Migration and isolation during the turbulent Ponto-Caspian Pleistocene create high diversity in the crustacean Paramysis lacustris.

The Ponto-Caspian brackish-water fauna inhabits estuaries and rivers of the Black, Azov and Caspian seas and is fragmented by higher salinity waters and a major interbasin watershed. The fauna is known for the high levels of endemism, complex zoogeographic histories, and as a recent source of successful invasive species. It remains debated whether the Black and Azov Sea brackish-water populations survived unfavourable Pleistocene conditions in multiple separate refugia or whether the two seas were (repeatedly) recolonized from the Caspian. Using microsatellite and mtDNA markers, we demonstrate deep among- and within-basin subdivisions in a widespread Ponto-Caspian mysid crustacean Paramysis lacustris. Five genetic clusters were identified, but their relationships did not reflect the geography of the region. The Azov cluster was the most distinct (4-5% COI divergence), despite its geographic position in the corridor between Black and Caspian seas, and may represent a new species. In the northern Black Sea area, the Dnieper cluster was closer to the Caspian cluster than to the neighbouring Danube-Dniester-Bug populations, suggesting separate colonizations of the Black Sea. Overall, the data implied a predominant gene flow from the east to the Black Sea and highlight the importance of Caspian Sea transgressions in facilitating dispersal. Yet, the presence of distinct lineages in the Black Sea points to the persistence of isolated refugial populations that have gained diagnostic differences under presumably high mutation rates and large population sizes. The unfavourable Pleistocene periods in the Black Sea therefore appear to have promoted diversification of the brackish-water lineages, rather than extirpated them.

Pollution patterns and their effects on biota within lotic and lentic freshwater ecosystems: How well contamination and response indicators correspond?

Aquatic environments are often severely polluted with chemical substances of anthropogenic origin, which can pose a potential threat to aquatic organisms and human health. In this study, patterns and sources of heavy metals (HMs, 6 metals) and polycyclic aromatic hydrocarbons (PAHs, 16 hydrocarbons), contamination indicators, environmental genotoxicity measures and metrics of ecological status in lotic and lentic ecosystems were collated for the first time. Chemical analysis has confirmed previously reported long-term contamination at certain study sites. The sediments of Lake Talksa, located in a city and characterized by exclusive anthropogenic pressure, exhibited the highest levels of contamination by both HMs and PAHs. Through positive matrix factorization (PMF) analysis, vehicle and industrial emissions were identified as the primary sources of HMs and PAHs. Our results revealed that frequencies of genotoxic aberrations were higher in river sites compared to lakes, with the highest genotoxic risk observed in the Nemunas River below industrial cities Alytus and Kaunas. Surprisingly, even the severely contaminated Lake Talksa showed only a "moderate" grade of genotoxic risk, highlighting the potential for adaptation of biota to long-term contamination especially in lentic ecosystems. The ecological quality status assessed by macroinvertebrate metrics, which may be sensitive to observed high biological contamination, appeared to be unrelated to contamination patterns. Consequently, to obtain the robust information on anthropogenic contamination and its effects, a combination of various assessment methods and metrics should be employed.

A DNA barcode reference library for endemic Ponto-Caspian amphipods.

The Ponto-Caspian region is an endemicity hotspot that harbours several crustacean radiations, among which amphipods are the most diverse. These poorly known species are severely threatened in their native range, while at the same time they are invading European inland waters with significant ecological consequences. A proper taxonomic knowledge of this fauna is paramount for its conservation within the native region and monitoring outside of it. Here, we assemble a DNA barcode reference library for nearly 60% of all known Ponto-Caspian amphipod species. We use several methods to define molecular operational taxonomic units (MOTUs), based on two mitochondrial markers (COI and 16S), and assess their congruence with current species-level taxonomy based on morphology. Depending on the method, we find that 54-69% of species had congruent morpho-molecular boundaries. The cases of incongruence resulted from lumping distinct morphospecies into a single MOTU (7-27%), splitting a morphospecies into several MOTUs (4-28%), or both (4-11%). MOTUs defined by distance-based methods without a priori divergence thresholds showed the highest congruence with morphological taxonomy. These results indicate that DNA barcoding is valuable for clarifying the diversity of Ponto-Caspian amphipods, but reveals that extensive work is needed to resolve taxonomic uncertainties. Our study advances the DNA barcode reference library for the European aquatic biota, paving the way towards improved taxonomic knowledge needed to enhance monitoring and conservation efforts.

Contamination and genotoxicity biomarker responses in bivalve mussels from the major Lithuanian rivers.

