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Article

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

by
Geize Aparecida Deon
1,2,
Larissa Glugoski
1,2,
Marcelo Ricardo Vicari
2,
Viviane Nogaroto
2,
Francisco de Menezes Cavalcante Sassi
1,
Marcelo de Bello Cioffi
1,
Thomas Liehr
3,*,
Luiz Antonio Carlos Bertollo
1 and
Orlando Moreira-Filho
1
1
Laboratório de Citogenética de Peixes, Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos SP 13565-905, Brazil
2
Laboratório de Biologia Cromossômica, Estrutura e Função, Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa PR 84030-900, Brazil
3
Institute of Human Genetics, University Hospital Jena, 07747 Jena, Germany
*
Author to whom correspondence should be addressed.
Genes 2020, 11(11), 1366; https://0-doi-org.brum.beds.ac.uk/10.3390/genes11111366
Submission received: 19 October 2020 / Revised: 15 November 2020 / Accepted: 16 November 2020 / Published: 18 November 2020
(This article belongs to the Special Issue Vertebrate Karyotype Evolution: How Far Have We Come, and Where Are We Going?)
Browse Figures
Versions Notes

Abstract

:
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.

1. Introduction

Fishes exhibit the greatest biodiversity among the vertebrates, constituting a useful model for studying several evolutionary questions [1]. Particularly, the large river networks found in the Neotropical region contain the world’s richest biodiversity. Despite the large geographic distribution of the Neotropical fish families, different species are found inhabiting adjacent river basins split by vicariant events millions of years ago [2]. In the same way, species inhabiting small streams, with limited migration opportunities, tend to present an increased rate of speciation [3], even in parapatric populations [4].
One of these examples relies on the genus Harttia (Siluriformes, Loricariidae, Loricariinae), an endemic and widespread group throughout many South American river basins [5]. These species have a sedentary lifestyle and reduced vagility, leading them to inhabit specific sections of the river and to form small local populations [6].
Studied species indicate a wide variation on the diploid number (2n) in Harttia, ranging from 2n = 52 to 62 chromosomes, with the occurrence of interstitial telomeric sites (ITS) as vestiges of chromosomal changes, different ribosomal genes distributions on the karyotypes, occurrence of B chromosomes, and multiple sex chromosome systems (Table 1). Based on the molecular phylogenetic inferences in the Harttiini tribe [7], a scenario for Harttia karyotype diversification was proposed by Blanco et al. [8]. In this scenario, a putative ancestral karyotype would have 2n = 58 chromosomes and no heteromorphic sex chromosomes, such as that found in Harttia kronei [8]. From such a karyotype, reductions in 2n number by chromosome fusions were proposed in the diversification of lineages, until the lowest number of chromosomes, 2n = 52, found in H. carvalhoi females was reached [8].
Furthermore, the chromosomal rearrangements in Harttia species culminated in different kinds of sex chromosome systems: (i) an XX/XY1Y2 system in H. carvalhoi [9]; (ii) an X1X1X2X2/X1X2Y system in Harttia punctata, Harttia duriventris, and Harttia villasboas [10,11]; and (iii) a neo XX/XY system in Harttia rondoni [11]. In accordance with the two branches of Harttia species [7], these sex chromosome systems followed independent evolutionary origins [8,11]. While H. carvalhoi is grouped with H. kronei, H. longipinna, H. loricariformis, and some other species distributed in southern and southeastern Brazil, H. punctata, H. rondoni, H. duriventris, and H. villasboas are grouped in a different branch with other species from the north and northeast Brazilian drainages [7].
Simple sex chromosome systems are proposed to have originated by an inversion event or by the accumulation of transposable elements in one homologue of a proto sex chromosome pair, thus promoting a cross-over restricted region able to differentiate in a sex-specific chromosomal segment [12,13,14]. Additionally, with regard to multiple sex chromosomes, different types of chromosomal rearrangements (such as centric fusions, centric fissions, pericentric inversions, translocations, tandem translocations), usually associated with autosomes and proto-sex chromosomes, have already been proposed to explain the origin of different types of systems [9,15,16,17,18,19]. More recently, and with the aim of discovering the evolutionary origin of the sex chromosomes systems, molecular cytogenetics approaches such as whole chromosome painting (WCP) and comparative genome hybridization (CGH) have been successfully used in some Neotropical fish species [19,20,21,22].
In this study, we provide a set of conventional and molecular cytogenetic approaches (Giemsa staining, C-banding, repetitive DNA mapping by FISH, comparative genomic hybridization (CGH)), in an attempt to advance the knowledge of the processes that have shaped the chromosomal evolution in the genus Harttia. Data allowed for a comprehensive perspective of the chromosomal diversity and evolutionary trends inside this group, in addition to a description of two other new rare occurrences of the XX/XY1Y2 sex chromosome system among fishes.

2. Materials and Methods

2.1. 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. Figure 1 also depicts the Brazilian distribution of other Harttia species previously studied. Fishes were collected with the authorization of the Chico Mendes Institute for Biodiversity Conservation (ICMBIO), System of Authorization and Information about Biodiversity (SISBIO-License Nso. 10538-3 and 15117-1), and National System of Genetic Resource Management and Associated Traditional Knowledge (SISGEN-A96FF09). The species were properly identified by Dr. Oswaldo Oyakawa (curator of the fish collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP), with expertise on Harttia taxonomy. One of the three species corresponds to Harttia intermontana, and the other two correspond to new species that have not yet been described, here named as Harttia sp. 1 and Harttia sp. 2.

2.2. 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].

2.3. 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].

2.4. 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 XY1Y2 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 C0t-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 C0t-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 XY1Y2 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 C0t-1 DNA of each species. The chosen ratio of probe vs. C0t-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].

2.5. 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.

