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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.

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.

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.

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.

Cochlear morphology has been regarded as one of the key traits to understand the origin and evolution of echolocation in bats, given its functionality and performance for receiving echolocation sonar. While numerous researchers have compared adult-stage morphology, few have studied the prenatal development of the cochlea. Here, we provide the first detailed three-dimensional description of the prenatal cranial development in bats, using Rhinolophus thomasi as a model, with particular interest to the petrosal which houses the cochlea. Results revealed that among all cranial bones the onset of the ossification of the petrosal is earlier in R. thomasi when compared to other reported mammals. Generally, the cochlea reaches adult size and shape before or around birth in placental mammals including bats, but we found that its shape and size growths continue until maturity in Rhinolophus species. The relationship of cochlear size and skull size is maintained constant throughout the postnatal ontogeny to adulthood in Rhinolophus, a pattern previously reported neither in any other bats nor other mammals. The peculiar developmental pattern in Rhinolophus possibly allows them to form their characteristically large cochlea and facilitate their distinctive echolocation behavior. A recent study reported that non-echolocating Pteropodidae shares a similar prenatal cochlear size to laryngeal echolocating bats. The apparent resemblance of fetal cochlear size was proposed to be a vestigial signal of large cochlear size in the last common ancestor of bats and thus as supporting evidence for the single origin of laryngeal echolocation. However, results from the present observations suggest that limited aspects of the cochlear development were captured in this previous investigation and that the resulting interpretations may be questionable. We point out that diversity and patterns of cochlear development among bats are still not resolved, and the controversy on the origins of laryngeal echolocation is still open to discussion.

Drift, selection, or their combined effects commonly drive geographic variation in traits. Clarifying the relative roles of each process is a long-standing research goal in evolutionary biology. Acoustic signals of bats are a phenotypic characteristic that plays an important role in social organization and species recognition. We extensively sampled the Chinese rufous horseshoe bat (Rhinolophus sinicus) throughout China and Vietnam and reconstructed a species phylogeny to better understand the patterns and causes of the geographic variation of acoustic signals. Our results showed that the resting frequency (RF) of calls varied with latitude, sex, and distance among different colony locations. RF differences were not correlated with genetic distance (based on only one nuclear locus and the mitochondrial locus), climatic factors (mean annual temperature and mean annual precipitable water), or body size, although differences in calls increased with distance among various populations. This suggests that cultural drift may play more important roles than genetic drift and acoustic adaptation in shaping acoustic differences within regions in R. sinicus.

© 2018 The Authors Global Change Biology Published by John Wiley & Sons Ltd The rapid global spread of artificial light at night is causing unprecedented disruption to ecosystems. In otherwise dark environments, street lights restrict the use of major flight routes by some bats, including the threatened lesser horseshoe bat Rhinolophus hipposideros, and may disrupt foraging. Using radio tracking, we examined the response of individual female R.hipposideros to experimental street lights placed on hedgerows used as major flight routes. Hedgerows were illuminated on one side over four nights using lights with different emission spectra, while the opposite side of the hedge was not illuminated. Automated bat detectors were used to examine changes in overall bat activity by R.hipposideros and other bat species present. R.hipposideros activity reduced significantly under all light types, including red light, challenging a previously held assumption that red light is safe for bats. Despite this, R.hipposideros rapidly adapted to the presence of lights by switching their flight paths to the dark side of the hedgerow, enabling them to reach foraging sites without restriction. Red light had no effect on the activity of the other species present. Slow-flying Myotis spp. avoided orange, white and green light, while more agile Pipistrellus spp. were significantly more active at these light types compared to dark controls, most probably in response to accumulations of insect prey. No effect of any light type was found for Nyctalus or Eptesicus spp. Our findings demonstrate that caution must be used when promoting forms of lighting that are thought to be safe for wildlife before they are tested more widely. We argue that it is essential to preserve dark corridors to mitigate the impacts of artificial light at night on bat activity and movements.

Artificial nighttime lighting has many effects on biodiversity. A proposed environmental management option, primarily to save energy, is to alter the duration of night lighting. Using the greater horseshoe bat Rhinolophus ferrumequinum as an example of a photophobic species, we explored roadside behaviour patterns throughout the night to assess the potential impact of part-night lighting. We found a large primary peak in activity 1 h after sunset, followed by a smaller secondary peak before sunrise. Simulated part-night lighting scenarios reveal that to capture a large proportion of bat activity, streetlights should be switched off before midnight. Current proposed uses of part-night lighting are unlikely to capture natural peaks in activity for nocturnal species.

