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Emerging infectious diseases pose a major threat to human, animal, and plant health. The risk of species-extinctions increases when pathogens can survive in the absence of the host, for example in environmental reservoirs. However, identifying such reservoirs and modes of infection is often highly challenging. In this study, we investigated the presence and nature of an environmental reservoir for the ascomycete fungus Pseudogymnoascus destructans, the causative agent of white-nose disease. We also characterised the modes and timing of transmission of the pathogen; key elements to better understand the disease dynamics. Using 18 microsatellite markers, we determined the genotypic and genic (based on allele frequencies) differentiation between 1,497 P. destructans isolates collected from nine closely situated hibernacula in North-Eastern Germany. One hibernaculum was the focus of intensive sampling in which both the bats and walls of the site were sampled at regular intervals over five consecutive winter seasons (1,062 isolates). We found significant genic differentiation between sites and few multi-locus genotypes shared across hibernacula (genotypic differentiation). This demonstrates that each hibernaculum has an essentially unique population of the fungus. This would be expected if bats purge viable P. destructans over the summer, preventing the mixing and exchange of the pathogen in maternity colonies, where bats from all of the studied hibernacula meet. Results from the intensively sampled site show higher measures of genotypic richness on walls compared to bats, the absence of genic differentiation between bats and walls, and stable relative abundance of multi-locus genotypes over multiple winter seasons. This clearly implicates hibernacula walls as the main environmental reservoir of the pathogen, from which bats become re-infected annually.

Hibernation, a period where bats have suppressed immunity and low body temperatures, provides the psychrophilic fungus Pseudogymnoascus destructans the opportunity to colonise bat skin, leading to severe disease in susceptible species. Innate immunity, which requires less energy and may remain more active during torpor, can control infections with local inflammation in some bat species that are resistant to infection. If infection is not controlled before emergence from hibernation, ineffective adaptive immune mechanisms are activated, including incomplete Th1, ineffective Th2, and variable Th17 responses. The Th17 and neutrophil responses, normally beneficial antifungal mechanisms, appear to be sources of immunopathology for susceptible bat species, because they are hyperactivated after return to homeothermy. Non-susceptible species show both well-balanced and suppressed immune responses both during and after hibernation.

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats’ unique traits.

The development of methods for individual identification in wild species and the refinement of Capture-Mark-Recapture (CMR) models during the past decades has greatly improved the assessment of population demographic rates to answer ecological and conservation questions. In particular, multistate models, with their flexibility for the analysis of complex study systems, have become popular in the ecological community. However, despite the extensive use of these models, little attention has been paid to the effect of common violations of the CMR model assumptions, such as mark loss and the often-associated recycling of remarked individuals.To explore this knowledge gap we used a wide range of simulation scenarios reflecting frequently encountered real case studies inspired from the survival rates of 700 vertebrates’ species. We estimated the effects of mark loss and recycled individuals on parameter estimates using a multi-state Cormack-Jolly-Seber (MSCJS) framework. We explored parameter bias through simulations of a metapopulation system with different capture and survival rates. We also illustrated how mark loss can be easily estimated and accounted for using an empirical long-term (10 years) CMR dataset of bats, individually identified using both PIT tag technology as marks that can be lost, and multi-locus genotypes as ‘permanent marks’.The results from our simulated scenarios demonstrated that the occurrence of bias and the parameters concerned were highly dependent on the study system, and no general rules on parameter behaviour can be established a priori. The model structure and the interdependency among parameters make it challenging to predict how bias could affect estimates.Our results highlight the need to assess the effect of mark loss when using MSCJS models. Ignoring such violations of model assumptions can have important implications for ecological inferences and conservation policies. In general, the use of permanent marks (e.g. genotype), should always be preferred when modelling of population dynamics and if not possible, combining two types of temporary marks (e.g. PIT tags, bands) should be considered.

International audience; Within-species genetic diversity is crucial for the persistence and integrity of populations and ecosystems. Conservation actions require an understanding of factors influencing genetic diversity, especially in the context of global change. Both population size and connectivity are factors greatly influencing genetic diversity; the relative importance of these factors can, however, change through time. Hence, quantifying the degree to which population size or genetic connectivity are shaping genetic diversity, and at which ecological time scale (past or present), is challenging, yet essential for the development of efficient conservation strategies. In this study, we estimated the genetic diversity of 42 colonies of Rhinolophus hipposideros, a long-lived mammal vulnerable to global change, sampling locations spanning its continental northern range. Here, we present an integrative approach that disentangles and quantifies the contribution of different connectivity measures in addition to contemporary colony size and historic bottlenecks in shaping genetic diversity. In our study, the best model explained 64% of the variation in genetic diversity. It included historic bottlenecks, contemporary colony size, connectivity and a negative interaction between the latter two. Contemporary connectivity explained most genetic diversity when considering a 65 km radius around the focal colonies, emphasizing the large geographic scale at which the positive impact of connectivity on genetic diversity is most profound and hence, the minimum scale at which conservation should be planned. Our results highlight that the relative importance of the two main factors shaping genetic diversity varies through time, emphasizing the relevance of disentangling them to ensure appropriate conservation strategies.

Emerging infectious diseases may become serious threats to wildlife, a prominent example being the white-nose disease (WND). In case of WND, the cold-loving fungus Pseudogymnoascus destructans colonizes bats during hibernation, invades the skin and has already lead to the death of millions of bats in North America. P. destructans most likely originated from Europe, where it also causes lesions but without associated mortalities. However, it is still unclear how European bats cope with the fungus. Hence, it is important to have tools that precisely characterise disease progression. Because hibernation is a physiological state during which bats are vulnerable to disturbance, in-situ assessments of the clinical status should be carried out minimal-invasively to avoid detrimental impacts on bats. However, currently available disease assessment methods require handling/touching bats and are therefore invasive: i) UV-light trans-illumination of wing membranes to detect lesions and ii) a qPCR-based quantification of fungal material from wing membrane swabs. Since P. destructans (‘Pd') becomes visible on all furless skin with distinct distribution patterns, we investigated the use of visible symptoms to assess levels of infections without handling/touching bats. We introduce a technique which we termed ‘Visual Pd-score’ (a visual classification scheme), which can be applied without disturbing the animals. To assess its reliability, we used P. destructans infected greater mouse-eared bats (Myotis myotis) to compare the novel method with the two existing golden-standard techniques. Our results show that infection levels obtained from all three techniques are correlated. Importantly, the information carried by the Visual Pd-score is most similar to a composite index combining the information from the qPCR-based and UV-light quantification methods. We conclude that the Visual Pd-score represents a promising index to better characterise disease severity as it is simultaneously representative for fungal colonization and wing damage. We discuss advantages and disadvantages of the applied techniques and conclude that the Visual Pd-score is particularly useful for routine hibernacula counts or large-scale P. destructans-surveillance. In combination with the lesion detection technique the new method is also applicable to immunological studies where both fungal colonization and associated damage have to be investigated, while qPCRs from swabs of all body parts are especially useful if it is necessary to detect cryptic infections, e.g. during the early hibernation period.

