Chromosomes of Asian Cyprinid Fishes: Genomic Differences in Conserved Karyotypes of ‘Poropuntiinae’ (Teleostei, Cyprinidae)

Simple Summary All Poropuntiinae fish species are diploid and have 50 chromosomes in their cells; however, their karyotypes differ (the organization of chromosomes according to size and shape). The goal of this study is to compare the genomic differences between their conserved karyotypes using conventional and molecular cytogenetic methods. We found distinct patterns in the distribution of ribosomal DNA and microsatellites, indicating that, while their karyotypes are conserved, these fishes have species-specific patterns. Our comparative genomic hybridization experiment reveals that any of their repetitive DNA content matches, highlighting the differences between such species. This study adds to our understanding of chromosome evolution in Cyprinidae fishes, which include diploid, tetraploid, and hexaploid species. Abstract The representatives of cyprinid lineage ‘Poropuntiinae’ with 16 recognized genera and around 100 species form a significant part of Southeast Asian ichthyofauna. Cytogenetics are valuable when studying fish evolution, especially the dynamics of repetitive DNAs, such as ribosomal DNAs (5S and 18S) and microsatellites, that can vary between species. Here, karyotypes of seven ‘poropuntiin’ species, namely Cosmochilus harmandi, Cyclocheilichthys apogon, Hypsibarbus malcomi, H. wetmorei, Mystacoleucus chilopterus, M. ectypus, and Puntioplties proctozysron occurring in Thailand were examined using conventional and molecular cytogenetic protocols. Variable numbers of uni- and bi-armed chromosomes indicated widespread chromosome rearrangements with a stable diploid chromosome number (2n) of 50. Examination with fluorescence in situ hybridization using major and minor ribosomal probes showed that Cosmochilus harmandi, Cyclocheilichthys apogon, and Puntioplites proctozystron all had one chromosomal pair with 5S rDNA sites. However, more than two sites were found in Hypsibarbus malcolmi, H. wetmorei, Mystacoleucus chilopterus, and M. ectypus. The number of chromosomes with 18S rDNA sites varied amongst their karyotypes from one to three; additionally, comparative genomic hybridization and microsatellite patterns varied among species. Our results reinforce the trend of chromosomal evolution in cyprinifom fishes, with major chromosomal rearrangements, while conserving their 2n.

structural genes or between other repetitive sequences [10]. By this way, the mapping of such sequences provides insights into intrachromosomal rearrangements and evolution of related species karyotypes. By identifying chromosomal markers associated with desirable traits and facilitating the production of genetically improved fish stocks, cytogenetic studies have also contributed to fish breeding and aquaculture. Table 1. Review of available cytogenetic data for representatives of 'Poropuntiinae' species analyzed up to now. The species analyzed in this study are highlighted in red. Chromosomes were classified following their arm ratios in m = metacentric, sm = submetacentric, st = subtelocentric, and a = acrocentric, and their fundamental number (NF, i.e., number of arms) are also displayed [24]. Nucleolar organizer regions (NORs)/18S rDNA carrying pairs are highlighted.

Species 2n NF Karyotype NORs/18S rDNA Pairs Reference
The present study examined the chromosomal diversity of the 'poropuntiinae' fishes/ species from Thailand, namely Cosmochilus harmandi, Cyclocheilichthys apogon, Hypsibarbus malcomi, H. wetmorei, Mystacoleucus chilopterus, M. ectypus, and Puntioplties proctozysron, using conventional (Giemsa staining and Ag-NOR impregnation) and molecular (distribution of repetitive DNA sequences using FISH with respective probes and CGH) cytogenetic tools. Our findings support the idea that cyprinifom fishes have undergone significant chromosomal rearrangements while maintaining their 2n. Overall, the findings demonstrated that the evolution of this cyprinid lineage was significantly influenced by structural chromosomal rearrangements such as pericentric inversions.