Divergence in phenotypic traits may arise from the interaction of different evolutionary forces, including different kinds of selection (e.g., ecological), genetic drift, and phenotypic plasticity. Sensory systems play an important role in survival and reproduction, and divergent selection on such systems may result in lineage diversification. Such diversification could be largely influenced by selection in different environments as a result of isolation by environment (IbE). We investigated this process using geographic variation in the resting echolocation frequency of the horseshoe bat species, Rhinolophus damarensis, as a test case. Bats were sampled along a latitudinal gradient ranging from 16°S to 32°S in the arid western half of southern Africa. We measured body size and peak resting frequencies (RF) from handheld individual bats. Three hypotheses for the divergence in RF were tested: (1) James’ Rule, (2) IbE, and (3) genetic drift through isolation by distance (IbD) to isolate the effects of body size, local climatic conditions, and geographic distance, respectively, on the resting frequency of R. damarensis. Our results did not support genetic drift because there was no correlation between RF variation and geographic distance. Our results also did not support James' Rule because there was no significant relationship between (1) geographic distances and RF, (2) body size and RF, or (3) body size and climatic variables. Instead, we found support for IbE in the form of a correlation between RF and both region and annual mean temperature, suggesting that RF variation may be the result of environmental discontinuities. The environmental discontinuities coincided with previously reported genetic divergence. Climatic gradients in conjunction with environmental discontinuities could lead to local adaptation in sensory signals and directed dispersal such that gene flow is restricted, allowing lineages to diverge. However, our study cannot exclude the role of processes like phenotypic plasticity in phenotypic variation.

The repeated formation and loss of land-bridges during the Pleistocene have had lasting impacts on population genetic structure. In the tropics, where island populations persisted through multiple glacial cycles, alternating periods of isolation and contact are expected to have driven population and taxonomic divergence. Here, we combine mitochondrial and nuclear sequence data with microsatellites to dissect the impact of Pleistocene climate change on intra-specific diversification in the horseshoe bat Rhinolophus affinis. This taxon shows considerable morphological and acoustic variation: two parapatric subspecies (himalayanus and macrurus) occur on mainland China and a third (hainanus) on Hainan Island. Our phylogeographic reconstruction and coalescent analyses suggest the island subspecies formed from an ancestral population of himalayanus via two colonization events c. 800,000 years before present. R. a. hainanus then recolonized the mainland, forming macrurus and thus a secondary contact zone with himalayanus. Finally, macrurus recolonized Hainan following the LGM. We found that all three biological events corresponded to known periods of land-bridge formation. Evidence of introgression was detected between macrurus and both its sister taxa, with geographical proximity rather than length of separation appearing to be the biggest determinant of subsequent genetic exchange. Our study highlights the important role of climate-mediated sea level changes have had in shaping current processes and patterns of population structure and taxonomic diversification.

Phylogeographical studies are typically based on haplotype data, occasionally on nuclear markers such as microsatellites, but rarely combine both. This is unfortunate because the use of markers with contrasting modes of inheritance and rates of evolution might provide a more accurate and comprehensive understanding of a species' history. Here we present a detailed study of the phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum, using 1098 bp of the mitochondrial ND2 gene from 45 localities from across its Palaearctic range to infer population history. In addition, we re-analysed a large microsatellite data set available for this species and compared the results of both markers to infer population relationships and the historical processes influencing them. We show that mtDNA, the most popular marker in phylogeography studies, yielded a misleading result, and would have led us to conclude erroneously that a single expansion had taken place in Europe. Only by combining the mitochondrial and microsatellite data sets are we able to reconstruct the species' history and show two colonization events in Europe, one before the Last Glacial Maximum (LGM) and one after it. Combining markers also revealed the importance of Asia Minor as an ancient refugium for this species and a source population for the expansion of the greater horseshoe bat into Europe before the LGM.

The origin and meaning of echolocation call frequency variation within rhinolophid bats is not well understood despite an increasing number of allopatric and sympatric examples being documented. A bimodal distribution of mean regional call frequency within the Okinawa-jima Island population of Rhinolophus cornutus pumilus (Rhinolophidae) provided a unique opportunity to investigate geographic call frequency variation early in its development. Individual resting echolocation frequencies, partial mitochondrial DNA D-loop sequences and genotypes from six microsatellite loci were obtained from 288 individuals in 11 colonies across the entire length of the island, and nearby Kume-jima Island. Acoustic differences (5-8 kHz) observed between the north and south regions have been maintained despite evidence of sufficient nuclear gene flow across the middle of the island. Significant subdivision of maternally inherited D-loop haplotypes suggested a limitation of movement of females between regions, but not within the regions, and was evidence of female philopatry. These results support a 'maternal transmission' hypothesis whereby the difference in the constant frequency (CF) component between the regions is maintained by mother-offspring transmission of CF, the restricted dispersal of females between regions and small effective population size. We suggest that the mean 5-8 kHz call frequency difference between the regions might develop through random cultural drift.

The distribution of genetic variability across a species' range can provide valuable insights into colonization history. To assess the relative importance of European and Asian refugia in shaping current levels of genetic variation in the greater horseshoe bats, we applied a microsatellite-based approach to data collected from 56 localities ranging from the UK to Japan. A decline in allelic richness from west Asia to the UK and analyses of F(ST) both imply a northwestward colonization across Europe. However, sharp discontinuities in gene frequencies within Europe and between the Balkans and west Asia (Syria/Russia) are consistent with suture zones following expansion from multiple refugia, and a lack of recent gene flow from Asia Minor. Together, these results suggest European populations originated from west Asia in the ancient past, and experienced a more recent range expansion since the Last Glacial Maximum. Current populations in central Europe appear to originate from the Balkans and those from west Europe from either Iberia and/or Italy. Comparisons of R(ST )and F(ST) suggest that stepwise mutation has contributed to differentiation between island and continental populations (France/UK and China/Japan) and also among distant samples. However, pairwise R(ST) values between distant populations appear to be unreliable, probably due to size homoplasy. Our findings also highlight two priorities for conservation. First, stronger genetic subdivision within the UK than across 4000 km of continental Eurasia is most likely the result of population fragmentation and highlights the need to maintain gene flow in this species. Second, deep splits within China and between Europe and China are indicative of cryptic taxonomic divisions which need further investigation.

