A collaborative backbone resource for comparative studies of subterranean evolution: The World Asellidae database.

Transition to novel environments, such as groundwater colonization by surface organisms, provides an excellent research ground to study phenotypic evolution. However, interspecific comparative studies on evolution to groundwater life are few because of the challenge in assembling large ecological and molecular resources for species-rich taxa comprised of surface and subterranean species. Here, we make available to the scientific community an operational set of working tools and resources for the Asellidae, a family of freshwater isopods containing hundreds of surface and subterranean species. First, we release the World Asellidae database (WAD) and its web application, a sustainable and FAIR solution to producing and sharing data and biological material. WAD provides access to thousands of species occurrences, specimens, DNA extracts and DNA sequences with rich metadata ensuring full scientific traceability. Second, we perform a large-scale dated phylogenetic reconstruction of Asellidae to support phylogenetic comparative analyses. Of 424 terminal branches, we identify 34 pairs of surface and subterranean species representing independent replicates of the transition from surface water to groundwater. Third, we exemplify the usefulness of WAD for documenting phenotypic shifts associated with colonization of subterranean habitats. We provide the first phylogenetically controlled evidence that body size of males decreases relative to that of females upon groundwater colonization, suggesting competition for rare receptive females selects for smaller, more agile males in groundwater. By making these tools and resources widely accessible, we open up new opportunities for exploring how phenotypic traits evolve in response to changes in selective pressures and trade-offs during groundwater colonization.

Regional climate contributes more than geographic distance to beta diversity of copepods (Crustacea Copepoda) between caves of Italy.

Despite the study of subterranean biodiversity facing harsh sampling and mapping challenges, the huge diversity of taxa, ecological adaptations and evolutionary trajectories in subterranean environments is gaining increasing attention. Yet, the spatial and environmental factors driving the composition of groundwater communities are still poorly understood. To partially fill this knowledge gap, we collected copepod crustaceans from 12 caves along the Italian peninsula between 2019 and 2022, sampling each cave twice. The resulting presence-absence data were analysed to assess: (i) between-cave taxonomic beta diversity, also partitioning between turnover and nestedness-resultant dissimilarity; (ii) the relative weight of geographic distance and climatic differences in shaping observed beta diversity. Seventy-one species of copepods were collected overall. Pairwise beta diversity was high for most pairs of caves, with turnover being the major component. Geographic distance-decay models partially explained total beta diversity and turnover patterns. However, in Generalized Dissimilarity Models (GDM), including surface climatic conditions as predictors, the contribution of seasonal temperature averages was generally higher than that of geographic distance. Further, the explanatory and predictive performance of the GDMs notably increased, along with temperature contribution, when widening the spatial extent from which climate data were gathered. Our results confirmed a high spatial turnover in groundwater copepods' assemblages and strengthened the link between regional climate and subterranean biodiversity.

Some like it hot: Thermal preference of the groundwater amphipod Niphargus longicaudatus (Costa, 1851) and climate change implications.

Groundwater is a crucial resource for humans and the environment, but its global human demand currently exceeds available volumes by 3.5 times. Climate change is expected to exacerbate this situation by increasing the frequency of droughts along with human impacts on groundwater ecosystems. Despite prior research on the quantitative effects of climate change on groundwater, the direct impacts on groundwater biodiversity, especially obligate groundwater species, remain largely unexplored. Therefore, investigating the potential impacts of climate change, including groundwater temperature changes, is crucial for the survival of obligate groundwater species. This study aimed to determine the thermal niche breadth of the crustacean amphipod species Niphargus longicaudatus by using the chronic method. We found that N. longicaudatus has a wide thermal niche with a natural performance range of 7-9 °C, which corresponds to the thermal regime this species experiences within its distribution range in Italy. The observed range of preferred temperature (PT) was different from the mean annual temperature of the sites from which the species has been collected, challenging the idea that groundwater species are only adapted to narrow temperature ranges. Considering the significant threats of climate change to groundwater ecosystems, these findings provide crucial information for the conservation of obligate groundwater species, suggesting that some of them may be more resilient to temperature changes than previously thought. Understanding the fundamental thermal niche of these species can inform conservation efforts and management strategies to protect groundwater ecosystems and their communities.

Ingestion of microplastics and textile cellulose particles by some meiofaunal taxa of an urban stream.

