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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.

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.

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.

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.

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.

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.

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.

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.

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.

Diurnal flight and foraging activity in insectivorous bats are atypical behaviours that have been recorded from islands with few avian predators and from locations with extended daylight hours. We present the first known observations of diurnal activity of Rhinolophus lepidus in forests on Tioman Island, Malaysia, recorded using visual surveys and acoustic monitoring. The bats were flying during the day and at night, and feeding buzzes detected suggest that they were actively foraging during the day. This appears to be a regular phenomenon on Tioman Island. The absence of resident diurnal avian predators that hunt below the forest canopy may account for the diurnal activity of R. lepidus in forests there.

Morphological changes of testes and the annual cycle of the seminiferous epithelium were observed by optical and transmission electron microscopy to determine the male reproductive cycle of the Korean greater horseshoe bat, Rhinolophus ferrumequinum korai. In this study, R. ferrumequinum korai showed a distinct reproductive cycle, unlike other hibernating bat species. The testicular reproductive cycle of R. ferrumequinum korai consists of three main stages. The first is the spermatogenesis stage (from April to September), including spermatocytogenesis (which appears from April to May) and spermiogenesis (from June to September). The activity of spermatogenesis was highest in August. The lumen of seminiferous tubules was open from mid-April to mid-October. It was closed from November to March of the following year. The second stage is the phagocytosis stage (from mid-October to mid-November), which is a purification process to prepare for new spermatogenesis the following year. This period is called the cleansing period. The third is the dormant stage – that is, a state of holding only spermatogonia and Sertoli cells. It is an adaptation strategy to store energy for long periods of hibernation. Compared to previous studies, spermatogenesis period preceded 1 month earlier. This suggests that temperature increase can impact reproductive development and spermatogenesis.

During the winter, bats use hibernation as a means of surviving the period of low prey offer. However, the Mediterranean horseshoe bat (Rhinolophus euryale) arouses from torpor quite frequently. Based on the actual climatic conditions, it can profit from occasional foraging oportunities, when they occur. We analysed faeces collected on four nights during the period from November 2012 to February 2013 from the Domica-Baradla cave system (Slovakia and Hungary). In mid-November, the largest proportion of faecal contents were from Lepidoptera. Later on, the proportion of non-consumptive mass in the faeces increased and prey remnants disappeared. We analysed the activity of digestive enzymes (amylase, chitobiase, endochitinase and glukosaminidase) in faeces. The activity of these enzymes was detected in fresh faeces throughout the whole winter. The faecal activity of the chitinases was relatively stable during the monitored period, whilst the activity of amylase was highest during late November and December. Some level of active digestive enzymes during the winter could be an adaptation to occasional winter foraging.

Bat wings are characterized by high endurance, and these mammals have developed a number of adaptations that protect them from falling into obstacles and potential injuries. However, in bat populations, there are individuals with visible fresh or healed injuries to the flight-enabling body parts. The aim of this research was to determine the differences in the occurrence of wing membrane damages among species of bats that differ in ecology and behavior. The study was conducted in southern and western Poland in the years 2000-2016 and included 3,525 individuals of six species: lesser horseshoe bat Rhinolopus hipposideros, Daubenton's bat Myotis daubentonii, Natterer's bat Myotis nattereri, greater mouse-eared bat Myotis myotis, western barbastelle Barbastella barbastellus, and brown long-eared bat Plecotus auritus. In all, 2.9% of the bats studied showed damage to the flight-enabling body parts. Natterer's bat was the species with the highest number of injured individuals (21.74%). The lowest number of injured individuals (0.3%) was found in the brown long-eared bat. The most frequently observed type of damage was loss of an edge of the wing membrane (29.3%). The bat species studied differed significantly in the occurrence and location of flight enabling body parts damages. Certain behavioral and ecological factors like foraging mode, foraging habitats and habitat types of bat species determine the number of wing and tail membrane damages.

