Comparative chromosomal mapping of microsatellite repeats reveals divergent patterns of accumulation in 12 Siluridae (Teleostei: Siluriformes) species

Abstract The freshwater family Siluridae occurs in Eurasia and is especially speciose in South and Southeast Asia, representing an important aquaculture and fishery targets. However, despite the restricted cytogenetic data, a high diploid number variation (from 2n=40 to 92) characterizes this fish group. Considering the large genomic divergence among its species, silurid genomes have experienced an enormous diversification throughout their evolutionary history. Here, we aim to investigate the chromosomal distribution of several microsatellite repeats in 12 Siluridae species and infer about their possible roles in the karyotype evolution that occurred in this group. Our results indicate divergent patterns of microsatellite distribution and accumulation among the analyzed species. Indeed, they are especially present in significant chromosome locations, such as the centromeric and telomeric regions, precisely the ones associated with several kinds of chromosomal rearrangements. Our data provide pieces of evidence that repetitive DNAs played a direct role in fostering the chromosomal differentiation and biodiversity in this fish family.


Introduction
The freshwater family Siluridae ranges in Eurasia, but containing the higher number of species in South and Southeast Asia (Bornbusch, 1995;Kottelat, 2013), with 103 recognized species (Fricke et al., 2019), thus representing important aquaculture and fishery targets. Silurid species show a significant size diversity, such as Silurus glanis, reaching over 300 kg in weight and 2m in size (Linhart et al., 2002) and Silurus soldatovi, that reaches 400 kg in weight and up to 4m in size (Berg, 1964), while others are much smaller, being used as ornamental fishes (Ng et al., 1994;Chapman et al., 1997;Ng and Ng, 1998;) or biological indicators (Ng and Lim, 1992;Ng and Ng, 1998). The monophyletic status of Siluridae is supported and confirmed by both morphological and molecular data (Bornbusch, 1995;Hardman, 2005), and, altough their phylogenetic position was doubtful for many years (Bornbusch, 1995;Hardman, 2005;Sullivan et al., 2006), a recentl study placed this family in the root of taxon Siluroidea (Kappas et al., 2016).
phaiosoma (Ditcharoen et al., 2019) to 92 in Kryptopterus cryptopterus (Donsakul and Magtoon, 1996) and Kryptopterus geminus (Ditcharoen et al., 2019). It is also known that Phalacronotus is the only genus that maintains the diploid number conservation with 2n = 64 in all analyzed species, while other genera in this family display a substantial variation (Ditcharoen et al., 2019). On the mapping of highly repetitive sequences, the high 2n variation also appears to be followed by a large variation of ribosomal DNAs loci among silurid species (Ditcharoen et al., 2019). Considering the extensive genomic reorganization, as revealed by CGH (comparative genomic hybridization), it is evident that silurid genomes have experienced an enormous diversification throughout their evolutionary history (Ditcharoen et al., 2019).
Microsatellites are repetitive DNA sequences, varying from one to six nucleotides, found in genomes of all eukaryotic organisms (Cioffi and Bertollo, 2012;López-Flores and Garrido Ramos, 2012). These repeats can also be associated with coding regions of structural genes and between other repetitive sequences (Tautz and Renz, 1984), contributing to the functional and structural organization of the genome (Schueler et al., 2001). Fish genomes usually have microsatellites distributed throughout telomeric and centromeric regions of autosomal and sex chromosomes, associated with other repetitive DNA sequences (Cioffi and Bertollo, 2012). Additionally, repetitive DNAs have an important role in speciation, differentiation of sex-specific regions, and promotion of biodiversity (Vicari et al., 2005;Cioffi et al., 2009;Sember et al., 2018). Therefore, here we analyzed the chromosomal location several microsatellites repeats to explore the intergenomic divergence at the chromosomal level in 12 Silurid species; the sampling resembles the one previously analyzed by Ditcharoen et al. (2019) with different cytogenetic methods. Indeed, our recent result provided new insights into the karyotype differentiation of this fish group, with a better understanding of the chromosomal organization of repetitive DNAs and uncovering chromosome homologies and differences among the studied species.

Material and Methods
Twelve silurid species were collected in the river basins of Thailand ( Figure 1, Table 1). All individuals were deposited in the fish collection of the Cytogenetic Laboratory, Department of Biology, Faculty of Science (Khon Kaen University). The procedures followed ethical protocols and anesthesia was conducted with clove oil before euthanasia, as approved by the Institutional Animal Care and Use Committee of Khon Kaen University, based on the Ethics of Animal Experimentation of the National Research Council of Thailand IACUC-KKU-10/62.

Results
The microsatellite (CA) 15 revealed a telomeric pattern of accumulation in all chromosomes of all species ( Figure  2), except for Kryptopterus geminus, where small telomeric signals occured in addition to strong centromeric ones in some other chromosomes. Similarly, the microsatellite (CAC) 10 also had a telomeric distribution on chromosomes ( Figure 3), but again with an exception, in this case for Silurichthys phaiosoma which had only strong centromeric and telomeric signals in two acrocentric pairs, a larger and a 2 Ditcharoen et al. smaller one, respectively. However, separate scattered signals were also observed in several other chromosomes. As to the microsatellite (CAT) 10 , all species have scattered telomeric hybridization signals ( Figure 4). In turn, a very diverse distribution pattern was observed for the microsatellite (GC) 15 (Figure 5) 10 had a very contrasting distribution compared to the other microsatellites. In this case, only one chromosome pair has telomeric signals in the p arms, in all twelve species analyzed ( Figure 6). The microsatellite (A) 30 was the only one not found in any of the examined species (data not shown).