3. Results

3.1. Karyotypes, C-Banding, and Sex Chromosomes

All H. intermontana females have 2n = 52 chromosomes (14m + 12sm + 12st + 14a; NF = 90) and all males have 2n = 53 chromosomes (13m + 12sm + 13st + 15a, NF = 91). Similarly, Harttia sp. 1 also differs in female and male karyotypes, with 2n = 56 (14m + 14sm + 10st + 18a; NF = 94) and 2n = 57 (13m + 14sm + 10st + 20a; NF = 94), respectively. In both cases, the sex-specific karyotypes are due the occurrence of an XX/XY1Y2 multiple sex chromosome system, where the X chromosome corresponds to a large metacentric, and the Y1 to a medium-size acrocentric. In its turn, the Y2 chromosome corresponds to a medium-size subtelocentric in H. intermontana and to an acrocentric chromosome in Harttia sp. 1 (Figure 2a,c). Additionally, Harttia sp. 2 has 2n = 62 chromosomes (16m + 14sm + 12st + 20a; NF = 104) in both sexes, without morphologically differentiated sex chromosomes (Figure 2e).
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).

3.2. 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).

3.3. Intraspecific and Interspecific Comparative Genomic Hybridizations

Intraspecific genomic comparisons between males (Figure 4b,f) and females (Figure 4c,g) of H. intermontana and Harttia sp. 1 showed an overlapped hybridization, mainly in the centromeric and pericentromeric regions of almost all chromosomes (Figure 4d,h). A strong binding preference for the 18S rDNA cluster occurs in H. intermontana (Figure 4b,c) and no sex-specific region was evidenced in both experiments. Interspecific comparisons of the gDNA of H. intermontana, H. carvalhoi, and Harttia sp. 1, all of them bearing an XY1Y2 sex system, did not detect species-specific regions in the sex chromosomes (Figure 5).

4. Discussion

4.1. 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.

4.2. 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 Harttia species: In the first, a syntenic condition for both 5S and 45S rDNAs occurs, as found in H. carvalhoi, H. loricariformis, and Harttia sp. 1, where the first acrocentric chromosome pair is the carrying one, although with Harttia sp. 1 showing a particular syntenic configuration (Figure 3 and Figure 6). Yet, H. carvalhoi and Harttia sp. 1 present an extra 5S rDNA locus. The second condition includes H. gracilis, H. kronei, H. longipinna, H. punctata, H. villasboas, H. duriventris, H. torrenticola, and Harttia sp. 2, 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 (Figure 3 and Figure 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].

4.3. The Rare XX/XY1Y2 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 ♀X1X1X2X2/♂X1X2Y; ♀XX/♂XY1Y2; ♀X1X1X2X2/♂X1Y1X2Y2; ♂ZZ/♀ZW1W2, and ♂Z1Z1Z2Z2/♀Z1W1Z2W2, 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 X1X1X2X2/X1X2Y one in H. punctata, H. duriventris, and H. villasboas and the XX/XY1Y2 system in H. carvalhoi [8,11]. While the first one is well-represented among a variety of fish families [18], the XX/XY1Y2 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/XY1Y2 sex chromosome systems are proposed to have originated by one bi-armed chromosome fission leading to Y1 and Y2 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 Y1 and Y2 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 XY1Y2 system. However, the occurrence of H. torrenticola (without differentiated sex chromosomes) and H. carvalhoi (XY1Y2) 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/XY1Y2), 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 Y2 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.

5. 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/XY1Y2 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 X1X1X2X2/X1X2Y 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.

Author Contributions

Conceptualization, G.A.D., F.d.M.C.S., O.M.-F., M.R.V., M.d.B.C.; Formal analysis, G.A.D., L.G.; F.d.M.C.S., O.M.-F., M.R.V., T.L., V.N., M.d.B.C.; Funding acquisition, O.M.-F., L.A.C.B., M.d.B.C.; Investigation, G.A.D., M.R.V., O.M.-F., M.d.B.C.; Methodology, G.A.D., L.G., F.d.M.C.S., V.N.; Project administration, L.A.C.B., T.L., M.d.B.C.; Resources, T.L.; Supervision, M.R.V., O.M.-F., M.d.B.C.; Validation, T.L., V.N., L.G.; Visualization, G.A.D., F.d.M.C.S., O.M.F., M.R.V., L.A.C.B., T.L., V.N., L.G., M.d.B.C.; Writing-original draft, G.A.D., F.d.M.C.S., M.R.V., M.d.B.C.; Writing-review and editing, G.A.D., F.d.M.C.S., O.M.-F., M.R.V., L.A.C.B., T.L., V.N.; L.G., M.d.B.C. All authors have read and agreed to the published version of the manuscript.

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.

