Genomic organization of repetitive DNAs highlights chromosomal evolution in the genus Clarias (Clariidae, Siluriformes)

Karyotype variability among Clarias species

Clariidae is a very well defined monophyletic family based on the presence of a unique
arborescent suprabranchial organ that enables the species to breathe atmospheric oxygen
4]. Nevertheless, Clariidae species are remarkable for the considerable variation in
their external morphology 7]–9]. Therefore, the chromosomal divergence among Clarias species (Table 1) also parallels the morphological differentiation of the clariid catfishes. In fact,
the remarkable variability of the 2n and NF values in different Clarias species indicates that distinct chromosomal rearrangements occur during the evolution
of their karyotypes. Karyotypes and other chromosomal characteristics of the three
species in this study confirmed the patterns found for other Clarias species, except C. batrachus (2n?=?104). However, Clarias and related cytogenetic parameters warrant deeper discussion.

The walking catfish C. batrachus is native to Southeast Asia, but has been introduced outside its native range, where
it is considered an invasive species responsible for invading aquaculture farms and
preying on fish stocks 10], 11]. Indeed, C. batrachus has type locality in Java 12], while the populations from Indochina either represent introduced stocks or belong
to other Clarias species, as a number of different species have been recently identified both in Indochina
and in the Sunda islands 13]. Accordingly, cytogenetic data also point to very distinct karyotypes for this species:
i) 2n?=?50 in Malaysia, ii) 2n?=?50–54 in India, iii) 2n?=?56 in China and 2n?=?100–104
in Thailand, in addition to sex and B chromosomes in some of these populations (Table 1). This brief overview of species suggests that the C. batrachus in Thailand, with 2n?=?104 and a karyotype dominated by one-armed chromosomes, may
represent a different unnamed species.

The hypothetical 2n for Siluriformes, as described in studies of different species
of this order, was proposed to be 2n?=?56, with a karyotype composed mainly by m-sm
chromosomes 14]–16]. Chromosomal studies of species in the group Heteropneustidae, which is phylogenetically
considered a sister-group to Clariidae 17], report that most of its members also have 2n?=?56 18]. Clarias gariepinus, with 2n?=?56 chromosomes, as well as a higher number of two-armed chromosomes and
few acrocentric chromosomes, retains the karyotype considered basal for Siluriformes.
These data support the phylogeny proposed for this family based on mtDNA analysis
9], in which C. gariepinus is placed together with C. anguillaris, as both species contain 2n?=?56 chromosomes 19]. The decrease in the 2n of other Clariidae species (such as C. macrocephalus – Fig. 2), suggests that chromosomal fusions also participated in the karyotypic differentiation
in the family.

Phylogenetically the species C. batrachus shows a derived position in the family 9]. In the present study, this species presented an unusual 2n?=?104 and a karyotype
dominated by acrocentric chromosomes. The occurrence of such a high 2n could be indicative
of a polyploidization event. However, when considering the large number of acrocentric
chromosomes and their relatively small size compared to the chromosomes found in the
other Clarias species, the present data suggest that multiple centric fissions are, in fact, the
most plausible explanation for karyotype diversification in this species. A similar
process culminating in an increased 2n number has also been reported for species of
the genus Potamorhina. One species – P. altamazonica – with a 2n?=?102, diverged from the most frequent 2n (54) found in other congeneric
species through a process of multiple centric fissions. In that case, meiotic analysis
showed only bivalents at metaphase I and confirmed a large scale occurrence of extensive
chromosomal fissions 20]. Additionally, extensive centric fission and heterochromatinization have been proposed
in the karyotype diversification of the Alaska black fish (Dallia pectoralis) 21].

The chromosomal distribution of repetitive DNA elements revealed remarkable differences
among the analyzed species. Both C. gariepinus and C. macrocephalus presented two 18S rDNA sites but were located in distinct chromosomal pairs, while
four 5S rDNA sites were present in C. gariepinus and only two in C. macrocephalus. In addition, analysis of C. batrachus revealed six chromosomal pairs harbouring 18S rDNA sites and 27 chromosomal pairs
harbouring 5S rDNA sites, including a synteny case. Though rDNAs are among the most
conservative components of the eukaryotic genome, undergoing minimal changes over
hundreds of millions of years, this conservatism appears to be a powerful source for
genome instability 22]. Due to high similarity among clusters, chromosomes that carry extended rDNA arrays
could be involved in heterologous synapses and recombination 23], providing variations of these sites inside the karyotypes.