Intraspecific phylogenies can provide useful insights into how populations have been shaped by historical and contemporary processes. Taiwan formed around 5 million years ago from tectonic uplift, and has been connected to mainland Asia several times since its emergence. A central mountain range runs north to south, bisecting the island, and potentially impedes gene flow along an east-west axis. The Formosan lesser horseshoe bat (Rhinolophus monoceros) is endemic to Taiwan, where it is found mainly at low altitude. To determine the population structure and the demographic and colonization history of this species, we examined variation in the mitochondrial DNA control region in 203 bats sampled at 26 sites. We found very high haplotype and nucleotide diversity, which decreased from the centre to the south and north. Population differentiation followed a pattern of isolation by distance, though most regional genetic variance was attributable to differences between the relatively isolated southern population and those from other regions. A haplotype network was consistent with these findings and also suggested a southward colonization, followed by subsequent secondary contact between the south and other regions. Mismatch distributions were used to infer a past population expansion predating the last glacial maximum, and a neighbour-joining tree showed that R. monoceros formed a monophyletic grouping with respect to its sister taxa. Taken together, our results suggest that this taxon arose from a single period of colonization, and that demographic growth followed in the late Pleistocene. Current genetic structure reflects limited gene flow, probably coupled with stepwise colonization in the past. We consider explanations for the persistence of the species through multiple glacial maxima.

The predation pressure of the greater horseshoe bat, Rhinolophus ferrumequinum, on the diapausing population of the noctuid moth, Goniocraspidum preyeri, was examined at an abandoned mine in central Japan. These bats did not prey on the moths in summer. The number of moths preyed on was largest in March. More than 60% of the diapausing moths were eaten by only three or four bats, which accounted for over 90% of all the moth deaths. This moth was an important source of food at the end of the hibernating period when the bat had already used most of its stored subcutaneous fat. The predation on G. preyeri may have raised the overwintering success rate of R. ferrumequinum.

Most regions of the world support moth species that use caves as diurnal roosts and/or as overwintering and mating sites. Although subterranean roosts are often also occupied by insectivorous bats, nothing is known about moth–bat interaction at these sites. The frequency of cohabitation and the dynamics of moth–bat interaction at roosts were examined during surveys at 30 known bat roosts in eastern Australia; 15 disused mines and 15 natural caves. Moths and bats cohabited in 20 roosts (67%); however, only two species of eared moths, Speiredonia spectans and S. mutabilis (Noctuidae), were observed. These large (wingspan up to 75 mm) species show a classic escape response to ultrasound. Moths used roosts occupied by bats with both frequency modulated (FM) (four species) and constant frequency (CF) (two species) echolocation calls. Signal frequencies of these bats had ranges of 23–25 and 44–71 kHz. Moths were absent from roosts occupied by high-frequency (> 140 kHz) CF bats. Predator–prey interaction was assessed by studying a sub-sample of six roosts occupied by S. spectans and the horseshoe bat Rhinolophus megaphyllus. Speiredonia spectans and R. megaphyllus cohabited roosts throughout the year. Bats captured and consumed moths in these roosts. The numbers of moth wings on the floor of roosts were counted to assess rates of predation. Bats consumed 209 moths at the six roosts over a 12-month period, representing 19% of the moth population at the start of the study. The study establishes that moth–bat cohabitation of roosts is relatively common in eastern Australia and that bats do capture moths in these sites. Bat predation seems to be a significant source of mortality of moths that occupy subterranean roosts.

We have isolated 14 polymorphic greater horseshoe bat, Rhinolophus ferrumequinum microsatellite loci. The number of alleles varied from two to 12 in 58 individuals. These loci will be used to assign paternity in order to characterize patterns of breeding and reproductive success in a wild R. ferrumequinum population. Loci were also tested in 17 other bat species. Twelve loci cross-amplified in other species and three loci were polymorphic in all eight Rhinolophus species tested.

Factors influencing prey selection by insectivorous bats are poorly understood but may be related to interspecific differences in echolocation call structure. The diet of Eptesicus fuscus was compared with prey abundance across a foraging season to examine prey selection by a bat species which has an echolocation call dominated by a frequency-modulated (FM) component. Guano samples were collected on 18 occasions from a single colony between May and September 2000. Abundance of aerial insects was measured using a black-light trap on nights when guano was collected. Regression analysis was used to test the hypotheses that E. fuscus: (1) does not take prey in proportion to their relative abundance; (2) forages preferentially for beetles in a manner predicted by foraging theory. Relative abundance (= proportion in relation to other prey types) of prey in trap samples was not a good predictor of prey in the diet across periods or within periods, which suggests selective foraging. Beetles (42–96% of the diet by per cent volume) were consistently eaten in higher proportions than their relative abundance. The absolute abundance of beetles (= abundance irrespective of the abundance of other prey types) explained 60% of the variation in beetle use with more beetles eaten when they were abundant. Absolute beetle abundance explained 66%, 53% and 47% of the variation in the use of three other prey types, with more of these prey eaten when beetle abundance was low. Finally, absolute beetle abundance explained 60% of the variation in diet breadth, which increased when beetle abundance was low. Our results agree with predictions derived from foraging theory and support the hypothesis that E. fuscus forages selectively for beetles, consuming a wider variety of prey when beetle abundance is low. These results are similar to those found for the greater horseshoe bat Rhinolophus ferrumequinum which has an echolocation call dominated by a constant-frequency (CF) component, and suggest that some FM bats may discriminate among prey taxa, at least at the ordinal level.