Microplastics (MPs) and textile cellulose are globally pervasive pollutants in freshwater. In-situ studies assessing the ingestion of MPs by freshwater meiofauna are few. Here, we evaluated MP and textile cellulose ingestion by some meiofaunal taxa and functional guilds of a first-order stream in the city of Florence (Italy) by using a tandem microscopy approach (fluorescence microscopy and µFTIR). The study targeted five taxa (nematodes, oligochaetes, copepods, ephemeropterans and chironomids), three feeding (scrapers, deposit-feeders, and predators), and three locomotion (crawlers, burrowers, and swimmers) guilds. Fluorescent particles related to both MPs and textile cellulose resulted in high numbers in all taxa and functional guilds. We found the highest number of particles in nematodes (5200 particles/ind.) and deposit-feeders (1693 particles/ind.). Oligochaetes and chironomids (burrowers) ingested the largest particles (medium length: 28 and 48 µm, respectively), whereas deposit-feeders ingested larger particles (medium length: 26 µm) than scrapers and predators. Pellets were abundant in all taxa, except for Chironomidae. Textile cellulose fibers were present in all taxa and functional guilds, while MP polymers (EVA, PET, PA, PE, PE-PP) differed among taxa and functional guilds. In detail: EVA and PET particles were found only in chironomids, PE particles occurred in chironomids, copepods and ephemeropterans, PA particles were found in all taxa except in nematodes, whereas particles made of PE-PP blend occurred in oligochaetes and copepods. Burrowers and deposit-feeders ingested EVA, PET, PA, PE and PE-PP, while crawlers and scrapers ingested PE and PA. Swimmers and predators ingested PE, PA and PE-PP. Our findings suggest a pervasive level of plastic and textile cellulose pollution consistent with an urban stream which propagates in the meiofaunal assemblage of the stream ecosystem.

The impact of nitrate on the groundwater assemblages of European unconsolidated aquifers is likely less severe than expected.

In this study, we analyzed the structure of the stygobiotic copepod assemblages of an unconsolidated European aquifer (VO), in southern Italy, that has been subject to persistent nitrate contamination for over 15 years. To this end, we monitored 25 bores where groundwater was contaminated only by nitrate, and no other chemical pollutants were reported as being above detection limits from 2009 to 2014. We monitored these bores three times, namely in autumn 2014 and in spring and autumn 2015. We expected that the chronic exposure to high nitrate concentrations had a significant and evident impact on the stygobiotic copepod assemblages. Unexpectedly, the assemblages were highly diversified. The stygobiotic species richness (SSR) accounted 17 species, a value that exceeded the European mean value (SSR = 12 species). However, the species density was only 0.6 species/km2, lower than the European mean value (= 1.6 species/km2). Moreover, the juvenile copepods were numerically less abundant than the adults and the biomass-abundance model showed signs of alteration of the structure of the copepod assemblages. This study highlighted that (i) nitrates, even at high concentrations, probably have a less severe impact on groundwater assemblages of unconsolidated aquifers than expected and (ii) the analysis of population traits and biomasses can detect signs of alteration of these assemblages that would, otherwise, not be visible from the analysis of the sole species richness and abundances.

Recommendations for ecotoxicity testing with stygobiotic species in the framework of groundwater environmental risk assessment.

As a consequence of the growing global dependence on groundwater resources, environmental risk assessments (ERA) for groundwater are increasingly required and, with that, ecotoxicological studies with groundwater fauna (stygofauna). However, the literature on the ecotoxicological studies with stygobiotic species (i.e. species that complete their life cycle exclusively in groundwater) has not expanded significantly since the first paper published in this field. The limitations regarding the use of stygobiotic species for ecotoxicological testing are clear and consistent across the globe; stygobiotic species are often 1) naturally present in low numbers, 2) difficult to collect, and 3) difficult to culture under laboratory conditions. This paper reviews the methods used in ecotoxicological studies performed with stygobiotic species, and provides ten recommendations for Good Laboratory Practice (GLP) for such tests. The recommendations focused on the following 10 points: 1) the taxonomic identification, the life stage/size and gender of the test organisms; 2) collection methodology of the organisms, including collection location, conditions and methods; 3) holding and acclimation conditions in the laboratory; 4) exposure conditions such as test set up and exposure time, number of replicates and densities of organisms in tests and in test vessels; 5) range-finding test set up and schedule; 6) final test design, including details of controls and treatments, and replication options; 7) incubation conditions, specifying temperature, pH and oxygenation levels throughout the test; 8) test duration; 9) observations and endpoints; 10) test validity criteria and compliance. The recommendations were developed for the purpose of supporting future short-term ecotoxicological testing with stygofauna through providing consistency in the protocols. A discussion of the potential implications for groundwater managers and decision-makers committed to ERA for groundwater is included.