Acknowledgments

The authors are grateful to. Oswaldo Oyakawa (curator of the fish collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP) for the proper specimens’ identification.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Nelson, J.S.; Grand, T.C.; Wilson, M.V.H. Fishes of The World, 5th ed.; John Wiley & Sons: Hoboken, NJ, USA, 2016. [Google Scholar]
  2. Albert, J.S.; Tagliacollo, V.A.; Dagosta, F. Diversification of Neotropical Freshwater Fishes. Annu. Rev. Ecol. Evol. Syst. 2020, 51, 27–53. [Google Scholar] [CrossRef]
  3. Reis, R.E.; Kullander, S.O.; Ferraris, C.J., Jr. Check List of The Freshwater Fishes of South and Central America; Edipucrs: Porto Alegre, Brazil, 2003. [Google Scholar]
  4. Do Nascimento, V.D.; Coelho, K.A.; Nogaroto, V.; de Almeida, R.B.; Ziemniczak, K.; Centofante, L.; Pavanelli, C.S.; Torres, R.A.; Moreira-Filho, O.; Vicari, M.R. Do multiple karyomorphs and population genetics of freshwater darter characines (Apareiodon affinis) indicate chromosomal speciation? Zool. Anz. 2018, 272, 93–103. [Google Scholar] [CrossRef]
  5. De Oliveira, J.C.; Oyakawa, O.T. New loricariid fishes from headwaters on serra da mantiqueira and complexo do espinhaço, Minas Gerais state, Brazil (Teleostei: Siluriformes: Loricariidae). Zootaxa 2019, 4586, 401–424. [Google Scholar] [CrossRef] [PubMed]
  6. Buckup, P.A. Sistemática e biogeografia de peixes de riachos. Oecologia Aust. 1999, 06, 91–138. [Google Scholar] [CrossRef]
  7. Covain, R.; Fisch-Muller, S.; Oliveira, C.; Mol, J.H.; Montoya-Burgos, J.I.; Dray, S. Molecular phylogeny of the highly diversified catfish subfamily Loricariinae (Siluriformes, Loricariidae) reveals incongruences with morphological classification. Mol. Phylogenet. Evol. 2016, 94, 492–517. [Google Scholar] [CrossRef]
  8. Blanco, D.R.; Vicari, M.R.; Lui, R.L.; Traldi, J.B.; Bueno, V.; Martinez, J.d.F.; Brandão, H.; Oyakawa, O.T.; Moreira Filho, O. Karyotype Diversity and Evolutionary Trends in Armored Catfish Species of the Genus Harttia (Siluriformes: Loricariidae). Zebrafish 2017, 14, 169–176. [Google Scholar] [CrossRef]
  9. Blanco, D.R.; Vicari, M.R.; Lui, R.L.; Bertollo, L.A.C.; Traldi, J.B.; Moreira-Filho, O. The role of the Robertsonian rearrangements in the origin of the XX/XY1Y2 sex chromosome system and in the chromosomal differentiation in Harttia species (Siluriformes, Loricariidae). Rev. Fish Biol. Fish. 2013, 23, 127–134. [Google Scholar] [CrossRef]
  10. Blanco, D.R.; Vicari, M.R.; Lui, R.L.; Artoni, R.F.; de Almeida, M.C.; Traldi, J.B.; Margarido, V.P.; Moreira-Filho, O. Origin of the X1X1X2X2/X1X2Y sex chromosome system of Harttia punctata (Siluriformes, Loricariidae) inferred from chromosome painting and FISH with ribosomal DNA markers. Genetica 2014, 142, 119–126. [Google Scholar] [CrossRef]
  11. Sassi, F.d.M.C.; Deon, G.A.; Moreira-filho, O.; Vicari, M.R.; Bertollo, L.A.C.; Liehr, T.; de Oliveira, E.A.; Cioffi, M.B. Multiple Sex Chromosomes and Evolutionary Relationships in Amazonian Catfishes: The Outstanding Model of the Genus Harttia (Siluriformes: Loricariidae). Genes (Basel) 2020, 11, 1179. [Google Scholar] [CrossRef]
  12. Charlesworth, D.; Charlesworth, B.; Marais, G. Steps in the evolution of heteromorphic sex chromosomes. Heredity (Edinb) 2005, 95, 118–128. [Google Scholar] [CrossRef] [Green Version]
  13. Steinemann, S.; Steinemann, M. Retroelements: Tools for sex chromosome evolution. Cytogenet. Genome Res. 2005, 110, 134–143. [Google Scholar] [CrossRef] [PubMed]
  14. Schemberger, M.O.; Nascimento, V.D.; Coan, R.; Ramos, É.; Nogaroto, V.; Ziemniczak, K.; Valente, G.T.; Moreira-Filho, O.; Martins, C.; Vicari, M.R. DNA transposon invasion and microsatellite accumulation guide W chromosome differentiation in a Neotropical fish genome. Chromosoma 2019, 128, 547–560. [Google Scholar] [CrossRef]
  15. Moreira-Filho, O.; Bertollo, L.A.C.; Junior, P.M.G. Evidences for a multiple sex chromosome system with female heterogamety in Apareiodon affinis (Pisces, Parodontidae). Caryologia 1980, 33, 83–91. [Google Scholar] [CrossRef]
  16. Bertollo, L.A.C.; Fontes, M.S.; Fenocchio, A.S.; Cano, J. The X1X2Y sex chromosome system in the fish Hoplias malabaricus. I. G-, C- and chromosome replication banding. Chromosom. Res. 1997, 5, 493–499. [Google Scholar] [CrossRef]
  17. Schemberger, M.O.; Bellafronte, E.; Nogaroto, V.; Almeida, M.C.; Schühli, G.S.; Artoni, R.F.; Moreira-Filho, O.; Vicari, M.R. Differentiation of repetitive DNA sites and sex chromosome systems reveal closely related group in Parodontidae (Actinopterygii: Characiformes). Genetica 2011, 139, 1499–1508. [Google Scholar] [CrossRef] [PubMed]