The diversity of bats worldwide includes large numbers of cryptic species, partly because divergence in acoustic traits such as echolocation calls are under stronger selection than differences in visual appearance in these nocturnal mammals. Island faunas often contain disproportionate numbers of endemic species, and hence we might expect cryptic, endemic species to be discovered relatively frequently in bats inhabiting islands. Species are best defined when multiple lines of evidence supports their diagnosis. Here we use morphometric, acoustic, and molecular phylogenetic data to show that a horseshoe bat in the Andaman Islands is distinct in all three aspects, supporting its description as a newly described endemic species. We recommend investigation into possible new and endemic bat species on islands by using integrated approaches that provide independent lines of evidence for taxonomic distinctiveness. We provide a formal description of the new species –Rhinolophus andamanensisDobson, 1872.

Hibernating bats exhibit ubiquitous winter activity in temperate zones, but there is considerable between- and within-species variety in their intensity and purpose. Bats may fly during winter for sustenance or travel to other hibernacula. This study compared inter-regional variation in the winter activity of the greater horseshoe bat (Rhinolophus ferrumequinum). We predicted that weather and hibernacula-environmental conditions would influence winter activity patterns. Winter activity patterns differed between regions. In the Anseong area, we confirmed movement inside the hibernaculum, but in Hampyeong, we observed movement both inside and between hibernacula. The two regions differ by 4 °C in average winter temperatures. Anseong experiences 22 days during which average daily temperatures exceeded 5 °C, whereas Hampyeong experienced 50 such days. During the hibernating period, bat body weight decreased by approximately 17–20% in both regions. Ambient temperatures and winter-roost environments appear to be behind regional differences in hibernating bat activity. As winter temperatures in Korea do not favor insect activity, feeding probability is low for bats. However, bats may need to access water. At Anseong, underground water flows inside the hibernaculum when the reservoir outside is frozen. At Hampyeong, the hibernaculum does not contain a water source, but the reservoir outside does not freeze during winter. In conclusion, water-source location is the most likely explanation for regional variation in the winter activity of hibernating bats.

Migratory behaviour, sociality and roost selection have a great impact on the population structure of one species. Many bat species live in groups, and movements between summer and hibernation sites are common in temperate bats. The Mediterranean horseshoe bat Rhinolophus euryale is a cave-dwelling species that exhibits roost philopatry and undertakes seasonal movements which are usually shorter than 50 km. Its distribution in Serbia is restricted to karstic areas in western and eastern parts of the country, with a lack of known roosts between them. In this study, microsatellite markers were used to evaluate genetic variation in this species in the Central Balkans. Specifically, spatial genetic structuring between geographic regions and relatedness within different colony types were assessed. All analysed loci were polymorphic, and there was no significant inbreeding coefficient recorded. A moderate degree of genetic differentiation among the sampled colonies was found, and significant isolation by distance was recorded. Our results revealed that populations show a tendency to segregate into three clusters. Unexpectedly, populations from Montenegro and Eastern Serbia tended to group into one cluster, while populations from Western Serbia and Slovenia represented second and third cluster, respectively. The majority of variance was partitioned within colonies, and only a small but significant portion among clusters. Average relatedness within colony members was close to zero, did not differ significantly between the different colony types, and kinship is unlikely to be a major grouping mechanism in this species.

Herbivorous arthropods cause immense damage in crop production annually. Consumption of these pests by insectivorous animals is of significant importance to counteract their adverse effects. Insectivorous bats are considered amongst the most voracious predators of arthropods, some of which are known crop pests. In vineyard-dominated Mediterranean agroecosystems, several crops are damaged by the attack of insect pests. In this study we aimed 1) to explore the diet and pest consumption of the lesser horseshoe bat Rhinolophus hipposideros and 2) analyse whether the composition of pest species in its diet changes throughout the season. We employed a dual-primer DNA metabarcoding analysis of DNA extracted from faeces collected in three bat colonies of a wine region in Southwestern Europe during the whole active period of most pest species. Overall, 395 arthropod prey species belonging to 11 orders were detected; lepidopterans and dipterans were the most diverse orders in terms of species. Altogether, 55 pest species were identified, 25 of which are known to cause significant agricultural damage and 8 are regarded as pests affecting grapevines. The composition of pest species in faeces changed significantly with the season, thus suggesting several periods should be sampled to assess the pest consumption by bats. As a whole, the results imply that R. hipposideros acts as a suppressor of a wide array of agricultural pests in Mediterranean agroecosystems. Therefore, management measures favouring the growth of R. hipposideros populations should be considered. The research was supported by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (CGL-2015-69069P), as well as the Government of the Basque Country (IT754-13). The Basque Government granted UB (PRE_2016_1_0139). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In Europe, 2 different diploid chromosome numbers, 2n = 54 and 2n = 56, have been described in the lesser horseshoe bat (Rhinolophushipposideros). The eastern form with 2n = 56 extends from the Czech Republic to Greece. To date, specimens with 54 chromosomes have been reported only from Spain and Germany. This study expands the distributional area of the western variant to Ireland. Strikingly, this distribution of European chromosomal variants is in contrast to the available molecular data that indicate little genetic differentiation of R. hipposideros populations spanning Northwestern to Central Europe. Further, we have developed an optimized protocol for establishing fibroblast cell cultures, suitable for karyotype analyses, from 3-mm wing membrane biopsies. This is a useful technique for cytogenetic studies of endangered bat species, as this non-lethal sampling method imposes only minimum stress to the animal without lasting adverse effects and is routinely used to sample tissue probes for molecular genetic studies in bats.

