Arthropod diversity in the alpine tundra using metabarcoding: Spatial and temporal differences in alpha- and beta-diversity.

All ecosystems face ecological challenges in this century. Therefore, it is becoming increasingly important to understand the ecology and degree of local adaptation of functionally important Arctic-alpine biomes by looking at the most diverse taxon of metazoans: the Arthropoda. This is the first study to utilize metabarcoding in the Alpine tundra, providing insights into the effects of micro-environmental parameters on alpha- and beta-diversity of arthropods in such unique environments. To characterize arthropod diversity, pitfall traps were set at three middle-alpine sampling sites in the Scandinavian mountain range in Norway during the snow-free season in 2015. A metabarcoding approach was then used to determine the small-scale biodiversity patterns of arthropods in the Alpine tundra. All DNA was extracted directly from the preservative EtOH from 27 pitfall traps. In order to identify the controlling environmental conditions, all sampling locations were equipped with automatic data loggers for permanent measurement of the microenvironmental conditions. The variables measured were: air temperature [°C] at 15 cm height, soil temperature [°C] at 15 cm depth, and soil moisture [vol.%] at 15 cm depth. A total of 233 Arthropoda OTUs were identified. The number of unique OTUs found per sampling location (ridge, south-facing slope, and depression) was generally higher than the OTUs shared between the sampling locations, demonstrating that niche features greatly impact arthropod community structure. Our findings emphasize the fine-scale heterogeneity of arctic-alpine ecosystems and provide evidence for trait-based and niche-driven adaptation. The spatial and temporal differences in arthropod diversity were best explained by soil moisture and soil temperature at the respective locations. Furthermore, our results show that arthropod diversity is underestimated in alpine-tundra ecosystems using classical approaches and highlight the importance of integrating long-term functional environmental data and modern taxonomic techniques into biodiversity research to expand our ecological understanding of fine- and meso-scale biogeographical patterns.

Updating International Society for Biological and Environmental Repositories Best Practices, Fifth Edition: A New Process for Relevance in an Evolving Landscape.

The recently published fifth edition of the International Society for Biological and Environmental Repositories (ISBER) Best Practices signifies a pivotal milestone in navigating the complexities of repository management. Repositories operate within a constantly evolving landscape influenced by the changing fields of biospecimen science, technology, legal requirements, and ethical considerations. This dynamic is further amplified by unprecedented local and global challenges, such as pandemics, conflicts, and supply chain disruptions. Creating this new edition required a comprehensive approach capable of delivering a focused and coherent resource reflecting the broad horizon of its diverse users. The innovative approach used the existing phased development process and integrated the canvassing of opinions, formal evaluation, and real-time collaboration platforms. Merging these techniques enabled efficient collection and effective distillation of the latest in biobanking practices, enhancing the value of the fifth edition for repositories of specimens and associated data. The expanded document is a testament to the collective efforts of many dedicated individuals who have built upon the foundations of prior editions.

Molecular Weevil Identification Project: A thoroughly curated barcode release of 1300 Western Palearctic weevil species (Coleoptera, Curculionoidea).

The Molecular Weevil Identification project (MWI) studies the systematics of Western Palearctic weevils (superfamily Curculionoidea) in an integrative taxonomic approach of DNA barcoding, morphology and ecology. This barcode release provides almost 3600 curated CO1 sequences linked to morphological vouchers in about 1300 weevil species. The dataset is presented in statistical distance tables and as a Neighbour-Joining tree. Bayesian Inference trees are computed for the subfamilies Cryptorhynchinae, Apioninae and Ceutorhynchinae. Altogether, 18 unresolved taxonomic issues are discussed. A new barcode primer set is presented. Finally, we establish group-specific genetic distances for many weevil genera to serve as a tool in species delineation. These values are statistically based on distances between "good species" and their congeners. With this morphologically calibrated approach, we could resolve most alpha-taxonomic questions within the MWI project.

The dark side of pseudoscorpion diversity: The German Barcode of Life campaign reveals high levels of undocumented diversity in European false scorpions.

