Hybridogenesis and a potential case of R2 non-LTR retrotransposon horizontal transmission in Bacillus stick insects (Insecta Phasmida)

Abstract

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.

Figure 1. Schematic representation of presently characterized R2 elements in the Bacillus genus.

For comparison, also the fragment representing R2Br^del has been reported. Black boxes indicate the open reading frame (ORF) with the zinc finger (ZF), reverse transcriptase (RT) and restriction enzyme-like endonuclease (RLE) domains. Vertical grey lines indicate frameshift mutations; ovals represent stop codon(s); grey squares represent duplications and triangles represent large deletions.

Figure 2. Phylogenetic position of Bacillus R2 elements.

Maximum Likelihood tree (-ln L = 35666.30) built on RT amino acid sequence of Bacillus elements and of elements representative of the four main clades (R2-A, R2-B, R2-C and R2-D). Numbers at nodes represent bootstrap values ≥50%.

Figure 3. Bacillus R2 elements phylogeny.

(a) Dated phylogeny based on R2 nucleotide sequences; numbers at nodes indicate, above, Maximum Likelihood (-ln L = 2449.07) bootstrap values ≥50%/Bayesian posterior probabilities ≥0.90 and, below, age estimate 95% high posterior density interval. Nodes with black dots are those where age calibration was applied. The R2Bgg^B element was not included due to its extreme nucleotide divergence. (b) Schematic drawing of Bacillus taxa phylogeny as derived from ref. .

Figure 4. Age versus divergence analysis.

Plot of host age vs. nucleotide divergence (a) and vs. amino acid divergence (b). Red diamonds indicate comparisons relative to putative horizontal transfers (HTs). Two trend-lines are reported: the dotted one is relative to the analysis including all comparisons; the filled one is relative to the analysis that exclude putative HT comparisons. Correlation coefficients (R²), together with probability (ns: not significant; *p < 0.001), are reported near the respective trend-line.

Figure 5. Possible models of R2 horizontal transfer (HT) in Bacillus stick insects.

(a) HT of an element from the paternal to the maternal species haploset. Via a standard hybridogenetic path followed by a backcross with a male of the maternal species, the element can invade the genome of the maternal species. (b) Horizontal transfer of an element from the maternal to the paternal species haploset. Following the standard hybridogenetic path, the element has no chance to enter into the paternal species genome given that the paternal haploset carrying the transferred element is lost. Only instances of maternal haploset exclusion may lead to offspring with fully paternal genome either through gynogenesis or backcross. Also androgenesis produces offspring with fully paternal genome in a maternal cytoplasm. In this case, though, the HT event must be mediated by the element RNA intermediate that allows the transfer from the excluded hybridogenetic genomes to the one derived from two sperm nuclei mixis. Dashed circles within cells are nuclei; red chromosomes represent the maternal haploset and green chromosomes represent the paternal haploset. Red and green small lines within cell cytoplasm represent transcribed RNAs. Thick and thin arrows indicate transcription and re-integration, respectively. Excluded haplosets are shaded in grey.