As a K-strategist and comparatively sedentary species, the lesser horseshoe bat Rhinolophus hipposideros is considered sensitive to changes in habitat quality. Knowledge of the species' dietary requirements and use of foraging habitats is thus considered an essential prerequisite to manage its habitats adequately. Based on four large annual samples of faecal pellets from three different nursery colonies, including two consecutive years of sampling from one colony, we studied the diet of Central German populations of R. hipposideros. Consistent with findings of similar studies carried out in other parts of the distribution range of R. hipposideros, in our study, Diptera, Lepidoptera, and Neuroptera represented the most important groups of prey for the studied colonies. However, Hemiptera made a comparatively larger contribution in our study than in others, and so did Hymenoptera in one of the colonies. We found seasonal compositional variation in all four annual samples, as well as compositional variation ...

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 that R. ferrumequinum feed 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 that R. ferrumequinum diet 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 between R. ferrumequinum dietary plasticity and fitness, to better assess the impact of core prey decline on R. ferrumequinum populations viability.

Although bats are increasingly recognised as potential reservoir hosts of human zoonotic pathogens, bacteria in bats are still poorly studied. To investigate the DNA faecal prevalence of the bacterium Anaplasma phagocytophilum, we sampled 23 lesser horseshoe bat (Rhinolophus hipposideros) maternity colonies located in buildings (churches, barns) in rural villages of eastern France. A total of 552 faecal samples were collected from 278 individuals. Anaplasma phagocytophilum DNA was detected in the faeces of 63 individuals (22.7%). Such high prevalence might suggest persistent infection in bats and/or a frequent consumption of insect preys carrying bacteria. Faecal DNA prevalence varied highly among colonies but was not related to the colony size. Faecal DNA prevalence was the highest in the Jura Department, where the density of ticks is known to be the highest across the study area. Because the sampled bats live in close proximity to humans, we discuss how concerning the presence of A. phagocytophilum DNA in bat guano is for humans frequenting places of worship that shelter bats. We also advocate future research to understand what a high faecal DNA prevalence in bat guano really implicates in terms of bacteria transmission.

Geographic variation in sensory traits is usually influenced by adaptive processes because these traits are involved in crucial life-history aspects including orientation, communication, lineage recognition and mate choice. Studying this variation can therefore provide insights into lineage diversification. According to the Sensory Drive Hypothesis, lineage diversification may be driven by adaptation of sensory systems to local environments. It predicts that acoustic signals vary in association with local climatic conditions so that atmospheric attenuation is minimized and transmission of the signals maximized. To test this prediction, we investigated the influence of climatic factors (specifically relative humidity and temperature) on geographic variation in the resting frequencies of the echolocation pulses of Geoffroy's horseshoe bat, Rhinolophus clivosus. If the evolution of phenotypic variation in this lineage tracks climate variation, human induced climate change may lead to decreases in detection volumes and a reduction in foraging efficiency. A complex non-linear interaction between relative humidity and temperature affects atmospheric attenuation of sound and principal components composed of these correlated variables were, therefore, used in a linear mixed effects model to assess their contribution to observed variation in resting frequencies. A principal component composed predominantly of mean annual temperature (factor loading of -0.8455) significantly explained a proportion of the variation in resting frequency across sites (P < 0.05). Specifically, at higher relative humidity (around 60%) prevalent across the distribution of R. clivosus, increasing temperature had a strong negative effect on resting frequency. Climatic factors thus strongly influence acoustic signal divergence in this lineage, supporting the prediction of the Sensory Drive Hypothesis. The predicted future increase in temperature due to climate change is likely to decrease the detection volume in echolocating bats and adversely impact their foraging efficiency.