The greater horseshoe bat(Rhinolophus ferrumequinum)is among the most widespread bat species in Europe but it has experienced severe declines, especially in Northern Europe. This species is listed Near Threatened in the EuropeanIUCN Red List of Threatened Animalsand it is considered to be highly sensitive to human activities and particularly to habitat fragmentation. Therefore, understanding the population boundaries and demographic history of populations of this species is of primary importance to assess relevant conservation strategies. In this study, we used 17 microsatellite markers to assess the genetic diversity, the genetic structure and the demographic history ofR. ferrumequinumcolonies in the Western European part of its distribution. We found high levels of genetic diversity and large population size on the European mainland and lower estimates in England and Northern France. Analyses of clustering and isolation by distance showed a barrier effect of the Channel and potentially of the Mediterranean Sea onR. ferrumequinumbat dispersal. Conversely, we could not reveal any gene flow disruption from both sides of the Western Pyrenees. These results provide important information to improve the delineation ofR. ferrumequinummanagement units in its western range. We suggest that a large management unit corresponding to the European mainland population must be considered. Particular attention should be given to mating territories as they seem to play a key role in maintaining the high levels of genetic mixing between colonies. Smaller management units corresponding to English and Northern France colonies must also be implemented. These insular or peripheral colonies could be at higher risk of extinction in a near future.

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.

Bats are the longest-lived mammals, given their body size. However, the underlying molecular mechanisms of their extended healthspans are poorly understood. To address this question we carried out an eight-year longitudinal study of ageing in long-lived bats (Myotis myotis). We deep-sequenced ~1.7 trillion base pairs of RNA from 150 blood samples collected from known aged bats to ascertain the age-related transcriptomic shifts and potential microRNA-directed regulation that occurred. We also compared ageing transcriptomic profiles between bats and other mammals by analysis of 298 longitudinal RNA sequencing datasets. Bats did not show the same transcriptomic changes with age as commonly observed in humans and other mammals, but rather exhibited a unique, age-related gene expression pattern associated with DNA repair, autophagy, immunity and tumour suppression that may drive their extended healthspans. We show that bats have naturally evolved transcriptomic signatures that are known to extend lifespan in model organisms, and identify novel genes not yet implicated in healthy ageing. We further show that bats’ longevity profiles are partially regulated by microRNA, thus providing novel regulatory targets and pathways for future ageing intervention studies. These results further disentangle the ageing process by highlighting which ageing pathways contribute most to healthy ageing in mammals. RNA-Seq analysis of an 8-year longitudinal study of long-lived bats reveals unique transcriptomic signatures and novel candidate genes associated with healthy ageing.

Significance Forecasts of species vulnerability and extinction risk under future climate change commonly ignore local adaptations despite their importance for determining the potential of populations to respond to future changes. We present an approach to assess the impacts of global climate change on biodiversity that takes into account adaptive genetic variation and evolutionary potential. We show that considering local climatic adaptations reduces range loss projections but increases the potential for competition between species. Our findings suggest that failure to account for within-species variability can result in overestimation of future biodiversity losses. Therefore, it is important to identify the climate-adaptive potential of populations and to increase landscape connectivity between populations to enable the spread of adaptive genetic variation., Local adaptations can determine the potential of populations to respond to environmental changes, yet adaptive genetic variation is commonly ignored in models forecasting species vulnerability and biogeographical shifts under future climate change. Here we integrate genomic and ecological modeling approaches to identify genetic adaptations associated with climate in two cryptic forest bats. We then incorporate this information directly into forecasts of range changes under future climate change and assessment of population persistence through the spread of climate-adaptive genetic variation (evolutionary rescue potential). Considering climate-adaptive potential reduced range loss projections, suggesting that failure to account for intraspecific variability can result in overestimation of future losses. On the other hand, range overlap between species was projected to increase, indicating that interspecific competition is likely to play an important role in limiting species’ future ranges. We show that although evolutionary rescue is possible, it depends on a population’s adaptive capacity and connectivity. Hence, we stress the importance of incorporating genomic data and landscape connectivity in climate change vulnerability assessments and conservation management.

International audience; ge-related telomere shortening is considered a hallmark of the ageing process. However, a recent cross-sectional ageing study of relative telomere length (rTL) in bats failed to detect a relationship between rTL and age in the long-lived genus Myotis (M. myotis and M. bechsteinii), suggesting some other factors are responsible for driving telomere dynamics in these species. Here, we test if longitudinal rTL data show signatures of age-associated telomere attrition in M. myotis and differentiate which intrinsic or extrinsic factors are likely to drive telomere length dynamics. Using quantitative polymerase chain reaction, rTL was measured in 504 samples from a marked population, from Brittany, France, captured between 2013 and 2016. These represent 174 individuals with an age range of 0 to 7+ years. We find no significant relationship between rTL and age (p = .762), but demonstrate that within-individual rTL is highly variable from year to year. To investigate the heritability of rTL, a population pedigree (n = 1744) was constructed from genotype data generated from a 16-microsatellite multiplex, designed from an initial, low-coverage, Illumina genome for M. myotis. Heritability was estimated in a Bayesian, mixed model framework, and showed that little of the observed variance in rTL is heritable (h(2) = 0.01-0.06). Rather, correlations of first differences, correlating yearly changes in telomere length and weather variables, demonstrate that, during the spring transition, average temperature, minimum temperature, rainfall and windspeed correlate with changes in longitudinal telomere dynamics. As such, rTL may represent a useful biomarker to quantify the physiological impact of various environmental stressors in bats.

Emerging infectious diseases rank among the most important threats to human and wildlife health. A comprehensive understanding of the mode of infection and presence of potential reservoirs is critical for the development of effective counter strategies. Fungal pathogens can remain viable in environmental reservoirs for extended periods of time before infecting susceptible individuals. This may be the case for Pseudogymnoascus destructans ( Pd ), the causative agent of bat white-nose disease. Owing to its cold-loving nature, this fungal pathogen only grows on bats during hibernation, when their body temperature is reduced. Bats only spend part of their life cycle in hibernation and do not typically show signs of infection in summer, raising the question of whether Pd remains viable in hibernacula during this period (roughly six months). If so, this could facilitate the re-infection of bats when they return to the sites the following winter. In a laboratory experiment, we determined the germination rate of Pd spores kept under constant conditions on a wall-like substrate, over the course of two years. Results showed that the seasonal pattern in Pd germination mirrored the life cycle of the bats, with an increased germination rate at times when hibernating bats would naturally be present and lower germination rates during their absence. We suggest that Pd is dependent on the presence of hibernating bats and has therefore coupled its germination rate to host availability. Furthermore, we demonstrate that Pd spores survive extended periods of host absence and can remain viable for at least two years. There is, however, a strong decrease in spore viability between the first and second years (98%). Pd viability for at least two years on a solid mineral-based substrate establishes the potential for environmental reservoirs in hibernacula walls and has strong implications for the efficacy of certain management strategies (e.g. bat culling).