DNA barcoding is particularly useful for identification and species delimitation in taxa with conserved morphology. Pseudoscorpions are arachnids with high prevalence of morphological crypsis. Here, we present the first comprehensive DNA barcode library for Central European Pseudoscorpiones, covering 70% of the German pseudoscorpion fauna (35 out of 50 species). For 21 species, we provide the first publicly available COI barcodes, including the rare Anthrenochernes stellae Lohmander, a species protected by the FFH Habitats Directive. The pattern of intraspecific COI variation and interspecific COI variation (i.e., presence of a barcode gap) generally allows application of the DNA barcoding approach, but revision of current taxonomic designations is indicated in several taxa. Sequences of 36 morphospecies were assigned to 74 BINs (barcode index numbers). This unusually high number of intraspecific BINs can be explained by the presence of overlooked cryptic species and by the accelerated substitution rate in the mitochondrial genome of pseudoscorpions, as known from previous studies. Therefore, BINs may not be an appropriate proxy for species numbers in pseudoscorpions, while partitions built with the ASAP algorithm (Assemble Species by Automatic Partitioning) correspond well with putative species. ASAP delineated 51 taxonomic units from our data, an increase of 42% compared with the present taxonomy. The Neobisium carcionoides complex, currently considered a polymorphic species, represents an outstanding example of cryptic diversity: 154 sequences from our dataset were allocated to 23 BINs and 12 ASAP units.

Towards retrieving the Promethean treasure: a first molecular assessment of the freshwater fish diversity of Georgia.

In this study, we provide a first estimation of the molecular diversity of the freshwater fishes of Georgia. In addition to field collections, we integrated DNA barcode data obtained from recent works and public databases (BOLD and NCBI GenBank). Currently, the DNA barcode reference library for freshwater fishes of Georgia comprises 352 DNA barcodes for 50 species, 36 genera and 15 families (52% of total Georgian freshwater fish diversity), from which 162 DNA barcodes belonging to 41 species were newly generated as part of this study. A total of 22 species are reported from the Caspian Sea basin and 31 from the Black Sea basin. Amongst the studied taxa, seven species were found with large interspecific divergences (> 2%) while 11 species were found to share DNA barcodes within our dataset. In the course of the study, we found the first evidence of the existence of Gymnocephalus cernua (Linnaeus, 1758) and also confirm the second occurrence of invasive Rhinogobius lindbergi (Berg, 1933) in Georgia. Based on the evaluation of currently-available barcode data for Georgian fishes, we highlighted major gaps and research needs to further progress DNA-based biodiversity studies in Georgia. Though this study lays a solid base for DNA, based biodiversity assessment and monitoring approaches, further efforts within the recently started CaBOL (Caucasus Barcode Of Life) project are needed to obtain reference data for the species still lacking DNA barcodes.

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes.

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence-capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence-capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence-capture recovered 80%-90% of the control sample species. DNA sequence-capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low-cost, whereas DNA-sequence capture for biodiversity assessment is still in its infancy, is more time-consuming but provides more taxonomic assignments.

New records of bats and terrestrial small mammals from the Seli River in Sierra Leone before the construction of a hydroelectric dam.

Sierra Leone is situated at the western edge of the Upper Guinean Forests in West Africa, a recognised biodiversity hotspot which is increasingly threatened by habitat degradation and loss through anthropogenic impacts. The small mammal fauna of Sierra Leone is poorly documented, although bats and rodents account for the majority of mammalian diversity. Based on morphological, genetic and echolocation data, we recorded 30 bat (Chiroptera), three shrew (Soricomorpha) and eleven rodent (Rodentia) species at the Seli River in the north of the country in 2014 and 2016, during a baseline study for the Bumbuna Phase II hydroelectric project. In 2016, 15 bat species were additionally documented at the western fringe of the Loma Mountains, a recently established national park and biodiversity offset for the Bumbuna Phase I dam. Three bat species were recorded for the first time in Sierra Leone, raising the total number for the country to 61. Further, two bat species are threatened and endemic to the Upper Guinean Forest and several taxa of small mammals are poorly known or represent undescribed species. Overall, the habitats of the project area supported a species-rich small mammal fauna including species of global conservation concern. Suitable mitigation measures and/or offsets are necessary to maintain biodiversity and ecosystems in a region that is under high human pressure.