Individuals, Mitotic Chromosome Preparation and Ag-NOR Banding
Individuals/fishes of seven representative 'poropuntiins' were collected from different natural ecosystems of wild regions in Thailand ( Figure 1). Table 2 lists the numbers, sex, and locations of the individuals investigated. The specimens as vouchers were deposited in the fish collection of the Cytogenetic Laboratory, Department of Biology, Faculty of Science (KhonKaen University). All species analyzed here were properly identified using morphological criteria [46]. Mitotic chromosomes were obtained from the anterior kidney [47] and stained with 5% Giemsa. In brief, the animals were first injected in the abdomen with a 0.025% aqueous colchicine solution at a dose of 1 mL/100 g of weight.
Then, specimens were euthanized after 50-60 min for the obtention of the rear kidney. Cells were dissociated with a sterile syringe in 5 mL of 0.075 M potassium chloride (KCl) and left for hypothonization at 37 • C for 25 min. Finally, turgid cells were fixed in Carnoy 2 (methanol 3:1 acetic acid) before being dropped into slides. The distribution of nucleolar organizer regions (Ag-NOR) was visualized according to the classical protocol, using silver nitrate (AgNO 3 ) [48]. The fishes were collected with the authorization of the Animal Ethics

Fluorescence In Situ Hybridization (FISH)
FISH experiments were performed under high stringency conditions [49] to identify both classes of ribosomal DNA (5S and 18S) and microsatellites (CA)15, (GC)15, (TA)15, and (CGG)10 sequences. The first ribosomal probe contained a 5S rDNA repeat copy and included 120 base pairs (bp) of the 5S rRNA transcribing gene in addition to 200 bp of the non-transcribed spacer (NTS) [50]. The second one corresponded to the 1400 bp segment of the 18S rRNA gene obtained via PCR from the nuclear DNA of the wolf fish Hoplias malabaricus [51]. Both probes were directly labeled with the Nick-Translation mix kit (Jena Bioscience, Jena, Germany), where 5S rDNA was labeled in red with Atto550-dUTP and

Fluorescence In Situ Hybridization (FISH)
FISH experiments were performed under high stringency conditions [49] to identify both classes of ribosomal DNA (5S and 18S) and microsatellites (CA) 15 , (GC) 15 , (TA) 15 , and (CGG) 10 sequences. The first ribosomal probe contained a 5S rDNA repeat copy and included 120 base pairs (bp) of the 5S rRNA transcribing gene in addition to 200 bp of the non-transcribed spacer (NTS) [50]. The second one corresponded to the 1400 bp segment of the 18S rRNA gene obtained via PCR from the nuclear DNA of the wolf fish Hoplias malabaricus [51]. Both probes were directly labeled with the Nick-Translation mix kit (Jena Bioscience, Jena, Germany), where 5S rDNA was labeled in red with Atto550-dUTP and the 18S rDNA was labeled in green with Atto448-dUTP, according to the manufacturer's instructions. The microsatellite sequences were directly labeled with Cy-3 during the synthesis, as described by [52]. Slides were aged at 60 • C for 1h before being treated with RNAse solution (1.5 µL RNase A (10 mg/mL) in 1.5 mL 2 × SSC) at 37 • C also for 1 h. Chromosomes were denatured in 70% Formamide/2 × SSC solution at 72 • C for 3.15 min, whereas probes at 85 • C for 10 min then cooled at 4 • C before the application onto the slides. Hybridization occurred in a dark moist chamber overnight and then ended by a 1 × SSC wash at 65 • C for 5 min, followed by a 5 min wash with 4 × SSC/Tween and 1 min with 1 × PBS. Chromosomes were counterstained with DAPI diluted in Vectashield (Vector Laboratories, Burlingame, CA, USA).

