Theoretical and empirical studies have shown differential management of transposable elements in organisms with different reproductive strategies. To investigate this issue, we analysed the R2 retroelement structure and variability in parthenogenetic and bisexual populations of Bacillus rossius stick insects, as well as insertions inheritance in the offspring of parthenogenetic isolates and of crosses. The B. rossius genome hosts a functional (R2Br(fun) ) and a degenerate (R2Br(deg) ) element, their presence correlating with neither reproductive strategies nor population distribution. The median-joining network method indicated that R2Br(fun) duplicates through a multiple source model, while R2Br(deg) is apparently still duplicating via a master gene model. Offspring analyses showed that unisexual and bisexual offspring have a similar number of R2Br-occupied sites. Multiple or recent shifts from gonochoric to parthenogenetic reproduction may explain the observed data. Moreover, insertion frequency spectra show that higher-frequency insertions in unisexual offspring significantly outnumber those in bisexual offspring. This suggests that unisexual offspring eliminate insertions with lower efficiency. A comparison with simulated insertion frequencies shows that inherited insertions in unisexual and bisexual offspring are significantly different from the expectation. On the whole, different mechanisms of R2 elimination in unisexual vs bisexual offspring and a complex interplay between recombination effectiveness, natural selection and time can explain the observed data.

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5’ end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution.

Transposable elements (TEs) are selfish genetic elements whose self-replication is contrasted by the host genome. In this context, host reproductive strategies are predicted to impact on both TEs load and activity. The presence and insertion distribution of the non-LTR retrotransposon R2 was here studied in populations of the strictly bisexual Bacillus grandii maretimi and of the obligatory parthenogenetic Bacillus atticus atticus. Furthermore, data were also obtained from the offspring of selected B. a. atticus females. At the population level, the gonochoric B. g. maretimi showed a significantly higher R2 load than the obligatory parthenogenetic B. a. atticus. The comparison with bisexual and unisexual Bacillus rossius populations showed that their values were higher than those recorded for B. a. atticus and similar, or even higher, than those of B. g. maretimi. Consistently, an R2 load reduction is scored in B. a. atticus offspring even if with a great variance. On the whole, data here produced indicate that in the obligatory unisexual B. a. atticus R2 is active and that mechanisms of molecular turnover are effective. Furthermore, progeny analyses show that, at variance of the facultative parthenogenetic B. rossius, the R2 activity is held at a lower rate. Modeling parental-offspring inheritance, suggests that in B. a. atticus recombination plays a major role in eliminating insertions rather than selection, as previously suggested for unisexual B. rossius progeny, even if in both cases a high variance is observed. In addition to this, mechanisms of R2 silencing or chances of clonal selection cannot be ruled out.

Horizontal transfer (HT) is an event in which the genetic material is transferred from one species to another, even if distantly related, and it has been demonstrated as a possible essential part of the lifecycle of transposable elements (TEs). However, previous studies on the non-LTR R2 retrotransposon, a metazoan-wide distributed element, indicated its vertical transmission since the Radiata-Bilateria split. Here we present the first possible instances of R2 HT in stick insects of the genus Bacillus (Phasmida). Six R2 elements were characterized in the strictly bisexual subspecies B. grandii grandii, B. grandii benazzii and B. grandii maretimi and in the obligatory parthenogenetic taxon B. atticus. These elements were compared with those previously retrieved in the facultative parthenogenetic species B. rossius. Phylogenetic inconsistencies between element and host taxa, and age versus divergence analyses agree and support at least two HT events. These HT events can be explained by taking into consideration the complex Bacillus reproductive biology, which includes also hybridogenesis, gynogenesis and androgenesis. Through these non-canonical reproductive modes, R2 elements may have been transferred between Bacillus genomes. Our data suggest, therefore, a possible role of hybridization for TEs survival and the consequent reshaping of involved genomes.

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