Physiological markers can be used in conservation studies if they reliably reflect environmental perturbations. Here, two oxidative markers reflected environmental conditions experienced by greater mouse-eared bats. However, the magnitude of the variation in oxidative markers differed among nearby colonies, suggesting that, if necessary, a one-size-fits-all conservation approach would be inappropriate., To increase the applicability and success of physiological approaches in conservation plans, conservation physiology should be based on ecologically relevant relationships between physiological markers and environmental variation that can only be obtained from wild populations. Given their integrative and multifaceted aspects, markers of oxidative status have recently been considered in conservation physiology, but still need to be validated across environmental conditions and locations. Here, we examined whether inter-annual variation in two oxidative markers, plasma antioxidant capacity and plasma hydroperoxides, followed inter-annual variation in temperature anomalies and associated vegetation changes in four colonies of long-lived greater mouse-eared bats (Myotis myotis) monitored over five consecutive years. We found that the plasma antioxidant capacity of bats decreased while plasma hydroperoxide concentrations increased with increasing temperature anomalies occurring in the two weeks before blood sampling. Moreover, the antioxidant defences of these bats reflected vegetation indices, which themselves reflected the thermal conditions experienced by bats in their foraging habitat. Variation in oxidative markers therefore appears to be due to variation in thermoregulatory costs and to indirect changes in foraging costs. Overall, these results validate the use of markers of oxidative status in conservation physiology to monitor thermal perturbations recently experienced by animals in their natural habitat. However, even though oxidative markers varied in the same direction in all four bat colonies across years, the amplitude of their response differed. If these different physiological responses reflect different performances (e.g. productivity, survival rate) between colonies, this implies that, if necessary, conservation measures may need to be applied at the local scale.

Emerging infectious diseases are a severe conservation threat for a variety of plants and animals. In North America, several bat species are threatened by white-nose disease, which has caused an unprecedented mass mortality of > 6 million bats since 2006. The fungus Pseudogymnoascus (Geomyces) destructans is the causative agent of the disease. Though asexual reproduction is the norm, sexual reproduction is possible as two mating types exist. Sexual reproduction has been implicated in the emergence of virulent strains of fungi but to date no rapid means of mating type characterisation was available for P. destructans. In this study, three mating type-specific primer sets were designed and tested on 80 isolates. The primers were multiplexed with microsatellite loci allowing for rapid and concurrent genotyping and mating type assignment. These markers will have great utility in better understanding and predicting the population dynamics and evolutionary potential of this fungus, including the emergence of virulent strains.

Bats are the only mammals capable of true, powered flight, which drives an extremely high metabolic rate. The “Free Radical Theory of Ageing” (FTRA) posits that a high metabolic rate causes mitochondrial heteroplasmy and the progressive ageing phenotype. Contrary to this, bats are the longest-lived order of mammals given their small size and high metabolic rate. To investigate if bats exhibit increased mitochondrial heteroplasmy with age, we performed targeted, deep sequencing of mitogenomes and measured point heteroplasmy in wild, long lived Myotis myotis. Blood was sampled from 195 individuals, aged between

International audience; Gene flow, maintained through natal dispersal and subsequent mating events, is one of the most important processes in both ecology and population genetics. Among mammalian populations, gene flow is strongly affected by a variety of factors, including the species’ ability to disperse, and the composition of the environment which can limit dispersal. Information on dispersal patterns is thus crucial both for conservation management and for understanding the social system of a species. We used 16 polymorphic nuclear microsatellite loci in addition to mitochondrial DNA sequences (1.61 kbp) to analyse the population structure and the sex‐specific pattern of natal dispersal in the frog‐eating fringe‐lipped bat, Trachops cirrhosus, in Central Panama. Our study revealed that—unlike most of the few other investigated Neotropical bats—gene flow in this species is mostly male‐mediated. Nevertheless, distinct genetic clusters occur in both sexes. In particular, the presence of genetic differentiation in the dataset only consisting of the dispersing sex (males) indicates that gene flow is impeded within our study area. Our data are in line with the Panama Canal in connection with the widening of the Río Chagres during the canal construction acting as a recent barrier to gene flow. The sensitivity of T. cirrhosus to human‐induced habitat modifications is further indicated by an extremely low capture success in highly fragmented areas. Taken together, our genetic and capture data provide evidence for this species to be classified as less mobile and thus vulnerable to habitat change, information that is important for conservation management.

Bat population trends are particularly affected by adult mortality, especially when large numbers of individuals die, as evidenced by white‐nose syndrome in North America. We obtained baseline mortality data from 318 European hibernacula. Mortality was low and negatively associated with elevation but not with fungal infestation. Mortality events involving more than seven bats at a hibernaculum should be considered unusual, and above this threshold, pathological or microbiological analysis should be carried out. To increase understanding of mortality in bats, there is an urgent need to develop and co‐ordinate national and international programs for monitoring and investigating mortality and diseases.

Bats are unique among mammals given their ability to fly, apparent tolerance of deadly viruses and extraordinary longevity. We propose that these traits are linked and driven by adaptations of the innate immune system. To explore this hypothesis we challenged macrophages from the greater mouse-eared bat, Myotis myotis and the house mouse, Mus musculus with Toll Like Receptors (TLRs) ligands, lipopolysaccharides, LPS and polyinosinic-polycytidylic acid, Poly(I:C). Macrophages from both species presented a high level of mRNA induction of inferon ß ( INF-ß) , tumor necrosis factor ( TNF) and interleukin-1ß (Il- ß) . However, in bat macrophages, this antiviral, proinflammatory response was balanced by a sustained high-level transcription of the anti-inflammatory cytokine Il-10, which was not observed in mouse, potentially resulting from adaptive regulation in bats. Additionally, phylogenomic selection tests across the basal divergences in mammals (n = 39) uncovered bat-specific adaptations in six genes involved in antiviral and proinflammatory signalling. Based on this pilot study, we put forward a hypothesis that bats may have evolved unique anti-inflamma tory responses to neutralize proinflammatory stimuli resulting from flight. This in turn may drive their extraordinary longevity and viral tolerance by limiting inflammation driven ageing and infection-induced immunopathology. Further data from other individuals and bat species are required to advance this intriguing hypothesis. European Research Council University College Dublin http://www.bioone.org/loi/acta

