Lake Skadar (LS) is the largest and the youngest Balkan lake shared by Montenegro and Albania. A characteristic feature of its basin is the presence of numerous geologically old karstic spring systems. The lake and its basin are known for rich biodiversity and endemism. Recent studies show that local aquatic invertebrates, particularly crenic fauna, are far from being well-documented. The lake is protected and included in the Ramsar list of wetlands of international importance. However, the ecosystem of the LS basin is highly endangered due to recent development of tourism industry and associated anthropogenic pressure. Thus, a rapid survey of the local aquatic biodiversity is of utter importance. Our study, done in May/June 2018, encompassed 58 sites in springs, streams and lake coastal waters, scattered throughout the LS basin. Using the PacBio Sequel II System in CBG (Guelph), we obtained over 4,500 COI barcodes for aquatic insects, mostly Diptera, Ephemeroptera, Plecoptera, Trichoptera, Odonata and Coleoptera, belonging to 60 families and, based on the Barcode Index Number algorithm, clustered to over 500 BINs that can be treated roughly as imperfect yet handy species-equivalents. In numerous cases, particularly aquatic insects (e.g., Ephemeroptera, Plecoptera, Trichoptera, Coleoptera, and Diptera), the survey provided records of species new to LS basin, Montenegro, Albania and also some potentially new to science. Regarding spatial distribution of BIN diversity, our results not only showed high BIN-level diversity in the relatively small area, but also high distinctiveness of BIN composition and little genetic connectivity, hence isolation, also between localities close to each other. It points to the importance of preserving the regional waterbodies at a very local scale, as even degradation of single waterbodies can potentially lead to irreversible biodiversity loss. The barcodes will provide a possibility for employing cost-effective methods such as DNA metabarcoding and, in perspective, ecologically harmless eDNA sampling in bioassessment and biomonitoring of local freshwater ecosystems. The work was supported by the Montenegrin Ministry of Sciences (“DNA‐Eco“ project), by National Agency for Academic Exchange (NAWA, PL) and National Science Centre (NCN, PL).

The Bieszczady Mountains and Przemyskie Foothills are characterised by the occurrence of remnants of Carpathian wildernesses recognised as one of the most important pristine mountain landscapes in Poland. The biodiversity of aquatic insects in the streams of the Bieszczady Mountains and Przemyskie Foothills has not been studied extensively yet. These regions are an attractive tourist destination, and therefore the fauna of their streams is potentially exposed to habitat loss and water pollution. Learning more about the biodiversity of both regions can be crucial in implementing plans for their protection. This project focuses on aquatic insects from EPT index groups (Ephemeroptera, Plecoptera, and Trichoptera) as an indicator of the potential biodiversity of streams and their water quality. The DNA barcoding approach followed by morphological identification was used for estimating cryptic diversity and to extend the Polish barcode library (PolBOL) in the Carpathians. The invertebrates were collected from different dominating mesohabitats, namely stones, gravel, wood, and detritus, to ensure the biodiversity of the streams was fully captured. The results will give an insight into the level of diversity of aquatic insects in both regions, as well as help dispel the discussion on the desirability of expanding the buffer zone of Bieszczadzki National Park and the establishment of the Turnicki National Park, which could offer additional protection for the streams of the Przemyskie Foothills.

Research is financed by the University of Lodz Students Grant “Integrative taxonomy of mayflies and stoneflies of Przemyskie Foothills and Bieszczady Mountains” no. SGB_647 and FAN(B) project “Benthos of streams in Pogórze Przemyskie – issues for planned Turnicki National Park”.

A total of 275 larvae, pupae, and adults of Simuliidae from different watercourses in Romania were examined to test the utility of the standard DNA barcode region of the mtCOI gene as a species-level identification tool for all life stages. 29 morphospecies from 3 genera were analysed: genus Prosimulium Roubaud, 1906 with one subgenus and 4 species, genus Twinnia Stone et Jamnback, 1955 with a genus Simulium Latreille, 1802 with 4 subgenera and 24 species. The Neighbor-Joining tree, inferred based on the DNA barcode sequences, grouped most specimens according to species or species-groups recognised by morphometric studies. Intraspecific sequence differences within morphospecies ranged from 0.00% to 5.96%. In some cases, however, the larger differences (2.33–5.96%) indicated the presence of important cryptic diversity. In total, we generated 233 DNA barcode sequences during our study, thus obtaining a total of 30 different BINs and 26 species. During the present work, 8 species are recorded for the first time from Romania: Twinnia hydroides Novák, 1956, Simulium petricolum (Rivosecchi, 1963), S. beltukovae (Rubtsov, 1956), S. cryophilum (Rubtsov, 1959), S. craigi Adler & Currie, 1986, S. trifasciatum Curtis, 1839, S. reptantoides Carlsson, 1962 and S. aff. monticola. The Simuliidae fauna from Romania is now represented by 72 different species. The DNA barcode data combined with morphology are a useful tool for the identification of Simuliidae species in Romania, however, we noticed large gaps in the case of species that are range restricted or regionally distributed, including some endemic species. In the case of the latter category, the lack of molecular data makes species-level identification difficult, which is further complicated by the high cryptic diversity characteristic for the region.

