Population genetic structure of the Daubenton’s bat (<Emphasis Type="Italic">Myotis daubentonii</Emphasis>) in western Europe and the associated occurrence of rabies

The Daubenton’s bat is widespread and common in the UK and countries bordering the English Channel and North Sea. European bat lyssavirus 2 (EBLV-2), a rabies virus, has been detected in Daubenton’s bats in the UK and continental Europe. Investigating the relatedness of colonies and gene flow between these regions would allow regional estimates of the movement of Daubenton’s bats and thus the potential for disease transmission. The genetic structure of the Daubenton’s bat in western Europe was investigated by analysing variability at eight microsatellite loci. Genetic diversity was found to be high at all sites (H _E = 0.73–0.84), with little differentiation between bats sampled in the UK and continental Europe. Mantel tests indicated a significant correlation between geographic distance and pair-wise F _ST (P = 0.000), between colonies sampled in Scotland and northern England. However, this was not continuous throughout the sampled range, with evidence of panmixia within the area sampled in continental Europe. Assignment tests show no evidence that the (potential) EBLV-2 sero-positive and virus positive bats were more likely to have originated from the continental rather than UK populations. There is no sufficient significant genetic differentiation amongst most UK and continental colonies to conclude that EBLV-2 is maintained in the UK by immigration. Results show that it is likely to be maintained at a low endemic level within the UK. The relative genetic uniformity of UK and continental populations implies that there is no migration barrier to EBLV-2, between these regions.     

Spatial distribution of mitochondrial and microsatellite DNA variation in Daubenton's bat within Scotland

Daubenton's bat ( Myotis daubentonii ) is a known reservoir for European bat lyssavirus type 2 (EBLV-2). An appreciation of the potential for epidemiological spread and disease risk requires an understanding of the dispersal of the primary host, and any large-scale geographical barriers that may impede gene flow. The spatial pattern of microsatellite and mitochondrial DNA variation was examined to infer patterns of dispersal of bats among 35 populations across Scotland. DNA sequence variation at the mitochondrial control region and ND1 genes revealed two distinct phylogeographical clades, with generally nonoverlapping geographical distributions except for a small number of populations where both matrilines were found in sympatry. Such discontinuity suggests that Scotland was recolonised twice following the retreat of the Pleistocene ice sheet with little subsequent matrilineal introgression. However, eight microsatellite loci showed low levels of genetic divergence among populations, even between populations from the two distinct mitochondrial DNA clades. An overall, macrogeographical genetic isolation-by-distance pattern was observed, with high levels of gene flow among local populations. Apparently contrasting patterns of mitochondrial and microsatellite divergence at different scales could be explained by sex-specific differences in gene flow at large scales.     

Genetic connectivity among swarming sites in the wide ranging and recently declining little brown bat (Myotis lucifugus)

Characterizing movement dynamics and spatial aspects of gene flow within a species permits inference on population structuring. As patterns of structuring are products of historical and current demographics and gene flow, assessment of structure through time can yield an understanding of evolutionary dynamics acting on populations that are necessary to inform management. Recent dramatic population declines in hibernating bats in eastern North America from white‐nose syndrome have prompted the need for information on movement dynamics for multiple bat species. We characterized population genetic structure of the little brown bat, Myotis lucifugus, at swarming sites in southeastern Canada using 9 nuclear microsatellites and a 292‐bp region of the mitochondrial genome. Analyses of F_ST, Φ_ST, and Bayesian clustering (STRUCTURE) found weak levels of genetic structure among swarming sites for the nuclear and mitochondrial genome (Global F_ST = 0.001, P < 0.05, Global Φ_ST = 0.045, P < 0.01, STRUCTURE K = 1) suggesting high contemporary gene flow. Hierarchical AMOVA also suggests little structuring at a regional (provincial) level. Metrics of nuclear genetic structure were not found to differ between males and females suggesting weak asymmetries in gene flow between the sexes. However, a greater degree of mitochondrial structuring does support male‐biased dispersal long term. Demographic analyses were consistent with past population growth and suggest a population expansion occurred from approximately 1250 to 12,500 BP, following Pleistocene deglaciation in the region. Our study suggests high gene flow and thus a high degree of connectivity among bats that visit swarming sites whereby mainland areas of the region may be best considered as one large gene pool for management and conservation.     