Over the past 50 years European populations of the lesser horseshoe bat Rhinolophus hipposideros have severely declined, probably because of the loss of foraging habitat. To date, studies of the foraging behaviour of this species have been limited as its low mass (4–8 g) precluded the use of radio-telemetry because commercially available radio-transmitters exceeded 10% of its body mass. In this study, radio-transmitters weighing < 0.35 g were built. These increased the body mass of the animals from 4.5% to 8.1%, with no demonstrable adverse effect on their flight behaviour. The habitat selection of eight female lesser horseshoe bats was studied in Monmouthshire, U.K. The bats had foraging ranges between 12 and 53 ha (100% kernel). Although one bat foraged 4.2 km from the roost, for 50% of the time tracked bats were recorded within 600 m of the nursery roost. The estimated density within 200 m of the roost was 5.8 foraging bats/ha. This decreased to 0.01 bats/ha at 1200 m. Compositional analysis revealed that this species used woodlands, predominately broadleaf, more than any other habitat. In addition, the bats foraged in areas of high habitat diversity. Conservation management of this species should concentrate on such areas within 2.5 km of the nursery roost.

A new species of horseshoe bat, Rhinolophus sakejiensis, is described from south-central Africa, near the source of the Zambezi River in north-west Zambia. A distinct combination of noseleaf, cranial and baculum characters are diagnostic of the species. It is a member of the ferrumequinum group, and its evolutionary affinities lie closest to R. clivosus Cretzschmar, 1828 and particularly the West African R. hillorum Koopman, 1989. This discovery of a new species of Rhinolophus in the clivosus complex required reappraisal of these and other related taxa. Known only from high forest in West Africa, hillorum is the closest relative of sakejiensis collected in mesic savanna in south-central Africa. Comparisons of this new species with other large Afrotropical Rhinolophus shows that hillorum is specifically distinct from clivosus, and endorses the specific status of deckeni and silvestris. The contemporary taxonomy of Afrotropical Rhinolophus was incapable of accommodating this new taxon, and the latter part of this paper argues for a more objective characterization of rhinolophids as evolutionary species.

Summary While bats are important reservoirs and vectors of pathogenic agents with zoonotic potential, their skin-associated microbial community could harbour organisms relevant to bat health as well as those associated with bat habitats in general. A total of 140 skin swabs collected from bats in the Czech Republic were cultured for bacteria from 2012 to 2013. A phytopathogen, isolated from a lesser horseshoe bat (Rhinolophus hipposideros), was identified as Lonsdalea quercina by MALDI-TOF (matrix-assisted laser desorption–ionization time-of-flight mass spectrometry) and characterized as Lonsdalea quercina subsp. britannica using phylogenetic analysis based on a 16S rRNA and two housekeeping (infB and atpD) genes. Lonsdalea quercina subsp. britannica is a gram-negative facultative anaerobic bacterium reported to cause bark canker and drippy nut disease in oaks. Our finding provides evidence of Lonsdalea quercina in the Czech Republic (Moravian Karst). As the lesser horseshoe bat is a sedentary species, it may bio-indicate the presence of the phytopathogenic bacterium in the broadleaved forests of the habitat of pathogen recovery. We therefore suggest active surveillance of the area of the Moravian Karst for signs of oak disease and/or decline.

This study examined the influence of inhibition on motion-direction-sensitive responses of neurons in the dorsal fields of auditory cortex of the rufous horseshoe bat. Responses to auditory apparent motion stimuli were recorded extracellularly from neurons while microiontophoretically applying gamma-aminobutyric acid (GABA) and the GABAA receptor antagonist bicuculline methiodide (BMI). Neurons could respond with a directional preference exhibiting stronger responses to one direction of motion or a shift of receptive field (RF) borders depending on direction of motion. BMI influenced the motion direction sensitivity of 53% of neurons. In 21% of neurons the motion-direction sensitivity was decreased by BMI by decreasing either directional preference or RF shift. In neurons with a directional preference, BMI increased the spike number for the preferred direction by a similar amount as for the nonpreferred direction. Thus, inhibition was not direction specific. BMI increased motion-direction sensitivity by either increasing directional preference or magnitude of RF shifts in 22% of neurons. Ten percent of neurons changed their response from a RF shift to a directional preference under BMI. In these neurons, the observed effects could often be better explained by adaptation of excitation rather than inhibition. The results suggest, that adaptation of excitation, as well as cortex specific GABAergic inhibition, contribute to motion-direction sensitivity in the auditory cortex of the rufous horseshoe bat.