Amphipods are among the most abundant and diverse crustaceans in Palearctic freshwaters. This group is extensively used in applied and basic research, including biodiversity assessments, ecotoxicology, and evolutionary ecology. Nevertheless, even well-studied model organisms may exhibit overlooked cryptic diversity. DNA barcoding, based on partial cytochrome c oxidase subunit 1 (COI) sequences, has been proven to be a valuable tool for species determination and revealing the cryptic diversity in many taxa, including amphipods.

Here, we present studies on epigean freshwater and brackishwater amphipods from Poland. Recent findings and descriptions of new species enhanced the Polish checklist to at least 27 species. Nine species (33%) of Polish Amphipoda fauna are invasive species, seven of which have Ponto-Caspian origin. We can also find five brackish species (G. zaddachi, G. salinus, G. locusta, G. oceanicus, Apocorophium lacustre) and one semi-terrestrial talitrid — Cryptorchestia garbinii.

Here, DNA barcodes from 993 individuals and 26 species were analysed, including sequences from previous studies and more than 400 newly generated DNA barcodes. We studied representatives of the six families: Gammaridae (831 ind., 19 species); Pontogammaridae (118 ind., 2 species), Corophiidae (20 ind., 2 species), Talitridae (12 ind., 1 species), Pallaseidae (3 ind., 1 species), Crangonyctidae (1 ind., 1 species). We found 38 BINs and exact correspondence between BINs and traditionally recognised species matched for 21 species (78%). Deep intraspecific sequence divergences with distinct lineages were revealed within six species, with two (Gammarus pulex, G. salinus, G. tigrinus), three (Echinogammarus ischnus, G. fossarum, G. tatrensis), and four (G. jazdzewskii) BINS, respectively.

Our findings are the first, almost complete, country-scaled attempt to provide a DNA barcode reference library of Amphipoda.

The Pannonian Basin, nestled in the heart of Europe, boasts characteristic habitats that harbour diverse wildlife shaped by its distinctive geological history and unique geographical and climatic features. From a phylogeographical perspective, this region stands out due to its abundance of endemic species and its role as a refugium and a gateway to the Balkan and eastern steppe fauna. Encompassing 133,000 km², the Pannonian ecoregion includes the entire Hungary and extends also across a part of Serbia, Romania, Slovakia, Croatia, Czechia, and Ukraine, and is classified by some to include minor portions of Austria and Slovenia.

The water beetle fauna of the Pannonian Basin is relatively well-documented, with records of approximately 350 species in 14 families and over two hundred thousand occurrence data entries in databases. Over the past two decades, DNA barcoding has been crucial in studying European water beetles, with more than half of the species barcoded and possessing at least one reference barcode. However, systematic barcoding remains essential at local levels to uncover hidden molecular diversity, spatial distribution patterns and elucidate phylogenetic relationships.

Compared to other regions, the Pannonian ecoregion lags behind in public reference barcoding data. It can be described as a large white spot, with only Hungary and Slovakia contributing sequences for less than 4% of known species (14 and 7 sequences for 6 and 7 species, respectively). These sequences originated from only 7 sites; most of the species only possess a single sequence. We initiated a project last year to address this deficiency by providing numerous and comprehensive barcode records. As a start, ~200 sequences representing ~70 species collected from ~30 sites across Hungary were provided. In this talk, we highlight key aspects of our dataset, showcasing unique BINs and intriguing case studies that contribute to a deeper understanding of aquatic beetle diversity within the Pannonian ecoregion.

