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Understanding the development of functional attributes of host-associated microbial communities is essential for developing novel microbe-based solutions for sustainable animal production. We applied multi-omics to 388 broiler chicken caecal samples to characterise and model the functional dynamics of 822 bacterial strains. Although microbial community diversity metrics increased with chicken age as expected, the overall metabolic capacity and activity of the microbiota exhibited an unexpected decrease. This drop occurred due to the spread of non-culturable clades with small genomes and low metabolic capacities, including RF39, RF32, and UBA1242. The intensity of this decrease was associated with animal growth, whereby chickens with higher abundances of low-capacity bacteria exhibited higher body weights. This previously unreported link between metabolic capacity of microbes and animal body weight suggests a relevant role of non-culturable bacteria with reduced-genomes for host biology, and opens new avenues in the search for microbe-based solutions to improve sustainability of animal production.

Freshwater habitats are under increasing pressure from numerous anthropogenic forces, including the introduction of alien species capable of altering ecosystems and threatening native species. Although alien species themself are likely to experience loss of genetic diversity when colonising novel environments, some manage to become invasive, suggesting that other factors might facilitate their adaptive capacity. Using a hologenomic approach, we elucidate population genomic trends, the gut microbiota composition and genome-environment-microbiota interaction in the endemic and endangered Spanish toothcarp (Aphanius iberus) and the highly invasive Eastern mosquitofish (Gambusia holbrooki). We found clear genetic signatures of captive breeding in the populations of A. iberus, while G. holbrooki are characterised by an overall low level of heterozygosity and likely signs of multiple introductions. Gut microbial communities of the two species differed significantly across locations, but no sign of increased microbial plasticity was detected in G. holbrooki. However, we report that the genetic profile of each fish was able to explain a considerable part of the microbiota variation measured across individuals. Using shotgun metagenomics, we observed an overall high functional capacity of the microbiota in both species, but we identified no significant differences in the functional capacity between them. The role of the gut microbiota in invasive species and conservation warrant further research using direct comparisons or controlled mesocosm setups, but based on the results of the current study, the gut microbiota of invasive species

The gut microbiomes that associate with animals can represent labile units of cooperating and competing microbes. This lability, sometimes referred to as metagenomic plasticity, has been posited to have an important role as an additional axis of hosts’ phenotypic plasticity. However, whether and how metagenomic plasticity varies across hosts with different ecological and evolutionary features remains unclear. To address this, we utilised faecal-derived genome-resolved metagenomics and compared how the taxonomic, phylogenetic and functional microbial dynamics varied across a series of disturbances in two mammal species; namely, the insectivorous-specialist, Crocidura russula (N = 29) and the omnivorous-generalist Apodemus sylvaticus (N = 22). Although faecal microbial diversity of both species remained stable, compositional dynamics differed significantly. C. russula exhibited substantially higher variability and directionality of microbial responses, with higher predictability associated with each disturbance, compared to A. sylvaticus. Predictions of functional traits using joint-species distribution modelling supported these observations. C. russula showed strong functional response to perturbations, with marked directional variation of various metabolic functions. In contrast, the significantly higher functional diversity and redundancy of the A. sylvaticus microbiome likely buffered its functional response to perturbations, which remained more constant across time. Our results indicate that the intrinsic properties (e.g., diversity, redundancy) of gut microbiomes associated with animals with different biological attributes shape the taxonomic, phylogenetic, and functional response to environmental stressors. This level of plasticity might affect the capacity of animal hosts to acclimate and adapt to changing environments.

Inferring the functional capabilities of bacteria from metagenome-assembled genomes (MAGs) is becoming a central process in microbiology. Here we show that the completeness of genomes has a significant impact on the recovered functional signal, spanning all domains of metabolic functions. We identify factors that affect this relationship between genome completeness and function fullness, and provide baseline knowledge to guide efforts to correct for this overlooked bias in metagenomic functional inference.

Background Understanding the complex structures and interactions of the bacterial communities inhabiting the upper (URT) and lower (LRT) respiratory tract of pigs is at an early stage. The objective of this study was to characterize the bacterial topography of three URT (nostrils, choana, and tonsils) and LRT (proximal trachea, left caudal lobe and secondary bronchi) sites in pigs. Thirty-six post-mortem samples from six pigs were analysed by 16S rRNA gene quantification and sequencing, and the microbiota in nostrils and trachea was additionally profiled by shotgun sequencing. Results The bacterial composition obtained by the two methods was congruent, although metagenomics recovered only a fraction of the diversity (32 metagenome-assembled genomes) due to the high proportion (85–98%) of host DNA. The highest abundance of 16S rRNA copies was observed in nostrils, followed by tonsils, trachea, bronchi, choana and lung. Bacterial richness and diversity were lower in the LRT compared to the URT. Overall, Firmicutes and Proteobacteria were identified as predominant taxa in all sample types. Glasserella (15.7%), Streptococcus (14.6%) and Clostridium (10.1%) were the most abundant genera but differences in microbiota composition were observed between the two tracts as well as between sampling sites within the same tract. Clear-cut differences were observed between nasal and tonsillar microbiomes (R-values 0.85–0.93), whereas bacterial communities inhabiting trachea and lung were similar (R-values 0.10–0.17). Moraxella and Streptococcus were more common in bronchial mucosal scraping than in lavage, probably because of mucosal adherence. The bacterial microbiota of the choana was less diverse than that of the nostrils and similar to the tracheal microbiota (R-value 0.24), suggesting that the posterior nasal cavity serves as the primary source of bacteria for the LRT. Conclusion We provide new knowledge on microbiota composition and species abundance in distinct ecological niches of the pig respiratory tract. Our results shed light on the distribution of opportunistic bacterial pathogens across the respiratory tract and support the hypothesis that bacteria present in the lungs originate from the posterior nasal cavity. Due to the high abundance of host DNA, high-resolution profiling of the pig respiratory microbiota by shotgun sequencing requires methods for host DNA depletion.

