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High-throughput sequencing (HTS) studies on invertebrates commonly use ethanol as the main sample fixative (upon collection) and preservative (for storage and curation). However, alternative agents exists, which should not be automatically neglected when studies are newly designed. This review provides an overview of the application of propylene glycol (PG) in DNA-based studies of invertebrates, thus to stimulate an evidence-based discussion. The use of PG in DNA-based studies of invertebrates is still limited (n = 79), but a steady increase has been visible since 2011. Most studies used PG as a fixative for passive trapping (73%) and performed Sanger sequencing (66%; e.g. DNA barcoding). More recently, HTS setups joined the field (11%). Terrestrial Coleoptera (30%) and Diptera (20%) were the most studied groups. Very often, information on the grade of PG used (75%) or storage conditions (duration, temperature) were lacking. This rendered direct comparisons of study results difficult, and highlight the need for further systematic studies on these subjects. When compared to absolute ethanol, PG can be more widely and cheaply acquired (e.g. as an antifreeze, 13% of studies). It also enables longer trapping intervals, being especially relevant at remote or hard-to-reach places. Shipping of PG-conserved samples is regarded as risk-free and is authorised, pinpointing its potential for larger trapping programs or citizen science projects. Its property to retain flexibility of morphological characters as well as to lead to a reduced shrinkage effect was especially appraised by integrative study designs. Finally, the so far limited application of PG in the context of HTS showed promising results for short read amplicon sequencing and reduced representation methods. Knowledge of the influence of PG fixation and storage for long(er) read HTS setups is currently unavailable. Given our review results and taking difficulties of direct methodological comparisons into account, future DNA-based studies of invertebrates should on a case-by-case basis critically scrutinise if the application of PG in their anticipated study design can be of benefit.

In the European Union, all bat species are strictly protected and member states must ensure their conservation. However, if populations are genetically structured, conservation units that correspond to whole countries may be too large, putting small populations with specific conservation requirements at risk. Geoffroy’s bat (Myotis emarginatus) has undergone well-documented declines at its north-western European range edge between the 1960 and 1990s and is considered to be negatively affected by habitat fragmentation. Here we analysed the species’ genetic population structure and diversity to identify subpopulations with reduced genetic diversity and to scientifically inform conservation management. We generated 811 microsatellite-based genetic profiles obtained from 42 European nursery colonies and analysed a total of 932 sequences of the hypervariable region II of the mitochondrial control region sampled from across Europe. While two geographically widespread genetic populations were inferred to be present in north-western Europe, both nuclear and mitochondrial genetic diversity were lowest in the areas that had experienced a decline during the last century. A microsatellite-based analysis of demographic history did not permit, however, to unequivocally link that reduced genetic diversity to the population contraction event. Given the large geographic extent of the genetic populations, preserving the connectivity of mating sites requires concerted conservation efforts across multiple political jurisdictions. Genetic monitoring ought to be done on a regular basis to ensure that large-scale connectivity is maintained and further loss of genetic diversity is prevented.

The common house mosquito, Culex pipiens s. l. is part of the morphologically hardly or non-distinguishable Culex pipiens complex. Upcoming molecular methods allowed us to identify members of mosquito populations that are characterized by differences in behavior, physiology, host and habitat preferences and thereof resulting in varying pathogen load and vector potential to deal with. In the last years, urban and surrounding periurban areas were of special interest due to the higher transmission risk of pathogens of medical and veterinary importance. Recently, surveys of underground habitats were performed to fully evaluate the spatial distribution of rare members of the Cx. pipiens complex in Europe. Subterranean environments and their contribution to mosquito-borne pathogen transmission are virtually unknown. Herein, we review the underground community structures of this species complex in Europe, add new data to Germany and provide the first reports of the Cx. pipiens complex and usually rarely found mosquito taxa in underground areas of Luxembourg. Furthermore, we report the first finding of Culiseta glaphyroptera in Luxembourg. Our results highlight the need for molecular specimen identifications to correctly and most comprehensively characterize subterranean mosquito community structures.

Museums and other institutions curating natural history collections (NHCs) are fundamental entities to many scientific disciplines, as they house data and reference material for varied research projects. As such, biological specimens preserved in NHCs represent accessible physical records of the living world's history. They provide useful information regarding the presence and distribution of different taxonomic groups through space and time. Despite the importance of biological museum specimens, their potential to answer scientific questions, pertinent to the necessities of our current historical context, is often under-explored. The currently-known wild bee fauna of Luxembourg comprises 341 registered species distributed amongst 38 different genera. However, specimens stored in the archives of local NHCs represent an untapped resource to update taxonomic lists, including potentially overlooked findings relevant to the development of national conservation strategies. We re-investigated the wild bee collection of the Zoology Department of the National Museum of Natural History Luxembourg by using morphotaxonomy and DNA barcoding. The collection revision led to the discovery of four species so far not described for the country: Andrena lagopus (Latreille, 1809), Nomada furva (Panzer, 1798), Hoplitis papaveris (Latreille, 1799) and Sphecodes majalis (Pérez, 1903). Additionally, the presence of Nomada sexfasciata (Panzer, 1799), which inexplicably had been omitted by the most current species list, can be re-confirmed. Altogether, our findings increase the number of recorded wild bee species in Luxembourg to 346. Moreover, the results highlight the crucial role of NHCs as repositories of our knowledge of the natural world.

