Karyotype diversity and evolutionary trends in the Asian swamp eel Monopterus albus (Synbranchiformes, Synbranchidae): a case of chromosomal speciation?

Background Synbranchidae or swamp eels are fishes belonging to the order Synbranchiformes that occur in both freshwater and occasionally in brackish. They are worldwide distributed in tropical and subtropical rivers of four different continents. A large degree of chromosomal variation has been found in this family, mainly through the use of conventional cytogenetic investigations. Inside this group, a still almost unexplored species under the cytogenetic point of view is the Asian swamp eel Monopterus albus, a widely distributed species throughout Asia. Here, we tested the hypothesis of chromosomal speciation, where a case of sympatric speciation may occur as the primary consequence of chromosomal rearrangements. We performed a comparative chromosomal analysis of M. albus from 22 different localities in Thailand, using distinct staining methods (C-banding, Ag-NO3, and Chromomycin A3), and FISH with repetitive DNA probes (5S rDNA, 18S rDNA, Rex1 element and microsatellite repeats). Results This approach evidenced two contrasting karyotypes (named karyomorphs A and B) that varied concerning their 2n and repetitive DNAs distribution, where chromosomal fusions and pericentric inversions were involved in such differentiation. While the karyomorph A has 2n = 24 chromosomes, the karyomorph B has only 2n = 18, both with NF = 24. In addition, karyomorph A contains only acrocentric chromosomes, while karyomorph B contains three unique metacentric pairs. These features highlight that M. albus has already gone through a significant genomic divergence, and may include at least two cryptic species. Conclusions This marked chromosomal differentiation, likely linked to the lifestyle of these fishes, point to the occurrence of a chromosomal speciation scenario, in which fusions and inversions had a prominent role. This highlights the biodiversity of M. albus and justifies its taxonomic revision, since this nominal species may constitute a species complex.


Background
Freshwater habitats make up less than 0.01% of available aquatic habitat but contain almost half of all 34,000 valid fish species, making freshwater fishes an excellent model for studying speciation events [1,2]. However, although freshwater environments are largely fragmented and isolated, which means allopatric speciation events are more frequently found, several known cases of sympatric speciation have already been identified [3,4].
In recent years, cytogenetic studies have made important contributions toward a better understanding of recent speciation events, since chromosomal rearrangements can act as genetic barriers to gene flow, thus facilitating reproductive isolation [5][6][7][8]. The chromosomal rearrangements promote the reorganization of the genetic structure, and the evolutionary impact and the consequences at the speciation level can vary according to the rearrangement type, that is, inversion, fusion, fission or translocation [7][8][9][10][11][12]. Such chromosomal rearrangements can facilitate adaptation to heterogeneous environments by limiting genomic recombination [10].
Molecular cytogenetic studies using fluorescence in situ hybridization (FISH) to map repetitive DNA sequences have provided important contributions to the characterization of chromosomal rearrangements and the evolution of distinct fish groups (reviewed in [13]). Repetitive DNAs, widely distributed in the eukaryotic genomes, are generally divided into two classes, one comprising tandem sequences (satellite DNAs, minisatellites and microsatellites), and the other comprising interspersed sequences, such as transposons and retrotransposons [14].
Synbranchidae or swamp eels are fishes belonging to the order Synbranchiformes; they occur in freshwater and occasionally in brackish water. They are distributed worldwide in tropical and subtropical Asia, the Indo-Australian Archipelago, West Africa (Liberia), Mexico and Central and South America (Fig. 1) [17][18][19]. However, Donsakul and Magtoon [17] have also reported that M. albus from the central part of Thailand differs from the other populations, as it has 2n = 18 and NF = 24 (Table 1). Similarly, the Venezuelan Ophisternon aenigmaticum also highlights a karyotype diversification, including 2n = 45 and NF = 51 and 2n = 46 and NF = 52 [20], as well as the Synbranchus marmoratus species from Brazil and Argentina, which presents 2n ranging from 42 to 46 [21][22][23][24][25]. The Synbranchidae family has high karyotype diversity among its species, with 2n ranging from 18 to 46, mainly due to extensive chromosomal rearrangements (fusions/fissions and inversions) ( Table 1).
This study presents a comparative chromosomal analysis of Monopterus albus from 22 different localities in Thailand (Fig. 2), using distinct staining methods (C-banding, Ag-NO 3, and chromomycin A 3 ) as well as FISH with repetitive DNA probes (5S rDNA, 18S rDNA, Rex1 element and microsatellite repeats). We tested the hypothesis of chromosomal speciation, where a case of sympatric speciation may occur as the primary consequence of chromosomal rearrangements. This approach provided an in-depth karyotype characterization of this taxon, evidencing the presence of two contrasting karyotypes occurring in sympatry and the occurrence of likely distinct species in this nominal species. This marked chromosomal differentiation, likely linked to the lifestyle of these fishes and their population fragmentation, protects from gene flow and therefore promotes speciation.

