100Effective identification of species using short DNA fragments (DNA barcoding and DNA 101 metabarcoding) requires reliable sequence reference libraries of known taxa. Both 102 taxonomically comprehensive coverage and content quality are important for sufficient 103 accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are 104 particularly important if molecular identification tools are to be implemented in biomonitoring 105 and reports in the context of the EU Water Framework Directive (WFD) and the Marine 106Strategy Framework Directive (MSFD). We analysed gaps in the two most important 107 reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a 108 focus on the taxa most frequently used in WFD and MSFD. Our analyses show that 109 coverage varies strongly among taxonomic groups, and among geographic regions. In 110 general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, 111 caddisflies and vascular plants) are well represented in the barcode libraries, while others 112 have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also 113 found that species monitored in several countries often are represented by barcodes in 114 reference libraries, while species monitored in a single country frequently lack sequence 115 records. A large proportion of species (up to 50%) in several taxonomic groups are only 116represented by private data in BOLD. Our results have implications for the future strategy to 117 fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the 118 monitoring of European aquatic biota under the WFD and MSFD. For example, missing 119 species relevant to monitoring in multiple countries should be prioritized. We also discuss 120 why a strategy for quality control and quality assurance of barcode reference libraries is 121 needed and recommend future steps to ensure full utilization of metabarcoding in aquatic 122 biomonitoring. 123 124

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61 sequences of cytochrome oxidase subunit 1 (COI), 570 bp long, from Central Europe (8), Slovenia (2), and from the Balkans (51: 6 from, Romania, 15 from Greece, 15 from Bulgaria, 6 from Serbia, and 9 from Montenegro), 33 of them new, together with 61 sequences of the ribosomal 18S, 450 bp long, all of them new, were analysed to infer the pattern of radiation of Bythinella in the Balkans. Thirty two nominal taxa of Bythinella (22 nominal species: B. austriaca, B. calimanica, B. charpentieri, B. compressa, B. dacica, B. dispersa, B. grossui, B. hansboetersi, B. luteola, B. micherdzinskii, B. molcsanyi, B. nonveilleri, B. pannonica, B. pesterica, B. radomani, B. rhodopensis, B. robiciana, B. schmidti, B. slaveyae, B. srednogorica, B. taraensis and B. viseuiana; one nominal subspecies: B. austriaca ehrmanni; and nine Greek species not yet described) were included, represented mostly by paratypes or at least topotypes, collected at 31 Balkan localities. The phylogeny, inferred on the combined data set with the ML approach, showed two large clades, although they were weakly supported. One of them comprised the Romanian and Montenegro populations, and one Serbian population, the other (less genetically diversified) consisted of one Serbian and all the Bulgarian/Greek populations. The origin of those two clades was dated, with the external data, to be more than 4.341±0753 MYA old, thus its origin was assigned to isolation by the Dacic Basin (part of Paratethys). All the Bulgarian populations presumably belong to one species, which may be assigned to the recent recolonisation of this territory from the south.

More than half of the global river network is composed of intermittent rivers and ephemeral streams (IRES), which are expanding in response to climate change and increasing water demands. After years of obscurity, the science of IRES has bloomed recently and it is being recognised that IRES support a unique and high biodiversity, provide essential ecosystem services and are functionally part of river networks and groundwater systems. However, they still lack protective and adequate management, thereby jeopardizing water resources at the global scale. This Action brings together hydrologists, biogeochemists, ecologists, modellers, environmental economists, social researchers and stakeholders from 14 different countries to develop a research network for synthesising the fragmented, recent knowledge on IRES, improving our understanding of IRES and translating this into a science-based, sustainable management of river networks. Deliverables will be provided through i) research workshops synthesising and addressing key challenges in IRES science, supporting research exchange and educating young researchers, and ii) researcher-stakeholder workshops translating improved knowledge into tangible tools and guidelines for protecting IRES and raising awareness of their importance and value in societal and decision-maker spheres. This Action is organized within six Working Groups to address: (i) the occurrence, distribution and hydrological trends of IRES; (ii) the effects of flow alterations on IRES functions and services; (iii) the interaction of aquatic and terrestrial biogeochemical processes at catchment scale; (iv) the biomonitoring of the ecological status of IRES; (v) synergies in IRES research at the European scale, data assemblage and sharing; (vi) IRES management and advocacy training.

1. Springs are distinct freshwater habitats that are threatened globally by human impacts, but are not included in standard freshwater assessment tools. This study analysed how the standard macroinvertebrate metrics currently used in biomonitoring programmes for running waters perform in springs with similar ecological qualities but with a different degree of hydromorphological modification.Macroinvertebrate assemblages were analysed in 50 riparian springs along the Crvcka River in the Dinaric karst.2. Most metrics analysed (ASPT, EPT fam , PTH fam , BMWP, STAR_ICMi, and Spring_ICMi) showed significant differences between hydromorphologically modified and natural, i.e. unmodified, springs.3. A self-organizing map (SOM), an unsupervised artificial neural network, was used for patterning and visualizing 10 environmental parameters and the metrics analysed.Most macroinvertebrate metrics, besides IBE (which did not show any pattern) and PTH fam (which showed an opposite trend), had significantly lower values in the spring group with the highest electrical conductivity and the lowest discharge.4. The study showed that hydromorphological modification does not necessarily lead to a loss of crenobiontic species, provided that the discharge and substrate composition remain suitable. Future coordinated conservation strategies must take into account the fact that spring species assemblages are sensitive to habitat structure and organic pollution, as has been achieved in Europe for rivers assessed under the Water Framework Directive. 5. Using multimetric indices, such as the new Spring_ICMi, may help to provide a framework for assessing the response of macroinvertebrate assemblages to human impacts. They may also help in assessing the success of measures used for the conservation and restoration of these threatened but highly valuable habitats. Further investigations should verify the suitability of such metrics in assessing the deterioration of springs globally.

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The biodiversity of the entrance zones of the Montenegro caves is barely studied, therefore the purpose of this study was to assess the biodiversity of several caves in Montenegro. The samples of phototrophs were taken from various substrates of the entrance zone of 7 caves in July 2017. A total of 87 species of phototrophs were identified, including 64 species of algae and Cyanobacteria, and 21 species of Bryophyta. Comparison of biodiversity was carried out using Jacquard and Shorygin indices. The prevalence of cyanobacteria in the algal flora and the dominance of green algae were revealed. The composition of the phototrophic communities was influenced mainly by the morphology of the entrance zones, not by the spatial proximity of the studied caves.

Using published records and original data from recent field work and revision of Iranian material of certain species deposited in the collections of the Natural History Museum Basel, the Zoological Museum Berlin, and Natural History Museum Vienna, a checklist of the freshwater gastropod fauna of Iran was compiled. This checklist contains 73 species from 34 genera and 14 families of freshwater snails; 27 of these species (37%) are endemic to Iran. Two new genera, Kaskakia and Sarkhia, and eight species, i.e., Bithynia forcarti, Bithynia starmuehlneri, Bithynia mazandaranensis, Pseudamnicola georgievi, Kaskakia khorrasanensis, Sarkhia sarabensis, Valvata nowsharensis and Acroloxus pseudolacustris are described as new to science; Ecrobia grimmi (Clessin & Dybowski, 1888), Heleobia dalmatica (Radoman, 1974) and Hippeutis complanatus (Linnaeus, 1758) are reported for the first time from Iran. Additional field work is highly desirable for a more appropriate evaluation of the extant freshwater snail biodiversity in Iran.