Research on animal-microbiota interactions has become a central topic in biological sciences because of its relevance to basic eco-evolutionary processes and applied questions in agriculture and health. However, animal hosts and their associated microbial communities are still seldom studied in a systemic fashion. Hologenomics, the integrated study of the genetic features of a eukaryotic host alongside that of its associated microbes, is becoming a feasible - yet still underexploited - approach that overcomes this limitation. Acknowledging the biological and genetic properties of both hosts and microbes, along with the advantages and disadvantages of implemented techniques, is essential for designing optimal studies that enable some of the major questions in biology to be addressed.

Over 40% of the Earth’s surface has been converted to agricultural use and agroecosystems have become important habitats for wildlife. In arid regions, intensive agriculture creates artificial oasis-like habitats due to their high irrigation inputs. Date production is one of the primary agricultural practices in the deserts of the Middle East and North Africa. Insectivorous bats are known to use agricultural areas, but the role of date plantations as their foraging habits and the importance of insectivorous bats as date bio-pest control agents are still unknown. We assessed the role of date plantations as foraging habitats for local desert bat species by acoustically recording bat activity in conventional and organic date plantations in the southern Arava Valley, Israel. In addition, we captured bats in the plantations and collected feces for DNA metabarcoding analysis to investigate the presence of pest species in their diets. We found that 12 out of the 16 known species of bats in this region frequently used both conventional and organic date plantations as foraging habitats. Species richness was highest in the organic plantation with complex ground vegetation cover. Foraging activity was not affected by plantation type or management. However, bat species richness and activity increased in all plantations during summer date harvesting. Molecular analysis confirmed that bats feed on a variety of important date pests, but the particular pests consumed and the extent of consumption varied among bat species. Our results highlight a win–win situation, whereby date plantations are an important foraging habitat for desert bats, while bats provide bio-pest control services that benefit the date plantations. Therefore, date farmers interested in bio-pest control should manage their plantations to support local desert bat populations.

Research on animal-microbiota interactions has become a central topic in biological sciences because of its relevance to basic eco-evolutionary processes and applied questions in agriculture and health. However, animal hosts and their associated microbial communities are still seldom studied in a systemic fashion. Hologenomics, the integrated study of the genetic features of a eukaryotic host alongside that of its associated microbes, is becoming a feasible - yet still underexploited - approach that overcomes this limitation. Acknowledging the biological and genetic properties of both hosts and microbes, along with the advantages and disadvantages of implemented techniques, is essential for designing optimal studies that enable some of the major questions in biology to be addressed.

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.

With the advent of DNA sequencing-based techniques, the way we detect and measure biodiversity is undergoing a radical shift. There is also an increasing awareness of the need to employ intuitively meaningful diversity measures based on unified statistical frameworks, so that different results can be easily interpreted and compared. This article aimed to serve as a guide to implementing biodiversity assessment using the general statistical framework developed around Hill numbers into the analysis of systems characterized using DNA sequencing-based techniques (e.g., diet, microbiomes and ecosystem biodiversity). Specifically, we discuss (a) the DNA-based approaches for defining the types upon which diversity is measured, (b) how to weight the importance of each type, (c) the differences between abundance-based versus incidence-based approaches, (d) the implementation of phylogenetic information into diversity measurement, (e) hierarchical diversity partitioning, (f) dissimilarity and overlap measurement and (g) how to deal with zero-inflated, insufficient and biased data. All steps are reproduced with real data to also provide step-by-step bash and R scripts to enable straightforward implementation of the explained procedures.