With the 4th Joint Danube Survey in 2019 (JDS4), for the first time, DNA- and environmental DNA-based approaches were integrated into the program of the JDS, focussing on the three biological quality elements of fish, macrozoobenthos and phytobenthos, and additionally on the sediment community (Liska et al. 2021, Weigand and Astrin 2021). The rationals for including (e)DNA-based tools into the survey's program were that i) many hard-to-identify organism groups can be assessed down to their species level, ii) taxonomic information can be unlocked even in cases where morphotaxonomic knowledge and expertise are limited, iii) all developmental stages, cryptic species and indeterminable sexes can be potentially identified, iv) taxalists relying on sequence-based information are reproducible and comparable in space and time (aspects, which are particularly important for a longitudinal survey involving many countries), and, v) this additional line of taxonomic evidence will help to draw a more precise and comprehensive picture of the Danubian biota. The target groups were assessed using group-specific (e)DNA-based metabarcoding approaches (for fish: Pont et al. 2021; macrozoobenthos: Beermann et al. 2021; phytobenthos: Zimmermann et al. 2021; sediment community: Cordier et al. 2021). The coverage of barcode libraries for Danubian biota were checked prior to conducting the metabarcoding approaches. Coverage values based on JDS3 outcomes were >90% for fish (12S, but depending on reference database), 84% for macrozoobenthos (COI) and 69% and 88% for all, respectively, only abundant phytobenthos species (18S + rbcL), so that (e)DNA-based approaches were expected to be implemented effectively from this perspective (Weigand and Astrin 2021). Although still a certain degree of methodological variation exists, the outcomes clearly demonstrate the huge potential of (e)DNA-based approaches for complementary biodiversity and ecological status class assessments: eDNA water analysis of fish revealed most of the taxa also detected by the traditional fish survey, but was particularly effective in detecting hard-to-capture benthic taxa (including endangered sturgeon species) and fish traces originating from waste water treatment plants (Pont et al. 2021). Many of the traditionally assigned macrozoobenthos species were detected by DNA metabarcoding as well, but sequence data allowed to add a plethora of new chironomid and oligochaete species to the taxalist. Molecular ecological status class assessments based on presence-absence values of macrozoobenthos species were largely congruent to traditional abundance or presence-absence-based outcomes (Beermann et al. 2021, Weigand 2021). Although traditional light microscopy, which is based on identifying phytobenthos species by their frostules, revealed a higher number of diatom species, the molecular assessment detected much more taxa (i.e. MOTUs), which await species-level taxonomic annotation in the future (Zimmermann et al. 2021). Metabarcoding of the sediment community was particularly effective to assess meiofaunal species and allowed the molecular inference of fine sediment quality based on local community structures of vulnerable nematode species (Cordier et al. 2021). Finally, all (e)DNA-based taxalists were compiled to inform invasive alien species detection in the Danube River Basin. However, despite their promising performance and large coherence with traditional outcomes during JDS4, the full potential of (e)DNA-based approaches in the context of larger environmental surveys might be further released by developing and curating catchment-specific DNA barcode reference libraries, focussing on a small(er) set of standardized (e)DNA-based approaches, integrating genetic diversity (and spatiotemporal changes thereof) in ecological status class assessments, installing a dense, large-scale environmental DNA-based screening, based on which traditional surveys can be performed at conspicuous sites, and, educating and training national authorities in state-of-the-art molecular tools. developing and curating catchment-specific DNA barcode reference libraries, focussing on a small(er) set of standardized (e)DNA-based approaches, integrating genetic diversity (and spatiotemporal changes thereof) in ecological status class assessments, installing a dense, large-scale environmental DNA-based screening, based on which traditional surveys can be performed at conspicuous sites, and, educating and training national authorities in state-of-the-art molecular tools. Already today, (e)DNA-based methods can be seen as an effective and complementary tool to provide consolidated results for biodiversity and ecological status class assessments in a highly integrative and international setup, as pursued during JDS4.

