp. 1 (56\/57_ -XX/XY 1 Y 2 ), the HCA-X probe detected the X chromosome pair in females and the X, Y 1 , and Y 2 chromosomes in males, besides small centromeric signals in both 20 and 24 acrocentric pairs ( Figures 2C,D). The HCA-9 hybridized to 21 and 26 acrocentric pairs ( Figures 2C,D). In females of H. intermontana (52\/53_ -XX/XY 1 Y 2 ), the HCA-X probe stained the long (q) arms of the chromosomes X and 2 ( Figure 2E). In males, this probe gave signals on Xq, the Y 2 chromosome, and the 2q ( Figure 2F), as well as in the centromeric region of the pair 24 in both males and females ( Figures 2E,F). The HCA-9 probe detected the short (p) arms of the X chromosome and the 20q distal region in females, and the Xp arms, the Y 1 chromosome, and the 20q distal region in males ( Figures  2E,F). In H. punctata (58\/57_ -X 1 X 1 X 2 X 2 /X 1 X 2 Y), the HCA-X probe hybridized on the submetacentric pairs 9 and 1 | Collection sites of the studied species, diploid chromosome number (2n), and sample size (N).
The HTO-1 probe, derived from H. torrenticola, showed the same results obtained with the HCA-X probe when tested on those species with an identical large metacentric pair (Supplementary Figure S1). In contrast, the HIN-X probe, from H. intermontana, showed different results than those obtained applying HCA-X and HTO-1 probes ( Figure 3). In H. carvalhoi, HIN-X hybridized on the Xq and 9q arms in females ( Figure 3A), and on the Xq arms, Y 2 chromosome, and 9q arms in males ( Figure 3B). In Harttia sp. 1, HIN-X labeled the Xq arms and the acrocentric 21 pair in females ( Figure 3C), and these same chromosomes, as well as the Y 2 chromosome, in males ( Figure 3D). In H. torrenticola, HIN-X stained the 1q arms and the acrocentric 23 pair ( Figures 3E,F).

DISCUSSION
In Harttia species, diploid numbers range from 52 to 62 chromosomes Sassi et al., 2020;Sassi et al., 2021). Data from phylogeny reconstructions indicate that 58 chromosomes and no large biarmed chromosomes could correspond to a plesiomorphic karyotype condition for species distributed on south and southeast Brazilian drainages-the clade III (Deon et al., submitted). These chromosomal features ( Figure 5) include the absence of morphologically differentiated sex chromosomes and a single location of the 5S and 45S rDNA sites in medium-sized bi-armed chromosomes Deon et al., submitted). Here, the ancestral reconstructions of the Harttia karyotype, using both HCA-X and HCA-9 probes, demonstrated that two chromosome pairs were probably related to the origin of the H. carvalhoi chromosomes X and 9 ( Figure 6). Thus, in situ localizations also reaffirm the role of Robertsonian fusions as the main rearrangements responsible for reducing the diploid number in H. carvalhoi. These homeologous chromosome pairs (unfused chromosomes) are shared by H. kronei, H. loricariformis, H. longipinna, and H. gracilis ( Figure 6). As common features, H. kronei, H. longipinna, and H. gracilis kept 2n = 58 chromosomes and the absence of morphologically differentiated SCSs (Blanco et al., 2017), with chromosomal diversification events mainly occurring by repositioning of the rDNA sites in their karyotypes Deon et al., submitted). Although H. loricariformis decreased the diploid number to 2n = 56, this species shares the homologous chromosome pairs to HCA-X and HCA-9 as highlighted in H. kronei by WCP-FISH. The presence of interstitial telomeric sites in a subtelocentric chromosome of H. loricariformis karyotype suggests an origin by Robertsonian fusions (Blanco et al., 2017). The current data thus support the hypothesis on the occurrence of a chromosomal fusion event in H. loricariformis karyotype, and that this corresponds to an independent evolutionary event being not associated with the chromosomes X and 9 of H. carvalhoi.
