The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from Portugal.

Background: The InBIO Barcoding Initiative (IBI) Orthoptera dataset contains records of 420 specimens covering all the eleven Orthoptera families occurring in Portugal. Specimens were collected in continental Portugal from 2005 to 2021 and were morphologically identified to species level by taxonomists. A total of 119 species were identified corresponding to about 77% of all the orthopteran species known from continental Portugal. New information: DNA barcodes of 54 taxa were made public for the first time at the Barcode of Life Data System (BOLD). Furthermore, the submitted sequences were found to cluster in 129 BINs (Barcode Index Numbers), 35 of which were new additions to the Barcode of Life Data System (BOLD). All specimens have their DNA barcodes publicly accessible through BOLD online database. Stenobothruslineatus is recorded for the first time for continental Portugal. This dataset greatly increases the knowledge on the DNA barcodes and distribution of Orthoptera from Portugal. All DNA extractions and most specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources.

Detection of hepatitis E virus genotype 3 in an Algerian mouse (Mus spretus) in Portugal.

Virus monitoring in small mammals is central to the design of epidemiological control strategies for rodent-borne zoonotic viruses. Synanthropic small mammals are versatile and may be potential carriers of several microbial agents. In the present work, a total of 330 fecal samples of small mammals were collected at two sites in the North of Portugal and screened for zoonotic hepatitis E virus (HEV, species Paslahepevirus balayani). Synanthropic small mammal samples (n = 40) were collected in a city park of Porto and belonged to the species Algerian mouse (Mus spretus) (n = 26) and to the greater white-toothed shrew (Crocidura russula) (n = 14). Furthermore, additional samples were collected in the Northeast region of Portugal and included Algerian mouse (n = 48), greater white-toothed shrew (n = 47), wood mouse (Apodemus sylvaticus) (n = 43), southwestern water vole (Arvicola sapidus) (n = 52), Cabrera's vole (Microtus cabrerae) (n = 49) and Lusitanian pine vole (Microtus lusitanicus) (n = 51). A nested RT-PCR targeting a part of open reading frame (ORF) 2 region of the HEV genome was used followed by sequencing and phylogenetic analysis. HEV RNA was detected in one fecal sample (0.3%; 95% confidence interval, CI: 0.01-1.68) from a synanthropic Algerian mouse that was genotyped as HEV-3, subgenotype 3e. This is the first study reporting the detection of HEV-3 in a synanthropic rodent, the Algerian mouse. The identified HEV isolate is probably the outcome of either a spill-over infection from domestic pigs or wild boars, or the result of passive viral transit through the intestinal tract. This finding reinforces the importance in the surveillance of novel potential hosts for HEV with a particular emphasis on synanthropic animals.

The European Nucleotide Archive in 2023.

The European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena) is maintained by the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI). The ENA is one of the three members of the International Nucleotide Sequence Database Collaboration (INSDC). It serves the bioinformatics community worldwide via the submission, processing, archiving and dissemination of sequence data. The ENA supports data types ranging from raw reads, through alignments and assemblies to functional annotation. The data is enriched with contextual information relating to samples and experimental configurations. In this article, we describe recent progress and improvements to ENA services. In particular, we focus upon three areas of work in 2023: FAIRness of ENA data, pandemic preparedness and foundational technology. For FAIRness, we have introduced minimal requirements for spatiotemporal annotation, created a metadata-based classification system, incorporated third party metadata curations with archived records, and developed a new rapid visualisation platform, the ENA Notebooks. For foundational enhancements, we have improved the INSDC data exchange and synchronisation pipelines, and invested in site reliability engineering for ENA infrastructure. In order to support genomic surveillance efforts, we have continued to provide ENA services in support of SARS-CoV-2 data mobilisation and have adapted these for broader pathogen surveillance efforts.

The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered.

UNITE (https://unite.ut.ee) is a web-based database and sequence management environment for molecular identification of eukaryotes. It targets the nuclear ribosomal internal transcribed spacer (ITS) region and offers nearly 10 million such sequences for reference. These are clustered into ∼2.4M species hypotheses (SHs), each assigned a unique digital object identifier (DOI) to promote unambiguous referencing across studies. UNITE users have contributed over 600 000 third-party sequence annotations, which are shared with a range of databases and other community resources. Recent improvements facilitate the detection of cross-kingdom biological associations and the integration of undescribed groups of organisms into everyday biological pursuits. Serving as a digital twin for eukaryotic biodiversity and communities worldwide, the latest release of UNITE offers improved avenues for biodiversity discovery, precise taxonomic communication and integration of biological knowledge across platforms.

