Phylogeny And Adaptive Evolution Of The Bats Within Rhinolophus Philippinensis Group

Adaptive evolution has become the most important content of biological research since Darwin period.Accurate phylogenetic relationship and taxonomic is an important basis for studying the adaptive evolution.Chiroptera(bat)is the secondlargest order for mammals.They have a high diversity of species and occupy a unique niche in the night sky.There has a high level of morphological convergence in bats,especially in the morphology related to vocalization.And the phylogenetic relationships are not clear among most species.Bats rely mainly on echolocation for navigation and foraging,and this important phenotypic feature is the key to bat adaptation.The study of echolocation,an important phenotype,helps to elucidate the molecular mechanisms underlying the acoustic evolution of bats.Flight of bats consumes a lot of energy.A study on the mitochondrial encoding genes of the oxidative phosphorylation(OXPHOS)system can show some evidence of the adaptive evolution of energy-related genes in bat evolution.The Rhinolophus philippinensis group,is a specific group among all the rhinolophid groups.They are characterized with huge ears and low echolocation frequency compared with other rhinolophidea with the same body size.There may be a special evolutionary process of the species within the philippinensis group.However,the taxonomic of species of the philippinensis group is still controversial,and the phylogeny of this group is poorly understood.Although this deviation has been described,the molecular mechanism of the adaptation remains rarely documented.Therefore,the philippinensis group is an ideal group for studying the phylogeny and adaptive evolution.We selected the philippinensis group as the research object.And we conduct this study based on molecular markers,mitochondrial genome(mitogenome),and transcriptome data,and combined both morphological and acoustic data.We delimited the species boundary of species in the philippinensis group,reconstructed the phylogenetic relationship between species,and further analyzed characteristics of the adaptive evolution of species with low echolocation frequencies in the process of evolution.The results can provide important basic data onto the study of phenotype differences in bats.The main contents and results as follows:The estimated phylogenies revealed the polyphyly of the philippinensis group for multilocus marker,mitogenomes and transcriptomes data.Except for R.philippinensis,all the other species in this group formed a monophyletic cluster,named herewith as “R.macrotis” species group.We promoted the former philippinensis group to two groups,philippinensis and macrotis groups.In the macrotis group,the macrotis complex formed one clade,except the concatenated mitochondrial tree.And different from phylogenetic analysis on mitogenomes,macrotis complex was close to R.rex.Phylogeny based on transcriptomes indicated the macrotis group formed a monophyletic clade,and this clade formed paraphyletic with the clade containing R.rex and R.marshalli.Rhinolophus macrotis formed paraphyletic in the concatenated mitochondrial gene tree and the concatenated multilocus tree,but formed one clade in the concatenated nuclear gene tree.We performed species delimitation and evolution analyses using the integrated data of morphology,acoustic,and genetic data,and the molecular marker includes four mitochondrial genes(CO1?Cytb?ND2 and 16 S rRNA),four autosomal introns(THY?PRKC1?ACOX2 and COPS7A)and one Y-linked intron(SMCY7)in 95 specimens representing nine taxa.Phylogenies,genetic and phenotypic divergence,and species delimitation analyses supported the revised status of R.paradoxolophus as a subspecies of R.rex,R.cf.macrotis as a subspecies of R.macrotis,and R.huananus and R.cf.siamensis as junior synonyms of R.siamensis.Significant discordances were found between the mitochondrial and nuclear gene trees,suggesting incomplete lineage sorting or ancient introgression events within the philippinensis group.The macrotis group appeared to have undergone a rapid radiation approximately 2.57 million years ago during the early Pleistocene period due to palaeoclimatic oscillations.Reconstruction of the ancestral ranges suggested a wide distribution of the common ancestor for the philippinensis and macrotis groups.We used mitochondrial genomes(mitogenomes)from twelve individuals,eight of which were newly determined sequences for comparative studies on adaptive evolution of those bats with low frequencies relative to body size.Based on codon models and physicochemical profiles of amino acid replacements,we found the evidence of positive selection on two amino acids,which were encoded by genes of OXPHOS complex I,NADH dehydrogenase(ND5 and ND6),with a background of widespread purifying selection on all mitochondrial genes.The ND2 gene(also in complex I),showed the relaxed purifying selection.The signal of positive selection was found in R.philippinensis,who distribute in tropics regions compared with other species with low frequencies.These results indicate that mitochondrial protein-coding genes were targets of adaptive evolution during the evolution of the philippinensis and macrotis groups.We concluded that the natural selection driving the evolution of mitochondrial OXPHOS genes might contribute to the particular characteristics of those two groups.We sequenced transcriptomes data of brain,liver,and cochlea for five species of the macrotis group and its closely related species,R.pusillus,to explore the molecular basis of their adaptations.We used comparative transcriptomics to study the special evolutionary process of species in the macrotis group at the sequence level.The results of selection pressure analysis showed that a total of seven positively selected genes(PSGs)(CRYM,FOXM1,MAP6,PYCARD,SLC35A2,WRB and SPRY2)linked to the hearing were found in the species of the macrotis group and 1,274 species-specific selection sites were detected.Among the PSGs,SPRY2,associated with hearing loss and bone development,was identified in R.osgoodi,who emit echolocation with normal frequency in the macrotis group,and the change of amino acid sites was the same as those of R.pusillus.Unexpectedly,we also found five PSGs(ARRDC3,LZTFL1,RAB8 A,IGFBPL1 and TRNT1)linked to vision in branches of species with low frequencies.These results showed the positively selected signals of some sensory genes were robustly correlated with the strength of natural selection.Furthermore,the PSGs identified in the branches of the macrotis group significantly enriched in GO categories related to metabolism(e.g.catalytic activity and oxidoreductase activity).Thus,positive selection in genes relevant to hearing and vision,and significant enrichment in metabolism-related GO categories at the molecular level provide evidence to parse the genetic adaptations of the species with low frequencies within the macrotis group.Based on the above analysis,the taxonomic were resolved,the phylogenetic relationship between species was clarified,and a new species group,the macrotis group was proposed by using multilocus markers.The mitogenome related to energy metabolism and multi-tissue transcriptomes data were used to analyze the adaptive evolution of the species with low frequencies.Explore the role of natural selection in the evolution of species at the molecular level is helpful to reveal the intrinsic genetic mechanism of complex adaptive phenotypes in species.This paper first reveals the genetic basis of low frequency species from the molecular level,and provide a new perspective to study the evolution of the bat development.The important genes and mutation loci we screened in this study are of great significance in the study of bats adaptive evolution for the future.Study the function and potential related pathways of these genes,such as the evolution of the auditory,will help to better understand the phenotypic evolution of bats,especially the acoustic evolution.