Museums hold most of the world’s most valuable biological specimens and tissues collected, including type material that is often decades or even centuries old. Unfortunately, traditional museum collection and storage methods were not designed to preserve the nucleic acids held within the material, often reducing its potential viability and value for many genetic applications. High-throughput sequencing technologies and associated applications offer new opportunities for obtaining sequence data from museum samples. In particular, target sequence capture offers a promising approach for recovering large numbers of orthologous loci from relatively small amounts of starting material. In the present study, we test the utility of target sequence capture for obtaining data from museum-held material from a speciose mammalian genus: the horseshoe bats (Rhinolophidae: Chiroptera). We designed a ‘bait’ for capturing > 3600 genes and applied this to 10 species of horseshoe bat that had been collected between 93 and 7 years ago and preserved using a range of methods. We found that the mean recovery rate per species was approximately 89% of target genes with partial sequence coverage, ranging from 3024 to 3186 genes recovered. On average, we recovered 1206 genes with ≥ 90% sequence coverage, per species. Our findings provide good support for the application of large-scale bait capture across congeneric species spanning approximately 15 Myr of evolution. On the other hand, we observed no clear association between the success of capture and the phylogenetic distance from the bait model, although sample sizes precluded a formal test. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 00, 000–000. ADDITIONAL KEYWORDS: DNA – high-throughput sequencing – phylogenomics – pull-down – Rhinolophus – systematics – target enrichment.

A new species of woolly horseshoe bat in the Rhinolophus trifoliatus species group is described from Sabah in Malaysian Borneo. Two specimens from Central and West Kalimantan, Indonesia are referred to this species. A fourth specimen from western Thailand is referable to this species but on the basis of ~10% genetic divergence at the cytochrome oxidase-I gene is described as a separate subspecies. Morphologically and acoustically the two subspecies are similar. With a forearm length of 52.90–54.70 mm, a skull length of 24.27–26.57 mm and a call frequency of 49.2–50.0 kHz, the new species overlaps in size and call frequency with the sympatric R. trifoliatus. However, it differs significantly in having a dark noseleaf and a uniformly dark brown pelage, resembling, but being intermediate in size between R. sedulus and R. luctus, which have a skull length of 18.99–20.17 and 26.35–32.07 mm, respectively. It also differs from R. trifoliatus in the shape and size of the rostral inflation. It can be distinguished from R. beddomei (forearm length 55.00–63.44 mm) and R. formosae (forearm length 53.85–62.40 mm), which are endemic to the Indian Subcontinent and Taiwan, respectively, by its relatively smaller body size. Acoustic and genetic data are included in the comparison between the species. Both character states support the conclusions based on morphology. Further surveys in intact evergreen forest together with a re-examination of museum specimens may reveal that this species is widespread in Southeast Asia.

We obtained cytochrome b complete sequences (1140 bp) of the greater horseshoe bat Rhinolophus ferrumequinum from Korea (Jeju Island and mainland Korea), and these sequences were compared to corresponding sequences of R. ferrumequinum, obtained from GenBank, in order to examine genetic divergence among populations within R. ferrumequinum from far-eastern Asia and other parts of the range. Specimens from four populations in far-eastern Asia (Jeju, mainland Korea, northeastern China, and Japan) formed a far-eastern clade and were little differentiated, indicating that our results support a former subspecies classification, recognized R. f. korai and R. f. quelpartis as synonyms of R. f. nippon. In addition, we found that the eastern China clade from Henan is genetically distinct from the far-eastern clade, although individuals from Japan, northeastern China, and eastern China are known as R. f. nippon, and we propose further analyses with additional specimens from China to examine whether or not the eastern China clade is a subpopulation within R. f. nippon. On the other hand, R. ferrumequinum from Eurasia and northern Africa was found to be composed of four clades (far-eastern Asia, eastern China, central China, and western Asia - Europe - northern Africa), and average nucleotide distances between the first and other three clades were 2.15%, 4.10%, and 5.37% respectively. Thus, we found that genetic distances between these clades are correlated with geographic distances between them, and we concluded that further analysis of cytochrome b and other markers from additional specimens of R. ferrumequinum across distributional range is necessary to reexamine its population structure and subspecies classification.

Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8 % identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.

To examine the level of genetic differentiation in the sequences of the mitochondrial D-loop gene of Rhinolophus ferrumequinum, and to evaluate the current taxonomic status of this species, 50 tissue samples of greater horseshoe bats were collected in 2011-2015 from 21 different localities in northwest, northeast, west, central, and south regions of Iran. Twenty-two published D-loop sequences from Europe (Switzerland, United Kingdom, Bulgaria, and Tunisia), and Anatolia (south, west, and east Turkey) were downloaded from GenBank. Molecular genetic analyses revealed remarkable variation among populations of R. ferrumequinum. Two major clades with strong support were identified within the greater horseshoe bat. One of these clades consists of individuals of R. ferrumequinum from Iran and eastern Turkey, and is further subdivided into two subclades. A second clade includes samples from western Turkey and Europe. The two subclades from Iran and Turkey and the second clade from western Turkey and Europe represent three diagnosable categories, which most probably warrant three subspecies for the species. Thus, based on genetic differences, it is clear that two subspecific populations are found in Iran: R. f. irani (southern Iran) and R. f. proximus (northern Iran).

Liang Bua is a limestone cave on the island of Flores, Indonesia. Palaeontological and archaeological excavations at this site have unearthed one of the best insular records of fossil bats to date. The assemblage is numerically dominated by the horseshoe bat species Rhinolophus euryotis, followed by the leafnosed bat Hipposideros diadema. In addition to these species, remains of Rhinolophus simplex, Hipposideros sp. cf. H. sumbae, Murina sp. aff. M. florium, Taphozous sp., Kerivoula sp. and Miniopterus sp. have also been recovered. Two taxa found in Liang Bua, Rhinolophus euryotis and Miniopterus sp., are not apparent in the modern bat fauna of Flores. Some specimens, found during the more recent excavations, are known from specific depths. Two age-groups are represented. The stratigraphically oldest is 74–61 ka and includes Murina sp. aff. M. florium and Rhinolophus euryotis. Conversely, the younger, Holocene, assemblage is much more diverse. Despite the relatively small amount of microbat material colle...

The aim of this study was to describe the dynamics of the long-legged bat tick Ixodes vespertilionis infestation on the lesser horseshoe bat Rhinolophus hipposideros in 2 nursery colonies roosting in attics. Out of a total of 810 lesser horseshoe bats examined, 217 (26.8%) were found to be infested with a total of 464 I. vespertilionis individuals. The developmental stage most frequently found was the larva, followed by the nymph, and the adult female. Bats were significantly more frequently infested with I. vespertilionis ticks in the period April to May than in other months. In these months, all tick developmental stages were observed. During summer and autumn, only immature developmental stages were recorded, whilst in September and October larvae predominated. Considerable differences in tick load between nursery colonies were observed. The length of seasonal presence on bats, prevalence, and infestation intensity of I. vespertilionis on lesser horseshoe bats were higher in the nursery colony situated in close vicinity of a cave than in the colony situated far from the caves. The results suggest that the pattern of seasonal infestation of ticks on bats roosting in nursery colonies coincides with the seasonal activity of Rh. hipposideros in the caves. The first case of mixed infestation of the lesser horseshoe bat with I. vespertilionis and I. ricinus were also recorded.

Mutual recognition is the product of species coexistence, and has direct effects on survival and reproduction of animals. Bats are able to discriminate between sympatric different heterospecifics based on their echolocation calls, which has been shown both in free-flying and captive bats. To date, however, the factors that may determine the behavioral responses of bats to echolocation calls from sympatric heterospecifics have rarely been tested, especially under well-controlled conditions in captive bats. Hence, we aimed at tackling this question by performing playback experiments (habituation–dishabituation) with three horseshoe bat species within the constant-frequency bat guild, which included big-eared horseshoe bats (Rhinolophus macrotis), Blyth’s horseshoe bats (Rhinolophus lepidus), and Chinese horseshoe bats (Rhinolophus sinicus). We studied the behavioral responses of these three species to echolocation calls of conspecifics, to other two species, and to another heterospecifics bat, Stoliczka’s trident bat (Asellisus stoliczkanus), which also belongs to this guild. We found that the three rhinolophid species displayed a series of distinct behaviors to heterospecific echolocation but few to conspecific calls after habituation, suggesting that they may have been able to discriminate sympatric heterospecific echolocation calls from those of conspecifics. Interestingly, the behavioral responses to heterospecific calls were positively correlated with the interspecific overlap index in trophic niche, whereas call design had only a minor effect. This implies that the behavioral responses of these bats to heterospecific echolocation calls may be related to the degree of interspecific food competition.

