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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.

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 Due to its central role in animal nutrition, the gut microbiota is likely a relevant factor shaping dietary niche shifts. We analysed both the impact and contribution of the gut microbiota to the dietary niche expansion of the only four bat species that have incorporated fish into their primarily arthropodophage diet. Results We first compared the taxonomic and functional features of the gut microbiota of the four piscivorous bats to that of 11 strictly arthropodophagous species using 16S rRNA targeted amplicon sequencing. Second, we increased the resolution of our analyses for one of the piscivorous bat species, namely Myotis capaccinii, and analysed multiple populations combining targeted approaches with shotgun sequencing. To better understand the origin of gut microorganisms, we also analysed the gut microbiota of their fish prey (Gambusia holbrooki). Our analyses showed that piscivorous bats carry a characteristic gut microbiota that differs from that of their strict arthropodophagous counterparts, in which the most relevant bacteria have been directly acquired from their fish prey. This characteristic microbiota exhibits enrichment of genes involved in vitamin biosynthesis, as well as complex carbohydrate and lipid metabolism, likely providing their hosts with an enhanced capacity to metabolise the glycosphingolipids and long-chain fatty acids that are particularly abundant in fish. Conclusions Our results depict the gut microbiota as a relevant element in facilitating the dietary transition from arthropodophagy to piscivory.

International audience; 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.

Summary In an attempt to explore the role of the gut microbiome during recent canine evolutionary history, we sequenced the metagenome of 13 canine coprolites dated ca. 3,600–3,450 years 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. Of interest, the Solarolo dog microbiome was particularly enriched in sequences encoding alpha-amylases and complemented a low copy number of the host amylase 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, thus compensating for delayed host response., Graphical abstract, Highlights • Ancient DNA of Bronze Age canine coprolites from Solarolo was sequenced • Solarolo dogs share gut microbiome features with modern wolves and dogs • The gut microbiome of Solarolo dogs shows high number of reads for alpha-amylase • Neolithic canine gut microbiome complemented delay in host genome adaptation, Biological sciences; Microbiome; Evolutionary biology; Evolutionary processes; Phylogenetics; Phylogeny; Evolutionary history; Genomic analysis; Sequence analysis; Omics; Genomics

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.

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.

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.

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.

Alberdi, Antton et al., 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.

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

Large-scale species’ distributions have been traditionally attributed to physiological traits related to abiotic factors, while behavioural features linked to biotic interactions have received little attention. We tested the relationship between trophic and spatial niche breadths through combining species distribution modelling with dietary DNA metabarcoding of over 400 bats sampled across Europe belonging to seven species. Our results point to a causality cascade between hunting plasticity, trophic niche breadth and spatial niche breadth, and thus indicate that behavioral plasticity and dietary diversification can contribute to shaping broad-scale species distributions.

Hill numbers provide a powerful framework for measuring, comparing and partitioning the diversity of biological systems as characterised using high throughput DNA sequencing approaches. In order to facilitate the implementation of Hill numbers into such analyses, whether focusing on diet reconstruction, microbial community profiling or more general ecosystem characterisation analyses, we present a new R package. ‘Hilldiv’ provides a set of functions to assist analysis of diversity based on Hill numbers, using count tables (e.g. OTU, ASV) and associated phylogenetic trees as inputs. Multiple functionalities of the library are introduced, including diversity measurement, diversity profile plotting, diversity comparison between samples and groups, multi-level diversity partitioning and (dis)similarity measurement. All of these are grounded in abundance-based and incidence-based Hill numbers, and can accommodate phylogenetic or functional correlation among OTUs or ASVs. The package can be installed from CRAN or Github, and tutorials and example scripts can be found in the package’s page (https://github.com/anttonalberdi/hilldiv).

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.

