We generated reduced representation genome sequencing (RADseq) data for more than 500 individuals sampled during several field trips to the mediterranean with a focus on sicily. Based on the data we first described all parental lineages and their hybrids and tested for losses of heterozygosity which revealed the absence of large scale losses. Further, we have generated genomic resources of eight (sub)species of Bacillus stick insects using long read and short read sequencing, and chromosome conformation capture. We utilized the whole-genome data to phase the genomes of all hybrid Bacillus lineages, i.e. separate sequences derived from the mother and the father, and reconstruct a genome-wide phylogenetic tree including the phased haplosets of the hybrids and the parental species. Further, we phased the RADseq data and reconstructed a maternal and a paternal phylogeny including all maternal and paternal haplosets, respectively. Both trees comprise > 350 individual haplosets. The results indicate a single, common origin of all hybrid lineages and showcase a scenario in which a transition to hybridogenesis was likely followed by a host switch and a transition to parthenogenesis. Such a transition between alternative reproductive strategies in the absence of sex has not been previously described.
Further, we conducted a series of crosses aimed at revealing associations between hybridogenesis and genomic regions. We first crossed hybridogenetic females with males of the maternal ancestral species. We backcrossed the resulting offspring (F1; here the hybridogenetic genome and the “sexual” maternal genome undergo meiosis) with males of the paternal species to reestablish the hybrid state (F2). This has resulted in rearing ~100 F2 individuals. In order to narrow down the genomic regions associated with hybridogenesis, we continued the crossing experiments by mating the obtained F2 females again with males of the maternal species (F3) and their offspring again with males of the paternal species (F4). We determined the reproductive mode of the F2 generation by genotyping their unfertilised eggs. We successfully detected the two expected reproductive phenotypes among the F2 generation: hyridogenesis was characterized by complete absence of the paternal genome in unfertilized eggs while meiotic production of eggs was characterized by presence of both parental haplotypes with distinct recombination break points visible in genotyping data. Surprisingly, we also detected a transition to an effectively clonal transmission of both parental haplosets that was characterized by presence of maternal and paternal haplotypes along the entire length of the genome. In addition to the whole genome sequencing data of the maternal haploset of the hybridogenetic lineages (see above) we generated whole genome sequencing data of the males of the maternal species that were used to initiate the crossing experiment. Presence of both datasets enables us to test for recombination between the hybridogenetic and the paternal haploset and ultimately identify the regions associated with hybridogenesis. These analyses are in progress.
We have engaged in several activities designated to disseminate our results including publication in eminent journals, several presentations at international conferences, presentations at (invited) seminars, dissemination via social media, a personal homepage and at workshops. The results of the project have stimulated several ongoing collaborative actions and grant applications.
