Species diversity and origin of non-biting midges (Chironomidae) from a geologically young lake and its old spring system

In the present thesis, using midges (Diptera Chironomidae) as flagship taxa of freshwater ecology, I am focusing on the interesting research model represented by the Skadar Lake system. It is a well-known hot-spot of freshwater biodiversity consisting of the geologically young lake Skadar (originated ca. 1200 years BP) and by its ancient system of springs (originated in the Pliocene). The main aim of my thesis was to reveal and compare the morphological and molecular species diversity of non-biting midges (Diptera, Chironomidae) inhabiting Skadar Lake and its spring system. Using a taxonomy-based approach for adult males and pupal exuviae, I identified 164 Chironomidae taxa providing the first insight into species diversity of the Skadar Lake basin. Results presented in my thesis extending the existing checklist with 152 taxa newly found in the Skadar Lake basin. DNA barcoding of larvae and mature males revealed a total of 168 Operational Taxonomic Units which is a higher result than the number of morphotypes obtained during morphological identification. Pursuing this goal, I additionally compared the level of species diversity with other central and southern European lakes. A comparison of species checklists from 13 other well-studied European lakes resulted that Lake Constance (Switzerland/Germany/ Austria) is the richest in species number, followed by the Skadar Lake. The second aim was to investigate the influence of physical-chemical conditions on composition and distribution of chironomid assemblages in Skadar Lake basin. The obtained results suggest that shallow, coastal parts of the lake covered with macrophytes are inhabited by a higher number of species. As a third aim, I developed and evaluated the first reference barcode library for Chironomidae from Skadar Lake basin. Moreover, using an expanded reference library and records deposited in Barcode of Life Database (BOLD), I estimated DNA barcoding efficiency for the European Chironomidae. My study provides COI barcodes for 770 Chironomidae individuals assigned, based on morphology, to 75 species collected in the Skadar Lake basin (all records from this area are new for online repositories) and confirms the usefulness of DNA barcoding for the identification of non-biting midges. My fourth aim was to explore chironomid species distribution patterns in Europe using universal Barcode Index Number (BIN) with a discussion of problematic species groups, both for traditional taxonomy and DNA barcoding. The results of my PhD thesis provide the first insight into the factual chironomid species diversity of the Lake Skadar basin, in comparison with chironomid fauna at the European scale. The results fill a significant gap in knowledge of biodiversity in the Balkan region. Based on the results of Chironomidae fauna investigation, I can conclude that the Skadar Lake basin is now well sampled and such a high representation of species from various sampling sites provides reliable estimation of the local chironomid fauna. Based on obtained results it is hard to predict the origin of the chironomids inhabiting the Skadar Lake basin based on the sequences uploaded so far to BOLD and on their known geographic distribution. The still insufficient number of sequences is distributed between the well-studied European regions and Skadar Lake basin. Additionally, the Skadar Lake basin could be recognized as a hot-spot of freshwater biodiversity but without species-level endemism

DNA barcoding of Chironomidae from the Lake Skadar region: Reference library and a comparative analysis of the European fauna

Aims: The main aim of this study was to fill a gap in barcoding data of the European Chironomidae of the Balkan region, developing and testing the efficiency of a reference DNA barcode library for ancient Lake Skadar basin species (Montenegro/Albania), a region of Europe never before subjected to barcoding studies on Chironomidae. Another aim was to test the efficiency of DNA barcoding for the identification of European Chironomidae, including the estimation of optimal identification thresholds, using >12,000 barcodes. Location: Lake Skadar basin and adjacent area (Montenegro/Albania). Methods: Through this study, 770 individuals of Chironomidae from the Lake Skadar region were barcoded, both at adult and pre-imaginal stages. Adults were morphologically identified, while larvae were assigned to species by molecular identification, using different methods, of which the efficiency was tested, for a total of 97 different barcoded species. Results: The identification efficiency of the reference dataset developed for the Lake Skadar region was 98.6%, a value in line with that obtained when the identification efficiency for European Chironomidae was evaluated (95.8%), which confirms the accuracy of DNA barcoding for the identification of these insects. Moreover, we found that the optimal threshold for the molecular identification of the family is 1.6% nucleotide distance, though more specific thresholds are suggested for the identification of species belonging to Chironomidae subfamilies, since they are related to lower identification errors than to the use of a general threshold. The analysis of inconsistency between molecular and morphological identification shed light on taxonomic issues within European Chironomidae. Previously postulated species synonyms were confirmed, and also further cases requiring deeper investigation were detected. Main conclusions: Our de novo DNA barcode library was shown to have a high identification efficiency. Taxon-specific thresholds increase the efficacy of molecular identification. Hypothesized species synonyms could be validated through molecular techniques

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.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)

A new species of freshwater Chaetonotidae (Gastrotricha, Chaetonotida) from Obodska Cave (Montenegro) based on morphological and molecular characters

