Research On Species Delimitation And Speciation Within Rhinolophus Macrotis Complex

Understanding the genetic mechanisms and evolutionary processes under speciation has been a central goal of evolutionary biology.In recent years,rapid changes in climate have caused impacts on biodiversity.It becomes more important to understand the influence of natural selection,drift and other factors on the process of speciation as well as the response mechanisms under local adaptation.Bats(Chiroptera)are widely distributed with rich diversity and play important roles in maintaining the stability of the ecosystem.This study targeted on the big-eared horseshoe bat complex(Rhinolophus macrotis complex)to demonstrate the important factors and evolutionary processes involved in interspecific divergence and speciation from four aspects,species delimitation,evolutionary reconstruction,local adaptation and driving forces.By collecting R.macrotis complex and “R.sp1” samples from southern China and Vietnam,and integrating genetic markers with different modes of inheritance(mitochondrial genes,nuclear introns,and microsatellite loci),genome-wide SNPs,phenotypic characteristics(external morphological,craniodental and acoustic parameters)and several topotypes,we clarified the presence of three closely related species,R.episcopus,R.siamensis and R.osgoodi within this complex.Although the genetics and phenotypes diverged among the allopatric populations within R.episcopus,no speciation events were detected by BPP,thus we conservatively classified them into three subspecies,R.e.episcopus,R.episcopus spp.and R.e.caldwelli.Based on the newly delimited species,we reconstructed the evolution process of three closely related horseshoe bats.Through a series of hybridization and introgression tests,we detected F2 hybrids and different levels of introgression among species,suggested hybridization and introgression may be the main reason for low levels of mitochondrial divergence among three species.Demographic analyses found the most recent common ancestor of three species could be traced back to ca.1.91 million years ago(Ma).Rhinolophus osgoodi firstly diverged at ca.1.59 Ma and then expanded;R.episcopus and R.siamensis diverged at ca.1.51 Ma,and then R.episcopus expanded,while the populations of R.siamensis were relatively stable.Combined with the geographic distribution and paleogeographical climate events,we deduced that glacial-interglacial cycles drove geographic isolation and secondary contacts of these species,and then promoted hybridization and lineage fusion among them,resulting in a reticulate evolutionary pattern.After evolutionary reconstruction,we studied the local adaptation under heterogeneous environments for each species by incorporating genotyping-by-sequencing method and environmental association analysis.Based on the simulation of allele frequencies turnover along the environmental gradients,we found R.episcopus which distributed widely in southern China,is mainly influenced by the lowest precipitation in dry season;R.siamensis which distributed in Southern Coast,is mostly influenced by altitude and maximum precipitation in wet season;and R.osgoodi which distributed in high altitude area,is mainly influenced by the lowest temperature.Enrichment analysis found the potential climate-adaptative SNPs mainly enriched in biological processes of neurodevelopment and signal transduction.In R.episcopus,we also found biological processes of angiogenesis,locomotory behavior and sensory perception of sound and light stimulus.The above processes might have related to circadian rhythms adjustment,acoustic regulation and dietary resource obtainment during the process of local adaptation in bats.For the purpose of demonstrating the importance of genetic drift,environmental selection and sensory variation in bat intraspecific divergence and incipient speciation,we focused on11 populations of R.episcopus,and tested the correlation among geographic distance,environmental distance,acoustic variation and genetic divergence.The results showed significant pattern of isolation by geographic distance in microsatellites and SNPs,suggesting that genetic drift had an important impact on nuclear DNA;divergence in microsatellites and mt DNA were significantly correlated with acoustic variation,indicating that sensory variation may have played roles in bat intraspecific divergence.While the causal models supported that geographic distance affects nuclear divergence primarily,then the genetic divergence influences acoustic variation.Mantel tests and MRM analyses didn't find significant correlation between environmental variable and genetic divergence,but the results of RDA showed that environments contributed to genetic divergence.In conclusion,this study has provided reliable evidence for demonstrating interspecific phylogenetic relationships and taxonomic status,has provided a case study for understanding the importance of hybridization,selection,genetic drift,and sensory variation on speciation,has provided insight into the process of local adaptation,and has practical significance for species diversity conservation.