Detection and Molecular Characterization of Giardia and Cryptosporidium spp. Circulating in Wild Small Mammals from Portugal.

Cryptosporidium spp. and Giardia spp. are important diarrhea-causing protozoan parasites worldwide that exhibit broad host ranges. Wild small mammals can harbor host-adapted and potentially zoonotic species of both parasites. The aim of this study was to investigate Cryptosporidium spp. and Giardia spp. in wild rodents and shrews in Portugal, focusing on the protist's occurrence and genetic diversity. Molecular screening by PCR at the small subunit (SSU) rRNA gene locus of 290 fecal samples from wood mice (Apodemus sylvaticus), southwestern water voles (Arvicola sapidus), Cabrera's voles (Microtus cabrerae), Lusitanian pine voles (Microtus lusitanicus), Algerian mice (Mus spretus) and greater white-toothed shrews (Crocidura russula) in Northeast Portugal revealed the low occurrence of Cryptosporidium spp. (1%) and high occurrence of Giardia spp. (32.8%). The analysis revealed that "species" was the only significant factor associated with the increasing probability of Giardia spp. infection, with the highest prevalence reported in southwestern water voles and Lusitanian pine voles. Cryptosporidium and Giardia species determination at the SSU rRNA gene locus revealed C. muris and G. microti as the only circulating species, respectively. Subtyping of the glutamate dehydrogenase (gdh) and beta-giardin (bg) genes provided evidence of the high genetic diversity within the G. microti clade. This study suggests that rodent-adapted G. microti occurs to a large extent in cricetid hosts and supports the limited role of wild rodents and shrews as natural sources of human infections in Northeast Portugal regarding the investigated parasites. Moreover, this is the first record of G. microti in southwestern water voles, Lusitanian pine voles, Algerian mice, wood mice and Cabrera's voles and C. muris in Cabrera's voles. Finally, this study improves the database of sequences relevant for the sequence typing of G. microti strains and provides new insights about the epidemiology of Giardia spp. and Cryptosporidium spp. in wild rodents and shrews, two parasite genera of high importance for public and animal health.

Fine scale genetics reveals the subtle negative effects of roads on an endangered bat.

The effective management of species with small and fragmented populations requires an in-depth understanding of how the effects of human-induced habitat disturbance shape the structure and gene flow at fine spatial scales. Identification of putative environmental barriers that affect individual exchange among subpopulations is imperative to prevent extinction risks. Here, we investigated how landscape affects the gene flow and relatedness structure of a population of the endangered lesser horseshoe bat (Rhinolophus hipposideros). We also assessed the effects of sexbiased dispersal on genetic relatedness. We genotyped 287 bat samples collected across southern Portugal and developed resistance surfaces for landscape variables hypothesized to affect gene flow. Then, we used spatially explicit models to fit relatedness distance through the resistance surfaces. We found genetic evidence of sex-biased dispersal and identified a significant fine scale structuring in the relatedness regarding females, the philopatric sex. Males displayed uniform levels of relatedness throughout the landscape. The results indicated less relatedness between the female´ from roosts located on proximity of roads than in roosts away from roads. Also, when analysing the sexes together the relatedness on roosts separated by highway were subtly less related in comparison to those occurring on the same side. Roads seem to be major shapers of the contemporary population structure of females, regardless of being relatively recent structures in the landscape. Furthermore, the relatedness patterns detected suggested that high tree density among roosts and continuity of forest patches in broader surrounding areas, promotes the relatedness among individuals. Landscape heterogeneity among roosts slightly decreases genetic relatedness. Nevertheless, those relationships are still weak, suggesting that population structuring driven by those factors is slowly ongoing. Thus, effective management measures should focus on issues for promoting safe road passages and suitable habitat corridors, allowing for the exchange of individuals and gene flow among lesser horseshoe bat roosts.

The European Nucleotide Archive in 2022.

The European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena), maintained by the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI), offers those producing data an open and supported platform for the management, archiving, publication, and dissemination of data; and to the scientific community as a whole, it offers a globally comprehensive data set through a host of data discovery and retrieval tools. Here, we describe recent updates to the ENA's submission and retrieval services as well as focused efforts to improve connectivity, reusability, and interoperability of ENA data and metadata.

The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, documenting biodiversity for freshwater biomonitoring in a Mediterranean hotspot.