  18. Kitano, J.; Peichel, C.L. Turnover of sex chromosomes and speciation in fishes. Environ. Biol. Fishes 2012, 94, 549–558. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. De Oliveira, E.A.; Sember, A.; Bertollo, L.A.C.; Yano, C.F.; Ezaz, T.; Moreira-Filho, O.; Hatanaka, T.; Trifonov, V.; Liehr, T.; Al-Rikabi, A.B.H.; et al. Tracking the evolutionary pathway of sex chromosomes among fishes: Characterizing the unique XX/XY1Y2 system in Hoplias malabaricus (Teleostei, Characiformes). Chromosoma 2017, 127, 115–128. [Google Scholar] [CrossRef]
  20. Cioffi, M.d.B.; Yano, C.F.; Sember, A.; Bertollo, L.A.C. Chromosomal evolution in lower vertebrates: Sex chromosomes in neotropical fishes. Genes (Basel). 2017, 8, 258. [Google Scholar] [CrossRef]
  21. Yano, C.F.; Bertollo, L.A.C.; Ezaz, T.; Trifonov, V.; Sember, A.; Liehr, T.; Cioffi, M.d.B. Highly conserved Z and molecularly diverged W chromosomes in the fish genus Triportheus (Characiformes, Triportheidae). Heredity (Edinb). 2017, 118, 276–283. [Google Scholar] [CrossRef] [Green Version]
  22. De Moraes, R.L.; Sember, A.; Bertollo, L.A.; de Oliveira, E.A.; Rab, P.; Hatanaka, T.; Marinho, M.M.; Liehr, T.; Al-Rikabi, A.B.; Feldberg, E.; et al. Comparative cytogenetics and neo-Y formation in small-sized fish species of the genus Pyrrhulina (Characiformes, Lebiasinidae). Front. Genet. 2019, 10, 1–13. [Google Scholar] [CrossRef] [Green Version]
  23. Rodrigues, R.M. Estudos Cromossômicos e Moleculares em Loricariinae com ênfase em espécies de Rineloricaria (Silurifomes, Loricariidae): Uma perspectiva evolutiva. Ph.D. Thesis, Universidade de São Paulo, São Paulo, Brazil, 2010. [Google Scholar]
  24. Blanco, D.R.; Vicari, M.R.; Artoni, R.F.; Traldi, J.B.; Moreira-Filho, O. Chromosomal characterization of armored catfish Harttia longipinna (Siluriformes, Loricariidae): First report of B chromosomes in the genus. Zoolog. Sci. 2012, 29, 604–609. [Google Scholar] [CrossRef] [PubMed]
  25. Kavalco, K.F.; Pazza, R.; Bertollo, L.A.C.; Moreira-Filho, O. Heterochromatin characterization of four fish species of the family Loricariidae (Siluriformes). Hereditas 2004, 141, 237–242. [Google Scholar] [CrossRef] [PubMed]
  26. Bertollo, L.A.C.; de Bello Cioffi, M.; Galetti, P.M.; Moreira-Filho, O. Contributions to the cytogenetics of the Neotropical fish fauna. Comp. Cytogenet. 2017, 11, 665–690. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Sumner, A.T. A simple technique for demonstrating centromeric heterochromatin. Exp. Cell Res. 1972, 75, 304–306. [Google Scholar] [CrossRef]
  28. Lui, R.L.; Blanco, D.R.; Moreira-Filho, O.; Margarido, V.P. Propidium iodide for making heterochromatin more evident in the C-banding technique. Biotech. Histochem. 2012, 87, 433–438. [Google Scholar] [CrossRef]
  29. Pendas, A.M.; Moran, P.; Freije, J.P.; Garcia-Vazquez, E. Chromosomal mapping and nucleotide sequence of two tandem repeats of Atlantic salmon 5S rDNA. Cytogenet. Genome Res. 1994, 67, 31–36. [Google Scholar] [CrossRef]
  30. Cioffi, M.d.B.; Martins, C.; Centofante, L.; Jacobina, U.; Bertollo, L.A.C. Chromosomal variability among allopatric populations of Erythrinidae fish Hoplias malabaricus: Mapping of three classes of repetitive DNAs. Cytogenet. Genome Res. 2009, 125, 132–141. [Google Scholar] [CrossRef]
  31. Yano, C.F.; Bertollo, L.A.C.; Cioffi, M.d.B. Fish-FISH: Molecular Cytogenetics in Fish Species. In Fluorescence In Situ Hybridization (FISH) Application Guide, 2nd ed.; Liehr, T., Ed.; Springer: Berlin, Germany, 2017; ISBN 9783662529577. [Google Scholar]
  32. Sambrook, J.; Russell, D.W. Molecular Cloning: A laboratory Manual; Sambrook, J., Russell, D.W., Eds.; Cold Spring Harbor Laboratory Press: New York, NY, USA, 2001. [Google Scholar]
  33. Zwick, M.S.; Hanson, R.E.; McKnight, T.D.; Islam-Faridi, M.N.; Stelly, D.M.; Wing, R.A.; Price, H.J. A rapid procedure for the isolation of C0t-1 DNA from plants. Genome 1997, 40, 138–142. [Google Scholar] [CrossRef] [Green Version]
  34. De Moraes, R.L.R.; Bertollo, L.A.C.; Marinho, M.M.F.; Yano, C.F.; Hatanaka, T.; Barby, F.F.; Troy, W.P.; Cioffi, M.d.B. Evolutionary relationships and cytotaxonomy considerations in the genus Pyrrhulina (Characiformes, Lebiasinidae). Zebrafish 2017, 14, 536–546. [Google Scholar] [CrossRef]
  35. Sassi, F.d.M.C.; de Oliveira, E.A.; Bertollo, L.A.C.; Nirchio, M.; Hatanaka, T.; Marinho, M.M.F.; Moreira-Filho, O.; Aroutiounian, R.; Liehr, T.; Al-Rikabi, A.B.H.; et al. Chromosomal evolution and evolutionary relationships of Lebiasina species (Characiformes, Lebiasinidae). Int. J. Mol. Sci. 2019, 20, 2944. [Google Scholar] [CrossRef] [Green Version]
  36. Toma, G.A.; de Moraes, R.L.R.; Sassi, F.d.M.C.; Bertollo, L.A.C.; de Oliveira, E.A.; Rab, P.; Sember, A.; Liehr, T.; Hatanaka, T.; Viana, P.F.; et al. Cytogenetics of the small-sized fish, Copeina guttata (Characiformes, Lebiasinidae): Novel insights into the karyotype differentiation of the family. PLoS ONE 2019, 14, e0226746. [Google Scholar] [CrossRef]