Insights into Persistence Mechanisms of a Zoonotic Virus in Bat Colonies Using a Multispecies Metapopulation Model

Rabies is a worldwide zoonosis resulting from Lyssavirus infection. In Europe, Eptesicus serotinus is the most frequently reported bat species infected with Lyssavirus, and thus considered to be the reservoir of European bat Lyssavirus type 1 (EBLV-1). To date, the role of other bat species in EBLV-1 epidemiology and persistence remains unknown. Here, we built an EBLV-1−transmission model based on local observations of a three-cave and four-bat species (Myotis capaccinii, Myotis myotis, Miniopterus schreibersii, Rhinolophus ferrumequinum) system in the Balearic Islands, for which a 1995–2011 serological dataset indicated the continuous presence of EBLV-1. Eptesicus serotinus was never observed in the system during the 16-year follow-up and therefore was not included in the model. We used the model to explore virus persistence mechanisms and to assess the importance of each bat species in the transmission dynamics. We found that EBLV-1 could not be sustained if transmission between M. schreibersii and other bat species was eliminated, suggesting that this species serves as a regional reservoir. Global sensitivity analysis using Sobol''s method revealed that following the rate of autumn−winter infectious contacts, M. schreibersii''s incubation- and immune-period durations, but not the infectious period length, were the most relevant factors driving virus persistence.     

Phylogeography, population dynamics, and molecular evolution of European bat lyssaviruses

European bat lyssaviruses types 1 and 2 (EBLV-1 and EBLV-2) are widespread in Europe, although little is known of their evolutionary history. We undertook a comprehensive sequence analysis to infer the selection pressures, rates of nucleotide substitution, age of genetic diversity, geographical origin, and population growth rates of EBLV-1. Our study encompassed data from 12 countries collected over a time span of 35 years and focused on the glycoprotein (G) and nucleoprotein (N) genes. We show that although the two subtypes of EBLV-1--EBLV-1a and EBLV-1b--have both grown at a low exponential rate since their introduction into Europe, they have differing population structures and dispersal patterns. Furthermore, there were strong constraints against amino acid change in both EBLV-1 and EBLV-2, as reflected in a low ratio of nonsynonymous to synonymous substitutions per site, particularly in EBLV-1b. Our inferred rate of nucleotide substitution in EBLV-1, approximately 5 x 10(-5) substitutions per site per year, was also one of the lowest recorded for RNA viruses and implied that the current genetic diversity in the virus arose 500 to 750 years ago. We propose that the slow evolution of EBLVs reflects their distinctive epidemiology in bats, where they occupy a relatively stable fitness peak.     

High genetic diversity and low population structure in red foxes (Vulpes vulpes) from Croatia

The red fox (Vulpes vulpes) is a highly adaptable omnivorous mammal distributed across all continents on the northern hemisphere. Although the red fox is present throughout Europe, where it plays an important ecological and socio-economic role not only as a game species but also as a rabies reservoir, few studies have examined its population-level mitochondrial DNA variability. In this study, 27 mitochondrial DNA control region haplotypes were identified in 229 red fox samples taken from four regions in Croatia. Haplotype diversity of Croatian red foxes (0.901) was found to be among the highest of all European red fox populations studied to date. Genetic differentiation among regions was quite low, and statistically significant estimates of differentiation were obtained only when comparing the population from the peninsular region of Istria with the three continental populations. It seems that landscape barriers like rivers and small mountains do not restrict gene flow among foxes in the continental part of Croatia, while the combination of a narrow land bridge and altitudes exceeding 1000 m limit fox migration between Istria and the rest of the continent. Better understanding of small-scale population structure will require analysis of highly variable nuclear markers like microsatellites.     

