Cryptic hybridization between the ancient lineages of Natterer's bat (Myotis nattereri).

Studying hybrid zones that form between morphologically cryptic taxa offers valuable insights into the mechanisms of cryptic speciation and the evolution of reproductive barriers. Although hybrid zones have long been the focus of evolutionary studies, the awareness of cryptic hybrid zones increased recently due to rapidly growing evidence of biological diversity lacking obvious phenotypic differentiation. The characterization of cryptic hybrid zones with genome-wide analysis is in its early stages and offers new perspectives for studying population admixture and thus the impact of gene flow. In this study, we investigate the population genomics of the Myotis nattereri complex in one of its secondary contact zones, where a putative hybrid zone is formed between two of its cryptic lineages. By utilizing a whole-genome shotgun sequencing approach, we aim to characterize this cryptic hybrid zone in detail. Demographic analysis suggests that the cryptic lineages diverged during the Pliocene, c. 3.6 million years ago. Despite this ancient separation, the populations in the contact zone exhibit mitochondrial introgression and a considerable amount of mixing in nuclear genomes. The genomic structure of the populations corresponds to geographic locations and the genomic admixture changes along a geographic gradient. These findings suggest that there is no effective hybridization barrier between both lineages, nevertheless, their population structure is shaped by dispersal barriers. Our findings highlight how such deeply diverged cryptic lineages can still readily hybridize in secondary contact.

Cryptic species Hydatigera kamiyai and other taeniid metacestodes in the populations of small mammals in Serbia.

BACKGROUND: Hydatigera (Cestoda: Taeniidae) is a recently resurrected genus with the description of a new species, Hydatigera kamiyai, a cryptic entity within the Hydatigera taeniaeformis species complex. Rodents are intermediate hosts and correct taxonomic identification of H. taeniaeformis sensu lato (s.l.) species is difficult without the use of molecular methods. The aim of this study was to identify and explore the genetic diversity of Hydatigera and other taeniid species. METHODS: Ten different small mammals species (856 individuals) (Rattus rattus, three Apodemus, three Arvicolinae and three Soricidae species) were examined from 2013 to 2023. Captured animals were visually examined for cysts and visible lesions. Two markers were used for amplification and sequencing: cox1 and 12S rDNA. RESULTS: Molecular analysis of cysts and visible lesions revealed four taeniid species: Hydatigera kamiyai, H. taeniaeformis sensu stricto (s.s.), Taenia martis and T. crassiceps. Hydatigera kamiyai was found in Apodemus flavicollis, A. agrarius, Microtus arvalis and Crocidrua leucodon, while H. taeniaeformis s.s. is registered in R. rattus. Hydatigera kamiyai cox1 sequences clustered with European populations and showed at least 25 nucleotid differences compared to Asian, African, Australian and one of our isolates of H. taeniaeformis s.s acquired from a rat, followed by large sequence distances (9.4% to 12.9%), indicating clear molecular distinction of two species. CONCLUSIONS: This is one of the few mitochondrial gene-based studies performed after the description of cryptic entities within the Hydatigera taeniaeformis s.l. complex and represents a valuable contribution to understanding of genetic diversity, host suitability and geographic distribution of these tapeworm species. Also, our study provides an important basis of molecular data from this part of Europe for further studies. We emphasize the importance of additional studies of intermediate hosts, especially rats from Europe and Apodemus spp. and voles from Asia and Africa.

Non-invasive investigation of Polychromophilus parasite infections in bat populations in Serbia using bat flies.

