bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of the macroinvertebrate community of the Vjosa river through non-destructive DNA metabarcoding of preservative ethanol
Authors: Brasseur Marie , Vitecek Simon , Zizka Vera , Martini Jan , Wüthrich Remo , Birnstiel Emil , Wilfling Olivia , Singer Gabriel , Leese Florian 
Published: March 2021

Streams and rivers represent hotspots of biodiversity in their natural state. This biodiversity is declining worldwide due to pollution, exploitation and hydromorphological degradation of these systems. One of the last big, natural rivers in Europe is the Vjosa in the Balkan region. The catchment is characterized by natural flow dynamics, resulting in high habitat diversity and turnover, and hosts several sensitive and endemic species (e.g. Isoperla vjosae). DNA metabarcoding represents a promising approach to characterize this biodiversity but methodological drawbacks such as primer bias or incomplete reference databases limit the application, particularly in taxonomically underexplored regions.
Here, we assessed stream biodiversity with a focus on macrozoobenthic (MZB) taxa via a non-destructive, voucher-preserving DNA metabarcoding protocol. In this approach, ethanol used for preservation of multi-habitat samples in the field was used as DNA template, allowing to retain the integrity of the original sample and further comparison of molecular and morphological taxa lists. Samples were taken in spring and autumn 2018 at 48 sites allocated over the Vjosa catchment. The preservative ethanol was filtered through 0.43 um nitrocellulose membranes from which DNA was extracted. Subsequently, a 421 bp fragment of the COI gene was amplified with the primer pair BF2/BR2 and Illumina sequenced. After filtering for sequences with similarity to reference entries &gt;85%, 4,123 OTUs were obtained, of which 921 were identified as MZB taxa. Dipterans and ephemeropterans were most abundant, followed by plecopterans. Some taxa (e.g. molluscs) were not identified due to a known primer bias and &gt;7,000 OTUs could not be assigned above 85 % similarity. Using the here presented voucher-preserving approach allowed us to identify the pitfalls of DNA metabarcoding as tool for biodiversity assessment in taxonomically unexplored regions such as the Vjosa catchment. However, the comparison of specimen abundance data and molecular data showed the power of non-destructive fixative metabarcoding for detecting MZB communities with highly increased taxonomic resolution.
Dots on Earth with huge genetic diversity: relict flatworm Crenobia alpinain the alpine lakes of Tatra Mountains
Authors: Tuhrinová Kornélia , Bozáňová Jana , Čiampor Jr Fedor , Čiamporová-Zaťovičová Zuzana 
Published: March 2021

Mountain lakes, typical signs of an alpine landscape all around the world, were formed in the western part of the Carpathian Arch about 10,000-15,000 years ago, as remnants of the last continental glaciation. In the Tatra Mountains (Slovak-Polish border), more than 110 permanent lakes of glacial origin and many other small ponds exist. These (sub)alpine lakes, with their specific conditions, are very vulnerable and any change in their environment can largely impact their biodiversity, which is, despite long-term faunistic and ecological research, still not fully recognized. This shortcoming could be strongly improved by molecular approaches, which, even within population-genetic studies, often reveal unsuspected cryptic lineages or potential new species.
Our long-term research is aimed at revealing the genetic diversity of aquatic macroinvertebrate species in the alpine environment of the Western Carpathians. One of the currently studied species is the glacial relict flatworm taxon Crenobia alpina (Dana, 1766) (Tricladida: Planariidae), representing an element of the permanent aquatic fauna. Totally, 88 COI haplotypes of C. alpina forming at least five well-separated genetic lineages were identified within 348 individuals collected from 45 lakes and ponds in theTatra Mts. Based on tests of selective neutrality and population stability, C. alpina populations in the Tatra lakes did not overcome recent population expansions or contractions. It seems that great genetic differences between localities and their population stability could be a consequence of the presence of natural topographic barriers (ridges, mountain peaks) dividing the mountains into small and relatively isolated valleys. Moreover, other important factors that could play a significant role are the limited dispersal ability of the species as well as its ability to reproduce asexually. Our findings were also compared with all publicly available sequences (Barcoding of Life Datasystems - BOLD and GenBank) in order to shift our data from a local to a holistic view on C. alpina. Our resultssupport the assumption of Brändle et al. 2017 that C. alpina represents a complex of cryptic lineages or species in Europe. As part of the AquaBOL.sk initiative, the data obtained contribute to the knowledge of genetic variability and barcoding of aquatic organisms in Slovakia.
Year-round monitoring and large-scale screening of native and invasive crayfishes in lotic systems
Authors: Chucholl Franziska , Epp Laura , Segelbacher Gernot 
Published: March 2021