Despite the long persistence of many mutualisms, it is largely unknown which mechanisms stabilize these interactions. This is especially true if only one mutualism partner can choose alternative partners while the other cannot, resulting in a power asymmetry. According to biological market theory the choosing partner should prefer the more dependent partner if the latter offers commodities of higher quality than its competitors. We tested this prediction using Bornean carnivorous pitcher plants (Nepenthes hemsleyana) that strongly rely on faecal nitrogen of bats (Kerivoula hardwickii) which roost inside the pitchers. The bats also roost in furled leaves of various plants. Surprisingly, during field observations the bats did not always choose N. hemsleyana pitchers despite their superior quality but were generally faithful either to pitchers or to furled leaves. In behavioural experiments 21% of the leaf-roosting bats switched to pitchers, while the majority of these bats and all pitcher-roosting individuals were faithful to the roost type in which we had found them. Genetic differentiation cannot explain this faithfulness, which likely results from different roosting traditions. Such traditions could have stabilizing or destabilizing effects on various mutualisms and should be investigated in more detail.

Sharing resources with conspecifics or heterospecifics can involve costs like increased competition or higher pathogen infection risks as well as benefits such as information on the location, quality and availability of resources. Depending on the consequences of sharing resources, the responses of individuals towards resources used by conspecifics and heterospecifics can range from ignoring them through avoidance to attraction. Within bats it is well known that colony members share information about day roosts and roost switching is often coordinated within the colony. However, little is known about roosting interactions between distinct colonies of conspecifics or heterospecifics. In this study, we investigated roosting interactions between five co-occurring bat colonies that belong to three forest-living species (Myotis bechsteinii, Myotis nattereri, Plecotus auritus). Occupied roosts were continuously monitored with an automatic RFID system over three maternity seasons. Furthermore, we used simulations to test whether colonies preferentially occupied recently used roosts of other colonies. We found no evidence that the roosting behaviour of the M. bechsteinii colony was influenced by the co-occurring heterospecific colonies. In contrast, P. auritus and M. nattereri frequently explored roosts of conspecific and heterospecific colonies, respectively. Nevertheless, with largely separated roosting ranges, the three P. auritus colonies avoided occupying roosts that had been inhabited by conspecific colonies. In contrast, M. nattereri specifically occupied recent roosts of all three P. auritus colonies. Our results give evidence that co-occurring colonies of conspecific and heterospecific bats can influence each other's roost usage. Our findings have implications for both our understanding of inter- and intraspecific resource sharing among distinct social groups and the management of forest-living bats that are of conservation concern.

Bats account for ~20% of all extant mammal species and are considered exceptional given their extraordinary adaptations, including biosonar, true flight, extreme longevity, and unparalleled immune systems. To understand these adaptations, we generated reference-quality genomes of six species representing the key divergent lineages. We assembled these genomes with a novel pipeline incorporating state-of-the-art long-read and long-range sequencing and assembly techniques. The genomes were annotated using a maximal evidence approach, de novo predictions, protein/mRNA alignments, Iso-seq long read and RNA-seq short read transcripts, and gene projections from our new TOGA pipeline, retrieving virtually all (>99%) mammalian BUSCO genes. Phylogenetic analyses of 12,931 protein coding-genes and 10,857 conserved non-coding elements identified across 48 mammalian genomes helped to resolve bats’ closest extant relatives within Laurasiatheria, supporting a basal position for bats within Scrotifera. Genome-wide screens along the bat ancestral branch revealed (a) selection on hearing-involved genes (e.g LRP2, SERPINB6, TJP2), which suggest that laryngeal echolocation is a shared ancestral trait of bats; (b) selection (e.g INAVA, CXCL13, NPSR1) and loss of immunity related proteins (e.g. LRRC70, IL36G), including pro-inflammatory NF-kB signalling; and (c) expansion of the APOBEC family, associated with restricting viral infection, transposon activity and interferon signalling. We also identified unique integrated viruses, indicating that bats have a history of tolerating viral pathogens, lethal to other mammal species. Non-coding RNA analyses identified variant and novel microRNAs, revealing regulatory relationships that may contribute to phenotypic diversity in bats. Together, our reference-quality genomes, high-quality annotations, genome-wide screens and in-vitro tests revealed previously unknown genomic adaptations in bats that may explain their extraordinary traits.

Miniopterus schreibersii is a cave-dwelling bat species with a wide distribution in the western Palearctic spanning southern and central Europe, North Africa, Anatolia, the Middle East, and the Caucasus. We investigated the social structure and its effects on the genetic makeup of this species, using 10 nuclear microsatellite markers and a partial fragment of the mitochondrial cytochrome b gene. Samples were examined from the species' entire circum-Mediterranean range. Local structuring that was previously detected among populations of M. schreibersii using mitochondrial markers was not observed for microsatellite markers, indicating male-biased dispersal for the species. Some support was found for postglacial expansions in Europe, with Anatolia potentially acting as the primary refugium during the Last Glacial Maximum (LGM). However, support for this hypothesis is not as strong as that previously detected using mitochondrial DNA markers. This is likely due to the diminishing effect of male-mediated dispersal, replenishing the nuclear diversity faster than the mitochondrial diversity in regions that are relatively far from the glacial refugia.

Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols1 to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus). We integrated gene projections from our ‘Tool to infer Orthologs from Genome Alignments’ (TOGA) software with de novo and homology gene predictions as well as short- and long-read transcriptomics to generate highly complete gene annotations. To resolve the phylogenetic position of bats within Laurasiatheria, we applied several phylogenetic methods to comprehensive sets of orthologous protein-coding and noncoding regions of the genome, and identified a basal origin for bats within Scrotifera. Our genome-wide screens revealed positive selection on hearing-related genes in the ancestral branch of bats, which is indicative of laryngeal echolocation being an ancestral trait in this clade. We found selection and loss of immunity-related genes (including pro-inflammatory NF-κB regulators) and expansions of anti-viral APOBEC3 genes, which highlights molecular mechanisms that may contribute to the exceptional immunity of bats. Genomic integrations of diverse viruses provide a genomic record of historical tolerance to viral infection in bats. Finally, we found and experimentally validated bat-specific variation in microRNAs, which may regulate bat-specific gene-expression programs. Our reference-quality bat genomes provide the resources required to uncover and validate the genomic basis of adaptations of bats, and stimulate new avenues of research that are directly relevant to human health and disease1., Reference-quality genomes for six bat species shed light on the phylogenetic position of Chiroptera, and provide insight into the genetic underpinnings of the unique adaptations of this clade.