Dear participants of DNAQUA2021 International Conference, Undoubtedly, DNAQUA2021 is a major highlight of the EU COST Action DNAqua-Net (CA15219). Even though we cannot claim that the organisation of DNAQUA2021 was a piece of cake, it is simply wonderful to see the great interest in this event. With 1,498 registered participants from 79 nations, 204 contributed talks and posters for only two and a half days, the conference shows how timely and relevant research on DNA-based aquatic bioassessment and monitoring is. As the managing team of DNAqua-Net, we could have hardly imagined the impact of DNAqua-Net back in 2015, when we wrote the proposal (Leese et al. 2016). Yet, the more we are now delighted and thankful to see the success. Together with many experts from many different countries, taxonomists, ecologists, geneticists and bioinformaticians, we have made significant methodological progress. Above all, we have succeeded in connecting biomonitoring experts all across Europe and beyond. With more than 100 scientific publications from DNAqua-Net's five working groups, the research impact of the network is obvious. Furthermore, with "Metabarcoding and Metagenomics" (MBMG), we have established an international journal for basic and applied aspects of genetic bioassessment and monitoring. However, in many ways, the impact of DNAqua-Net goes far beyond the mere scientific progress. Capacity building e.g. via barcoding projects have been initiated in many countries, validation studies were co-designed by researchers and stakeholders from the applied sector and launched - even across several countries as for example the SCANDNAnet project shows. DNAqua-Net has supported over 50 research exchanges that fostered close cooperation among the institutions and countries. Also, DNAqua-Net accompanied the fourth Joint Danube Survey (JDS4) and conducted the (e)DNA-based surveys for fish, benthic invertebrates, phytobenthos and the sediment community. Last but not least, we have developed many essential pieces of an applied concept for future implementation of DNA-based methods together with various stakeholders at national and international level. Here, of particular importance was the establishment of a working group within the European Standardisation Organisation CEN on DNA and eDNA-based methods (CEN/TC230/WG28). We are particularly grateful also to our colleagues from 'beyond Europe' that have supported us, participated in workshops, discussions and training schools, invited us to their national meetings on DNA and eDNA-based biomonitoring on five continents. The implementation of (e)DNA-based methods into bioassessment and monitoring programs of our rivers, lakes, oceans and the groundwater, will be particularly successful if we sustainably stay connected across countries, generations, cultures and disciplines (Fig. 1). Many of the findings from basic to applied research will be presented at DNAQUA2021. We are particularly pleased that so many early career researchers present their findings. Please take the chance and discuss with them (but not only with them) about their findings. With "Spatial.Chat" we offer you a nice and intuitive environment that allows for some 'real' conference spirit even in these COVID-19 virtual meeting times. Now enjoy two and a half days packed with fascinating insights from (e)DNA-based aquatic biomonitoring. Take the chance, foster and extend your collaborations. We hope to see and discuss with you over the next days at DNAQUA2021 and beyond. THANK YOU! Florian, Agnès, Charly & Alex (Fig. 2)

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data, (undefined)

The Joint Danube Survey (JDS) is a multinational effort in monitoring Danube’s water quality, including its major tributaries. The Danube river stretches over a distance of 2,800 km and flows through or borders 10 different countries to which it is of utter importance as a source of potable water and hydrodynamic power. The JDS is conducted every 6 years and provides a unique opportunity to collect comprehensive data on both abiotic parameters and organisms and to raise awareness of the importance of water as a natural resource. As part of JDS and as a biological quality element in many monitoring programs worldwide, macroinvertebrates are monitored as indicators for various environmental conditions. However, due to their diverse taxonomic composition, associated difficulties with their morphology-based identification as well as their sheer abundance, macroinvertebrates are often analysed with a low taxonomic resolution (i.e., above species level). As an alternative, DNA metabarcoding offers a promising approach to capture this species diversity more accurately. Here, we used DNA metabarcoding to investigate the macrozoobenthic diversity of 46 sites from the latest JDS sampling campaign in 2019. To analyse macroinvertebrate diversity, bulk samples were taken by kick-net sampling and analysed using two different approaches, analysing the bulk sample fixative and analysing homogenised organisms from complete bulk samples. DNA metabarcoding of the sample fixative revealed 1,146 Operational Taxonomic Units (OTUs) and 231 species compared to 833 OTUs and 333 species from homogenised sample analysis. While more dipterans, in particular Chironomidae, were detected in fixative (136 species) than homogenised bulk (90 species) analyses, the latter picked up more Trichoptera (19 vs. 2), Amphipoda (10 vs. 4) and Bivalvia species (13 vs. 5). Even though these results of a DNA-based assessment deliver new insights into species richness and composition of Danube’s macroinvertebrate communities from the Danube source to its delta already, it is evident that the majority of OTUs was not assigned to species. While filling this lack of reference sequences poses a major challenge, the JDS consortium also offers a unique opportunity to complement reference databases in a multinational effort towards a more comprehensive Danube assessment and monitoring.