  37. Symonová, R.; Sember, A.; Maitánová, Z.; Ráb, P. Characterization of Fish Genome by GISH and CGH. In Fish Cytogenetic Techniques. Ray-Fin Fishes and Chondrichthyans; Ozouf-Costaz, C., Pisano, E., Foresti, F., Almeida Toledo, L.F., Eds.; CRC Press: Boca Ranton, FL, USA, 2015; pp. 118–131. [Google Scholar]
  38. Levan, A.; Fredga, K.; Sandberg, A.A. Nomenclature for Centromeric Position on Chromosomes. Hereditas 1964, 52, 201–220. [Google Scholar] [CrossRef]
  39. Mariotto, S.; Centofante, L.; Vicari, M.R.; Artoni, R.F.; Moreira-Filho, O. Chromosomal diversification in ribosomal DNA sites in Ancistrus Kner, 1854 (Loricariidae, Ancistrini) from three hydrographic basins of Mato Grosso, Brazil. Comp. Cytogenet. 2011, 5, 31–42. [Google Scholar] [CrossRef] [PubMed]
  40. Barros, A.V.; Wolski, M.A.V.; Nogaroto, V.; Almeida, M.C.; Moreira-Filho, O.; Vicari, M.R. Fragile sites, dysfunctional telomere and chromosome fusions: What is 5S rDNA role? Gene 2017, 608, 20–27. [Google Scholar] [CrossRef] [PubMed]
  41. Glugoski, L.; Giuliano-Caetano, L.; Moreira-Filho, O.; Vicari, M.R.; Nogaroto, V. Co-located hAT transposable element and 5S rDNA in an interstitial telomeric sequence suggest the formation of Robertsonian fusion in armored catfish. Gene 2018, 650, 49–54. [Google Scholar] [CrossRef]
  42. Glugoski, L.; Deon, G.; Schott, S.; Vicari, M.R.; Nogaroto, V.; Moreira-Filho, O. Comparative cytogenetic analyses in Ancistrus species (Siluriformes: Loricariidae). Neotrop. Ichthyol. 2020, 18, 1–16. [Google Scholar] [CrossRef]
  43. Pinna, M.C.C. Phylogenetic Relationships of Neotropical Siluriformes (Teleostei: Ostariophysi): Historical Overview and Synthesis of Hypotheses. In Phylogeny and Classification of Neotropical Fishes; Malabarba, L.R., Reis, R.E., Vari, R.P., Lucena, Z.M., Lucena, C.A.S., Eds.; Edipucrs: Porto Alegre, Brazil, 1998; pp. 279–330. [Google Scholar]
  44. Wright, S. Breeding Structure of Populations in Relation to Speciation. Amer Natur 1940, 74, 232–248. [Google Scholar] [CrossRef]
  45. King, M. Species Evolution; Cambridge University Press: Cambridge, UK, 1993. [Google Scholar]
  46. Rieseberg, L.H. Chromosomal rearrangements and speciation. TRENDS Ecol. Evol. 2001, 16, 351–358. [Google Scholar] [CrossRef]
  47. Faria, R.; Navarro, A. Chromosomal speciation revisited: Rearranging theory with pieces of evidence. Trends Ecol. Evol. 2010, 25, 660–669. [Google Scholar] [CrossRef]
  48. Rosa, K.O.; Ziemniczak, K.; de Barros, A.V.; Nogaroto, V.; Almeida, M.C.; Cestari, M.M.; Artoni, R.F.; Vicari, M.R. Numeric and structural chromosome polymorphism in Rineloricaria lima (Siluriformes: Loricariidae): Fusion points carrying 5S rDNA or telomere sequence vestiges. Rev. Fish Biol. Fish. 2012, 22, 739–749. [Google Scholar] [CrossRef]
  49. Porto, F.E.; de Rossi Vieira, M.M.; Barbosa, L.M.; Borin-Carvalho, L.A.; Vicari, M.R.; De Brito Portela-Castro, A.L.; Martins-Santos, I.C. Chromosomal polymorphism in Rineloricaria lanceolata Günther, 1868 (Loricariidae: Loricariinae) of the paraguay basin (Mato Grosso do Sul, Brazil): Evidence of fusions and their consequences in the population. Zebrafish 2014, 11, 318–324. [Google Scholar] [CrossRef] [PubMed]
  50. Primo, C.C.; Glugoski, L.; Almeida, M.C.; Zawadzki, C.H.; Moreira-Filho, O.; Vicari, M.R.; Nogaroto, V. Mechanisms of Chromosomal Diversification in Species of Rineloricaria (Actinopterygii: Siluriformes: Loricariidae). Zebrafish 2017, 14, 161–168. [Google Scholar] [CrossRef] [PubMed]
  51. Ruiz-Herrera, A.; Nergadze, S.G.; Santagostino, M.; Giulotto, E. Telomeric repeats far from the ends: Mechanisms of origin and role in evolution. Cytogenet. Genome Res. 2008, 122, 219–228. [Google Scholar] [CrossRef] [PubMed]
  52. Marajó, L.; Viana, P.F.; Ferreira, M.; Py-Daniel, L.H.R.; Feldberg, E. Cytogenetics of two Farlowella species (Loricariidae: Loricariinae): Implications on the taxonomic status of the species. Neotrop. Ichthyol. 2018, 16, 1–8. [Google Scholar] [CrossRef] [Green Version]
  53. Ocalewicz, K. Telomeres in fishes. Cytogenet. Genome Res. 2013, 141, 114–125. [Google Scholar] [CrossRef]
  54. Garagna, S.; Broccoli, D.; Redi, C.A.; Searle, J.B.; Cooke, H.J.; Capanna, E. Robertsonian metacentrics of the house mouse lose telomeric sequences but retain some minor satellite DNA in the pericentromeric area. Chromosoma 1995, 103, 685–692. [Google Scholar] [CrossRef]
  55. Slijepcevic, P. Telomere length and telomere-centromere relationships? Mutat. Res.-Fundam. Mol. Mech. Mutagen. 1998, 404, 215–220. [Google Scholar] [CrossRef]
  56. Bolzán, A.D. Interstitial telomeric sequences in vertebrate chromosomes: Origin, function, instability and evolution. Mutat. Res.-Rev. Mutat. Res. 2017, 773, 51–65. [Google Scholar] [CrossRef]