Comparative Genomic Hybridization (CGH)
As substantial variation in karyotype structures was observed among 'poropuntiin' species, we selected those from distinct clades that exhibit different karyotype compositions to be compared. Total genomic DNA (gDNAs) from the males of C. harmandi and M. chilopterus was extracted from liver tissue using a purification DNA/RNA standard kit (Cellco Biotech, São Carlos, Brazil). The gDNA of C. harmandi and M. chilopterus were compared with that of M. chilopterus on metaphase chromosomes. For this purpose, gDNAs of C. harmandi and M. chilopterus were, respectively, directly labeled with Atto488-dUTP (green) and Atto550-dUTP (red) using the Nick-translation Labeling Kit (Jena Bioscience, Jena, Germany) at 15 • C for 3 h. To block common genomic repetitive sequences, we used unlabeled C 0 t-1 DNA (i.e., a subset of genomic DNA from each species that is enriched for highly and moderately repetitive sequences), prepared according to [53]. The final hybridization mixture for each experiment was composed of 500 ng labeled DNA of each compared species, plus 15 µg of male-derived C 0 t-1 DNA from the respective species and the hybridization buffer (50% formamide, 2 × SSC, 10% SDSC 10% dextran sulfate and Denhardt's solution, pH 7.0). The CGH experiments were performed according to previous reports in related fish groups [18]. The probe mix with C 0 t-1 was denatured at 86 • C for 8 min, cooled at 4 • C and prehybridized at 37 • C for 1h. Hybridization occurred for 48h at 37 • C in a dark moist chamber. Post-hybridization washes were performed two times for 5 min in 1 × SSC at 65 • C, then in 4 × SSC/Tween at room temperature for 5min, following a short wash in 1 × PBS for 1 min. Slides were dehydrated in ethanol series (70%, 85%, 100%) for 2 min each before the application of DAPI solution as mentioned above in the FISH experiment.

Karyotyping and Image Processing
To confirm the 2n, karyotype structure, and FISH results, at least 20 metaphase spreads were analyzed per individual. Images were captured with an Axioplan II microscope (Carl Zeiss Jena GmbH, Jena, Germany) with CoolSNAP, and processed using Image-Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, USA). Chromosomes were classified according to their arm ratios as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a) [24].

Karyotypes and Ag-NOR Phenotypes
All seven studied species had 2n = 50 in both females and males, but different karyotype compositions (Figures 2-4 and Table 1). We were unable to detect sex chromosomes in any of the species examined. Ag-NORs were always found near the terminal region of all chromosomes of all species, except for H. malcolmi, in which they were located in the pericentromeric area of the first chromosome pair (Figures 2 and 3).

FISH-Mapping
The 18S rDNA probe hybridized to a single chromosome pair in H. wetmorei, M. chilopterus, M. ectypus and P. proctozystron. Two chromosome pairs carried these sites in C. harmandi and H. malcolmi, whereas three pairs were found in C. apogon. Except for H. malcolmi, which was found in both the centromeric and telomeric regions of the p arms, the 18S rDNA was found in the telomeric region of the p arms (Figures 2 and 3). The distribution of the 5S rDNA site, on the other hand, varied significantly, ranging from one chromosomal pair in C. harmandi, C. apogon, and P. proctozystron to two chromosome pairs in H. wetmorei and H. malcolmi, three in M. chilopterus, and four pairs in M. ectypus. Except for H. malcolmi and M. ectypus, where the 5S rDNA sites were located in both the pericentromeric and telomeric regions, they were present in the telomeric region of the p arms in nearly all species (Figures 2-4).
The chromosomal mapping of (CA)n revealed the same hybridization pattern in the telomeric regions of many chromosomal pairs across all species. The same situation occurred with (GC)n, but H. wetmorei again experienced substantial hybridization in a single pair's telomeric region. (TA)n followed a similar pattern, being dispersed over all chromosomes but with significant signals in the telomeric region of a single pair in M. ectypus and H. wetmorei. Furthermore, (CGG)n accumulates in the telomeric regions of all species, in

FISH-Mapping
The 18S rDNA probe hybridized to a single chromosome pair in H. wetmorei, M. chilopterus, M. ectypus and P. proctozystron. Two chromosome pairs carried these sites in C. harmandi and H. malcolmi, whereas three pairs were found in C. apogon. Except for H. malcolmi, which was found in both the centromeric and telomeric regions of the p arms, the 18S rDNA was found in the telomeric region of the p arms (Figures 2 and 3). The distribution of the 5S rDNA site, on the other hand, varied significantly, ranging from one chromosomal pair in C. harmandi, C. apogon, and P. proctozystron to two chromosome pairs in H. wetmorei and H. malcolmi, three in M. chilopterus, and four pairs in M. ectypus. Except for H. malcolmi and M. ectypus, where the 5S rDNA sites were located in both the pericentromeric and telomeric regions, they were present in the telomeric region of the p arms in nearly all species (Figures 2-4).
The chromosomal mapping of (CA)n revealed the same hybridization pattern in the telomeric regions of many chromosomal pairs across all species. The same situation occurred with (GC)n, but H. wetmorei again experienced substantial hybridization in a single pair's telomeric region. (TA)n followed a similar pattern, being dispersed over all chromosomes but with significant signals in the telomeric region of a single pair in M. ectypus and H. wetmorei. Furthermore, (CGG)n accumulates in the telomeric regions of all species, in addition to two pairs and in the pericentromeric region of a single chromosomal pair (Figures 5 and 6).