Understanding the relationship between habitat quality and population dynamics is fundamental for long-term management and range predictions in ecology. However, habitat quality is generally only investigated at the individual scale, as it is the case for the lesser horseshoe bat (Rhinolophus hipposideros), a species of conservation concern. Using a statistical modelling approach and census data of 94 lesser horseshoe bat colonies located in Brittany (France), we analysed the effect of landscape composition and configuration on the demographic components of surveyed maternity colonies (i.e. colony size, fecundity and growth rate), and compared our result to those provided by individual-based studies. Our results validated that the landscape in a 500-meter buffer around colonies (core foraging area) is crucial for population size and dynamics, and confirmed the positive influence of broadleaved woodland proportion on bat colony size. We revealed a positive effect of lakeshores and riverbanks on colony size and growth rate, underlying the importance of these habitats for the long-term conservation of this non-migratory forest species. Importantly, our results refine previous knowledge concerning the threat posed by the intensification of human activities (e.g. urbanization, agriculture, habitat fragmentation), and highlight the negative effect of large and regular patches of artificial and crop lands and of open land patches shape complexity on all demographic variables investigated. While our results support the dependence of population dynamics and associated conservation management to individual behaviour and sensitivity, environmental responses differed between the population metrics investigated, showing that efficient range prediction will require to fully grasp the complexity of the interaction between landscape and the different population dynamic parameters.

The speed and dynamics of range expansions shape species distributions and community composition. Despite the critical impact of population growth rates for range expansion, they are neglected in existing empirical studies, which focus on the investigation of selected life-history traits. Here, we present an approach based on non-invasive genetic capture–mark–recapture data for the estimation of adult survival, fecundity and juvenile survival, which determine population growth. We demonstrate the reliability of our method with simulated data, and use it to investigate life-history changes associated with range expansion in 35 colonies of the bat species Rhinolophus hipposideros . Comparing the demographic parameters inferred for 19 of those colonies which belong to an expanding population with those inferred for the remaining 16 colonies from a non-expanding population reveals that range expansion is associated with higher net reproduction. Juvenile survival was the main driver of the observed reproduction increase in this long-lived bat species with low per capita annual reproductive output. The higher average growth rate in the expanding population was not associated with a trade-off between increased reproduction and survival, suggesting that the observed increase in reproduction stems from a higher resource acquisition in the expanding population. Environmental conditions in the novel habitat hence seem to have an important influence on range expansion dynamics, and warrant further investigation for the management of range expansion in both native and invasive species.

International audience; The development of transportation infrastructure has been identified as one of the main pressures on biodiversity. The effects of transport infrastructure are more documented for terrestrial mammals, birds and amphibians than for bats. To assess the impacts of roads on bat activity, we carried out full-night acoustic recordings of bat calls at 306 sampling points at different distances from a major road at three study sites in France. To assess the relationship between bat activity and the distance to the major road, we performed generalized linear mixed model analyses for thirteen different species or groups and additionally explored the non-linear effect with generalized additive mixed models. Our results showed that low-flying species are more affected than high-flying species. Indeed, we found a significant negative effect of major roads on bat activity for the 'clutter-adapted' species, Eptesicus serotinus, Myotis spp., Pipistrellus pipistrellus and Rhinolophus hipposideros. These results demonstrate that the road-effect zone of major roads extends up to five kilometres. Extrapolating those road-effects zones to the major roads in the European Union, we estimated that 35% of the European Union is potentially negatively impacted. Finally, it seems urgent to consider these road effects with the cumulative effects of other roads by improving habitat connectivity and foraging areas in land use policies. Additionally, to implement drastic conservation practices for species of conservation concern in environmental impact assessment studies, efficient mitigation and offset measures implemented should be sized proportionally to the disturbance caused.

International audience; Introduced pathogens causing emerging infectious diseases (EIDs) are serious contemporary threats to animal, plant, and ecosystem health. The invasive fungus, Pseudogymnoascus destructans, has established populations of European origin in North America, resulting in mass mortality of several hibernating bat species. Extensive monitoring for this pathogen exists in Europe and North America, but limited screening is taking place elsewhere. We report results from cave surveys on Hawai‘i Island. Substrates in 10 lava-tube caves with elevations up to 3,045 m were swabbed providing samples for screening P. destructans. Interior cave air temperatures spanned temperatures suitable for the growth and survival of P. destructans. Using quantitative PCR, all 85 samples tested were negative for the presence of P. destructans. The biology of the Hawaiian hoary bat (Lasiurus cinereus semotus) in relation to its unusual use of high elevation caves is discussed because these bats could come into contact with P. destructans should it arrive in Hawai‘i. Large numbers of cave enthusiasts visit Hawaiian caves from across the world after having been inside caves elsewhere including areas with P. destructans. Thus, resource managers in Hawai‘i and other remote areas may want to consider the potential for P. destructans to arrive unintentionally via human activities. Biosecurity measures and periodic screening for P. destructans are especially important in Hawai‘i given the presence of high elevation caves with suitable temperatures for its growth. If P. destructans was introduced to Hawaiian caves, it could affect the local fauna but also act as a source population for colonisations elsewhere.

International audience; Roads have a multitude of negative effects on wildlife, including their prominent role in habitat fragmentation. Habitat fragmentation particularly affects bats during their nightly movements between roosts and foraging areas. Bat overpasses are among the proposed improvements intended to reduce the fragmentation impact of roads, but they have rarely been tested. In this study, we performed a Before-After Control-Impact analysis (BACI) to assess the efficiency of one bat overpass on the number of bat crossings, by using an acoustic flight path reconstruction (AFPR) approach. We obtained 888 bat crossings of five taxa. Our results showed that the number of bat crossings increased significantly after the installation of the bat overpass. Finally, we demonstrated that an overpass correctly placed with respect to a narrow commuting route (a twenty-meter-wide hedgerow) could efficiently restore bat habitat connectivity.