Results
Karyotypes M. albus from the populations analyzed presented two distinct karyotype forms, one with 2n = 24 (24a) and NF = 24 (karyomorph A) and the other with 2n = 18 (6 m + 12a) and NF = 24 (karyomorph B) (Fig. 3a). The first karyomorph was present in 21 localities (183 specimens), while the latter was found in five (17 specimens). Sympatry of both karyomorphs was observed in four localities, namely Nakhon Nayok, Kanchanaburi (Sri Yok), Chon Buri and Sa Kaeo Provinces (Table 2 and Fig. 2). No heterozygous karyotype forms were observed in the four localities of sympatry.  C-banding, ag-NORs and Chromomycin A3 staining C-positive heterochromatic bands were observed in the centromeric/pericentromeric region of all chromosomes as well as in the telomeric region of several pairs in both karyomorphs. Interstitial heterochromatic blocks were also found in chromosome pair no. 4 in both karyomorphs (Fig. 3a). Ag-NORs sites were present in the centromeric region of pair no. 7 and on the telomeric region of pair no. 3 in karyomorphs A and B, respectively. These Ag-NORs were the only observed GC-rich regions in the karyotype (Fig. 3a, boxed).
Chromosome mapping of 5S and 18S rDNAs 5S rDNA sequences were found in the pericentromeric region of the q arms of chromosome pair no. 7 in karyomorph A and in the pericentromeric region of the p arms of chromosome pair no. 3 in karyomorph B (Fig. 3b).
Concerning the 18S rDNA sequences, chromosome pair no. 7 of karyomorph A also displayed sites in the pericentromeric region of the q arms, while in karyomorph B, in addition to the signals in the pericentromeric region, chromosomal pair no. 3 also showed telomeric markings (Fig. 3b). Therefore, the 5S and 18S rDNAs are located together in one chromosome pair in both karyomorphs.

Discussion
The occurrence of distinct M. albus karyomorphs living in sympatry with the absence of natural hybrids, as found in the present study in Nakhon Nayok, Kanchanaburi (Sri Yok), Chon Buri and Sa Kaeo Provinces, reinforces the hypothesis that these karyomorphs represent two reproductively isolated biological units. Although easily distinguishable through cytogenetic analysis, specimens from both karyomorphs have the same morphology, making difficult the identification of such probable new species. The integration of both conventional and molecular cytogenetic approaches allowed the proposal of some chromosomal rearrangements probably related to the differentiation of both M. albus karyomorphs, where centric fusions appear as the main evolutionary sources shaping such a process (Fig. 3c). The different karyotype composition among individuals allowed us to identify two distinct karyomorphs, named A and B, which presented 2n of 24 and 18 chromosomes, respectively, both with NF = 24. The chromosomal divergence as well as the relation between the karyomorphs is clearly evidenced by their same NF and different karyotype formulas. This indicates that karyomorph B originated from karyomorph A (with only acrocentric chromosomes), where centric fusions were the most probable mechanism behind the presence of six metacentric chromosomes in karyomorph B (Fig. 3c). However, another scenario can also not be ruled out, in which fission-type rearrangements would have originated the additional 12 acrocentric chromosomes in karyomorph A. Once the majority of chromosomal rearrangements involve heterochromatic regions, especially in fish species [26,27], this explains the centromeric rearrangements in M. albus. The co-localization of CMA 3 positive heterochromatin with the NOR loci also occurs in other synbranchids [25,28], probably because of local changes in base composition (increase in GC content) due to the so-called GC-biased gene conversion that involves rDNA in many vertebrates, including ray-finned fishes [29].
Although the 18S and 5S sites are located in an acrocentric pair in karyomorph A and in a metacentric pair in karyomorph B, it is possible to infer that both pairs are related in both karyomorphs. As karyomorph B contains additional 18S rDNA signals in the telomeric region in pair no. 3, it is likely that a pericentric inversion has divided the pericentromeric 18S rDNA loci into two parts and has transposed one of them near the telomere. Centric fusions would be the most suitable rearrangements to illustrate this scenario.
The differential distribution of the microsatellite motifs (CA) 15 and (TA) 15 between the two karyomorphs reinforces the interpretation that they represent, in fact, different species. A genome-wide analysis found that microsatellites have a repeat-and chromosome-biased distribution in M. albus from China, mainly located in noncoding regions (98,602, 99%) [30]. Differences both in the abundance and in the chromosomal location of several microsatellite motifs have been reported among closely related fish species also involved in recent speciation events [31,32]. Similar to microsatellites, the Rex1 sequences are also resolutive markers for comparative genomic studies, as was already shown for several Asian fish species [19,30,31]. In the present study, the interspersed distribution of the retroelement Rex1 contrasts with that reported by [19].
Inter-and/or intrapopulation diversity has also been found in the karyotypes of other Synbranchidae species. For example, Ophisternon aenigmaticum and Synbranchus marmoratus possess high karyotype variability, with several karyomorphs described among their populations; they are also considered species complexes, where independent and bidirectional rearrangements, such as fusion and fission events, were responsible for the distinct 2n and karyotypic characteristics observed (Table 1). Biological, physiological and/or reproductive characteristics of Synbranchidae fishes may facilitate the intra-and interspecific karyotype variability observed among species and populations, especially in Monopterus and Synbranchus. These species tolerate a wide range of water oxygen levels, being able to obtain up to 25% of air oxygen by the cutaneous surface, allowing them to survive up to nine months in a drying burrow [33]. They also form small populations, and although they prefer freshwater habitats, these fishes tolerate brackish and saline conditions [1]. On the other hand, as many individuals can be isolated in small lakes during dry years, the capacity of sex reversal observed in synbranchids could contribute to the viability of such populations, increasing the probability of the fixation of genetic differences and speciation processes [25,28].