European inland waters are under continuous threat of anthropogenic pollution. Determination of background level of biomarker response and subsequent classification of the impact increases the applicability of results. In the current study, we evaluate the range of chemical contamination by measuring the concentrations of metals, polycyclic aromatic hydrocarbons and polychlorinated biphenyls, and the levels of environmental genotoxicity by using the micronuclei and nuclear bud tests in bivalve mussels of the major Lithuanian rivers. Second, we aimed to evaluate the association between chemical contamination and genotoxicity biomarker responses. Finally, we set to determine the background level of genotoxic effects. Such value (summed frequency of MN and NB) was assessed-6‰. On that basis, we develop a scale of potential genotoxic impact and perform ranging sites into five categories. The results clearly indicate the existence of significant differences in the levels of chemical pollution and genotoxicity in different sites. Increased levels of studied parameters were assessed at the areas affected by municipal and industrial effluents, road runoff, combustion products, and in the area contaminated by accidental spillage. On the other hand, downstream decrease of contamination level, presumably associated with biological degradation and photochemical oxidation, were also observed. Genotoxicity parameters were associated with PAH and metal concentrations measured in mussel tissues as well as in sediments. Results also indicate that in situ genotoxicity assessment performed in the areas affected by long-term contamination of municipal origin might be not sufficiently precise. Study highlights the necessity to combine genotoxicity assessment with chemical analysis. Environ. Mol. Mutagen. 61:338-354, 2020. © 2019 Wiley Periodicals, Inc.

Trend in ambient ozone and an attempt to detect its effect on biota in forest ecosystem. Step I of Lithuanian studies.

The presented study aimed to explore the relationships between ambient ozone (O3) and tree defoliation, specific diversity, and abundance of soil microarthropods, stream macroinvertebrates, and small mammals (mainly rodents) in order to test the hypothesis that changes in the considered objects of the forest ecosystem could be related to changes in ambient O3, concentration of which is below critical level. The observations were carried out from 1994 at three integrated monitoring stations. The obtained data revealed that only peak O3 concentrations (from 125-215 microg x m(-3)) had significant effect on changes in the considered components of forest biota.

Benthic macroinvertebrates in lake ecological assessment: A review of methods, intercalibration and practical recommendations.

Legislation in Europe has been adopted to determine and improve the ecological integrity of inland and coastal waters. Assessment is based on four biotic groups, including benthic macroinvertebrate communities. For lakes, benthic invertebrates have been recognized as one of the most difficult organism groups to use in ecological assessment, and hitherto their use in ecological assessment has been limited. In this study, we review and intercalibrate 13 benthic invertebrate-based tools across Europe. These assessment tools address different human impacts: acidification (3 methods), eutrophication (3 methods), morphological alterations (2 methods), and a combination of the last two (5 methods). For intercalibration, the methods were grouped into four intercalibration groups, according to the habitat sampled and putative pressure. Boundaries of the 'good ecological status' were compared and harmonized using direct or indirect comparison approaches. To enable indirect comparison of the methods, three common pressure indices and two common biological multimetric indices were developed for larger geographical areas. Additionally, we identified the best-performing methods based on their responsiveness to different human impacts. Based on these experiences, we provide practical recommendations for the development and harmonization of benthic invertebrate assessment methods in lakes and similar habitats.

Analysis of DNA Methylation and Hydroxymethylation in the Genome of Crustacean Daphnia pulex.

The aim of our study was to analyze the presence of 5-methyl-cytosine (5-mC) and 5-hydroxymethyl-cytosine (5-hmC) in the genome of crustacean Daphnia pulex. First, the presence of 5-mC and 5-hmC in genomic DNA was demonstrated by using antibodies specific to either 5-mC or 5-hmC. Then, analysis of 5-mC and 5-hmC using pairs of restriction enzymes with different sensitivity to methylation and hydroxymethylation confirmed the presence of both modifications in selected regions of three genes (Cox4, Cand2 and Ephx1). To get a detailed picture of 5-hmC distribution over the D. pulex genome, we performed 5-hmC enrichment and sequenced the enriched fraction using next generation sequencing and non-enriched library (input) as a control. Comparison of input and enriched libraries showed that 5-hmC in exons is twice as frequent as in introns. Functional analysis indicated that 5-hmC abundance is associated with genes that are involved in the adenylate cyclase-activating G-protein-coupled receptor signaling pathway, molting cycles, morphogenesis and cell fate determination. Genes that lack 5-hmC tend to be involved in the regulation of the transforming growth factor beta receptor signaling pathway and in many mRNA-related processes. Our results suggest that epigenetic modifications are present in the genome of D. pulex and most likely are involved in the regulation of gene expression of this crustacean.

Assessing the risks of aquatic species invasions via European inland waterways: from concepts to environmental indicators.

Over the past century, the potential for aquatic species to expand their ranges in Europe has been enhanced both as a result of the construction of new canals and because of increased international trade. A complex network of inland waterways now connects some previously isolated catchments in southern (Caspian, Azov, Black, Mediterranean seas) and northern (Baltic, North, Wadden, White seas) Europe, and these waterways act as corridors for nonnative species invasions. We have developed a conceptual risk assessment model for invasive alien species introductions via European inland waterways, with specific protocols that focus on the development of environmental indicators within the socioeconomic context of the driving forces-pressures-state-impact-response framework. The risk assessment protocols and water quality indicators on alien species were tested for selected ecosystems within 3 main European invasion corridors, and these can be recommended for application as part of the Common Implementation Strategy of the European Commission Water Framework Directive, which aims to provide a holistic risk-based management of European river basins. The conceptual structure of the online Risk Assessment Toolkit for aquatic invasive alien species is provided and includes 3 main interlinked components: online risk assessment protocols, an early warning system, and an information transmitter for risk communication to end users.