Freshwater crayfish are among the most threatened animal taxa in Central Europe. Effective conservation measures for endangered native and management of invasive alien crayfishes requires knowledge about distribution, monitoring of existing and early detection of newly established populations. eDNA has recently emerged as a promsing, highly sensitive, and non-invasive detection tool in this regard.
To evaluate eDNA as detection tool for freshwater crayfish, we developed a novel set of specific eDNA-assays for all native (Austropotamobius torrentium, Austropotamobius pallipes, Astacus astacus) and the most relevant invasive crayfish species (Pacifastacus leniusculus, Faxonius limosus, Faxonius immunis) in Central Europe. To ensure specificity each primer pair was tested in silico, in vitro, and in situ, including a total of 13 lotic and lentic waterbodies (Fig. 1). Moreover, we assessed the influence of spatio-temporal variables (distance to upstream population, season, and stream size) on eDNA detection in seven streams using two different detection methods (qualitative endpoint PCR and quantitative droplet digital PCR, ddPCR).
The newly developed eDNA assays successfully detected all crayfish species across different lotic and lentic habitats. Multiple linear mixed-effects analysis revealed a significant effect of distance and season on eDNA detection rate (endpoint PCR) and eDNA-concentration (ddPCR). Year-round detection was successful up to 7 km downstream of the source population, although detectability was lowest in winter. eDNA detection rate further decreased with increasing stream size. Finally, eDNA-concentration correlated positively with estimated upstream population size.
Overall, our study provides easily applicable eDNA assays for six crayfish species, enabling year-round detection, which represents a clear benefit over conventional methods. Due to its high sensitivity, eDNA detection is also suitable for the targeted search of as-yet unrecorded or newly emerging populations. Using quantitative ddPCR might further allow for a rough estimation of population size, provided that the identified spatio-temporal factors are accounted for. We therefore recommend implementing eDNA-detection as a complementary survey tool, particularly for a large-scale screening of data-deficient catchments or a year-round monitoring (Chucholl et al. 2021).
eDNA metabarcoding survey of fish communities in the Danube and its tributaries
Authors: Pont Didier , Meulenbroek Paul , Bammer Vinzenz , Dejean Tony , Eros Tibor , Hein Thomas , Jean Pauline , Lenhardt Mirjana , Nagel Christoffer , Pekárik Ladislav , Schabuss Michael , Stoeckle Bernhard , Weigand Alexander , Zornig Horst , Valentini Alice 
Published: March 2021

