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DNA barcodes (COI) of Troglocobitis starostini, endemic to a single site in Turkmenistan, were analysed and put into the taxonomic context of the large group of nemacheilid loaches known from Western and Central Asia. All applied phylogenetic tree-based analyses place the species into the genus Paracobitis. This finding supports previous morphological studies. While the exact position of Troglocobitis starostini within Paracobitis was not resolved unambiguously, it was constantly recovered within Paracobitis, irrespective of the tree reconstruction method applied. With a minimum interspecific K2P distance of 7.19% P. persa was the closest hit in our dataset, which comprised a total of ten species of Paracobitis, which showed an average interspecific K2P distance of 5.43% (range 2.78–9.44%).

Insect declines and biodiversity loss have attracted much attention in recent years, but lack of comprehensive data, conflicting interests among stakeholders and insufficient policy guidance hinder progress in preserving biodiversity. The project DINA (Diversity of Insects in Nature protected Areas) investigates insect communities in 21 nature reserves in Germany. All selected conservation sites border arable land, with agricultural practices assumed to influence insect populations. We taught citizen scientists how to manage Malaise traps for insect collection, and subsequently used a DNA metabarcoding approach for species identification. Vegetation surveys, plant metabarcoding as well as geospatial and ecotoxicological analyses will help to unravel contributing factors for the deterioration of insect communities. As a pioneering research project in this field, DINA includes a transdisciplinary dialogue involving relevant stakeholders such as local authorities, policymakers, and farmers, which aims at a shared understanding of conservation goals and action pathways. Stakeholder engagement combined with scientific results will support the development of sound policy recommendations to improve legal frameworks, landscape planning, land use, and conservation strategies. With this transdisciplinary approach, we aim to provide the background knowledge to implement policy strategies that will halt further decline of insects in German protected areas.

The Oxynoemacheilus bergianus species group is revised based on tree topology (ML, NJ, MP), distance (K2P and ASAP) and Poisson tree process analyses of DNA barcode data tested against morphometric and morphological characters including colour patterns. The O. bergianus species group is distinguished from other Oxynoemacheilus groups based on morphological characters: its constituent species have a slender caudal peduncle, a suborbital flap in the male, a mottled or blotched colour pattern, and lack bold, black spots on the caudal-fin base. It is also supported as a monophyletic unit in our molecular analysis. The O. bergianus group includes 10 molecular clades following congruently well-supported NJ, MP and ML based entities. Species described as O. bergianus, O. banarescui, O. erdali, O. fatsaensis, O. samanticus, and O. simavicus from Turkey, O. lenkoranensis from Azerbaijan, and O. longipinnis and O. parvinae from Iran belong to this species group. The group includes also four unnamed molecular clades. We were unable to detect external differences between any of the molecular clades in colour pattern or any morphometric or morphological characters examined. In the 10 molecular clades in the O. bergianus species group, the intraclade K2P distance ranges from 0.0–1.8% while the distances between molecular clades ranges from 0.6–5.9%. To resolve the species diversity of this group, we also analysed the intraspecific and interspecific variability in the K2P distance of DNA barcode data from 53 other Oxynoemacheilus species. Here, the intraspecific variability ranges from 0.0–2.4% while the interspecific K2P distance ranges from 1.2–20.8%. In the O. bergianus species group, only four groups are detected by the mPTP species delimitation approach distinguished by a K2P distance of 2.9% or more. We treat these four groups as valid species, corresponding to O. banarescui, O. bergianus, O. fatsaensis, and O. simavicus. Oxynoemacheilus samanticus from the Kızılırmak and Seyhan drainages, O. lenkoranensis from the Caspian basin, O. erdali from the Euphrates, and O. longipinnis and O. parvinae from the Tigris drainage are treated as synonyms of O. bergianus. Fishes from an unnamed molecular clade from the upper Tigris, and from a second unnamed clade from the upper Euphrates, are both identified as O. bergianus. Oxynoemacheilus bergianus might be a junior synonym of O. bergi from the Kura. The distribution range of O. simavicus, described from the Simav drainage in the Marmara basin, is expanded to the east and two molecularly differentiated population groups occur in the Sakarya drainage, the Büyük Melen River and potentially in other adjacent coastal streams. Oxynoemacheilus fatsaensis, described from the coastal stream Elekçi in northern Anatolia, is also widespread in the Yeşilırmak drainage. Morphological characters proposed to distinguish O. fatsaensis from the other species of the O. bergianus group could not be confirmed by our data on fishes from the Yeşilırmak. This study also discusses the theoretical background, our reasons for conducting this revision in the way we did, and what the alternatives would be.

With increased application of DNA metabarcoding in fast and high-resolution biodiversity assessment, various laboratory protocols have been optimised in recent years and their further evaluation is subject of current research. Homogenisation of bulk samples and subsequent DNA extraction from destructed tissue is one way of starting the metabarcoding process. This essential step in the protocol can either be conducted from wet sample material (e.g. bulk insect samples) soaked in fixative or from completely dried individuals. While the latter method appears to produce more consistent results, it is time consuming and more prone to cross-contamination. We tested both homogenisation approaches with regard to time efficiency and biodiversity assessment of complex arthropod bulk samples, in particular how the amount of processed tissue affects taxon recovery. Both approaches reveal similar taxa compositions and detect a similar total OTU diversity in a single extraction reaction. Increased amounts of tissue used in DNA extraction improved OTU diversity detection and recovered particularly specific low-biomass taxa, making this approach valuable for samples with high biomass and/or diversity. Due to less handling time and lower vulnerability for cross-contamination we recommend the processing of wet material when sample homogenisation is applied.