  57. Slijepcevic, P.; Hande, M.P.; Bouffler, S.D.; Lansdorp, P.; Bryant, P.E. Telomere length, chromatin structure and chromosome fusigenic potential. Chromosoma 1997, 106, 413–421. [Google Scholar] [CrossRef]
  58. Fumagalli, M.; Rossiello, F.; Clerici, M.; Barozzi, S.; Cittaro, D.; Kaplunov, J.M.; Bucci, G.; Dobreva, M.; Matti, V.; Beausejour, C.M.; et al. Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation. Nat. Cell Biol. 2012, 14, 355–365. [Google Scholar] [CrossRef] [Green Version]
  59. Sfeir, A.; Kosiyatrakul, S.T.; Hockemeyer, D.; MacRae, S.L.; Karlseder, J.; Schildkraut, C.L.; de Lange, T. Mammalian Telomeres Resemble Fragile Sites and Require TRF1 for Efficient Replication. Cell 2009, 138, 90–103. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Perry, J.; Slater, H.R.; Choo, K.H.A. Centric fission—Simple and complex mechanisms. Chromosom. Res. 2004, 12, 627–640. [Google Scholar] [CrossRef] [PubMed]
  61. Slijepcevic, P. Mechanisms of the evolutionary chromosome plasticity: Integrating the “centromere-from-telomere” hypothesis with telomere length regulation. Cytogenet. Genome Res. 2016, 148, 268–278. [Google Scholar] [CrossRef] [PubMed]
  62. Navarro, A.; Barton, N.H. Accumulating postzygotic isolation genes in parapatry: A new twist on chromosomal speciation. Evolution (N. Y.) 2003, 57, 447–459. [Google Scholar] [CrossRef]
  63. Lin, K.W.; Yan, J. Endings in the middle: Current knowledge of interstitial telomeric sequences. Mutat. Res.-Rev. Mutat. Res. 2008, 658, 95–110. [Google Scholar] [CrossRef] [PubMed]
  64. Ruiz-Herrera, A.; Castresana, J.; Robinson, T.J. Is mammalian chromosomal evolution driven by regions of genome fragility? Genome Biol. 2006, 7, 1–16. [Google Scholar] [CrossRef] [Green Version]
  65. Carbone, L.; Harris, R.A.; Vessere, G.M.; Mootnick, A.R.; Humphray, S.; Rogers, J.; Kim, S.K.; Wall, J.D.; Martin, D.; Jurka, J.; et al. Evolutionary breakpoints in the gibbon suggest association between cytosine methylation and karyotype evolution. PLoS Genet. 2009, 5, 1–10. [Google Scholar] [CrossRef]
  66. Longo, M.S.; Carone, D.M.; Green, E.D.; O’Neill, M.J.; O’Neill, R.J. Distinct retroelement classes define evolutionary breakpoints demarcating sites of evolutionary novelty. BMC Genom. 2009, 10, 1–14. [Google Scholar] [CrossRef] [Green Version]
  67. Farré, M.; Bosch, M.; López-Giráldez, F.; Ponsà, M.; Ruiz-Herrera, A. Assessing the role of tandem repeats in shaping the genomic architecture of great apes. PLoS ONE 2011, 6, e27239. [Google Scholar] [CrossRef] [Green Version]
  68. Arai, R. Fish Karyotypes: A Check List; Springer Science & Business Media: Tokyo, Japan, 2011. [Google Scholar]
  69. Pennell, M.W.; Kirkpatrick, M.; Otto, S.P.; Vamosi, J.C.; Peichel, C.L.; Valenzuela, N.; Kitano, J. Y Fuse? Sex Chromosome Fusions in Fishes and Reptiles. PLoS Genet. 2015, 11, 1–18. [Google Scholar] [CrossRef] [Green Version]
  70. Mank, J.E.; Avise, J.C. Evolutionary diversity and turn-over of sex determination in teleost fishes. Sex. Dev. 2009, 3, 60–67. [Google Scholar] [CrossRef] [PubMed]
  71. Frolov, S.V. Differentiation of sex chromosomes in the Salmonidae. III. Multiple sex chromosomes in Coregonus sardinella. Tsitologiya 1990, 32, 659–663. [Google Scholar]
  72. Ozouf-Costaz, C.; Hureau, J.; Beaunier, M. Chromosome studies on fish of the suborder Notothenioidei collected in the weddell sea during epos 3 cruise. Cybium (Paris) 1991, 15, 271–289. [Google Scholar]
  73. Sember, A.; Bohlen, J.; Šlechtová, V.; Altmanová, M.; Symonová, R.; Ráb, P. Karyotype differentiation in 19 species of river loach fishes (Nemacheilidae, Teleostei): Extensive variability associated with rDNA and heterochromatin distribution and its phylogenetic and ecological interpretation. BMC Evol. Biol. 2015, 15, 1–22. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. Bertollo, L.A.C.; Filho, O.M.; Takahashi, C.S. Multiple Sex Chromosomes in the Genus Hoplias (Pisces: Erythrinidae). Cytologia (Tokyo). 1983, 48, 1–12. [Google Scholar] [CrossRef] [Green Version]
  75. De Oliveira, R.R.; Feldberg, E.; Dos Anjos, M.B.; Zuanon, J. Occurrence of multiple sexual chromosomes (XX/XY1Y2 and Z1Z1Z2Z2/Z1Z2W1W2) in catfishes of the genus Ancistrus (Siluriformes: Loricariidae) from the Amazon basin. Genetica 2008, 134, 243–249. [Google Scholar] [CrossRef]
  76. Almeida, J.S.; Migues, V.H.; Diniz, D.; Affonso, P.R.A.M. A unique sex chromosome system in the knifefish Gymnotus bahianus with inferences about chromosomal evolution of gymnotidae. J. Hered. 2015, 106, 177–183. [Google Scholar] [CrossRef] [Green Version]
  77. Favarato, R.M.; Da Silva, M.; De Oliveira, R.R.; Artoni, R.F.; Feldberg, E.; Matoso, D.A. Cytogenetic Diversity and the Evolutionary Dynamics of rDNA Genes and Telomeric Sequences in the Ancistrus Genus (Loricariidae: Ancistrini). Zebrafish 2016, 13, 103–111. [Google Scholar] [CrossRef]