Data also showed that the chromosomal rearrangements that led to the XX/XY 1 Y 2 SCS were triggered within the branch with H. torrenticola ( Figure 5). The phylogenetic branch grouping H. carvalhoi and H. torrenticola (Covain et al., 2016; Londoño-Burbano and Reis, 2021; Figure 5) was diversified by Robertsonian fusions, initially giving rise to a large metacentric pair, like that found in the H. torrenticola karyotype. Indeed, the large homeologous chromosome  Deon et al. (2020) proposed the same origin of the XX/XY 1 Y 2 system in H. carvalhoi, Harttia sp. 1, and H. intermontana. However, the use of the HCA-X, HCA-9, and HIN-X probes enabled now to evidence that additional rearrangements are associated with the XX/XY 1 Y 2 system of H. intermontana. The X chromosome of this species comprises the 9q arms and one arm of the X chromosome of H. carvalhoi, indicating a reciprocal translocation between these two chromosome pairs in its origin ( Figure 6). After that, centric fission in one of the X chromosomes, followed by a pericentric inversion in one of the resulted elements, generated the Y 1 and Y 2 chromosomes in males (Figure 7). It is relevant to notice that the Y 1 chromosome of H. intermontana is derived from chromosome 9 of H. carvalhoi, thus different from the Y 1 chromosome of H. carvalhoi and Harttia sp. 1. In the same way, the metacentric pair 2 of H. intermontana was originated from species-specific chromosomal rearrangements, implying a translocation between the acrocentric chromosome bearing the 45S site and one X chromosome arm. Indeed, the chromosome pair of H. intermontana bears the 45S rDNA locus , a site prone to breaks in Harttia karyotypes, leading to extensive chromosomal remodeling events Deon et al., submitted). According to molecular-phylogenetic reconstructions, H. punctata -2n = 58\/57_, X 1 X 1 /X 2 X 2 /X 1 X 2 Y (Blanco et al., 2014) -belongs to Harttia's clade II (Covain et al., 2016;Londoño-Burbano and Reis, 2021), and the WCP results here obtained evidenced a similar hybridization condition to those found in H. kronei, i.e., the HTO-1, HCA-X, HCA-9, and HIN-X chromosomes were not related to the karyotype diversification of H. punctata, highlighting a probable plesiomorphic condition.
It was demonstrated that sex chromosomes could emerge independently and follow distinct differentiation patterns, even among closely related species (Cioffi et al., 2013). Our WCP-FISH data also indicated independent origins for the X 1 X 2 Y and XY 1 Y 2 SCSs of Harttia lineage, as previously proposed Sassi et al., 2020). The X chromosome of the XX/XY 1 Y 2 system originated by fusion of two autosome pairs, leading to the largest metacentric in the karyotype. This fusion could set up a putative homomorphic XX/XY SCS, with subsequent centric fission originating the Y 1 and Y 2 chromosomes, as proposed by Blanco et al. (2013). Thus, a set of diverse chromosomal rearrangements probably triggered the differentiation of the same or different SCSs within the Harttia lineage, suggesting that sex chromosome turnover may play an important role in the speciation processes of this group.
Evolutionarily conserved breakpoint regions (ECBRs), inside or adjacent to rDNA clusters, were proposed to occur in some Loricariidae lineages, leading to extensive chromosomal remodeling (Barros et al., 2017;Glugoski et al., 2018;Deon et al., 2020;Deon et al., submitted). In the Harttia clade III from the south/southeast Brazilian region, several rearrangements adjacent to the rDNAs sites have been extensively reused in the chromosomal diversification Deon et al., submitted), including the origin of the X 1 X 1 X 2 X 2 /X 1 X 2 Y SCS in Harttia clade II (Deon et al., submitted). In contrast, as rDNAs were not involved in the  origin of the XX/XY 1 Y 2 system, other unstable sites likely occur in the genomes of the species from Harttia clade III, as well.