The hyporheic zone, i.e. the ecotone between surface water and the groundwater, is a rarely studied freshwater ecosystem. Hyporheic taxa are often meiofaunal (

The bird-infecting acanthocephalanPolymorphus minutushas been suggested to comprise different lineages or even cryptic species using different intermediate hosts. To clarify this open question, we investigatedPolymorphuscf.minutuscystacanths originating from amphipod intermediate hosts from 27 sites in Germany and France. Parasites and hosts were identified using integrated datasets (COI and/or morphology for hosts and COI + ITS1-5.8S-ITS2 for parasites).Mitochondrial and nuclear data (ITS1) strongly support the existence of three cryptic species inPolymorphuscf.minutus(type 1-3). These three types reveal a high degree of intermediate host specificity, withPolymorphustype 1 only encountered inGammarus fossarumtype B,Polymorphustype 2 inEchinogammarussp. andEchinogammarus berilloni, andPolymorphustype 3 inGammarus pulexandGammarus roeselii. Our results point to a so far neglected cryptic diversity of the genusPolymorphusin Central Europe. Furthermore,Polymorphustype 2 is most likely a non-native parasite in Germany that co-invaded withE. berillonifrom the Mediterranean area. Potentially, type 3 originates from South-East Europe and migrated to Germany byG. roeselii, where it might have capturedG. pulexas an intermediate host. Therefore, our findings can be seen in the context of ecological globalization in terms of the anthropogenic displacement of intermediate hosts and its impact on the genetic divergence of the parasites.

DNA (meta)barcoding is applied to study biodiversity and is available for standardized biodiversity assessments. However, it suffers from PCR bias, which can lead to the loss of specific taxa. PCR-free techniques such as shotgun metagenomics are therefore thought to be more suited for biodiversity assessments, but are currently limited by incomplete reference libraries. The technique of “mitogenome-skimming” or “mitogenomics,” in which complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria, roughly 99% of all sequencing reads are of non-mitochondrial origin and mostly useless for common applications, e.g. species identification. Here, we present a simple centrifugation protocol that leads to an average 140-fold enrichment of mitochondrial DNA. By sequencing six ‘mock’-communities—comprising the freshwater taxa Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata each—we recovered whole mitochondrial genomes of these species and the acanthocephalan endoparasite Pomphorhynchus laevis. The enrichment protocol will speed up building reference libraries for whole mitochondrial genomes, as dozens of species can be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments, using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without prior enrichment for mitochondria.

DNA barcoding utilizes short standardized DNA sequences to identify species and is increasingly used in biodiversity assessments. The technique has unveiled an unforeseeably high number of morphologically cryptic species. However, if speciation has occurred relatively recently and rapidly, the use of single gene markers, and especially the exclusive use of mitochondrial markers, will presumably fail in delimitating species. Therefore, the true number of biological species might be even higher. One mechanism that can result in rapid speciation is hybridization of different species in combination with polyploidization, that is, allopolyploid speciation. In this study, we analyzed the population genetic structure of the polyploid freshwater snail Ancylus fluviatilis, for which allopolyploidization was postulated as a speciation mechanism. DNA barcoding has already revealed four cryptic species within A. fluviatilis (i.e., A. fluviatilis s. str., Ancylus sp. A–C), but early allozyme data even hint at the presence of additional cryptic lineages in Central Europe. We combined COI sequencing with high-resolution genome-wide SNP data (ddRAD data) to analyze the genetic structure of A. fluviatilis populations in a Central German low mountain range (Sauerland). The ddRAD data results indicate the presence of three cryptic species within A. fluviatilis s. str. occurring in sympatry and even syntopy, whereas mitochondrial sequence data only support the existence of one species, with shared haplotypes between species. Our study hence points to the limitations of DNA barcoding when dealing with organismal groups where speciation is assumed to have occurred rapidly, for example, through the process of allopolyploidization. We therefore emphasize that single marker DNA barcoding can underestimate the true species diversity and argue in strong favor of using genome-wide data for species delimitation in such groups. OA gold - CA Weiss

Cirolana troglexuma Botosaneanu & Iliffe, 1997 is redescribed and a Lucayalana Bruce & Brix, gen. n. established for the species. In total 38 specimens were collected from Hatchet Bay Cave, Eleuthera. Specimens on which previous records of L. troglexuma (from Exuma Cays, Cat Island, and Eleuthera) were based have been re-examined when possible. The diagnostic identifying characters and purported apomorphies for Lucayalana gen. n. are: frontal lamina short, narrow, less than 7% width of labrum, not extending to anterior margin of head; pleonite 3 extending posteriorly to posterior of pleonite 5, laterally overlapping pleonites 4 and 5; ventrally broad, forming a strong ventrally directed blade; pereopods 1–3 merus inferior margin RS not molariform. Mitochondrial COI and 16S loci and the nuclear 18S locus data show that all specimens are the one species. Comparison to additional cirolanid COI sequence data (BOLD, GenBank) show that Lucayalana troglexuma is genetically distinct to all other cirolanid genera with available COI sequences. The single male and females have shared COI (with three females), 16S (eight females) and 18S sequences (two females).