In complement to the JDS4 traditional fish survey (TFS, mainly by electrofishing), a fish eDNA metabarcoding-based survey has been implemented within the framework of the monitoring program organized by DNAqua-Net and in collaboration with the INTERREG "MEASURES" program. Water samples were collected at 29 sites from the source to the mouth of the Danube River, and at 18 tributaries. Two water samples (mean volume 29 L per sample) were collected at each site and the water filtered in situ. Twelve PCR replicates were performed per sample using teleo primers.
In total, 80 taxa were detected, of which 19 corresponded mainly to farmed fish or food fish due to eDNA release in waste waters, most often downstream of large cities. Of the remaining 61 taxa, 50 taxa were identified at the species level, six taxa at the genus level, and five taxa at a higher taxonomic level. Concerning the Danube river itself, 69 and 57 species/markers were detected along the Danube River by TFS and eDNA surveys, respectively. 50 of these taxa were detected by both methods. Nine species were captured by TFS alone, mainly due to gaps in reference librairies and too colosely related barcodes. Eight species were only detected by eDNA. Except for the Salvelinus group, these were all benthic species, which are difficult to catch by electrofishing in large rivers (Acipenser ruthenus, Acipenser stellatus, Benthophilus sp., Romanogobio uranoscopus, Sabanejewia balcanica, Umbra krameri).
The species richness tended to increase from upstream to downstream and the relative number of DNA sequences per taxa showed a clear succession of species along the river. Barbatula barbatula, Cottus gobio, Hucho hucho, Lampletra planeri, Phoxinus phoxinus and Thymallus thymallus were restricted to the Upper Danube whereas A. ruthenus, Neogobius fluviatilis, S. balcanica and Scardinius erythrophtalmus were detected from Vienna to the Danube river mouth. Abramis brama, Alburnus alburnus, Cyprinus carpio, Silurus glanis, Sander spp, Zingel streber were detected all along the river course; Alosa spp. and Syngnathus abaster downstream from the Iron Gate; A. stellatus and U. krameri only on the most downstream site (Danube delta).
The calculation of a fish index, based on the common metrics used to intercalibrate national fish assessment methods on a European scale, classified most of the sites as being in moderate ecological status. Comparison of the indicative ecological status calculated using the same assessment method based on TSF and eDNA data at the common Danube sites showed a similar classification for six of the 13 sites and a difference of one class for the remaining seven sites.
Development of a set of oligonucleotides for the identification of the Sakhalin sturgeon Acipenser mikadoi Hilgendorf, 1892 by PCR
Authors: Turanov Sergei 
Published: March 2021

Sturgeons (family Acipenseridae) are valuable commercial fish and aquaculture resources. The Acipenser genus includes about 20 species, 12 of which are recorded for the Russian territory. One of the rarest is the Sakhalin sturgeon A. mikadoi Hilgendorf, 1892. At present, its population size is significantly decreased and the species is close to extinction. Natural populations of the species have survived in the Tumnin River in Khabarovsk Region and the Viakhtu River in Sakhalin Region. Vihtu River in Sakhalin Region. Due to the small numbers of rare and endangered fish species, noninvasive approaches, including the use of DNA from the environment, appear to be the most suitable for monitoring their species and genetic diversity. The method is well established in this area and has been successfully tested for the monitoring of several species of rare and endangered sturgeons. This paper presents the results of the development of primers specific for the mitochondrial DNA of the species A. mikadoi.
A matrix of 15 sequences of complete mitochondrial genomes from 3 sturgeon species whose ranges may overlap - A. mikadoi (KX276658), A. dauricus (KJ402277) and A. schrenckii (MH973728- MH973734, KX276660, KX276659, KF150287, KC905169, KC820796) - was generated. A. medirostris (NC_028405), which is genetically closest to the Sakhalin sturgeon, was also included in the analysis. A sliding window algorithm was applied to the sequence matrix. A graph showing the distribution of divergence values along the mitochondrial genome of sturgeon was plotted based on the results of the analysis Fig. 1.
Based on matrices of individual genome fragments (16S rRNA, COI, ND1, and D-loop), a search for species-specific primers was performed in the DECIPHER program. A Primer-BLAST algorithm was run based on each pair of primers. Taxa Actinopterygii and Acipenseridae were used as references separately in different runs. In addition, primers were checked using local reference sequences of the complete mitochondrial genome of representatives of the genus Acipenser. The results of primer evaluation are summarized in the table Table 1. As a result, a combination of primer pairs developed on the basis of D-loop and COI fragments seems to be the most successful for error-free identification of Sakhalin sturgeon.
Strategy for Successful Integration of eDNA-based Methods in Aquatic Monitoring
Authors: Blancher Philippe , Lefrançois Estelle , Rimet Frederic , Bouchez Agnès 
Published: March 2021

