Groundwater is a hidden global keystone ecosystem.

Groundwater is a vital ecosystem of the global water cycle, hosting unique biodiversity and providing essential services to societies. Despite being the largest unfrozen freshwater resource, in a period of depletion by extraction and pollution, groundwater environments have been repeatedly overlooked in global biodiversity conservation agendas. Disregarding the importance of groundwater as an ecosystem ignores its critical role in preserving surface biomes. To foster timely global conservation of groundwater, we propose elevating the concept of keystone species into the realm of ecosystems, claiming groundwater as a keystone ecosystem that influences the integrity of many dependent ecosystems. Our global analysis shows that over half of land surface areas (52.6%) has a medium-to-high interaction with groundwater, reaching up to 74.9% when deserts and high mountains are excluded. We postulate that the intrinsic transboundary features of groundwater are critical for shifting perspectives towards more holistic approaches in aquatic ecology and beyond. Furthermore, we propose eight key themes to develop a science-policy integrated groundwater conservation agenda. Given ecosystems above and below the ground intersect at many levels, considering groundwater as an essential component of planetary health is pivotal to reduce biodiversity loss and buffer against climate change.

Integrating citizen science and environmental DNA metabarcoding to study biodiversity of groundwater amphipods in Switzerland.

Groundwater is the physically largest freshwater ecosystem, yet one of the least explored habitats on earth, both because of accessing difficulties and the scarcity of the organisms inhabiting it. Here, we demonstrate how a two-fold approach provides complementary information on the occurrence and diversity of groundwater amphipods. Firstly, we used a citizen science approach in collaboration with municipal water providers who sampled groundwater organisms in their spring catchment boxes over multiple weeks, followed by DNA barcoding. Secondly, we collected four 10 L water samples at each site, in one sampling event, for environmental DNA (eDNA) metabarcoding. We found that citizen science was very effective in describing the distribution and abundance of groundwater amphipods. Although the single time-point of eDNA sampling did not detect as many amphipods, it allowed the assessment of the entire groundwater community, including microorganisms. By combining both methods, we found different amphipod species co-occurring with distinct sequences from the eDNA-metabarcoding dataset, representing mainly micro-eukaryotic species. We also found a distinct correlation between the diversity of amphipods and the overall biodiversity of groundwater organisms detected by eDNA at each site. We thus suggest that these approaches can be used to get a better understanding of subterranean biodiversity.

Groundwater environmental DNA metabarcoding reveals hidden diversity and reflects land-use and geology.

Despite being the most important source of liquid freshwater on the planet, groundwater is severely threatened by climate change, agriculture, or industrial mining. It is thus extensively monitored for pollutants and declines in quantity. The organisms living in groundwater, however, are rarely the target of surveillance programmes and little is known about the fauna inhabiting underground habitats. The difficulties accessing groundwater, the lack of expertise, and the apparent scarcity of these organisms challenge sampling and prohibit adequate knowledge on groundwater fauna. Environmental DNA (eDNA) metabarcoding provides an approach to overcome these limitations but is largely unexplored. Here, we sampled water in 20 communal spring catchment boxes used for drinking water provisioning in Switzerland, with a high level of replication at both filtration and amplification steps. We sequenced a portion of the COI mitochondrial gene, which resulted in 4917 ASVs, yet only 3% of the reads could be assigned to a species, genus, or family with more than 90% identity. Careful evaluation of the unassigned reads corroborated that these sequences were true COI sequences belonging mostly to diverse eukaryotic groups, not present in the reference databases. Principal component analyses showed a strong correlation of the community composition with the surface land-use (agriculture vs. forest) and geology (fissured rock vs. unconsolidated sediment). While incomplete reference databases limit the assignment of taxa in groundwater eDNA metabarcoding, we showed that taxonomy-free approaches can reveal large hidden diversity and couple it with major land-use drivers, revealing their imprint on chemical and biological properties of groundwater.

Optimizing laboratory cultures of Gammarus fossarum (Crustacea: Amphipoda) as a study organism in environmental sciences and ecotoxicology.

Amphipods are among the most abundant macroinvertebrates in freshwater ecosystems of the Palaearctic and crucial for ecosystem functioning. Furthermore, their high sensitivity to environmental change and pollutants makes them widely used model organisms in environmental sciences and ecotoxicology. In field studies and surveys across Eurasia, species of the genus Gammarus are commonly used, yet laboratory-based studies and ecotoxicological tests are often restricted to the in most parts of the world non-native Hyalella azteca, as Gammarus is much harder to breed and maintain under laboratory conditions. However, for direct comparisons and extrapolations of results of field- vs. laboratory-based studies, the use of the same species would be desirable. Here, we investigated different settings with respect to feeding, shelter and day length to successfully increase survival, juvenile production and their respective growth and survival, and ultimately multi-generation breeding of the amphipod Gammarus fossarum. Amphipod populations persisted and reproduced successfully under optimized husbandry conditions for 12 months and were partially maintained for another year in populations up to a few hundred individuals. Specifically, supplementing diet with protein-rich food sources as well as the provisioning of shelters improved survival rate of G. fossarum significantly. However, we found no significant effect of different day length treatments on the overall relative reproductive activity or on the total amphipod abundance maintained. We conclude that G. fossarum can be kept and reared under standardized conditions. Despite the longer generation times of G. fossarum and higher effort required for maintenance compared to H. azteca, direct ecological relevance and comparability of results to natural systems may justify its future use and development as a study organism for environmental sciences and ecotoxicology.