Background: The Trichoptera are an important component of freshwater ecosystems. In the Iberian Peninsula, 380 taxa of caddisflies are known, with nearly 1/3 of the total species being endemic in the region. A reference collection of morphologically identified Trichoptera specimens, representing 142 Iberian taxa, was constructed. The InBIO Barcoding Initiative (IBI) Trichoptera 01 dataset contains records of 438 sequenced specimens. The species of this dataset correspond to about 37% of Iberian Trichoptera species diversity. Specimens were collected between 1975 and 2018 and are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources, Portugal) or in the collection Marcos A. González at the University of Santiago de Compostela (Spain). New information: Twenty-nine species, from nine different families, were new additions to the Barcode of Life Data System (BOLD). A success identification rate of over 80% was achieved when comparing morphological identifications and DNA barcodes for the species analysed. This encouraging step advances incorporation of informed Environmental DNA tools in biomonitoring schemes, given the shortcomings of morphological identifications of larvae and adult Caddisflies in such studies. DNA barcoding was not successful in identifying species in six Trichoptera genera: Hydropsyche (Hydropsychidae), Athripsodes (Leptoceridae), Wormaldia (Philopotamidae), Polycentropus (Polycentropodidae) Rhyacophila (Rhyacophilidae) and Sericostoma (Sericostomatidae). The high levels of intraspecific genetic variability found, combined with a lack of a barcode gap and a challenging morphological identification, rendered these species as needing additional studies to resolve their taxonomy.

Characterising Mitochondrial Capture in an Iberian Shrew.

Mitochondrial introgression raises questions of biogeography and of the extent of reproductive isolation and natural selection. Previous phylogenetic work on the Sorex araneus complex revealed apparent mitonuclear discordance in Iberian shrews, indicating past hybridisation of Sorex granarius and the Carlit chromosomal race of S. araneus, enabling introgression of the S. araneus mitochondrial genome into S. granarius. To further study this, we genetically typed 61 Sorex araneus/coronatus/granarius from localities in Portugal, Spain, France, and Andorra at mitochondrial, autosomal, and sex-linked loci and combined our data with the previously published sequences. Our data are consistent with earlier data indicating that S. coronatus and S. granarius are the most closely related of the three species, confirming that S. granarius from the Central System mountain range in Spain captured the mitochondrial genome from a population of S. araneus. This mitochondrial capture event can be explained by invoking a biogeographical scenario whereby S. araneus was in contact with S. granarius during the Younger Dryas in central Iberia, despite the two species currently having disjunct distributions. We discuss whether selection favoured S. granarius with an introgressed mitochondrial genome. Our data also suggest recent hybridisation and introgression between S. coronatus and S. granarius, as well as between S. araneus and S. coronatus.

Enriching captivity conditions with natural elements does not prevent the loss of wild-like gut microbiota but shapes its compositional variation in two small mammals.

As continued growth in gut microbiota studies in captive and model animals elucidates the importance of their role in host biology, further pursuit of how to retain a wild-like microbial community is becoming increasingly important to obtain representative results from captive animals. In this study, we assessed how the gut microbiota of two wild-caught small mammals, namely Crocidura russula (Eulipotyphla, insectivore) and Apodemus sylvaticus (Rodentia, omnivore), changed when bringing them into captivity. We analyzed fecal samples of 15 A. sylvaticus and 21 C. russula, immediately after bringing them into captivity and 5 weeks later, spread over two housing treatments: a "natural" setup enriched with elements freshly collected from nature and a "laboratory" setup with sterile artificial elements. Through sequencing of the V3-V4 region of the 16S recombinant RNA gene, we found that the initial microbial diversity dropped during captivity in both species, regardless of treatment. Community composition underwent a change of similar magnitude in both species and under both treatments. However, we did observe that the temporal development of the gut microbiome took different trajectories (i.e., changed in different directions) under different treatments, particularly in C. russula, suggesting that C. russula may be more susceptible to environmental change. The results of this experiment do not support the use of microbially enriched environments to retain wild-like microbial diversities and compositions, yet show that specific housing conditions can significantly affect the drift of microbial communities under captivity.

Trust your guts? The effect of gut section on diet composition and impact of Mus musculus on islands using metabarcoding.