Alpine regions cover less than 3% of the Earth’s land surface, excluding Antarctica, but they represent a particularly unique ecosystem with a high degree of endemism and many endangered species. During the last glacial maximum, most of the alpine areas were covered by glaciers and firms, the gradual melting of which created ~50,000 alpine lakes and ponds only within Europe. At a time of increasing global warming, these unique freshwater habitats are an important refuge for many freshwater cold-tolerant species but also for those that are not adapted to rising temperatures. However, their remoteness, short ice-free period, and frequently difficult accessibility make research on these ecosystems difficult. The aim of this study, as a part of a broader research initiative on barcoding Tatra Mts freshwater fauna, was to analyse in detail the genetic diversity of common inhabitants of alpine lakes and ponds, the water beetles.

Within 387 sequences from 105 localities sampled between 2007–2018, 68 morphologically identified species in 27 genera and eight families were recorded, of which 1/3 was found for the first time in this area. To evaluate the genetic uniqueness of the Tatra beetle fauna, we compared our data with those available in the BOLD database (>1,700 sequences from 26 countries). The total alignment consisted of 657 haplotypes representing 106 BINs. Additional delimitation approaches resulted in 81 (ASAP) and 92 (mPTP) MOTUs. On average, ~75% of the globally captured putative species/BINs were also recorded within the Tatra Mountains. The recorded species consisted of 179 haplotypes, of which 30% were found exclusively here, confirming the important role of the Tatra Mt lakes in maintaining unique genetic information. Relatively high intraspecific genetic diversity was also confirmed, especially in three recorded species, each consisting of two phylogenetically distant, likely Pleistocene lineages. 

Our study confirmed that even relatively small but richly diversified alpine lakes can accumulate high genetic diversity and be an essential source of unique genetic information. Our data also significantly enriched the regional DNA barcode database, enabling more effective future monitoring of valuable alpine freshwater habitats. The study was supported by the project VEGA 2/0084/21.

Springs possess several unique environmental characteristics, including thermal and chemical stability, typically low oxygen content, and isolation by geographical or physicochemical barriers. This can result in low gene flow between springs and local episodes of adaptive radiation, potentially leading to significant differences in biodiversity between different springs and high levels of endemism. Moreover, strong directional selection in isolated habitats greatly reduces the range of morphological variation, potentially favouring speciation, including cryptic species. The study of cryptic diversity is crucial for a full understanding of species distribution, ecology, assessment of endemism patterns, as well as the development of appropriate biomonitoring and conservation strategies. All these characteristics make springs hotspots of biodiversity and endemism. Paradoxically, although springs are among the key ecosystems for the functioning of the biosphere, they remain understudied. Our international project aims to provide original and detailed information on the location, environmental properties and biodiversity of different types of springs in five model Carpathian ranges with different geographical locations and geological structures in Poland and Slovakia: 1. the Orava Beskid (northern Slovakia, flysch rocks) and the Żywiec Beskid (southern Poland, flysch rocks); 2. the Považský Inovec (western Slovakia, carbonate rocks); 3. Bukovec Mountains (north-eastern Slovakia, flysch rocks), Bieszczady (south-eastern Poland, flysch rocks); 4. Veporskie Mountains (central Slovakia, crystalline rocks); 5. Beskid Wyspowy (southern Poland, flysch, volcanic rocks). During the two-year study, we collected samples of macroinvertebrates from 52 sites located in the above mountain ranges. The dominant groups were Oligochaeta, Bivalvia, Gastropoda, Amphipoda, Isopoda, Ephemeroptera, Odonata, Plecoptera, Trichoptera, Coleoptera, and Diptera. DNA was extracted from more than 1,500 individuals. Approximately 900 DNA barcodes, representing 14 orders and four classes of invertebrates, have been obtained so far. They have been deposited in the Barcode of Life Data System (BOLD) public repository as the basis for a reference DNA barcode library for Carpathian springs. So far, BOLD algorithms grouped the DNA sequences into 74 molecular operational taxonomic units (BINs – Barcode Index Numbers), which are rough equivalents of species, of which 23 BINs are new to BOLD. We will provide an overview of the project as an example of a successful bottom-up international initiative focusing on poorly studied Carpathian aquatic habitats and funded by both national and international channels, as well as the first results on faunal and genetic connectivity between springs.

Funding provided by APVV SK-PL-21-0023, NAWA BPN/BSK/2021/1/00084/U/00001, UniLodz IDUB NR4/ML/2022, Eramsus+ BIP: Biodiversty of the Carpathian Springs.