This study summarizes results of a DNA barcoding campaign on German Diptera, involving analysis of 45,040 specimens. The resultant DNA barcode library includes records for 2,453 named species comprising a total of 5,200 barcode index numbers (BINs), including 2,700 COI haplotype clusters without species‐level assignment, so called “dark taxa.” Overall, 88 out of 117 families (75%) recorded from Germany were covered, representing more than 50% of the 9,544 known species of German Diptera. Until now, most of these families, especially the most diverse, have been taxonomically inaccessible. By contrast, within a few years this study provided an intermediate taxonomic system for half of the German Dipteran fauna, which will provide a useful foundation for subsequent detailed, integrative taxonomic studies. Using DNA extracts derived from bulk collections made by Malaise traps, we further demonstrate that species delineation using BINs and operational taxonomic units (OTUs) constitutes an effective method for biodiversity studies using DNA metabarcoding. As the reference libraries continue to grow, and gaps in the species catalogue are filled, BIN lists assembled by metabarcoding will provide greater taxonomic resolution. The present study has three main goals: (a) to provide a DNA barcode library for 5,200 BINs of Diptera; (b) to demonstrate, based on the example of bulk extractions from a Malaise trap experiment, that DNA barcode clusters, labelled with globally unique identifiers (such as OTUs and/or BINs), provide a pragmatic, accurate solution to the “taxonomic impediment”; and (c) to demonstrate that interim names based on BINs and OTUs obtained through metabarcoding provide an effective method for studies on species‐rich groups that are usually neglected in biodiversity research projects because of their unresolved taxonomy.

It was reported that the complex geological history of the southern Balkans caused diversification of terrestrial species. On the other hand, knowledge of the evolutionary history of freshwater biota is scarce. In order to contribute to an understanding of the historical biogeography of the region’s freshwaters, as well as to describe intraspecific diversity and structure of a species distributed in the Ionian Ecoregion, we have analysed the population genetic structure and evolutionary history of Telestes pleurobipunctatus. Analyses were based on the mitochondrial gene for cytochrome b from samples covering the whole distribution range of this species. A high level of intraspecific structuring was revealed inside T. pleurobipunctatus. Its evolutionary history corroborates the complexity of historical geologic events in the area. Telestes pleurobipunctatus comprises two main clades (northern and southern), which have been separated from each other for about seven million years and that are further divided. The Louros and Arachthos Rivers were denoted as ancestral geographic ranges of the northern clade. The distribution range of the ancestor of the southern clade seems to comprise rivers flowing into the Patraikos Gulf. Structures within the two main clades, as well as timing of diversification events are different, implying separate evolutionary courses.

The Glyptothorax species inhabiting the Euphrates and Tigris drainages are reviewed and six species are recognised, one of which is described herein as new species. Glyptothorax armeniacus is endemic to headwater streams in the Euphrates drainage. Glyptothorax kurdistanicus is endemic to the upper Tigris downstream to the Lesser Zab drainage. Glyptothorax cous and G. steindachneri are riverine species widespread in both the Euphrates and Tigris drainages. Glyptothorax silviae is endemic to Iran. Glyptothorax daemon, new species, from the Greater Zab and Yanarsu in the upper Tigris drainage, is distinguished by having the thoracic adhesive apparatus strongly elevated, 1.1–1.2 times longer than wide, without tubercles on the head, well developed anteromedial striae, the medial pit without striae, and a short adipose fin. Glyptothorax daemon is separated into two mitochondrial lineages, externally indistinguishable and separated by a minimum K2P distance of 2.0% in the DNA barcode region. These lineages are paraphyletic in our analysis indicating past introgressive hybridisation with G. cous. All six species are diagnosed and all, except unstudied G. steindachneri, form distinct mitochondrial clades with between 1.2% and 3.4% minimum K2P distance between them. Species from the Euphrates and Tigris form a monophyletic mitochondrial group separated from 53 other Glyptothorax species studied from India and areas further east.

Oxynoemacheilus phasicus, new species, is described from the Rioni and Enguri River drainages in Georgia. It is distinguished from other Oxynoemacheilus species in the O. brandtii group by a mottled or marbled flank pattern in adults, a slightly emarginate caudal fin, and a deep caudal peduncle. Molecular data suggest that the new species is characterized by a minimum K2P distance of 7.5% from O. brandtii from the Kura drainage in the mtDNA COI barcode region. Oxynoemacheilus brandtii and O. elsae are re-diagnosed. A very slender Oxynoemacheilus from the Aras drainage clusters as sister to O. elsae in our molecular analysis and not with O. brandtii from the Kura River. However, it is identified as O. brandtii as it is indistinguishable from this species in morphological characters.

Oxynoemacheilus shehabi, new species, is described from the upper Orontes in southern Syria. It is distinguished from other Oxynoemacheilus species in the Levant by possession of a complete lateral line, a deeply emarginate caudal fin, a narrow caudal peduncle, a suborbital groove in male individuals, a well-developed pelvic axillary lobe, and 5–7 dark-brown bars on the flank. Molecular data suggest that the new species is characterised by a minimum K2P distance of 3.8% to O. “seyhanicola” in the COI mtDNA barcode region.

