General aspects of heterochromatin and repeated DNAs organization
The presence of large blocks of heterochromatin and C
t-1 DNA fraction in the studied species suggests the occurrence of amplification of repetitive DNAs and/or heterochromatin transfer between the chromosomes during the karyotype differentiation of species as previously observed in other animals[28–31]. This statement is supported by the common pattern of heterochromatic blocks mainly located in pericentromeric areas in relates species, including coleopterans[32–34]. Most information concerning heterochromatin in coleopterans is focused on the description of chromosomal distribution with few data regarding its molecular content. The C
t-1 DNA fraction hybridization showed a general pattern coinciding with the data generated by C-banding[25, 27], indicating that the heterochromatin is enriched in highly repetitive DNA. The presence of large blocks of C
t-1 suggests an abundance of repetitive sequences, and cross-species hybridization analysis among Phanaeini species evidences high conservation between the fractions of repetitive DNA within genera and divergence between the two different studied genera. However, the use of C
t-1 DNA fractions as probes in Dichotomius species (Coleoptera, Scarabaeidae) allowed the observation of heterochromatin distribution patterns highly conserved in the terminal/sub-terminal region and an extensive variation in relation to the pericentromeric heterochromatin; which contrasts with the Phanaeini species studied. These data reinforce the intense evolutionary dynamics of the repeated DNA fraction by mutation, gene conversion, unequal crossing-over, circular replication and slippage replication[36–38] generating high divergence among taxa above the genus level.
Chromosomal organization of Mariner transposable elements
It is a common observation that some transposable elements may be overabundant in specific regions of chromosomes, and the results obtained with the mapping of Mariner shows that these sequences are not randomly distributed and have accumulated in the heterochromatic areas. However, the accumulation of this element in euchromatic areas was recently reported in Eyprepocnemis plorans. The accumulation of a large amount of copies in the heterochromatic regions can indicates a selection against insertions of TEs in euchromatin based on ectopic exchanges. Different major forces can affect TEs in heterochromatin and euchromatin regions of the genome, being that accumulation in heterochromatin regions explained by the absence of selection against insertional mutations in genetically inert regions, and stochastic accumulation of deleterious elements in regions with no recombination[1, 2, 39].
Possibly the absence of labeling in three autosomal pairs of C. cyanescens indicates that the evolutionary history of these sequences within the genome of the species follows a distinct pattern; possibly including suppression of recombination between these chromosomes with the other autosomes.
The accumulation of Mariner sequences in the pericentromeric regions is possibly due to the low rate of recombination characteristic of these regions, and could indicates that this element is enriched in regions where the damage of its insertion is reduced[22, 40]. Although it is not possible to predict the possible role of these elements in Coleoptera they may be involved with the chromosomal rearrangements, as the occurrence of pericentromeric inversion observed in D. mimas. This species presents meta-submetacentric (pairs 1, 2, 3 and 7) and acrocentric (pairs 5, 6, 8 and 9) autosomal chromosomes, while C. cyanescens and C. ensifer have meta-submetacentric morphology for all autosomal chromosomes. In D. mimas the presence of four acrocentric autosomal pairs indicates the occurrence of pericentromeric inversions unlike the standard meta-submetacentric karyotype described for the family Scarabaeidae. Chromosomal rearrangements, as that observed in D. mimas, are possible a consequence of transposable elements, that were reported to be involved with various types of rearrangements by transposition and recombination[42–44].
Another approach to the accumulation of transposable elements is that the Mariner transposon could have been maintained in the pericentromeric region by presenting any functional role in the maintenance of this region. For example, during the evolution of the genome, heterochromatic transposable elements may lose the ability to transpose and accumulate mutations and structural rearrangements, acquiring new functions[46, 47]. Feschotte proposed that the movement and accumulation of TEs, as well their derived proteins, have played an important role in the evolution of the genome. The association of TEs and the structure and/or function of centromeres seems to be an usual occurrence, and have been observed in diverse species[47, 48].
The mapping of Mariner in the sex chromosomes of the three species could be related to the common spreading of the TEs in most heterochromatic areas of the genome, or the sex chromosomes can act as a refuge for transposable elements as previously reported[49–51]. Several genetic processes can cause an accumulation of TEs in genomic regions where crossing over is reduced or absent. In some cases, for example, the sex chromosomes show the tendency of non-recombining in the genomic regions to accumulate transposable elements[52, 53]. Another possibility is that, the recombination suppression itself could inhibit recombination in nearby regions of the sex chromosomes.
The transposition/selection model establishes that the distribution and abundance of TEs are indicative of their evolutionary history[36, 54]. This process involves three stages: (i) invasion of the host genome, (ii) rapid spread by replicative transposition, and (iii) vertical inactivation and accumulation in the heterochromatin. Considering that hypothesis, the Mariner present in C. cyanescens, C. ensifer and D. mimas could be considered ancient because active and recently acquired elements are expected to be preferentially located in euchromatin. TEs are expected to be overabundant in the heterochromatin where recombination is strongly reduced, and because the TEs cannot be easily removed from heterochromatin once they have been inserted[55, 56].
Besides the accumulation in the heterochromatin, the Mariner sequences hybridization patterns are quite different between the three species. This suggests that the chromosomes do not share a general pool of Mariner sequences, and could indicate a different evolutionary path after the emergence within each species.
Mariner transposable elements in Scarabaeinae coleopterans
The Mariner sequences of Scarabaeinae coleopterans branched out into two groups, showing a relatively high genetic distance between them, indicating an early divergence from an ancestral element. This is consistent with previous studies, proposing that members of the Tc1/Mariner are probably monophyletic in origin, and diversified in various groups by accumulation of modifications and/or horizontal transfer mechanisms[9, 57, 58].
Probably, each TE copy of beetles has evolved independently of each other, according to the pattern of molecular evolution related for Mariner transposon. When divergent elements do exist, they display, as observed, a low percentage of similarity to the full-length sequences. This suggests that TEs are highly active within the genome, and that the highly divergent copies reflect relics of ancient mobilizations, as described to Drosophila melanogaster.
Mariner horizontal transfer
Mariner transposable elements have been described in many arthropods, possibly spread by HT[17, 20, 59]. In general, the phylogenies based on Mariner sequences are not always congruent with the phylogenies of the taxa, suggesting the occurrence of HT[14, 60].
The high sequence similarity between sequences from distantly related organisms, the incongruence between TE distribution and phylogeny, and the unequal distribution of some Mariner subfamilies among closely related taxa indicate that the HT contributed to this widespread distribution[18, 20, 61]. Several TEs have been introduced into mammal lineages through HT[62–65], including Mariner[19, 66, 67]). Comparative analyses of mammalian genomes show the presence of high amount of TEs, but their content could vary among the different lineages[18, 68]. The genetic distance within Mariner1_Tbel sequences between mammals and insects species were relatively low, consistent with the phylogenetic distances between them and reinforces the occurrence of HT in the spread of these elements to different taxa.
Considering the Mariner tree topology clearly indicates the involvement of HT during the evolutionary history of insects and mammals, although it is not possible to show in which evolutionary moment this transfer occurred. Multiple mechanisms may be related to the spread of TE by horizontal transfer, using different types of vectors (external parasites, infectious agents, intracellular parasites and symbionts, DNA viruses, RNA viruses, retroviruses)[13, 69, 70]. Thus, for each described case of a proposed HT, could be implemented a model of transfer. Our results are consistent with the criteria of HT, and reveal interesting patterns of patchy distribution among animals, suggesting a repeated invasion of Mariner from insects to mammals.