Final Report Summary - RHINOSPEC (Dissecting speciation using a genomics approach) It is widely accepted that when different species or subspecies (sometimes called ‘taxa’) interbreed (or ‘hybridise’) successfully, their genes can become mixed up. However, less well understood is whether all genes are equally likely to flow between different taxa, or whether some genes are resilient to this process of so-called ‘introgression’. Indeed, quite often after such interbreeding, taxa are seen to retain their distinct physical appearance, in which case we might expect that genes encoding taxonomic differences have not undergone introgression. On the other hand, foreign genes that offer advantages might be likely to introgress more easily and subsequently spread in the population.Our project focused on the process and the consequence of hybridisation among wild horseshoe bats in China, known by their scientific names of Rhinolophus pearsoni pearsoni, R. p. chinensis and R. yunanensis. Our earlier work showed that these taxa sometime hybridise in areas of range overlap, and that introgression has taken place as a result. The principle aim of the Marie Curie grant was to determine the extent to which this introgression has occurred and, specifically, to assess which genes or larger ‘genomic islands’ have resisted mixing. To do this, originally we proposed to collect low coverage (2x depth) genome sequence data from three focal taxa, and to use a statistical pipeline to identify genes that have crossed taxonomic barriers. The pipeline exploits that fact those taxa that swap genes will appear to be more related based on these genes or genomic islands than they would if other parts of their genomes were examined. We then planned to screen a small subset of around 15 introgressed and non-introgressed genes across the populations in China. During the early stages of our project, newly available Next Generation Sequencing (NGS) technologies became available, which were also associated with lower costs. In order to harness the power of these new technologies we made some strategic modifications to our original methods. Briefly, we combined two recent NGS strategies together: transcriptome sequencing (i.e. RNAseq) and targeted resequencing. First, we generated RNAseq data for each of our three focal taxa and one other species (as a comparison or ‘outgroup’), which yielded much greater coverage (>100X) of around 10,000 genes per taxon. Analysing these data revealed a total of 3655 genes that were detected across our focal taxa and the outgroup. We then built multiple gene alignments, and ran our pipeline to identify loci that either show interesting signatures of introgression (hereafter called putative introgression genes) or selection, as well as genes known to be involved in reproductive isolation in other vertebrates based on the results of other studies (hereafter called putative speciation gene). Second, for each of these putative introgression and/or speciation genes (134 genes), together with genes from other categories (e.g. genes function in echolocation/hearing, vision, sexual conflict, and reproduction; in total 1800 genes) we designed “baits” that are also based on RNA. The function of these baits is to anneal to our targeted gene sequences in other individuals, thus allowing these genes to be “captured”, and then to be sequenced. Using this state-of-the-art target capture method, we resequenced over 2000 genes in 14 R. p. chinensis, 49 R. p. pearsoni and 19 R. yunanensis. The data are being processed at the moment and look to be of very high quality.Our current study system and sequencing strategy provide us opportunities to test several hypothesises on speciation. First, among those chosen genes, ~80 are putative echolocation/hearing genes, which will allow us to test whether echolocation plays an important role in reproductive isolation of horseshoe bats compared with other sensory (i.e. vision) genes or non-sensory genes. If the answer is yes, we will expect more echolocation genes to be observed in the category of reproductive isolation compared with other genes. Second, ~200 genes from the X chromosome were resequenced across our three focal taxa, which can be used to test whether X chromosome genes contribute greatly to reproductive isolation in bats, as suggested for other mammals (e.g. mouse). This is the first study to test this hypothesis in bats based on the data from the whole X chromosome. Our second objective in our original proposal is to determine whether there is a relationship between the extent of genomic divergence/genetic mixing and the presence, nature and frequency of large mutations known as chromosomal rearrangements. This can be examined at three levels: between the two species (see objective 1), in the chromosomal hybrid zone between R. p. pearsoni and R. p. chinensis. Now new chromosome data (i.e. G-band) of R. p. pearsoni from Hunan (i.e. parapatric region with R. p. chinensis) have been generated, which can be compared with the published chromosome data of R. p. pearsoni from Guizhou (i.e. outside of the hybrid zone between R. p. pearsoni and R. p. chinensis) and of R. p. chinensis from Anhui. As a consequence of using NGS technologies, we have successfully generated a very large volume of data. Dr Mao’s analysis of the dataset is ongoing, and is also a main task during the third year (reintegration year in China) of this project. We strongly believe that we will be able to characterise genes with differential introgression rates at a scale that was not envisioned in our original proposal, and we anticipate at least five further papers in peer-review journals (we have written two in the past year). Our findings will undoubtedly contribute greatly to current knowledge and understanding of the nature and genetic consequences of interbreeding in wild animal populations.