DNA metabarcoding is widely used to characterize the diet of species, and it becomes very relevant for biodiversity conservation, allowing the understanding of trophic chains and the impact of invasive species. The need for cost-effective biodiversity monitoring methods fostered advances in this technique. One question that arises is which sample type provides a better diet representation.Therefore, with this study, we intended to evaluate if there were differences in diet estimates according to the section of the gastrointestinal tract analysed and which section(s) provided the best diet representation. Additionally, we intended to infer the ecological/economic impacts of an invader as a model of the potential effects in an originally mammal-free ecosystem.We examined the gut contents of the house mouse Mus musculus introduced to Cabo Verde, considering three sections: stomach, small intestine, and large intestine. We applied a DNA-metabarcoding approach using two genetic markers, one specific for plants and another for invertebrates.We showed that this invader consumed 131 taxa (73 plants and 58 invertebrates). We obtained significant differences in the composition of two of the three sections, with a higher incidence of invertebrates in the stomach and plants in the intestines. This may be due to stomach inhibitors acting on plants and/or to faster absorption of soft-body invertebrates compared to the plant fibers in the intestines. We verified that the impact of this invader in the ecosystem is predominantly negative, as at least 50% of the ingested items were native, endemic, or economically important taxa, and only 19% of the diet items were exotics.Overall, results showed the need to analyse only two gastrointestinal tract sections to obtain robust diet data, increasing the cost-effectiveness of the method. Furthermore, by uncovering the native taxa most frequently preyed on by mice, this DNA-metabarcoding approach allowed us to evaluate efficiently which are at the highest risk.

O metabarcoding de ADN é amplamente utilizado para a caracterização da dieta de espécies, e tornou­se bastante relevante para a conservação da biodiversidade, permitindo a compreensão sobre cadeias tróficas e o impacto de espécies invasoras. A necessidade de métodos de monitorização da biodiversidade com uma boa relação custo­benefício fomentaram avanços nesta técnica. Uma questão que se coloca é qual o tipo de amostra que fornece uma melhor representação da dieta. Deste modo, com este estudo, pretendemos avaliar se existem diferenças nas estimativas da dieta de acordo com a secção do tracto gastrointestinal analisada e qual(is) a(s) secção(ões) que proporciona(m) uma melhor representação da dieta. Adicionalmente, pretendemos inferir os impactos ecológicos/ económicos de um invasor como um modelo dos efeitos potenciais que este pode ter num ecossistema originalmente sem mamíferos. Analisámos os conteúdos gastrointestinais do rato doméstico Mus musculus introduzido em Cabo Verde, considerando três secções: estômago, intestino delgado e intestino grosso. Aplicámos uma abordagem de metabarcoding de ADN usando dois marcadores genéticos, um específico para plantas e outro para invertebrados. Mostrámos que este invasor consumiu 131 taxa (73 plantas e 58 invertebrados). Obtivemos diferenças significativas na composição de duas das três secções, com maior incidência de invertebrados no estômago e de plantas nos intestinos. Isto pode dever­se a inibidores estomacais que agem sobre as plantas e/ ou à absorção mais rápida de invertebrados de corpo mole em comparação com as fibras vegetais nos intestinos. Verificámos que o impacto deste invasor no ecossistema é predominantemente negativo, pois pelo menos 50% dos itens ingeridos eram nativos, endémicos ou economicamente importantes e apenas 19% dos itens da dieta eram exóticos. De modo geral, os resultados mostraram a necessidade de analisar apenas duas secções do tracto gastrointestinal para obter dados robustos da dieta, aumentando a relação custo­eficácia deste método. Além disso, ao descobrir os taxa nativos mais frequentemente predados por ratos, a abordagem de metabarcoding de ADN permitiu­nos avaliar com eficiência quais estão sob maior risco.

The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese Diptera 02 - Limoniidae, Pediciidae and Tipulidae.

BACKGROUND: The InBIO Barcoding Initiative (IBI) Diptera 02 dataset contains records of 412 crane fly specimens belonging to the Diptera families: Limoniidae, Pediciidae and Tipulidae. This dataset is the second release by IBI on Diptera and it greatly increases the knowledge on the DNA barcodes and distribution of crane flies from Portugal. All specimens were collected in Portugal, including six specimens from the Azores and Madeira archipelagos. Sampling took place from 2003 to 2019. Specimens have been morphologically identified to species level by taxonomists and belong to 83 species in total. The species, represented in this dataset, correspond to about 55% of all the crane fly species known from Portugal and 22% of crane fly species known from the Iberian Peninsula. All DNA extractions and most specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. NEW INFORMATION: Fifty-three species were new additions to the Barcode of Life Data System (BOLD), with another 18 species' barcodes added from under-represented species in BOLD. Furthermore, the submitted sequences were found to cluster in 88 BINs, 54 of which were new to BOLD. All specimens have their DNA barcodes publicly accessible through BOLD online database and its collection data can be accessed through the Global Biodiversity Information Facility (GBIF). One species, Gonomyiatenella (Limoniidae), is recorded for the first time from Portugal, raising the number of crane flies recorded in the country to 145 species.