Climate, immigration and speciation shape terrestrial and aquatic biodiversity in the European Alps.

Quaternary climate fluctuations can affect speciation in regional biodiversity assembly in two non-mutually exclusive ways: a glacial species pump, where isolation in glacial refugia accelerates allopatric speciation, and adaptive radiation in underused adaptive zones during ice-free periods. We detected biogeographic and genetic signatures associated with both mechanisms in the assembly of the biota of the European Alps. Age distributions of endemic and widespread species within aquatic and terrestrial taxa (amphipods, fishes, amphibians, butterflies and flowering plants) revealed that endemic fish evolved only in lakes, are highly sympatric, and mainly of Holocene age, consistent with adaptive radiation. Endemic amphipods are ancient, suggesting preglacial radiation with limited range expansion and local Pleistocene survival, perhaps facilitated by a groundwater-dwelling lifestyle. Terrestrial endemics are mostly of Pleistocene age and are thus more consistent with the glacial species pump. The lack of evidence for Holocene adaptive radiation in the terrestrial biome is consistent with faster recolonization through range expansion of these taxa after glacial retreats. More stable and less seasonal ecological conditions in lakes during the Holocene may also have contributed to Holocene speciation in lakes. The high proportion of young, endemic species makes the Alpine biota vulnerable to climate change, but the mechanisms and consequences of species loss will likely differ between biomes because of their distinct evolutionary histories.

Dispersal behaviour and riverine network connectivity shape the genetic diversity of freshwater amphipod metapopulations.

Theory predicts that the distribution of genetic diversity in a landscape is strongly dependent on the connectivity of the metapopulation and the dispersal of individuals between patches. However, the influence of explicit spatial configurations such as dendritic landscapes on the genetic diversity of metapopulations is still understudied, and theoretical corroborations of empirical patterns are largely lacking. Here, we used microsatellite data and stochastic simulations of two metapopulations of freshwater amphipods in a 28,000 km2 riverine network to study the influence of spatial connectivity and dispersal strategies on the spatial distribution of their genetic diversity. We found a significant imprint of the effects of riverine network connectivity on the local and global genetic diversity of both amphipod species. Data from 95 sites showed that allelic richness significantly increased towards more central nodes of the network. This was also seen for observed heterozygosity, yet not for expected heterozygosity. Genetic differentiation increased with instream distance. In simulation models, depending on the mutational model assumed, upstream movement probability and dispersal rate, respectively, emerged as key factors explaining the empirically observed distribution of local genetic diversity and genetic differentiation. Surprisingly, the role of site-specific carrying capacities, for example by assuming a direct dependency of population size on local river size, was less clear cut: while our best fitting model scenario included this feature, over all simulations, scaling of carrying capacities did not increase data-model fit. This highlights the importance of dispersal behaviour along spatial networks in shaping population genetic diversity.

Species multidimensional effects explain idiosyncratic responses of communities to environmental change.

Environmental change can alter species' abundances within communities consistently; for example, increasing all abundances by the same percentage, or more idiosyncratically. Here, we show how comparing effects of temperature on species grown in isolation and when grown together helps our understanding of how ecological communities more generally respond to environmental change. In particular, we find that the shape of the feasibility domain (the parameter space of carrying capacities compatible with positive species' abundances) helps to explain the composition of experimental microbial communities under changing environmental conditions. First, we introduce a measure to quantify the asymmetry of a community's feasibility domain using the column vectors of the corresponding interaction matrix. These column vectors describe the effects each species has on all other species in the community (hereafter referred to as species' multidimensional effects). We show that as the asymmetry of the feasibility domain increases the relationship between species' abundance when grown together and when grown in isolation weakens. We then show that microbial communities experiencing different temperature environments exhibit patterns consistent with this theory. Specifically, communities at warmer temperatures show relatively more asymmetry; thus, the idiosyncrasy of responses is higher compared with that in communities at cooler temperatures. These results suggest that while species' interactions are typically defined at the pairwise level, multispecies dynamics can be better understood by focusing on the effects of these interactions at the community level.

Biodiversity increases and decreases ecosystem stability.

