Horseshoe Bat Species Research Articles

Systematics of the Rhinolophus landeri complex, with evidence for 3 additional Afrotropical bat species

Abstract Roughly a third of all horseshoe bat species (Rhinolophidae: Rhinolophus) are found in Africa, where a recent continent-wide genetic survey suggested the presence of both undescribed and apparently invalid species. Here, we focus on the R. landeri species complex and the recent elevation of R. lobatus Peters, 1852, to species rank. That action created ambiguity in the taxonomy of East African members of the group—are both R. landeri Martin, 1838, and R. lobatus sympatric in East Africa or is another, unnamed species present there? Here, we refine genetic, morphological, and behavioral characterizations of R. landeri and its erstwhile synonyms with samples from the vicinity of their type localities. The distribution of R. landeri appears to be limited to Central and West Africa; existing genetic records attributed to this species from Mali clearly represent another taxon. We marshal genetic evidence for the species-level distinction of R. dobsoni Thomas, 1904, from Sudan, which was previously considered a synonym of R. landeri. We reject R. axillaris J. A. Allen, 1917, as a synonym of the R. landeri complex, provisionally regarding it as a valid member of the landeri species group. Finally, we demonstrate that East Africa is home to a fourth species of the landeri complex that is named herein. Final resolution of the systematics of this species complex awaits expanded characterizations (especially of genetics, vocalizations, and noseleaves) and studies of variation in regions of contact.

Rapid and reliable method for identification of three medium-sized horseshoe bat species in Europe

Rapid and reliable method for identification of three medium-sized horseshoe bat species in Europe

Sarbecoviruses of British horseshoe bats; sequence variation and epidemiology.

Horseshoe bats are the natural hosts of the Sarbecovirus subgenus that includes SARS-CoV and SARS-CoV- 2. Despite the devastating impact of the COVID-19 pandemic, there is still little known about the underlying epidemiology and virology of sarbecoviruses in their natural hosts, leaving large gaps in our pandemic preparedness. Here we describe the results of PCR testing for sarbecoviruses in the two horseshoe bat species (Rhinolophus hipposideros and R. ferrumequinum) present in Great Britain, collected in 2021-22 during the peak of COVID-19 pandemic. One hundred and ninety seven R. hipposideros samples from 33 roost sites and 277 R. ferrumequinum samples from 20 roost sites were tested. No coronaviruses were detected in any samples from R. ferrumequinum whereas 44 and 56 % of individual and pooled (respectively) faecal samples from R. hipposideros across multiple roost sites tested positive in a sarbecovirus-specific qPCR. Full genome sequences were generated from three of the positive samples (and partial genomes from two more) using Illumina RNAseq on unenriched samples. Phylogenetic analyses showed that the obtained sequences belong to the same monophyletic clade, with >95 % similarity to previously-reported European isolates from R. hipposideros. The sequences differed in the presence or absence of accessory genes ORF 7b, 9b and 10. All lacked the furin cleavage site of SARS-CoV-2 spike gene and are therefore unlikely to be infective for humans. These results demonstrate a lack, or at least low incidence, of SARS-CoV-2 spill over from humans to susceptible GB bats, and confirm that sarbecovirus infection is widespread in R. hipposideros. Despite frequently sharing roost sites with R. ferrumequinum, no evidence of cross-species transmission was found.

Sex differences in gene expression and alternative splicing in the Chinese horseshoe bat.

Sexually dimorphic traits are common in sexually reproducing organisms and can be encoded by differential gene regulation between males and females. Although alternative splicing is common mechanism in generating transcriptional diversity, its role in generating sex differences relative to differential gene expression is less clear. Here, we investigate the relative roles of differential gene expression and alternative splicing between male and female the horseshoe bat species, Rhinolophus sinicus. Horseshoe bats are an excellent model to study acoustic differences between sexes. Using RNA-seq analyses of two somatic tissues (brain and liver) from males and females of the same population, we identified 3,471 and 2,208 differentially expressed genes between the sexes (DEGs) in the brain and liver, respectively. DEGs were enriched with functional categories associated with physiological difference of the sexes (e.g.,gamete generation and energy production for reproduction in females). In addition, we also detected many differentially spliced genes between the sexes (DSGs, 2,231 and 1,027 in the brain and liver, respectively) which were mainly involved in regulation of RNA splicing and mRNA metabolic process. Interestingly, we found a significant enrichment of DEGs on the X chromosome, but not for DSGs. As for the extent of overlap between the two sets of genes, more than expected overlap of DEGs and DSGs was observed in the brain but not in the liver. This suggests that more complex tissues, such as the brain, may require the intricate and simultaneous interplay of both differential gene expression and splicing of genes to govern sex-specific functions. Overall, our results support that variation in gene expression and alternative splicing are important and complementary mechanisms governing sex differences.