Discussion
The role of repetitive DNAs in the genome evolution has been documented for different fish groups (Cioffi et al., 2010;Cioffi and Bertollo, 2012;Terencio et al., 2013;Yano et al., 2014;Cioffi et al., 2015;Moraes et al., 2017Moraes et al., , 2019Sassi et al., 2019). Worthy of note is the great evolutionary diversification that Siluriformes fishes have experienced, es-Microsatellite distribution in Siluridae 3 pecially at the chromosomal level. Here, six mono-, bi-and tri-nucleotide microsatellite sequences were mapped on chromosomes of twelve Siluridae species. Except for (A) 30-, which was not found to occur in any of the analyzed species, all other probes generated ell visible hybridization patterns. However, highly divergent distributions have been found, even among congeneric species, as observed in Kryptopterus. Accordingly, this genus displays different 2n, karyotypes and an extensive variation of their repetitive DNA con-tent (Ditcharoen et al., 2019). On the other hand, the Phalacronotus species had a similar distribution pattern among chromosomes, probably linked to their chromosomal-conserved characteristics, since they share similarities in both karyotype and genome features (Ditcharoen et al., 2019). It is also remarkable that the same kind of microsatellite did not present the same pattern among silurids. Indeed, very different hybridization patterns for the same microsatellite occur among distinct species, as for the (CA) 15 , (GC) 15 and 4 Ditcharoen et al.  (7); Ompok siluroides (8); Phalacronotus apogon (9); Phalacronotus bleekeri (10); Silurichthys phaiosoma (11) and Wallago attu (12). Scale bar = 5 mm.
(CAC) 10 probes, for example, although mostly restricted to centromeric and telomeric regions, where a significant fraction of repetitive DNA is localized (Cioffi and Bertollo, 2012).
(CA) 15 and (GA) 15 , showed strong hybridization signals at subtelomeric and heterochromatic regions of several autosomes, in addition to a strong accumulation on the sex chromosomes (Cioffi et al., 2011, Supiwong et al., 2014. In fact, for most of these species, the 18S rDNA repeats are found in the short arms of a single chromosome pair (Ditcharoen et al., 2019), and this region matches the position of the (CGG)n marks found in our experiments. Similarities of both microsatellite and ribosomal DNA location do not seem to be a rare event among fishes, as they are also found in other species, such as Lebiasina bimaculata (Sassi et al., 2019) and Hepsetus odoe (Carvalho et al., 2017), for example. Indeed, G+C rich motifs are common in exons of all vertebrates (Chistiakov et al., 2006). Since higher recombination rates can be found near the telomeric region (Jensen-Seaman et al., 2004), the physical proximity of microsatellite and rDNA repeats could favor the evolutionary spreading of both sequences together, as triplet se-   (7); Ompok siluroides (8); Phalacronotus apogon (9); Phalacronotus bleekeri (10); Silurichthys phaiosoma (11) and Wallago attu (12). Scale bar = 5 mm.
quences are particularly able to stabilize, by hairpin, some alternative structures generated from DNA polymerase slippage (Sinden, 1999). Reinforcing the above considerations, Silurichthys phaiosoma has a very particular distribution of the (CAC)n repeats, accumulated in the centromeric and telomeric regions of two acrocentric pairs, respectively. Accordingly, this species also presents a unique pattern of 5S rDNA distribution concerning the other silurids, with the spreading of multiple loci in the karyotype. Besides, a 5S rDNA site is found in the telomeric region of the long arms of the 18 th chromosome pair (Ditcharoen et al., 2019), the same one that harbors a conspicuous (CAC)n site.
2006), it is noteworthy the predominance of (GC) rich microsatellites in the heterochromatic regions of fishes (Artoni and Bertollo, 1999;Kavalco et al., 2005;Sassi et al., 2019). Accordingly, at least six Siluridae species now investigated (Micronema cheveyi, Ompok fumidus, O. siluroides, Phalacronotus apogon, P. bleekeri, and Wallago attu) have (GC)n pericentromeric signals for almost half chromosomes, in addition to other species, like Kryptopterus limpok and Silurichthys phaiosoma, that have a single labeled chromosome pair but also in this same region. These findings suggest an association and accumulation of such sequences in this relevant chromosome region, as observed in several other fish species (reviewed in Cioffi and Bertollo, 2012).
structural maintenance of chromosomes (Dernburg et al., 1996). Therefore, great variations in the amount and position of these sequences could create fertility barriers by fostering the occurrence of chromosomal rearrangements (Cioffi and Bertollo, 2012). Indeed, the distribution of microsatellite motifs in fish genomes could be biased to some specific noncoding regions, as found in the Asian swamp eel Monopterus albus (Li et al., 2017). Additionally, closely related fish species involved in recent speciation events could present a differential pattern in the distribution and quantity of microsatellite sequences on chromosomes, as demonstrated for naked catfishes (Supiwong et al., 2014), channid fishes  and Siluridae species in this paper.
Our results indicate that microsatellite sequences have divergent patterns of distribution and accumulation among Siluridae fishes, probably fostering the chromosomal differentiation and biodiversity in this fish family. Indeed, they are especially present in especific chromosome locations, such as the centromeric and telomeric regions, precisely the ones that are associated with several kinds of chromosomal rearrangements. In addition to their probable roles during chromosomal diversification, it is also highlighted that microsatellites can have a close association with other important classes of repetitive sequences, like ribosomal DNAs. This association can represent a good strategy for increasing biodiversity, facilitating a combined distribution of distinct DNA sequences along with the evolutionary divergence.