Phenotypic diversity plays a crucial role in facilitating the establishment and expansion of invasive populations in new environments. Particularly high levels of plasticity can be observed among the diverse Ponto-Caspian fauna that invaded inland European waters. However, the precise influence of the environment on shaping the phenotypic variation of these successful invaders remains inadequately understood. A good model species for such studies is Dikerogammarus villosus (Sovinsky, 1894) (Gammaridae, Amphipoda). This highly predatory and voracious species invaded a notable part of Europe within a few decades. It originated in the Ponto-Caspian region and the two isolated populations from the Danube and the Dnieper deltas colonised Europe through two phylogenetic lineages – the Western (via Danube) and the Eastern (via Dnieper). Although this species has a broad distribution and is currently expanding in Europe, its phenotypic diversity related to traits that determine invasive success was not studied. Therefore, we aimed to test morphological variation of functional traits that directly or indirectly reflect the diet of D. villosus across the European continent. Our results show that D. villosus exhibits a high morphological variability across Europe and that it is mainly shaped by the type of environment. We discovered an enhancement of predation-related traits in brackish waters compared to freshwaters. These results suggest that D. villosus could exert a greater threat on macroinvertebrate communities in brackish environments. Morphological differentiation among phylogenetic lineages and native vs. invaded ranges is weaker and mainly concerns locomotion and food processing traits (i.e., mouthparts and stomachs). Both lineages display a unique pattern of morphospace change between native and invaded ranges. A strong morphospace expansion in the invaded range of the Eastern Lineage suggests a higher plasticity of this lineage and possibly higher success in establishment in new habitats. Our results highlight the importance of accounting for ecological, evolutionary, and biogeographical factors when studying the phenotypic diversity of invasive species at broad continental scales. The results will be an important input to understanding the invasion dynamics of Ponto-Caspian amphipods in Europe.

Riverine ecosystems are dendritic, linear networks that are connected longitudinally, laterally, and vertically. Environmental gradients, as a result of uni-directional flow, create a great number of diverse but still interconnected microhabitats, making stream ecosystems the most heterogeneous, most complex, hierarchically nested systems where ecological processes both in the upstream reaches and the adjacent catchments govern local conditions. Examining, explaining, and especially modelling the relationships between riverine species and their environment are therefore particularly challenging and require a multi-scale and multi-model approach.

In the present study, our aims were to build an ensemble of base learner machine learning (ML) models based on selected multi-scale environmental parameters shaping the sequential distribution of ten Central European species of the genus Hydropsyche that could explain and effectively predict the occurrence of species and/or groups of them with similar ecological preferences.

Our results show that several gradient-like and pollution related factors that are usually measured as part of monitoring programmes (such as those of Water Framework Directive) at finer spatial scales and in higher detail could be considered suitable to complement or substitute those (e.g., topographic, geographic or precipitation data) that are routinely included in species distribution models (SDMs). It is also revealed that an ensemble of multi-scale models can outperform single modelling algorithms in mapping the longitudinal distribution of species and species groups.

Vegetation in the riverine ecosystems plays a crucial role in providing food and shelter for the invertebrates. Not only the vascular plants but also the aquatic mosses are important habitats for the benthos. The common water moss (Fontinalis antipyretica Hedw.) occurrence is limited by a presence of solid substrates, e.g., stones or submerged wood to which plant can attach. F. antipyretica occurs in highly oxygenated and clear rivers. Therefore, the common water moss as the microhabitats for Chironomidae in the lowland river Rawka (central Poland) was studied. Rawka is protected as a nature reserve, being one of the few natural rivers of the Polish Lowlands. Its benthic assemblages have not been studied extensively so far, only preliminary research conducted indicates that Chironomidae are the least-known and most underestimated group. As the riverbed of the Rawka is not regulated or dredged, it exhibits a variety of moss microhabitats. The material was collected from 22 sites, including samples of Chironomidae larvae and algal periphyton, and a control sample from the bottom. The physicochemical conditions have been examined. As the diatoms are the main component of periphytic invertebrates’ diet, their species composition and richness were analysed. The preliminary results indicate 20 species of Chironomidae, including 3 new species to the Polish fauna. Most of the recorded species are rheobiontic and small-sized, i.e., Rheotanytarsus photophilus, Eukiefferiella claripennis and Thienemanniella majuscula. Eighteen Chironomidae species have been recorded for the first time from this river which leads to the hypothesis that aquatic moss microhabitats hide a substantial diversity of Chironomidae. The new data contribute to the knowledge concerning the aquatic fauna of Rawka Nature Reserve and shed new light on aquatic moss as an important habitat for macroinvertebrates.

