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Environmental DNA metabarcoding is increasingly implemented in biodiversity monitoring, including phytoplankton studies. Using 21 mock communities composed of seven unicellular diatom and dinoflagellate algae, assembled with different composition and abundance by controlling the number of cells, we tested the accuracy of an eDNA metabarcoding protocol in reconstructing patterns of alpha and beta diversity. This approach allowed us to directly evaluate both qualitative and quantitative metabarcoding estimates. Our results showed non-negligible rates (17–25%) of false negatives (i.e., failure to detect a taxon in a community where it was included), for three taxa. This led to a statistically significant underestimation of metabarcoding-derived alpha diversity (Wilcoxon p  = 0.02), with the detected species richness being lower than expected (based on cell numbers) in 8/21 mock communities. Considering beta diversity, the correlation between metabarcoding-derived and expected community dissimilarities was significant but not strong (R 2  = 0.41), indicating suboptimal accuracy of metabarcoding results. Average biovolume and rDNA gene copy number were estimated for the seven taxa, highlighting a potential, though not exhaustive, role of the latter in explaining the recorded biases. Our findings highlight the importance of mock communities for assessing the reliability of phytoplankton eDNA metabarcoding studies and identifying their limitations.

About 100 km east of Rome, in the central Apennine Mountains, a critically endangered population of ∼50 brown bears live in complete isolation. Mating outside this population is prevented by several 100 km of bear-free territories. We exploited this natural experiment to better understand the gene and genomic consequences of surviving at extremely small population size. We found that brown bear populations in Europe lost connectivity since Neolithic times, when farming communities expanded and forest burning was used for land clearance. In central Italy, this resulted in a 40-fold population decline. The overall genomic impact of this decline included the complete loss of variation in the mitochondrial genome and along long stretches of the nuclear genome. Several private and deleterious amino acid changes were fixed by random drift; predicted effects include energy deficit, muscle weakness, anomalies in cranial and skeletal development, and reduced aggressiveness. Despite this extreme loss of diversity, Apennine bear genomes show nonrandom peaks of high variation, possibly maintained by balancing selection, at genomic regions significantly enriched for genes associated with immune and olfactory systems. Challenging the paradigm of increased extinction risk in small populations, we suggest that random fixation of deleterious alleles (i) can be an important driver of divergence in isolation, (ii) can be tolerated when balancing selection prevents random loss of variation at important genes, and (iii) is followed by or results directly in favorable behavioral changes.

The European beech ( Fagus sylvatica L.) is one of the most widespread forest trees in Europe whose distribution and intraspecific diversity has been largely shaped by repeated glacial cycles. Previous studies, mainly based on palaeobotanical evidence and a limited set of chloroplast and nuclear genetic markers, highlighted a complex phylogeographic scenario, with southern and western Europe characterized by a rather heterogeneous genetic structure, as a result of recolonization from different glacial refugia. Despite its ecological and economic importance, the genome of this broad-leaved tree has only recently been assembled, and its intra-species genomic diversity is still largely unexplored. Here, we performed whole-genome resequencing of nine Italian beech individuals sampled from two stands located in the Alpine and Apennine mountain ranges. We investigated patterns of genetic diversity at chloroplast, mitochondrial and nuclear genomes and we used chloroplast genomes to reconstruct a temporally-resolved phylogeny. Results allowed us to test European beech differentiation on a whole-genome level and to accurately date their divergence time. Our results showed comparable, relatively high levels of genomic diversity in the two populations and highlighted a clear differentiation at chloroplast, mitochondrial and nuclear genomes. The molecular clock analysis indicated an ancient split between the Alpine and Apennine populations, occurred between the Günz and the Riss glaciations (approximately 660 kyrs ago), suggesting a long history of separation for the two gene pools. This information has important conservation implications in the context of adaptation to ongoing climate changes.

Windstorms and salvage logging lead to huge soil disturbance in alpine spruce forests, potentially affecting soil-living arthropods. However, the impacts of forest loss and possible interactions with underlying ecological gradients on soil microarthropod communities remain little known, especially across different environmental conditions. Here we used DNA metabarcoding approach to study wind-induced disturbances on forest communities of springtails and soil mites. In particular, we aimed to test the effect of forest soil disturbance on the abundance, richness, species composition, and functional guilds of microarthropods. We sampled 29 pairs of windfall-forest sites across gradients of elevation, precipitation, aspect and slope, 2 years after a massive windstorm, named Vaia, which hit North-Eastern Italy in October 2018. Our results showed that wind-induced disturbances led to detrimental impacts on soil-living communities. Abundance of microarthropods decreased in windfalls, but with interacting effects with precipitation gradients. Operative Taxonomic Units (OTU) richness strongly decreased in post-disturbance sites, particularly affecting plant-feeder trophic guilds. Furthermore, species composition analyses revealed that communities occurring in post-disturbance sites were different to those in undisturbed forests ( i.e ., stands without wind damage). However, variables at different spatial scales played different roles depending on the considered taxon. Our study contributes to shed light on the impacts on important, but often neglected arthropod communities after windstorm in spruce forests. Effects of forest disturbance are often mediated by underlying large scale ecological gradients, such as precipitation and topography. Massive impacts of stronger and more frequent windstorms are expected to hit forests in the future; given the response we recorded, mediated by environmental features, forest managers need to take site-specific conservation measures.