Population structure of a widespread bat (Tadarida brasiliensis) in an island system

Dispersal is a driving factor in the creation and maintenance of biodiversity, yet little is known about the effects of habitat variation and geography on dispersal and population connectivity in most mammalian groups. Bats of the family Molossidae are fast‐flying mammals thought to have potentially high dispersal ability, and recent studies have indicated gene flow across hundreds of kilometers in continental North American populations of the Brazilian free‐tailed bat, Tadarida brasiliensis. We examined the population genetics, phylogeography, and morphology of this species in Florida and across islands of The Bahamas, which are part of an island archipelago in the West Indies. Previous studies indicate that bats in the family Phyllostomidae, which are possibly less mobile than members of the family Molossidae, exhibit population structuring across The Bahamas. We hypothesized that T. brasiliensis would show high population connectivity throughout the islands and that T. brasiliensis would show higher connectivity than two species of phyllostomid bats that have been previously examined in The Bahamas. Contrary to our predictions, T. brasiliensis shows high population structure between two groups of islands in The Bahamas, similar to the structure exhibited by one species of phyllostomid bat. Phylogenetic and morphological analyses suggest that this structure may be the result of ancient divergence between two populations of T. brasiliensis that subsequently came into contact in The Bahamas. Our findings additionally suggest that there may be cryptic species within T. brasiliensis in The Bahamas and the West Indies more broadly.     

Genetic structure and diversity of a rare woodland bat, <Emphasis Type="Italic">Myotis bechsteinii</Emphasis>: comparison of continental Europe and Britain

The Bechstein’s bat (Myotis bechsteinii) is a rare sedentary bat considered to be highly reliant on the presence of ancient woodland. Understanding the genetic connectivity and population structure of such elusive mammals is important for assessing their conservation status. In this study, we report the genetic diversity and structure of M. bechsteinii across Britain and Europe. Assessments were made using 14 microsatellite markers and a 747 bp region of the mitochondrial cytochrome b gene. Nuclear DNA (microsatellites) showed high levels of genetic diversity and little inbreeding across the species range, though genetic diversity was slightly lower in Britain than in mainland Europe. Bayesian and spatial PCA analysis showed a clear separation between the British and European sites. Within Europe, the Italian population south of the Alps was isolated from the other sites. In Britain, there was genetic structuring between the northern and southern part of the geographical range. Despite there being little genetic divergence in mitochondrial DNA (mtDNA) sequences throughout most of Europe, the mtDNA patterns in Britain confirmed this separation of northern and southern populations. Such genetic structuring within Britain—in the absence of any obvious physical barriers—suggests that other factors such as land-use may limit gene-flow.     

New insights on postglacial colonization in western Europe: the phylogeography of the Leisler's bat (Nyctalus leisleri)

Despite recent advances in the understanding of the interplay between a dynamic physical environment and phylogeography in Europe, the origins of contemporary Irish biota remain uncertain. Current thinking is that Ireland was colonized post-glacially from southern European refugia, following the end of the last glacial maximum (LGM), some 20 000 years BP. The Leisler''s bat (Nyctalus leisleri), one of the few native Irish mammal species, is widely distributed throughout Europe but, with the exception of Ireland, is generally rare and considered vulnerable. We investigate the origins and phylogeographic relationships of Irish populations in relation to those across Europe, including the closely related species N. azoreum. We use a combination of approaches, including mitochondrial and nuclear DNA markers, in addition to approximate Bayesian computation and palaeo-climatic species distribution modelling. Molecular analyses revealed two distinct and diverse European mitochondrial DNA lineages, which probably diverged in separate glacial refugia. A western lineage, restricted to Ireland, Britain and the Azores, comprises Irish and British N. leisleri and N. azoreum specimens; an eastern lineage is distributed throughout mainland Europe. Palaeo-climatic projections indicate suitable habitats during the LGM, including known glacial refugia, in addition to potential novel cryptic refugia along the western fringe of Europe. These results may be applicable to populations of many species.     

Italy as a major Ice Age refuge area for the bat Myotis myotis (Chiroptera: Vespertilionidae) in Europe