  78. Bertollo, L.A.C.; Born, G.G.; Dergam, J.A.; Fenocchio, A.S.; Moreira-Filho, O. A biodiversity approach in the neotropical Erythrinidae fish, Hoplias malabaricus. Karyotypic survey, geographic distribution of cytotypes and cytotaxonomic considerations. Chromosom. Res. 2000, 8, 603–613. [Google Scholar] [CrossRef]
  79. Centofante, L.; Bertollo, L.A.C.; Moreira-Filho, O. Cytogenetic characterization and description of an XX/XY1Y2 sex chromosome system in catfish Harttia carvalhoi (Siluriformes, Loricariidae). Cytogenet. Genome Res. 2006, 112, 320–324. [Google Scholar] [CrossRef]
  80. Traldi, J.B.; Lui, R.L.; Martinez, J.d.F.; Vicari, M.R.; Nogaroto, V.; Moreira-Filho, O.; Blanco, D.R. Chromosomal distribution of the retroelements Rex1, Rex3 and Rex6 in species of the genus Harttia and Hypostomus (Siluriformes: Loricariidae). Neotrop. Ichthyol. 2019, 17, 1–10. [Google Scholar] [CrossRef] [Green Version]
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Figure 1. Partial map of South America highlighting the Brazilian collection sites of the three Harttia species analyzed in the present work (color circles) named: 1. H. intermontana (red circle); 2. Harttia sp. 1 (yellow circle); and 3. Harttia sp. 2 (green circle). The white circles represent other Harttia species previously studied: 4. H. absaberi, 5. H. carvalhoi, 6. H. gracilis, 7. H. loricariformis, 8. H. kronei, 9. H. torrenticola, 10. H. longipinna, 11. H. punctata; 12. H. rondoni; 13. H. villasboas, and 14. H. duriventris. Map created using QGis 3.4.3 and Adobe Photoshop CC 2020.
Figure 1. Partial map of South America highlighting the Brazilian collection sites of the three Harttia species analyzed in the present work (color circles) named: 1. H. intermontana (red circle); 2. Harttia sp. 1 (yellow circle); and 3. Harttia sp. 2 (green circle). The white circles represent other Harttia species previously studied: 4. H. absaberi, 5. H. carvalhoi, 6. H. gracilis, 7. H. loricariformis, 8. H. kronei, 9. H. torrenticola, 10. H. longipinna, 11. H. punctata; 12. H. rondoni; 13. H. villasboas, and 14. H. duriventris. Map created using QGis 3.4.3 and Adobe Photoshop CC 2020.
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Figure 2. Karyotypes of H. intermontana (a,b), Harttia sp. 1 (c,d), and Harttia sp. 2 (e,f), showing sequentially Giemsa-stained (a,c,e) and C-banded (b,d,f) chromosomes. Insets depict the male sex chromosomes. Bar = 5 μm.
Figure 2. Karyotypes of H. intermontana (a,b), Harttia sp. 1 (c,d), and Harttia sp. 2 (e,f), showing sequentially Giemsa-stained (a,c,e) and C-banded (b,d,f) chromosomes. Insets depict the male sex chromosomes. Bar = 5 μm.
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Figure 3. Karyotypes of H. intermontana (a,b), Harttia sp. 1 (c,d), and Harttia sp. 2 (e,f) arranged after FISH with 5S rDNA (red) and 18S rDNA (green) probes (a,c,e), and telomeric (TTAGGG)n probe (b,d,f). Inserts depict the male sex chromosomes. Arrowheads indicate the interstitial telomeric sites (ITS) locations and the (*) signals the joint localization of the 18S and ITS sites. Bar = 5 μm.
Figure 3. Karyotypes of H. intermontana (a,b), Harttia sp. 1 (c,d), and Harttia sp. 2 (e,f) arranged after FISH with 5S rDNA (red) and 18S rDNA (green) probes (a,c,e), and telomeric (TTAGGG)n probe (b,d,f). Inserts depict the male sex chromosomes. Arrowheads indicate the interstitial telomeric sites (ITS) locations and the (*) signals the joint localization of the 18S and ITS sites. Bar = 5 μm.
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Figure 4. Mitotic chromosome spreads of males H. intermontana (A–D) and Harttia sp. 1 (E–H) after intraspecific genomic hybridizations, with male- and female-derived genomic probes hybridized together for each species. The first column (A,E): DAPI images (blue); Second column (B,F): hybridization pattern for the male-derived probe (red); Third column (C,G): hybridization pattern for the female-derived probe (green); Fourth column (D,H): merged images for both genomic probes and DAPI staining. The common genomic regions for males and females are depicted in yellow. Arrows indicate the sex chromosomes. Bar = 10 μm.
Figure 4. Mitotic chromosome spreads of males H. intermontana (A–D) and Harttia sp. 1 (E–H) after intraspecific genomic hybridizations, with male- and female-derived genomic probes hybridized together for each species. The first column (A,E): DAPI images (blue); Second column (B,F): hybridization pattern for the male-derived probe (red); Third column (C,G): hybridization pattern for the female-derived probe (green); Fourth column (D,H): merged images for both genomic probes and DAPI staining. The common genomic regions for males and females are depicted in yellow. Arrows indicate the sex chromosomes. Bar = 10 μm.