The Graphoderus bilineatus, a highly protected species listed as Vulnerable (VU) by the IUCN Red List and designated under Natura 2000 in Hungary, holds significant conservation importance at both national and European levels. Despite increasing literature on its ecology, knowledge gaps persist, as the life history, development, and ecological preferences of the G. bilineatus exhibit considerable variability across regions and over time. Understanding these variations is crucial for developing effective long-term conservation strategies targeting both the species and its habitats. Decades of field observations in Hungary have revealed a consistent pattern: the abundance and presence of G. bilineatus are closely linked to spring floods, particularly within floodplain habitats, a phenomenon less observed in other European countries to the north. We quantified the time intervals between the occurrence of spring floods and the presence or absence of the species in known habitats along the Drava, Danube, Bodrog, and Tisza rivers. Our findings demonstrate a significant correlation between the occurrence and appearance of the species and spring floods. Furthermore, our data suggest that these floods may act as a triggering mechanism, prompting the G. bilineatus to initiate flight and potentially colonise new water bodies within its range. Understanding the interplay between spring flood dynamics and the ecology of G. bilineatus is essential for informing targeted conservation efforts aimed at preserving this species and its associated habitats.

Species coexistence has been one of the focal points in ecology. Particularly, the coexistence of closely related taxa occupying similar niches provides an interesting research topic. According to ecological theory, such an event is unlikely. However, recent evidence indicates that this phenomenon emerges across a wide range of phylogenetically distinct taxa and from all types of habitats. Nevertheless, the stability and the mechanisms facilitating such coexistence remain unknown. Therefore, a multitude of evidence is needed to better understand the dynamics and eco-evolutionary drivers underlying this ecological paradox. Here, we focused on a unique case on the island of Sicily where two local amphipod species, Homeogammarus sicilianus and Homeogammarus adipatus, co-occur in a single river system, Fosso del Tempio. Despite being widespread across the island, they solely co-occur in the studied system. This raises the question of whether this coexistence is stable and supported by niche differentiation or reflects an unstable, possibly seasonal, co-occurrence. By applying an array of DNA-based methods and seasonal fine-scale sampling, we aimed to investigate potential niche differentiation by studying the microhabitat preferences, dietary habits, reproductive activity, and potential influence of parasites on the co-occurring amphipod species. Our results indicate that both species co-occur at nearly all studied sampling sites across the entire river system, regardless of the season. We also found significant differences in microhabitat distribution amongst both species, with H. sicilianus occurring more in biotic and H. adipatus found more in abiotic microhabitats. DNA metabarcoding results revealed different feeding strategies between the two species, with H. adipatus being a more specialised feeder and H. sicilianus having a more generalistic diet. We also found differences in species’ reproductive activity over the year. Finally, even though both species shared the same parasite lineages, the parasite composition and prevalence patterns significantly differed between the two host species. The obtained results support a stable coexistence between the two studied species. However, based on the observed niche differences, H. sicilianus seems better suited to outcompete H. adipatus in a longer evolutionary timescale, challenging the stability of observed coexistence. Our results indicate that a highly integrative framework is needed to disentangle drivers of species coexistence, supporting its use for studying similar cases.

Gammarus fossarum, a wide-ranging cryptic species complex, has exceptional diversity in the Western Carpathians, whereat local syntopy of distinct lineages is common. Previous research from Western Europe, as well as the Carpathians, has indicated that at least some of G. fossarum lineages differ ecologically (e.g., in resistance to anthropogenic pollution, or interactions with parasites). Within the Vsetín region, Czechia, three highly divergent, reproductively isolated lineages of the complex, i.e., biological species, commonly co-occur. In this project, we explore the puzzling nature of this coexistence, specifically to what extent the lineage composition is influenced by the local conditions. Conveniently, many streams of the area exhibit a downstream environmental gradient, leading to a gradual change in conditions from forested headwaters to an urbanised stream mouth. We expect that local conditions promote dominance of certain lineages, and thus the compositions of the Gammarus communities change gradually along the stream path. We hypothesise that this pattern should be consistent between streams, and temporally stable. Three streams were selected for their moderate length, downstream environmental gradient, and the known presence of all three studied lineages. We sampled ten sites along the entirety of each stream during the spring, summer, and fall over two years. Mitochondrial 16S rDNA sequencing of 30 individuals per site was used to determine lineage composition. Preliminary results show a gradual change in dominance along and temporal stability between the spring season of two years in one of the streams. On the contrary, two other streams exhibited an irregular pattern, though this pattern was similar between them. So far, it seems that the environmental gradient of anthropogenic pressure is not the key driver of Gammarus lineage distribution in the studied area, but more data is needed to support or reject this conclusion. As our research progresses, it will enable us to compare the dynamic environment of streams with more stable habitat of spring fens, where alternative food source use and body size differences seem to promote the syntopy of Gammarus lineages. Interestingly, we did not detect body size differentiation among syntopic lineages in the streams. This may indicate that the lineage syntopy is driven by alternative mechanisms in different environments.