This report presents the outcome of the joint work of PhD students and senior researchers working with DNA-based biodiversity assessment approaches with the goal to facilitate others the access to definitions and explanations about novel DNA-based methods. The work was performed during a PhD course (SLU PNS0169) at the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. The course was co-organized by the EU COST research network DNAqua-Net and the SLU Research Schools Focus on Soils and Water (FoSW) and Ecology - basics and applications. DNAqua-Net (COST Action CA15219, 2016-2020) is a network connecting researchers, water managers, politicians and other stakeholders with the aim to develop new genetic tools for bioassessment of aquatic ecosystems in Europe and beyond. The PhD course offered a comprehensive overview of the paradigm shift from traditional morphology-based species identification to novel identification approaches based on molecular markers. We covered the use of molecular tools in both basic research and applied use with a focus on aquatic ecosystem assessment, from species collection to the use of diversity in environmental legislation. The focus of the course was on DNA (meta)barcoding and aquatic organisms. The knowledge gained was shared with the general public by creating Wikipedia pages and through this collaborative Open Access publication, co-authored by all course participants.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring., This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER).

Niphargus puteanus is the oldest described species of its genus and, in the past, was used as a taxonomic annotation for any subterranean amphipod record. For that reason, no clear knowledge exists about its actual range size and habitat preferences. We here applied a molecular taxonomic and phylogeographical approach based on mitochondrial and nuclear DNA to shed light on its distribution and to infer its demographic history. Furthermore, we analysed aquifer types and water flow regimes to provide a clearer picture of the species’ ecological requirements. Our results indicate that N. puteanus is widely distributed north of the Alps, having its core range in the geomorphological natural region of the ‘South German Scarplands’ (SGS). Additionally, isolated satellite populations exist in the Taunus and the Sauerland, and two single individuals were collected in Luxembourg and in Austria, respectively. The species’ maximal distribution range reaches 756 km between the two single-specimen records and 371 km within the SGS. A very high haplotype diversity was observed, revealing the presence of seven haplotype groups. All the haplogroups were present in the SGS and exhibited distinct spatial-genetic patterns. We thus inferred a high degree of population isolation, with the SGS being a potential long-term refugium for N. puteanus. Historical drift of specimens along major riverine networks (Rhine, Danube) may provide an evolutionary dispersal mechanism explaining the formation of satellite populations. From an ecological perspective, populations of N. puteanus were found to be closely associated with fast-flowing spring environments within fissured aquifers. This species should therefore be classified as both crenophile and stygophile, effectively exploiting resources of surface as well as subsurface spring waters., SCOPUS: ar.j, info:eu-repo/semantics/published

Traditionally, taxonomic characterisation of organisms has relied on their morphology; however, molecular methods are increasingly used to monitor and assess biodiversity and ecosystem health. Approaches such as DNA amplicon diversity assessments are a particularly useful tool when morphology-based taxonomy is difficult or taxa are morphologically ambiguous, for example for freshwater bacteria and fungi as well as many freshwater invertebrate species. DNA metabarcoding provides the ability to distinguish cryptic taxa (which can differ markedly in their ecological requirements and tolerances) and in addition it can provide valuable insights into the genetic and ecological diversity of taxa and ecosystems. While DNA metabarcoding has been used mostly on tissue of sampled specimens, recent years have seen an increased use of metabarcoding on environmental DNA samples: DNA extracted not from sampled specimens, but from the surrounding soil or water. However, the ability of metabarcoding of specimens and metabarcoding of environmental DNA (eDNA) to assess biodiversity and the impact of anthropogenic stressors on freshwater ecosystems is largely understudied. In this talk, several studies that document the advantages and still open challenges of (e)DNA metabarcoding for assessing impacts of environmental stressors on aquatic ecosystems will be presented. These studies, performed in Europe and New Zealand, integrate impacts across different biotic groups, i.e. look at stressor effects on bacterial, protist, fungal and macroinvertebrate communities. Specifically, we use various case studies from freshwater ecosystems to address the following questions: whether eDNA samples, which can be relatively quickly obtained from the water, can act as reliable proxies for catchment-level stressor impacts by comparing these to DNA obtained from local bulk samples, and whether DNA metabarcoding data can also provide quantitative information rather than only presence-absence data. In view of the case studies presented, a perspective on the urgent next steps that need to be taken in order to include genetic tools in routine biomonitoring will be derived and linked to the vision of the international network DNAqua-Net.

Aquatic biomonitoring has become an essential task in Europe and many other regions as a consequence of strong anthropogenic pressures affecting the health of lakes, rivers, oceans and groundwater. A typical assessment of the environmental quality status, such as it is required by European but also North American and other legislation, relies on matching the composition of assemblages of organisms identified using morphological criteria present in aquatic ecosystems to those expected in the absence of anthropogenic pressures. Through decade-long and difficult intercalibration exercises among networks of regulators and scientists in European countries, a pragmatic biomonitoring approach was developed and adopted, which now produces invaluable information. Nonetheless, this approach is based on several hundred different protocols, making it susceptible to issues with comparability, scale and resolution. Furthermore, data acquisition is often slow due to a lack of taxonomic experts for many taxa and regions and time-consuming morphological identification of organisms. High-throughput genetic screening methods such as (e)DNA metabarcoding have been proposed as a possible solution to these shortcomings. Such "next-generation biomonitoring", also termed "biomonitoring 2.0", has many advantages over the traditional approach in terms of speed, comparability and costs. It also creates the potential to include new bioindicators and thereby further improves the assessment of aquatic ecosystem health. However, several major conceptual and technological challenges still hinder its implementation into legal and regulatory frameworks. Academic scientists sometimes tend to overlook legal or socioeconomic constraints, which regulators have to consider on a regular basis. Moreover, quantification of species abundance or biomass remains a significant bottleneck to releasing the full potential of these approaches. Here, we highlight the main challenges for next-generation aquatic biomonitoring and outline principles and good practic, COST - European Cooperation in Science and Technology(CA15219). COST Action DNAqua-Net (CA15219), supported by the COST (European Cooperation in Science and Technology) programme