The distribution of biota from the temperate regions changed considerably during the climatic fluctuations of the Quaternary. This is especially true for many bat species that depend on warm roosts to install their nursery colonies. Surveys of genetic variation among European bats have shown that the southern peninsulas (Iberia and the Balkans) harbour endemic diversity, but to date, no such surveys have been conducted in the third potential glacial refuge area, the Apennine peninsula. We report here the phylogeographical analysis of 115 greater mouse-eared bats ( Myotis myotis ) sampled throughout Italy, and show that 15 of the 18 different haplotypes found in the mitochondrial control region of these bats were unique to the Apennine peninsula. Colonies within this region also showed substantial genetic structure at both mitochondrial ( Φ_ST  = 0.47, P  < 0.001) and nuclear markers ( F _ST = 0.039, P  < 0.001). Based on a comprehensive survey of 575 bats from Europe, these genetic markers further indicate that central Italian populations of M. myotis are more closely related to Greek samples from across the Adriatic Sea, than to other Italian bats. Mouse-eared bat populations from the Apennine peninsula thus represent a complex mixture of several endemic lineages, which evolved in situ , with others that colonized this region more recently along an Adriatic route. Our broad survey also confirms that the Alps represent a relatively impermeable barrier to gene flow for Apennine lineages, even for vagile animals such as bats. These results underline the conservation value of bats from this region and the need to include the Apennine peninsula in phylogeographical surveys in order to provide a more accurate view of the evolution of bats in Europe.     

Bat rabies surveillance in France: first report of unusual mortality among serotine bats

Background

Rabies is a fatal viral encephalitic disease that is caused by lyssaviruses which can affect all mammals, including human and bats. In Europe, bat rabies cases are attributed to five different lyssavirus species, the majority of rabid bats being attributed to European bat 1 lyssavirus (EBLV-1), circulating mainly in serotine bats (Eptesicus serotinus). In France, rabies in bats is under surveillance since 1989, with 77 positive cases reported between 1989 and 2016.

Case presentation

In the frame of the bat rabies surveillance, an unusual mortality of serotine bats was reported in 2009 in a village in North-East France. Six juvenile bats from an E. serotinus maternity colony counting ~200 individuals were found to be infected with EBLV-1. The active surveillance of the colony by capture sessions of bats from July to September 2009 showed a high detection rate of neutralising EBLV-1 antibodies (≈ 50%) in the colony. Moreover, one out of 111 animals tested was found to shed viable virus in saliva, while lyssavirus RNA was detected by RT-PCR for five individuals.

Conclusion

This study demonstrated that the lyssavirus infection in the serotine maternity colony was followed by a high rate of bat rabies immunity after circulation of the virus in the colony. The ratio of seropositive bats is probably indicative of an efficient virus transmission coupled to a rapid circulation of EBLV-1 in the colony.

     

Genetic structure of winter populations of the endangered Indiana bat (<Emphasis Type="Italic">Myotis sodalis</Emphasis>) prior to the white nose syndrome epidemic: implications for the risk of disease spread

The spread of white nose syndrome raises serious concerns about the long-term viability of affected bat species. Here we examine the geographic distribution of genetic variation, levels of population connectivity that may influence the spatial spread of WNS, and the likelihood that recent population declines in regions affected by WNS have led to the loss of unique genetic variation for the endangered Indiana bat (Myotis sodalis). We amplified a fragment of the mitochondrial control region for 375 individuals and genotyped 445 individuals at 10 microsatellite loci from 18 sampling sites distributed across the majority of the species’ range. Analysis of mitochondrial DNA indicated the presence of at least five distinct matrilineal clusters, with the most pronounced differences between northeastern sites and those in the rest of the range. The majority of individuals in the Ozark-Central, Midwest, and Appalachian recovery units fell into a single cluster. Significant differentiation also was observed between one Appalachian and one Midwestern site and the majority of other sites. However, using nuclear microsatellites we observed the absence of differentiation and widespread gene flow among all hibernacula, suggesting the occurrence of extensive gene flow through dispersal and mating. The absence of genetically distinct populations within the range of Indiana bats indicates a lack of barriers to WNS transmission, and it is unlikely that significant portions of the hibernating population of Indiana bats will remain disease free into the future. Further, while matrilineal gene flow was restricted among some sites and regions, we found no genetic evidence to support the division of Indiana bats into separate recovery units.     