Recent advances in DNA sequencing techniques have greatly accelerated the acquisition of large-scale data. Among others, access to the information on complete mitogenomes has been greatly facilitated, improving the resolution of phylogenetic or other analyses requiring robust and reliable DNA data. Among the most promising technology in this regard is long-read nanopore sequencing, developed by Oxford Nanopore Technologies. This study aims to elucidate the composition features of the mitogenome of A. bipustulatus, achieved through long-read nanopore sequencing. 

Samples were collected from the Tatra Mountain glacial lakes (Western Carpathians, Slovakia), and High-Molecular-Weight DNA was utilised for library preparation. Sequencing was performed using the Ligation sequencing gDNA – Native Barcoding Kit 96 V14 (SQK-NBD114.96). Bioinformatic processing of reads for mitogenome generation involved several programs, including Flye, MitoFinder, Minimap2, Medaka and Mitos2. Nucleotide composition was analysed using MegaX, and skewness was calculated utilising standard formulae. Start and stop codons were determined using exPasy. The resulting mitogenome was 17,876 base pairs (bp) long, with sequencing data achieving 139× coverage. Among the 13 protein-coding genes (PCGs), three (nad6, nad3, nad1) initiated with an ATA start codon, five (cox2, atp6, cox3, cob, nad4) with an ATG start codon, four (nad2, atp8, nad5, nad4l) with an ATT start codon, while the cox1 gene commenced with a CCG. Codon usage analysis revealed Leu and Asn as the most frequent, while Cys and Arg were the least common. The stop codon TAA predominated among PCGs, with only 3 (nad3, nad1, cob) ending with TAG. The overall base composition of the 13 PCGs, which had a total length of 11,181 bp, was 34.40% for A, 43.56% for T, 11.58% for G, and 10.48% for C. The total length of tRNAs was 1,460 bp, with individual lengths ranging from 63 to 71 bp (the overall base composition of tRNAs was A: 39.66%, T: 38.91%, G: 11.58%, C: 9.74%). The total length of the 2 rRNAs was 2049 bp, with an overall base composition of 38.54% A, 43.72% T, 11.47% G, and 6.31% C. The length of the control region was 2,955 bp (A: 49.60%, C: 3.90%, T: 42.80%, G: 3.70%). Skewness analysis revealed a positive AT skew in the control region (0.074) and tRNAs (0.010), while PCGs (-0.118) and rRNAs (-0.063) exhibited a negative AT skew. The GC skewness was negative in the control region (-0.026) and positive for PCGs (0.050), tRNAs (0.092), and rRNAs (0.290). Compared to previously published diving beetle mitogenomes, the sequence obtained by nanopore-based long-read sequencing is significantly more reliable concerning problematic regions of mtDNA. Data obtained in this way can be a reliable basis for subsequent comparative molecular studies, examining gene arrangement or differences in mitogenome size and composition. The study was supported by the European Union NextGenerationEU project No 09I03-03-V01-00075, titled “Scholarships for excellent researchers threatened by the conflict in Ukraine“ under the Recovery and Resilience Plan of the Slovak Republic, and by the Slovak National Grant Agency (VEGA), project no. 2/0084/21.

According to the original concept of Illies (1978), adopted by the EU Water Framework Directive (WFD 2000), the areas of North Macedonia and Greece are divided longitudinally along the Vardar/Axios River as the border between two ecoregions: Ecoregion 6 – Greek Western Balkans (right tributaries) and Ecoregion 7 – Eastern Balkans (left tributaries). To date, there are no macroinvertebrate data that would support an adjustment of the boundaries between the two hydrofaunal ecoregions for the area of North Macedonia. The aim of this study is an approach to a more realistic definition of the hydrofaunistic boundaries in the territory of North Macedonia, with respect to aquatic macroinvertebrate communities, based on the semi-mountainous and mountainous river types. To achieve this goal, a comparative analysis (PCA, Principal Component Analysis) of the macroinvertebrate communities from the studied areas (94 sites from eight river basins belonging to the three ecoregions 5, 6 and 7) was performed to verify their affiliation to a specific ecoregion. The results showed that the strict delineation of hydrofaunistic boundaries between the studied ecoregions is complex, mainly due to the peculiarities in the geographical distribution of macroinvertebrates and the biotypic characteristics of the rivers themselves, which show greater similarities in the ecoregions’ border zones. As the selected study sites from all three ecoregions were chosen based on their categorisation as semi-mountainous and mountainous river types, one would expect greater similarity between areas of similar elevation and substrate. Regarding the distribution of the studied watersheds of the selected semi-mountainous and mountainous rivers, the obtained results indicate the shift of the border to the west, where the entire Vardar watershed belongs to Ecoregion 6 – Greek Western Balkans. These results would be a good basis for future research in relation to the other biological quality elements in order to find the best approach to support the proposed delineation between ecoregions 6 and 7.