Responses of neurons to apparent auditory motion in the azimuth were recorded in three different fields of auditory cortex of the rufous horseshoe bat. Motion was simulated using successive stimuli with dynamically changing interaural intensity differences presented via earphones. Seventy-one percent of sampled neurons were motion-direction-sensitive. Two types of responses could be distinguished. Thirty-four percent of neurons showed a directional preference exhibiting stronger responses to one direction of motion. Fifty-seven percent of neurons responded with a shift of spatial receptive field position depending on direction of motion. Both effects could occur in the same neuron depending on the parameters of apparent motion. Most neurons with contralateral receptive fields exhibited directional preference only with motion entering the receptive field from the opposite direction. Receptive field shifts were opposite to the direction of motion. Specific combinations of spatiotemporal parameters determined the motion-direction-sensitive responses. Velocity was not encoded as a specific parameter. Temporal parameters of motion and azimuth position of the moving sound source were differentially encoded by neurons in different fields of auditory cortex. Neurons with a directional preference in the dorsal fields can encode motion with short interpulse intervals, whereas direction-preferring neurons in the primary field can best encode motion with medium interpulse intervals. Furthermore, neurons with a directional preference in the dorsal fields are specialized for encoding motion in the midfield of azimuth, whereas direction-preferring neurons in the primary field can encode motion in lateral positions. The results suggest that motion information is differentially processed in different fields of the auditory cortex of the rufous horseshoe bat.

Doppler shift compensation (DSC) behaviour in horseshoe bats is a remarkable example of sensorimotor feedback that stabilizes the echo frequency at the bat's optimum hearing range regardless of motion-induced frequency shifts in the echoes. Searching for a related neural interface, the nucleus of the central acoustic tract (NCAT) was investigated in the echolocating horseshoe bat, Rhinolophus rouxi, using various neurophysiological and tracer methods. The NCAT receives bilateral auditory input from the cochlear nuclei and sends projections to regions outside the classical acoustic pathway like the pretectal area or the superior colliculus. The binaural input is excitatory from the contralateral and inhibitory from the ipsilateral ear to 53% of the units, and auditory responses were biased to frontal and contralateral directions. The best frequencies of NCAT neurons match a narrow range above the main frequency component of the bat's species-specific echolocation call (62% of the units), and the neurons exhibit extremely sharp tuning (Q10dB up to 632). DSC is degraded by unilateral electrical or pharmacological microstimulation of the NCAT, and heavily impaired by unilateral lesion of the region. Altogether, the efferents of the NCAT to prevocal areas, the tuning of its neurons to the DSC-relevant echo frequency range, and the possibility to affect DSC by manipulation of the NCAT, support the assumption that the nucleus plays an important role in audio-vocal control in the horseshoe bat.

We isolated nine polymorphic microsatellite markers from the Chinese rufous horseshoe bat (Rhinolophus sinicus) using an enriched library method. We assessed genetic polymorphism at these loci in 42 individuals from a single population. We recorded high genetic diversity with four to 17 alleles per locus, and estimated expected and observed heterozygosity values ranging from 0.492 to 0.910 and from 0.462 to 0.881, respectively. No locus departed from Hardy-Weinberg equilibrium following Bonferroni correction, and no linkage disequilibrium was detected. Most loci successfully cross-amplified congeneric species. These loci will be used to characterize phylogeographical history of Rhinolophus sinicus in China.

The present study sheds light on species delimitation in what has been previously described as Rhinolophus arcuatus, a morphologically conservative bat species complex nominally distributed throughout archipelagic South-East Asia from New Guinea to Sumatra. Given that rhinolophids tend to be relatively weak fliers, hence have low vagility, we hypothesized that some specimens attributed to R. arcuatus, but originating from geographically disjunct populations, may in fact represent distinct species. To test this hypothesis, we examined specimens attributed to R. arcuatus as well as to other species in the Rhinolophus euryotis species group using both morphological techniques and mitochondrial cytochrome b and control region sequences. Careful morphological analysis reveals heretofore cryptic but nevertheless distinct, species-level morphological differences among specimens derived from geographically isolated locations. Furthermore, molecular data illuminate the existence of several species-level sequence divergences among specimens heretofore attributed to R. arcuatus. These analyses similarly suggest the existence of additional species in other South-East Asian Rhinolophus taxa previously considered monotypic. We suggest at least one description to be undertaken of a previously unrecognized species as well as the elevation of several others from sub-specific to specific status.

Double staining immunohistochemistry was used to investigate the origin and projection of nerves with substance P (SP) immunoreactivity (-IR) in the walls of the major cerebral arteries in two microchiropteran species. In the greater horseshoe bat, most of the cerebral perivascular nerves with SP-IR did not exhibit calcitonin gene-related peptide (CGRP)-IR, but emitted bright immunofluorescence for vasoactive intestinal polypeptide (VIP). In this species, a large number of cell bodies with both SP- and VIP-IR were observed in many cranial ganglia along various branches of the facial and glossopharyngeal nerves. There were no cell bodies immunoreactive for either SP and VIP in the two sensory (trigeminal and upper cervical dorsal root), two sympathetic (superior cervical and stellate), or two vagal (superior and jugular) ganglia. In addition, several thick fiber bundles with both SP- and VIP-IR were present in the wall of the cerebral carotid artery, and descended progressively reaching as far as the middle part of the basilar artery (BA). These and other findings suggest that SP-immunoreactive nerves with VIP-IR but not CGRP-IR, which contribute to the rich innervation of the vertebrobasilar system in the greater horseshoe bat, originate from neurons with the same combination of peptide-IR in the major or local facial or glossopharyngeal parasympathetic ganglia, and enter the cranial cavity along the internal carotid artery. In the bent-winged bat, however, cerebral perivascular SP-immunoreactive nerves, as well as SP-immunoreactive neurons within the trigeminal and upper cervical dorsal root ganglia (uCDRG), showed neither CGRP-IR nor VIP-IR, and were mostly confined to the caudal BA and the vertebral artery (VA). These observations, in addition to the projection of this nerve type to the BA via the VA as fiber bundles, or through the meninges, indicate that the principal source of the cerebrovascular SP-immunoreactive innervation in this species is the uCDRG. © 1994 Wiley-Liss, Inc.