Cave-dwelling microbats are known to occupy abandoned mines, which can be important habitat for threatened species. Surveys and monitoring between 2012 and 2014 in Mugii Murum-ban State Conservation Area identified use of three adits from historic mine workings by three species: Chalinolobus dwyeri, Miniopterus orianae oceanensis and Rhinolophus megaphyllus. One of the adits is a potential small maternity roost for the threatened M. o. oceanensis, as indicated by captures of pregnant females in December 2014, as well as increased emergence counts and call activity in spring compared with autumn. While there were some signs of reproducing R. megaphyllus at the adits (a single pregnant female in late October, and postlactating females and juveniles in February) the complete absence of females during December trapping indicated otherwise. Use of the three adits was typical for roost and access preferences of the species, with only R. megaphyllus occupying a doored adit and all species recorded at a large unobstructed adit, and a shallow adit likely only used as a night roost by all species.

Three different diploid chromosome numbers (2n = 54, 56 and 58) have been reported in the lesser horseshoe bat, Rhinolophus hipposideros. Asia Minor and the Middle East are inhabited by R. hipposideros specimens with 58 chromosomes. In Europe, specimens with 56 chromosomes have been recorded from several localities in the Czech Republic, Slovakia, Italy and Greece. Up to now, specimens with 54 chromosomes have been reported only from Spain and possibly from Switzerland. With the record of 54 chromosomes in specimens from Germany presented here, the distributional area of this variant is expanded into Central Europe. According to the cytogenetic data presently available, we presume that the European R. hipposideros population is divided into a western form (from Spain to Germany) with a 2n = 54 karyotype and an eastern form (from the Czech Republic to Greece) with a 2n = 56 karyotype. This study presents banded karyotypes for the 2n = 54 and 2n = 56 variants for the first time. In addition, chromosomal arm homology to the vespertilionid bat species Myotis myotis revealed by chromosome painting is reported. Whether the variants could represent separate species is also discussed.

Geographic variation can be an indicator of still poorly understood evolutionary processes such as adaptation and drift. Sensory systems used in communication play a key role in mate choice and species recognition. Habitat-mediated (i.e. adaptive) differences in communication signals may therefore lead to diversification. We investigated geographic variation in echolocation calls of African horseshoe bats, Rhinolophus simulator and R . swinnyi in the context of two adaptive hypotheses: 1) James' Rule and 2) the Sensory Drive Hypothesis. According to James' Rule body-size should vary in response to relative humidity and temperature so that divergence in call frequency may therefore be the result of climate-mediated variation in body size because of the correlation between body size and call frequency. The Sensory Drive Hypothesis proposes that call frequency is a response to climate-induced differences in atmospheric attenuation and predicts that increases in atmospheric attenuation selects for calls of lower frequency. We measured the morphology and resting call frequency (RF) of 111 R . simulator and 126 R . swinnyi individuals across their distributional range to test the above hypotheses. Contrary to the prediction of James' Rule, divergence in body size could not explain the variation in RF. Instead, acoustic divergence in RF was best predicted by latitude, geography and climate-induced differences in atmospheric attenuation, as predicted by the Sensory Drive Hypothesis. Although variation in RF was strongly influenced by temperature and humidity, other climatic variables (associated with latitude and altitude) as well as drift (as suggested by a positive correlation between call variation and geographic distance, especially in R . simulator ) may also play an important role.

