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Our study addresses how large-scale hydrological alterations shape zooplankton biodiversity in floodplain ecosystems, which are highly sensitive to changes in river connectivity. Following the operation of the Gabčíkovo hydroelectric power plant in the Danube inland delta, we examined the long-term responses of crustacean zooplankton communities, as these organisms are key indicators of hydromorphological disturbance. Based on previous evidence that river regulation often reduces habitat heterogeneity, we hypothesized that hydrological alterations in the Danube riverscape would promote increasing taxonomic and functional homogenization within sites, while simultaneously enhancing differentiation between sites over the past three decades. A total of 121 planktonic crustacean species were recorded across six monitored sites between 1991 and 2020, comprising 49 copepods and 72 cladocerans. Communities showed rising species richness, especially during the first decade of the hydropower plant’s operation. While overall richness increased, dam-induced hydromorphological changes triggered habitat-specific community shifts. In the main channel and adjacent parapotamal arm, taxonomic and functional homogenization occurred, dominated by resilient tychoplanktonic species with a gathering or secondary filter-feeding strategy. In contrast, isolated side arms experienced gradual eutrophication, favoring euplanktonic and primary filter-feeding taxa. The observed taxonomic and functional convergence within both habitat groups reflects the loss of connectivity and the cessation of artificial flooding.

Routine biomonitoring under the Water Framework Directive (WFD) follows a conventional methodology primarily based on the morphological identification of taxa within the five biological quality elements (phytoplankton, phytobenthos, macrozoobenthos, macrophytes and fish). This identification is particularly challenging for macrozoobenthos due to their high taxonomic diversity. Moreover, this approach is time-consuming and resource intensive. Our current study aims to: (i) evaluate the implementation of DNA metabarcoding in long-term monitored sites to assess the effectiveness of three sample types (bulk samples of macrozoobenthos, and eDNA from water and sediments), (ii) evaluate the ecological status of water bodies using metabarcoding data, and (iii) compare ecological metrics values using conventional and molecular approaches. Sampling was conducted at 17 localities in Slovakia, covering eight stream types. DNA metabarcoding detected 30% more species than conventional monitoring performed over 15 years without needing time-intensive sample processing and morphological identification. At the same time, the bulk samples captured more macrozoobenthos taxa than the other sample types. On average, the bulk samples detected 85% of the taxa captured by DNA metabarcoding. The differences in values of ecological metrics obtained within the conventional approach and between the conventional and metabarcoding approaches were relatively small, typically corresponding to differences within one ecological quality class. However, more pronounced differences were observed in non-abundance-based metrics, such as EPT and BMWP, indicating higher sensitivity of species detection with DNA metabarcoding. This study demonstrates the effectiveness of DNA metabarcoding across multiple metrics and highlights its potential to enhance routine biomonitoring.

Assessments of biodiversity and ecosystem status can benefit from DNA metabarcoding as a means to streamline sample processing and specimen identification. Moreover, processing the fixation medium instead of the precious material introduces straightforward protocols that allow subsequent focus on certain organisms detected among the preserved specimens. In this study, we present a proof of concept via the analysis of freshwater invertebrate samples from the Tatra Mountain lakes (Slovakia). Besides highlighting a match between the lake-specific environmental conditions and the results of our fixative DNA metabarcoding, we observed an option to fine-tune the fixation time: to prefer two weeks over a day or a month. This effect emerged from the presence/absence of individual taxa rather than from coarse per-sample records of taxonomic richness, demonstrating that metabarcoding studies—and efforts to optimize their protocols—can use the robust metrics to explore even subtle trends. We also provide evidence that fixative DNA might better capture large freshwater species than terrestrial or meiofauna.

Springs as ecotones represent a suitable environment for testing hypotheses regarding the principles of a benthic community assembly. In this study, we aimed to uncover the processes which form the oligochaete assemblages in the karst springs environment. To uncover the assembly mechanism of the oligochaete community, we applied a multidimensional approach which included taxonomic, trait, and phylogenetic data. In total, 37 aquatic/semiaquatic oligochaete taxa were found. Null model analyses did not uncover any clear evidence of a nonrandom functional/phylogenetic structure of spring oligochaete fauna. Although, for all of the springs, the observed functional/phylogenetic diversity did not differ from that of null models, the position of the majority of observed values under the mean value of null models suggests that environmental filtering may be a more important driver in the community assembly. The environmental parameters that significantly drive the three biodiversity facets of the oligochaetes were spring temperature (functional and phylogenetic diversity), trophic status (taxonomic a phylogenetic diversity), spring connection (taxonomic diversity), and substrate, as well as oxygen conditions (functional diversity). Our findings provide, therefore, robust evidence that the ecological drivers of biodiversity differ among different scales, and future research should apply the multifacet approach to determine biodiversity conservation management.