Summary The gastrointestinal tract of the horseshoe bat, Rhinolophus hildebrandti (Peters), was studied macroscopically, with the light microscope and with the scanning electron microscope. Macroscopically, the stomach was of the simple type and the intestine was a short convoluted tube with an almost uniform diameter. A caecum, an appendix and a colon were absent, and the only portion of the large intestine observed was a short rectum which had a distinctly greater diameter than the rest of the intestine. The proximal part of the intestine showed a brief ‘honeycomb’ segment preceding the ridge-like transverse villi that occupied the rest of the foregut. In the region immediately proximal to the rectum the villi joined in a geometrical pattern to form chambers resembling ‘reticular cells’ of the ruminant stomach. The villi were covered with a prominent columnar epithelium occasionally interrupted by goblet cells. Generally, the intestine of Rhinolophus resembles that of the other bats which have been studied, but showed structural details suggestive of an increased surface area. Resume Le tractus intestinal du Rhinolophe, Rhinolophus hildebrandti (Peters) a eteetudie au moyen d'un microscope simple et d'un microscope electronique. Macroscopiquement, l'estomac est de type simple, et l'intestin est un tube court enroule d'un diametre presque constant. Il n'y a ni caecum, ni appendice, ni colon, et la seule portion de gros intestin observeeetait un rectum court d'un diametre nettement plus grand que le reste de l'intestin. La partie proximate de l'intestin presente un court segment en “nid d'abeille” qui precede le reste de l'intestin strie transversalement. Dans la partie qui precede directement le rectum, les villosites transversales rejoignent un schema geometrique pour former des chambres semblables aux “cellules reticulaires” de l'estomac des ruminants. Les villosites etaient couvertes d'un epithelium en colonnes tres visible, interrompu ca et la par des cellules en gobelet. Globalement, l'intestin du Rhinolophe ressemble a celui des autres-chauves souris qui ont dejaeteetudiees mais presente des detais de structure qui suggerent une augmentation de la surface.

A comparative study of the immunostain to antibodies directed against glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in the ascending auditory pathway was carried out in horseshoe bats (Rhinolophus rouxi) and mustached bats (Pteronotus parnellii). In both species GAD/GABA-positive puncta (presumed axonal boutons) and GAD/GABA-positive cells were found in the cochlear nucleus, the superior olivary complex, the nuclei of the lateral lemsiscus, the inferior colliculus, and the medial geniculate body. General features of the immunostaining pattern in the auditory pathway agree with observations in other mammals, Quantitative analysis of puncta distribution shows that many auditory centers are characterized by subregional differences in puncta density and distribution. This indicates local differences in putatively inhibitory input related to connectivity and tonotopic organization. The following species characteristic features were found: 1) The dorsal non-laminated portion of the dorsal cochlear nucleus in horseshoe bats lacks the GAD/GABA-immunoreactive cells typical for the ventral laminated portion and the dorsal cochlear nucleus of other species. Clearly, a cytoarchitectonic specialization is accompanied by a loss of putatively GABAergic local inhibitory circuits. 2) The ventral division of the medial geniculate body of the mustached bat lacks GAD/GABA-immunopositive cells. Such cells are present in the horseshoe bat and other mammals. This finding implies functional differences in the organization of the medial geniculate body within the same mammalian order. © 1992 Wiley-Liss, Inc.

The fine structure of the organ of Corti was investigated in the echolocating horseshoe bat (Rhinolophus rouxi) by transmission electron microscopy. Particular emphasis was placed on the receptor cells and their supporting cells. The receptor cells, inner hair cells (IHC) and outer hair cells (OHC), possess the typical mammalian shape, but OHCs are extremely short (length: 12–15 μm in the basal turn and up to 28–30 μm in the apical turn). The afferent innervation of both types of receptor cells and the efferent innervation of the IHC system conform to the general mammalian scheme; however, confirming earlier reports, an efferent innervation to the OHCs is absent. Throughout the cochlea, IHCs and OHCs possess a single layer of subsurface cisternae. Above the level of the nucleus of the OHCs, the arrangements of the subsurface cisternae and their connection to the lateral cell membrane via pillars are highly regular, whereas in IHCs, the cisternae are of irregular shape and the pillar system is much less distinct. In the basal turn of the cochlea, the attachment sites of the OHCs to the supporting cells possess specialized features: (a) in the reticular lamina, the contact sites of the cuticular plates of OHCs with the outer pillar cells and the Deiters cell phalanges are of exaggerated length, and (b) the cup formation of the Deiters cell body, which houses the bottom of the OHC, has a specialized shape and is packed with electron-dense material and microtubules. The results are discussed in relation to cochlear ultrastructure in other mammals and in the context of active processes in cochlear mechanics.