Previous studies on the phylogenetic relationships between lamprey species relied either on a low number of morphological characters related to the feeding apparatus, or on a low number of molecular mitochondrial DNA markers. Here, we apply a multilocus approach to assess the phylogenetic relationships of northern hemisphere lampreys, with a special emphasis on the 17 European species. The study comprises two mitochondrial (cytochrome c oxidase subunit 1 gene—DNA barcodes, and cytochrome b gene) a nd t wo n uclear (internal transcribed spacers I and II) markers to investigate species' phylogenetic affinities. The phylogeny obtained with mitochondrial markers revealed a clear and highly supported separation of all northern hemisphere lampreys. Among those, our multilocus results show several polyphyletic genera, stressing the need for a taxonomic revision in a near future. Lampetra morii (Berg, 1931) from East Asia, often included in Eudontomyzon, is placed in the genus Lethenteron. Lampetra richardsoni Vladykov & Follett, 1965 and Entosphenus hubbsi (Vladykov & Kott, 1976) should be placed in a new genus, as well as the southern populations of Lethenteron camtschaticum (Tilesius, 1811) and Lethenteron reissneri (Dybowski, 1869). Considering European species, our results argue for a taxonomic revision of Eudontomyzon, with emphasis on Eudontomyzon vladykovi Oliva & Zanandrea, 1959. RESUMEN: Estudios anteriores sobre las relaciones filogenéticas entre especies de lampreas, se basan en un escaso número de caracteres morfológicos relacionados con las estructuras de alimentación, o en un bajo número de marcadores moleculares de ADN mitocondrial. Aquí aplicamos un enfoque multilocus para evaluar las relaciones filogenéticas de las lampreas del hemisferio norte, con especial énfasis en 17 especies europeas. Este estudio comprende dos marcadores mitocondriales (citocromo oxidasa c subunidad 1 —ADN barcodes, y citocromo oxidasa b) y dos marcadores nucleares (espaciadores transcritos internos I y II (ITS) ) para investigar la afinidad filogenética de las especies. La filogenia obtenida con losmarcadores mitocondriales reveló una separación clara y altamente apoyada de todas las lampreas del hemisferio norte. Entre ellas, nuestros resultados multilocus muestran varios géneros polifiléticos, lo que resalta la necesidad de una revisión taxonómica en el futuro. Lampetra morii (Berg, 1931) de Asia oriental, a menudo incluida en Eudontomyzon, se sitúa en el género Lethenteron. Lampetra richardsoni Vladykov & Follett, 1965 y Entosphenus hubbsi (Vladykov & Kott, 1976) deben colocarse en un nuevo género, así como las poblaciones meridionales de Lethenteron camtschaticum (Tilesius, 1811) y Lethenteron reissneri (Dybowski, 1869). Considerando las especies europeas, nuestros resultados abogan por una revisión taxonómica de Eudontomyzon, con énfasis en Eudontomyzon vladykovi Oliva & Zanandrea, 1959. Fundação para a Ciência e Tecnologia - FCT info:eu-repo/semantics/publishedVersion

BackgroundDragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches.MethodsThis study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD.ResultsOver 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation inCordulia aenea(Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database are highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.

The nemacheilid genus Seminemacheilus is revised. Six species are recognised, three of them described herein as new. All species are endemic to Central Anatolia. Seminemacheilus lendlii from the Sakarya River drainage and the endorheic Lake Aksehir and Eber basins, S. ispartensis from Lake Eğirdir basin and S. ahmeti from Sultan Sazlığı are valid species. Although Seminemacheilus lendlii and S. ispartensis have almost identical COI sequences, they are distinguished by the shape of the caudal peduncle and the presence of scales on the caudal peduncle in S. ispartensis (vs. absent in S. lendlii). Seminemacheilus attalicus, new species, from Kırkgöz drainage, is distinguished by having a slightly emarginated caudal fin, a central pore in the supratemporal canal and a marbled flank pattern. Seminemacheilus ekmekciae, new species, from Lake Tuz basin is distinguished by having a roundish caudal fin, a short post-dorsal length and large brown blotches fused into stripes on the flank. Seminemacheilus tubae, new species, from Lake Beyşehir basin, is distinguished by having a truncate caudal fin and 2–5 (6) supraorbital head pores. Seminemacheilus dursunavsari from the Göksu River drainage is not a valid name and this population is identified as S. tubae.

Although aquatic macroinvertebrates and freshwater fishes are important indicators for freshwater quality assessments, the morphological identification to species-level is often impossible and thus especially in many invertebrate taxa not mandatory during Water Framework Directive monitoring, a pragmatism that potentially leads to information loss. Here, we focus on the freshwater fauna of the River Sieg (Germany) to test congruence and additional value in taxa detection and taxonomic resolution of DNA barcoding vs. morphology-based identification in monitoring routines. Prior generated morphological identifications of juvenile fishes and aquatic macroinvertebrates were directly compared to species assignments using the identification engine of the Barcode of Life Data System. In 18% of the invertebrates morphology allowed only assignments to higher systematic entities, but DNA barcoding lead to species-level assignment. Dissimilarities between the two approaches occurred in 7% of the invertebrates and in 1% of the fishes. The 18 fish species were assigned to 20 molecular barcode index numbers, the 104 aquatic invertebrate taxa to 113 molecular entities. Although the cost-benefit analysis of both methods showed that DNA barcoding is still more expensive (5.30-8.60€ per sample) and time consuming (12.5h), the results emphasize the potential to increase taxonomic resolution and gain a more complete profile of biodiversity, especially in invertebrates. The provided reference DNA barcodes help building the foundation for metabarcoding approaches, which provide faster sample processing and more cost-efficient ecological status determination.

