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The exact origin of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) and source of introduction into humans has not been established yet, though it might be originated from animals. Therefore, we conducted a study to understand the putative reservoirs, transmission dynamics, and susceptibility patterns of SARS‐CoV‐2 in animals. Rhinolophus bats are presumed to be natural progenitors of SARS‐CoV‐2‐related viruses. Initially, pangolin was thought to be the source of spillover to humans, but they might be infected by human or other animal species. So, the virus spillover pathways to humans remain unknown. Human‐to‐animal transmission has been testified in pet, farmed, zoo and free‐ranging wild animals. Infected animals can transmit the virus to other animals in natural settings like mink‐to‐mink and mink‐to‐cat transmission. Animal‐to‐human transmission is not a persistent pathway, while mink‐to‐human transmission continues to be illuminated. Multiple companions and captive wild animals were infected by an emerging alpha variant of concern (B.1.1.7 lineage) whereas Asiatic lions were infected by delta variant, (B.1.617.2). To date, multiple animal species – cat, ferrets, non‐human primates, hamsters and bats – showed high susceptibility to SARS‐CoV‐2 in the experimental condition, while swine, poultry, cattle showed no susceptibility. The founding of SARS‐CoV‐2 in wild animal reservoirs can confront the control of the virus in humans and might carry a risk to the welfare and conservation of wildlife as well. We suggest vaccinating pets and captive animals to stop spillovers and spillback events. We recommend sustainable One Health surveillance at the animal–human–environmental interface to detect and prevent future epidemics and pandemics by Disease X.

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., The relative contributions of adaptation and drift to morphological diversification of the crania of echolocating mammals were investigated using two horseshoe bat species, Rhinolophus simulator and R. cf. simulator, as test cases. Shape variation was predominantly attributed to selective forces. 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.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing tens of millions of infections and over a million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species’ ACE2 and human ACE2. As cattle are a known reservoir for coronaviruses with previous human zoonotic transfer, and has a relatively divergent ACE2 sequence, we compared the binding kinetics of bovine and human ACE2 to SARS-CoV-2 RBD. This revealed a nanomolar binding affinity for bovine ACE2 but an approximate ten-fold reduction of binding compared to human ACE2. Since cows have been experimentally infected by SARS-CoV-2, this lower affinity sets a threshold for sequences with lower homology to human ACE2 to be able to serve as a productive viral receptor for SARS-CoV-2.

Here we discuss the issues for attributing the new coronavirus infection COVID-19 to zoonoses based on the data on probable origin of the SARS-CoV-2 virus, the possible formation of its reservoir in animals (bats) as well as human susceptibility. Today, the dominant point of view is that the outbreak of COVID-19 arose as a result of the SARS-CoV-2 coronavirus overcoming the interspecies barrier, acquiring ability to infect and spread in human population. Comparative phylogenetic analysis at the molecular level showed that SARS-CoV-2 is genetically closest to bat coronaviruses, particularly to the RmYN02 and RaTG13 strains isolated from the horseshoe bat, a species considered to be the main host of SARSCoV and MERS-CoV coronaviruses. The ability of the SARS-CoV-2 coronavirus to infect various wild animal has been revealed. SARS-CoV-2 has been found in minks on farms in the Netherlands with mortality rates ranging from 1.2 to 2.4%. While infecting rhesus monkeys with the SARS-CoV-2, it resulted in productive infection and detected viremia. Cats have been found to be susceptible hosts for the human SARS-CoV-2 virus. A likely explanation for this lies in the high similarity between the human and feline counterpart of the ACE2 receptor. It has been shown that dogs can become infected but transmit no virus to other animals. To date, over the entire period of the pandemic the World Organization for Animal Health provides no information about cases of human infection transmitted from pets. Thus, there is no evidence that animals play a role in the spread of SARS-CoV-2 among people during the current period of the pandemic. Human outbreaks are caused by human-to-human virus transmission, and based on the currently available information, the risk of spreading COVID-19 from animals is considered low. More research is needed to understand how COVID-19 can affect animals of a wide variety of species and how big might be the risks of infection transmission from them to humans.

In Australia, there are at least 50 000 derelict mines, many of which provide habitat for cave-roosting bats. Grating of derelict mines, be it horizontal (adits) or vertical (shafts) drives, is commonly undertaken to prevent human access, though longer-term responses of bats are largely unknown. We assessed the long-term (2–20 years) effects of grating on bats by documenting trends in emergence activity and bat abundance at grated and ungrated derelict mines and quantified behavioural responses of bats in autumn and winter. Emergence activity was dominated by the eastern horseshoe bat (Rhinolophus megaphyllus) with limited activity of other less manoeuvrable species. Both emergence activity and minimum colony size at horizontal adits were 8–9 times greater than at vertical shafts, with bats observed emerging from only 2 of 13 shafts. Emergence activity and minimum colony size were 7–10 times greater at adits with ‘bat friendly’ grating (horizontal bars with spacing >125 mm) than at other treatments (ungrated adits and adits with standard grating). In winter, there were 4–11 times more aborted exit attempts per bat at adits with ‘bat friendly’ grating compared with other treatments, which corresponded to greater emergence activity. Emergence activity and minimum colony size were not related to spacing between bars or time since grating, indicating rapid habituation by R. megaphyllus. However, circling at grates continued for many years and bentwing bats (Miniopterus spp.) made little use of these sites. Bat-friendly grates appear to be an effective management option for R. megaphyllus, but alternatives need to be trialled for other species.