A New Species of Horseshoe Bat (Chiroptera: Rhinolophidae) from Mount Namuli, Mozambique

The ecology of the high-altitude mountains of northern Mozambique is understudied in comparison to surrounding countries. A series of biological surveys have focused on filling this data gap, with Mount Namuli in Zambezia Province one of the focal sites of these expeditions. A biological survey of Mount Namuli in 2009 resulted in the collection of five specimens of a horseshoe bat species (Rhinolophidae) that is here described as a new species from Mozambique. Morphologically, the new species is very similar to Rhinolophus maendeleo Kock, Csorba and Howell, 2000 of the adami-group, but lacks some key morphological characters of this group (large ears, narrow skull, long palate). Molecular reconstructions clearly suggest the new species belongs to the capensis-group, but no members of the adami-group were included in this analysis (due to lacking data). It is thus unclear whether this unexpected phylogenetic position reflects morphological convergences between members of the adami- and capensis-groups, or whether the morphology-based adami-group should be reconsidered. The new species and R. maendeleo share similar external and craniodental measurements, but can be distinguished based on a number of key characters. These include the presence of a bony bar forming the interorbital foramena, rostrum shape, ear length and highly differing bacular morphologies. It also differs from the genetically closely related R. denti Thomas, 1904, R. swinnyi Gough, 1908 (including two recently described cryptic species) and R. simulator Andersen, 1904 by non-overlapping external and cranial measurements. The new species echolocates at a mean peak frequency of 76.9 kHz and shows an affinity to forest habitats, which are highly threatened in the surrounding region. It joins other coastal and montane forest endemics in defining the bat fauna of south-eastern Africa.

Geographic variation in the skulls of the horseshoe bats, Rhinolophus simulator and R. cf. simulator: Determining the relative contributions of adaptation and drift using geometric morphometrics.

The relative contributions of adaptation and genetic drift to morphological diversification of the skulls of echolocating mammals were investigated using two horseshoe bat species, Rhinolophus simulator and R. cf. simulator, as test cases. We used 3D geometric morphometrics to compare the shapes of skulls of the two lineages collected at various localities in southern Africa. Size and shape variation was predominantly attributed to selective forces; the between‐population variance (B) was not proportional to the within‐population variance (W). Modularity was evident in the crania of R. simulator but absent in the crania of R. cf. simulator and the mandibles of both species. The skulls of the two lineages thus appeared to be under different selection pressures, despite the overlap in their distributions. Difference in the crania of R. cf. simulator was centered largely on the nasal dome region of R. cf. simulator but on the cranium and mandibles of R. simulator. It is likely that the size and shape of the nasal dome, which acts as a frequency‐dependent acoustic horn, is more crucial in R. cf. simulator than in R. simulator because of the higher echolocation frequencies used by R. cf. simulator. A larger nasal dome in R. cf. simulator would allow the emission of higher intensity pulses, resulting in comparable detection distances to that of R. simulator. In contrast, selection pressure is probably more pronounced on the mandibles and cranium of R. simulator to compensate for the loss in bite force because of its elongated rostrum. The predominance of selection probably reflects the stringent association between environment and the optimal functioning of phenotypic characters associated with echolocation and feeding in bats.

Horseshoe bat species recorded in the mangrove ecosystem of the Cat Ba National Park, Northern Vietnam

Cat Ba National Park is the core zone of the Cat Ba Archipelago Biosphere Reserve. It contains quite diverse and unique ecosystems including 776 ha of mangrove forest. Although bats of the park were included in many publications, none were recorded from the mangrove ecosystem. We recently conducted field surveys through the wetland areas of the park and recorded three horseshoe bat species: Marshall's horseshoe bat (Rhinolophus marshalli), Pearson’s horseshoe bat (Rhinolophus pearsonii), and the least horseshoe bat (R. pusillus). Of these, R. pearsonii is the most common species. We here provide morpholocal and acoustic features of each species with notes on the importance of the mangrove forest in the park for bat research and conservation.

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.

Bat-borne polyomaviruses in Europe reveal an evolutionary history of intrahost divergence with horseshoe bats distributed across the African and Eurasian continents.