There are particular challenges in defining the taxonomic status of recently radiated groups due to the low level of phylogenetic signal. Members of the Salmo trutta species-complex, which mostly evolved during and following the Pleistocene, show high morphological and ecological diversity that, along with their very wide geographic distribution, have led to morphological description of 47 extant nominal species. However, many of these species have not been supported by previous phylogenetic studies, which could be partly due to lack of significant genetic differences among them, the limited resolution offered by molecular methods previously used, as well as the often local scale of these studies. The development of next-generation sequencing (NGS) and related analytical tools have enhanced our ability to address such challenging questions. In this study, Genotyping-by-Sequencing (GBS) of 15,169 filtered SNPs and mitochondrial DNA (mtDNA) D-loop sequences were combined to assess the phylogenetic relationships among 166 brown trouts representing 21 described species and three undescribed groups collected from 84 localities throughout their natural distribution in Europe, west Asia, and North Africa. The data were analysed using different clustering algorithms (admixture analysis and discriminant analysis of principal components-DAPC), a Bayes Factor Delimitation (BFD) test, species tree reconstruction, gene flow tests (three- and four-population tests), and Rogue taxa identification tests. Genomic contributions of the Atlantic lineage brown trout were found in all major sea basins excluding the North African and Aral Sea basins, suggesting introgressive hybridization of native brown trouts driven by stocking using strains of the Atlantic lineage. After removing the phylogenetic noise caused by the Atlantic brown trout, admixture clusters and DAPC clustering based on GBS data, respectively, resolved 11 and 13 clusters among the previously described brown trout species, which were also supported by BFD test results. Our results suggest that natural hybridization between different brown trout lineages has probably played an important role in the origin of several of the putative species, including S. marmoratus, S. carpio, S. farioides, S. pellegrini, S. caspius (in the Kura River drainage) and Salmo sp. in the Danube River basin. Overall, our results support a multi-species taxonomy for brown trouts. They also resolve some species in the Adriatic-Mediterranean and Black Sea drainages as members of very closely related genomic clusters that may need taxonomic revision. However, any final conclusions pertaining to the taxonomy of the brown trout complex should be based on an integrative approach combining genomic, morphological, and ecological data. To avoid challenges in taxonomy and conservation of species complexes like brown trouts, it is suggested to describe species based on genomic clusters of populations instead of describing species based only on morphologically differentiated single type populations.

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 ofGymnocephalus cernua(Linnaeus, 1758) and also confirm the second occurrence of invasiveRhinogobius 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.

DNA metabarcoding of macroinvertebrates is increasingly used for aquatic bioassessment and -monitoring. A major strength of metabarcoding is the high taxonomic resolution provided while the inability to deliver reliable abundance data is regarded a main drawback. Data on the potential of metabarcoding to disentangle site-specific and seasonal variation with presence/absence data have been poorly explored. In addition, the robustness of ecological status assessments compliant with the European Water Framework Directive (Directive 2000/60/EG) have only been compared in few studies, none of which adressed changes across seasons. Therefore, we here investigated seasonal as well as site-specific effects on macroinvertebrate communities at six river sites in a near-natural (Sieg) and urban (Emscher) German river through metabarcoding. Furthermore, we evaluated the usability of the method for ecological status assessments. Our data showed distinct seasonal effects on OTU composition at the near-natural river Sieg. However, ecological status assessment was constant through seasons and comparable to assessments based on available morphological identification. In contrast to the river Sieg, we found strong site and stressor-specific impacts in the heterogeneous urban river Emscher. Here, ecological status assessment varied between sampling sites ranging from good to bad status but was largely consistent between seasons. Our study demonstrates the ability of presence/absence metabarcoding data to reliably assess invertebrate community composition in streams and infer environmental (natural or anthropogenic) impacts. Together with the technical robustness, our study encourages the wider adoption of the technique in stream bioassessment and -monitoring.

Today, one of the challenges facing modern biology is to develop an accurate, reliable and cost- efficient method for the rapid identification of organisms to species level. Here, we combine high resolution melting (HRM) analysis with DNA barcoding (Bar-HRM) and evaluated its efficiency to discriminate between 215 specimens of 22 common European cyprinid species. After screening the standard barcode region of the mitochondrial cytochrome oxidase gene subunit I (COI) for a distinct fragment, a short region (142 bp) with sufficient sequence diversity was chosen. Using the selected mini-barcode, Bar-HRM analysis was performed to test the potential of this closed- tube method to reveal genetic variation between the cyprinids. To evaluate inter-run reproducibility several Bar-HRM runs were performed on different days using between 8 and 10 specimens per species, resulting in only minor variations in the amplification curves and melting temperatures. The technique was also found to be highly sensitive to single base changes, allowing the detection of geographic variants. 92% of the specimens were unambiguously assigned to the correct cyprinid species whereas in seven individuals the presence of ambiguities prevents the assignment; in case of Barbus barbus the method failed. We provide a guideline for setting up a simple molecular identification tool for unknown samples from a pool of target species with one pair of mini-barcode primers and reduced contamination risk. We demonstrate that Bar-HRM may provide a reliable and simple alternative to Sanger-sequencing based identification in certain applications with the potential to significantly reduce time (up to 241 h) and labor costs (up to 88%) during the process of identification.

1.In recent years, large-scale DNA barcoding campaigns have generated an enormous amount of COI barcodes, which are usually stored in NCBI's GenBank and the official Barcode of Life database (BOLD). BOLD data are generally associated with more detailed and better curated meta-data, because a great proportion is based on expert-verified and vouchered material, accessible in public collections. In the course of the initiative German Barcode of Life (GBOL), data were generated for the reference library of 2,846 species of Coleoptera from 13,516 individuals. 2.Confronted with the high effort associated with the identification, verification and data validation, a bioinformatic pipeline, “TaxCI” was developed that i) identifies taxonomic inconsistencies in a given tree topology (optionally including a reference data set), ii) discriminates between different cases of incongruence in order to identify contamination or misidentified specimens, iii) graphically marks those cases in the tree, which finally can be checked again and, if needed, corrected or removed from the dataset. For this, “TaxCI” may use DNA-based species delimitations from other approaches (e.g., mPTP) or may perform implemented threshold-based clustering. 3.The data-processing pipeline was tested on a newly generated set of barcodes, using the available BOLD records as a reference. A data revision based on the first run of the TaxCI tool resulted in the second TaxCI analysis in a taxonomic match ratio very similar to the one recorded from the reference set (92 vs 94%). The revised dataset improved by nearly 20% through this procedure compared to the original, uncorrected one. 4.Overall, the new processing pipeline for DNA barcode data allows for the rapid and easy identification of inconsistencies in large datasets, which can be dealt with before submitting them to public data repositories like BOLD or GenBank. Ultimately, this will increase the quality of submitted data and the speed of data submission, while primarily avoiding the deterioration of the accuracy of the data repositories due to ambiguously identified or contaminated specimens. This article is protected by copyright. All rights reserved.