Dispersal plays an important role in the ecological and evolutionary processes of natural populations. Mating behavior (or mating system) is a critical factor shaping dispersal patterns and extents in social mammals, sometimes driving the evolution of sex-biased dispersal. Using molecular markers with contrasting modes of inheritance (mitochondrial DNA and nuclear microsatellites), we determined the population genetic structure and evolutionary history of the great horseshoe bat, Rhinolophus ferrumequinum inhabiting eleven national parks of South Korea, being known as a biodiversity hotspot. Despite apparent matrilineal structure observed over space, there was weak nuclear geographic structure, suggesting female philopatry with male-biased dispersal. The analyses indicated the signal of nonrandom mating (i.e. inbreeding), which is at least partly due to female’s sedentary lifestyle. The large-scale phylogenetic analysis revealed unexpected deep divergence among three distinct clades (Southwest China, East China, and Northeast Asia including South Korea), suggesting these may possibly represent cryptic species complex in R. ferrumequinum. Our findings of sex differences in dispersal in R. ferrumequinum inform conservation managements to enhance the population connectivity among the national parks through promoting female dispersal. Our study also highlights cryptic species diversity in a temperate bat that should have a priority for conservation.

Anatomy of bat genital organs has been conventionally studied by gross and microscopic observations to date. Here, we employ both histological observation and diceCT (diffusible iodine-based contrast-enhanced computed tomography) to study the detailed three-dimensional morphological structure of the male genital organs in bats, using the greater horseshoe bat, Rhinolophus ferrumequinum. This is the first study to three-dimensionally describe the whole reproductive organs of bats in detail. Our highly resolved three-dimensional reconstruction reveals that the male organs of R. ferrumequinum consist of paired testes, epididymides, deferent ducts, and five accessory genital glands. The boundary between the ampullary and vesicular glands has been difficult to identify in previous observations, but our diceCT imaging allowed us to clearly differentiate the two. We found that the ampullary gland is located at the terminal part of the deferent ducts, and the vesicular gland lies distal to the ampullary glands. This species possesses a single and carrot-shaped urethral gland, which is not found in most chiropteran families. The presence of the urethral gland in this species and its secretions suggest that after copulation this species is capable of forming a vaginal plug, which can seal the female’s vaginal orifice to block the entrance of spermatozoa from other males. The presence of the urethral gland and elongated epididymal tail and the fact that some individuals can terminate their hibernation and reactivate imply forced copulation on hibernating females can occur in R. ferrumequinum.

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.

The greater horseshoe bat (Rhinolophus ferrumequinum), distributed from Europe and Northeast Africa through the temperate regions of Asia to Japan, is regarded as either a single, albeit polytypic, species or as representing a complex of two species, R. ferrumequinum sensu stricto in the western part and R. nippon in the eastern part of the seemingly continuous range. To evaluate patterns of geographic variation within the complex and to determine if distinguishing morphological characters exist between R. ferrumequinum and R. nippon, we performed a comprehensive comparative study of skull morphology, combining traditional linear measurements, geometric morphometric methods, and evaluation of non-quantitative discriminant characters. We analyzed 230 specimens from 19 localities in 10 geographic regions sampled from central Europe, Iraq, Nepal, China, North Korea, South Korea, and Japan. Our results revealed remarkable differences in skull morphology between the European-West Asian and East Asian groups; variation between these two groups was considerably larger than within-group variation. The two groups were separable by three main features: 1) the position of the anterior palatal edge in relation to the cusps of the upper first molar; 2) the position of the glenoid cavity; and 3) the robustness of the skull as expressed by the ratio of the zygomatic and mastoid widths. In line with recent molecular biological studies, we recommend recognizing R. ferrumequinum (Schreber, 1774) and R. nippon Temminck, 1835 as two distinct species. Our results also revealed unique size variations in island populations of R. nippon, presumably shaped by the island rule and the founder effect.

Flycatching is relatively uncommon in insectivorous bats, yet members of the family Rhinolophidae constitute over one-half of the documented flycatching species. The Formosan woolly horseshoe bat, Rhinolophus formosae, is among the largest in size and relies primarily on flycatching for foraging. We assessed perch use of flycatching R. formosae in relation to vegetation structure in tropical monsoon forests in southern Taiwan. We located bats using acoustic detectors in forest interior and edge-open forest sites, and measured perch features, dispersion of the nearest trees, and vegetation structure within a 5-m radius of each perch. The same measurements were applied to randomly selected perches in both habitats where bats were not detected. We found no seasonal effects or differences between used and random perches in perch features, dispersion of neighboring trees, or vegetation structure surrounding the perches. Perches used at edge-open forest sites were farther from the perch tree trunk and neighboring trees, and surrounded by larger trees than in forest interiors. In contrast, perches in forest interiors were surrounded by higher shrub and reef layers and greater canopy, shrub, and reef layer cover, than those at edge-open forests. Overall, perches in forest interiors were in more cluttered settings, containing higher vegetation obstacles than edge-open habitats. In both habitats, vegetation obstacles generally increased in a curvilinear manner when moving horizontally and downward from the perch. However, in forest interiors perches used by bats had significantly lower vegetation obstacles horizontally and downwardly and were less cluttered than randomly selected perches. Overall, our results indicate that R. formosae in forest interiors selectively used perches associated with more open space that allows for more maneuverable sally flights and a longer detection range suitable for its exceptionally low constant frequency calls to explore less cluttered environments.

The Mediterranean horseshoe bat, Rhinolophus euryale, creates in the pre-hibernation period an aggregation in the Domica cave (Slovakia) and despite the harsh winter conditions displays a certain amount of flying activity. The goal of the study was to find out whether some environmental factors influence these conspicuous aggregation dynamics. We used image and statistical analysis of pictures of the aggregation taken automatically in the cave along with atmospheric pressure, humidity, outside and inside cave temperature and oxygen and carbon dioxide contents in the cave. These environmental variables were assumed to explain the observed activity. We hypothesised that the number of bats in particular time periods would correlate with some of these variables, but this was not confirmed. The only significant relations confirmed were those between bat numbers and humidity and carbon dioxide values in the cave. No trend in the spatial positioning of the bats was observed, and no environmental factors influenced the numbers, movements or groupings of bats. On the contrary, bats by their presence (breathing), activity and production of excrements may significantly influence the microclimate of the cave environment.