BACKGROUND: Haemosporidian parasites of the genus Polychromophilus infect bats worldwide. They are vectored by obligate ectoparasitic bat flies of the family Nycteribiidae. Despite their global distribution, only five Polychromophilus morphospecies have been described to date. The two predominant species, Polychromophilus melanipherus and Polychromophilus murinus, are broadly distributed and mainly infect miniopterid and vespertilionid bats, respectively. In areas where species from different bat families aggregate together, the infection dynamics and ability of either Polychromophilus species to infect other host families is poorly characterized. METHODS: We collected 215 bat flies from two bat species, Miniopterus schreibersii and Rhinolophus ferrumequinum, which sometimes form mixed clusters in Serbia. Miniopterus schreibersii is known to be frequently infected with P. melanipherus, whereas R. ferrumequinum has been observed to be incidentally infected with both Polychromophilus species. All flies were screened for Polychromophilus infections using a PCR targeting the haemosporidian cytb gene. Positive samples were subsequently sequenced for 579 bp of cytochrome b (cytb) and 945 bp of cytochrome oxidase subunit 1 (cox1). RESULTS: Polychromophilus melanipherus DNA was detected at six out of nine sampling locations and in all three examined bat fly species collected from M. schreibersii (Nycteribia schmidlii, n = 21; Penicillidia conspicua, n = 8; Penicillidia dufourii, n = 3). Four and five haplotypes were found for cytb and cox1, respectively. Evidence for multiple Polychromophilus haplotypes was found in 15 individual flies. These results point to a high diversity of P. melanipherus parasites in Miniopterus hosts and efficient transmission throughout the study area. A single Phthiridium biarticulatum bat fly collected from R. ferrumequinum screened positive for P. melanipherus, but only yielded a partial cox1 sequence fragment. Nevertheless, this result suggests that secondary hosts (both bat and fly species) are regularly confronted with this parasite. CONCLUSIONS: The results of this study provide new insights into the prevalence and distribution of Polychromophilus parasites in European bats and their nycteribiid vectors. The use of bat flies for the non-invasive investigation of Polychromophilus infections in bat populations has proven to be efficient and thus represents an alternative for large-scale studies of infections in bat populations without the need to invasively collect blood from bats.

Newspaper Coverage and Framing of Bats, and Their Impact on Readership Engagement.

The media is a valuable pathway for transforming people's attitudes towards conservation issues. Understanding how bats are framed in the media is hence essential for bat conservation, particularly considering the recent fearmongering and misinformation about the risks posed by bats. We reviewed bat-related articles published online no later than 2019 (before the recent COVID19 pandemic), in 15 newspapers from the five most populated countries in Western Europe. We examined the extent to which bats were presented as a threat to human health and the assumed general attitudes towards bats that such articles supported. We quantified press coverage on bat conservation values and evaluated whether the country and political stance had any information bias. Finally, we assessed their terminology and, for the first time, modelled the active response from the readership based on the number of online comments. Out of 1095 articles sampled, 17% focused on bats and diseases, 53% on a range of ecological and conservation topics, and 30% only mention bats anecdotally. While most of the ecological articles did not present bats as a threat (97%), most articles focusing on diseases did so (80%). Ecosystem services were mentioned on very few occasions in both types (< 30%), and references to the economic benefits they provide were meagre (< 4%). Disease-related concepts were recurrent, and those articles that framed bats as a threat were the ones that garnered the highest number of comments. Therefore, we encourage the media to play a more proactive role in reinforcing positive conservation messaging by presenting the myriad ways in which bats contribute to safeguarding human well-being and ecosystem functioning.

Sharing roosts but not ectoparasites: high host-specificity in bat flies and wing mites of Miniopterus schreibersii and Rhinolophus ferrumequinum (Mammalia: Chiroptera).

Schreiber's bent-winged bat Miniopterus schreibersii and the greater horseshoe bat Rhinolophus ferrumequinum are widespread and common cavernicolous species across southern Europe that host numerous specialized ectoparasite species. The objective of this study was to characterize the species assemblage, genetic diversity, and host specificity of bat flies (Nycteribiidae, Diptera) and wing mites (Spinturnicidae, Acari) found on these bat hosts in Serbia and Bosnia and Herzegovina. Notably, while bat flies lay puparia on the cave walls and can thus be transmitted indirectly, wing mites require direct body contact for transmission. Morphological identification and sequencing of a 710-bp fragment of cytochrome oxidase I gene of 207 bat flies yielded 4 species, 3 on M. schreibersii and 1 on R. ferrumequinum. Sequencing of a 460-bp small subunit ribosomal RNA fragment, in all 190 collected wing mites revealed 2 species, 1 per host. In no case was a parasite associated with 1 host found on the other host. Species and genetic diversity of flies were higher in M. schreibersii, likely reflecting their host's larger colony sizes and migratory potential. Mite species of both hosts showed similarly low diversity, likely due to their faster life history and lower winter survival. Our findings highlight a remarkably high host-specificity and segregation of ectoparasite species despite direct contact among their hosts in the roost, suggesting a defined host preference in the investigated ectoparasite species. Furthermore, the differences in ectoparasite genetic diversity exemplify the interplay between host and parasite life histories in shaping parasite population genetic structure.

Host-Parasite Relationship-Nematode Communities in Populations of Small Mammals.