Based on a cave survey, we present the first substantiated data of two bat species from Iraq. Both Mediterranean horseshoe bat (Rhinolophus euryale) and Geoffroy’s bat (Myotis emarginatus) were mentioned in the earlier literature, although none of them was accepted and listed by subsequent authors. We overview the controversial history of the Mediterranean and Mehely’s horseshoe bat (Rhinolophus mehelyi) records, provide details of the diagnostic characters of vouchered specimens of the former species, and present photographic evidence of the presence of Geoffroy’s bat. Both species are rare in the Middle East, and the territory of Iraq was a gap in their distribution.

Bats (order Chiroptera) form a large proportion of the species-rich mammalian fauna of the Philippines, and while the threats posed to these animals are well documented, for many species there is currently insufficient data to enable even a basic assessment of their conservation status. This is true for Panay Island, located in the Western Visayas region of the archipelago, where the need for surveying remaining suitable bat habitat has been identified as a priority. Between 5 April and 9 May 2011 a survey of 21 caves was undertaken on Panay, along the central section of the west coast of the island and within the southern section of the Northwest Panay Peninsula. Survey methods included visual observations, emergence counts and the recording of echolocation calls. Of the caves surveyed, 19 were found to support bats or show signs of their use, and at least 12 different species were observed. Three large maternity colonies of the Common Rousette Rousettus amplexicaudatus and two of the Dusky Roundleaf Bat Hipposideros ater were noted as having particular significance in terms of their conservation value for local populations. Potential maternity colonies of Asian Lesser False Vampire Megaderma spasma, Black-bearded Tomb Bat Taphozous melanopogon and Diadem Roundleaf Bat Hipposideros diadema were also observed but not confirmed. M. spasma was the most frequently encountered species, occurring in small numbers at five different caves. Other species/genera encountered in small numbers were the Arcuate Horseshoe Bat Rhinolophus arcuatus, Common Short-nosed Fruit Bat Cynopterus brachyotis, Philippine Sheath-tailed Bat Emballonura alecto, Yellow-faced Horseshoe Bat Rhinolophus virgo, Bent-wing (Miniopterus) and Myotis bat species, and at least one other Horseshoe (Rhinolophus) bat species. Ten of the caves were confirmed to support multiple bat species. An indication of current threats and recommendations for further survey and management priorities are provided.

Echolocation calls produced by bats contain information on the emitter that could be used in social interactions. Nevertheless, little is known about the whether such information actually is being used by bats. Here we tested the ability of two horseshoe bat species (Rhinolophus mehelyi and R. euryale; n = 20 each) to discriminate conspecific male from conspecific female echolocation calls and vice versa. Behavioural data from a habituation-discrimination paradigm revealed that both species were able to recognize the sex of conspecifics from their calls, providing evidence that cues on the sex of the emitter are encoded in echolocation signals. While our data support that the sex-specific acoustic cues are not (just) the horseshoe bats' constant-frequency call part, their nature remains enigmatic. This is the second study to show that bats can recognize the caller's gender from echolocation calls and the first to do so for ‘high-duty-cycle bats’; those that use calls with a prominent constant-frequency (C...

Transposition of mitochondrial DNA into the nucleus, which gives rise to nuclear mitochondrial DNAs (NUMTs), has been well documented in eukaryotes. However, very few studies have assessed the frequency of these transpositions during the evolutionary history of a specific taxonomic group. Here we used the horseshoe bats (Rhinolophus) as a case study to determine the frequency and relative timing of nuclear transfers of mitochondrial control region sequences. For this, phylogenetic and coalescent analyzes were performed on NUMTs and authentic mtDNA sequences generated from eight horseshoe bat species. Our results suggest at least three independent transpositions, including two ancient and one more recent, during the evolutionary history of Rhinolophus. The two ancient transpositions are represented by the NUMT-1 and -2 clades, with each clade consisting of NUMTs from almost all studied species but originating from different portions of the mtDNA genome. Furthermore, estimates of the most recent common ancestor for each clade corresponded to the time of the initial diversification of this genus. The recent transposition is represented by NUMT-3, which was discovered only in a specific subgroup of Rhinolophus and exhibited a close relationship to its mitochondrial counterpart. Our similarity searches of mtDNA in the R. ferrumequinum genome confirmed the presence of NUMT-1 and NUMT-2 clade sequences and, for the first time, assessed the extent of NUMTs in a bat genome. To our knowledge, this is the first study to report on the frequency of transpositions of mtDNA occurring before the common ancestry of a genus.