WOS: 000375896000026, PubMed ID: 27001602, The isolation of populations in the Iberian, Italian and Balkan peninsulas during the ice ages define four main paradigms that explain much of the known distribution of intraspecific genetic diversity in Europe. In this study we investigated the phylogeography of a wide-spread bat species, the bent-winged bat, Miniopterus schreibersii around the Mediterranean basin and in the Caucasus. Environmental Niche Modeling (ENM) analysis was applied to predict both the current distribution of the species and its distribution during the last glacial maximum (LGM). The combination of genetics and ENM results suggest that the populations of M. schreibersii in Europe, the Caucasus and Anatolia went extinct during the LGM, and the refugium for the species was a relatively small area to the east of the Levantine Sea, corresponding to the Mediterranean coasts of present-day Syria, Lebanon, Israel, and northeastern and northwestern Egypt. Subsequently the species first repopulated Anatolia, diversified there, and afterwards expanded into the Caucasus, continental Europe and North Africa after the end of the LGM. The fossil record in Iberia and the ENM results indicate continuous presence of Miniopterus in this peninsula that most probably was related to the Maghrebian lineage during the LGM, which did not persist afterwards. Using our results combined with similar findings in previous studies, we propose a new paradigm explaining the general distribution of genetic diversity in Europe involving the recolonization of the continent, with the main contribution from refugial populations in Anatolia and the Middle East. The study shows how genetics and ENM approaches can complement each other in providing a more detailed picture of intraspecific evolution. (C) 2016 Elsevier Inc. All rights reserved., Research Fund of Bogazici University, Istanbul [08M104, 09S101, 11Y00P2]; Scientific and Technological Research Council of Turkey, TUBITAK [112T698]; Fundacao para a Ciencia e Tecnologia [IF/0497/2013]; IRCSET-Marie Curie International Mobility Fellowship in Science, Engineering and Technology; Ministry of Culture of the Czech Republic [DKRVO 00023272], We would like to thank Elizabeth Hemond and three anonymous reviewers for their comments on an earlier version of the manuscript. We also would like to thank Simos Demetropoulos, Petros Lymperakis, Dina Kovac, Vida Zrncic, Darija Josic, Sanja Drakulic, Tea Knapic, Yannis Kazoglou, Elena Papadatou, Xavier Gremillet, Monika Podgorelec, Peter Vallo, Thierry Disca, Vincent Prie, members of the Win-timdouine expedition (Morocco), Wassim M. Hizem, Pr. Said Nouira, and the "Direction generale des fork de Tunisie" for their assistance with the fieldwork. This study was supported by grants from the Research Fund of Bogazici University, Istanbul (08M104, 09S101 and 11Y00P2) and the Scientific and Technological Research Council of Turkey, TUBITAK (112T698) to RB, from Fundacao para a Ciencia e Tecnologia (research contract IF/0497/2013) to HR, from the IRCSET-Marie Curie International Mobility Fellowship in Science, Engineering and Technology to SJP, and from the Ministry of Culture of the Czech Republic (# DKRVO 00023272) to PB.

The New Zealand long-tailed bat (Chalinolobus tuberculatus) is an endemic species threatened with extinction. Since the arrival of humans, massive deforestation has occurred and invasive mammalian predators were introduced. As a result, C. tuberculatus’ distribution shrank dramatically and became fragmented. To aid the management of the remaining populations, two Evolutionary Significant Units (ESUs) were designated: one on each of New Zealand’s main islands. We utilised mitochondrial sequence data (cytb, 703 bp) and 10 nuclear DNA microsatellite loci to reconstruct the demographic history of this species, to characterise the level of genetic diversity in remaining populations, and to assess the current connectivity between them. Our results indicate that the North Island, with the highest genetic diversity, served as a glacial refuge, with a loss of diversity following the path recolonization to the south of the South Island. However, our data are also consistent with continued, or at least very recent, genetic exchange between colonies across the species distribution. The only exception is the Hanging Rock colony on the east coast of the South Island, which appears to be isolated. Thus, there was no support for the previously designated ESUs. Signatures of past population declines were found in three colonies, the most extreme of which was found in Hanging Rock. Consequently, we recommend that it be genetically rescued via translocation from a donor population. In general, future management priorities should treat Chalinolobus tuberculatus as a single unit, focusing on maintaining connectivity between remaining populations, together with continued roost protection and pest control.

Despite many studies illustrating the perils of utilising mitochondrial DNA in phylogenetic studies, it remains one of the most widely used genetic markers for this purpose. Over the last decade, nuclear introns have been proposed as alternative markers for phylogenetic reconstruction. However, the resolution capabilities of mtDNA and nuclear introns have rarely been quantified and compared. In the current study we generated a novel ∼5kb dataset comprising six nuclear introns and a mtDNA fragment. We assessed the relative resolution capabilities of the six intronic fragments with respect to each other, when used in various combinations together, and when compared to the traditionally used mtDNA. We focused on a major clade in the horseshoe bat family (Afro-Palaearctic clade; Rhinolophidae) as our case study. This old, widely distributed and speciose group contains a high level of conserved morphology. This morphological stasis renders the reconstruction of the phylogeny of this group with traditional morphological characters complex. We sampled multiple individuals per species to represent their geographic distributions as best as possible (122 individuals, 24 species, 68 localities). We reconstructed the species phylogeny using several complementary methods (partitioned Maximum Likelihood and Bayesian and Bayesian multispecies-coalescent) and made inferences based on consensus across these methods. We computed pairwise comparisons based on Robinson-Foulds tree distance metric between all Bayesian topologies generated (27,000) for every gene(s) and visualised the tree space using multidimensional scaling (MDS) plots. Using our supported species phylogeny we estimated the ancestral state of key traits of interest within this group, e.g. echolocation peak frequency which has been implicated in speciation. Our results revealed many potential cryptic species within this group, even in taxa where this was not suspected a priori and also found evidence for mtDNA introgression. We demonstrated that by using just two introns one can recover a better supported species tree than when using the mtDNA alone, despite the shorter overall length of the combined introns. Additionally, when combining any single intron with mtDNA, we showed that the result is highly similar to the mtDNA gene tree and far from the true species tree and therefore this approach should be avoided. We caution against the indiscriminate use of mtDNA in phylogenetic studies and advocate for pilot studies to select nuclear introns. The selection of marker type and number is a crucial step that is best based on critical examination of preliminary or previously published data. Based on our findings and previous publications, we recommend the following markers to recover phylogenetic relationships between recently diverged taxa (

The emergence of SARS-CoV and MERS-CoV, triggered the discovery of a high diversity of coronaviruses in bats. Studies from Europe have shown that coronaviruses circulate in bats in France but this reflects only a fraction of the whole diversity. In the current study the diversity of coronaviruses circulating in western Europe was extensively explored. Ten alphacoronaviruses in eleven bat species belonging to the Miniopteridae, Vespertilionidae and Rhinolophidae families and, a SARS-CoV-related Betacoronavirus in Rhinolophus ferrumequinum were identified. The diversity and prevalence of bat coronaviruses presently reported from western Europe is much higher than previously described and includes a SARS-CoV sister group. This diversity demonstrates the dynamic evolution and circulation of coronaviruses in this species. That said, the identified coronaviruses were consistently associated with a particular bat species or genus, and these relationships were maintained no matter the geographic location. The observed phylogenetic grouping of coronaviruses from the same species in Europe and Asia, emphasizes the role of host/pathogen coevolution in this group., Graphical abstract fx1, Highlights • A SARS-CoV sister clade member, Betacoronavirus EPI1, found in Western Europe. • Betacoronavirus EPI1 circulates in Rhinolophus ferrumequinum bat in Western Europe. • 9 alphacoronaviruses species found in Vespertillionidae and Miniopteridae bats. • Rhinolophus ferrumequinum hosts Betacov EPI1 and Alphacov EPI4, EPI6 and EPI7. • Alphacoronavirus EPI6 is strictly associated with Rhinolophus ferrumequinum.