Dipterans are key players in the assessment of the ecological integrity of freshwaters. However, species-level identification of most taxa remains challenging, especially in morphologically cryptic larvae. To improve these shortcomings, 1,149 mtCOI barcode sequences were generated from a bulk sample of macroinvertebrates, collected in different mountainous headwaters and large lowland rivers in the Romanian Carpathians. These corresponded to 177 morphologically identified species and 77 undetermined taxonomic units, represented mostly by larvae. Analysis of the Barcode Index Number (BIN) System revealed 287 BINs, of which 82.5% coincide strongly with known species boundaries. However, DNA gap analyses yielded significant intraspecific differences, whereby K2P distances at this level ranged from 0% to 16.22%, with an average of 2.81%, whereas interspecific distances within a genus ranged from 0% to 26.97%, with an average of 13.15%, revealing the likely occurrence of overlooked cryptic or pseudo-cryptic species, especially in mountainous headwaters. 95 BINs were assigned for the first time in BOLD, with 31 representing unknown operational taxonomic units. Species-level identification was successful or confirmed for 531 larvae, based on genetic similarity to corresponding adults, from which 48 larvae (28 taxa) were assigned to species-level exclusively using their genetic similarity in the BOLD database. Our data considerably improved the species-level identification effort of macroinvertebrates in a region with exceptionally high aquatic biodiversity and a considerable number of regionally restricted or endemic taxa. Our results support the utility of DNA barcodes to delimit species boundaries for the majority of Diptera groups, especially in morphologically cryptic larvae, and provide an effective taxonomic tool to improve routine bio-assessment protocols with high-quality data.

Epikarst, the uppermost zone of the karst landscape, functions as a critical interface between soil and carbonate rocks, forming a perched aquifer that stores and gradually releases water infiltrated from precipitation. This unique environment serves as both a water reservoir and a subterranean aquatic habitat for minute invertebrates, yet its biological and ecological processes remain significantly understudied. Between 2019 and 2020, we conducted extensive sampling of epikarst invertebrates using 27 filtering devices in the Demänová Cave System, northern Slovakia. Our integrative approach employed both conventional morphological techniques and molecular tools, specifically DNA barcoding, to evaluate the invertebrate diversity of this understudied subterranean ecosystem. Utilising the mitochondrial cytochrome oxidase subunit I (COI) gene as the standard barcoding marker, we successfully generated 784 DNA barcodes representing 36 morphospecies from 17 orders and 6 animal classes, with a predominant representation of Arthropoda (98.3%). Morphological identification revealed 10 obligatory aquatic species, including four stygobionts limited to groundwaters and nine amphibiotic species, as well as 17 terrestrial species, including five troglobionts associated with the cave environment. Our study provides a unique documentation of the genetic and species diversity of Metazoa within the epikarst, with more than 70% of Barcode Index Numbers (BINs) being new to the Barcode of Life Data Systems (BOLD). Importantly, 24 out of 28 morphologically identified subterranean species lacked sequence coverage in BOLD, emphasising the novelty of our findings. Notable cases of intraspecific divergence were observed, particularly in subterranean taxa, showcasing the existence of morphologically indistinguishable cryptic species within the fragmented karst system. Our data provide evidence for undescribed species of harpacticoid copepod Elaphoidella and suggest the potential presence of a new-to-science family within the order Amphipoda. In the case study of the Demänovský cave system, we demonstrate that the genetic and species diversity of invertebrates associated with seeping epikarst waters is vastly understudied. Our findings shed light on the overlooked realm of subterranean biodiversity, emphasising the need for future extensive investigations into groundwater life diversity in the Western Carpathians and adjacent regions. This study serves as a crucial foundation for understanding and conserving the unique ecosystems thriving in the depths of the epikarst.

Lake Ohrid, located in the Balkan Peninsula at the Albanian/Macedonian border, is the oldest European lake (ca. 1.3 My old), one of the world’s smallest ancient lakes and an important biodiversity hotspot. At the same time, considering the lake surface, it harbours the highest level of endemism, especially in the case of amphipod crustaceans (ca. 90%). Most of Lake Ohrid amphipods belong to the local endemic Gammarus (Amphipoda, Crustacea) species flock. Still, the evolutionary processes underlying speciation within this group in ancient lakes are weakly understood. Therefore, the Ohridian Gammarus species flock, encompassing about a dozen species, is an exceptional model for testing adaptive radiation compared to other such systems. The current study aims to unravel the evolutionary processes behind the origins of the endemic Gammarus species flock in this ancient lake using an integrative approach. We incorporate molecular study through extensive COI barcoding and transcriptome sequencing as well as a detailed examination of the functional morphology of the species flock and relatives.