The 2018 Revision of the ISBER Best Practices: Summary of Changes and the Editorial Team's Development Process.

An increased need for specimens of reliable and consistent quality for research purposes requires the development of standardized policies and practices for the collection, handling, storage, retrieval, and distribution of specimens and specimen-related data. Providers of specimen resources should strive to incorporate new technologies and state-of-the-science approaches and thus ensure the availability of fit-for-purpose research specimens. Strategies to achieve quality outcomes and performance improvements often include adherence to established standards and implementation of best practices. Although standards represent a rigid set of guidelines that define exactly how a task should be completed, best practices are recommended actions and principles that demonstrate an awareness of standards, solve problems, can be replicated, and work in a given context. Adoption of best practice elements will vary based on the goals and circumstances of a given initiative, and in some instances, may not be possible to implement or may represent an aspirational achievement. In an effort to harmonize the scientific, technical, legal, and ethical issues relevant to repositories of biological and environmental specimens, the International Society for Biological and Environmental Repositories (ISBER) has released the updated ISBER Best Practices: Recommendations for Repositories (ISBER Best Practices). The document provides a comprehensive tool to guide repository professionals in both managerial and technical aspects such as practical details on repository governance, development, and operation; regulatory compliance; and ethical, legal, and social issues relevant to repositories. This summary describes the process for revising the document and summarizes the new topics, updates, and areas of expansion included in the fourth edition of ISBER Best Practices.

Timing and host plant associations in the evolution of the weevil tribe Apionini (Apioninae, Brentidae, Curculionoidea, Coleoptera) indicate an ancient co-diversification pattern of beetles and flowering plants.

Host plant shifts of insects can lead to a burst of diversification driven by their arrival in a new adaptive zone. In this context, our study aims to explore timing and patterns in the evolution of the weevil tribe Apionini (Brentidae, Curculionoidea, Coleoptera), particularly in relation to affiliations with their host plants. The classification of Apionini is difficult because of their relatively uniform appearance. Most taxa live mono- or oligophagously on members of Asteraceae or Fabaceae, but many are associated with other plant families, like Lamiaceae, Malvaceae and Polygonaceae. However, a comprehensive hypothesis of the phylogenetic relationships within the tribe Apionini is still missing. In the present study, we reconstructed trees and estimated divergence times among tribes. These results were further used to reconstruct the ancestral host plant use in Apionini weevils and to infer if the divergence timing of putative subtribes corresponds with the occurrence and radiation of their specific host plant groups. Phylogenetic analyses confirm the monophyly of most subtribes, with the exceptions of Oxystomatina, Kalcapiina and Aspidapiina. The subribe Aplemonina is inferred to be sister to all remaining Apionini. Divergence time estimates indicate the first occurrence of Apionini in the Upper Cretaceous and a simultaneous occurrence of several families of flowering plants and the occupation by Apionini weevil herbivores. These conspicuous coincidences support either an ancient co-diversification scenario or an escalating diversification in weevils induced by the radiation of flowering plants.

Towards a DNA Barcode Reference Database for Spiders and Harvestmen of Germany.

As part of the German Barcode of Life campaign, over 3500 arachnid specimens have been collected and analyzed: ca. 3300 Araneae and 200 Opiliones, belonging to almost 600 species (median: 4 individuals/species). This covers about 60% of the spider fauna and more than 70% of the harvestmen fauna recorded for Germany. The overwhelming majority of species could be readily identified through DNA barcoding: median distances between closest species lay around 9% in spiders and 13% in harvestmen, while in 95% of the cases, intraspecific distances were below 2.5% and 8% respectively, with intraspecific medians at 0.3% and 0.2%. However, almost 20 spider species, most notably in the family Lycosidae, could not be separated through DNA barcoding (although many of them present discrete morphological differences). Conspicuously high interspecific distances were found in even more cases, hinting at cryptic species in some instances. A new program is presented: DiStats calculates the statistics needed to meet DNA barcode release criteria. Furthermore, new generic COI primers useful for a wide range of taxa (also other than arachnids) are introduced.