Recent developments in the use of environmental DNA are opening up new horizons for the assessment of the quality of aquatic environments. These rapid and cost-effective methods, in very swift progress, will potentially offer the opportunity to identify all the taxa present in an environmental sample (water or biota) by the use of complementary markers. The produced inventories can then be used for the assessment of biodiversity and ecological quality. However, the inclusion of these new DNA-based methods in monitoring practices is not straightforward and requires harmonised actions in the coming years at national and international levels.
In order to foresee and stimulate such a harmonised implementation, the European network DNAqua-Net (COST Action CA15219) brought together some of its members, experts of ECOSTAT and other environmental biomonitoring stakeholders from different European countries. Through workshops, bringing together 51 participants in 7 sub-groups in April 2020, an implementation roadmap was designed. The coordinated actions to be taken in the different countries, and the possible collaborations and steps to be taken at the EU level were identified.
This presentation will give an overview of all discussions (Lefrançois et al. 2020) reflecting the diversity of situations in Europe, as well as common views. We will highlight important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems to the horizon of 2030.
Macroinvertebrate community assessment and biomonitoring of European water bodies. Is a multimarker approach necessary?
Authors: Villaescusa Juan Antonio , Villena Mª José , González Raquel , Picazo Antonio , Rojo Verónica , Nieminem Marko , Classen Silke , Pujante Ana , Camacho Antonio 
Published: March 2021

The present work, included in the European Project BIOWAT, aims to develop and validate the use of genomic tools or metabarcoding for its utilization as a routine method for river biomonitoring in different European Biogeographical regions. The project included sampling points in three biogeographic regions, Mediterranean (Spain), Continental (Germany) and Boreal (Finland). The current development of the study was designed using mock communities obtained from the three mentioned areas and different aspects were tested: DNA extraction methods, selection of informative region (16S vs COI), design and performance of primers, bioinformatic pipeline, etc…Although the use of COI has become very popular, and its barcode database is more complete, the use of mitochondrial 16S as taxonomic marker can provide similar or even better results when accompanied by a rich local barcode database Elbrecht 2016. In this presentation, the results and conclusions obtained for the biomonitoring of nine rivers (3 for each of the biogeographic regions) using 16S as DNA marker and a local barcode database are shown. The results of ecological status assignment using 16S marker were promising, showing a good correlation between morphological determinations and metabarcoding data for most of the studied rivers. However, in some cases, the metabarcoding data showed a jump in the ecological status class (to better or worst status), highlighting the existence of some problems related with primers (for some taxonomic groups) or missing taxa in the barcode database that still need to be solved prior the utilization of this method on a routine basis.Additionally, for the studied Mediterranean rivers, a complementary analysis using COI as marker was made, using the universal primers developed by Elbrecht and Leese 2017. In general, this marker showed better results in the identification for some taxa, whereas other included in the mock communities were not identified showing important problems that could be related with primers (sometimes not well covering characteristic taxa present in other biogeographic regions) or the lack of a complete COI macroinvertebrate barcode databases in the Iberian Peninsula for the case of Spain Múrria 2020.Our results show that both markers have the potential to produce a good identification of benthic macroinvertebrates, showing an acceptable correlation between morphology and metabarcoding approaches. However, none of them is able to amplify all of the present groups, so the parallel use of both markers (mitochondrial 16S and COI) in a multimarker approach could solve some of the problems, giving a more complete profile of the macroinvertebrate community. This approach has already been proposed and can lead the future of macroinvertebrate community assessment Ficetola 2020, Martins 2020.
Quantitative eDNA estimates show a strong correlation with Northern Pike biomass in a controlled environment
Authors: Karlsson Erik , Sundblad Göran , Ogonowski Martin , Sundin Josefin , Svensson Ofir , Vasemägi Anti 
Published: March 2021