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)

Behavioral plasticity is a key feature allowing animals to broaden their dietary niche when novel food resources become available, and long-fingered bats provide an appropriate model system to study the underpinnings of behavioral plasticity, since although generally being an insectivorous species, some individuals have been reported to catch fish. Aiming to get insight into the origin of fishing behavior in long-fingered bats, we studied in the field the differences in sensorial and mechanical reactions to insect-like (stationary) and fish-like (temporary) prey stimuli between well-known piscivorous and strictly insectivorous individuals. Both piscivorous and insectivorous individuals exhibited a qualitatively similar reaction to temporary target stimuli (longer and deeper dips and terminal echolocation phase skewed towards buzz I compared to stationary stimuli). Nevertheless, the quantitative differences observed in the sensorial and mechanical features (the intensity of the shift was significantly greater in piscivorous than in insectivorous individuals) show that piscivorous individuals have honed their capture technique likely enhancing the fishing success. Thus, our results suggest that the fishing technique was developed from a primary reaction shared by all long-fingered bats. All individuals seem to be mechanically and sensorially adapted to detect and capture fish, although under appropriate environmental conditions, they would further improve their technique by experience and/or social learning. 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 Ikertraile doktoreak espezializatreko kontratatzeko laguntzak). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

After the dispersal of modern humans (Homo sapiens) Out of Africa, hominins with a similar morphology to that of present-day humans initiated the gradual demographic expansion into Eurasia. The mitogenome (33-fold coverage) of the Peştera Muierii 1 individual (PM1) from Romania (35 ky cal BP) we present in this article corresponds fully to Homo sapiens, whilst exhibiting a mosaic of morphological features related to both modern humans and Neandertals. We have identified the PM1 mitogenome as a basal haplogroup U6*, not previously found in any ancient or present-day humans. The derived U6 haplotypes are predominantly found in present-day North-Western African populations. Concomitantly, those found in Europe have been attributed to recent gene-flow from North Africa. The presence of the basal haplogroup U6* in South East Europe (Romania) at 35 ky BP confirms a Eurasian origin of the U6 mitochondrial lineage. Consequently, we propose that the PM1 lineage is an offshoot to South East Europe that can be traced to the Early Upper Paleolithic back migration from Western Asia to North Africa, during which the U6 lineage diversified, until the emergence of the present-day U6 African lineages.

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).

Several alpine vertebrates share a distribution pattern that extends across the South-western Palearctic but is limited to the main mountain massifs. Although they are usually regarded as cold-adapted species, the range of many alpine vertebrates also includes relatively warm areas, suggesting that factors beyond climatic conditions may be driving their distribution. In this work we first recognize the species belonging to the mentioned biogeographic group and, based on the environmental niche analysis of Plecotus macrobullaris, we identify and characterize the environmental factors constraining their ranges. Distribution overlap analysis of 504 European vertebrates was done using the Sorensen Similarity Index, and we identified four birds and one mammal that share the distribution with P. macrobullaris. We generated 135 environmental niche models including different variable combinations and regularization values for P. macrobullaris at two different scales and resolutions. After selecting the best models, we observed that topographic variables outperformed climatic predictors, and the abruptness of the landscape showed better predictive ability than elevation. The best explanatory climatic variable was mean summer temperature, which showed that P. macrobullaris is able to cope with mean temperature ranges spanning up to 16 degrees C. The models showed that the distribution of P. macrobullaris is mainly shaped by topographic factors that provide rock-abundant and open-space habitats rather than climatic determinants, and that the species is not a cold-adapted, but rather a cold-tolerant eurithermic organism. P. macrobullaris shares its distribution pattern as well as several ecological features with five other alpine vertebrates, suggesting that the conclusions obtained from this study might be extensible to them. We concluded that rock-dwelling and open-space foraging vertebrates with broad temperature tolerance are the best candidates to show wide alpine distribution in the Western Palearctic. We wish to thank Orly Razgour for her insightful comments on the manuscript and for proofreading the text. The Basque Government (research projects IT385-07, IT301-10 and IT754-13, and predoctoral grants BFI-2010-190 and BFI-2009-252) and the University of the Basque Country UPV/EHU supported this work.

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.