Metagenomic data sets of host-associated microbial communities often contain host DNA that is usually discarded because the amount of data is too low for accurate host genetic analyses. However, if a reference panel is available, genotype imputation can be employed to reconstruct host genotypes and maximise the use of such a priori useless data. We tested the performance of a two-step strategy to input genotypes from four types of reference panels, comprised of deeply sequenced chickens to low-depth host genome (~2x coverage) data recovered from metagenomic samples of chicken intestines. The target chicken population was formed by two broiler breeds and the four reference panels employed were (i) an internal panel formed by population-specific individuals, (ii) an external panel created from a public database, (iii) a combined panel of the previous two, and (iv) a diverse panel including more distant populations. Imputation accuracy was high for all tested panels (concordance >0.90), although samples with coverage under 0.28x consistently showed the lowest accuracies. The best imputation performance was achieved by the combined panel due to the high number of imputed variants, including low-frequency ones. However, common population genetics parameters measured to characterise the chicken populations, including observed heterozygosity, nucleotide diversity, pairwise distances and kinship, were only minimally affected by panel choice, with all four panels yielding suitable results for host population characterization and comparison. Likewise, genome scans between the two studied broiler breeds using imputed data with each panel consistently identified the same sweep regions. In conclusion, we show that the applied imputation strategy enables leveraging insofar discarded host DNA to get insights into the genetic structure of host populations, and in doing so, facilitate the implementation of hologenomic approaches that jointly analyse host genomic and microbial metagenomic data.Author summaryWe introduce and assess a methodological approach that enables recovering animal genomes from complex mixtures of metagenomic data, and thus expand the portfolio of analyses that can be conducted from samples such as faeces and gut contents. Metagenomic data sets of host-associated microbial communities often contain DNA of the host organism. The principal drawback to use this data for host genomic characterisation is the low percentage and quality of the host DNA. In order to leverage this data, we propose a two-step imputation method, to recover high-density of variants. We tested the pipeline in a chicken metagenomic dataset, validated imputation accuracy statistics, and studied common population genetics parameters to assess how these are affected by genotype imputation and choice of reference panel. Being able to analyse both domains from the same data set could considerably reduce sampling and laboratory efforts and resources, thereby yielding more sustainable practices for future studies that embrace a hologenomic approach that jointly analyses animal genomic and microbial metagenomic features.

The application of high-throughput sequencing-based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic composition and diversity. Here, we review these studies in the light of high-throughput sequencing-based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing-based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimize the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing-based diet analyses. In doing so, we aim to aid end-users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing-based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity.

There is an open debate on whether and how captivity alters the gut microbiota of vertebrates, due to the contrasting results reported in different taxa and the absence of systematic multi-species analyses. We performed a meta-analysis of gut microbiota profiles of 322 captive and 322 wild specimens from 24 vertebrate species, including fish, reptiles, amphibians and mammals. We found no evidence that captivity either systematically depletes or increases their gut microbiota. In 88% of the species analysed, although captivity entailed a loss of a fraction of the diversity found in the wild, this was compensated through recruitment of a proportionally similar amount of new taxa only found in captivity. We show such compositional changes can impact evolutionary and ecological inferences that rely on hierarchical clustering-based comparative analyses of gut microbial communities across species.

Dog evolution has long been debated, particularly with regards to the origin of domestication and its effects on the genome, but little is known about the role of the gut microbiome during the recent canine evolutionary history. We sequenced the metagenome of 13 canine coprolites dated ca. 3,600-3,450 y ago from the Bronze Age archaeological site of Solarolo (Italy), which housed a complex farming community. The microbiome structure of Solarolo dogs revealed continuity with that of modern dogs, but it also shared some features with the wild wolf microbiome, as a kind of transitional state between them. The dietary niche, as also inferred from the microbiome composition, was omnivorous, with evidence of consumption of starchy agricultural foods. Interestingly, the microbiome from Solarolo dogs was particularly enriched in sequences encoding alpha-amylases and complemented a low copy number of the host amylase (AMY2B) gene. These findings suggest that Neolithic dogs could have responded to the transition to a starch-rich diet by expanding microbial functionalities devoted to starch catabolism. Such a microbiome adaptive mechanism would have compensated for delayed host response to the new primarily abundant plant food resources, available from companionship with Bronze Age agriculturalists.

The microbial gut communities of fish are receiving increased attention for their relevance, among others, in a growing aquaculture industry. The members of these communities are often split into resident (long-term colonisers specialised to grow in and adhere to the mucus lining of the gut) and transient (short-term colonisers originated from food items and the surrounding water) microorganisms. Separating these two communities in small fish are impeded by the small size and fragility of the gastrointestinal tract. With the aim of testing whether it is possible to recover two distinct communities in small species of fish using a simple sampling technique, we used 16S amplicon sequencing of paired intestinal wall and digesta samples from three small Cyprinodontiformes fish. We examined the diversity and compositional variation of the two recovered communities, and we used joint species distribution modelling to identify microbes that are most likely to be a part of the resident community. For all three species we found that the diversity of intestinal wall samples was significantly lower compared to digesta samples and that the community composition between sample types was significantly different. Across the three species we found seven unique families of bacteria to be significantly enriched in samples from the intestinal wall, encompassing most of the 89 ASVs enriched in intestinal wall samples. We conclude that it is possible to characterise two different microbial communities and identify potentially resident microbes through separately analysing samples from the intestinal wall and digesta from small species of fish. We encourage researchers to be aware that different sampling procedures for gut microbiome characterization will capture different parts of the microbiome and that this should be taken into consideration when reporting results from such studies on small species of fish.

