Evolutionary Genomics Of Pteropodidae And Rhinolophidae Bats

Bats(Chiroptera)are the second most diverse group of mammals and can asymptomatically carry several virulent viruses.Among bats,Pteropodidae and Rhinolophidae bats are associated with most of the natural virus hosts that have attracted the attention of international public health organizations.Therefore,the study of their unique immune evolution mechanism is of great scientific significance to biomedicine.In this study,we developed a new assembly pipeline to generate five high-quality chromosome-level bat genomes,including Cynopterus sphinx in Pteropodidae,Rhinolophus sinicus and Rhinolophus pearsonii in Rhinolophidae,and Hipposideros armiger and Hipposideros pratti in Hipposideridae.Through genomic comparative methods,we clarified the evolutionary genomic characteristics of Pteropodidae and Rhinolophidae,and revealed the lineage-specific genetic changes in bat immunity-related genes.We further verified downregulation of the systemic pro-inflammatory response in Pteropodidae through cell experiments.The main findings of this study are as follows:1.An improved assembly pipeline generates five high-quality chromosome-level bat genomesWe employed Hi-C interaction pairs to cluster Nanopore long sequences that possess potential linkages,thereby avoiding any erroneous overlap caused by long-distance repetitive sequences during string graph assembly.In contrast to the currently commonly used two-step assembly pipeline(assembly first,followed by chromosome anchoring),this study presents a four-step assembly approach that yields better-quality genome assemblies with greater integrity and continuity.The four steps encompass global assembly,clustering,local assembly,and chromosome anchoring.Using this approach,we assembled five chromosome-level genomes for Cynopterus sphinx,Rhinolophus sinicus,Rhinolophus pearsonii,Hipposideros armiger and Hipposideros pratti.These genomes range in size from 1.86 to 2.14 Gb,with contig N50 values spanning 41.00 to84.24 Mb.This study provides the five chromosome-level bat genomes with the highest level of completeness and contiguity to date.2.Exploring the genome evolutionary feature of Pteropodidae and their unique genetic mechanism of immune evolutionComparative genomics analyses revealed that the Pteropodidae-specific r RNAderived SINE/5S elements frequently insert in coding genes related to important adaptive traits.Moreover,immunity-related genes in Pteropodidae show significantly higher evolutionary rates and undergo stronger positive selection than those of other bat species.Specifically,our study identified the lineage-specific genetic changes in immunity-related genes in pteropodid bats,including the loss of the inflammasome gene NLRP1,duplication of the complement receptor C5 a R2 and the peptidoglycan recognition gene PGLYRP1,and four amino acid replacements in the My D88 gene,which encodes My D88– a key signal transducer involve in signaling pathways of the innate immune response.We experimentally tested the impact of My D88 mutants on TLR2-dependent inflammation in both P.altcto kidney-derived Paki cells and human PEAKrapid cells.First,we have applied a co-immunoprecipitation approach to provide direct evidence that pteropodid-specific mutations in My D88 reduce its binding affinity with TLR2 when coexpressed in Pa Ki cells.Then,we expressed human and pteropodid My D88 transgenes containing these four residue mutations,confirming that proteins with pteropodid-specific replacements dampen the inducing of TLR2-dependent inflammation in human and bat cells.Additionally,we also revealed that the N-and C-terminus of cyclic GMP-AMP synthase(c GAS)gene in Pteropodidae enhance sensing of viral DNA and attenuate inflammatory responses to viruses,respectively.In summary,our findings suggest that Pteropodidae have evolved a diversity of functional pathways for immune and inflammatory dampening responses to microbial infection.3.Revealing the evolution of chromosome rearrangements and the unique genetic mechanism of immune evolution in RhinolophidaeBy conducting comparative genomics analyses,we discovered that the evolution of segmental duplications(SDs)is conducive to the innovation of immune gene families in Rhinolophidae.SDs have promoted the functional expansion of annexin II receptor genes,which could potentially inhibit viral replication by enhancing the induction of apoptosis,thus helping to maintain viral tolerance in Rhinolophus bats.We analyzed the genomic distribution of type I IFN(IFN-I)genes in bats,and observed that bats have a contracted IFN-α subfamily,while exhibiting varying degrees of expansion in IFN-ω and IFN-δsubfamilies,accompanied by the SD evolution.Especially in Rhinolophus bats,the IFN-δ and IFN-ω subfamilies have simultaneously undergone the most extensive gene expansion evolution,and the IFN-β subfamily have undergone positive selection.These results demonstrate a compensatory evolutionary mechanism of the IFN-I subfamilies,with the contraction of the IFN-α subfamily and the expansion or positive selection of other IFN-I subfamilies,which promotes the unique virus infection tolerance of the immune system in Rhinolophus bats.Finally,we uncovered evidence that positive selection of immune genes in Rhinolophus bats could suppress inflammation,such as the amino acid replacements in Rhinolophus C5 a R1 that can alter ligand binding affinity of complement cascade related to C5a-C5 a R1 axis,thereby attenuating inflammatory responses to viruses in bats.In conclusion,we present an improved four-step genome assembly pipeline that has the potential to improve upon current approaches in producing genome assemblies with higher integrity and contiguity.The five chromosome-level genomes generated provide valuable genomic resources for the systematic study of distinctive adaptations in bats.Comparative genomics analyses have revealed a parallel evolutionary mechanism of the immune system in Pteropodidae and Rhinolophidae bats,including the adaptive evolution of the C5 a complement cascade signaling pathway,an enhanced apoptotic program,and compensatory evolution among the different subfamilies of antiviral immunity-related IFN-I genes.These findings may help to explain why bats have been frequently identified as viral hosts.Particularly in the current pandemic of coronavirus disease 2019(COVID-19),our results will serve as a reference for how bats tolerate coronavirus infection,and provide ideas for antiviral therapy in humans.