In a result, we see that the distribution of the Gammarus species flock is vertically structured, reflecting habitat zonation and suggesting parapatric speciation as one of the possible mechanisms behind the flock’s diversification. Two new molecular units representing putatively new species are revealed. The onset of flock radiation overlaps with the time of lake formation, while the subsequent speciation events and demographic changes relate presumably to glacial and postglacial water level changes and to the colonisation of new depth ranges and the associated springs. The transcriptomic data show complex patterns of diversification, probably resulting from hybridisation during the flock radiation, while morphological variability shows relatively high diversity and divergence between species. All this suggests that adaptive radiation was the main force behind origins of the Gammarus species flock in ancient Lake Ohrid.

This work is funded by the Polish National Science Centre (OPUS16 grant Nb. UMO-2018/31/B/NZ8/03103).

Lake Ohrid, located at the border of North Macedonia and Albania, is considered the oldest lake in Europe (1.5 My). It has been designated a UNESCO World Heritage Site for its high level of endemism. Its biodiversity is still not fully known, and only few taxonomic groups have been analysed using an integrative approach that includes DNA methods (e.g., freshwater snails, leeches from the genus Dina, asselids and gammarids). What is more, some taxa are particularly challenging in identification, contain undescribed species or represent complexes of cryptic species so the DNA barcoding comes in handy for such.

The main aim of our study is to create a reliable publicly accessible reference library of macroinvertebrates inhabiting Lake Ohrid basin and its surrounding springs. The samples were collected in 2019 and 2022 by dredging or kick-sampling and preserved in 96% ethanol. The material was sorted into higher taxonomic groups. The animals belonging to Diptera, Gastropoda, Bivalvia, Turbelaria, Trichoptera, Hemiptera, Plecoptera, Oligochaeta, Coleoptera, Hydracarina, Ephemeroptera, Isopoda and Odonata were divided into morphotypes. From one to five individuals (1920 in total) of each morphotype the cytochrome c oxidase subunit I (COI) DNA barcode was obtained. The PCR products were sequenced using the Sanger and Oxford Nanopore platform. All obtained sequences were deposited in the Barcode of Life Database (BOLD). The final dataset contained 1460 barcode sequences belonging to 322 Barcode Index Numbers (BINs) of which 47 were new for BOLD. Additionally, to provide information about distribution of microorganisms in the Lake, substrate from different habitat zones was processed using a metabarcoding approach with 7 markers. Our study suggests that there are still some undescribed invertebrate species inhabiting Lake Ohrid basin and its springs. There is a need for further investigation of Lake Ohrid fauna to fully understand this unique ecosystem and protect it from human impacts and degradation.

This work is funded by the Polish National Science Centre (OPUS16 grant Nb. UMO-2018/ 31/B/NZ8/03103).

The lateral line organ is an intriguing sensory structure of gammarids, presumed to function as chemo-, mechano-, and/or electro-receptors. This organ is composed of two rows of dorsal and lateral units that are spread along the body. These structures are often found on each somite and telson, and each unit is characterised by a nearly straight or curved excavation on the cuticle with an arrangement of flattened microtrichs inserted within a rounded concentric “ring”. Despite some previous studies shedding light on the lateral line organ, many aspects of its composition and function are still unclear. As such, our study aims to investigate the structure and variation of this organ in amphipod crustaceans from diverse habitats and modes of life, with emphasis on the Gammarus species flock from the ancient Lake Ohrid. Preliminary analyses of species of 20 amphipod families from a broad geographic range show remarkable differentiation of the lateral line structure, making it possible to assume it as a potential morphological marker of adaptive radiation. As a study case, specimens collected from Lake Ohrid across a depth gradient from 5 to 240 meters were compared with sister species living in local springs and streams, all exposed to varied water current intensities, predation pressure and chemical composition. For the initial analysis, 10 males and 10 females of similar sizes from five species (Gammarus parechiniformis, G. roeselii, G. sketi, G. solidus and G. stankokaramani) were selected, coated with gold, and analysed through Scanning Electron Microscopy. The results show clear interspecific differences in the morphology of the lateral lines, including the degree of curvature of the excavations and the number of microtrichs of species from the same sampling site. Additionally, size- and sex-specific changes were observed. Based on these observations, we hypothesize that, in different environmental conditions, even the conspecific individuals might show divergent patterns in the structure of the lateral line. This could suggest that the organ is a specialised and adaptable structure, helping the gammarids in spatial orientation.