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Figure 5. Mitotic chromosome spreads of males from H. intermontana (A–D) after comparative genomic hybridization (CGH): interspecific hybridizations probed with a male-derived genomic probe from H. intermontana (B), Harttia sp. 1 (C), and H. carvalhoi (D). (A) depicts the merged images of the genomic probes and DAPI staining. The common genomic regions for male and female are depicted in yellow. Sex chromosomes are indicated. Bar = 10 μm.
Figure 5. Mitotic chromosome spreads of males from H. intermontana (A–D) after comparative genomic hybridization (CGH): interspecific hybridizations probed with a male-derived genomic probe from H. intermontana (B), Harttia sp. 1 (C), and H. carvalhoi (D). (A) depicts the merged images of the genomic probes and DAPI staining. The common genomic regions for male and female are depicted in yellow. Sex chromosomes are indicated. Bar = 10 μm.
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Genes 11 01366 g006 550
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.
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Table
Table 1. Available cytogenetic data for Harttia species.
Table 1. Available cytogenetic data for Harttia species.
Species2nKaryotypeFN 1References
Harttia absaberi♀♂6213m + 23sm + 16st + 10a114[23]
Harttia carvalhoi52♀, 53♂16m + 16sm + 12st + 8a ♀
15m + 16sm + 12st + 10a ♂		96
96		[9]
Harttia duriventris56♀, 55♂16m + 16sm + 16st + 8a ♀
17m + 16sm + 16st + 6a ♂		104
104		[11]
Harttia gracilis♀♂5820m + 22sm + 8st + 8a108[8]
Harttia intermontana52♀, 53♂14m + 12sm + 12st + 14a ♀
13m + 12sm + 13st + 15a ♂		90
91		Present study
Harttia kronei♀♂5816m + 16sm + 16st + 10a106[8]
Harttia longipinna♀♂58 + 0 − 2 Bs16m + 12sm + 16st + 14a102[24]
Harttia loricariformis♀♂5616m + 22sm + 10st + 8a104[25]
Harttia punctata58♀, 57♂16m + 20sm + 12st + 10a ♀
16m + 21sm + 12st + 8a ♂		106
106		[10]
Harttia rondoni♀♂5420m + 26sm + 4st + 4a104[11]
Harttia torrenticola♀♂5616m + 10sm + 16st + 14a98[9]
Harttia villasboas56♀, 55♂18m + 24sm + 6st + 8a ♀
19m + 24sm + 6st + 6a ♂		104
104		[11]
Harttia sp. 1 (Macacos stream)56♀, 57♂14m + 14sm + 10st + 18a ♀
13m + 14sm + 10st + 20a ♂		94
94		Present study
Harttia sp. 2 (Barra Grande river)♀♂6216m + 14sm + 12st + 20a104Present study
1 FN = fundamental number.
Table
Table 2. Collection sites, sample sizes (n), and sex of the Harttia species analyzed.
Table 2. Collection sites, sample sizes (n), and sex of the Harttia species analyzed.
SpeciesLocalityn
1. 
Harttia intermontana
Piranga river, Carandaí, MG (Brazil) (20°59’34.0″ S, 43°43′30.0″ W)20♀, 13♂
2. 
Harttia sp. 1
Macacos stream, Silveira, SP (Brazil) (22°40’43.0″ S, 44°51′25.0″ W)10♀, 7♂
3. 
Harttia sp. 2
Barra Grande river, Prudentópolis, PR (Brazil) (24°58′40.72″ S, 51°7′34.25″ W)17♀, 11♂
Table
Table 3. Multiple XX/XY1Y2 sex chromosome systems currently found in teleosts.
Table 3. Multiple XX/XY1Y2 sex chromosome systems currently found in teleosts.
Species2nMechanism of OriginReference
Bathydraco marri38♀, 39♂Y-chromosome fission[72]
Coregonus sardinella80♀, 81♂Y-chromosome fission[71]
Schistura cf. fasciolata50♀, 51♂Y-chromosome fission[73]
Hoplias malabaricus (karyomorph G)40♀, 41♂Tandem fusion X-A[19,74,78]
Gymnotus bahianus36♀, 37♂Tandem fusion X-A[76]
Ancistrus dubius38♀, 39♂X-A tandem fusion and further neo-Y chromosome fission[75,77]
Harttia carvalhoi52♀, 53♂Y-chromosome fission[8,9,79,80]
Harttia intermontana52♀, 53♂X-A tandem fusion and further neo-Y chromosome fissionPresent study
Harttia sp. 156♀, 57♂X-A tandem fusion and further neo-Y chromosome fissionPresent study
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Deon, G.A.; Glugoski, L.; Vicari, M.R.; Nogaroto, V.; Sassi, F.d.M.C.; Cioffi, M.d.B.; Liehr, T.; Bertollo, L.A.C.; Moreira-Filho, O. Highly Rearranged Karyotypes and Multiple Sex Chromosome Systems in Armored Catfishes from the Genus Harttia (Teleostei, Siluriformes). Genes 2020, 11, 1366. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11111366

AMA Style

Deon GA, Glugoski L, Vicari MR, Nogaroto V, Sassi FdMC, Cioffi MdB, Liehr T, Bertollo LAC, Moreira-Filho O. Highly Rearranged Karyotypes and Multiple Sex Chromosome Systems in Armored Catfishes from the Genus Harttia (Teleostei, Siluriformes). Genes. 2020; 11(11):1366. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11111366

Chicago/Turabian Style

Deon, Geize Aparecida, Larissa Glugoski, Marcelo Ricardo Vicari, Viviane Nogaroto, Francisco de Menezes Cavalcante Sassi, Marcelo de Bello Cioffi, Thomas Liehr, Luiz Antonio Carlos Bertollo, and Orlando Moreira-Filho. 2020. "Highly Rearranged Karyotypes and Multiple Sex Chromosome Systems in Armored Catfishes from the Genus Harttia (Teleostei, Siluriformes)" Genes 11, no. 11: 1366. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11111366

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