Genetic diversity and population structure of wolverine (Gulo gulo) populations at the southern edge of their current distribution in North America with implications for genetic viability

The current range of wolverines (Gulo gulo) within the lower 48 states includes small, remnant populations in Idaho, Washington, Wyoming and Montana. The size and trend of each of these populations and connectivity to adjacent populations in the contiguous United States and Canada are poorly understood. In this study, levels of genetic diversity and population genetic structure were examined in three states (Idaho, Wyoming, and Montana) and two Canadian provinces (Alberta and British Columbia) using both mitochondrial (mtDNA) and nuclear microsatellite DNA. Restricted levels of gene flow were detected among these populations with mitochondrial and nuclear DNA and our observations suggest a pattern of male-mediated gene flow. Populations in the United States appear to be receiving migrants from Canada, however, substantial genetic differentiation suggests that gene flow may not be high enough to prevent genetic drift. Our analyses suggest that at least 400 breeding pairs or 1–2 effective migrants per generation would be needed to ensure genetic viability in the long-term for each of the populations in the United States. Significant matrilineal structuring and restricted female gene flow indicates that demographic viability will depend upon the movement of female wolverines into new territories. Results from this study provide guidelines for conservation and management and indicate the need for more ecological data.     

Mitochondrial DNA variation and population structure of the island endemic Azorean bat (Nyctalus azoreum)

The Azorean bat Nyctalus azoreum is the only endemic mammal native to the remote archipelago of the Azores. It evolved from a continental ancestor related to the Leisler's bat Nyctalus leisleri and is considered threatened because of its restricted and highly fragmented distribution. We studied the genetic variability in 159 individuals from 14 colonies sampled throughout the archipelago. Sequences of the D-loop region revealed moderate but highly structured genetic variability. Half of the 15 distinct haplotypes were restricted to a single island, but the most common was found throughout the archipelago, suggesting a single colonization event followed by limited interisland female gene flow. All N. azoreum haplotypes were closely related and formed a star-like structure typical of expanded populations. The inferred age of demographic expansions was consistent with the arrival of founder animals during the Holocene, well before the first humans inhabited the Azores. Comparisons with a population of N. leisleri from continental Portugal confirmed not only that all N. azoreum lineages were unique to the archipelago, but also that the current levels of genetic diversity were surprisingly high for an insular species. Our data imply that the Azorean bat has a high conservation value. We argue that geographical patterns of genetic structuring indicate the existence of two management units.     

Temporal dynamics of European bat Lyssavirus type 1 and survival of Myotis myotis bats in natural colonies

Many emerging RNA viruses of public health concern have recently been detected in bats. However, the dynamics of these viruses in natural bat colonies is presently unknown. Consequently, prediction of the spread of these viruses and the establishment of appropriate control measures are hindered by a lack of information. To this aim, we collected epidemiological, virological and ecological data during a twelve-year longitudinal study in two colonies of insectivorous bats (Myotis myotis) located in Spain and infected by the most common bat lyssavirus found in Europe, the European bat lyssavirus subtype 1 (EBLV-1). This active survey demonstrates that cyclic lyssavirus infections occurred with periodic oscillations in the number of susceptible, immune and infected bats. Persistence of immunity for more than one year was detected in some individuals. These data were further used to feed models to analyze the temporal dynamics of EBLV-1 and the survival rate of bats. According to these models, the infection is characterized by a predicted low basic reproductive rate (R(0) = 1.706) and a short infectious period (D = 5.1 days). In contrast to observations in most non-flying animals infected with rabies, the survival model shows no variation in mortality after EBLV-1 infection of M. myotis. These findings have considerable public health implications in terms of management of colonies where lyssavirus-positive bats have been recorded and confirm the potential risk of rabies transmission to humans. A greater understanding of the dynamics of lyssavirus in bat colonies also provides a model to study how bats contribute to the maintenance and transmission of other viruses of public health concern.     

Phylogeography of the Japanese greater horseshoe bat Rhinolophus nippon (Mammalia: Chiroptera) in Northeast Asia: New insight into the monophyly of the Japanese populations

The Japanese greater horseshoe bat (Rhinolophus nippon) is distributed widely in East Asia. Within the species, R. nippon in Northeast Asia is regarded as the lineage that diverged most recently. However, the monophyly of the Japanese populations is unclear due to insufficient data about phylogenetic relationship of the western Japanese populations. To test the monophyly of the Japanese populations of R. nippon, we sampled R. nippon from western Japan and performed a phylogeographic analysis based on mitochondrial DNA cytochrome b and the D‐loop. The Northeast Asian lineage consisted of three main clades in eastern Japan (clade I), western Japan (clade II), and the continent as well as the Kumamoto population in westernmost Japan (clade III). The results of this study do not support the monophyly of the Japanese population. The findings suggest the “reverse colonization” of R. nippon from the Japanese Archipelago to the Eurasian continent, and provide important insight into the role of the island system in creation and supply of diversity to the continent.     