DNA Barcoding of Portuguese Lacewings (Neuroptera) and Snakeflies (Raphidioptera) (Insecta, Neuropterida).

The orders Neuroptera and Raphidioptera include the species of insects known as lacewings and snakeflies, respectively. In Portugal, these groups account for over 100 species, some of which are very difficult to identify by morphological analysis. This work is the first to sample and DNA sequence lacewings and snakeflies of Portugal. A reference collection was built with captured specimens that were identified morphologically. DNA barcode sequences of 658 bp were obtained from 243 specimens of 54 species. The results showed that most species can be successfully identified through DNA barcoding, with the exception of seven species of Chrysopidae (Neuroptera). Additionally, the first published distribution data are presented for Portugal for the neuropterans Gymnocnemiavariegata (Schneider, 1845) and Myrmecaelurus (Myrmecaelurus) trigrammus (Pallas, 1771).

The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese Hemiptera 01.

BACKGROUND: The InBIO Barcoding Initiative (IBI) Hemiptera 01 dataset contains records of 131 specimens of Hemiptera. Most specimens have been morphologically identified to species or subspecies level and represent 88 species in total. The species of this dataset correspond to about 7.3% of continental Portuguese hemipteran species diversity. All specimens were collected in continental Portugal. Sampling took place from 2015 to 2019 and specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. NEW INFORMATION: This dataset increases the knowledge on the DNA barcodes and distribution of 88 species of Hemiptera from Portugal. Six species, from five different families, were new additions to the Barcode of Life Data System (BOLD), with another twenty five species barcodes' added from under-represented taxa in BOLD. All specimens have their DNA barcodes publicly accessible through BOLD online database and the distribution data can be accessed through the Global Biodiversity Information Facility (GBIF). Eutettix variabilis and Fieberiella florii are recorded for the first time for Portugal and Siphanta acuta, an invasive species, previously reported from the Portuguese Azores archipelago, is recorded for the first time for continental Portugal.

The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera.

BACKGROUND: The use of DNA barcoding allows unprecedented advances in biodiversity assessments and monitoring schemes of freshwater ecosystems; nevertheless, it requires the construction of comprehensive reference collections of DNA sequences that represent the existing biodiversity. Plecoptera are considered particularly good ecological indicators and one of the most endangered groups of insects, but very limited information on their DNA barcodes is available in public databases. Currently, less than 50% of the Iberian species are represented in BOLD. NEW INFORMATION: The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera dataset contains records of 71 specimens of Plecoptera. All specimens have been morphologically identified to species level and belong to 29 species in total. This dataset contributes to the knowledge on the DNA barcodes and distribution of Plecoptera from the Iberian Peninsula and it is one of the IBI database public releases that makes available genetic and distribution data for a series of taxa.The species represented in this dataset correspond to an addition to public databases of 17 species and 21 BINs. Fifty-eight specimens were collected in Portugal and 18 in Spain during the period of 2004 to 2018. All specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources and their DNA barcodes are publicly available in the Barcode of Life Data System (BOLD) online database. The distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).

The InBIO Barcoding Initiative Database: DNA barcodes of Portuguese Diptera 01.

BACKGROUND: The InBIO Barcoding Initiative (IBI) Diptera 01 dataset contains records of 203 specimens of Diptera. All specimens have been morphologically identified to species level, and belong to 154 species in total. The species represented in this dataset correspond to about 10% of continental Portugal dipteran species diversity. All specimens were collected north of the Tagus river in Portugal. Sampling took place from 2014 to 2018, and specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. NEW INFORMATION: This dataset contributes to the knowledge on the DNA barcodes and distribution of 154 species of Diptera from Portugal and is the first of the planned IBI database public releases, which will make available genetic and distribution data for a series of taxa. All specimens have their DNA barcodes made publicly available in the Barcode of Life Data System (BOLD) online database and the distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).

Evolutionary history of two cryptic species of northern African jerboas.