This study was funded by “Programme for Support of Young Researchers and PhD Students at the Bulgarian Academy of Sciences” (Grant No. DFNP-17-108/28.07.2017 – “Implementation of biotic indices BMWP and ASPT in order to evaluate the ecological status of mountain and semi-mountain rivers in the 7th Ecoregion (Eastern Balkans)”) and by the “Japan Funds-in-Trust Project” (REF.: ERI/MSP/PPF/LZF/15.037) – “Management of freshwater bodies with implementation of EU-Water Framework Directives using aquatic macroinvertebrates as the necessary measure for sustainable development of freshwater ecosystems in less developed countries”.

The Grand Duchy of Luxembourg is located in the transition area between Western and Central Europe. A team has been established at the National Museum of Natural History Luxembourg that focuses on recording subterranean life. The project “Scientific findings, basic data collection and monitoring of subterranean habitats in the Grand Duchy of Luxembourg” focuses on recording all plant, fungal and animal species living in underground environments. Luxembourg is rich in natural caves but also artificial cavities, e.g., mines for iron ore, copper, antimony, slate, gypsum, dolomite. Also, subterranean military installations such as the casemates of the City of Luxembourg comprise prominent objects. However, running or standing waters in underground environments being directly human-accessible are scarce. Therefore, groundwater measuring points and shaft wells are sampled, in addition to springs as outflows of groundwater. Interesting results were also obtained from the investigation of interstitials close to riverine shorelines.

Specimens were collected mainly by sieving with four sieves with mesh sizes of 5,000 μm, 1,000 μm, 600 μm and 200 μm. Meat-baited tin cans were placed in mines. Numerous experts carry out the morphological identification of various animal groups. Questionable results and species thought to be new to science are genetically analysed in the museum’s own laboratories.

An overview of the proven classes and orders is given. As can be expected, the focus of subterranean aquatic fauna is on crustaceans. Ostracods (12 species) and copepods (27 species) are frequently found. The malacostracans are represented by the orders of isopods (two species) and amphipods, the latter by the families Crangonyctidae (one species), Pseudoniphargidae (one species), and Niphargidae (eight species). Two Niphargus species are new to science. They will be described in 2024 and one even bears the name of the Grand Duchy.

The faunistic outcomes from caves are used for the Habitats Directive assessment of the Habitat type 8310. In addition, artificial cavities with an unusual species inventory are protected, such as the slate mine Laura in Haut-Martelange in which Niphargus boulangei was found has already been categorised as being of high conservation value. So far, only one locality of this species (northern France), which has probably been destroyed in the interim, has been published. In the meanwhile, there are sufficient records to analyse them biogeographically, then in connection with neighbouring regions. Furthermore, previously neglected biotope groups, such as castle wells or groundwater measuring points, are to be intensively investigated. We also hope that new collection techniques such as downpipes, net collectors and wood washing will yield previously unknown results.

Niphargus elegans Garbini, 1894 is one of the few surface species in the predominantly groundwater genus Niphargus. Described on material from the surroundings of Verona, Italy, it shows a wide distribution in the Padanian Plain and along the Adriatic coast where it inhabits surface streams, rivers, and ponds. As for many niphargid species, the taxonomy of N. elegans is intricate. Considered by some authors as a subspecies of the central-European N. puteanus (Koch, 1836), it was later enlarged to include spring-dwelling subspecies in Slovenia (N. illidzensis slovenicus S. Karaman, 1932, subsequently transferred to N. elegans by Gordan Karaman) and Croatia (N. elegans zagrebensis S. Karaman, 1950); furthermore, it was reported from springs and caves in Romania.