Trends in Biobanking: A Bibliometric Overview.

Biobanks have become indispensable tools for a wide array of life and environmental sciences, and biotechnology. To evaluate trends in biobanking, 20,000 bibliographic records were retrieved and analyzed between 1939 and 2014 from the Scopus database using a series of biobank-related search terms within titles and keywords. Since the 1990s, the field of biobanking has been, and still is, experiencing above-average growth in terms of publications, journals, and thematic orientations. Almost two-thirds of all indexed biobanking documents have been published in the last decade, with now >1,000 publications in 600 distinct journals per year. Around 50,000 individual authors can be identified who have so far contributed to the field of biobanking, with an average of 1.5 publications per author. Author affiliations follow an uneven distribution: 42% of the authors are based in Europe, 33% in North America, 13% in Asia, 5% in South America, 4% in Australasia, and 2% in Africa. Analyzing the most common title words revealed (as did the journals) a strong focus on blood banking, but other biospecimen types-especially seeds, cells, and tissues-have been gaining increasing weight recently. Among medical applications, transfusion dominates, followed by transplantation. While a noticeable increase in disease and especially health occurred at the beginning of the millennium, cohort and consent seem to have become high-relevance topics only in this decade. In terms of banked organisms, human dominates, followed by viruses and plants (especially represented through seed banking). A very rough estimate based on subject categories suggests that a third of all publications in biobanking focus on organisms other than humans. However, animal, fungal, and microbial biobanking are still underrepresented, especially when considering their shares in global biodiversity.

Morphological and Molecular Revision of the Genus Ozirhincus (Diptera: Cecidomyiidae)-Long-Snouted Seed-Feeding Gall Midges on Asteraceae.

The Palaearctic gall-midge genus Ozirhincus is unique among the Cecidomyiidae for its morphology and biology. Unlike most other phytophagous gall midges, species in this genus do not induce galls but develop inside achenes of Asteraceae plants. The heads of adults are characterized by an unusually elongate proboscis, the function of which is unclear. Despite a lot of attention from taxonomists in the 19th and early 20th century, a proper revision of the genus has been hindered by complex host associations, the loss of most relevant type material, and the lack of a thorough comparative study of all life stages. The present revision integrated morphological, molecular, and life-history data to clearly define species boundaries within Ozirhincus, and delimit host-plant ranges for each of them. A phylogenetic analysis based on the mitochondrial COI and 16S genes confirmed the validity of four distinct species but did not resolve the relationships among them. All species are oligophages, and some may occur together on the same host plant. Species with wider host-plant ranges have wider European and circum-Mediterranean distribution ranges, whereas species with narrower host ranges are limited to Europe and the Russian Far East. As part of the present work, O. hungaricus is reinstated from synonymy, O. tanaceti is synonymized under O. longicollis, neotypes are designated for O. longicollis and O. millefolii, and a lectotype is designated for O. anthemidis.

The Global Genome Biodiversity Network (GGBN) Data Portal.

The Global Genome Biodiversity Network (GGBN) was formed in 2011 with the principal aim of making high-quality well-documented and vouchered collections that store DNA or tissue samples of biodiversity, discoverable for research through a networked community of biodiversity repositories. This is achieved through the GGBN Data Portal (http://data.ggbn.org), which links globally distributed databases and bridges the gap between biodiversity repositories, sequence databases and research results. Advances in DNA extraction techniques combined with next-generation sequencing technologies provide new tools for genome sequencing. Many ambitious genome sequencing projects with the potential to revolutionize biodiversity research consider access to adequate samples to be a major bottleneck in their workflow. This is linked not only to accelerating biodiversity loss and demands to improve conservation efforts but also to a lack of standardized methods for providing access to genomic samples. Biodiversity biobank-holding institutions urgently need to set a standard of collaboration towards excellence in collections stewardship, information access and sharing and responsible and ethical use of such collections. GGBN meets these needs by enabling and supporting accessibility and the efficient coordinated expansion of biodiversity biobanks worldwide.

