Ponticolaalasanicus sp. n. (Gobiiformes, Gobiidae) from the Alazani River Basin, Georgia.

Background: The South Caucasus Region and Georgia, in particular, is a biodiversity hotspot and characterised by high diversity of landscapes and ecosystems, as well as high levels of endemism. At the same time, diversity of freshwater organisms in the region remains poorly studied, including fishes. The freshwater fish fauna of the South Caucasus Region consists of 119 fish species, of which 13 species belong to the order Gobiiformes. It should be noted that gobies are amongst the poorly studied taxa in Georgia and probably unknown/undescribed species still living in the Georgian freshwater ecosystems which requires further research. New information: Ponticolaalasanicus, a new species is described from the Alazani River, western Caspian Sea Basin, Georgia. It is distinguished from its congeners in the Caspian and Black Sea Basins by having the following features: dorsal fin with VI-VII spines and 15½-16½ branched rays, anal fin with 10½-12½ branched rays; lateral line with 48-55 scales; laterally compressed body with dark brown and black blotches - scales ctenoid; first and second dorsal fins almost touching with dorsal fins bases; head large, depressed, wider than deep, its length approaches almost 3.4th of standard length; nape scaled completely; cycloid scales cover upper part of opercle, cheeks noticeably swollen; snout longer than eye, eye diameter 4.5 times its head length; lower jaw slightly protruding; upper lip is uniform; pelvic disc short, elongated and flat, not reaching the anus; the pectoral fins extends vertically through first branched dorsal fin; caudal fin rounded. Ponticolaalasanicus sp. n. belongs to P.syrman group and it is separated by a minimum Kimura 2-parameter distance of 3.5, 3.6 and 4.8% from P.syrman, P.iranicus and P.patimari, respectively.

Diatom endemism and taxonomic turnover: Assessment in high-altitude alpine lakes covering a large geographical range.

Diatoms are widely used as ecological indicators and show various degrees of endemism. Many studies that support the idea of endemic species integrate several climate zones, a variety of ecosystem types, and often focus on a global scale. Here, we investigated whether endemism could be detected when considering a homogeneous type of ecosystem in a single climate zone. We sampled stone biofilms at 40-50 cm depth in high-altitude lakes in the Alpine climate zone. A total of 149 samples were obtained from the French and Georgian mountains, two areas separated by ∼3000 km. Using Amplicon Sequence Variants derived from DNA metabarcoding, we assessed taxonomic turnover and Zeta-diversity (a measure of endemism). We ran haplotype networks and phylogenetic tests to measure geographical signal in the phylogenies of dominant taxa. The French and Georgian communities shared 51 % of species. Species that were not shared across both regions were mostly rare, and often not characteristic of lakes but of neighboring habitats instead. In contrast, at the sub-species level, 87 % of the genotypes showed restricted distributions. Whereas endemism was the rule at sub-species level, most species were shared across both French and Georgian lakes, suggesting that geographic barriers strongly limited dispersal at the sub-species level but not species level. Dominant species hosted higher levels of sub-specific diversity than rare species. In contrast to global-scale studies, we did not find any significant geographical structuring in the phylogeny of the investigated species. This could indicate ongoing dispersal at a frequency fast enough to prevent allopatric divergence, yet slow enough to prevent sharing most haplotypes between France and Georgia. These results have implications for biomonitoring: depending on the taxonomic level chosen, robust generic tools (species level) or tools dedicated to a region able to discriminate fine pressures differences (sub-species level) may be developed.

Species diversity and DNA barcode library of freshwater Molluscs of South Caucasus.

This study provides the first attempt to investigate the molecular diversity of South Caucasian freshwater molluscs (Mollusca, Gastropoda) and lay down the first bricks to build up a DNA-barcode library. In total, 289 COI barcode sequences were obtained from 33 morpho-species belonging to 24 molluscan genera and 10 families that represent nearly 30% of known freshwater molluscan diversity of the South Caucasus region. DNA barcodes were analysed by means of the Barcode Index Number (BIN) and the other tools available in BOLD Systems. Results showed that the knowledge of freshwater molluscs diversity in the South Caucasus is far from comprehensive. For the studied 33 morpho-species, 289 barcodes were clustered into 40 BINs, from which unique BINs were defined for 12 species and five species were characterised with more than a single BIN. From the studied taxa, 60% were characterised larger than 2.2% sequence divergence indicating high genetic variation or cryptic diversity. Within our limited taxonomic coverage, we found one new species for the Republic of Georgia (Galbaschirazensis) and at least three undescribed species belonging to the genera Stagnicola, Segmentina and Anisus. Uniqueness and high molecular diversity of the studied species emphasise the need for further intensive morphological and molecular investigations of the South Caucasian freshwater molluscan fauna.