We conducted a DNA-based species delimitation using four molecular markers (two nuclear rRNA gene fragments – ITS and 28S, the nuclear histone H3 gene and a fragment of the mitochondrial COI) to investigate the relationships among the alleged N. elegans subspecies as well with Italian and Romanian populations attributed to N. elegans. A phylogenetic analysis based on a concatenated dataset of four markers was also performed to investigate the relationships between N. elegans and two other surface niphargids, N. valachicus Dobreanu & Manolache, 1933 and N. hrabei Karaman, 1932, both widely distributed in the Danubian basin in western Europe. We tested (i) whether Niphargus elegans is monophyletic and whether its alleged subspecies are actually distinct species, (ii) whether its phylogenetic position suggests a secondary colonisation of the epigean environment and, in that case, (iii) whether N. elegans, N. hrabei and N. valachicus independently colonised surface waters from groundwaters.

Results show that despite their morphological similarities, the three N. elegans populations from Italy, Slovenia, and Croatia are three distinct, closely related species. By contrast, Romanian populations previously called N. elegans belong to a different clade (close to N. valachicus) that is a complex of new or poorly described species, including N. pater Mehely, 1941. Italian N. elegans, N. hrabei and N. valachicus, despite their ecological similarity, are not closely related and independently colonised surface waters from three different groundwater ancestors. These results highlight that although the genus Niphargus shows major adaptations to groundwater life (such as eye and pigment loss), it repeatedly colonised surface waters in different geographical areas in Europe.

The release of ornamental pets and associated pathogens outside their native range might directly or indirectly impact the recipient community. In central Europe, feral freshwater species originating in tropical and sub-tropical regions are typically restricted to thermally altered environments such as heated waters and thermal springs. However, species like the shrimp Neocaridina davidi have high environmental plasticity and may acclimate to cooler temperatures. A widening thermal niche may eventually overcome thermal barriers, further expanding the range, and enhancing transmission opportunities for host generalist parasites. This study investigates the observed (field observations) and theoretical (species distribution models) range expansion of N. davidi and associated parasites across Europe. We report the establishment of three new N. davidi populations in thermally altered waters in Germany, Hungary, and Slovakia, and a range expansion into colder environments. Species distribution models predict suitable thermal habitats in Mediterranean regions, with a foreseeable expansion towards Western Europe and the Balkans by 2050.

Furthermore, we confirm the presence of the microsporidian parasite Ecytonucleospora hepatopenaei in feral N. davidi populations throughout Europe and expand the list of microsporidians found in this host from two to four. We present the first evidence of parasite transmission between N. davidi and the invasive crayfish Procambarus clarkii, indicating a potential for parasite exchange with native fauna. Such possibility, coupled with an ongoing range expansion of N. davidi bolstered by human-mediated introductions and climate change, will likely exacerbate the impact on native biota.

Niphargids (Malacostraca: Niphargidae: Niphargus sp.) constitute a family of crustaceans, with over 450 species described to date, predominantly inhabiting subterranean environments. As a result, they exhibit cavernicolous adaptations, including depigmentation and eyelessness. These crustaceans are widespread, ranging from Spain to Iran, with approximately 30 species identified in Germany. However, evidence of their occurrence north of the glacial boundary from the last ice age glaciations is scarce. Previous records from the United Kingdom, the Netherlands, and Poland either turned out to be misidentified or lacked sufficient recollection.

This study presents, for the first time, DNA barcoding confirmed records of Niphargus from three German springs, specifically rheocrenes, north of the former glaciation, situated approximately 70 km north of the glacier edge associated with the Elster and Saale glaciations. These springs are approximately 60 kilometres apart from each other.

All three springs harbour Niphargus aquilex, a species widely distributed from England to northern France, Belgium, Luxembourg, and Germany, albeit not commonly found. The Folmer fragment of the Cytochrome c Oxidase Subunit 1 (COI) gene was sequenced for 19 individuals, revealing two haplotypes. The more common haplotype is also present in the southern distribution area of the species, providing evidence of post-glacial migration to the north. Contrary to initial assumptions, the species did not survive the ice age in nunataks or under the ice sheet.

The new findings, however, prompt several questions. Although 18 of the 19 sequences are identical and match sequences found in the south, one sequence is missing in the south. Could this indicate a population that survived under the ice? Alternatively, is it a recent mutation involving at least two base pairs within the presumed 100,000 years? The N. aquilex group is a species complex comprising 10 to 20 species, the majority of which are cryptic, while the rest are pseudocryptic. Why, then, has only one species from this complex migrated northward? Unlike other species (e.g., Niphargus puteanus, Niphargus fontanus), which have migrated northwards parallel to the rivers, N. aquilex’s colonisation of the north poses unique questions about its adaptive mechanisms.