Natural predators of arthropods provide an important ecosystem service by preying on crop-damaging species. However, measuring the positive impact of natural pest predators is still challenging. We present a framework to estimate the pest-consumption pressure by natural predators across space and project it onto geographical maps. We use DNA metabarcoding and species distribution modelling to integrate predator density estimations with their energetic requirements and direct pest predation, which yields a comprehensive measure of pest-consumption pressure per time-frame and area. We showcase it on a European bat assemblage, and show that bats consume a variety of pests whose predation pressure varies throughout space. We also report that the impact of different predators depends on spatial scale, and that pest predation pressure is negatively correlated with agricultural intensity. Our framework can be used to estimate broad-scale effects of natural predators on pest arthropods as well as to design research and management strategies.

Summary From ontogenesis to homeostasis, the phenotypes of complex organisms are shaped by the bidirectional interactions between the host organisms and their associated microbiota. Current technology can reveal many such interactions by combining multi-omic data from both hosts and microbes. However, exploring the full extent of these interactions requires careful consideration of study design for the efficient generation and optimal integration of data derived from (meta)genomics, (meta)transcriptomics, (meta)proteomics, and (meta)metabolomics. In this perspective, we introduce the holo-omic approach that incorporates multi-omic data from both host and microbiota domains to untangle the interplay between the two. We revisit the recent literature on biomolecular host-microbe interactions and discuss the implementation and current limitations of the holo-omic approach. We anticipate that the application of this approach can contribute to opening new research avenues and discoveries in biomedicine, biotechnology, agricultural and aquacultural sciences, nature conservation, as well as basic ecological and evolutionary research., Graphical Abstract, Microbiome; Evolutionary Biology

Plecotus macrobullaris (Kuzjakin, 1965) is a medium-sized chiropteran commonly called the alpine long-eared bat, and 1 of the 11 Palearctic species recognized in the genus Plecotus. It has the characteristic long ears of plecotine bats, and externally its most conspicuous diagnostic characteristic is the inverted triangular-shaped pad most individuals exhibit in the chin. It is distributed across the mountain environments in the southwestern and central regions of the Palearctic. It is the most abundant bat species in European alpine environments, although it can be also found at lower elevations. It roosts on both natural (rock crevices, caves, and scree deposits) and artificial shelters (buildings), and it mainly feeds on moths. P. macrobullaris is currently listed as “Least Concern” by the International Union for Conservation of Nature and Natural Resources, although actual population sizes and demographic trends are unknown.

1.Metabarcoding of environmental samples has many challenges and limitations that require carefully considered laboratory and analysis pipelines to ensure reliable results. We explore how decisions regarding study design, laboratory work and bioinformatic processing affect the final results, and provide guidelines for reliable study of environmental samples. 2.We evaluate the performance of four primer sets targeting COI and 16S regions characterising arthropod diversity in bat faecal samples, and investigate how metabarcoding results are affected by parameters including: i) number of PCR replicates per sample, ii) sequencing depth, iii) PCR replicate processing strategy (i.e. either additively, by combining the sequences obtained from the PCR replicates, or restrictively, by only retaining sequences that occur in multiple PCR replicates for each sample), iv) minimum copy number for sequences to be retained, v) chimera removal, and vi) similarity thresholds for OTU clustering. Lastly, we measure within- and between-taxa dissimilarities when using sequences from public databases to determine the most appropriate thresholds for OTU clustering and taxonomy assignment. 3.Our results show that the use of multiple primer sets reduces taxonomic biases and increases taxonomic coverage. Taxonomic profiles resulting from each primer set are principally affected by how many PCR replicates are carried out per sample and how sequences are filtered across them, the sequence copy number threshold and the OTU clustering threshold. We also report considerable diversity differences between PCR replicates from each sample. Sequencing depth increases the dissimilarity between PCR replicates unless the bioinformatic strategies to remove allegedly artefactual sequences are adjusted according to the number of analysed sequences. Finally, we show that the appropriate identity thresholds for OTU clustering and taxonomy assignment differ between target markers. 4.Metabarcoding of complex environmental samples ideally requires i) investigation of whether more than one primer sets targeting the same taxonomic group is needed to offset the effect of primer biases, ii) more than one PCR replicate per sample, iii) bioinformatic processing approaches of sequences that balance diversity detection with removal of artificial sequences, and iv) empirical selection of OTU clustering and taxonomy assignment thresholds tailored to each genetic marker and the obtained taxa. This article is protected by copyright. All rights reserved.