The performance of vocal motor control is crucial for vocal communication; however, its evolution and ecological drivers are largely unknown. Doppler shift compensation (DSC), which has been found only in bats, represents an excellent model for studying the mechanisms and evolution of vocal control. We tested whether species that are more dependent on DSC for pulse–echo separation have a more precise DSC and whether changes in environmental clutter would result in adjustments in DSC performance. We recorded the echolocation and flight behaviour of the greater horseshoe bat, Rhinolophus ferrumequinum, and the great leaf-nosed bat, Hipposideros armiger, flying through a corridor and a window in turn. The DSC precision in H. armiger was significantly lower than that of R. ferrumequinum; the offset between the reference frequency and resting frequency, however, was not significantly different between the two species. We also studied the DSC of H. armiger flying through windows of different sizes. Their flight speed decreased significantly with reduced window size. However, DSC precision and the offset did not vary significantly with window size. Our results suggest that DSC is more precise in rhinolophids, which have a higher demand for pulse–echo separation by DSC, than in hipposiderids. Changes in environmental clutter do not necessarily result in adjustments of DSC performance. Functional importance associated with high-performance vocal control may be critical in shaping the precision of vocal control in the course of evolution. Instead, habitat clutter encountered by vocalizing animals may not play an important role.

The objective of this study was to investigate morphological characteristics and functional roles of vaginal plug formation and release in female hibernating Korean greater horseshoe bat (Rhinolophus ferrumequinum korai) using optical microscope and scanning electron microscope. Vaginal plug formation begins by part of semen first jetted from cervical region and secretions of vaginal epithelium immediately after mating (at the end of August). These secretions are gradually solidified, accumulated, and moved toward the vaginal introitus while filling the vaginal lumen. This formation process continues through the copulation period and the hibernation period (from end of October to middle of March), maintaining vaginal plug throughout the next pregnancy period (from end of March to end of May). Finally, vaginal plug is discharged to the outside of vagina immediately before giving birth. Normal vaginal structures are taken from the nurturing period (from the beginning of June to the middle of August) and before mating period. Therefore, vaginal plug generated immediately after mating is important as a protective barrier to prevent re-mating with male. In addition, vaginal plug during pregnancy can protect fetus in the uterus from physical stimulation by preventing re-mating with male as a kind of defense mechanism to maintain healthy development of fetus in uterus.

Bats form the second most diverse mammalian order (Chiroptera), after rodents, and vary widely in their physiology and ecology. Those species that live in temperate climates are generally insectivorous and nocturnal or crepuscular, sheltering in tree hollows, caves, or buildings during the day. They are potentially valuable ecological indicators, due to their dependence on suitable roosting sites and arthropod food, both of which are commonly affected by human activities. Identification of bats from ancient assemblages that are found in caves could therefore provide useful data for palaeoenvironmental reconstructions and show the effect of habitat loss. Here, we apply the recently developed approach of collagen fingerprinting by soft ionisation mass spectrometry to the identification of ancient bat remains in an archaeological assemblage from Pin Hole Cave (Derbyshire, England). Our results show that a simple set of markers can distinguish all seven genera of bats known to be present in either modern or ancient Britain (Myotis, Nyctalus, Pipistrellus, Barbastella, Plecotus, Eptesicus, and Rhinolophus). Further analysis indicates that species‐level determination is possible in some of these taxa, but it would more readily be achieved using the more advanced methods of collagen sequence analysis by liquid chromatography coupled with tandem mass spectrometry. Within our assemblage yielding ~6,800 ancient bone collagen fingerprints, we identified only ~1% that derived from chiropterans, and these were predominantly derived from Myotis (two apparent Brandt's bat fingerprints but the majority indistinguishable between the whiskered, Daubenton's and Natterer's bats), Barbastella (the western barbastelle being the only member of this genus known within Europe), and Rhinolophus (identified as the lesser horsehoe bat R. hipposideros rather than the rare greater horseshoe bat R. ferrumequinum). We infer that the site was likely used by roosting bats throughout the year, and the accumulation of these remains was probably not the result of predator activity. More importantly, the peptide biomarkers provided here could proove valuable in the more systematic analysis of microfaunal remains across many European archaeological and palaeontological sites, preferably those that are collected with well curated stratigraphical information and chronological frameworks.

Mehely’s horseshoe bat, Rhinolophus mehelyi Matschie 1901, is a medium-sized rhinolophid with a discontinuous distribution from North Africa and southern Europe through Asia Minor, Anatolia, to Transcaucasia and Iran. Here, we present a detailed study of the phylogeography and population genetics of this species using 745 bp of the mitochondrial cytochrome b and 522 bp of the mitochondrial D-loop genes in 46 bats in eight localities in western and southwesternIran. Based on the mtDNA sequences, we found a low degree of genetic diversity in the Iranian populations of R. mehelyi (π = 0.0032 for Cytb and π = 0.0064 for D-loop gene), that show a close relationship among the haplotypes. With a K2P genetic distance (0.38%–0.97% on D-loop and 0.22%–0.69% on Cytb), and the phylogenetic reconstruction in the Eurasia lineage two major clades are introduced. Here, the phylogenetic trees and statistical parsimony network showed all Iranian samples were grouped in the same clade, but southern Europe and North Africa samples belonged to another clade. The average genetic divergence between reciprocally monophyletic clade of the R. mehelyi group in Iran and southern Europe and North Africa were 0.69% for Cytb and 4.09% for D-Loop respectively. The analysis of population structure suggests an incongruent pattern of genetic differentiation. Mismatch distributions and neutrality tests showed recent historical demographic events as indicated by significant deviations in Fu’s FS from neutrality. The hypothesis of sudden-expansion was not rejected by analyses of mismatch distribution as the Raggedness and SSD were insignificant (p(r) = 0.11 and p (SSD) = 0.07). Similarly, the Bayesian skyline plot suggested a population expansion scenario that took place in the past 4.000 years.

