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Comparative genomics provides insights into chromosomal evolution and immunological adaptation in horseshoe bats

Nature Ecology & Evolution volume 9, pages 705–720 (2025)Cite this article

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Abstract

Horseshoe bats are natural hosts of zoonotic viruses, yet the genetic basis of their antiviral immunity is poorly understood. Here we generated two new chromosomal-level genome assemblies for horseshoe bat species (Rhinolophus) and three close relatives, and show that, during their diversification, horseshoe bats underwent extensive chromosomal rearrangements and gene expansions linked to segmental duplications. These expansions have generated new adaptive variations in type I interferons and the interferon-stimulated gene ANXA2R, which potentially enhance antiviral states, as suggested by our functional assays. Genome-wide selection screens, including of candidate introgressed regions, uncover numerous putative molecular adaptations linked to immunity, including in viral receptors. By expanding taxon coverage to ten horseshoe bat species, we identify new variants of the SARS-CoV-2 receptor ACE2, and report convergent functionally important residues that could explain wider patterns of susceptibility across mammals. We conclude that horseshoe bats have numerous signatures of adaptation, including some potentially related to immune response to viruses, in genomic regions with diverse and multiscale mutational changes.

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Fig. 1: Genome assembly quality and phylogenetic relationships in the Rhinolophidae.
Fig. 2: Chromosomal evolution in the Rhinolophidae.
Fig. 3: Expansion of the ANXA2R gene.
Fig. 4: Diversity of type I IFN genes.
Fig. 5: Evaluation of ACE2 critical sites determining SARS-CoV-2 binding and entry among species and individuals.
Fig. 6: Putative genetic introgression in Rhinolophidae.

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Data availability

New genome sequence data for the five bats are deposited at the Genome Sequence Archive in National Genomics Data Center (https://ngdc.cncb.ac.cn) under accession code CRA018832. Genome assemblies are deposited at the NGDC GenBank under accessions GWHFDMV00000000.1 (https://ngdc.cncb.ac.cn/gwh/Assembly/86071/show, R. sinicus), GWHFDMW00000000.1 (https://ngdc.cncb.ac.cn/gwh/Assembly/86072/show, R. pearsonii), GWHFDMX00000000.1 (https://ngdc.cncb.ac.cn/gwh/Assembly/86073/show, H. armiger), GWHFDMY00000000.1 (https://ngdc.cncb.ac.cn/gwh/Assembly/86073/show, H. pratti) and GWHFDMZ00000000.1 (https://ngdc.cncb.ac.cn/gwh/Assembly/86075/show, M. lyra). RNA-seq data of RfKT cells can be accessed at the NGDC (accession number: PRJCA023723). SARS-CoV-2 S protein sequence was obtained from GenBank (accession number: MN908947). Human ACE2 protein sequence was obtained from GenBank (accession number: NP_001358344.1). The data files used for the population genomics analyses, gene family analysis and PAML analysis are available via Figshare at https://doi.org/10.6084/m9.figshare.27612597 (ref. 124).

Code availability

The code and pipelines used for the analyses are available via Zenodo at https://doi.org/10.5281/zenodo.13690583 (ref. 125) and GitHub (https://github.com/SLbio/Comparative-genomics-of-horseshoe-bats).

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant no. 32270436), National Key Research and Development Program of China (grant no. 2021YFF0702004), R&D Program of Guangzhou National Laboratory (grant no. SRPG22-001), Fundamental Research Funds for the Central Universities (grant no. 2042022dx0003) and Natural Science Foundation of Hubei Province (grant no. 2023AFA015) to H.Z. S.T. was supported in part by the National Natural Science Foundation of China (grant no. 32471689), the Beijing Nova Program (grant nos Z211100002121022 and 20230484446) and the Open Fund of Key Laboratory of Biodiversity and Environment on the Qinghai-Tibet Plateau, Ministry of Education (grant no. KLBE2024009). L.Z. was supported in part by the Guangdong Provincial Science and Technology Program (grant no. 2021B1212050021).

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Author notes

  1. These authors contributed equally: Shilin Tian, Junyu Si, Libiao Zhang.

Authors and Affiliations

  1. State Key Laboratory of Virology and Biosafety, Key Laboratory of Biodiversity and Environment on the Qinghai-Tibetan Plateau, Ministry of Education, Frontier Science Center for Immunology and Metabolism, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China

    Shilin Tian, Junyu Si, Jiaming Zeng, Xiangyi Zhang, Chen Huang, Caoqi Lei, Huan Yan & Huabin Zhao

  2. Novogene Bioinformatics Institute, Beijing, China

    Shilin Tian

  3. Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, China

    Libiao Zhang

  4. College of Life Sciences, Shaanxi Normal University, Xi’an, China

    Gang Li

  5. CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Xuming Zhou

  6. Guangzhou National Laboratory, Guangzhou International Bio Island, Guangzhou, China

    Rong Geng & Peng Zhou

  7. School of Biological and Behavioural Sciences, Queen Mary University of London, London, UK

    Stephen J. Rossiter

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  1. Shilin Tian
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Contributions

H.Z. conceived and designed the research. S.T. designed and performed analyses. J.S., J.Z., X. Zhang, X. Zhou, C.H., C.L. and H.Y. performed the functional experiments. L.Z. collected the samples. G.L. provided the genome assembly of R. affinis. R.G. and P.Z. provided RNA-seq data of RfKT cells. S.T., S.J.R. and H.Z. analysed data, discussed results and wrote the manuscript. All authors have read and approved the paper.