Conclusions
In summary, the present scenario points to the occurrence of a chromosomal speciation scenario, in which fusions and inversions had a prominent role in the diversification of two distinct karyomorphs that differ with respect to diploid number, chromosome features and repetitive DNA distributions. However, this does not necessarily mean that a sympatric speciation is the only viable alternative, since such karyomorphs could have been established in allopatry, where a secondary join between them originated the present distribution. The karyotypic features highlight the biodiversity of M. albus and justify a taxonomic revision, since this nominal species may actually constitute a species complex.

Individuals examined
Two hundred specimens of M. albus were collected in 22 localities from distinct Thai regions ( Fig. 2 and Table 2). The specimens were caught using traps, and after capture, the animals were placed in sealed plastic bags containing oxygen and were transported to the research station. The specimens were deposited in the fish collection of the Cytogenetic Laboratory, Department of Biology, Faculty of Science, Khon Kaen University. All the experiments followed ethical protocols, and anesthesia with clove oil was used prior to sacrificing the animals to minimize suffering. The fishes were then immersed in an ice-slurry to achieve death by hypothermia. The process was approved by the Animal Ethics Committee of Khon Kaen University based on the Chromosome preparation and C-banding, ag-and CMA 3 staining Mitotic chromosomes were obtained from the cell suspensions of the anterior kidney, using the conventional air-drying method [34]. Conventional staining was done using 5% Giemsa solution in phosphate buffer, pH 6.8, for 10 min. Chromosomes were analyzed after silver nitrate staining [35] in order to visualize the nucleolar organizing regions (Ag-NORs), and C-banding was also employed to detect the C-positive heterochromatin [36]. GC-specific fluorochrome chromomycin A 3 (CMA 3 ) was carried out following the method of Amemiya and Gold [37] to detect CG-rich regions on the chromosomes.

Detection of repetitive DNA sequences by FISH
All FISH experiments with repetitive DNA probes were essentially carried out according to the protocol described in Yano et al. [42]. The first post-hybridization wash was performed with 2x SSC for 5 min at 42°C, and a final wash was performed at room temperature in 1x SSC for 5 min. Finally, the slides were counterstained with DAPI and mounted in an antifade solution (Vectashield from Vector Laboratories).

Microscopic analysis and image processing
At least 30 metaphase spreads per individual were analyzed to confirm the 2n, karyotype structure and FISH results. Images were captured using an Olympus BX50 microscope (Olympus Corporation, Ishikawa, Japan) with Cool SNAP, and the images were processed using Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, USA). Chromosomes were classified as acrocentric (a) or metacentric (m), according to their arm ratios [43].