Freshwater biomonitoring under the EU Water Framework Directive (WFD) relies on multiple biological quality elements, with macrozoobenthos being one of the most widely used, although still constrained by time-consuming morphological identification and the continued decline in taxonomic expertise. These constraints, together with ongoing rapid biodiversity loss, create an urgent need for high-resolution, scalable assessment methods such as DNA metabarcoding. Here, we evaluated this molecular approach as a WFD-compatible method and compared the performance of bulk and environmental DNA (eDNA) sampling for ecological quality classification in Slovakia. During spring 2022–2023, we sampled 34 WFD long-term monitoring sites using (i) bulk macroinvertebrate kick-samples, and (ii) water and (iii) sediment eDNA. A ~420 bp fragment of the mitochondrial COI gene was amplified using the BF3/BR2 primers, and sequenced. Filtered reads were clustered into OTUs based on 97% identity and taxonomically assigned using the BOLD database. Raw read counts were used as abundance proxies to calculate 12 ASTERICS metrics and multimetric Ecological Quality Ratios (EQRs). Sequencing yielded > 32.1 million reads and 1,205 target OTUs, of which 702 corresponded to 558 Linnaean species. Bulk samples captured the highest richness, followed by water and then sediment eDNA. Bulk samples classified 22 sites as very good Ecological Quality Class (EQC), whereas water and sediment samples frequently reduced EQC by two to three classes. EQC differences were driven mainly by presence/absence metrics (EPT taxa and BMWP), while several abundance-dependent metrics remained stable. Bulk metabarcoding provides a comprehensive signal but still inflates presence/absence metrics, highlighting the need for recalibration and optimised eDNA sampling, including primer selection, to enable comparable non-invasive assessments.

Monitoring changes in freshwater fauna requires timely, efficient, and comprehensive data collection, needs that are increasingly met by molecular methods. This is especially important in remote regions along environmental gradients, such as the Tatra Mountain lakes in Slovakia. In this study, DNA metabarcoding was applied to littoral benthic invertebrates collected from lakes in three valleys, spanning a gradient from the subalpine to alpine zones. Ordination analyses grouped the lakes based on community composition, and these groups were evaluated in relation to altitude, physico-chemical properties, indicator species, and previous data. To assess their additional value, surface sediment samples from the littoral zone were also analyzed using metabarcoding. The results revealed two distinct lake groups, primarily differentiated by altitude, alkalinity, and other correlated environmental parameters. The placement of specific lakes within these groups corresponded with known patterns of recovery from acidification, catchment characteristics, and potentially climate change or lake connectivity. Metabarcoding-based indicator species analysis successfully identified even taxa that had been previously overlooked or misidentified in morphology-based studies. While sediment metabarcoding provided limited supplementary information, its ordination patterns aligned with those from invertebrate analyses. Overall, our study of an alpine lake system supports broader trends: species richness declines with increasing elevation; community composition reflects environmental gradients; and DNA metabarcoding offers high taxonomic resolution and valuable insights into freshwater invertebrate biodiversity.

Throughout the ages, geological bedrock has significantly determined the structure of the Tatra stonefly taxocoenoses. Because of the high altitude and discharge of the streams, stoneflies are mainly affected by alkalinity, Ca, and Si. Statistical analysis has previously shown that alkalinity, Ca and Si content, catchment area, and altitude are correlated with the stonefly Tatrataxocenoses. In the present study, we recorded two different stonefly taxocenoses attached to different types of bedrock. For example, the stonefly species Amphinemura sulcicollis (Stephens, 1835), Protonemura auberti Illies, 1954, Protonemura nitida (Pictet, 1835), Leuctra braueri Kempny, 1898, and Dinocras cephalotes (Curtis, 1827) are characteristic for limestone brooks, while the genera Brachyptera Newport, 1848, Rhabdiopteryx Klapálek, 1902, Capnia Pictet, 1841, Perlodes Banks, 1903, Leuctra nigra (Olivier, 1811), and Perla grandis Rambur, 1842 are characteristic for crystalline streams. Furthermore, the Amphinemura sulcicollis species is characteristic of carbonate streams of the Carpathian Mts., while Brachyptera seticornis (Klapálek, 1902) is typical of crystalline streams. In crystalline streams, Perloidae represent 24–25%, however, just 13–15% in carbonate streams. Taeniopterygidae represent 11–14% in crystalline streams, while only 1–4% in carbonate streams. Finally, we recorded Nemouridae and Leuctridae as the dominate species in carbonate streams.