Gastrotricha is a cosmopolitan phylum of aquatic and semi-aquatic invertebrates that comprises about 820 described species. Current knowledge regarding freshwater gastrotrichs inhabiting caves is extremely poor and there are no extant data regarding freshwater Gastrotricha from Montenegro. We describe a new species from Obodska Cave, which is also the first record of a gastrotrich from this region. Due to its unusual habitat and morphological characteristics, this species may be important when considering the evolution and dispersion routes of Chaetonotidae Gosse, 1864 (sensu Leasi & Todaro 2008). We provide morphometic, molecular and phylogenetic data for the new species, together with photomicrographs and drawings

Morphology and molecules say: Tanytarsus latens, sp. nov. from Finland (Diptera: Chironomidae)

Giłka, Wojciech, Paasivirta, Lauri, Gadawski, Piotr, Grabowski, Michał (2018): Morphology and molecules say: Tanytarsus latens, sp. nov. from Finland (Diptera: Chironomidae). Zootaxa 4471 (3): 569-579, DOI: 10.11646/zootaxa.4471.3.

The Chironomidae (Diptera) diversity in the Balkan Peninsula spring systems and other small water bodies

Chironomidae are known to occur in small, even astatic water bodies like seepages, rheocrens, pools and wells. The Balkan Peninsula reveals a high variability of small water bodies, with springs (rheocrens, limnocrens and helocrens) being the most widely distributed. In this review, we give a brief presentation of the Chironomidae communities in valley and mountain small water bodies, and in Lake Skadar (Shkodra) spring system. River valleys offer a large variety of small freshwater habitats. Their presence strongly increases of midge (Chironomidae) diversity, providing a habitat for the cold-stenotherms and semiterrestrial taxa that do not occur in rivers and lakes. The species richness in small water bodies strongly depends on their hydrological conditions (i.e. perennial vs. astatic water bodies), size and microhabitat complexity. Mountain spring communities depend on precipitation and exhibit altitudinal zonation. The higher mountain zones (1400–1500 m a.s.l.) have the most diverse midge assemblages, due to their stable perennial hydrological conditions. Human activity may alter species composition in riparian springs, favouring taxa that are not typical to the local fauna. By studying these small aquatic habitats, the significance of their Chironomidae fauna is being recognised, thereby filling a gap in the knowledge of freshwater insects biodiversity in the Balkan region.There are many types of small waters in the Balkans. They are habitats for Chironomidae. Mountain rheocrenes are inhabited by cold-adapted midges. The fauna of springs in the river valleys is diverse. Midges living in the Skodra L.’ spring system prefer higher water temperatures and hard substrates

DNA barcoding reveals an unknown Chironomidae diversity from the freshwater biodiversity hot-spot: comparison between local and the European datasets

In the present study, we developed and evaluated the first reference barcode library for non-biting midges (Diptera: Chironomidae) as flagship taxa of freshwater ecology from Skadar Lake system (Montenegro and Albania), a well-known hotspot of freshwater biodiversity composed by the young lake Skadar (originated 1200 before present) and by its old system of springs (originated during Pliocene). Using an expanded reference library and records deposited in Barcode of Life Database (BOLD), we estimated DNA barcoding efficiency for the European Chironomidae. Study provides COI barcodes for 770 Chironomidae individuals assigned, based on morphology, to 77 species collected in the Skadar Lake basin. Molecular analyses assigned sequences to 100 BINs and 104 OTUs (all records from this area are new for online repositories) and confirms the usefulness of DNA barcoding for the identification of non-biting midges. Additionally, we explored chironomid species distribution patterns in Europe using universal Barcode Index Number (BIN) with a discussion of problematic species groups, both for traditional taxonomy and DNA barcoding. The results of our study provide the first insight into the factual chironomid species diversity of the Lake Skadar basin, in comparison with chironomid fauna at the European scale. The results fill a significant gap in knowledge of biodiversity in the Balkan region. Based on the results of Chironomidae fauna investigation, we conclude that the Skadar Lake basin is now well sampled and such a high representation of species from various sampling sites provides reliable estimation of the non-biting midges fauna

DNA barcode library revealed unknown diversity of chironomid midges in Montenegro

We present the first results of the study aiming to investigate the diversity of the non-biting midges (Diptera: Chironomidae) fauna of the Skadar Lake system (Montenegro and Albania), a well-known hotspot of freshwater biodiversity composed by the young lake Skadar (originated 1200 before present) and by its old system of springs (originated during Pliocene). During the study, 8,147 COI barcodes were obtained and revealed the presence of 474 BINs and 586 OTUs assigned to 148 species. Our results provide the first insight into the factual molecular diversity among chironomids inhabiting Skadar Lake basin and fill a significant gap in the knowledge of the biodiversity in the Balkan region. With 219 (46.2%) unique BINs from the Skadar Lake basin new for BOLD, we can expect that further development of barcode reference libraries will help to bind unidentified developmental stages with those identified based on morphology and will reveal hidden Chironomidae species diversity. Further studies should be focused on sampling developmental stages which provides the best species-level resolution, such as mature males. It will help to develop a reliable reference barcode library - fundamental during further assessments