In addition, some hybridization signals besides those indicating the discussed main rearrangements were also detected in small chromosomal regions. They correspond to a highly rearranged repetitive DNA unit shared among Harttia species. In H. carvalhoi, H. gracilis, H. longipinna, H. torrenticola, and Harttia sp. 1, they localize close to the nucleolar organizer region (NOR) and in a small acrocentric pair. In H. loricariformis and H. intermontana only the signal close to the NOR site and in the small acrocentric chromosome were detected, respectively. The mechanisms responsible for the instability of Harttia genome are not fully understood Deon et al., submitted). However, repetitive DNA clusters scattered at some genome locations are likely candidates for chromosomal breaks and rearrangements. Cytogenetic data indicate that these sequences are reused in several chromosome rearrangements, including the Robertsonian ones responsible for the origin of the SCSs and a 2n decrease in Harttia.
Interstitial telomeric sites (ITS) are common features in some Harttia genomes (Blanco et al., 2017;Deon et al., 2020). H. carvalhoi and H. torrenticola, for example, present an ITS in the large metacentric chromosome (Blanco et al., 2017), indicating its origin by Robertsonian fusion. In contrast, this ITS was lost during the chromosomal evolution of Harttia sp. 1. It is known that ITS are hotspots for breakages (Slijepcevic et al., 1997) and that telomeric DNA damages can be irreparable, causing persistent DNA-damageresponse activation (Fumagalli et al., 2012), or remaining as fragile sites (Sfeir et al., 2009). According to Slijepcevic (2016), both ITS and terminal telomeric sequences are naturally prone to breakage, leading to chromosome plasticity. Therefore, the rearrangements observed in the X and 2 chromosomes of H. intermontana may have been triggered by the instability generated by the ITS in the X chromosome of H. carvalhoi.

CONCLUSION
Data obtained by WCP-FISH allowed to highlight small pieces of the complex chromosomal evolution that has taken place in Harttia species, with a particular emphasis on the origin of a rare multiple SCS and diploid number decrease. We demonstrated the existence of unstable genomic sites promoting chromosomal differentiation and remodeling, where homeologous chromosome blocks were identified after WCP experiments. Besides, we highlighted the distinct Robertsonian fusions and fissions that were involved in the origin the sex chromosomes. In this context, the genus Harttia has proved to be an excellent model for the study of evolution of sexual chromosome systems among Neotropical fish species. Next steps now will include a fine-scale analysis of the genetic content of the sex chromosomes in this group aiming to discover novel sex-determining genes, which is an inevitable next step towards fully understating this puzzling scenario.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by All procedures agreed with the Ethics Committee of Animal Usage of the Universidade Federal de São Carlos (Process number CEUA 1853260315), Brazil.

AUTHOR CONTRIBUTIONS
GD, LG, FS, MC, and MV carried out the analysis and drafted the manuscript. TH, VN and AA-R helped in the analysis, drafted, and revised the manuscript. TL, LB, OM-F, MC and MV drafted and revised the manuscript. All authors read and approved the final version of the manuscript.

FUNDING
MC was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Proc. No. 302449/2018-3) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Proc. No. 2020/11772-8). FS was supported by Fundação de Amparo FIGURE 7 | Schematic model representing the rearrangements occurred from H. carvalhoi to H. intermontana, and the evolvement of HCA-X (yellow) and HCA-9 probes (light pink) on the origin of the XX/XY 1 Y 2 SCS. Considering H. intermontana, a derived species from H. carvalhoi, a centric fusion between the long arms of the chromosome 1 (X) and the long arms of the chromosome 9 of H. carvalhoi, lead the origin of the X chromosome in H. intermontana. An additional fusion with part of the X chromosome and part of the chromosome 23 (bearing the 18S rDNA site) gave rise to the second-largest metacentric chromosome pair in H. intermontana (pair 2). It is worth mentioning that the different origin of the X chromosome directly reflects on the genomic composition of the Y chromosomes: while the Y 1 chromosome corresponds to the long arms of the chromosome 9, the Y2 chromosome corresponds to a part of the X chromosome of H. carvalhoi.