Enhanced Passive Bat Rabies Surveillance in Indigenous Bat Species from Germany - A Retrospective Study

In Germany, rabies in bats is a notifiable zoonotic disease, which is caused by European bat lyssaviruses type 1 and 2 (EBLV-1 and 2), and the recently discovered new lyssavirus species Bokeloh bat lyssavirus (BBLV). As the understanding of bat rabies in insectivorous bat species is limited, in addition to routine bat rabies diagnosis, an enhanced passive surveillance study, i.e. the retrospective investigation of dead bats that had not been tested for rabies, was initiated in 1998 to study the distribution, abundance and epidemiology of lyssavirus infections in bats from Germany. A total number of 5478 individuals representing 21 bat species within two families were included in this study. The Noctule bat (Nyctalus noctula) and the Common pipistrelle (Pipistrellus pipistrellus) represented the most specimens submitted. Of all investigated bats, 1.17% tested positive for lyssaviruses using the fluorescent antibody test (FAT). The vast majority of positive cases was identified as EBLV-1, predominately associated with the Serotine bat (Eptesicus serotinus). However, rabies cases in other species, i.e. Nathusius'' pipistrelle bat (Pipistrellus nathusii), P. pipistrellus and Brown long-eared bat (Plecotus auritus) were also characterized as EBLV-1. In contrast, EBLV-2 was isolated from three Daubenton''s bats (Myotis daubentonii). These three cases contribute significantly to the understanding of EBLV-2 infections in Germany as only one case had been reported prior to this study. This enhanced passive surveillance indicated that besides known reservoir species, further bat species are affected by lyssavirus infections. Given the increasing diversity of lyssaviruses and bats as reservoir host species worldwide, lyssavirus positive specimens, i.e. both bat and virus need to be confirmed by molecular techniques.     

A tale of two genomes: contrasting patterns of phylogeographic structure in a widely distributed bat

One of the most widely distributed bats in the New World, the big brown bat (Eptesicus fuscus) exhibits well-documented geographic variation in morphology and life history traits, suggesting the potential for significant phylogeographic structure as well as adaptive differentiation among populations. In a pattern broadly consistent with morphologically defined subspecies, we found deeply divergent mitochondrial lineages restricted to different geographic regions. In contrast, sequence data from two nuclear loci suggest a general lack of regional genetic structure except for peripheral populations in the Caribbean and Mexico/South America. Coalescent analyses suggest that the striking difference in population structure between genomes cannot be attributed solely to different rates of lineage sorting, but is likely due to male-mediated gene flow homogenizing nuclear genetic diversity across most of the continental range. Despite this ongoing gene flow, selection has apparently been effective in producing and maintaining adaptive differentiation among populations, while strong female site fidelity, maintained over the course of millions of years, has produced remarkably deep divergence among geographically isolated matrilines. Our results highlight the importance of evaluating multiple genetic markers for a more complete understanding of population structure and history.     

Genetic and morphometric diversity of the goldcrest (Regulus regulus) populations in the Azores

We studied the genetic diversity and phylogeography of the goldcrest Regulus regulus from the archipelago of the Azores (North Atlantic Ocean) based on sequences of two mitochondrial genes (cytochrome b and the NADH dehydrogenase subunit 2) and one nuclear gene in the Z-chromosome (intron 9 of the aconitase 1) from 69 individuals, and 41 birds from the Canary Islands and continental Europe for outgroup comparison. To understand the level of concordance between the genetic data and possible morphometric variability, 197 adult living birds from the seven Azorean islands where the species breeds were analysed in terms of eight morphometric characters. Our results are in accordance with previous studies, indicating a recent expansion of goldcrests throughout the archipelago and a low divergence in relation to continental Europe. Within the Azores, there is evidence of historical and/or recent gene flow among the island's populations, revealing a lack of current genetic structure within the archipelago. Only goldcrests from Flores Island seem to be genetically distinct and showed significantly larger body mass and tarsus length than birds on the other islands.