CGH-Studies
The gDNA comparison of C. harmandi (Char gDNA, Figure 7B) and M. chilopterus (Mchi gDNA, Figure 7C), hybridized in male metaphase chromosomes of M. chilopterus ( Figure 7A) indicated a high degree of genomic divergence between species, as evidenced by the great number of non-overlapped signals ( Figure 7D). The Char gDNA was hybridized to many centromeric areas, the majority of which were shared with the Mchi gDNA. Furthermore, certain chromosomal pairs displayed unique hybridization signals in the telomeric region with Char gDNA, whereas Mchi gDNA presented exclusive sites in centromeres, as well as three chromosomes exhibiting strong hybridization in the telomeric region ( Figure 7D).

CGH-Studies
The gDNA comparison of C. harmandi (Char gDNA, Figure 7B) and M. chilopterus (Mchi gDNA, Figure 7C), hybridized in male metaphase chromosomes of M. chilopterus ( Figure 6A) indicated a high degree of genomic divergence between species, as evidenced by the great number of non-overlapped signals ( Figure 7D). The Char gDNA was hybridized to many centromeric areas, the majority of which were shared with the Mchi gDNA. Furthermore, certain chromosomal pairs displayed unique hybridization signals in the telomeric region with Char gDNA, whereas Mchi gDNA presented exclusive sites in centromeres, as well as three chromosomes exhibiting strong hybridization in the telomeric region ( Figure 7D).

Discussion
The family Cyprinidae s.str. (sensu [1]) includes 11 lineages, from which 8 altogether contain evolutionarily tetraploid and hexaploid forms beside diploid ones, whereas only 3 include exclusively diploid representatives, namely Acrossocheilinae, Labeoninae, and Poropuntinae. All Poropuntiinae species under investigation, as well as those previously studied (Table 1), have a diploid chromosomal number equal to 2n = 50, confirming their diploid status.
This chromosomal number is also seen in diploid members of other cyprinid lineages, in addition to diploid Acrossocheilinae and Labeoninae [2,3,54]. Indeed, 2n = 50 appears to be preserved in various cyprinid and cobitoid fish lineages [35]. However, such conserved 2n is evidently associated with extensive intrachromosomal variations, which stress the role of structural rearrangements, such as pericentric inversions, chromatin additions/deletions, transpositions, and non-Robertsonian translocations as, e.g., demonstrated by [55] in other cyprinoid lineage, chondrostomine species (Leuciscidae).
The Ag-NOR stained regions corresponded to the 18S rDNA loci in all studied species (Figures 2-4), except H. malcomi, which had an extra site in the pericentromeric region. This means that all 18S rDNA sites in poropuntiins were transcriptionally active, due to the presence of nucleolin and nucleophosmin, two argyrophilic proteins involved in rRNA transcription and processing, and the targets of the Ag-NOR stain approach [56]. The majority of cyprinoid species possess this pattern [33], hypothesized to be the ancestral pattern across cypriniform fishes, but several sites, as shown in C. harmandi, C. apogon, and H. malcomi, were classified as derived ones [35]. In contrast to 5S rDNA, where the number of loci is likely to be associated with the diversification of clades, 18S rDNA distribution pattern does not follow a phylogenetic trend (Figure 8). Therefore, we hypothesized that in comparison with its sister clades, the Hypsibarbus + Mystacoleucus clade had a greater number of chromosomal rearrangements. The dynamic of ribosomal gene clusters was known to promote large intragenomic diversification [6,[57][58][59][60]. The rDNA