Polyomaviruses (PyVs) are small, circular dsDNA viruses carried by diverse vertebrates, including bats. Although previous studies have reported several horseshoe bat PyVs collected in Zambia and China, it is still unclear how PyVs evolved in this group of widely dispersed mammals. Horseshoe bats (genus Rhinolophus) are distributed across the Old World and are natural reservoirs of numerous pathogenic viruses. Herein, non-invasive bat samples from European horseshoe bat species were collected in Hungary for PyV identification and novel PyVs with complete genomes were successfully recovered from two different European horseshoe bat species. Genomic and phylogenetic analysis of the Hungarian horseshoe bat PyVs supported their classification into the genera Alphapolyomavirus and Betapolyomavirus. Notably, despite the significant geographical distances between the corresponding sampling locations, Hungarian PyVs exhibited high genetic relatedness with previously described Zambian and Chinese horseshoe bat PyVs, and phylogenetically clustered with these viruses in each PyV genus. Correlation and virus-host relationship analysis suggested that these PyVs co-diverged with their European, African and Asian horseshoe bat hosts distributed on different continents during their evolutionary history. Additionally, assessment of selective pressures over the major capsid protein (VP1) of horseshoe bat PyVs showed sites under positive selection located in motifs exposed to the exterior of the capsid. In summary, our findings revealed a pattern of stable intrahost divergence of horseshoe bat PyVs with their mammalian hosts on the African and Eurasian continents over evolutionary time.

What We Need to Consider During and After the SARS-CoV-2 Pandemic.

Even though extreme containment and mitigation strategies were implemented by numerous governments around the world to slow down the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the number of critically ill patients and fatalities keeps rising. This crisis has highlighted the socioeconomic disparities of health care systems within and among countries. As new CoVID policies and responses are implemented to lessen the impact of the virus, it is imperative (1) to consider additional mitigation strategies critical for the development of effective countermeasures, (2) to promote long-term policies and strict regulations of the trade of wildlife and live animal markets, and (3) to advocate for necessary funding and investments in global health, specifically for the prevention of and response to natural and manmade pandemics. This document considers some of these challenges.

Extensive Genetic Diversity of Polyomaviruses in Sympatric Bat Communities: Host Switching versus Coevolution.

Polyomaviruses (PyVs) are small DNA viruses carried by diverse vertebrates. The evolutionary relationships of viruses and hosts remain largely unclear due to very limited surveillance in sympatric communities. In order to investigate whether PyVs can transmit among different mammalian species and to identify host-switching events in the field, we conducted a systematic study of a large collection of bats (n = 1,083) from 29 sympatric communities across China which contained multiple species with frequent contact. PyVs were detected in 21 bat communities, with 192 PyVs identified in 186 bats from 15 species within 6 families representing at least 28 newly described PyVs. Surveillance results and phylogenetic analyses surprisingly revealed three interfamily PyV host-switching events in these sympatric bat communities: two distinct PyVs were identified in two bat species in restricted geographical locations, while another PyV clustered phylogenetically with PyVs carried by bats from a different host family. Virus-host relationships of all discovered PyVs were also evaluated, and no additional host-switching events were found. PyVs were identified in different horseshoe bat species in sympatric communities without observation of host-switching events, showed high genomic identities, and clustered with each other. This suggested that even for PyVs with high genomic identities in closely related host species, the potential for host switching is low. In summary, our findings revealed that PyV host switching in sympatric bat communities can occur but is limited and that host switching of bat-borne PyVs is relatively rare on the predominantly evolutionary background of codivergence with their hosts.IMPORTANCE Since the discovery of murine polyomavirus in the 1950s, polyomaviruses (PyVs) have been considered highly host restricted in mammals. Sympatric bat communities commonly contain several different bat species in an ecological niche facilitating viral transmission, and they therefore represent a model to identify host-switching events of PyVs. In this study, we screened PyVs in a large number of bats in sympatric communities from diverse habitats across China. We provide evidence that cross-species bat-borne PyV transmission exists, though is limited, and that host-switching events appear relatively rare during the evolutionary history of these viruses. PyVs with close genomic identities were also identified in different bat species without host-switching events. Based on these findings, we propose an evolutionary scheme for bat-borne PyVs in which limited host-switching events occur on the background of codivergence and lineage duplication, generating the viral genetic diversity in bats.