Losses and gains in species diversity affect ecological stability1-7 and the sustainability of ecosystem functions and services8-13. Experiments and models have revealed positive, negative and no effects of diversity on individual components of stability, such as temporal variability, resistance and resilience2,3,6,11,12,14. How these stability components covary remains poorly understood15. Similarly, the effects of diversity on overall ecosystem stability16, which is conceptually akin to ecosystem multifunctionality17,18, remain unknown. Here we studied communities of aquatic ciliates to understand how temporal variability, resistance and overall ecosystem stability responded to diversity (that is, species richness) in a large experiment involving 690 micro-ecosystems sampled 19 times over 40 days, resulting in 12,939 samplings. Species richness increased temporal stability but decreased resistance to warming. Thus, two stability components covaried negatively along the diversity gradient. Previous biodiversity manipulation studies rarely reported such negative covariation despite general predictions of the negative effects of diversity on individual stability components3. Integrating our findings with the ecosystem multifunctionality concept revealed hump- and U-shaped effects of diversity on overall ecosystem stability. That is, biodiversity can increase overall ecosystem stability when biodiversity is low, and decrease it when biodiversity is high, or the opposite with a U-shaped relationship. The effects of diversity on ecosystem multifunctionality would also be hump- or U-shaped if diversity had positive effects on some functions and negative effects on others. Linking the ecosystem multifunctionality concept and ecosystem stability can transform the perceived effects of diversity on ecological stability and may help to translate this science into policy-relevant information.

Translating Niphargus barcodes from Switzerland into taxonomy with a description of two new species (Amphipoda, Niphargidae).

The amphipod genus Niphargus (Amphipoda: Niphargidae Bousfield, 1977) is the most species-rich genus of freshwater amphipods in the World. Species of this genus, which live almost exclusively in subterranean water, offer an interesting model system for basic and applied biodiversity science. Their use, however, is often limited due to the hitherto unresolved taxonomy within the whole genus. As a comprehensive taxonomic revision of the currently >425 Niphargus species is too demanding, it has been suggested that the taxonomy of the genus could be advanced in smaller steps, by reviewing regional faunas, that would eventually integrate into a global revision. In this study, we provide such a revision of Niphargus in Switzerland. First, we molecularly delimited, morphologically diagnosed, and formally described two new species, namely Niphargus luchoffmannisp. n. and Niphargus tonywhittenisp. n. Second, we updated and revised a checklist of Niphargus in Switzerland with new findings, and prepared a list of reference sequences for routine molecular identification, available at BOLD and GenBank. All available specimens of 22 known species from the area were morphologically examined, and their morphological variation was compiled in a data file of DEscription Language for TAxonomy, which can be used for automated generation of dichotomous or interactive keys. The data file is freely available at the World Amphipoda Database. Together, the checklist, the library of reference sequences, the DELTA file, but also a list of hitherto unresolved aspects are an important step towards a complete revision of the genus within a well-defined and biogeographically interesting area in Central Europe.

Spatial patterns of genetic diversity, community composition and occurrence of native and non-native amphipods in naturally replicated tributary streams.

BACKGROUND: Worldwide, natural communities are invaded by non-native species, with potentially devastating effects on the native communities. A large part of past research aimed at finding traits and characteristics of the invading species or the invaded community explaining observed invasions. Only recently, the focus shifted on the spatial patterns during invasions per se. Empirical data, however, are limited, as invasions are often unique incidences of a complex spatio-temporal process. In order to identify generalities of invasion patterns, we studied 13 naturally replicated tributary streams draining into Lake Constance, and studied the occurrence of native and non-native amphipods along linear transects from the stream outlets to the upstream headwater reaches. RESULTS: We found repeated spatial patterns of community composition and the occurrence of native and non-native amphipod species across two different years. Specifically, occurrence as well as abundance of two non-native amphipod species decreased from the stream outlets at the lake site towards upstream headwater reaches. Populations of the most common native amphipod species were largest at the uppermost headwater reaches. All populations of this native species, however, showed significant signals of recent genetic bottlenecks, irrespective of the stream position and occurrence of non-native species. Contrary to our expectations, this native species also showed no longitudinal genetic differentiation within individual tributaries as postulated for headwater versus outlet populations. CONCLUSIONS: Our results indicate that invasions of river-systems may overall follow predictable patterns on the level of spatial distributions and community composition. However, effects of invading organisms on the genetic diversity and genetic structure of native populations observed at larger scales may not necessarily be directly reflected at the scale of smaller tributaries.

Diversity and distribution of freshwater amphipod species in Switzerland (Crustacea: Amphipoda).

Amphipods are key organisms in many freshwater systems and contribute substantially to the diversity and functioning of macroinvertebrate communities. Furthermore, they are commonly used as bioindicators and for ecotoxicological tests. For many areas, however, diversity and distribution of amphipods is inadequately known, which limits their use in ecological and ecotoxicological studies and handicaps conservation initiatives. We studied the diversity and distribution of amphipods in Switzerland (Central Europe), covering four major drainage basins, an altitudinal gradient of>2,500 m, and various habitats (rivers, streams, lakes and groundwater). We provide the first provisional checklist and detailed information on the distribution and diversity of all amphipod species from Switzerland. In total, we found 29 amphipod species. This includes 16 native and 13 non-native species, one of the latter (Orchestia cavimana) reported here for the first time for Switzerland. The diversity is compared to neighboring countries. We specifically discuss species of the genus Niphargus, which are often receiving less attention. We also found evidence of an even higher level of hidden diversity, and the potential occurrence of further cryptic species. This diversity reflects the biogeographic past of Switzerland, and suggests that amphipods are ideally suited to address questions on endemism and adaptive radiations, post-glaciation re-colonization and invasion dynamics as well as biodiversity-ecosystem functioning relationships in aquatic systems.