Discussion
The family Cyprinidae s.str. (sensu [1]) includes 11 lineages, from which 8 altogether contain evolutionarily tetraploid and hexaploid forms beside diploid ones, whereas only 3 include exclusively diploid representatives, namely Acrossocheilinae, Labeoninae, and Poropuntinae. All Poropuntiinae species under investigation, as well as those previously studied (Table 1), have a diploid chromosomal number equal to 2n = 50, confirming their diploid status.
This chromosomal number is also seen in diploid members of other cyprinid lineages, in addition to diploid Acrossocheilinae and Labeoninae [2,3,54]. Indeed, 2n = 50 appears to be preserved in various cyprinid and cobitoid fish lineages [35]. However, such conserved 2n is evidently associated with extensive intrachromosomal variations, which stress the role of structural rearrangements, such as pericentric inversions, chromatin additions/deletions, transpositions, and non-Robertsonian translocations as, e.g., demonstrated by [55] in other cyprinoid lineage, chondrostomine species (Leuciscidae).
The Ag-NOR stained regions corresponded to the 18S rDNA loci in all studied species (Figures 2-4), except H. malcomi, which had an extra site in the pericentromeric region. This means that all 18S rDNA sites in poropuntiins were transcriptionally active, due to the presence of nucleolin and nucleophosmin, two argyrophilic proteins involved in rRNA transcription and processing, and the targets of the Ag-NOR stain approach [56]. The majority of cyprinoid species possess this pattern [33], hypothesized to be the ancestral pattern across cypriniform fishes, but several sites, as shown in C. harmandi, C. apogon, and H. malcomi, were classified as derived ones [35]. In contrast to 5S rDNA, where the number of loci is likely to be associated with the diversification of clades, 18S rDNA distribution pattern does not follow a phylogenetic trend (Figure 8). Therefore, we hypothesized that in comparison with its sister clades, the Hypsibarbus + Mystacoleucus clade had a greater number of chromosomal rearrangements. The dynamic of ribosomal gene clusters was known to promote large intragenomic diversification [6,[57][58][59][60]. The rDNA clusters in all eukaryotes were made up of four units: the 18S rDNA (40S ribosomal subunit), the 5S, 5.8S, and 25-25S (60S ribosomal subunit), and the 5S, 5.8S, and 25-25S (60S ribosomal subunit) (reviewed in [61]). The syntenic arrangement of both 5S and 18S rDNAs, as observed in H. malcomi, do not seem to have a functional role on ribosomes and then can be considered a simple result of the intrinsic high dynamic of those sequences [62,63].
in their repetitive DNA content, evidenced by a variety of non-overlapping signals reve ing sequence conservation among their genomes, particularly in centromeric regions (Fi ure 7). A similar dynamic situation was observed after microsatellite mapping (Figures  and 6). Microsatellite motifs are abundant in the heterochromatic regions of fish genom (telomeres, centromeres, and sex chromosomes) (reviewed in [10]). The genome of C. ha mandi had hybridizations in the centromeric areas, whereas other species showed signa in the telomeric regions in the previously examined species. The sequences (GC)n, (TA) and (CGG)n were found in the terminal region of several chromosomes of H. wetmorei, M ectypus, and C. harmandi (Figures 5 and 6), respectively. Because repetitive DNAs are abu dant in eukaryotic genomes and evolve more quickly, their role as the primary mechanis in inducing karyotype rearrangements has been intensively studied [10]. Figure 8. Adapted phylogenetic tree for the tribe Poropuntiinae, based on the molecular-phylog netic data generated by [2,3], with chromosomal data mapped on it. Superscript numbers cor spond, respectively, to the amount of 5S and 18S rDNA loci in their karyotypes.
Karyotypes of cyprinoid fishes usually contain a significantly higher proportion biarmed chromosomes than uniarmed ones [5,70]. However, the representatives Hypsiba bus + Mystacoleucus clade had a significantly higher number of acrocentric chromosom than its sister clade, which includes Cyclocheilichthys, Puntioplites, and Cosmochilus (Figu 8). The same was true for other poropuntiin genera such as Balantiocheilos, Barbonymu Figure 8. Adapted phylogenetic tree for the tribe Poropuntiinae, based on the molecular-phylogenetic data generated by [2,3], with chromosomal data mapped on it. Superscript numbers correspond, respectively, to the amount of 5S and 18S rDNA loci in their karyotypes.