This article is a comprehensive summary of the PhD presented by Antton Alberdi at the University of the Basque Country (UPV/EHU), on december 5th, 2014, under the supervision of Dr. Inazio Garin and Dr. Joxerra Aihartza. The thesis was awarded by the Spanish Society of Mammalogist (SECEM) in 2015, as the best thesis about Iberian mammals. DOI: 10.7325/Galemys.2016.A1

Multiple large-scale restoration strategies are emerging globally to counteract ecosystem degradation and biodiversity loss. However, restoration often remains insufficient to offset that loss. To address this challenge, we propose to focus restoration science on the long-term (centuries to millennia) re-assembly of degraded ecosystem complexity integrating interaction network and evolutionary potential approaches. This approach provides insights into eco-evolutionary feedbacks determining the structure, functioning and stability of recovering ecosystems. Eco-evolutionary feedbacks may help to understand changes in the adaptive potential after disturbance of metacommunity hub species with core structural and functional roles for their use in restoration. Those changes can be studied combining a restoration genomics approach based on whole-genome sequencing with replicated space-for-time substitutions linking changes in genetic variation to functions or traits relevant to the establishment of evolutionarily resilient communities. This approach may set the knowledge basis for future tools to accelerate the restoration of ecosystems able to adapt to ongoing global changes.

During times of rapid environmental change, survival of most vertebrate populations depends on their phenomic plasticity. Although differential gene-expression and post-transcriptional processes of the host genome receive focus as the main molecular mechanisms, growing evidence points to the gut microbiota as a key driver defining hosts' phenotypes. We propose that the plasticity of the gut microbiota might be an essential factor determining phenomic plasticity of vertebrates, and that it might play a pivotal role when vertebrates acclimate and adapt to fast environmental variation. We contemplate some key questions and suggest methodological approaches and experimental designs that can be used to evaluate whether gut microorganisms provide a boost of plasticity to vertebrates' phenomes, thereby increasing their acclimation and adaptation capacity.

Conservation efforts for endangered animals commonly focus on the protection of foraging habitats, aiming to ensure sufficient food availability. However, the diet of many species is based on animals that undergo habitat shifts across ontogenetic life stages, yielding considerable differences between the lifelong habitat requirements of both predator and prey. Consequently, prey availability may not only depend on the suitability of the foraging grounds where predator and prey coincide, but also on habitats where the ecological requirements of the non-prey stages are fulfilled. In this study we test to what extent prey of the insectivorous bat Rhinolophus euryale (Blasius 1853) originate either from the grounds where they are consumed, or in areas/habitats outside the bat's foraging sites. We analyzed the diet of R. euryale , by identifying its prey to the species level using DNA metabarcoding, and by searching for its prey's larval feeding requirements in the literature. We found that the larvae of the moth prey grow both inside and outside the grounds where they are hunted by the bats once the moths reach their adult stage. The importance of prey that originated from outside the bat's foraging grounds varied considerably across seasons. As a result, R. euryale does not only rely on the landscape elements where it hunts, but also on other source areas/habitats that supply it with food. This study shows that conservation measures that aim to address the foraging requirements of predatory species should not be limited to merely protecting their foraging grounds, but should also take into account the ecological requirements of their prey throughout their life stages.

Recent exploration into the interactions and relationship between hosts and their microbiota has revealed a connection between many aspects of the host’s biology, health and associated microorganisms. Whereas amplicon sequencing has traditionally been used to characterise the microbiome, the increasing number of published population genomics datasets offer an underexploited opportunity to study microbial profiles from the host shotgun sequencing data. Here, we use sequence data originally generated from killer whale Orcinus orca skin biopsies for population genomics, to characterise the skin microbiome and investigate how host social and geographic factors influence the microbial community composition. Having identified 845 microbial taxa from 2.4 million reads that did not map to the killer whale reference genome, we found that both ecotypic and geographic factors influence community composition of killer whale skin microbiomes. Furthermore, we uncovered key taxa that drive the microbiome community composition and showed that they are embedded in unique networks, one of which is tentatively linked to diatom presence and poor skin condition. Community composition differed between Antarctic killer whales with and without diatom coverage, suggesting that the previously reported episodic migrations of Antarctic killer whales to warmer waters associated with skin turnover may control the effects of potentially pathogenic bacteria such as Tenacibaculum dicentrarchi. Our work demonstrates the feasibility of microbiome studies from host shotgun sequencing data and highlights the importance of metagenomics in understanding the relationship between host and microbial ecology.

Climate change is a major threat to global biodiversity that will produce a range of new selection pressures. Understanding species responses to climate change requires an interdisciplinary perspective, combining ecological, molecular and environmental approaches. We propose an applied integrated framework to identify populations under threat from climate change based on their extent of exposure, inherent sensitivity due to adaptive and neutral genetic variation and range shift potential. We consider intraspecific vulnerability and population-level responses, an important but often neglected conservation research priority. We demonstrate how this framework can be applied to vertebrates with limited dispersal abilities using empirical data for the bat Plecotus austriacus. We use ecological niche modelling and environmental dissimilarity analysis to locate areas at high risk of exposure to future changes. Combining outlier tests with genotype–environment association analysis, we identify potential climate-adaptive SNPs in our genomic data set and differences in the frequency of adaptive and neutral variation between populations. We assess landscape connectivity and show that changing environmental suitability may limit the future movement of individuals, thus affecting both the ability of populations to shift their distribution to climatically suitable areas and the probability of evolutionary rescue through the spread of adaptive genetic variation among populations. Therefore, a better understanding of movement ecology and landscape connectivity is needed for predicting population persistence under climate change. Our study highlights the importance of incorporating genomic data to determine sensitivity, adaptive potential and range shift potential, instead of relying solely on exposure to guide species vulnerability assessments and conservation planning.