Nematode burdens and variation in morphological characteristics were assessed in eighty-eight animals from three host species (Apodemus sylvaticus, Apodemus flavicollis, and Myodes glareolus) from eight localities in Serbia. In total, 15 species of nematodes were identified, and the overall mean parasite species richness (IndPSR) was 1.61 per animal (1.98 in A. flavicollis, 1.43 in M. glareolus, and 0.83 in A. sylvaticus). Furthermore, the studied host species significantly differed in individual parasite load (IndPL) and in the following morphological characters: spleen mass, body condition index (BCI), and body mass. We aimed to analyze the relationship between the burden of intestinal nematodes, on one hand, and the body conditions of the host and its capability to develop immune defends on the other. Spleen mass was considered as a measure of immune response. In all host species, larger animals with a better condition (higher BCI) were infected with more parasites species (IndPSR), while parasite load was not related to BCI. Only in A. flavicollis were males significantly larger, but females of the same sizes were infected with more parasite species. This female-biased parasitism is contrary to the theoretical expectation that males should be more parasitized, being larger, more active, with a wider home range. Although the spleen size was significantly correlated with body condition and body mass, IndPSR was not related to spleen mass in any studied species, but in M. galareolus, we found that a smaller spleen was related to higher infection intensity (IndPL).

The new highest number of B chromosomes (Bs) in Leisler's bat Nyctalusleisleri (Kuhl, 1817).

B chromosomes (Bs) are supernumerary to the standard chromosome set, from which they prevalently derive. Variation in numbers both among individuals or populations and among cells within individuals is their constant feature. Leisler's bat Nyctalusleisleri (Kuhl, 1817) is one of only four species of Chiroptera with detected Bs. Four males of N.leisleri were collected from two localities on the territory of Serbia and cytogenetically analysed. All animals had Bs with interindividual variability ranging from two to five heterochromatic micro Bs. The highest number of Bs was detected in this species. Among mammals, Rodentia and Chiroptera are orders with the largest number of species, but Bs frequently appear in rodents and rarely in chiropterans. Possible explanations for this difference are offered.

Canalization and developmental stability of the yellow-necked mouse (Apodemus flavicollis) mandible and cranium related to age and nematode parasitism.

BACKGROUND: Mammalian mandible and cranium are well-established model systems for studying canalization and developmental stability (DS) as two elements of developmental homeostasis. Nematode infections are usually acquired in early life and increase in intensity with age, while canalization and DS of rodent skulls could vary through late postnatal ontogeny. We aimed to estimate magnitudes and describe patterns of mandibular and cranial canalization and DS related to age and parasite intensity (diversity) in adult yellow-necked mice (Apodemus flavicollis). RESULTS: We found the absence of age-related changes in the levels of canalization for mandibular and cranial size and DS for mandibular size. However, individual measures of mandibular and cranial shape variance increased, while individual measures of mandibular shape fluctuating asymmetry (FA) decreased with age. We detected mandibular and cranial shape changes during postnatal ontogeny, but revealed no age-related dynamics of their covariance structure among and within individuals. Categories regarding parasitism differed in the level of canalization for cranial size and the level of DS for cranial shape. We observed differences in age-related dynamics of the level of canalization between non-parasitized and parasitized animals, as well as between yellow-necked mice parasitized by different number of nematode species. Likewise, individual measures of mandibular and cranial shape FA decreased with age for the mandible in the less parasitized category and increased for the cranium in the most parasitized category. CONCLUSIONS: Our age-related results partly agree with previous findings. However, no rodent study so far has explored age-related changes in the magnitude of FA for mandibular size or mandibular and cranial FA covariance structure. This is the first study dealing with the nematode parasitism-related canalization and DS in rodents. We showed that nematode parasitism does not affect mandibular and cranial shape variation and covariance structure among and within individuals. However, parasite intensity (diversity) is related to ontogenetic dynamics of the levels of canalization and DS. Overall, additional studies on animals from natural populations are required before drawing some general conclusions.

Hydrogen isotopes reveal evidence of migration of Miniopterus schreibersii in Europe.

BACKGROUND: The Schreiber's bat, Miniopterus schreibersii, is adapted to long-distance flight, yet long distance movements have only been recorded sporadically using capture-mark-recapture. In this study, we used the hydrogen isotopic composition of 208 wing and 335 fur specimens from across the species' European range to test the hypothesis that the species migrates over long distances. RESULTS: After obtaining the hydrogen isotopic composition (δ2H) of each sample, we performed geographic assignment tests by comparing the δ2H of samples with the δ2H of sampling sites. We found that 95 bats out of 325 showed evidence of long-distance movement, based on the analysis of either fur or wing samples. The eastern European part of the species range (Greece, Bulgaria and Serbia) had the highest numbers of bats that had moved. The assignment tests also helped identify possible migratory routes, such as movement between the Alps and the Balkans. CONCLUSIONS: This is the first continental-scale study to provide evidence of migratory behaviour of M. schreibersii throughout its European range. The work highlights the need for further investigation of this behaviour to provide appropriate conservation strategies.