In contrast to the loci of 5S rDNA identified in the clade Hypsibarbus + Mystacoleucus, a single locus of 5S rDNA may indicate a derived trait, according to the phylogenetic reconstruction proposed by [3] (Figures 2 and 3). As a result, we selected a representative from each branch and compared their genomes using CGH to see if they also presented a genomic variation associated with their repetitive DNA content. This genome comparison technique has been applied to several teleost families, including the Salmonidae [64], Characidae [65], Cichlidae [66], Siluridae [67], and cyprinoids with small genome sizes, such as the Iberian Leuciscidae [68] or Carassius [69]. The remarkable chromosomal dynamism in both C. harmandi and M. chilopterus species corresponded with high dynamics in their repetitive DNA content, evidenced by a variety of non-overlapping signals revealing sequence conservation among their genomes, particularly in centromeric regions (Figure 7). A similar dynamic situation was observed after microsatellite mapping (Figures 5 and 6). Microsatellite motifs are abundant in the heterochromatic regions of fish genomes (telomeres, centromeres, and sex chromosomes) (reviewed in [10]). The genome of C. harmandi had hybridizations in the centromeric areas, whereas other species showed signals in the telomeric regions in the previously examined species. The sequences (GC)n, (TA)n, and (CGG)n were found in the terminal region of several chromosomes of H. wetmorei, M. ectypus, and C. harmandi (Figures 5 and 6), respectively. Because repetitive DNAs are abundant in eukaryotic genomes and evolve more quickly, their role as the primary mechanism in inducing karyotype rearrangements has been intensively studied [10].
Karyotypes of cyprinoid fishes usually contain a significantly higher proportion of biarmed chromosomes than uniarmed ones [5,70]. However, the representatives Hypsibarbus + Mystacoleucus clade had a significantly higher number of acrocentric chromosomes than its sister clade, which includes Cyclocheilichthys, Puntioplites, and Cosmochilus ( Figure 8).
The same was true for other poropuntiin genera such as Balantiocheilos, Barbonymus, Poropuntius, Puntioplties, Scaphognathops, and Sikukia (Table 1). Other populations of C. harmandi [26] exhibit similar characteristics, contradicting the reported trend. However, cypriniform chromosomes are noticeably small, making classification of the exact centromere position difficult and hence the proper assignment of chromosomes into chromosome categories problematic. This might explain why karyotype reports differ between populations and species [6,[70][71][72][73]. In the other sister lineage Smiliogastrinae [2,3], the genus Hampala, Puntius, and Systomus also have a high number of acrocentric chromosomes in their karyotypes (reviewed by [16]). Thus, a high number of acrocentric chromosomes might be a plesiomorphic feature of both the Poropuntiini and Smiliogastrini tribes. The presence of karyotypes composed primarily of mono-armed chromosomes (acrocentric) appears to be a feature of most derived fish clades, whereas the basal ones exhibit primarily biarmed ones (meta-, submetacentric) [74]. Aside from the differences found in fish phylogeny between basal and derived orders, the tendency towards chromosome acrocentrization appears to occur even within groups at the family level, as seem in some Neotropical and marine groups [15,75]. Considering the diversity of freshwater fishes, Cypriniformes can only be considered a basal clade when compared to Characiformes, Siluriformes and Gymnotiformes, but not within Acipenseriformes and Osteoglossomorpha [76]. It is important to note that a single karyotypic feature of a particular clade may not represent the entire evolutionary trend of cypriniforms. The high proportion of acrocentric chromosomes in this case could be due to pericentric inversions, which occur when a chromosome segment breaks off, rotates 180 degrees, and reattaches to the same chromosome in the opposite orientation. This inversion can result in the reversal of gene order along the chromosome and is a significant event in the diversification of karyotypes. While karyotypic features can be used to identify relationships between organisms, it is critical to consider all available evidence when determining the evolutionary history of a specific group.

Conclusions
Our findings have expanded the knowledge of karyotypes and chromosomal characteristics of 'poropuntiin' fishes. Its species had a conserved 2n of 50, a large number of acrocentric chromosomes in their karyotypes, and as result NF ranging between 62 and 92, indicating large intra-karyotype differentiation. Overall, these patterns suggest that structural chromosomal rearrangements such as pericentric inversions played an important role in the development of this cyprinid lineage. We also demonstrated that ribosomal DNAs and microsatellites have distinct patterns of accumulation in each species, suggesting a high variability of karyotypes while maintaining a level of 2n.