Chromosomes of Asian cyprinid fishes: Variable karyotype patterns and evolutionary trends in the genus Osteochilus (Cyprinidae, Labeoninae, “Osteochilini”)

Abstract The Cyprinidae family is a highly diversified but demonstrably monophyletic lineage of cypriniform fishes. Among them, the genus Osteochilus contains 35 recognized valid species distributed from India, throughout Myanmar, Laos, Thailand, Malaysia, Indonesian archipelago to southern China. In this study, karyotypes and other chromosomal characteristics of five Osteochilus species occurring in Thailand, namely O. lini, O. melanopleura, O. microcephalus, O. vittatus and O. waandersii were examined using conventional and molecular cytogenetic protocols. Our results showed they possessed diploid chromosome number (2n) invariably 2n = 50, but the ratio of uni- and bi-armed chromosomes was highly variable among their karyotypes, indicating extensive chromosomal rearrangements. Only one chromosome pair bearing 5S rDNA sites occurred in most species, except O. melanopleura, where two sites were detected. In contrast, only one chromosomal pair bearing 18S rDNA sites were observed among their karyotypes, but in different positions. These cytogenetic patterns indicated that the cytogenomic divergence patterns of these Osteochilus species were largely corresponding to the inferred phylogenetic tree. Similarly, different patterns of the distributions of rDNAs and microsatellites across genomes of examined species as well as their different karyotype structures indicated significant evolutionary differentiation of Osteochilus genomes.


Introduction
The Cyprinidae family (sensu Tan and Ambruster, 2018), i.e. sensu stricto, is now restricted to phylogenetically and taxonomically highly diversified but a demonstrably monophyletic lineage of cypriniform fishes (Yang et al., 2015) which itself encompasses eleven intra-clade monophyletic lineages taxonomically recently recognized as subfamilies by Tan and Ambruster (2018).One of these lineages, Labeoninae, was demonstrated as sister basal lineage of all remaining cyprinid subfamilies (Conway, 2011;Yang et al., 2015;Stout et al., 2016).Moreover, the lineage monophyly of labeonine cyprinids was supported by both morphological and molecular studies (see review by Yang et al., 2012).These authors also identified four monophyletic intra-lineage groups within Labeoninae, taxonomically recognized (Tan and Ambruster, 2018) as tribes Garrini, Labeonini and taxonomically informal "Osteochilini" and "Semilabeonini": Labeonine cyprinids are highly morphologically diversified and include altogether around 50 genera with more than 500 species (Eschmeyer Catalog of Fishes, 2020), "Osteochilini" itself contains eight genera with close to 100 recently recognized species.
The genus Osteochilus (Günther, 1868) contains 35 recognized valid species distributed from India, throughout Myanmar, Laos, Thailand, Malaysia, Indonesian archipelago to southern China (Karnasuta, 1993).Although three major systematic revisions have been performed for this genus (Karnasuta, 1993), just eight species were included in detailed molecular phylogenetic analyses performed by Yang et al. (2012).Although the cytogenetic analysis are restricted, up to now, to only three species, the results point for a quite large karyotype differentiation inside Osteochilus (Table 1).
This study aimed to analyze karyotypes and other chromosomal characteristics as revealed by conventional (Giemsa-staining and C-banding) and molecular (rDNA and microsatellite FISH) protocols in five species of the genus Osteocheilus occurring in Thailand, namely O. lini, O. melanoptera, O. microcephalus, O. vittatus, and O. waandersii together with a brief overlook of cytotaxonomy of "osteochiline" cyprinids.The results added new informative characters useful in comparative genomics at the chromosomal level and highlighted extensive diversity among the analyzed species.

Individuals, mitotic chromosome preparation and C-banding
Representatives of five Osteochilus species were collected from distinct natural ecosystems of wild regions in Thailand (Figure 1).The numbers and sexes of the individuals under study were presented in Table 2.The specimens were    deposited in the fish collections of the Cytogenetic Laboratory, Department of Biology, Faculty of Science (KhonKaen University).Mitotic chromosomes were obtained from anterior kidney, by the conventional air-drying method (Bertollo et al., 2015).The distribution of C-positive heterochromatin blocks was visualized according to Sumner (1972).All the experiments followed ethical protocols, and anesthesia was conducted with clove oil before the sacrifice of the animals.The process was approved by the Animal Ethics Committee of KhonKaen University based on the Ethics of Animal Experimentation of the National Research Council of Thailand AEKKU23/2558.

Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization experiments were performed under high stringency conditions (Yano et al., 2017) to identify both classes of ribosomal DNA and microsatellites (CA) 15 , (GA) 15 , (GC) 15 , (A) 30 , (CAC) 10 and (CGG) 10 sequences.Two tandemly-arrayed DNA sequences isolated from the genome of Hoplias malabaricus, previously cloned into plasmid vectors and propagated in competent cells of Escherichia coli DH5α (Invitrogen, San Diego, CA, USA), were used.The first probe contained a 5S rDNA repeat copy and included 120 base pairs (bp) of the 5S rRNA transcribing gene and 200 bp of the non-transcribed spacer (NTS) (Martins et al., 2006).The second probe corresponded to the 1400 bp segment of the 18S rRNA gene obtained via PCR from the nuclear DNA (Cioffi et al., 2009).Both probes were directly labeled with the Nick-Translation mix kit (Roche, Manheim, Germany).The 5S rDNA was labeled with Spectrum Orange-dUTP, and the 18S rDNA was labeled with Spectrum Green-dUTP(Vysis, Downers Grove, IL, USA), according to the manufacturer's manual.The microsatellite sequences were directly labeled with Cy-3 during the synthesis, as described by Kubat et al. (2008).

Karyotyping and image processing
To confirm the 2n and the results of hybridization experiments, at least 30 metaphase spreads were analyzed per individual.Images were captured with an Axioplan II microscope (Carl Zeiss Jena GmbH, Germany) with CoolSNAP, and processed using an 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) (Levan et al., 1964).
In general, a spreading pattern was a frequent feature for the microsatellites analyzed.However, some specific features could also be highlighted among species (Figures 3-7).In this sense, O. waandersi had small spread (GC) n signals in all chromosomes but a strong hybridization pattern in the pericentromeric region of a single pair.