DNA metabarcoding and spatial modelling link diet diversification with distribution homogeneity in European bats.

Inferences of the interactions between species' ecological niches and spatial distribution have been historically based on simple metrics such as low-resolution dietary breadth and range size, which might have impeded the identification of meaningful links between niche features and spatial patterns. We analysed the relationship between dietary niche breadth and spatial distribution features of European bats, by combining continent-wide DNA metabarcoding of faecal samples with species distribution modelling. Our results show that while range size is not correlated with dietary features of bats, the homogeneity of the spatial distribution of species exhibits a strong correlation with dietary breadth. We also found that dietary breadth is correlated with bats' hunting flexibility. However, these two patterns only stand when the phylogenetic relations between prey are accounted for when measuring dietary breadth. Our results suggest that the capacity to exploit different prey types enables species to thrive in more distinct environments and therefore exhibit more homogeneous distributions within their ranges.

The prevalence of pathogenic forms of Leptospira in natural populations of small wild mammals in Serbia.

The greatest epidemiological significance of leptospirosis in Europe comes from the fact that it is the most widespread zoonosis in the world. However, epizootiological data, especially information on maintenance hosts such as small wild mammals, are largely missing. To fill this gap in data in Serbia, we used RT-PCR for the detection of pathogenic Leptospira species and analysed 107 animals belonging to six species of small wild mammals (Apodemus agrarius, Apodemus flavicollis, Microtus arvalis, Myodes glareolus, Microtus subterraneus and Sorex araneus) collected from two localities. The animals from the first locality that was situated in a tourist area, were collected for four consecutive years (2014-2017). We found persistent incidence of infection from year to year ranging from 6.67% to 78.57%. The average frequency of infected animals was 33.3% with the highest frequency in 2014, the year characterised by a very high number of flooding days. All animals proved to be infected with pathogenic Leptospira species that were collected from the second locality situated in an agricultural area in a single year, 2014. The findings show a variable but constant presence of pathogenic Leptospira species in populations of small wild mammals in the studied areas, which indicates the need for constant monitoring.

Population genetic structure of the Mediterranean horseshoe bat Rhinolophus euryale in the central Balkans.

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

What is driving range expansion in a common bat? Hints from thermoregulation and habitat selection.

Human-induced alterations of ecosystems and environmental conditions often lead to changes in the geographical range of plants and animals. While modelling exercises may contribute to understanding such dynamics at large spatial scales, they rarely offer insights into the mechanisms that prompt the process at a local scale. Savi's pipistrelle (Hypsugo savii) is a vespertilionid bat widespread throughout the Mediterranean region. The species' recent range expansion towards northeastern Europe is thought to be induced by urbanization, yet no study actually tested this hypothesis, and climate change is a potential alternative driver. In this radio-telemetry study, set in the Vesuvius National Park (Campania region, Southern Italy) we provide insights into the species' thermal physiology and foraging ecology and investigate their relationships with potential large-scale responses to climate, and land use changes. Specifically, we test whether H. savii i) exploits urbanisation by selecting urban areas for roosting and foraging, and ii) tolerates heatwaves (a proxy for thermophily) through a plastic use of thermoregulation. Tolerance to heatwaves would be consistent with the observation that the species' geographic range is not shifting but expanding northwards. Tracked bats roosted mainly in buildings but avoided urban habitats while foraging, actively selecting non-intensive farmland and natural wooded areas. Hypsugo H. savii showed tolerance to heat, reaching the highest body temperature ever recorded for a free-ranging bat (46.5 °C), and performing long periods of overheating. We conclude that H. savii is not a strictly synurbic species because it exploits urban areas mainly for roosting, and avoids them for foraging: this questions the role of synurbization as a range expansion driver. On the other hand, the species' extreme heat tolerance and plastic thermoregulatory behaviour represent winning traits to cope with heatwaves typical of climate change-related weather fluctuations.

Agriculture shapes the trophic niche of a bat preying on multiple pest arthropods across Europe: Evidence from DNA metabarcoding.