Pseudophoxinus cilicicus, new species, is described from the Arsuz, Ceyhan and Seyhan river drainages in the Gulf of İskenderun. It is distinguished from other members of the Pseudophoxinus zeregi species group by having a complete lateral line with 38–45 + 2–3 scales, the lower lip usually slightly projecting beyond the tip of the upper lip, a prominent black stripe along the flank, and no black pigments below the lateral line. Pseudophoxinus cilicicus is distinguished from P. zekayi by a minimum K2P distance of 3.8% based on the mitochondrial DNA barcode region. Pseudophoxinus atropatenus and P. sojuchbulagi are returned to the genus Rutilus.

The Oxynoemacheilus tigris species group is reviewed, resulting in the recognition of six species, of which two are described herein as new. Oxynoemacheilus tigris is known from the endorheic Qweik River and the Merziman River, which is a tributary of the western Euphrates. Oxynoemacheilus ercisianus is endemic to the endorheic Lake Van basin and O. hazarensis is endemic to Lake Hazar basin in the upper Tigris drainage. Oxynoemacheilus kaynaki is widespread in the Euphrates drainage. The two undescribed species occur in the Euphrates drainage. Oxynoemacheilus arsaniasus, new species, from the Murat River and the upper Karasu (Muş) River drainage, is distinguished from other species of the O. tigris group by having a bold, black, irregularly-shaped bar at the caudal-fin base, an incomplete lateral line and a scaleless body. Oxynoemacheilus muefiti, new species, from the upper Murat River drainage and a tributary to the Atatürk reservoir, is most similar to O. ercisianus, from which it is distinguished by a more slender body and a shallower dorsal adipose crest. According to our molecular data, the Qweik population of O. tigris is suspected to be introgressed by O. namiri from the Orontes drainage. Oxynoemacheilus erdali is identified as a synonym of O. bergianus as we were unable to find differences between the two species.

Article 16.4. of the International Code for Zoological Nomenclature (ICZN, 1999) requires that the fixation of name-bearing types for a new species to be explicit: “Every new specific and subspecific name published after 1999, except a new replacement name…, must be accompanied in the original publication 16.4.1. by the explicit fixation of a holotype,…..and 16.4.2. where the holotype or syntypes are extant specimens, by a statement of intent that they will be (or are) deposited in a collection and a statement indicating the name and location of that collection.” That means that for species described after 1999, the holotype must be finally deposited in a collection and it is obligatory to indicate the name of the collection and where it is located.

Paracobitis abrishamchiani, new species, is described from the southern Caspian Sea basin, where it is found in the Babol, Haraz, Siah, Tajan, Talar and Kashpal River drainages. It is distinguished from P. hircanica and P. atrakensis, the two other Paracobitis species known from the Caspian Sea basin, by having scales on the flank posterior to the dorsal-fin origin and from its congeners in the Middle East by the position of its dorsal-fin origin, having a roundish posterior narial opening, and a colour pattern consisting of many widely spaced brown spots and blotches. It is further characterized by six diagnostic nucleotide substitutions and a minimum K2P distance of 4.3% to P. persa and 4.6% to P. malapterura in the mtDNA COI barcode region.

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).

Metabarcoding is a powerful, increasingly popular tool for biodiversity assessment, but it still suffers from some drawbacks (specimen destruction, separation, and size sorting). In the present study, we tested a non-destructive protocol that excludes any sample sorting, where the ethanol used for sample preserving is filtered and DNA is extracted from the filter for subsequent DNA metabarcoding. When tested on macroinvertebrate mock communities, the method was widely successful but was unable to reliably detect mollusc taxa. Three different protocols (no treatment, shaking, and freezing) were successfully applied to increase DNA release to the fixative. The protocols resulted in similar success in taxa detection (6.8–7 taxa) but differences in read numbers assigned to taxa of interest (33.8%–93.7%). In comparison to conventional bulk sample metabarcoding of environmental samples, taxa with pronounced exoskeleton and small-bodied taxa were especially underrepresented in ethanol samples. For EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa, which are important for determining stream ecological status, the methods detected 46 OTUs in common, with only 4 unique to the ethanol samples and 10 to the bulk samples. These results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. However, for a comprehensive assessment on total invertebrate biodiversity, the method may not be sufficient, and conventional bulk sample metabarcoding should be applied.

BACKGROUND: Biodiversity patterns are inherently complex and difficult to comprehensively assess. Yet, deciphering shifts in species composition through time and space are crucial for efficient and successful management of ecosystem services, as well as for predicting change. To better understand species diversity patterns, Germany participated in the Global Malaise Trap Program, a world-wide collection program for arthropods using this sampling method followed by their DNA barcode analysis. Traps were deployed at two localities: “Nationalpark Bayerischer Wald” in Bavaria, the largest terrestrial Natura 2000 area in Germany, and the nature conservation area Landskrone, an EU habitats directive site in the Rhine Valley. Arthropods were collected from May to September to track shifts in the taxonomic composition and temporal succession at these locations. NEW INFORMATION: In total, 37,274 specimens were sorted and DNA barcoded, resulting in 5,301 different genetic clusters (BINs, Barcode Index Numbers, proxy for species) with just 7.6% of their BINs shared. Accumulation curves for the BIN count versus the number of specimens analyzed suggest that about 63% of the potential diversity at these sites was recovered with this single season of sampling. Diversity at both sites rose from May (496 & 565 BINs) to July (1,236 & 1,522 BINs) before decreasing in September (572 & 504 BINs). Unambiguous species names were assigned to 35% of the BINs (1,868) which represented 12,640 specimens. Another 7% of the BINs (386) with 1,988 specimens were assigned to genus, while 26% (1,390) with 12,092 specimens were only placed to a family. These results illustrate how a comprehensive DNA barcode reference library can identify unknown specimens, but also reveal how this potential is constrained by gaps in the quantity and quality of records in BOLD, especially for Hymenoptera and Diptera. As voucher specimens are available for morphological study, we invite taxonomic experts to assist in the identification of unnamed BINs.