Our study provides insights into the spatio-temporal dynamics of macrophyte assemblages in the Danube Inland Delta, situated in the northwest part of the Pannonian lowland on the Slovak-Hungarian border. A total of 63 aquatic plant species were recorded at six monitored sites during the years 2003–2020, including 1 endangered, 2 vulnerable, and 1 neophyte species. Macrophyte data from long-term monitoring were used to test the hypothesis that aquatic plant assemblages in the hydrologically disturbed Danube riverscape have become more taxonomically and functionally similar over the past 17 years. Although a decline in the taxonomic and functional richness of macrophyte assemblages was observed at most monitoring sites, no evident year-to-year decrease in their heterogeneity was noted for either aspect of biodiversity. However, a significant decline in taxonomic and functional richness, as well as in the heterogeneity of the macrophyte community across the entire monitored area, was observed during the study period. While a decrease in species number represented approximately one species per year, the taxonomical beta diversity, including its taxonomical turnover component, also declined, with a mean annual decrease of 0.5% and 0.7%, respectively. Using abundance class data, the mean annual decrease in beta diversity was 0.6%. A time-series analysis of macrophyte functional traits (growth form, dispersal unit, and strategy) revealed that aquatic plant assemblages from different monitoring sites converged toward a narrower range of functional structure. The mean annual decline in macrophyte functional heterogeneity was 0.7% based on abundance class data and approximately 0.3% based on incidence data.

Our study examines community structure and nymphal biology (life cycles and secondary production) of stoneflies in two adjacent mountain streams with different degree of forest cover in the Prosiečanka River Basin (Chočské Vrchy Mts., West Carpathians). One of the streams has non-forested catchment, converted to meadows and pastures, while the other one has catchment with 60% covered by spruce forest. Differences in forest cover and in thermal regime of the streams were reflected by the difference of stonefly communities at their structural and functional level. Species Nemoura cinerea and Leuctra aurita created stonefly assemblage in non-forested stream, whereas Nemoura cinerea also occurred in naturally forested stream together with species Leuctra armata, Leuctra nigra, Leuctra prima, Siphonoperla neglecta and Arcynopteryx dichroa. All examined species had maximally annual life cycle and in eudominant species Nemoura cinerea one month shift was found in nymphal hatching and adult emergence between streams. Total secondary production of stoneflies in undisturbed stream (126.46 mg DW m-2 y-1) was more than two times higher than the production in non-forested stream (47.39 mg DW m-2 y-1). 

The Gabčíkovo Water Project, a major construction of damming and canalizing on the upper part of the middle Danube, significantly changed hydrological regime of the Danube inland delta, destroyed or affected most of the 230 km 2 of wetlands and directly disrupted the original and unique ecosystem along a 37 km long river stretch. The aim of this study was to describe the effect of the Gabčíkovo Waterworks on the taxonomical and functional structure of the molluscan assemblages in the Danube riparian floodplain forests. The results demonstrate that the Gabčíkovo Waterworks had a direct and long-lasting effect on the direction of the succession of terrestrial molluscan assemblages, especially in the area of the by-pass section. The changes in the soil moisture caused by the waterworks’ operation led to significant changes in the species and functional composition of these assemblages. More specifically, however, the proportion of the generalists which prefer dry biotopes increased, while the number of moisture-demanding species decreased. Our results indicate that the current artificial flooding system cannot fully replace previous natural floods in the Danube inland delta, and it is also insufficient for restoration and preservation of the humidity conditions in the softwood floodplain forests which would be similar to the pre-operation period of the Gabčíkovo Waterworks.