Telomeres do not shorten with age in longest-lived bats., Understanding aging is a grand challenge in biology. Exceptionally long-lived animals have mechanisms that underpin extreme longevity. Telomeres are protective nucleotide repeats on chromosome tips that shorten with cell division, potentially limiting life span. Bats are the longest-lived mammals for their size, but it is unknown whether their telomeres shorten. Using >60 years of cumulative mark-recapture field data, we show that telomeres shorten with age in Rhinolophus ferrumequinum and Miniopterus schreibersii, but not in the bat genus with greatest longevity, Myotis. As in humans, telomerase is not expressed in Myotis myotis blood or fibroblasts. Selection tests on telomere maintenance genes show that ATM and SETX, which repair and prevent DNA damage, potentially mediate telomere dynamics in Myotis bats. Twenty-one telomere maintenance genes are differentially expressed in Myotis, of which 14 are enriched for DNA repair, and 5 for alternative telomere-lengthening mechanisms. We demonstrate how telomeres, telomerase, and DNA repair genes have contributed to the evolution of exceptional longevity in Myotis bats, advancing our understanding of healthy aging.

Gene flow, maintained through natal dispersal and subsequent mating events, is one of the most important processes in both ecology and population genetics. Among mammalian populations, gene flow is strongly affected by a variety of factors, including the species’ ability to disperse, and the composition of the environment which can limit dispersal. Information on dispersal patterns is thus crucial both for conservation management and for understanding the social system of a species. We used 16 polymorphic nuclear microsatellite loci in addition to mitochondrial DNA sequences (1.61 kbp) to analyse the population structure and the sex‐specific pattern of natal dispersal in the frog‐eating fringe‐lipped bat, Trachops cirrhosus, in Central Panama. Our study revealed that—unlike most of the few other investigated Neotropical bats—gene flow in this species is mostly male‐mediated. Nevertheless, distinct genetic clusters occur in both sexes. In particular, the presence of genetic differentiation in the dataset only consisting of the dispersing sex (males) indicates that gene flow is impeded within our study area. Our data are in line with the Panama Canal in connection with the widening of the Río Chagres during the canal construction acting as a recent barrier to gene flow. The sensitivity of T. cirrhosus to human‐induced habitat modifications is further indicated by an extremely low capture success in highly fragmented areas. Taken together, our genetic and capture data provide evidence for this species to be classified as less mobile and thus vulnerable to habitat change, information that is important for conservation management.

International audience; The Myotis nattereri species complex consists of an entangled group of Western Palaearctic bats characterized by fringing hairs along the rear edge of their uropatagium. Some members are relatively common while others are rare but all forms are morphologically very similar and their taxonomy is unresolved. Recent studies based on different molecular markers have shown that several major and unexpected lineages exist within this group of forest-dwelling bats. All the mitochondrial and nuclear markers tested to date have shown that these major lineages evolved as fully independent and coherent units and therefore each qualifies as distinct species. In the absence of proper morphological diagnosis, these lineages are informally referred to in the literature under different names. We explore here the external and craniodental variation of these lineages. Although all morphological measurements were overlapping between these lineages, we show that lineages can be completely discriminated in a multivariate morphometric space. Consistent with previous molecular reconstructions, these four major lineages represent two pairs of related species, each represented by a named species (Myotis nattereri s. str. and M. escalerai, respectively) and by unnamed forms (Myotis sp. A and Myotis sp. B, respectively). Herein we describe formally these two unnamed forms to clarify the taxonomy within this species complex. This new taxonomic view has important implication for the protection of these species, as three of the four taxa must now be considered as range-restricted species in need of conservation actions.

International audience; Multiple biotic and abiotic parameters influence the dynamics of individual fungal species and entire communities. Major drivers for tropical plant endophytes are undoubtedly seasonality, local habitat conditions and biogeography. However, host specialization and tissue preferences also contribute to the structuring of endophytic mycobiomes. To elucidate such specializations and preferences, we sampled two commercially important, unrelated plant species, Amorphophallus albispathus and Camellia sinensis (tea plant) simultaneously at close proximity. The mycobiomes of different tissue types were assessed with high-throughput amplicon sequencing of the internal transcribed spacer DNA region. Both plants hosted different fungal communities and varied in α-and β-diversity, despite their neighboring occurrence. However, the fungal assemblages of Amorphophallus leaflets shared taxa with the mycobiomes of tea leaves, thereby suggesting common driving forces for leaf-inhabiting fungi irrespective of host plant identity. The mycobiome composition and diversity of tea leaves was clearly driven by leaf age. We suggest that the very youngest tea leaves are colonized by stochastic processes, while mycobiomes of old leaves are rather similar as the result of progressive succession. The biodiversity of fungi associated with A. albispathus was characterized by a large number of unclassified OTUs (at genus and species level) and by tissue-specific composition.This study is the first cultivation-independent high-throughput assessment of fungal biodiversity of an Amorphophallus species, and additionally expands the knowledge base on fungi associated with tea plants.

International audience; Population sex ratios are of high ecological relevance, but are challenging to determine in species lacking conspicuous external cues indicating their sex. Acoustic sexing is an option if vocalizations differ between sexes, but is precluded by overlapping distributions of the values of male and female vocalizations in many species. A method allowing the inference of sex ratios despite such an overlap will therefore greatly increase the information extractable from acoustic data. To meet this demand, we developed a novel approach using Approximate Bayesian Computation (ABC) to infer the sex ratio of populations from acoustic data. Additionally, parameters characterizing the male and female distribution of acoustic values (mean and standard deviation) are inferred. This information is then used to probabilistically assign a sex to a single acoustic signal. We furthermore develop a simpler means of sex ratio estimation based on the exclusion of calls from the overlap zone. Applying our methods to simulated data demonstrates that sex ratio and acoustic parameter characteristics of males and females are reliably inferred by the ABC approach. Applying both the ABC and the exclusion method to empirical datasets (echolocation calls recorded in colonies of lesser horseshoe bats, Rhinolophus hipposideros) provides similar sex ratios as molecular sexing. Our methods aim to facilitate evidence-based conservation, and to benefit scientists investigating ecological or conservation questions related to sex-or group specific behaviour across a wide range of organisms emitting acoustic signals. The developed methodology is non-invasive, low-cost and time-efficient, thus allowing the study of many sites and individuals. We provide an R-script for the easy application of the method and discuss potential future extensions and fields of applications. The script can be easily adapted to account for numerous biological systems by adjusting the type and number of groups to be distinguished (e.g. age, social rank, cryptic species) and the acoustic parameters investigated.