Niche partitioning through foraging is a mechanism likely involved in facilitating the coexistence of ecologically similar and co-occurring animal species by separating their use of resources. Yet, this mechanism is not well understood in flying insectivorous animals. This is particularly true of bats, where many ecologically similar or cryptic species coexist. The detailed analysis of the foraging niche in sympatric, cryptic sibling species provides an excellent framework to disentangle the role of specific niche factors likely involved in facilitating coexistence. We used DNA metabarcoding to determine the prey species consumed by a population of sympatric sibling Rhinolophus euryale and Rhinolophus mehelyi whose use of habitat in both sympatric and allopatric ranges has been well established through radio tracking. Although some subtle dietary differences exist in prey species composition, the diet of both bats greatly overlapped (Ojk = 0.83) due to the consumption of the same common and widespread moths. Those dietary differences we did detect might be related to divergences in prey availabilities among foraging habitats, which prior radio tracking on the same population showed are differentially used and selected when both species co-occur. This minor dietary segregation in sympatry may be the result of foraging on the same prey-types and could contribute to reduce potential competitive interactions (e.g., for prey, acoustic space). Our results highlight the need to evaluate the spatial niche dimension in mediating the co-occurrence of similar insectivorous bat species, a niche factor likely involved in processes of bat species coexistence.

Aim: We used an integrative approach to reconstruct the evolutionary history of the alpine long-eared bat, Plecotus macrobullaris, to test whether the variable effects of Pleistocene climatic oscillations across geographical regions led to contrasting population-level demographic histories within a single species. Location: The Western Palaearctic. Methods: We sequenced the complete mitochondrial genomes of 57 individuals from across the distribution of the species. The analysis integrated ecological niche modelling (ENM), approximate Bayesian computation (ABC), measures of genetic diversity and Bayesian phylogenetic methods. Results: We identified two deep lineages: a western lineage, restricted to the Pyrenees and the Alps, and an eastern lineage, which expanded across the mountain ranges east of the Dinarides (Croatia). ENM projections of past conditions predicted that climatic suitability was reduced during cold stages in the areas inhabited by the western lineage, while the opposite trend was observed in the mountains inhabited by the eastern lineage. The palaeodemographic scenario that best fitted our data is consistent with the western lineage population size having shrunk repeatedly because of the extensive glaciation events that occurred in the Alps and Pyrenees during the Pleistocene. In contrast, the eastern lineage maintained a constant population size as is consistent with more limited glaciation in the mountains of south-eastern Europe and the Middle East. Main conclusions: This study shows that the demographic response of populations to Pleistocene climatic oscillations depended on their geographical location, offering an example of population-level variations in the effects and long-term consequences of climate change.

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., We thank the staff at the Danish National High-Throughput DNASequencing Centre for generating the sequencing data. Furthermore,we thank Kristine Bohmann and the three anonymous reviewers foredits and comments on the manuscript. AA was supported by The Danish Council for Independent Research (5051-00033), and OAwas supported by the Carlsberg Foundation’s Distinguished Postdoc-toral Fellowship (CF15-0619). SG was supported by a Marie Skło-dowska-Curie Individual Fellowship (655732)

Littleis known about the alpinebat community, but recent studies suggest that the alpine long-eared bat, Plecotus macrobullaris, commonly forages in alpine habitats, although most of its known roosting records are from locations situated below the treeline. Aiming to contribute to resolving this seemingly contradictory pattern of ecological preferences of P. macrobullaris, we carried out a radio-tracking study to (1) identify its roosts and unveil its roosting habitat preferences, (2) determine whether bats found foraging in alpine habitats do actually roost and breed in such high-mountain environ- ments, and (3) test if any elevation-related sexual segregation occurs. We captured 117 alpine long-eared bats and radio- tracked 37 individuals to 54 roosts located at elevations be- tween 1,450 and 2,430 m, 46 of them above the treeline. Bats used rock crevices (30 roosts), scree deposits (21) and build- ings (3) for roosting, and most lactating and pregnant females relied on crevices. Bats selected areas with high meadow availability near the roost, while avoiding densely forested areas. Foraging areas and roosting sites were located at the same elevation, indicating that alpine long-eared bats use alpine areas for both roosting and foraging in the Pyrenees. Breeding females roosted at lower elevations than nulliparous females and males, though they remained above the treeline. Although being considerably different to the ecological pref- erencesdescribedsofar intheAlps,theroostingbehaviour we observed was consistent with some ecological traits, namely foragingand trophic behaviour, ofP. macrobullaris, as well as its distribution pattern linked to mountain regions.