As a popular game species, roe deer (Capreolus capreolus) have been subjected to a strong anthropogenic influence for centuries. While there have been attempts to introduce roe deer to some German islands, most of these initiatives were based on a small number of individuals and did not result in viable populations. The island population on Fehmarn became established, however, despite being founded by eight individuals with (supposedly) no subsequent restocking. Despite this strong founder effect, previous work has shown the contemporary population not to be genetically impoverished. The reasons for this were not entirely clear. Here, we use 13 microsatellite loci and population genetic techniques to test whether the high diversity resulted from a continuous genetic exchange with the mainland, whether a small number of immigrants maintained heterozygosity at a high level or whether the island was completely isolated and clandestine restocking by hunters another explanation for the observed high diversity. We confirmed that the genetic diversity on Fehmarn was high, but also show that the island population was genetically differentiated from the adjoining mainland. Results from different assignment methods identified a small number of mainland immigrants. Simulations provided support for the feasibility of a scenario of a small number of founders and limited natural immigration maintaining high genetic diversity despite population differentiation. Our simulation results also suggested that there will only be a relatively slight decrease in expected heterozygosity (He) over the next seventy generations and that there will be no need for restocking in the future.

Amphipoda from the superfamily Lysianassoidea Dana, 1849 play an important role in Southern Ocean benthic food webs due to their high biomass, abundance and predominantly scavenging mode of feeding. Our knowledge on the lysianassoid fauna, even in well-studied areas of the Western Antarctic Peninsula, is incomplete. Here we report the findings of an integrated study of lysianassoid amphipods of Potter Cove, King George Island/Isla 25 de Mayo (KGI), combining morphological and molecular species identification (COI barcoding) methods, investigating more than 41,000 specimens from baited traps. For comparison, 2,039 specimens from the adjacent Marian Cove were analysed. Ten lysianassoid species were recorded in the deeper outer Potter Cove, whereas the inner cove (

Background Transitions from marine to intertidal and terrestrial habitats resulted in a significant adaptive radiation within the Panpulmonata (Gastropoda: Heterobranchia). This clade comprises several groups that invaded the land realm independently and in different time periods, e.g., Ellobioidea, Systellomatophora, and Stylommatophora. Thus, mitochondrial genomes of panpulmonate gastropods are promising to screen for adaptive molecular signatures related to land invasions. Results We obtained three complete mitochondrial genomes of terrestrial panpulmonates, i.e., the ellobiid Carychium tridentatum, and the stylommatophorans Arion rufus and Helicella itala. Our dataset consisted of 50 mitogenomes comprising almost all major panpulmonate lineages. The phylogenetic tree based on mitochondrial genes supports the monophyly of the clade Panpulmonata. Terrestrial lineages were sampled from Ellobioidea (1 sp.) and Stylommatophora (9 spp.). The branch-site test of positive selection detected significant non-synonymous changes in the terrestrial branches leading to Carychium (Ellobiodea) and Stylommatophora. These convergent changes occurred in the cob and nad5 genes (OXPHOS complex III and I, respectively). Conclusions The convergence of the non-synonymous changes in cob and nad5 suggest possible ancient episodes of positive selection related to adaptations to non-marine habitats. The positively selected sites in our data are in agreement with previous results in vertebrates suggesting a general pattern of adaptation to the new metabolic requirements. The demand for energy due to the colonization of land (for example, to move and sustain the body mass in the new habitat) and the necessity to tolerate new conditions of abiotic stress may have changed the physiological constraints in the early terrestrial panpulmonates and triggered adaptations at the mitochondrial level. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0735-8) contains supplementary material, which is available to authorized users.

Three new species of the genus Carychium O.F. Müller, 1773, Carychium hardiei Jochum & Weigand, sp. n., Carychium belizeense Jochum & Weigand, sp. n. and Carychium zarzaae Jochum & Weigand, sp. n. are described from the Southeastern United States, Belize and Panama, respectively. In two consecutive molecular phylogenetic studies of worldwide members of Carychiidae, the North and Central American morphospecies Carychium mexicanum Pilsbry, 1891 and Carychium costaricanum E. von Martens, 1898 were found to consist of several evolutionary lineages. Although the related lineages were found to be molecularly distinct from the two nominal species, the consequential morphological and taxonomic assessment of these lineages is still lacking. In the present paper, the shells of these uncovered Carychium lineages are assessed by comparing them with those of related species, using computer tomography for the first time for this genus. The interior diagnostic characters are emphasized, such as columellar configuration in conjunction with the columellar lamella and their relationship in context of the entire shell. These taxa are morphologically described and formally assigned their own names.