Some species are difficult to observe and others, need to be not disturbed because of their vulnerability. In response to the difficulty of studying the dispersal behaviors of these species, some areas of biology have been combined in order to access the information despite practical limitations. Here we present the combination of several methodologies from landscape ecology to non- invasive population genetics that allow us to obtain important information onRinolophus hipposideros, a vulnerable European bat. We genotyped 18 georeferrenced colonies in Brittany (France) from droppings collected in their refuges. We used 6 microsatellite markers in order to obtain the genetic distances between them. On the other hand we calculated Euclidian distances between the refuges occupied by these colonies and some ecological distances with the Pathmatrix module of ArcGis 3.2. We tested hypothesis about the difficulty of dispersal of the species in areas without forest cover or with a low density of hedges. Thanks to the Monmonier algorithm we could infer possible genetic barriers between the colonies and we could compare their location to the presence of landscape barriers (areas with little tree cover). We detected a pattern of isolation by distance that reveals limited dispersal capacities in the species but no pattern linked to ecological distances. We found that some of the neighboring colonies with greater genetic distances between them were located in areas with low density of hedges which could suggest an impact of this landscape element in their movements. Finer studies should allow us to conclude on the need or not of forest cover in the dispersal of this species.Abstract Figure

T-cell antigen receptors (TRs) in vertebrates can be divided into αβ or γδ, encoded by TRA/D, TRG, or TRB loci. TRs play a central role in mammal cellular immunity, which occurs by rearrangement of V, D, J, and C genes in the loci. The bat is the only mammal with flying ability and is considered the main host of zoonotic viruses, an important public health concern. However, at present, little is known about the composition of bat TR genes. Based on the whole genome sequence of the greater horseshoe bat (Rhinolophus ferrumequinum) and referring to the TR/IG annotation rules formulated by the international ImMunoGeneTics information system (IMGT), we present a complete annotation of TRA/D, TRG, and TRB loci of R. ferrumequinum. A total of 128 V segments, three D segments, 85 J segments, and 6 C segments were annotated and compared with other known mammalian data. The characteristics of the TR locus and germline genes of R. ferrumequinum are analyzed.

Bats are the reservoir for numerous human pathogens including coronaviruses. The factors leading to the emergence and sustained transmission of coronaviruses in humans are poorly understood. An outstanding question is how coronaviruses can accomplish a host switch with a likely mismatch between the surface protein spike of a bat virus and the human cellular receptor at the time of zoonotic virus transmission. To identify potential novel evolutionary pathways for zoonotic virus emergence, we serially passaged six human 229E isolates in a newly established Rhinolophus lepidus (horseshoe bat) kidney cells and analyzed viral genetic changes. Here we observed extensive deletions within the spike and ORF4 genes of five 229E viruses after passaging in bat cells. As a result, spike protein expression and infectivity of human cells was lost in 5 of 6 viruses but the capability to infect bat cells was maintained. Only viruses that expressed the spike protein could be neutralized by 229E spike-specific antibodies in human cells, whereas there was no neutralizing effect on viruses that do not express the spike protein inoculated on bat cells. However, one isolate acquired an early stop codon abrogating spike expression but maintaining infection in bat cells. Upon passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions amongst virus subpopulations. Spike-independent infection of coronaviruses provides an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and cellular entry receptors.

The subgenus Sarbecovirus includes two human viruses, SARS-CoV and SARS-CoV-2, respectively responsible for the SARS epidemic and COVID-19 pandemic, as well as many bat viruses and two pangolin viruses.Here, the synonymous nucleotide composition (SNC) of Sarbecovirus genomes was analysed by examining third codon-positions, dinucleotides, and degenerate codons. The results show evidence for the eigth following groups: (i) SARS-CoV related coronaviruses (SCoVrC including many bat viruses from China), (ii) SARS-CoV-2 related coronaviruses (SCoV2rC; including five bat viruses from Cambodia, Thailand and Yunnan), (iii) pangolin viruses, (iv) three bat viruses showing evidence of recombination between SCoVrC and SCoV2rC genomes, (v) two highly divergent bat viruses from Yunnan, (vi) the bat virus from Japan, (vii) the bat virus from Bulgaria, and (viii) the bat virus from Kenya. All these groups can be diagnosed by specific nucleotide compositional features except the one concerned by recombination between SCoVrC and SCoV2rC. In particular, SCoV2rC genomes are characterised by the lowest percentages of cyosine and highest percentages of uracil at third codon-positions, whereas the genomes of pangolin viruses exhibit the highest percentages of adenine at third codon-positions. I suggest that latitudinal and taxonomic differences in the imbalanced nucleotide pools available in host cells during viral replication can explain the seven groups of SNC here detected among Sarbecovirus genomes. A related effect due to hibernating bats is also considered. I conclude that the two independent host switches from Rhinolophus bats to pangolins resulted in convergent mutational constraints and that SARS-CoV-2 emerged directly from a horseshoe bat virus.

The source of the COVID-19 pandemic is unknown, but the natural host of the progenitor sarbecovirus is thought to be Asian horseshoe (rhinolophid) bats. We identified and sequenced a novel sarbecovirus (RhGB01) from a British horseshoe bat, at the western extreme of the rhinolophid range. Our results extend both the geographic and species ranges of sarbecoviruses and suggest their presence throughout the horseshoe bat distribution. Within the spike protein receptor binding domain, but excluding the receptor binding motif, RhGB01 has a 77% (SARS-CoV-2) and 81% (SARS-CoV) amino acid homology. While apparently lacking hACE2 binding ability, and hence unlikely to be zoonotic without mutation, RhGB01 presents opportunity for SARS-CoV-2 and other sarbecovirus homologous recombination. Our findings highlight that the natural distribution of sarbecoviruses and opportunities for recombination through intermediate host co-infection are underestimated. Preventing transmission of SARS-CoV-2 to bats is critical with the current global mass vaccination campaign against this virus.