Ostreopsis is a benthic and epiphytic dinoflagellate producing potent toxins widespread in tropical and warm temperate coastal areas world-wide. We tested the hypothesis that as it is benthic, it would show distinct biogeographical patterns in comparison with planktonic species. Here, we analyse sequence variability in ribosomal DNA markers to provide the first phylogeographical study of this toxic benthic dinoflagellate. Mediterranean Sea, Atlantic Ocean, Pacific Ocean. Ribosomal DNA sequence data from partial nuclear LSU (D1/D2 domains) and 5.8S genes and non-coding internal transcribed spacer (ITS) regions were obtained from 82 isolates of Ostreopsis species, collected at 26 localities throughout the world. Molecular sequence data were analysed using maximum parsimony, maximum likelihood and Bayesian methods for phylogenetic inference. A statistical parsimony network was obtained based on concatenated LSU and 5.8S rDNA-ITS region sequences of the Mediterranean/Atlantic Ostreopsis cf. ovata isolates to infer haplotype distribution over their geographical range. Light epifluorescence microscopy analyses were performed on cultured and field Ostreopsis material for taxonomic identification, while laboratory experiments for encystment induction were carried out on selected O. cf. ovata isolates. Toxin assays of Ostreopsis species isolates were carried out using the haemolytic-based method. Analyses based on single and concatenated ribosomal genes gave substantially similar results. The rDNA phylogeny revealed different clades corresponding to different species within the genus Ostreopsis. In the species O. cf. ovata, different genetic lineages were correlated with macrogeographical distribution. A network of haplotypes inferred from the Atlantic and Mediterranean isolates of O. cf. ovata revealed that these two areas might host a single panmictic population. The Atlantic/Mediterranean population of O. cf. ovata was differentiated considerably from the Indo-Pacific populations. Other species of Ostreopsis were found, but they turned out to be restricted to just one of the two main warm-water oceanic basins, the Mediterranean/Atlantic and the Indo-Pacific. Ostreopsis cf. ovata was found to be widely dispersed throughout the coastal areas of tropical and some warm temperate seas. In the Atlantic/Mediterranean region it may constitute a panmictic population that is highly distinct from Indo-Pacific populations. Ostreopsis cf. siamensis was found only in the Mediterranean Sea, and strains identified as Ostreopsis lenticularis and Ostreopsis labens were found only in the Indo-Pacific region.

Marine sediments host heterogeneous protist communities consisting of both living benthic microorganisms and planktonic resting stages. Despite their key functions in marine ecosystem processes and biogeochemical cycles, their structure and dynamics are largely unknown. In the present study, with a spatially intensive sampling design we investigated benthic protist diversity and function of surface sediment samples from three subregions of the Mediterranean Sea, through an environmental DNA metabarcoding approach targeting the 18S V4 region of rRNA gene. Protists were characterized at the taxonomic level and trophic function, both in terms of alpha diversity and community composition, testing for potential differences among marine subregions and bathymetric groups. Overall, Alveolata and Stramenopiles were the two divisions that dominated the communities. These dominant groups exhibited significant differences among the three Mediterranean subregions in the alpha diversity estimates based on the detected ASVs, for all computed indices (ASV richness, Shannon and Simpson indices). Protist communities were also found to be significantly different in terms of composition at the order rank in the three subregions p-value < 0.01). These differences were mainly driven by Anoecales, Peridiniales, Borokales, Paraliales and Gonyaulacales, which together contributed almost 80% of the average dissimilarity. Anoecales was the dominant order in the Ionian – Central Mediterranean and Adriatic Sea, but with considerably different relative abundances (52% and 36%, respectively), while Borokales was the dominant order in the Western Mediterranean Sea (33%). Similarly, significant differences among the three marine subregions were also highlighted when protist assemblages were examined in terms of trophic function, both in terms of alpha diversity (calculated on the ASVs for each trophic group) and community composition p-value < 0.01. In particular, the Adriatic Sea stood out for having the highest relative abundance of autotrophic/mixotrophic components in the surface sediments analyzed. Conversely, no significant differences in protist assemblages were found among depth groups. This study provided new insights into the taxonomic and trophic composition of benthic protist communities found in Mediterranean surface sediments, revealing geographical differences among regional seas. The results were discussed in relation to the Mediterranean environmental features that could generate the differences among benthic protist communities.