Northern pike, Esox Lucius, is one of the most important species for recreational fisheries in Sweden and as a top-predator; it holds an important role in the food web. Despite its ecological and socioeconomic significance, pike is largely neglected in current monitoring programs because it is seldom caught by conventional monitoring methods. As a result, there is not sufficient data for management authorities to employ biologically justified management strategies. To be able to further understand pike ecology and monitor populations, new monitoring methods are needed. Recent developments in environmental DNA (eDNA) has shown the potential to produce (semi-)quantitative estimates of fish biomass. However, the amount of eDNA in water may vary depending on temperature, fish size, age and density, and it is therefore important to first evaluate the effect of biotic and abiotic factors on eDNA biomass relationships. Here, we evaluated the relationship between eDNA concentration and individual biomass of pike by keeping adult fish of varying size (0.75 – 3.41 kg, n = 11) individually in outdoor mesocosms (~7000 L) filled with water from the adjacent Lake Mälaren. Samples were collected by filtering water through a combination of cellulose nitrate and glass microfiber filter membranes. After a comparison of different extraction methods, the eDNA was extracted using Chelex 100 resin, a low-cost and fast method. Samples were subsequently diluted in 1:8 to alleviate problems from inhibition. A TaqMan assay targeting Cytochrome Oxidase I developed by Olsen et al.(Olsen et al., 2016, 2015) was used to quantify pike eDNA. The results show a strong positive relationship between pike biomass and pike eDNA indicating that the latter represents a promising tool for estimating pike biomass in natural waters.
References:
Olsen, J.B., Lewis, C.J., Massengill, R.L., Dunker, K.J., Wenburg, J.K., 2016. Erratum to: An evaluation of target specificity and sensitivity of three qPCR assay for detecting environmental DNA from Northern Pike (Esox lucius). Conservation Genetics Resources 8.
Olsen, J.B., Lewis, C.J., Massengill, R.L., Dunker, K.J., Wenburg, J.K., 2015. An evaluation of target specificity and sensitivity of three qPCR assays for detecting environmental DNA from Northern Pike (Esox lucius). Conservation Genetics Resources 7.
101 lakes - lentic fish community monitoring using eDNA metabarcoding
Authors: Hänfling Bernd , Willby Nigel , Lawson-Handley Lori , Sellers Graham 
Published: March 2021

We have recently developed and deployed methods for environmental DNA (eDNA) based monitoring of lake fish communities in the UK. This approach combines eDNA with modern High-Throughput-Sequencing technology, so-called eDNA metabarcoding. This non-invasive method has proven to be more effective at detecting elusive species than established invasive surveying techniques such as electro fishing or fyke netting and can provide meaningful semi-quantitative abundance estimates. The UK Environment Agencies have funded the collection of an eDNA meta-barcoding data set of vertebrates from 101 UK lakes covering a broad spectrum of environmental conditions Fig. 1. This dataset is based on analysing 20 water samples per lake and has successfully been used to develop a WFD compatible water quality assessment tool. In its current form this tool is suitable for reporting the status of fish in water bodies where eutrophication is the dominant pressure.
DNA is not homogeneously distributed in lentic environments and hence the detection of species relies on the collection of an adequate number of samples from a water body to capture the eDNA signal. Previous analyses on a subset of lakes have indicated that the number of samples used for the 101 lake fish data set is more than sufficient to reliably estimate species richness of lakes, but it is unclear how exactly reduced sampling effort affects other biodiversity estimates and inferences made about water quality. As the number of samples determines the cost of monitoring programmes it is essential that the sampling effort is optimised for a specific monitoring objective. The objective of this study was to explore the effect a reduced sampling effort would have on various biological inferences using algorithmic and statistical resampling techniques. with a much lower number of samples. For example, almost 90% of lakes achieved a sample coverage of 95% with only 10 samples. However, rare species are more often missed with fewer samples, with implications for monitoring programs of invasive or endangered species. Estimates of community composition and the ecological quality ratio (EQR) responded slowly to decreasing sampling effort. For example, subsets of 10 samples were in most cases much more closely related to each other than to sample sets from other lakes and showed very similar Ecological Quality Ratios. These results are able to inform the design of eDNA sampling strategies, so that these can be optimised to achieve specific monitoring goals.
Accès à l'énergie en Europe
Authors: Le Roux Dominique 
Published: March 2021

Résumé La transformation du secteur de l’énergie électrique et gazière en Europe sous l’effet des directives européennes a occasionné en France une série de changements considérables : c’est l’ensemble du modèle français développé sur le long terme et soutenu par la puissance publique, de même que sa conception du service public, qui ont été alors remis en cause. La France est passée en quelques années d’un monopole intégré et d’un système de planification à un modèle dominé par la stratégie commerciale et concurrentielle à l’échelle européenne et mondiale.