Our understanding of trophic interactions of small insectivorous mammals has been drastically improved with the advent of DNA metabarcoding. The technique has continued to be optimised over the years, with primer choice repeatedly being a vital factor for dietary inferences. However, the majority of dietary studies examining the effect of primer choice often rely on in silico analyses or comparing between species that occupy an identical niche type. Here, we apply DNA metabarcoding to empirically compare the prey detection capabilities of two widely used primer sets when assessing the diets of a flying (lesser horseshoe bat; Rhinolophus hipposideros) and two ground-dwelling insectivores (greater white-toothed shrew; Crocidura russula and pygmy shrew; Sorex minutus). Although R. hipposideros primarily rely on two prey orders (Lepidoptera and Diptera), the unique taxa detected by each primer shows that a combination of primers may be the best approach to fully describe bat trophic ecology. However, random forest classifier analysis suggests that one highly degenerate primer set detected the majority of both shrews’ diet despite higher levels of host amplification. The wide range of prey consumed by ground-dwelling insectivores can therefore be accurately documented from using a single broad-range primer set, which can decrease cost and labour. The results presented here show that dietary inferences will differ depending on the primer or primer combination used for insectivores occupying different niches (i.e., hunting in the air or ground) and demonstrate the importance of performing empirical pilot studies for novel study systems.

AIM: Geographical variations in endotherm body size (e.g. Bergmann's rule/James's rule and Allen's rule) have long been tested. However, the patterns and causes of geographical variation in body size within bat species, especially within widespread hibernating species, are little known. Here, we evaluated the possible causes of geographical size variation patterns in the greater horseshoe bat (Rhinolophus ferrumequinum), a bat species widely distributed across the Palearctic. LOCATION: Palearctic. METHODS: We collected body size data (body mass and forearm length) from 1,172 adult bats via direct measurements or from the literature. We used high‐resolution environmental data for the sampled sites. We applied multiple linear regressions and an information‐theoretic approach on separate female and male datasets to estimate the support for various hypotheses. RESULTS: Temperature and latitude failed to predict body mass in both females and males. The best model showed that populations with longer forearms in both sexes were associated with higher mean temperatures (in the warmest quarter) and lower mean temperatures (in the coldest quarter). The independent contribution of these temperature parameters to forearm length in both sexes was higher than that of all other variables. MAIN CONCLUSION: The observed relationship between temperature and body mass was not consistent with James's rule, and thus failed to support the heat conservation hypothesis. Our results are consistent with the predictions of Allen's rule and indicated that direct heat dissipation from the wing may be the most likely mechanism underlying geographical variation in forearm length. We suggest that the evaluation of the relative importance of multiple causal mechanisms may improve our understanding of patterns of geographical variation in endotherms.

Shortly after its emergence in southern China in 2002/2003, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) was confirmed to be the cause of SARS. Subsequently, SARS-related CoVs (SARSr-CoVs) were found in palm civets from live animal markets in Guangdong and in various horseshoe bat species, which were believed to be the ultimate reservoir of SARSr-CoV. Till November 2018, 339 SARSr-CoV genomes have been sequenced, including 274 from human, 18 from civets and 47 from bats [mostly from Chinese horseshoe bats (Rhinolophus sinicus), n = 30; and greater horseshoe bats (Rhinolophus ferrumequinum), n = 9]. The human SARS-CoVs and civet SARSr-CoVs were collected in 2003/2004, while bat SARSr-CoVs were continuously isolated in the past 13 years even after the cessation of the SARS epidemic. SARSr-CoVs belong to the subgenus Sarbecovirus (previously lineage B) of genus Betacoronavirus and occupy a unique phylogenetic position. Overall, it is observed that the SARSr-CoV genomes from bats in Yunnan province of China possess the highest nucleotide identity to those from civets. It is evident from both multiple alignment and phylogenetic analyses that some genes of a particular SARSr-CoV from bats may possess higher while other genes possess much lower nucleotide identity to the corresponding genes of SARSr-CoV from human/civets, resulting in the shift of phylogenetic position in different phylogenetic trees. Our current model on the origin of SARS is that the human SARS-CoV that caused the epidemic in 2002/2003 was probably a result of multiple recombination events from a number of SARSr-CoV ancestors in different horseshoe bat species., Highlights • 313 SARSr-CoV genomes have been sequenced (274 from human, 18 civets and 47 bats). • SARSr-CoV genomes of bats in Yunnan possess highest nt identity to those from civets. • The origin of human SARS-CoV was probably a result of multiple recombination events. • Recombination from a number of SARSr-CoV ancestors in different horseshoe bat species.

The order Chiroptera (bats) is the second largest group of mammals, composed of more than 1,300 species. Although powered flight and echolocation in bats have attracted many biologists, diversity in bat facial morphology has been almost neglected. Some bat species have a "nose leaf," a leaf-like epithelial appendage around their nostrils. The nose leaf appears to have been acquired at least three times independently in bat evolution, and its morphology is highly diverse among bats species. Internal tissue morphology of nose-leaves has been investigated through histological analyses of late-stage fetuses of some bat species possessing the nose leaf. However, the proximate factors that bring about chiropteran nose-leaves have not been identified. As an initial step to address the question above, we describe the normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, and examine development of the tissues associated with their nose leaf during embryogenesis through histological analyses. We found that the nose leaf of R. ferrumequinum is formed through two phases. First, the primordium of the nose leaf appears as two tissue bulges aligned top and bottom on the face at embryonic stages 15-16. Second, the sub-regions of the nose leaf are differentiated through ingrowth as well as outgrowth of the epithelium at stage 17. In embryogenesis of Carollia perspicillata, a phyllostomid species with a nose leaf, the nose leaf primordium is formed as a small tissue bulge on the nostril at stage 17. This tissue bulge grows into a dorsally projected thin epithelial structure. Such differences in the nose leaf developmental process between chiropteran lineages may suggest that distinct developmental mechanisms have been employed in each lineage's nose leaf evolution.