BACKGROUND: Climatic variation and geologic change both play significant roles in shaping species distributions, thus affecting their evolutionary history. In Sahara-Sahel, climatic oscillations shifted the desert extent during the Pliocene-Pleistocene interval, triggering the diversification of several species. Here, we investigated how these biogeographical and ecological events have shaped patterns of genetic diversity and divergence in African Jerboas, desert specialist rodents. We focused on two sister and cryptic species, Jaculus jaculus and J. hirtipes, where we (1) evaluated their genetic differentiation, (2) reconstructed their evolutionary and demographic history; (3) tested the level of gene flow between them, and (4) assessed their ecological niche divergence. RESULTS: The analyses based on 231 individuals sampled throughout North Africa, 8 sequence fragments (one mitochondrial and seven single copy nuclear DNA, including two candidate genes for fur coloration: MC1R and Agouti), 6 microsatellite markers and ecological modelling revealed: (1) two distinct genetic lineages with overlapping distributions, in agreement with their classification as different species, J. jaculus and J. hirtipes, with (2) low levels of gene flow and strong species divergence, (3) high haplotypic diversity without evident geographic structure within species, and (4) a low level of large-scale ecological divergence between the two taxa, suggesting species micro-habitat specialization. CONCLUSIONS: Overall, our results suggest a speciation event that occurred during the Pliocene-Pleistocene transition. The contemporary distribution of genetic variation suggests ongoing population expansions. Despite the largely overlapping distributions at a macrogeographic scale, our genetic results suggest that the two species remain reproductively isolated, as only negligible levels of gene flow were observed. The overlapping ecological preferences at a macro-geographic scale and the ecological divergence at the micro-habitat scale suggest that local adaptation may have played a crucial role in the speciation process of these species.

Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring.

DNA metabarcoding can contribute to improving cost-effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time-consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column-based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic-based enzymatic protocol (BEAD), and a 313-bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7-14 than 1-7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.

Glacial cycles drive rapid divergence of cryptic field vole species.

Understanding the factors that contribute to the generation of reproductively isolated forms is a fundamental goal of evolutionary biology. Cryptic species are an especially interesting challenge to study in this context since they lack obvious morphological differentiation that provides clues to adaptive divergence that may drive reproductive isolation. Geographical isolation in refugial areas during glacial cycling is known to be important for generating genetically divergent populations, but its role in the origination of new species is still not fully understood and likely to be situation dependent. We combine analysis of 35,434 single-nucleotide polymorphisms (SNPs) with environmental niche modeling (ENM) to investigate genomic and ecological divergence in three cryptic species formerly classified as the field vole (Microtus agrestis). The SNPs demonstrate high genomic divergence (pairwise F ST values of 0.45-0.72) and little evidence of gene flow among the three field vole cryptic species, and we argue that genetic drift may have been a particularly important mechanism for divergence in the group. The ENM reveals three areas as potential glacial refugia for the cryptic species and differing climatic niches, although with spatial overlap between species pairs. This evidence underscores the role that glacial cycling has in promoting genetic differentiation and reproductive isolation by subdivision into disjunct distributions at glacial maxima in areas relatively close to ice sheets. Future investigation of the intrinsic barriers to gene flow between the field vole cryptic species is required to fully assess the mechanisms that contribute to reproductive isolation. In addition, the Portuguese field vole (M. rozianus) shows a high inbreeding coefficient and a restricted climatic niche, and warrants investigation into its conservation status.

Integrative approaches to guide conservation decisions: Using genomics to define conservation units and functional corridors.

Climate change and increasing habitat loss greatly impact species survival, requiring range shifts, phenotypic plasticity and/or evolutionary change for long-term persistence, which may not readily occur unaided in threatened species. Therefore, defining conservation actions requires a detailed assessment of evolutionary factors. Existing genetic diversity needs to be thoroughly evaluated and spatially mapped to define conservation units (CUs) in an evolutionary context, and we address that here. We also propose a multidisciplinary approach to determine corridors and functional connectivity between CUs by including genetic diversity in the modelling while controlling for isolation by distance and phylogeographic history. We evaluate our approach on a Near Threatened Iberian endemic rodent by analysing genotyping-by-sequencing (GBS) genomic data from 107 Cabrera voles (Microtus cabrerae), screening the entire species distribution to define categories of CUs and their connectivity: We defined six management units (MUs) which can be grouped into four evolutionarily significant units (ESUs) and three (putatively) adaptive units (AUs). We demonstrate that the three different categories of CU can be objectively defined using genomic data, and their characteristics and connectivity can inform conservation decision-making. In particular, we show that connectivity of the Cabrera vole is very limited in eastern Iberia and that the pre-Pyrenean and part of the Betic geographic nuclei contribute the most to the species genetic diversity. We argue that a multidisciplinary framework for CU definition is essential and that this framework needs a strong evolutionary basis.