DNA (meta)barcoding is used to study biodiversity and is available for standardised assessments. However, it suffers from PCR bias, which can lead to the loss of specific taxa. PCR-free techniques such as shotgun metagenomics are therefore thought to be more suited for biodiversity assessments, but are currently limited by incomplete reference libraries.The technique of ‘mitogenome-skimming’ or ‘mitogenomics’, in which complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria, roughly 99 % of all sequencing reads are of non-mitochondrial origin and mostly useless for common applications, e.g. species identification.Here, we present a simple centrifugation protocol that leads to an average 140-fold enrichment of mitochondrial DNA. By sequencing six ‘mock’- communities – comprising the freshwater taxa Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata each – we recovered whole mitochondrial genomes of these species and the acanthocephalan endoparasite Pomphorhynchus laevis.The presented protocol will greatly speed up building reference libraries for whole mitochondrial genomes, as dozens of species could be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without prior enrichment for mitochondria.

A new species of Eupolybothrus Verhoeff, 1907 discovered in caves of Velebit Mountain in Croatia is described. E. liburnicus sp. n. exhibits a few morphological differences from its most similar congeners, all of which are attributed to the subgenus Schizopolybothrus Verhoeff, 1934, and two approaches to species delimitation using the COI barcode region identify it as distinct from the closely allied E. cavernicolus Stoev & Komerički, 2013. E. spiniger (Latzel, 1888) is redescribed and a lectotype is designated for it as well as E. caesar (Verhoeff, 1899) to stabilize their respective taxonomic status. The subspecies E. acherontis wardaranus Verhoeff, 1937, previously suspected to be a synonym of E. caesar (Verhoeff, 1899), is redescribed and its taxonomy revised after the study of type material whereas the identity of E. acherontis (Verhoeff, 1900) described from a female from southwest Trebinje (Bosnia and Herzegovina) remains unknown. Type material of E. stygis (Folkmanova, 1940) is confirmed to be lost and future designation of neotypes from topotypic specimens is necessary to stabilize its taxonomy. The importance of setal arrangement on the intermediate and 14th tergites and the sexual modifications on the male 15th prefemur for species identification is discussed in the light of present findings, and a review of the species of E. (Schizopolybothrus) that display these traits is also provided.

Ready for the deluge! Introducing the new Metabarcoding and Metagenomics (MBMG) journal

The terrestrial, ellobioid taxon, Carychiidae provides an excellent case study for testing integrative taxonomy and addressing the plethora of historical species designations based on vague morphological characters. Since the Carychiidae are ephemeral, hermaphroditic microgastropods (< 2mm) inhabiting permanently moist aphotic zones of epigean (Carychium O. F. Muller, 1773) and subterranean habitats (eutroglobiont Zospeum Bourguignat, 1856), studies investigating their reproductive system for species delimitation have been futile. Many carychiid species designations were established during the mid-18th to 20th Century heydays of species discovery. Naturalists either split (“splitters”) species by recognizing them according to trivial differences in shell morphology or grouped them (“lumpers”) based upon common morphological traits. The concept of phenotypic variability was not considered or greatly underestimated in these species hypotheses. Although integration of DNA barcodes has since enabled...

The Lusitanian slug, presumed to be native to south-west Europe, was ranked among the 100 worst invading species in Central Europe. However, from the very beginning of its recognition in the presumed invasion area, there was little evidence that the species was actually anthropogenically introduced. We investigated the invasive status of the species by comparing specific predictions on the population genetic structure in the invasion area with the pattern actually found. In a DNA-taxonomy approach, the species could not be found in its presumed native range. Using statistical phylogeographic techniques on a mitochondrial (COI) and nuclear (ZF) marker and species distribution modelling, we could show that the species is with very high probability not an invasor, but native to Central Europe. The study underlines the value of statistical phylogeography in rigorously testing hypotheses on the dynamics of biological invasions.

A new species of the eutroglobiont gastropod taxon Zospeum Bourguignat, 1856 is described. Zospeum tholussum sp. n. is characterized based on a population from the Lukina Jama–Trojama cave system (Ve­lebit Mts., Croatia). A single living specimen occurred at 980 m depth. The species is morphologically related to Zospeum amoenum (Frauenfeld, 1856), but can be readily distinguished from the latter by the presence of a weak columellar fold and its dome-like structured 2nd whorl. DNA barcoding is capable to clearly delineate Z. tholussum from other Zospeum spp. as well.