The interaction between agricultural production and wildlife can shape, and even condition, the functioning of both systems. In this study, we i) explored the degree to which a widespread European bat, namely the common bent-wing bat Miniopterus schreibersii, consumes crop-damaging insects at a continental scale, and ii) tested whether its dietary niche is shaped by the extension and type of agricultural fields. We employed a dual-primer DNA metabarcoding approach to characterize arthropod 16S and COI DNA sequences within bat faecal pellets collected across 16 Southern European localities, to first characterize the bat species' dietary niche, second measure the incidence of agricultural pests across their ranges and third assess whether geographical dietary variation responds to climatic, landscape diversity, agriculture type and vegetation productivity factors. We detected 12 arthropod orders, among which lepidopterans were predominant. We identified >200 species, 44 of which are known to cause agricultural damage. Pest species were detected at all but one sampling site and in 94% of the analysed samples. Furthermore, the dietary diversity of M. schreibersii exhibited a negative linear relation with the area of intensive agricultural fields, thus suggesting crops restrict the dietary niche of bats to prey taxa associated with agricultural production within their foraging range. Overall, our results imply that M. schreibersii might be a valuable asset for biological pest suppression in a variety of agricultural productions and highlight the dynamic interplay between wildlife and agricultural systems.

Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

Sociality influences thermoregulation and roost switching in a forest bat using ephemeral roosts.

In summer, many temperate bat species use daytime torpor, but breeding females do so less to avoid interferences with reproduction. In forest-roosting bats, deep tree cavities buffer roost microclimate from abrupt temperature oscillations and facilitate thermoregulation. Forest bats also switch roosts frequently, so thermally suitable cavities may be limiting. We tested how barbastelle bats (Barbastella barbastellus), often roosting beneath flaking bark in snags, may thermoregulate successfully despite the unstable microclimate of their preferred cavities. We assessed thermoregulation patterns of bats roosting in trees in a beech forest of central Italy. Although all bats used torpor, females were more often normothermic. Cavities were poorly insulated, but social thermoregulation probably overcomes this problem. A model incorporating the presence of roost mates and group size explained thermoregulation patterns better than others based, respectively, on the location and structural characteristics of tree roosts and cavities, weather, or sex, reproductive or body condition. Homeothermy was recorded for all subjects, including nonreproductive females: This probably ensures availability of a warm roosting environment for nonvolant juveniles. Homeothermy may also represent a lifesaver for bats roosting beneath loose bark, very exposed to predators, because homeothermic bats may react quickly in case of emergency. We also found that barbastelle bats maintain group cohesion when switching roosts: This may accelerate roost occupation at the end of a night, quickly securing a stable microclimate in the newly occupied cavity. Overall, both thermoregulation and roost-switching patterns were satisfactorily explained as adaptations to a structurally and thermally labile roosting environment.

Candidate new rotavirus species in Schreiber's bats, Serbia.

The genus Rotavirus comprises eight species designated A to H and one tentative species, Rotavirus I. In a virus metagenomic analysis of Schreiber's bats sampled in Serbia in 2014 we obtained sequences likely representing novel rotavirus species. Whole genome sequencing and phylogenetic analysis classified the representative strain into a tentative tenth rotavirus species, we provisionally called Rotavirus J. The novel virus shared a maximum of 50% amino acid sequence identity within the VP6 gene to currently known members of the genus. This study extends our understanding of the genetic diversity of rotaviruses in bats.

Possible influence of B chromosomes on genes included in immune response and parasite burden in Apodemus flavicollis.

Genetic background underlying wild populations immune response to different parasites is still not well understood. We studied immune response to multiple infections and to competition between different parasite species at different developmental stages in population of yellow-necked mouse, Apodemus flavicollis. Quantitative real-time PCR was used to investigate associations of MHC II-DRB, IL-10 and Tgf-ß genes expressions with presence of intestinal parasites at different developmental stages. Furthermore, we were interested whether the host related characteristics (sex, age, body condition, presence of B chromosomes or expression of other genes) or characteristics of present parasites (number of adult parasites of each identified species, egg count of each parasite genus, total number of nematode individuals) affect differential expression of the studied genes. A significant invert association between the expression of MHC II-DRB and Tgf-ß gene was found, which together with absence of IL-10 association confirmed modified Th2 as the main type of immune response to nematode infections. Effect of recorded parasites and parasite life-cycle stage on expression levels of MHC II-DRB gene was detected only through interactions with host-related characteristics such as sex, age, and the presence of B chromosomes. The presence of B chromosomes is associated with lower expression level of Tgf-ß gene. Although the influence of host genetic background on parasite infection has already been well documented, this is the first study in mammals that gave presence of B chromosomes on immune response full consideration.