Biodiversity loss is mainly driven by human activity. While concern grows over the fate of hot spots of biodiversity, contemporary species losses still prevail in industrialized nations. Therefore, strategies were formulated to halt or reverse the loss, driven by evidence for its value for ecosystem services. Maintenance of the latter through conservation depends on correctly identified species. To this aim, the German Federal Ministry of Education and Research is funding the GBOL project, a consortium of natural history collections, botanic gardens, and universities working on a barcode reference database for the country’s fauna and flora. Several noticeable findings could be useful for future campaigns: (i) validating taxon lists to serve as a taxonomic backbone is time-consuming, but without alternative; (ii) offering financial incentives to taxonomic experts, often citizen scientists, is indispensable; (iii) completion of the libraries for widespread species enables analyses of environmental samples, but the process may not hold pace with technological advancements; (iv) discoveries of new species are among the best stories for the media; (v) a commitment to common data standards and repositories is needed, as well as transboundary cooperation between nations; (vi) after validation, all data should be published online via the BOLD to make them searchable for external users and to allow cross-checking with data from other countries.

In the Middle East, species of Garra are believed to have invaded the area in two independent waves from the Indo-Malayan biogeographic region. This hypothesis is based on the structure of the mental disc, a unique specialization of the lower lip, which is believed to be an adaptation to fast-flowing waters. While several species have such a mental disc, others completely lack a mental disc, being adapted to slow-moving water or to subterranean life. In this study, the phylogenetic relationships of Middle Eastern Garra species, including 16 described and 4 undescribed species, were analysed using mitochondrial cytochrome c oxidase I sequences. The results are concordant with traditional hypotheses on two invasion events; however, these invasion events are independent from the presence, absence or shape of the mental disc. We postulate convergent reduction of the mental disc in 5–6 independent lineages of Garra in the Middle East.

This integrative study examined the morphological and genetic affinities of three endemic barbel species from Italy (brook barbel Barbus caninus, Italian barbel Barbus plebejus and horse barbel Barbus tyberinus) and of putative hybrid specimens to their species of origin. Two of the species frequently occur together with the non-native barbel Barbus barbus. DNA barcoding indicates that mitochondrial (mt) haplotypes often do not match the species expected from morphology. Linear distance measurements and meristics are not informative for discrimination of the species and putative hybrids, but a discriminant analysis of principal components (DAPC) of geometric landmark data produces reassignments largely in congruence with mt and nuclear genetic data. Cyto-nuclear conflicts confirm the presence of hybridization in B. plebejus and B. tyberinus and identify additional introgressed specimens. A comparison between mixed genotypes and their morphology-based assignment reveals no predictable pattern. The finding that most individuals of the morphologically similar B. plebejus and B. tyberinus have very high assignment probabilities to their respective species suggests that the presented approach may serve as a valuable tool to distinguish morphologically very similar taxa.

The diversity of Cobitis in the Middle East is reviewed, resulting in the recognition of 30 species, of which eight are described herein as new. Two species, C. amphilekta and C. kellei , seem to be extinct. Hypotheses on species-level diversity derived from distance and Poisson tree process analyses of DNA barcode data are tested against morphometric and morphological characters including colour patterns. For species pairs separated by small K2P distances in COI sequence data we follow a practitioner-oriented diagnostic species concept, in which we recognise species only if differentiated morphologically (including by colour pattern). For all 30 species we provide diagnoses and identification keys. Cobitis afifeae , new species, from the Buyuk Menderes River drainage in the Aegean Sea basin, is distinguished by having two laminae circularis in the male, a row of blotches below Z4, a small, roundish or comma-shaped black spot at the upper caudal-fin base, and elevated mental lobes. Cobitis aliyeae , new species, from the lower Seyhan and Ceyhan River drainages, is distinguished by having two laminae circularis in the male, the blotches in Z2 and Z4 anterior to the dorsal-fin origin usually well separated from each other, and the pigmentation in Z1 well distinguished from the pigmentation in Z2. Cobiti s anabelae , new species, from the lower Orontes River drainage, is distinguished by having two laminae circularis in the male, the pigmentation in Z2 formed by small, brown spots, always much smaller than blotches in Z3, much smaller than the pupil diameter, Z2 and Z3 well separated, and no pigmentation below Z4. Cobitis erkakanae , new species, from the Golbasi Lakes, adjacent to the Ceyhan River drainage, is distinguished by having two laminae circularis in the male, no blotches below Z4, the blotches in Z2 and Z4 being horizontally elongated and often fused with adjacent ones, and the caudal fin with 4 – 6 wide, regularly-shaped, brown bands . Cobitis emrei , new species, from the Lake Sapanca basin is distinguished by having one lamina circularis in the male, a large black spot at the upper caudal-fin base, and Z3 fully covered by very small spots forming a sand-like pattern. Cobitis joergbohleni , new species, from the Sultan marshes in Central Anatolia is distinguished by having two laminae circularis in the male, and the flank colour pattern being completely disorganised, not following the Gambetta zones. Cobitis pirii, new species, from the endorheic Lake Egirdir basin and the Mediterranean Aksu and Kopru Rivers, is distinguished by having two laminae circularis in the male, a simple external part of the suborbital spine and two distinct rows of small blotches in Z4, one along the lateral midline and one distinctly below. Cobitis troasensis, new species, from the Tuzla River drainage, is distinguished by having one lamina circularis in the male and 25–36 small, comma-shaped brown blotches in Z4. A lectotype is designated for Cobitis battalgilae . As First Revisers, priority is given to Cobitis fahireae over C. kurui . Cobitis damlae and C. kurui are treated as synonyms of C. fahireae . Cobitis strumicae and C. taenia are recorded for the first time from Anatolia and C. saniae is newly documented from the Black Sea basin in Georgia. The Poisson tree process analysis of COI data proposed 31 groups, most of which could be distinguished by morphological characters. Cobitis troasensis is described based on morphological data alone .