Hypervariability in the number and location of rDNA loci, as presently reported in
C. batrachus, has been previously described for several groups 24]–26]. Variability in the number and position of rDNA sites suggested that chromosomal
rearrangements played a role in the speciation of the plant Sideritis dendrochahorra6]. This species possesses a large number of acrocentric chromosomes and multiple terminal
45S rDNA sites in most of its chromosomes. It has been suggested that in some groups,
structural changes may be induced by selective pressures from ecological or environmental
stresses 6], 27]. Among fishes, the spreading of rDNA has reportedly affected the recombination rates
of two coexisting salmonid species, Coregonus albula and C. fontanae, leading to rapid genomic divergence and faster ecological speciation 28]. In some cases, transposable elements have been reported to play an important role
in spreading rDNA sequences over the genome 24], 25]. Some classes of transposons appear to be able to “capture” entire genes and move
them to other parts of the genome 29], 30]. Alternatively, several satellite DNA repeats may have originated from rDNA sequences
and thus facilitate their dispersal into different genomic regions. For example in
the fish Hoplias malabaricus, a highly amplified satellite repeat (5SHindIII-DNA) with sequence sharing similarity with 5S rDNA have been reported to exist
in the centromeric region of several chromosomes 31], 32]. However, the reasons for the higher number of rDNA sites in C. batrachus still need to be clarified.

Repetitive DNAs as a powerful tool for Clarias hybrids identification

Cultured catfishes are one of the most important commodities in Thailand’s domestic
freshwater fish market, where C. macrocephalus is always preferred for consumption due to its better taste 33]. However, this species has a very slow growth rate and low disease resistance. At
the end of 1980s, hybrid catfish production increased, with the most produced fish
derived by crossing the Asian catfish (C. macrocephalus) and the African catfish (C. gariepinus) 34]; fast growth and high disease resistance made these species attractive to farmers
33], 35]. However, this hybrid form is currently abundant in all Thailand’s rivers, threatening
wild catfish populations due to competition, predation, and genetic introgression
34], 36]. In fact, these hybrids are potentially able to interbreed with the parental species,
which can lead to gene pool introgression, as has been reported for C. macrocephalus33].

Karyotype similarity, such as that which exists between C. gariepinus and C. macrocephalus, enhances the success of hybridization and back cross of many species 37]. In nature, the occurrence of chromosome numbers around the modal values of the clariid
species may suggest that speciation within this group is related to a high rate of
hybridization that results from common spawning 19].

However, when hybrids and individuals from parental species have similar karyotype
structures, the use of differential cytogenetic techniques is required to provide
distinguishable chromosomal markers 38]. Indeed, several known hybrids can be precisely identified and clearly distinguished
from their parental species using cytogenetic markers 38]–40]. For example, conventional staining helped in the precise identification of the parental
chromosomal types of the artificial hybrid resulted from the cross between Colossoma macropomum and Piaractus brachypomus41]. In the present work, repetitive DNAs proved to be informative and allowed for precise
characterization of the hybridization process. The hybrid C. gariepinus x C. macrocephalus had two 18S rDNA sites in non-homologous chromosomes in addition to three 5S rDNA
sites; these were the exact intermediate numbers present in the parental species.
In addition to the rDNA markers, microsatellites were highly useful for confirming
the hybrid nature of the fish. These ubiquitous repeated sequences are present in
all eukaryotic genomes, either in euchromatin or in heterochromatin, inside coding
regions of structural genes or between other repetitive sequences 42]. In the current analyzed species, C. gariepinus presented faint hybridization signals of (CA)
₁₅
and (GA)
₁₅
at subtelomeric regions, while in C. batrachus and C. macrocephalus these sequences were highly accumulated in all chromosomes. Notably, the C. gariepinus x C. macrocephalus hybrid showed 27 chromosomes with the strong hybridization pattern, characteristic
of C. macrocephalus chromosomes and the other 28 elements presenting a weak accumulation, found in C. gariepinus.

However, what are the genomic and ecological consequences of such hybrid unbalance?
Interspecific hybrids not only led to diversification and speciation but also have
important ecological consequences 43]. Some hybrids appear prevalent in nature, suggesting an evolutionary advantage for
having different sets of chromosomes for adaptation and development 44]. It is known, for example, that hybridization can promote the activation of mobile
elements and rapid genomic changes 45], 46]. Among fishes, interspecific hybrids between the red crucian carp (Carassius auratus)?×?common carp (Cyprinus carpio) showed faster genomic changes compared to the parental species, facilitated by intron
gains and losses, homologous recombination and the formation of novel genes 47]. This ‘genomic shock’ has also been reported in many allopolyploid plants, translating
as gene loss, chromosome mispairing, retrotransposon activation, altered methylation
or rearrangements between parental genomes that could lead to novel gene sequences
or differential homologous gene expression in hybrids throughout evolution 48].