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.

Paper description: Large Mediterranean peninsulas (Iberian, Italian, Balkan) are the main glacial refugia for many European bat species. We analysed mitochondrial D-loop sequences of Rhinolophus euryale from the Balkans in order to examine its genetic diversity and demographic history. Our results yielded twenty new haplotypes that belonge to the same monophyletic clade. The star-like topology of haplotype network and shallow genetic differentiation support a scenario of population expansion. New data on the genetic diversity of this species are presented. We propose that the Balkan Peninsula was its glacial refugium during the Pleistocene. Abstract : The Balkan Peninsula is identified as one of the major glacial refugia in Europe during the Pleistocene, and it has served as a genetic source for post-glacial recolonization for many temperate species. The aim of this study was to investigate the genetic diversity and phylogeographic patterns of the Mediterranean horseshoe bat, Rhinolophus euryale Blasius 1853, on the Balkan Peninsula. We also analyzed its demographic history and tested the hypothesis that this region was a glacial refugium for this species. We collected 82 samples from 20 localities in the Balkans and Italy and sequenced the mitochondrial D-loop region. Our results revealed low nucleotide but high haplotype diversity, with 20 out of 24 haplotypes reported for the first time. All Balkan and Italian samples belonged to a single genetic clade in the phylogenetic reconstruction, where they clustered together with previously published samples from Turkey, southern France and North Africa. The haplotype network had a star-like pattern that is indicative of recent population expansion. Both mismatch distribution and shallow genetic differentiation also supported the scenario of a sudden demographic expansion. We estimated that expansion within this lineage commenced in the Late Pleistocene. We suggest that the Balkan Peninsula was a glacial refugium for R. euryale . https://doi.org/10.2298/ABS190529059B Received: May 29, 2019; Revised: August 5, 2019; Accepted: September 9, 2019; Published online: September 13, 2019 How to cite this article: Budinski I, Jovanovic VM, Pejic B, Blagojevic J, Rajicic M, Paunovic M, Presetnik P, Vujosevic M. Mitochondrial phylogeography of the Mediterranean horseshoe bat on the Balkan Peninsula. Arch Biol Sci. 2019;71(4):767-74.

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.

Bats may be natural reservoirs for a large variety of emerging viruses, including mammalian coronaviruses (CoV). The recent emergence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) in humans, with evidence that these viruses may have their ancestry in bats, highlights the importance of virus surveillance in bat populations. Here, we report the identification and molecular characterization of a bat β-Coronavirus, detected during a viral survey carried out on different bat species in the island of Sardinia (Italy). Cutaneous, oral swabs, and faecal samples were collected from 46 bats, belonging to 15 different species, and tested for viral presence. Coronavirus RNA was detected in faecal samples from three different species: the greater horseshoe bat (Rhinolophus ferrumequinum), the brown long-eared bat (Plecotus auritus), and the European free-tailed bat (Tadarida teniotis). Phylogenetic analyses based on RNA-dependent RNA polymerase (RdRp) sequences assigned the detected CoV to clade 2b within betacoronaviruses, clustering with SARS-like bat CoVs previously reported. These findings point to the need for continued surveillance of bat CoV circulating in Sardinian bats, and extend the current knowledge on CoV ecology with novel sequences detected in bat species not previously described as β-Coronavirus hosts.

Populations occupying peripheral areas of a current species range, particularly those located at rearing edges of the distributional ranges, have a high adaptive potential to environmental changes for the species, being of key conservation importance and, simultaneously, are heavily understudied. We provide the first description of the home-range size and foraging habitat preferences of the isolated northern-most population of the Mediterranean horseshoe bat (Rhinolophus euryale) living at the contact zone of the Carpathian Mountains and Pannonian Lowland, located ~300 km north of the core species range. The median size of the Minimum Convex Polygons (MCP) for all individuals throughout the study was 1.8 km2 and was in accordance with data reported for the southern populations from the range-core. The MCP median size varied between the reproductive periods, being the largest (3.3 km2) in the pregnancy period, decreased dramatically (0.8 km2) during the lactation period and then increased again considerably (7.5 km2) in the post-lactation period. The Ecological Niche Modelling revealed the affinity of the bats to forage in a close distance to riverine habitats, but also in villages and forest edges, while they avoided closed forest areas and open habitats. The differences in habitat use in the study area as compared to the core range populations bring an evidence of ecological plasticity of the northern marginal populations of this species and its potential to exploit human-altered habitats, such as village interiors.

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., Author summary The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like the closely related virus SARS-CoV (SARS-CoV-1), infects cells by interacting with the cellular receptor angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the viral spike (S) protein through an independently folded subdomain, described as its receptor-binding domain (RBD). The susceptibility of a species to SARS-CoV-1 or -2 infection correlates with the binding affinity of their respective RBD for the ACE2 orthologs of that species. We therefore investigated the binding affinity of the RBD regions of multiple SARS-like coronaviruses with a range of ACE2 orthologs. Our results are consistent with the hypothesis that pangolins serve as an intermediate between humans and horseshoe bats. We further observed a high level of variability in the ability of the SARS-CoV-2 RBD to bind horseshoe bat ACE2 orthologs, suggesting ongoing selection pressure on their receptor ACE2 proteins from SARS-like viruses. Indeed, mutations derived from different horseshoe bat orthologs introduced into a soluble form of human ACE2 differentially impacted SARS-CoV-1 and SARS-CoV-2 S-protein-mediated infection. A combination of five residues present in multiple horseshoe bats increased the ability of a soluble form of ACE2 to neutralize SARS-CoV-2 S-protein-mediated infection. Thus horseshoe bats ACE2 orthologs can provide insight useful to improving the potency of ACE2-based therapeutics.