CORRIGENDUM: Integrative taxonomy resolves three new cryptic species of small southern African horseshoe bats (Rhinolophus)

CORRIGENDUM: Integrative taxonomy resolves three new cryptic species of small southern African horseshoe bats (Rhinolophus)

Resolving evolutionary relationships among six closely related taxa of the horseshoe bats (Rhinolophus) with targeted resequencing data

Recently diverged taxa are often characterised by high rates of introgressive hybridization and incomplete lineage sorting, both of which can complicate phylogenetic reconstructions of species histories. Here we use a sequence capture approach to obtain genome-wide data to resolve the evolutionary relationships, and infer the extent and timescale of hybridization and introgression events, among six recently diverged taxa of the horseshoe bat species complexes Rhinolophus sinicus and R. thomasi. We show that two different methods of species tree reconstruction applied to a set of ~1500 nuclear loci all recover species trees with similar topologies, differing from the previous phylogeny based on two nuclear loci. By comparing the tree topology obtained from the nuclear loci with that inferred from the mitochondrial genome, we observed at least three cases of conflict, each of which likely results from past introgression. Of these, the occurrence of a highly similar mitogenome sequence shared by individuals of two taxa in a sympatric region points to very recent mtDNA introgression. The other cases are characterised by greater divergence and strong phylogeographic structure among putative introgressed individuals and their source populations, and thus likely reflect more ancient hybridization events. These results also suggest that two of the subspecies (R. s. septentrionalis and the undescribed taxon R. s. ssp) are likely to represent full species, warranting full taxonomic descriptions. This work adds a growing number of studies showing the potential problems of relying solely on mitochondrial sequences, or a limited number of loci, to infer phylogenetic relationships among recently diverged taxa.

Echolocation call frequency and mitochondrial control region variation in the closely related bat species of the genus Rhinolophus (Chiroptera: Rhinolophidae) occurring in Iran: implications for taxonomy and intraspecific phylogeny

There is a high level of morphological similarity among rhinolophid species leading to problematic taxonomic identifications. We undertook analyses of mitochondrial DNA gene sequences (D-loop), along with morphologic and acoustic examinations in order to evaluate taxonomic status and phylogenetic relationships of the horseshoe bat species in Iran. All analyses based on molecular, morphological, and sonar characteristics revealed five rhinolophid species including Rhinolophus mehelyi, R. euryale, R. ferrumequinum, R. blasii, and R. hipposideros in Iran. Genetic study revealed one lineage in R. mehelyi, two lineages in each of species R. euryale, R. ferrumequinum, and R. blasii, and three lineages in R. hipposideros. Our results showed high haplotype diversity (Hd) in the Iranian rhinolophid species. Compared with other studies of bat D-loop sequences, the genetic mean nucleotide diversity obtained for R. blasii in the current study (π = 0.0569) was the highest value and for R. euryale was found to be the lowest value (π = 0.0126) among nucleotide diversity values for other bat species. The average dominant frequency (peak frequency ± standard deviation) for R. euryale, R. ferrumequinum, R. mehelyi, R. blasii, and R. hipposideros was 106.57 ± 0.32, 82.02 ± 0.19, 105.57 ± 0.45, 92.85 ± 0.95, and 110.74 ± 1.93 respectively. As a conclusion, based on the present genetic study, there are one to three lineages including at least one southern and one northern in every rhinolophid species in Iran.

Maxilloturbinal Aids in Nasophonation in Horseshoe Bats (Chiroptera: Rhinolophidae).

Horseshoe bats (Family Rhinolophidae) show an impressive array of morphological traits associated with use of high duty cycle echolocation calls that they emit via their nostrils (nasophonation). Delicate maxilloturbinal bones inside the nasal fossa of horseshoe bats have a unique elongated strand-like shape unknown in other mammals. Maxilloturbinal strands also vary considerably in length and cross-sectional shape. In other mammals, maxilloturbinals help direct respired air and prevent respiratory heat and water loss. We investigated whether strand-shaped maxilloturbinals in horseshoe bats perform a similar function to those of other mammals, or whether they were shaped for a role in nasophonation. Using histology, we studied the mucosa of the nasal fossa in Rhinolophus lepidus, which we compared with Hipposideros lankadiva (Hipposideridae) and Megaderma lyra (Megadermatidae). Using micro-CT scans of 30 horseshoe bat species, we quantified maxilloturbinal surface area and skull shape within a phylogenetic context. Histological results showed horseshoe bat maxilloturbinals are covered in a thin, poorly vascularized, sparsely ciliated mucosa poorly suited for preventing respiratory heat and water loss. Maxilloturbinal surface area was correlated with basicranial width, but exceptionally long and dorsoventrally flat maxilloturbinals did not show enhanced surface area for heat and moisture exchange. Skull shape variation appears to be driven by structures linked to nasophonation, including maxilloturbinals. Resting echolocation call frequency better predicted skull shape than did skull size, and was specifically correlated with dimensions of the rostral inflations, palate, and maxilloturbinals. These traits appear to form a morphological complex, indicating a nasophonatory role for the strand-shaped rhinolophid maxilloturbinals. Anat Rec, 2018. © 2018 American Association for Anatomy.

