Marija Mladinić

Dragonflies and damselflies are important indicator species for quality and health of (semi-)aquatic habitats. Hitherto, 78 species of Odonata have been reported for Austria. Ecological data, Red List assessments, and a dragonfly association index exist, but population- and species-level genetic data are largely lacking. In this study, we establish a comprehensive reference DNA barcode library for Austrian dragonflies and damselflies based on the standard barcoding marker COI. Because of the increasing significance of environmental DNA (eDNA) analyses, we also sequenced a segment of the mitochondrial 16S rRNA gene, a marker often used in eDNA metabarcoding approaches. In total, we provide 786 new COI barcode sequences and 867 new 16S sequences for future applications. Sequencing success was >90 percent for both markers. Identification success was similar for both markers and exceeded 90 percent. Difficulties were only encountered in the genera Anax Leach, 1815, Chalcolestes Kennedy, 1920, Coenagrion Kirby, 1890 and Somatochlora Selys, 1871, with low interspecific genetic distances and, consequently, BIN (barcode index number) sharing. In Anax, however, individual sequences clustered together in species-specific groups in the COI tree. Irrespective of these challenges, the results suggest that both markers perform well within most odonate families in terms of sequencing success and species identification and can be used for reliably delimiting Austrian species, monitoring, and eDNA approaches.

The marine diatom genus Pseudo-nitzschia comprises cosmopolitan phytoplankton species commonly present in the Adriatic Sea. Species within the genus Pseudo-nitzschia have been of significant concern because they produce domoic acid (DA), which can cause amnesic shellfish poisoning (ASP). In this study, we identified Pseudo-nitzschia species along the Central and Southeastern Adriatic Sea, where monthly sampling carried out from February 2022 to February 2024 allowed for comprehensive species documentation. Pseudo-nitzschia species cell cultures isolated from the study areas were morphologically and molecularly analysed. Morphological analyses were performed using a scanning electron microscope (FE-SEM/STEM), while molecular analyses were conducted, targeting the ITS1-5.8S-ITS2, LSU, and rbcL regions, to confirm species identity. This integrative approach led to the identification of eight species: Pseudo-nitzschia allochrona, Pseudo-nitzschia calliantha, Pseudo-nitzschia delicatissima, Pseudo-nitzschia fraudulenta, Pseudo-nitzschia mannii, Pseudo-nitzschia multistriata, Pseudo-nitzschia pseudodelicatissima, and Pseudo-nitzschia subfraudulenta. Our findings underscore the value of a combined approach for reliable species identification and contribute to the development of genetic sequence databases that support the advancement of next-generation methods such as metabarcoding. This research emphasises the importance of combined morphological and molecular methods for the differentiation of the cryptic and pseudo-cryptic Pseudo-nitzschia species.

Austria is inhabited by more than 80 species of native and non-native freshwater fishes. Despite considerable knowledge about Austrian fish species, the latest Red List of threatened species dates back 15 years and a systematic genetic inventory of Austria’s fish species does not exist. To fulfill this deficit, we employed DNA barcoding to generate an up-to-date and comprehensive genetic reference database for Austrian fish species. In total, 639 newly generated cytochrome c oxidase subunit 1 (COI) sequences were added to the 377 existing records from the BOLD data base, to compile a near complete reference dataset. Standard sequence similarity analyses resulted in 83 distinct clusters almost perfectly reflecting the expected number of species in Austria. Mean intraspecific distances of 0.22% were significantly lower than distances to closest relatives, resulting in a pronounced barcoding gap and unique Barcode Index Numbers (BINs) for most of the species. Four cases of BIN sharing were detected, pointing to hybridization and/or recent divergence, whereas in Phoxinus spp., Gobio spp. and Barbatula barbatula intraspecific splits, multiple BINs and consequently cryptic diversity were observed. The overall high identification success and clear genetic separation of most of the species confirms the applicability and accuracy of genetic methods for bio-surveillance. Furthermore, the new DNA barcoding data pinpoints cases of taxonomic uncertainty, which need to be addressed in further detail, to more precisely assort genetic lineages and their local distribution ranges in a new National Red-List.