International audience; Roads have many negative effects on wildlife, including their role in habitat fragmentation. Habitat fragmentation affects bats during their daily movements between roosts and foraging areas. As bats are protected in Europe, developers must implement specific mitigation measures that are hierarchically structured to achieve a null net impact. However, very few specific mitigation measures have been undertaken specifically for bats. Bat overpasses (e.g. gantries) are among proposed improvements intended to reduce the impact of roads, but they have rarely been tested. The effectiveness of overpasses in facilitating safe road crossing of bats is critical for justifying the implementation of this mitigation measure. We therefore assessed whether bat overpasses are effectively used by bats. We studied three bat overpasses with different designs in France. We developed an innovative method to characterize bat crossings using acoustic flight path reconstruction (AFPR). We used six pairs of stereo acoustic recorders in different habitat types that were located on both sides of the road, and operated simultaneously throughout the night. Recording data contained 57,941 bat passes and 284 bat crossings from six species of bats at the three study sites. Our results suggest that crossings are more numerous if an overpass is located where bat commuting routes have been identified by environmental impact assessment. However, we found that the proportion of bat crossings along the commuting route was the same with or without an overpass; thus highlighting that bat overpasses do not fully restore habitat connectivity. Synthesis and applications. Our study demonstrates that acoustic flight path reconstruction (AFPR) is a useful approach to obtain information on bat flight behaviour. We also emphasize the importance of field testing the effectiveness of mitigation measures, such as those intended to offset the impact of roads on biodiversity, and highlight that such measures should not be implemented based on their theoretical effectiveness alone.

A changing microbiome has been linked to biological aging in mice and humans, suggesting a possible role of gut flora in pathogenic aging phenotypes. Many bat species have exceptional longevity given their body size and some can live up to ten times longer than expected with little signs of aging. This study explores the anal microbiome of the exceptionally long-lived Myotis myotis bat, investigating bacterial composition in both adult and juvenile bats to determine if the microbiome changes with age in a wild, long-lived non-model organism, using non-lethal sampling. The anal microbiome was sequenced using metabarcoding in more than 50 individuals, finding no significant difference between the composition of juvenile and adult bats, suggesting that age-related microbial shifts previously observed in other mammals may not be present in Myotis myotis. Functional gene categories, inferred from metabarcoding data, expressed in the M. myotis microbiome were categorized identifying pathways involved in metabolism, DNA repair and oxidative phosphorylation. We highlight an abundance of ‘Proteobacteria’ relative to other mammals, with similar patterns compared to other bat microbiomes. Our results suggest that M. myotis may have a relatively stable, unchanging microbiome playing a role in their extended ‘health spans’ with the advancement of age, and suggest a potential link between microbiome and sustained, powered flight.

The discovery of new species of mammals in Europe is a rare event, but owing to recent progress in genetic methods used to survey current biodiversity, such discoveries have been accumulating during the last decades. The naming of these new taxa has important bearings in conservation, as this is the first step needed for them to be recognised by the scientific community but also legally. The naming of new animal species, however, needs to follow the strict rules of the International Code of Zoological Nomenclature in order to be available and to provide a stable basis for further taxonomic research. We report here a case where new names have been improperly proposed to designate distinctive lineages in the Natterer's bat species complex. As these new names do not follow several mandatory rules imposed by the Code, they are declared unavailable and are not to be used for further taxonomy studies.

The acquisition of tissue samples from wild populations is a constant challenge in conservation biology, especially for endangered species and protected species where nonlethal sampling is the only option. Whole blood has been suggested as a nonlethal sample type that contains a high percentage of bodywide and genomewide transcripts and therefore can be used to assess the transcriptional status of an individual, and to infer a high percentage of the genome. However, only limited quantities of blood can be nonlethally sampled from small species and it is not known if enough genetic material is contained in only a few drops of blood, which represents the upper limit of sample collection for some small species. In this study, we developed a nonlethal sampling method, the laboratory protocols and a bioinformatic pipeline to sequence and assemble the whole blood transcriptome, using Illumina RNA-Seq, from wild greater mouse-eared bats (Myotis myotis). For optimal results, both ribosomal and globin RNAs must be removed before library construction. Treatment of DNase is recommended but not required enabling the use of smaller amounts of starting RNA. A large proportion of protein-coding genes (61%) in the genome were expressed in the blood transcriptome, comparable to brain (65%), kidney (63%) and liver (58%) transcriptomes, and up to 99% of the mitogenome (excluding D-loop) was recovered in the RNA-Seq data. In conclusion, this nonlethal blood sampling method provides an opportunity for a genomewide transcriptomic study of small, endangered or critically protected species, without sacrificing any individuals.

SummaryMutualisms between plants and animals shape the world’s ecosystems [1, 2]. In such interactions, achieving contact with the partner species is imperative. Plants regularly advertise themselves with signals that specifically appeal to the partner’s perceptual preferences [3–5]. For example, many plants have acquired traits such as brightly colored, fragrant flowers that attract pollinators with visual, olfactory, or—in the case of a few bat-pollinated flowers—even acoustic stimuli in the form of echo-reflecting structures [6–9]. However, acoustic attraction in plants is rare compared to other advertisements and has never been found outside the pollination context and only in the Neotropics. We hypothesized that this phenomenon is more widespread and more diverse as plant-bat interactions also occur in the Paleotropics. In Borneo, mutualistic bats fertilize a carnivorous pitcher plant while roosting in its pitchers [10, 11]. The pitcher’s orifice features a prolonged concave structure, which we predicted to distinctively reflect the bats’ echolocation calls for a wide range of angles. This structure should facilitate the location and identification of pitchers even within highly cluttered surroundings. Pitchers lacking this structure should be less attractive for the bats. Ensonifications of the pitchers around their orifice revealed that this structure indeed acts as a multidirectional ultrasound reflector. In behavioral experiments where bats were confronted with differently modified pitchers, the reflector’s presence clearly facilitated the finding and identification of pitchers. These results suggest that plants have convergently acquired reflectors in the Paleotropics and the Neotropics to acoustically attract bats, albeit for completely different ecological reasons.