Barbus anatolicus, new species, is described from the Kızılırmak and Yeşilırmak River drainages in the southern Black Sea basin. It is distinguished from other Barbus species in the Middle East by having 58–71 total lateral line scales, a moderately ossified last simple dorsal-fin ray, serrated along about 70–80% of its posterior margin, many small irregular shaped black or brown spots, smaller or as large as scales, often forming large, dark-brown blotches on the head, back and flank in adults and juveniles, and a concave posterior dorsal-fin margin. In addition, DNA barcode data reject the hypothesis that it belongs to one of the other species of the B. barbus species group. Barbus bergi from Bulgaria and adjacent Turkey is treated as synonym of B. tauricus. Barbus tauricus was previously believed to be restricted to the Crimean Peninsula but is found to be widespread in the Black Sea basin.

Alburnus goekhani, new species, is described from the Yeşilırmak and Kızılırmak River drainages. It belongs to the A. alburnus species group and is distinguished from other species in this group in Anatolia by having 48–56 + 2–3 scales in the lateral-line, 12–15 gill rakers, a distinct dark lateral stripe along the flank on live and preserved specimens and the anal-fin origin situated below the branched dorsal-fin ray 6–8. Alburnus goekhani is also well distinguished from other Alburnus species by its DNA barcode sequence.

The generic position of Leuciscus kurui Bogutskaya, 1995 is reviewed through a comparison of morphological and molecular characters (COI). The molecular data place L. kurui in Alburnus , close to Alburnus timarensis from the Lake Van basin. Alburnus kurui (Bogutskaya) is distinguished from this species by lacking a ventral keel and possessing both a very low number of gill rakers and midlateral scales. Alburnus selcuklui , from the upper Tigris drainage, cannot be distinguished from the widespread A. sellal and is therefore treated as a synonym of this species. Alburnus kurui Mangit & Yerli, 2018 is a junior secondary homonym of A. kurui (Bogutskaya, 1995) and A. carianorum is proposed as its replacement name. Several specimens of Alburnus caeruleus and Alburnus heckeli shared the same haplotypes as some A. sellal and therefore these species cannot always be distinguished by mitochondrial molecular characters. Alburnus caeruleus and A. heckeli are treated as valid species. Other individuals of A. caeruleus have haplotypes very different from A. sellal , and A. heckeli is well distinguished from A. sellal by having more gill rakers. The Lake Van basin as a separate freshwater ecoregion and the treatment of several species of Alburnus in synonymy of A. mento are discussed.

Metabarcoding is a powerful tool for biodiversity assessment and has become increasingly popular in recent years. Although its reliability and applicability have been proven in numerous scientific studies, metabarcoding still suffers from some drawbacks. One is the usually mandatory destruction of specimens before DNA extraction, which is problematic because it does not allow a later taxonomic evaluation of the results. Additionally, metabarcoding often implements a time-consuming step, where specimens need to be separated from substrate or sorted in different size classes. A non-destructive protocol, excluding any sorting step, where the extraction of DNA is conducted from a samples fixative (ethanol) could serve as an alternative. We test an innovative protocol, where the sample preserving ethanol is filtered and DNA extracted from the filter for subsequent DNA metabarcoding. We first tested the general functionality of this approach on 15 mock communities comprising one individual of eight different macroinvertebrate taxa each and tried to increase DNA yield through different treatments (ultrasonic irradiation, shaking, freezing). Application of the method was successful for most of the samples and taxa, but showed weaknesses in detecting mollusc taxa. In a second step, the community composition detected in DNA from ethanol was compared to conventional bulk sample metabarcoding of complex environmental samples. We found that especially taxa with pronounced exoskeleton or shells (Coleoptera, Isopoda) and small taxa (Trombidiformes) were underrepresented in ethanol samples regarding taxa diversity and read numbers. However, read numbers of Diptera (mainly chironomids) and Haplotaxida were higher in ethanol derived DNA samples, which might indicate the detection of stomach content, which would be an additional advantage of this approach. Concerning EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa which are decisive for the determination of ecological statuses, both methods had 46 OTUs in common with 4 unique to the ethanol samples and 10 to the bulk samples. Results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. For a comprehensive picture on total biodiversity, the method might however not be sufficient and conventional bulk sample metabarcoding should be applied.

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

Oxynoemacheilus zarzianus, new species, is described from the Lesser Zab River drainage, a tributary of the lower Tigris. It is distinguished from other Oxynoemacheilus species in the Tigris drainage by having a slightly emarginate caudal fin, no suborbital groove in males, a complete lateral line, the posterior process of the bony air-bladder capsule directed posteriorly, the flank and posterior part of back covered by scales, short barbels and a deep caudal peduncle. It is the fourth Oxynoemacheilus species known from the Lesser Zab drainage, where such loaches seem to be highly isolated in headwaters. Oxynoemacheilus species diversity in the Euphrates and Tigris drainage is exceptional high. Today 22 species are known from the entire Euphrates and Tigris drainage and 15 from the Tigris drainage alone.