Biodiversity hotspots are centers of biological diversity and particularly threatened by anthropogenic activities. Their true magnitude of species diversity and endemism, however, is still largely unknown as species diversity is traditionally assessed using morphological descriptions only, thereby ignoring cryptic species. This directly limits evidence-based monitoring and management strategies. Here we used molecular species delimitation methods to quantify cryptic diversity of the montane amphipods in the Irano-Anatolian and Caucasus biodiversity hotspots. Amphipods are ecosystem engineers in rivers and lakes. Species diversity was assessed by analysing two genetic markers (mitochondrial COI and nuclear 28S rDNA), compared with morphological assignments. Our results unambiguously demonstrate that species diversity and endemism is dramatically underestimated, with 42 genetically identified freshwater species in only five reported morphospecies. Over 90% of the newly recovered species cluster inside Gammarus komareki and G. lacustris; 69% of the recovered species comprise narrow range endemics. Amphipod biodiversity is drastically underestimated for the studied regions. Thus, the risk of biodiversity loss is significantly greater than currently inferred as most endangered species remain unrecognized and/or are only found locally. Integrative application of genetic assessments in monitoring programs will help to understand the true magnitude of biodiversity and accurately evaluate its threat status.

Only sparse knowledge exists on amphipod-infecting microsporidians in subterranean waters. Here, we DNA barcoded two sympatric amphipod populations (obligate subterranean Niphargus schellenbergi, facultative Gammarus fossarum) and their microsporidian parasites. Parasite prevalence was assessed by diagnostic PCR assays. Overall prevalence was 82.7 %. Both amphipod populations shared all four identified parasite species (Nosema granulosis, Microsporidium sp. I, Orthosomella sp., Microsporidium sp. BPAR3). Hence, we postulate underground transmission pathways of microsporidians mediated by subterranean amphipod hosts.

Valid taxonomic descriptions are paramount in evolutionary biology. Many date back centuries and are based on ambiguous morphological data. Microgastropods, in particular the taxon Carychiidae (Eupulmonata, Ellobioidea), demonstrate a paucity of informative conchological features. However, as exemplified by Carychium mariae Paulucci, 1878, their taxonomic classification is based almost entirely on these few features. Here we investigated the questionable taxonomic status of Carychium mariae combining DNA barcoding, field-emission scanning electron microscopy and conchological data. This taxon occurs in the Southern Alps, where it shows a sympatric distribution with two widely distributed members of Carychium—C. minimum Muller, 1774 and C. tridentatum (Risso, 1826). Our analyses do not support the species status of C. mariae. In contrast, DNA barcoding reveals a monophyletic grouping of C. minimum and C. mariae specimens with averaged intraspecific variability less than 3.2% (barcoding gap for Carychiidae). Hence, C. mariae is treated and should be regarded as a synonym of C. minimum, just representing a different morphotype. The differentiation and monophyletic status of C. tridentatum can be validated by showing an averaged interspecific variability of 5.9% to C. minimum. In general, we are critical of the sole use of conchological characters for microgastropod taxonomy and strongly recommend the implementation of molecular data (e.g., DNA barcoding) to reevaluate established species designations.

The freshwater amphipod Gammarus fossarum is widely distributed throughout Europe and an important species for stream biomonitoring. It is known to consist of several cryptic species. We here report the complete mitochondrial genome of G. fossarum clade 11/type B with a length of 15,989 bp, encoding for 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. Protein-coding and ribosomal genes have a similar arrangement as in other gammarid amphipods. A phylogenetic analysis clarifies the placement of G. fossarum within the Gammaridae.

The minute troglobitic species, Zospeum isselianum Pollonera, 1887 (Eupulmonata: Ellobioidea, Carychiidae) is widely distributed within its Southern Alpine-Dinaric range. Its broad distribution and highly variable shell has caused this species to be historically lumped into its current taxonomic state of ambiguity. In an integrative taxonomic approach, phenotypic and genotypic data are synthesized to assess the intraspecific variability recently inferred for this taxon. We collected 16 Zospeum specimens in the Slovenian Alpine Arc encompassing the type locality for Z. isselianum. The material comprises five morphologically recognized (sub)species. The species are re-evaluated using SEM, X-ray nanotomography (nano-CT), conchological, histological and molecular data. Four well-defined lineages are present, which can be attributed to i) Z. isselianum s.str. from its new type locality (Turjeva jama), ii) a highly morphologically variable lineage that so far cannot be attributed to a single morphospecies, iii) Z. kupitzense A. Stummer, 1984 (raised to species rank) and iv) a lineage comprising the two subspecies Z. alpestre alpestre (Freyer, 1855) and Z. alpestre bolei Slapnik, 1991 plus Z. isselianum individuals. The latter is treated as a single taxon Z. alpestre. After considering the severely degraded syntype material of Zospeum isselianum, we provide a taxonomic re-description and propose aneotype for this species. Furthermore, new diagnostic information is revealed regarding the columella of Zospeum isselianum and allied species. Detailed anatomical study reveals new structural aspects of Zospeum morphology and provides groundwork for future investigations.