Discussion
"Osteochilini" species possess 2n = 50 (Arai, 2011), which is also considered a basal pattern for cypriniform fishes (Chaiyasan et al., 2018).Our results showed that 2n = 50 is also a demonstrably conserved pattern for all Osteochilus species karyotyped to date.However, despite the conservative 2n, significant differences in the karyotype structures in all five species examined were observed.Hence, this species also had multiple 5S rDNA sites and a different hybridization pattern for (A) 30 , (CA) 15 and (CGG) 10 microsatellites.According to the phylogeny of the Labeonini tribe proposed by Yang and Mayden (2010), Osteochilus was recovered as a monophyletic genus, with three Labiobarbus species forming a sister basal clade (Figure 8).O. melanopleura was recognized as the oldest derived species of the genus and Labiobarbus lineatus possessed 20 acrocentric chromosomes composing its karyotype (Magtoon and Arai, 1990).This fact suggests that the acrocentric pair No. 25 of O. melanopleura could be a remnant of the common ancestor between both Osteochilus and Labiobarbus genera.Thus, the karyotype diversification in Osteochilus genus was probably accompanied by a series of structural chromosome rearrangements, with a special role of pericentric inversions or centromere reposition, as indicated by changes in karyotype structure and a constant 2n (Figure 8).
Many representatives of several fish orders, such as Characiformes, Cypriniformes, Siluriformes, and Gymnotiformes have karyotypes dominated by bi-armed chromosomes (Molina et al., 2014).Our data also demonstrated that Osteochilus species have more bi-armed elements in their karyotypes, suggesting that orthoselection and meiotic drift (White, 1973;Molina et al., 2014) could be strong evolutionary drivers for this group.Noteworthy, the karyotype now reported for O. waandersi was different from that reported by Magtoon and Arai (1993).Cypriniform chromosomes have notable small sizes (Sember et al., 2015;Saenjundaeng et al., 2018) Knytl et al., 2018), thus impairing the identification of the chromosomal morphology.
Microsatellite motifs had a preferential accumulation in heterochromatic regions (reviewed in Cioffi and Bertollo, 2012).However, the majority of the microsatellite sequences in Osteochilus showed a scattered pattern on chromosomes, without a specific relation with heterochromatic regions.Nevertheless, the (A) 30 motif presented a strong accumulation pattern in the pericentromeric regions of O. melanopleura, a species in which this same chromosomal region appeared strongly C-banded, i.e., with C-positive heterochromatin.Also, microsatellites are often embedded within rDNA clusters (Piscor and Parise-Maltempi, 2016), which can also explain the strong labeling in the (CGG) n motifs found in chromosomes of O. vittatus and O. melanopleura.
Usually, the 18S rDNA occupies a terminal position in chromosomes, in contrast to the more frequent interstitial position of the 5S rDNA (Sochorová et al., 2018).All the Osteochilus species under study had both ribosomal classes located in a terminal position in association with heterochromatin, suggesting that these regions were recombination hotspots (Salvadori et al., 1997;Sola et al., 2003;Gornung, 2013).Their terminal position may also facilitate the dispersion of these sequences to other chromosomes, according to Rabl's model, since higher recombination rates were found near the telomeric region (reviewed in Foster and Bridger, 2005).Besides that, the heterochromatinization of ribosomal loci was suggested to facilitate chromosomal heteromorphisms, by unequal crossing over between homologs and/or amplification of the heterochromatin between sister chromatids (Collares-Pereira and Ráb, 1999;Sola and Gornung, 2001;Gromicho et al., 2006).The presence of both rDNAs in different chromosomal pairs is a usual condition in fish species (Sochorová et al., 2018), as also observable for cyprinids in our study.Besides, it is noteworthy that O. melanopleura was recognized as a basal one in the genus (Karnasuta, 1993;Yang and Mayden, 2010; Figure 8), and this species had two chromosome pairs with 5S rDNA sites.In this sense, this could suggest that a single pair bearing such sites in the karyotypes of other Osteochilus species could be a derived pattern.However, this second pair with 5S sites in O. melanopleura was likely a particular pattern due to spreading events (Figure 8).Ribosomal clusters are characterized by its dynamism promoting significant intragenomic diversification (Gornung, 2013;Rebordinos et al., 2013;Cioffi et al., 2015;Sember et al., 2015;Symonová and Howell, 2018).
A general pattern on Osteochilus karyotypes with a fundamental number (NF) of 100 and a high variation on their karyotype macrostructure can generally be observed.This was somehow expected since Osteochilus is a specious genus, and it is known that the speciation process itself can be the result of high macrostructure karyotypic variation (White, 1973;Lowry and Willis, 2010).However, we cannot disregard the variation found in O. melanopleura, the variation that was also probably extended to the sister species O. schlegelii, but more studies are required to confirm this assumption.
In conclusion, our data have improved the data about the karyotypes and chromosome characteristics in the genus Osteochilus.Its species presented a conservative 2n = 50 and NF = 100, but with differentiation of their karyotypes.Altogether these features indicate that chromosomal rearrangements, particularly the structural ones as centromere reposition and pericentric inversions, have taken place a major role during the evolutionary history of this cyprinid genus.The detailed cytogenetic survey indicated that the cytogenomic divergence patterns of these Osteochilus species were largely corresponding to the inferred phylogenetic tree.Also, repetitive DNAs, such as ribosomal and microsatellite ones, showed specificities in their distribution among species, thus being shown as good markers and promoters of specific genomic differentiation inside the genus.
5 correspond to the localization of each collection region shown in Figure1.
For (A) 30 , O. melanopleura showed the pericentromeric region of 46 chromosomes hybridized, while all the other species had scattered signals in all 50 chromosomes.Concerning (CA) n , while O. microcephalus and O. waandersi had a scattered distribution in all chromosomes, O. lini and O. vittatus presented small telomeric signals and O. melanopleura had scattered signals except in the centromeric regions.Spreading signals were also observed for the (GA) n , (CAC) n , and (CGG) n probes in all chromosomes of all species.Additionally, O. melanopleura and O. vittatus had a strong (CGG) n signal in the telomeric region of a single chromosome pair.
and this feature can make it difficult to visualize the correct centromere position(Ráb and Collares-Pereira, 1995;Spoz et al., 2014;

Table 1 -
Available cytogenetic data for Osteochilus species.

Table 2 -
Species analyzed, collection sites and number of analyzed individuals (n).