In order to better understand the complex geologic history of the Mediterranean area, we have analysed evolutionary history, phylogeographic structure and molecular diversity of freshwater fishes belonging to the genus Telestes. As primary freshwater fishes distributed largely in the Mediterranean basin, this genus represents a suitable model system for investigating the historical biogeography of freshwater drainage systems in southern Europe. In this investigation we have included samples representing all Telestes species and based our analyses on one mitochondrial and one nuclear gene. We have investigated phylogenetic structure inside the genus Telestes, estimated divergence times, reconstructed ancestral distribution ranges and described intraspecific molecular diversity. Diversification of Telestes started in the Early Miocene, when the ancestors of T. souffia, lineage comprising T. croaticus and T. fontinalis, and the one comprising T. pleurobipunctatus and T. beoticus got isolated. The remaining species are genetically more closely related and form a common cluster in the recovered phylogenetic trees. Complex geological history of southern Europe, including formation of continental bridges, fragmentation of landmass, closing of the sea corridor, local tectonic activities, led to complicated biogeographical pattern of this genus, caused by multiple colonization events and passovers between ancient rivers and water basins. Especially pronounced diversity of Telestes found in the Adriatic watershed in Croatia and Bosnia and Herzegovina is a consequence of a triple colonization of this area by different lineages, which led to an existence of genetically distinct species in neighboring areas. Significant intraspecific structuring is present in T. souffia, T. muticellus, T. croaticus and T. pleurobipunctatus. Besides in well-structured species, elevated levels of genetic polymorphism were found inside T. turskyi and T. ukliva, as a consequence of their old origin and unconstrained evolutionary history., This work was supported by the Czech Science Foundation (project GA15-19382S). IB received support from the SYNTHESYS Programme project CZ-TAF-4228 at the National Museum Prague, financed by European Community Research Infrastructure Action under the FP7 "Capacities" Programme. RS also received support from the SYNTHESYS Programme project ES-TAF-1187 at the Museo Nacional de Ciencias Naturales, Madrid, financed by European Community Research Infrastructure Action under the FP6 "Structuring the European Research Area" Programme.

International audience; The taxonomy of French ninespined sticklebacks (Pungitius spp.) has long been controver- sial. To clarify the taxonomy in this group, we use mitochondrial (COI) and nuclear (RNF213) sequence markers, as well as morphological data. In France, both genetic mark- ers discriminate three evolutionary lineages. Morphological analysis on fresh and type specimens supports the different lineages and the existence of three species in France. Pungitius pungitius, occurring in the North of France and Rhone basin, is characterized by specimens longer than 35 mm SL, by a flat head with a straight or slightly concave snout, typically 9–10 dorsal spines, 10–11 dorsal soft rays, 9–10 anal soft rays, 0–12 scutes on the caudal peduncle with a keel reaching the last anal-fin ray, longer pelvic fin, post-dor- sal and caudal peduncle lengths, and a slim caudal peduncle (caudal peduncle depth/ length 11.8%–21.9%). Pungitius laevis, occurring in France, in the English Channel basins and Loire drainage, differs from the other species by a head rounded with concave snout in specimens longer than 35 mm SL, accentuating the impression of fleshy lips, 0–4 scutes on the caudal peduncle and a higher caudal peduncle depth/length ratio (15.7%– 34.5%). Finally, Pungitius vulgaris, endemic to the Vienne River and rivers of south-wes- tern France as far north as the Garonne estuary, is differentiated by a rounded head with a straight or slightly convex snout, the absence of scutes on the caudal peduncle and by having 11 pectoral-fin rays. Our data confirm the existence of a hybridization zone in the North of France between P. pungitius and P. laevis. As a result, Pungitius lotharingus is invalid, as it was described based on hybrid specimens. A lectotype for P. laevis was des- ignated because the syntypes included hybrids. This revision provides new perspectives for evolutionary biology studies and will have consequences for Pungitius conservation in France.

This study represents the first comprehensive molecular assessment of freshwater fishes and lampreys from Germany. We analysed COI sequences for almost 80% of the species mentioned in the current German Red List. In total, 1056 DNA barcodes belonging to 92 species from all major drainages were used to (i) build a reliable DNA barcode reference library, (ii) test for phylogeographic patterns, (iii) check for the presence of barcode gaps between species and (iv) evaluate the performance of the barcode index number (BIN) system, available on the Barcode of Life Data Systems. For over 78% of all analysed species, DNA barcodes are a reliable means for identification, indicated by the presence of barcode gaps. An overlap between intra- and interspecific genetic distances was present in 19 species, six of which belong to the genus Coregonus. The Neighbour-Joining phenogram showed 60 nonoverlapping species clusters and three singleton species, which were related to 63 separate BIN numbers. Furthermore, Barbatula barbatula, Leucaspius delineatus, Phoxinus phoxinus and Squalius cephalus exhibited remarkable levels of cryptic diversity. In contrast, 11 clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. The analysis of our barcode library together with public data resulted in 89 BINs, of which 56% showed taxonomic conflicts. Most of these conflicts were caused by the use of synonymies, inadequate taxonomy or misidentifications. Moreover, our study increased the number of potential alien species in Germany from 14 to 21 and is therefore a valuable groundwork for further faunistic investigations.

Eidinemacheilus proudlovei, new species, is described from subterranean waters in the Little Zab River drainage in Iraqi Kurdistan. After the discovery of E. smithi in 1976, E. proudlovei is the second troglomorphic nemacheilid loach found in the Middle East and the second species placed in Eidinemacheilus. Eidinemacheilus proudlovei is distinguished from E. smithi by having 8+8 or 8+7 branched caudal-fin rays, no adipose keel on the caudal peduncle, enlarged jaws and a fully developed head canal system. It furthers differs substantially in its DNA barcode (gt;8% K2P distance) from all other nemacheilid loaches in the Middle East, Europe and Western India.