Information on the population dynamics of a reservoir species have been increasingly adopted to understand and eventually predict the dispersal patterns of infectious diseases throughout an area. Although potentially relevant, to date there are no studies which have investigated the genetic structure of the red fox population in relation to infectious disease dynamics. Therefore, we genetically and spatially characterised the red fox population in the area stretching between the Eastern and Dinaric Alps, which has been affected by both distemper and rabies at different time intervals. Red foxes collected from north-eastern Italy, Austria, Slovenia and Croatia between 2006-2012, were studied using a set of 21 microsatellite markers. We confirmed a weak genetic differentiation within the fox population using Bayesian clustering analyses, and we were able to differentiate the fox population into geographically segregated groups. Our finding might be due to the presence of geographical barriers that have likely influenced the distribution of the fox population, limiting in turn gene flow and spread of infectious diseases. Focusing on the Italian red fox population, we observed interesting variations in the prevalence of both diseases among distinct fox clusters, with the previously identified Italy 1 and Italy 2 rabies as well as distemper viruses preferentially affecting different sub-groups identified in the study. Knowledge of the regional-scale population structure can improve understanding of the epidemiology and spread of diseases. Our study paves the way for an integrated approach for disease control coupling pathogen, host and environmental data to inform targeted control programs in the future.

The contribution of domestic cattle in human societies is enormous, making cattle, along with other essential benefits, the economically most important domestic animal in the world today. To expand existing knowledge on cattle domestication and mitogenome diversity, we performed a comprehensive complete mitogenome analysis of the species (802 sequences, 114 breeds). A large sample was collected in South‐east Europe, an important agricultural gateway to Europe during Neolithization and a region rich in cattle biodiversity. We found 1725 polymorphic sites (810 singletons, 853 parsimony‐informative sites and 57 indels), 701 unique haplotypes, a haplotype diversity of 0.9995 and a nucleotide diversity of 0.0015. In addition to the dominant T3 and several rare haplogroups (Q, T5, T4, T2 and T1), we have identified maternal line in Austrian Murbodner cattle that possess surviving aurochs’ mitochondria haplotype P1 that diverged prior to the Neolithization process. This is convincing evidence for rare female‐mediated adaptive introgression of wild aurochs into domesticated cattle in Europe. We revalidated the existing haplogroup classification and provided Bayesian phylogenetic inference with a more precise estimated divergence time than previously available. Occasionally, classification based on partial mitogenomes was not reliable; for example, some individuals with haplogroups P and T5 were not recognized based on D‐loop information. Bayesian skyline plot estimates (median) show that the earliest population growth began before domestication in cattle with haplogroup T2, followed by Q (~10.0–9.5 kyBP), whereas cattle with T3 (~7.5 kyBP) and T1 (~3.0–2.5 kyBP) expanded later. Overall, our results support the existence of interactions between aurochs and cattle during domestication and dispersal of cattle in the past, contribute to the conservation of maternal cattle diversity and enable functional analyses of the surviving aurochs P1 mitogenome.

Hybridization capture is an emerging method making use of short oligonucleotide baits to enrich DNA libraries for genomic fragments of specific organisms thus enabling detection of their presence in environmental samples. Although it offers a primer‐independent alternative to metabarcoding, little empirical work has been dedicated to characterizing the underlying biases and coupled implications for biological interpretation. Moreover, few published bioinformatic pipelines are available for designing polynucleotide capture baits from a reference sequence collection. We designed RNA‐baits specifically targeting two chloroplast barcoding genes matK and rbcL to reveal the plant taxonomic diversity present in a given environmental sample. Our approach leverages the sensitivity of hybridization capture and the capacity of high‐throughput DNA sequencing instruments. It builds on a new and universal method based on ancestral sequence reconstruction, ultimately limiting the number of bait‐probes required and reducing experimental costs, while accessing high levels of taxonomic diversity. Our bait‐set selectively targets four main plant orders (Fagales, Pinales, Asterales, and Poales), representing ~18% of all described vascular plants. This is achieved through the use of only 4084 baits, each 80 nucleotides in length (80‐mer), capturing ~1.0–1.6 k nucleotide sequences from each taxon. Tests on mock communities revealed important factors influencing capture efficiency and relative abundance estimates, including GC‐content, the overall target length per taxa, and the bait density and mean number of mismatches to the bait sequence. Our results show that hybridization capture, like metabarcoding, requires caution when interpreting results quantitatively within (paleo)‐ecological studies. Biases detected in this work have the potential to be mitigated with bait designs that avoid extreme base compositional biases and balancing bait targets across taxa. However, we strongly recommend the use of mock communities and read simulations to quantify the accuracy of taxonomic representation when using new bait designs. Hybridization capture to enrich environmental DNA libraries for specific organisms is offering an alternative to metabarcoding; however, little empirical work has addressed biases and implications for interpretations in molecular ecology. We used a phylogenetic‐based approach to reduce the number of probes, assessed capture bias, and provided recommendations, based on insights gained from mock communities.

In this essay we explore questions on how to increase the visibility and utility of genetic information for biodiversity managers and policy makers. This is discussed in the light of Aichi CBD Target 13, which for the first time impels signatories to minimise genetic erosion and safeguard genetic diversity. Drawing on qualitative results from a questionnaire sent to European conservation professionals by the ConGRESS Framework 7 Support Action ( www.congressgenetics.eu ), we summarise our preliminary findings on the attitudes and experiences of European conservation professionals in using genetics. We then discuss the implications of these findings for academics involved in conservation genetics and suggest that a much closer partnership between academic conservation geneticists and conservation practitioners is necessary if the full potential of genetic tools in conservation is to be realised.