Bats are particularly susceptible to environmental changes because of their low reproductive rate, longevity, and high metabolic rates, which lead to relatively high food requirements. Thus, bat populations take a relatively long time to recover from increased mortality rates, and monitoring schemes should cover long time periods. In this work we analyzed the population trajectories of two bat species, Rhinolophus hipposideros and Myotis myotis, the most numerous in five caves in southern Poland, which are known as important bat hibernacula on a continental scale. Data were collected by regular counts in 1985–2001, depending on the particular cave; in addition, previous data on the number of hibernating bats in these caves, available since 1951, were taken from existing publications. We analyzed time-series data using average locality indices and TRIM methods, and both produced similar results. Generally, the populations of the two studied bat species showed recent increasing trends, especially visible as an effect of recovery after years of decline. The situation recorded in southern Poland is very similar to that described in other places in Europe, where recoveries of bat populations have also been observed in the last decades. Although it is difficult to present results from formal analyses, because of the lack of good data, at least some factors—less exposure to contaminants (pesticides, heavy metals), improving food availability due to climate change, and a lower predation rate (including human pressure), both in the breeding season and during wintering—positively affected both species.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causing agent of the COVID-19 pandemic, has spread globally. Angiotensin-converting enzyme 2 (ACE2) has been identified as the host cell receptor that binds to receptor-binding domain (RBD) of the SARS-COV-2 spike protein and mediates cell entry. Because the ACE2 proteins are widely available in mammals, it is important to investigate the interactions between the RBD and the ACE2 of other mammals. Here we analyzed the sequences of ACE2 proteins from 16 mammals and predicted the structures of ACE2-RBD complexes. Analyses on sequence, structure, and dynamics synergistically provide valuable insights into the interactions between ACE2 and RBD. The comparison results suggest that the ACE2 of bovine, cat and panda form strong binding with RBD, while in the cases of rat, least horseshoe bat, horse, pig, mouse and civet, the ACE2 proteins interact weakly with RBD.

SummaryThe lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2, and the large geographical distance between Wuhan and where the closest evolutionary related coronaviruses circulating in horseshoe bats (Sarbecoviruses) have been identified, is fuelling speculation on the natural origins of SARS-CoV-2. We have comprehensively analysed phylogenetic relations between SARS-CoV-2, and the related bat and pangolin Sarbecoviruses sampled so far. Determining the likely recombination events reveals a highly reticulate evolutionary history within this group of coronaviruses. Clustering of the inferred recombination events is non-random with evidence that Spike, the main target for humoral immunity, is beside a recombination hotspot likely driving antigenic shift in the ancestry of bat Sarbecoviruses. Coupled with the geographic ranges of their hosts and the sampling locations, across southern China, and into Southeast Asia, we confirm horseshoe bats, Rhinolophus, are the likely SARS-CoV-2 progenitor reservoir species. By tracing the recombinant sequence patterns, we conclude that there has been relatively recent geographic movement and co-circulation of these viruses’ ancestors, extending across their bat host ranges in China and Southeast Asia over the last 100 years or so. We confirm that a direct proximal ancestor to SARS-CoV-2 is yet to be sampled, since the closest relative shared a common ancestor with SARS-CoV-2 approximately 40 years ago. Our analysis highlights the need for more wildlife sampling to (i) pinpoint the exact origins of SARS-CoV-2’s animal progenitor, and (ii) survey the extent of the diversity in the related Sarbecoviruses’ phylogeny that present high risk for future spillover.HighlightsThe origin of SARS-CoV-2 can be traced to horseshoe bats, genus Rhinolophus, with ranges in both China and Southeast Asia.The closest known relatives of SARS-CoV-2 exhibit frequent transmission among their Rhinolophus host species.Sarbecoviruses have undergone extensive recombination throughout their evolutionary history.Accounting for the mosaic patterns of these recombinants is important when inferring relatedness to SARS-CoV-2.Breakpoint patterns are consistent with recombination hotspots in the coronavirus genome, particularly upstream of the pike open reading frame with a coldspot in S1.

Background The SARS-CoV-2 pandemic is one of the greatest global medical and social challenges that have emerged in recent history. Human coronavirus strains discovered during previous SARS outbreaks have been hypothesized to pass from bats to humans using intermediate hosts, e.g. civets for SARS-CoV and camels for MERS-CoV. The discovery of an intermediate host of SARS-CoV-2 and the identification of specific mechanism of its emergence in humans are topics of primary evolutionary importance. In this study we investigate the evolutionary patterns of 11 main genes of SARS-CoV-2. Previous studies suggested that the genome of SARS-CoV-2 is highly similar to the horseshoe bat coronavirus RaTG13 for most of the genes and to some Malayan pangolin coronavirus (CoV) strains for the receptor binding (RB) domain of the spike protein. Results We provide a detailed list of statistically significant horizontal gene transfer and recombination events (both intergenic and intragenic) inferred for each of 11 main genes of the SARS-CoV-2 genome. Our analysis reveals that two continuous regions of genes S and N of SARS-CoV-2 may result from intragenic recombination between RaTG13 and Guangdong (GD) Pangolin CoVs. Statistically significant gene transfer-recombination events between RaTG13 and GD Pangolin CoV have been identified in region [1215–1425] of gene S and region [534–727] of gene N. Moreover, some statistically significant recombination events between the ancestors of SARS-CoV-2, RaTG13, GD Pangolin CoV and bat CoV ZC45-ZXC21 coronaviruses have been identified in genes ORF1ab, S, ORF3a, ORF7a, ORF8 and N. Furthermore, topology-based clustering of gene trees inferred for 25 CoV organisms revealed a three-way evolution of coronavirus genes, with gene phylogenies of ORF1ab, S and N forming the first cluster, gene phylogenies of ORF3a, E, M, ORF6, ORF7a, ORF7b and ORF8 forming the second cluster, and phylogeny of gene ORF10 forming the third cluster. Conclusions The results of our horizontal gene transfer and recombination analysis suggest that SARS-CoV-2 could not only be a chimera virus resulting from recombination of the bat RaTG13 and Guangdong pangolin coronaviruses but also a close relative of the bat CoV ZC45 and ZXC21 strains. They also indicate that a GD pangolin may be an intermediate host of this dangerous virus.

