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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.

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.

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.

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.

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.

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

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 micro-organisms. Whereas amplicon sequencing has traditionally been used to characterize the microbiome, the increasing number of published population genomics data sets offers 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 characterize the skin microbiome and investigate how host social and geographical 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 geographical 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. These authors contributed equally to this work: Rebecca Hooper, Jaelle C. Brealey, Katerina Guschanski, Andrew D. Foote

Aquaculture will play an essential role in feeding a growing human population, but several biological challenges impede sustainable growth of production. Emerging evidence across all areas of life has revealed the importance of the intimate biological interactions between animals and their associated gut microbiota. Based on challenges in aquaculture, we leverage current knowledge in molecular biology and host microbiota interactions to propose an applied holo-omic framework that integrates molecular data including genomes, transcriptomes, epigenomes, proteomes, and metabolomes for analyzing fish and their gut microbiota as interconnected and coregulated systems. With an eye towards aquaculture, we discuss the feasibility and potential of our holo-omic framework to improve growth, health, and sustainability in any area of food production, including livestock and agriculture.