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Correspondence to Stephen J. Rossiter or Huabin Zhao.

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Nature Ecology & Evolution thanks Zijun Xiong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Table 1 Global summary of genome assemblies for six bats
Full size table

Extended Data Fig. 1 Maximum likelihood (ML) tree of 13 bats and 7 other mammals, showing numbers of expanded (purple) and contracted (red) gene families.

Grey blocks indicate confidence intervals for divergence times for all nodes. Orange shading corresponds to Rhinolophidae (horseshoe bats), pink to Hipposideridae, blue to other bats, and green to non-bat mammals.

Extended Data Fig. 2 Chord diagram depicting genome synteny between R. sinicus and each of R. affinis, R. pearsonii, and R. ferrumequinum.

In each diagram, R. sinicus is shown on the right-hand side. Syntenic blocks are connected, with percentages indicating the overall synteny rates across the genomes.

Extended Data Fig. 3 Tracing chromosomal evolution of Rhinolophidae.

The top panel displays the heat map of the interaction signal after aligning the R. pearsonii Hi-C data to the R. ferrumequinum genome. The middle panel shows the collinearity of the sequences related to two ancestral chromosome fission events between R. pearsonii and R. ferrumequinum, and between R. affinis and R. ferrumequinum, respectively. The bottom panel displays the heat map of the interaction signals after aligning the R. affinis Hi-C data to the R. ferrumequinum genome. Abbreviations: RAC, Rhinolophus ancestral chromosome; Chr, Chromosome.

Extended Data Fig. 4 Evolution of ANXA2R genes.

(a) The significant change of ANXA2R loci in each branch. “1” represents expansion, “−1” denotes contraction, no marks mean no change. Orange shading corresponds to horseshoe bats (Rhinolophidae), pink to roundleaf bats (Hipposideridae), blue to other bats, and green to non-bat mammals. (b) Phylogenetic tree analysis of intact ANXA2R genes of Yinpterochiroptera bats. The human ANXA2R sequence is used as an outgroup. All nodes received 100% bootstrap support.

Extended Data Fig. 5 Collinear analysis of R. aegyptiacus between the chromosome-level genome and the scaffold-level genome (Raegyp2.0).

There were many duplicated scaffold sequences where IFN-ω was located when examining.

Extended Data Fig. 6 Evolutionary expansions and contractions of type I IFN genes across 20 focal mammals.

“1” represents an expansion event relative to its ancestral clades, “−1” denotes a contraction event relative to its ancestral clades. “0” and no marks mean no change. We used the conditional likelihoods to test the statistical significance for each lineage (P < 0.05). Orange shading corresponds to horseshoe bats (Rhinolophidae), pink to roundleaf bats (Hipposideridae), blue to other bats, and green to non-bat mammals.

Extended Data Fig. 7 Gene gain and loss events in the examined bats.

The intact IFN-δ (a) and IFN-ω (b) gene repertoires of bats, with humans serving as the outgroup. Gene gain and loss events are mapped to the species tree, marked by purple and red numbers, respectively. Orange shading corresponds to horseshoe bats (Rhinolophidae), pink to roundleaf bats (Hipposideridae), blue to other bats, and green to humans.

Extended Data Fig. 8 Molecular evolutionary changes in C5aR1 protein.

(a) Alignment of C5aR1 protein sequences. The bottom panel shows the alignment for 20 mammals, with dots representing amino acids identical to the human sequence, and dashes denoting alignment gaps. The top panel shows the genotypes for three Rhinolophus-specific residues in 10 Rhinolophus populations, with H. armiger for comparison. We assessed the predicted physicochemical impact and found that E199Q (R. ferrumequinum and R. pearsonii) and L278T (all Rhinolophus bats) alters hydrophilic affinity, and E199K (R. sinicus and R. affinis) alters negative charge. (b) 3D-structure predictions of C5aR1 proteins for humans, R. sinicus, R. pearsonii, and H. armiger.

Extended Data Fig. 9 Functional assays in Rhinolophus ACE2.

(a) Expression of Rhinolophus ACE2 orthologues. We conducted immunofluorescence of intracellular Rhinolophus ACE2 expression level by detecting the C-terminal 3×FLAG-tag. The label ‘Human’ represents human ACE2-expressing HEK293T cells, and the ‘Vector’ indicates the HEK293T control cells. The scale bar is shown. One time for this experiment. (b) Assessment of the interaction between various Rhinolophus ACE2 orthologues and the SARS-CoV-2 RBD (receptor binding domain). Results were consistent across two biological replicates. (c) Characterization of Rhinolophus ACE2 orthologues mediating entry of SARS-CoV-2 viruses as shown using intracellular EGFP (Enhanced Green Fluorescent Protein). One time for this experiment.

Supplementary information

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Supplementary Notes 1–8 and Figs. 1–12.

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Tian, S., Si, J., Zhang, L. et al. Comparative genomics provides insights into chromosomal evolution and immunological adaptation in horseshoe bats. Nat Ecol Evol 9, 705–720 (2025). https://doi.org/10.1038/s41559-025-02638-2

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