The role of freshwater copepods in the environmental risk assessment of caffeine and propranolol mixtures in the surface water bodies of Spain.

In this study we aimed at assessing: (i) the environmental risk posed by mixtures of caffeine and propranolol to the freshwater ecosystems of Spain; (ii) the sensitivity of freshwater copepod species to the two compounds; (iii) if the toxicity of caffeine and propranolol to freshwater copepods contributes to the environmental risk posed by the two compounds in the freshwater bodies of Spain. The environmental risk was computed as the ratio of MECs (i.e. the measured environmental concentrations) to PNECs (i.e. the respective predicted no-effect concentrations). The effects of caffeine and propranolol on the freshwater cyclopoid Diacyclops crassicaudis crassicaudis were tested both individually and in binary mixtures. Propranolol posed an environmental risk in some but not in all the surface water ecosystems of Spain investigated in this study, while caffeine posed an environmental risk to all the investigated freshwater bodies, both as single compound and in the mixture with propranolol. Propranolol was the most toxic compound to D. crassicaudis crassicaudis, while caffeine was non-toxic to this species. The CA model predicted the toxicity of the propranolol and caffeine mixture for this species. D. crassicaudis crassicaudis was much less sensitive than several other aquatic species to both compounds. The sensitivity of D. crassicaudis crassicaudis does not increase the environmental risk posed by the two compounds in the freshwater bodies of Spain, however, further testing is recommended since the effect of toxicants on freshwater copepods can be more pronounced under multiple stressors and temperature increasing due to climate change.

Ecological risk assessment of pesticide mixtures in the alluvial aquifers of central Italy: Toward more realistic scenarios for risk mitigation.

In this study we used the data of an extensive pesticide monitoring survey that took place in 11 alluvial aquifers of central Italy from 2010 to 2015 to explore 4 different scenarios of risk. The Scenarios 1 and 2 were used to depict the risk of failing to meet the good groundwater chemical status as defined by the Water Framework Directive. The Scenarios 3 and 4 were used to assess for the first time the ecological risk in groundwater bodies, defined as the likelihood of hazard to the groundwater communities stably residing in the 11 alluvial aquifers that may be affected by pesticide contamination. The ecological risk was assessed through a new procedure called GERAp (Groundwater Ecological Risk Assessment due to pesticides). The main results of this study highlighted that: 1) the Scenario 1 provided information of little use for risk managers; 2) more realistic information was provided by using the highest concentrations measured in the six-year monitoring period and considering the ecological risk in a combined scenario (Scenarios 2 and 4); 3) the achievement of the good chemical status by 2027 in 3 aquifers will be likely much more difficult than in the others because the ecosystem services, such as pesticide biodegradation, are likely less efficient in the 3 groundwater bodies; 4) some pesticides that were banned in Europe in 2009 should be kept monitored in the next surveys because they showed a persistent occurrence in the 11 aquifers; 5) DDT forms, Dieldrin and Heptachlor are expected to damage groundwater communities at concentrations that are lower than the present legal limits.

Test procedures for measuring the (sub)chronic effects of chemicals on the freshwater cyclopoid Eucyclops serrulatus.

The purpose of this study has been to describe test procedures for measuring the (sub)chronic effects of chemicals on the freshwater cyclopoid Eucyclops serrulatus. To this end we have adapted the setting of the standard full life-cycle protocol of the marine harpacticoid A. tenuiremis to E. serrulatus. We have tested the effects of 4 different diets, two temperatures and two rearing volumes on the survival, development, reproduction and population growth rates of this species. Our results have highlighted that full life-cycle tests can be run using 2 mL-glass vials, a diet consisting of a mixture of living cells of Chlorella sorokiniana and Scenedesmus quadricauda, at either 25 °C (test duration: 42 days) or 18 °C (test duration: 51 days). However, the best performance in terms of survival, development, reproducibility and population growth rates with this species was obtained at 18 °C, albeit with significantly longer test duration. Subchronic tests in 2 mL-glass vials with the mixture microalgal diet at both temperatures are available options if considered appropriate for the objectives of a given study. In particular, developmental tests from nauplius to copepodid may profitably be performed in about 11 days at 18 °C and in 6 days at 25 °C. Under the same test conditions, subchronic tests from copepodid to adult may be run in 19 days at 18 °C and in 16 days at 25 °C.

Genomic resources notes accepted 1 October 2014-30 November 2014.

This article documents the public availability of (i) RAD sequencing data and validated SNPs for the American mink Neovison vison and (ii) Transcriptome resources for two nonmodel freshwater crustacean species, the copepod Eucyclops serrulatus and the amphipod Echinogammarus veneris.