Populations of Myotis bechsteinii in Sierra de Cazorla, Segura y Las Villas Natural Park (southwestern Spain) survive in a large, old-growth coniferous woodland, which is in contrast with known ecological preferences of the species. We tracked ten lactating females and studied patterns of habitat selection by Bechstein's bat ( Myotis bechsteinii ) at two spatial scales: macrohabitat and microhabitat, regarding forest essence (coniferous vs. deciduous). The tracked individuals always foraged within forested areas and did not use areas devoid of trees. At the macrohabitat level, no positive selection of deciduous stands was apparent, suggesting selection studies of coarse resolution may not be able to capture subtle selection patterns. At the microhabitat level Myotis bechsteinii selected deciduous patches within the coniferous matrix, therefore, our results corroborate the perception of this species as dependent of services provided by deciduous woodlands. Larger foraging home ranges and commuting distances as compared with other Mediterranean localities suggest that our studied population inhabits a marginal or suboptimal environment in terms of habitat quality. We argue that this population of Bechstein's bat has survived as a relict one probably as a consequence of fragmentation and transformation of deciduous forest ecosystems in the Mediterranean range.

The mountain long-eared bat, Plecotus macrobullaris, is a recently described species, and characterisation of its geographic and elevational distribution is still in progress. Captures in various environments led to a controversial ecological perception, with P. macrobullaris initially defined as an alpine species but subsequently found in the Mediterranean seashore and other lowland areas. Sampling efforts hitherto were uneven; this bat has been studied more thoroughly in Western Europe than in the eastern portion of its range, and in lowlands more than highlands or alpine environments. For greater insight into its distribution pattern, we conducted a field survey in several mountain areas of its known range, using a novel mist-netting technique (described herein) that has proven very useful for targeting and capturing low-flying open-space bats in alpine environments. We also gathered all available distribution data on this species from published resources and by contacting researchers, obtaining record...

Mountain environments, characterized by high levels of endemism, are at risk of experiencing significant biodiversity loss due to current trends in global warming. While many acknowledge their importance and vulnerability, these ecosystems still remain poorly studied, particularly for taxa that are difficult to sample such as bats. Aiming to estimate the amount of cryptic diversity among bats of a Neotropical montane cloud forest in Talamanca Range—south-east Central America—, we performed a 15-night sampling campaign, which resulted in 90 captured bats belonging to 8 species. We sequenced their mitochondrial cytochrome c oxidase subunit I (COI) and screened their inter- and intraspecific genetic variation. Phylogenetic relations with conspecifics and closely related species from other geographic regions were established using Maximum Likelihood and Bayesian inference methods, as well as median-joining haplotype networks. Mitochondrial lineages highly divergent from hitherto characterized populations (> 9% COI dissimilarity) were found in Myotis oxyotus and Hylonycteris underwoodi. Sturnira burtonlimi and M. keaysi also showed distinct mitochondrial structure with sibling species and/or populations. These results suggest that mountains in the region hold a high degree of endemicity potential that has previously been ignored in bats. They also warn of the high extinction risk montane bats may be facing due to climatic change, particularly in isolated mountain systems like Talamanca Range. Los entornos de montaña, caracterizados por altos niveles de endemismo, corren el riesgo de experimentar una pérdida significativa de biodiversidad debido a las tendencias actuales en el calentamiento global. Si bien muchos reconocen su importancia y vulnerabilidad, estos ecosistemas siguen siendo poco estudiados, particularmente para los taxones que son difíciles de muestrear, como los murciélagos. Con el objetivo de estimar la cantidad de diversidad críptica entre los murciélagos de un bosque nublado neotropical montano en la Cordillera de Talamanca, en el sudeste de América Central, realizamos una campaña de muestreo de 15 noches, que resultó en 90 murciélagos capturados pertenecientes a 8 especies. Secuenciamos su subunidad mitocondrial citocromo c oxidasa I (COI) y seleccionamos su variación genética inter e intraespecífica. Las relaciones filogenéticas con especies específicas y estrechamente relacionadas de otras regiones geográficas se establecieron utilizando métodos de máxima verosimilitud y de inferencia bayesiana, así como redes de haplotipos de unión media. Se encontraron linajes mitocondriales altamente divergentes de las poblaciones caracterizadas hasta ahora (> 9% de disimilitud de COI) en Myotis oxyotus y Hylonycteris underwoodi. Sturnira burtonlimi y M. keaysi también mostraron una estructura mitocondrial distinta con especies y / o poblaciones hermanas. Estos resultados sugieren que las montañas en la región tienen un alto grado de potencial de endemicidad que previamente se había ignorado en los murciélagos. También advierten sobre el alto riesgo de extinción que los murciélagos montanos pueden enfrentar debido al cambio climático, particularmente en sistemas montañosos aislados como la Cordillera de Talamanca. Universidad Nacional, Costa Rica Escuela de Ciencias Biológicas