High Duty Cycle Echolocation May Constrain the Evolution of Diversity within Horseshoe Bats (Family: Rhinolophidae)

The phenotype of organisms is the net result of various evolutionary forces acting upon their lineages over time. When an innovative trait arises that confers a substantial advantage in terms of survival and reproduction, the evolution of adaptive complexes between such an innovation and other traits may constrain diversification of that lineage. The specialized echolocation system of the Rhinolophidae may represent such an innovation which affects other parts of the phenotype. We investigated a potential constraint on the diversity of phenotypes of several species of horseshoe bats within a phylogenetic framework. If phenotypic convergence stems from stasis as a result of the specialized echolocation system, phenotypes should converge not only among members of the same species and between sexes but also among species. We analyzed the phenotypic diversity of >800 individuals of 13–16 species. The phenotypes in the horseshoe bats did indeed converge. There was no sexual size dimorphism in mass, forearm length and wingspan within species and there was marked interspecific similarity in both wing and echolocation variables but marked variability in body mass. Furthermore, correlations of wing and echolocation variables with mass suggest that variability within horseshoe bats was largely the result of selection on body size with allometric responses in wing and echolocation parameters, a potential consequence of constraints imposed by their specialized echolocation.

Contribution to the Ectoparasite Fauna of Bats (Chiroptera: Vespertilionidae, Rhinolophidae) of Crimea

New data are presented on the ectoparasite fauna of several species of vesper and horseshoe bats (Chiroptera: Vespertilionidae, Rhinolophidae) of the Crimean Peninsula. In the studied territory, 11 species of ectoparasites (mites and insects) have been collected from 6 bat species; 2 of the ectoparasite species were new to Crimea. Findings of gamasid mites Ichoronyssus scutatus on an unusual host are discussed. The gamasid mite Spinturnix emarginatus (Acari: Mesostigmata: Gamasina) is described for the territory of Russia for the first time.

Integrative taxonomy resolves three new cryptic species of small southern African horseshoe bats (Rhinolophus)

AbstractExamination of historical and recent collections of small Rhinolophus bats revealed cryptic taxonomic diversity within southern African populations previously referred to as R. swinnyi Gough, 1908 and R. landeri Martin, 1832. Specimens from Mozambique morphologically referable to R. swinnyi were phylogenetically unrelated to topotypic R. swinnyi from the Eastern Cape Province of South Africa based on cytochrome b sequences and showed distinctive echolocation, baculum and noseleaf characters. Due to their genetic similarity to a previously reported molecular operational taxonomic unit (OTU) from north-eastern South Africa, Zimbabwe and Zambia, we recognize the available synonym (R. rhodesiae Roberts, 1946) to denote this distinct evolutionary species. This new taxon is genetically identical to R. simulator K. Andersen, 1904 based on mtDNA and nuclear DNA sequences but can easily be distinguished on morphological and acoustic grounds. We attribute this genetic similarity to historical introgression, a frequently documented phenomenon in bats. An additional genetically distinct and diminutive taxon in the swinnyi s.l. group (named herein, R. gorongosae sp. nov.) is described from Gorongosa National Park, central Mozambique. Specimens from Mozambique referable based on morphology to R. landeri were distinct from topotypic landeri from West Africa based on mtDNA sequences, and acoustic, noseleaf and baculum characters. This Mozambique population is assigned to the available synonym R. lobatus Peters, 1952.

Remnants of an Ancient Deltaretrovirus in the Genomes of Horseshoe Bats (Rhinolophidae).

Endogenous retrovirus (ERV) sequences provide a rich source of information about the long-term interactions between retroviruses and their hosts. However, most ERVs are derived from a subset of retrovirus groups, while ERVs derived from certain other groups remain extremely rare. In particular, only a single ERV sequence has been identified that shows evidence of being related to an ancient Deltaretrovirus, despite the large number of vertebrate genome sequences now available. In this report, we identify a second example of an ERV sequence putatively derived from a past deltaretroviral infection, in the genomes of several species of horseshoe bats (Rhinolophidae). This sequence represents a fragment of viral genome derived from a single integration. The time of the integration was estimated to be 11–19 million years ago. This finding, together with the previously identified endogenous Deltaretrovirus in long-fingered bats (Miniopteridae), suggest a close association of bats with ancient deltaretroviruses.

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus.

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.