Molecular phylogeny of the weevil genus Dichromacalles Stuben (Curculionidae: Cryptorhynchinae) and description of a new species.

A molecular phylogeny of the western Palearctic weevil genus Dichromacalles Stuben, 1998, is presented, combining two mitochondrial genes, COI and 16S, and the nuclear gene 28S in a Bayesian analysis of up to 1528 combined nucleotide positions. Based on this data we point out the putative ancestor of the currently known extant Dichromacalles species that initiated the unique radiation within the species of the formerly Acalles s.l. on the Canary Islands around 10 to 20 million years ago. Where morphology reaches its limits in species differentiation, molecular analysis can provide deeper insight. By combining morphology and molecular biology into an integrative taxonomy, new characters can be found, making phenotypic descriptions easier. Using this integrative taxonomy background, the new species Dichromacalles algecirasensis Stuben (Spain: Cadiz) is described here and D. lentisci (Chevrolat, 1861) is transferred into the subgenus Balcanacalles Stuben & Behne, 1998 following a molecular phylogenetic reconstruction. A catalogue of all 12 species of Dichromacalles is given and a key is presented, combined with image stackings of the habitus and aedeagus for all species.

Pholcid spider molecular systematics revisited, with new insights into the biogeography and the evolution of the group.

We analysed seven genetic markers sampled from 165 pholcids and 34 outgroups in order to test and improve the recently revised classification of the family. Our results are based on the largest and most comprehensive set of molecular data so far to study pholcid relationships. The data were analysed using parsimony, maximum-likelihood and Bayesian methods for phylogenetic reconstruction. We show that in several previously problematic cases molecular and morphological data are converging towards a single hypothesis. This is also the first study that explicitly addresses the age of pholcid diversification and intends to shed light on the factors that have shaped species diversity and distributions. Results from relaxed uncorrelated lognormal clock analyses suggest that the family is much older than revealed by the fossil record alone. The first pholcids appeared and diversified in the early Mesozoic about 207 Ma ago (185-228 Ma) before the breakup of the supercontinent Pangea. Vicariance events coupled with niche conservatism seem to have played an important role in setting distributional patterns of pholcids. Finally, our data provide further support for multiple convergent shifts in microhabitat preferences in several pholcid lineages. Our findings suggest that both adaptive and non-adaptive speciation may have played an important role in the diversification of pholcid lineages.

Exploring diversity in cryptorhynchine weevils (Coleoptera) using distance-, character- and tree-based species delineation.

Species boundaries are studied in a group of beetles, the western Palaearctic Cryptorhynchinae. We test for congruence of 'traditionally' identified morphospecies with species inferred through parsimony networks, distance-based clustering and the ultrametric tree-based generalized mixed yule-coalescent (GMYC) approach. For that purpose, we sequenced two variable fragments of mitochondrial DNA (CO1 and 16S) for a total of 791 specimens in 217 species of Cryptorhynchinae. Parsimony networks, morphology-calibrated distance clusters and the different tree-based species inferences all achieved low congruence with morphospecies, at best 60%. Although the degree of match with morphospecies was often similar for the different approaches, the composition of clusters partially varied. A barcoding gap was absent in morphospecies-oriented distances as well as for GMYC species clusters. This demonstrates that not only erroneous taxonomic assignments, incomplete lineage sorting, hybridization, or insufficient sampling can compromise distance-based identification, but also differences in speciation rates and uneven tree structure. The initially low match between morphospecies and the different molecular species delineation methods in this case study shows the necessity of combining the output of various methods in an integrative approach. Thereby we obtain an idea about the reliability of the different results and signals, which enables us to fine-tune sampling, delineation technique and data collection, and to identify species that require taxonomic revision.

Molecular species identification of Central European ground beetles (Coleoptera: Carabidae) using nuclear rDNA expansion segments and DNA barcodes.

BACKGROUND: The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI) gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous. RESULTS: We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97%) of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95%) of the studied Carabidae. CONCLUSION: Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.