Several insectivorous bats have included fish in their diet, yet little is known about the processes underlying this trophic shift. We performed three field experiments with wild fishing bats to address how they manage to discern fish from insects and adapt their hunting technique to capture fish. We show that bats react only to targets protruding above the water and discern fish from insects based on prey disappearance patterns. Stationary fish trigger short and shallow dips and a terminal echolocation pattern with an important component of the narrowband and low frequency calls. When the fish disappears during the attack process, bats regulate their attack increasing the number of broadband and high frequency calls in the last phase of the echolocation as well as by lengthening and deepening their dips. These adjustments may allow bats to obtain more valuable sensorial information and to perform dips adjusted to the level of uncertainty on the location of the submerged prey. The observed ultrafast regulation may be essential for enabling fishing to become cost-effective in bats, and demonstrates the ability of bats to rapidly modify and synchronise their sensorial and motor features as a response to last minute stimulus variations. This study was part of the Ministerio de Ciencia e Innovacion (MICINN) project CGL2009-12393. The University of The Basque Country (UPV/EHU) (INF09/15) and the Basque Government (IT385-07 and IT301-10) funded this study and provided grant support to O.A. and A.A (BFI-2009-252, BFI-2010-190, Doktore berriak eta Ikertzaile doktoreak espezializatzeko kontratatzeko laguntzak).

Formerly thought to be a strictly insectivorous trawling bat, recent studies have shown that Myotis capaccinii also preys on fish. To determine if differences exist in bat flight behaviour, prey handling, and echolocation characteristics when catching fish and insects of different sizes, we conducted a field experiment focused on the last stage of prey capture. We used synchronized video and ultrasound recordings to measure several flight and dip features as well as echolocation characteristics, focusing on terminal buzz phase I, characterized by a call rate exceeding 100 Hz, and buzz phase II, characterized by a drop in the fundamental well below 20 kHz and a repetition rate exceeding 150 Hz. When capturing insects, bats used both parts of the terminal phase to the same extent, and performed short and superficial drags on the water surface. In contrast, when preying on fish, buzz I was longer and buzz II shorter, and the bats made longer and deeper dips. These variations suggest that lengthening buzz I and shortening buzz II when fishing is beneficial, probably because buzz I gives better discrimination ability and the broader sonar beam provided by buzz II is useless when no evasive flight of the prey is expected. Additionally, bats continued emitting calls beyond the theoretical signal-overlap zone, suggesting that they might obtain information even when they have surpassed that threshold, at least initially. This study shows that M. capaccinii can regulate the temporal components of its feeding buzzes and modify prey capture technique according to the target.

and Inazio Garin 1,* 1 Department of Zoology and Animal Cell Biology , University of the Basque Country, Box 644, E-48080 Bilbao, Basque Country , Spain , e-mail: inazio.garin@ehu.es 2 Sodemasa , Avda. C e sar Augusto 14, 7, 50004 Zaragoza , Spain 3 Museu de Granollers-Ci e ncies Naturals , Francesc Maci a 51, 08402 Granollers, Catalonia , Spain 4 Galanthus Association , Ctra. de Jui a 46, 17460 Celr a , Catalonia , Spain

10 p. Molecular analysis of diet overcomes the considerable limitations of traditional techniques for identifying prey remains in bat faeces. We collected faeces from individual Mountain Long-eared Bats Plecotus macrobullaris trapped using mist nets during the summers of 2009 and 2010 in the Pyrenees. We analysed their diet using DNA mini-barcodes to identify prey species. In addition, we inferred some basic features of the bat's foraging ecology that had not yet been addressed. P. macrobullaris fed almost exclusively on moths (97.8%). As prey we detected one dipteran genus (Tipulidae) and 29 moth taxa: 28 were identified at species level (23 Noctuidae, 1 Crambidae, 1 Geometridae, 1 Pyralidae, 1 Sphingidae, 1 Tortricidae), and one at genus level (Rhyacia sp., Noctuidae). Known ecological information about the prey species allowed us to determine that bats had foraged at elevations between 1,500 and 2,500 m amsl (above mean sea level), mostly in subalpine meadows, followed by other open habitats such as orophilous grasslands and alpine meadows. No forest prey species were identified in the diet. As 96.4% of identified prey species were tympanate moths and no evidence of gleaning behaviour was revealed, we suggest P. macrobullaris probably forages by aerial hawking using faint echolocation pulses to avoid detection by hearing moths. As we could identify 87.8% of the analysed sequences (64.1% of the MOTUs, Molecular Operational Taxonomic Units) at species level, we conclude that DNA mini-barcodes are a very useful tool to analyse the diet of moth-specialist bats. This work was supported by Hezkuntza, Unibertsitate eta Ikerketa Saila, Eusko Jaurlaritza – The Basque Government (research projects GIC07/05 and IT301-10, and grants BFI-2010-190, and BFI-2009-252) and the University of The Basque Country, Euskal Herriko Uniberstiatatea UPV/EHU. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.