Dietary plasticity is an important issue for conservation biology as it may be essential for species to cope with environmental changes. However, this process still remains scarcely addressed in the literature, potentially because diet studies have long been constrained by methodological limits. The advent of molecular approaches now makes it possible to get a precise picture of diet and its plasticity, even for endangered and elusive species. Here we focused on the greater horseshoe bat (Rhinolophus ferrumequinum) in Western France, where this insectivorous species has been classified as ‘Vulnerable’ on the Regional Red List in 2016. We applied an eDNA metabarcoding approach on 1986 fecal samples collected in six maternity colonies at three sampling dates. We described its diet and investigated whether the landscape surrounding colonies and the different phases of the maternity cycle influenced the diversity and the composition of this diet. We showed thatR. ferrumequinumfeed on a highly more diverse spectrum of prey than expected from previous studies, therefore highlighting how eDNA metabarcoding can help improving diet knowledge of a flying elusive endangered species. Our approach also revealed thatR. ferrumequinumdiet is composed of two distinct features: the core diet consisting in a few preferred taxa shared by all the colonies (25% of the occurrences) and the secondary diet consisting in numerous rare prey that were highly different between colonies and sampling dates (75% of the occurrences). Energetic needs and constraints associated with the greater horseshoe bat life-cycle, as well as insect phenology and landscape features, strongly influenced the diversity and composition of both the core and whole diets. Further research should now explore the relationships betweenR. ferrumequinumdietary plasticity and fitness, to better assess the impact of core prey decline onR. ferrumequinumpopulations viability.

Eyndhovenia is one of the twelve genera of Spinturnicidae which are highly specialised parasites of bats. Previously known hosts of this genus comprised 17 species of Old World bats: Eptesicus serotinus, Hipposideros larvatus, Miniopterus schreibersi, Myotis blythi, M. emarginatus, Pipistrellus pipistrellus, P. gaisleri, Rhinolophus axillaris, R. blasii, R. clivosus, R. cornutus, R. euryale, R. ferrumequinum, R. hipposideros, R. megachyllus, R. mehelyi, R. rouxi. Within Asia, Eyndhovenia was only recorded from two countries, China and Thailand. Between 2018 and 2020, we conducted a series of bats surveys and recorded of this genus from intermediate horseshoe bat, Rhinolophus affinis, in Vietnam. The present study exhibits the new record in both parasitological and geographical aspects.

The relative contributions of adaptation and drift to morphological diversification of the crania of echolocating mammals was 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. 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. Selection acted mainly on the nasal dome region of R. cf. simulator whereas selection acted more on the cranium and mandibles than on the nasal domes of R. simulator. Probably the relatively higher echolocation frequencies used by R. cf. simulator, the shape of the nasal dome, which acts as a frequency dependent acoustic horn, is more crucial than in R. simulator, allowing maximization of the intensity of the emitted calls and resulting in comparable detection distances. 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.

Background The current coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome (SARS)-CoV-2, has become the most devastating public health emergency in the 21st century and one of the most influential plagues in history. Studies on the origin of SARS-CoV-2 have generally agreed that the virus probably comes from bat, closely related to a bat CoV named BCoV-RaTG13 taken from horseshoe bat (Rhinolophus affinis), with Malayan pangolin (Manis javanica) being a plausible intermediate host. However, due to the relatively low number of SARS-CoV-2-related strains available in public domain, the evolutionary history remains unclear. Methodology Nine hundred ninety-five coronavirus sequences from NCBI Genbank and GISAID were obtained and multiple sequence alignment was carried out to categorize SARS-CoV-2 related groups. Spike sequences were analyzed using similarity analysis and conservation analyses. Mutation analysis was used to identify variations within receptor-binding domain (RBD) in spike for SARS-CoV-2-related strains. Results We identified a family of SARS-CoV-2-related strains, including the closest relatives, bat CoV RaTG13 and pangolin CoV strains. Sequence similarity analysis and conservation analysis on spike sequence identified that N-terminal domain, RBD and S2 subunit display different degrees of conservation with several coronavirus strains. Mutation analysis on contact sites in SARS-CoV-2 RBD reveals that human-susceptibility probably emerges in pangolin. Conclusion and implication We conclude that the spike sequence of SARS-CoV-2 is the result of multiple recombination events during its transmission from bat to human, and we propose a framework of evolutionary history that resolve the relationship of BCoV-RaTG13 and pangolin coronaviruses with SARS-CoV-2. Lay Summary This study analyses whole-genome and spike sequences of coronavirus from NCBI using phylogenetic and conservation analyses to reconstruct the evolutionary history of severe acute respiratory syndrome (SARS)-CoV-2 and proposes an evolutionary history of spike in the progenitors of SARS-CoV-2 from bat to human through mammal hosts before they recombine into the current form.

Land use change, livestock production and human encroachment into wildlife habitats drive zoonotic emergence. Quantitative analyses of horseshoe bat populations provide evidence for how food systems may contribute to hotspots of potential zoonotic spillover.