Responses of auditory neurons to complex stimuli were recorded in the dorsal belt region of the auditory cortex of two taxonomically unrelated bat species, Rhinolophus rouxi and Pteronotus parnellii parnellii, both showing Doppler shift compensation behaviour. As in P.p.parnellii (Suga et al., J. Neurophysiol., 49, 1573 - 1626, 1983), cortical neurons of R.rouxi show facilitated responses to pairs of pure tones or frequency modulations. Best frequencies for the two components lie near the first and second harmonic of the echolocation call but are in most cases not harmonically related. Neurons facilitated by pairs of pure tones show little dependence on the delay between the stimuli, whereas pairs of frequency modulations evoke best facilitated responses at distinct best delays between 1 and 10 ms. Facilitated neurons are found in distinct portions of the dorsal cortical belt region, with a segregation of facilitated neurons responding to pure tones and to frequency modulations. Non-facilitated neurons are found throughout the field. Neurons are topographically aligned with increasing best delays along a rostrocaudal axis. The best delays between 2 and 4 ms are largely overrepresented numerically, and occupy approximately 56% of the cortical area containing facilitated neurons. A functional interpretation of the large overrepresentation of best delays approximately 3 ms is proposed. Facilitated neurons are located almost entirely within layer V of the dorsal field.

Dusk to dawn observations, using a bat detector and occasionally an image intensifier, were made outside two nursery roosts of lesser horseshoe bats Rhinolophus hipposideros from late May to September. Emergence was correlated with sunset but delayed by extended twilight. Light intensity was important in triggering departure and cloud cover advanced it. Light-testing behaviour was invariably undertaken, in the form of brief flights out and back into the roosts. The exit from one roost was shaded by trees and exploratory flights were generally more extended there. Heavy rain inhibited emergence. There was almost always intermittent activity throughout the night, with many individuals returning and departing, and no indication of seasonal or overnight peaks. A bat detector inside a third roost confirmed overnight observations at the other two. Some bats often returned to the roost for the night before dawn. Dawn return was linked to sunrise, prolonged twilight in midsummer hastening it. Colony size varied appreciably over periods of a few days and even overnight. There is some limited evidence that increased colony size, perhaps through social interaction, may have influenced timing of departure at dusk and return at dawn.

From the point of view of adaptation for flight in bats, we compared growth and LDH (lactate dehydrogenase) isozyme patterns in the pectoral and cardiac muscles of the Japanese Lesser horseshoe bat, Rhinolophus cornutus cornutus. In the pectoral muscles, the four LDH isozyme bands were recognized throughout life. In the embryo and newborn young the predominant isozyme was LDH-3 and the relative amount of LDH-1 was very low. The predominant isozyme of the infant in the flying stage was LDH-1. Moreover, the percentage of B subunit of LDH isozymes in the pectoral muscles increased remarkably during the transition period from the flapping to flying stage. Thus, it is concluded that there is a reversal of LDH isozyme activity toward more anodal side (cardiac muscle type) during the transition period, which is regarded as an adaptation to the metabolic requirement at isozyme level of the pectoral muscles for an adult. This species has a highly manoeuvrable flight with low speed, like that of a butterfly. On the other hand, most of the activity in the cardiac muscles, even in the embryo, was concentrated in more anodal isozymes with LDH-1 and LDH-2, and consequently the percentage of B subunit showed a considerably higher value, which continued to increase gradually until the adult stage.

The functional organization of the cochlear nucleus (CN) was studied with physiological recording and anatomical tracing techniques. Recordings were made from single CN neurons to examine their temporal firing patterns to tone burst stimuli and their frequency tuning characteristics. Recording loci of individual neurons were carefully monitored in order to understand how the functional properties of a cell relate to its location within the CN. We found that tonal frequencies were systematically represented in each of the three CN divisions (anteroventral, AVCN; posteroventral, PVCN; dorsal, DCN). Eight temporal response patterns were observed in CN neurons when stimulated at units' best excitatory frequencies (BF). With a few exceptions, neurons in each CN division could generate all eight firing patterns with different distributions for the three divisions. A focal injection of horseradish peroxidase (HRP), at the end of the physiological study, to a group of neurons possessing a similar BF in one CN division resulted in anterograde labeling of nerve terminals in the other two divisions at precisely the areas where the same frequency band was processed in these divisions. Labeled terminals in each division were closely congregated in the form of a thin slab. The slab orientation was division specific whereas its location was frequency specific, which could be predicted on the basis of physiological data. HRP injections into the DCN also resulted in retrograde labeling of somata in the AVCN and PVCN. On the other hand, only DCN neurons were retrogradely labeled when HRP was injected into the AVCN or the PVCN. These data showed how the three CN divisions are internally connected. Furthermore, retrogradely labeled cells occupied the same slabs where we found anterogradely labeled nerve terminals. Additionally, in a group of bats, HRP was injected into various functionally (i.e., BF) identified regions of the central nucleus of the inferior coliculus (IC) to clarify the type and location of CN projecting neurons. Retrogradely labeled cells in individual CN divisions likewise were arranged in slabs whose locations in the CN nuclei depended on the BFs of neurons at the injection site in the IC. These results show that slabs represent units of functional organization (i.e., tonal frequency, local connection and central projection) in the CN.