Genetic diversity, phylogenetic position and morphometric analysis of Astacus colchicus (Decapoda, Astacidae): a new insight into Eastern European crayfish fauna.

The phylogeny of European crayfish fauna, especially with respect to Eastern European species, is still far from being completely resolved. To fill this gap, we analyzed most of the European crayfish species focusing on the phylogenetic position of the endemic crayfish Astacus colchicus, inhabiting Georgia. Three mitochondrial and one nuclear marker were used to study evolutionary relationships among European crayfish species, resulting in the unique phylogenetic position of A. colchicus indicating independent species status to A. astacus. Phylogenetic analyses revealed a deep molecular divergence of A. colchicus in comparison to A. astacus (6.5-10.9% in mtDNA and 1.1% in nDNA) as well as to Pontastacus leptodactylus and P. pachypus (5.5-10.0% in mtDNA and 1.4-2.4% in nDNA). Absent ventral process on second male pleopod and abdominal somites II and III with pleura rounded lacking prominent spines clearly indicate taxonomic assignment to the genus Astacus; however, the species is distributed almost in the middle of Ponto-Caspian area typical by occurrence of the genus Pontastacus. Several morphological indices linked to head length, carapace, and total body length and width were found to demonstrate apparent differences between A. colchicus and A. astacus. Although this study provides a novel insight into European crayfish phylogeography, we also point out the gaps in comprehensive study of the P. leptodactylus species complex, which could reveal details about the potential species status of particular species and subspecies within this genus.

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

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

Rhodeus caspius, a new bitterling from Iran (Teleostei: Cypriniformes: Acheilognathidae).

A new bitterling from the Rhodeus amarus group is described based on morphological and molecular data. Rhodeus caspius sp. nov. from the Caspian Sea, Urmia Lake and the upper Tigris River drainages in Iran, is distinguished from its closest congeners (R. amarus and R. colchicus) by having short post-dorsal distance and caudal peduncle length and long head length. It is also characterized by one fixed diagnostic nucleotide substitutions and a K2P nearest neighbor distance of 1% to R. amarus and R. colchicus in the mtDNA COI barcode region.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

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.

Phylogeny, phylogeography and hybridization of Caucasian barbels of the genus Barbus (Actinopterygii, Cyprinidae).

Phylogenetic relationships and phylogeography of six species of Caucasian barbels, the genus Barbus s. str., were studied based on extended geographic coverage and using mtDNA and nDNA markers. Based on 27 species studied, matrilineal phylogeny of the genus Barbus is composed of two clades - (a) West European clade, (b) Central and East European clade. The latter comprises two subclades: (b1) Balkanian subclade, and (b2) Ponto-Caspian one that includes 11 lineages mainly from Black and Caspian Sea drainages. Caucasian barbels are not monophyletic and subdivided for two groups. The Black Sea group encompasses species from tributaries of Black Sea including re-erected B. rionicus and excluding B. kubanicus. The Caspian group includes B. ciscaucasicus, B. cyri (with B. goktschaicus that might be synonymized with B. cyri), B. lacerta from the Tigris-Euphrates basin and B. kubanicus from the Kuban basin. Genetic structure of Black Sea barbels was influenced by glaciation-deglaciation periods accompanying by freshwater phases, periods of migration and colonization of Black Sea tributaries. Intra- and intergeneric hybridization among Caucasian barbines was revealed. In the present study, we report about finding of B. tauricus in the Kuban basin, where only B. kubanicus was thought to inhabit. Hybrids between these species were detected based on both mtDNA and nDNA markers. Remarkably, Kuban population of B. tauricus is distant to closely located conspecific populations and we consider it as relic. We highlight revealing the intergeneric hybridization between evolutionary tetraploid (2n = 100) B. goktschaicus and evolutionary hexaploid (2n = 150) Capoeta sevangi in Lake Sevan.