The glyoxylic-acid-induced fluorescence technique was applied to demonstrate patterns of catecholaminergic innervation within the auditory brainstem of echolocating bats and the house mouse. In the cochlear nucleus of the rufous horseshoe bat (Rhinolophus rouxi) and the mustache bat (Pteronotus parnellii), species-specific catecholaminergic innervation patterns are found that contrast with the relatively homogeneous innervation in the rodent. In both bats the subnuclei of the cochlear nucleus receive a differentially dense supply of catecholaminergic fibers, and within the subnuclei, the catecholamine innervation densities can be correlated with the tonotopic frequency representation. The areas devoted to the high-frequency echolocation calls are less densely innervated than those regions which are responsive to lower frequencies. Apart from this common scheme, there are noteworthy distinctions between the two bats which correlate with specialized cytoarchitectural features of the cochlear nucleus. The marginal cell group, located medially to the anteroventral cochlear nucleus of Pteronotus, receives the densest supply of catecholaminergic fibers of all auditory nuclei. This plexus is formed by a morphologically distinct population of catecholaminergic fibers.

The diet of the lesser horseshoe bat Rhinolophus hipposideros was investigated over one season by analysing faeces and discarded insect fragments collected on polythene sheets at eight roosts. Remains of 23 insect families from seven orders (Lepidoptera, Neuroptera, Trichoptera, Hymenoptera, Coleoptera, Diptera and Hemiptera) and of spiders (Araneae: Arachnida) were identified. Nematoceran Diptera were the chief prey but Lepidoptera, Trichoptera and Neuroptera were also important. Both locational and seasonal variation were demonstrated for some food categories. The predicted seasonal availability of the different insect taxa is broadly reflected in the results: the question of possible prey selection is discussed. The bat fed successfully on three families of Lepidoptera known to possess hearing organs sensitive to bat ultrasounds. The possible mechanisms by which R. hipposideros might catch such prey are reviewed.

The connections of the inferior colliculus, the mammalian mid- brain auditory center, were determined in the greater horseshoe bat (Rhinolophus ferrumequinum), using the horseradish peroxidase method. In order to localize the auditory centers of this bat, brains were investigated with the aid of cell and fiber-stained material. The results show that most auditory centers are highly developed in this echo- locating bat. However, the organization of the central auditory system does not generally differ from the mammalian scheme. This holds also for the organization of the superior olivary complex where a well-developed medial superior olivary nucleus was found. In addition to the ventral and dorsal nuclei of the lateral lemniscus a third well-developed nucleus has been defined which projects ipsi- laterally to the inferior colliculus and which was called the intermediate nucleus of the lateral leminiscus. All nuclei of the central auditory pathway project ipsi-, contra-, or bilaterally to the central nucleus of the inferior colliculus with the exception of the medial nucleus of the trapezoid body and the medial geniculate body. The tonotopic organization of these projections and their possible functions are discussed in context with neurophysiological investigations. Insectivorous bats are mammals that hunt at night with the help of an active sonar sys- tem. They emit ultrasonic calls and extract from the returning echoes information about their environment and their prey with the aid of a highly developed auditory system. Since the discovery of this specialized orientation system in bats many neurophysiological ex- periments have been performed on acoustic processing in the central auditory system of these animals. The neurophysiological data show that the auditory system of echolocating bats is highly specialized and adapted to their specific orientation behavior (e.g., Neuweiler, '80; Pollack, '80; Suga and O'Neill, '80). Surprisingly, only a few anatomical data exist for the central auditory system in bats (for review see Henson, '70). Most anatomical investigations show that some of the auditory centers are highly developed. But, two main questions arise from earlier investigations: How is the auditory pathway organized up to the inferior colliculus and how well-developed

The ultrasonic emissions of both adult and juvenile Lesser Horseshoe bats have been studied. Two features were observed:—The range of frequencies used by adult bats when congregated in a crowd of 60 or more does not exceed 3 Kc/s, even though their frequency of emission is of the order of 113 Kc/s. The baby bats can make sounds of echo-location type within 3 days of birth; they open their mouths to do so when disturbed and the frequency of the sound at first is about 30 kc/s. Erratic variations in frequency take place during development.

Undisturbed hibernating Greater horseshoe bats were studied over three winters to find the effect of temperature on arousal frequency, timing of arousals and the selection of a hibernation site. The study showed that temperature is important in all three aspects and the ability to select a temperature zone accurately is an important factor in the survival of hibernation by a bat. The arousal frequency falls with falling ambient temperature then seems to rise again below 6°C. There is a clear seasonal effect, bats waking up on average once a day at 10.5°C in winter compared with 8.5°C in spring. An arousal frequency of once in six days occurs at 8°C in winter compared with 6°C in spring. This shift of 2°C is compensated for by a shift of 2°C in temperature selection by the bats in relation to the same external climatic temperatures on the day of arousal. It appears that bats may select the temperature zone which, if prolonged, will result in the most advantageous arousal frequency in relation to feeding experiences. The seasonal effect discovered indicates that inhibition of the arousal process is important in hibernation and a hormone is postulated.