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The Caucasus region harbors a rich subterranean amphipod fauna. However, it was rarely studied using molecular tools. Recent field explorations unveiled a number of populations that morphologically correspond to the little known amphipod Niphargus alasonius described from Georgia. The populations are distributed along the Kura River drainage, spanning a range that exceeds 300 km. This unusually broad distribution implies that N. alasonius is a good disperser, exploiting the riverine interstitial as a distributional corridor, or alternatively, that it is a complex of cryptic species. We tested these two hypotheses using a molecular genetic approach. Multilocus phylogenetic analysis suggested that N. alasonius is a monophyletic taxon, closely related to Crimean taxa. Species delimitations and molecular dating imply that N. alasonius comprises at least five closely related cryptic species that diverged during the Late Miocene/Early Pliocene. Since these putative species are distributed along the entire drainage system, we hypothesized that the Kura River played a crucial role in shaping biogeographic patterns of this species. To promote further research of subterranean amphipods in the region, we provide a checklist of Caucasian Niphargus, and define shared morphological traits of the N. alasonius species complex.

Aim Common species contribute more to species richness patterns (SRPs) than rare species in most studies. Our aim was to test this hypothesis using a novel model system, species living exclusively in subterranean habitats. They consist of mainly rare species (small ranges), only a few of them being common (large ranges), and challenge whether rare species are less important for the development of SRPs in this environment. We separately analyzed aquatic and terrestrial species. Location Western Balkans in southeastern Europe. Methods We assembled two datasets comprising 431 beetle and 145 amphipod species, representing the model groups of subterranean terrestrial and aquatic diversity, respectively. We assessed the importance of rare and common species using the stepwise reconstruction of SRPs and subsequent correlation analyses, corrected also for the cumulative information content of the subsets based on species prevalence. We applied generalized linear regression models to evaluate the importance of rare and common species in forming SRPs. Additionally, we analyzed the contribution of rare and common species in species‐rich cells. Results Patterns of subterranean aquatic and terrestrial species richness overlapped only weakly, with aquatic species having larger ranges than terrestrial ones. Our analyses supported higher importance of common species for forming overall SRPs in both beetles and amphipods. However, in stepwise analysis corrected for information content, results were ambiguous. Common species presented a higher proportion of species than rare species in species‐rich cells. Main Conclusion We have shown that even in habitats with the domination of rare species, it is still common species that drive SRPs. This may be due to an even spatial distribution of rare species or spatial mismatch in hotspots of rare and common species. SRPs of aquatic and terrestrial subterranean organisms overlap very little, so the conservation approaches need to be habitat specific., We studied whether species richness patterns develop due to rare or common species, using a novel model system, subterranean species. Subterranean species with extremely high endemism, challenge the view that common species constitute a backbone of species richness patterns. Although subterranean aquatic and terrestrial species differ in degrees of endemism and spatial patterns, our analyses consistently imply that also in such systems common species backbone the patterns.

We characterized taxonomically, ecologically, and phylogenetically the amphipod community of Melissotrypa Cave (Central Greece), which comprises both freshwater and sulphidic lakes. We found four amphipod species: Niphargus jovanovici, Niphargus lindbergi, Niphargus gammariformis sp. nov. and an unknown species of Bogidiella. The three Niphargus species form a well-supported monophylum but differ in their ecology and morphology: N. jovanovici is a small and slender species inhabiting small freshwater voids, N. lindbergi is a large and stout species living in freshwater lakes, whereas N. gammariformis sp. nov. is a small and stout species found predominantly in a sulphidic lake. Available evidence suggests that diversification may have happened in a geographically restricted area and was driven by ecological differentiation. Niphargus gammariformis sp. nov. shows morphological convergences in diagnostic traits with two species hitherto classified into the genus Pontoniphargus. As molecular phylogenies show Pontoniphargus nested within Niphargus, we synonymize here Pontoniphargus with Niphargus. The species originally named Pontoniphargus ruffoi needed to be renamed into Niphargus pontoruffoi.http://www.zoobank.org/urn:lsid:zoobank.org:pub:4F5EED3B-DEB4-4402-BCE8-6AE2B65CCC3A., info:eu-repo/semantics/published

Species that successfully colonized subterranean environments are subject to two opposing selection processes. Stringent abiotic factors select for convergent adaptations, such as loss of eyes and pigments, while interspecific competition drives between-species divergence. Subterranean species can resolve opposing selection by adaptation to physically different microhabitats. Yet, species frequently co-occur in physically homogeneous subterranean habitats, like interstitial. These co-occurrences in such a narrow ecological context can be explained either by equalizing mechanisms, in which neither of the co-occurring species has a competitive advantage, or by more complex niche models that include species’ differentiation along a trophic niche axis. We tested these hypotheses using the amphipod genus Niphargus. We analysed Europe-wide co-occurrence records of Niphargus species from interstitial habitats, split into six independent large-scale regions. Firstly, we addressed whether species’ pairwise co-occurrences are random using a probabilistic model. Secondly, we tested whether species cluster into distinct functional–morphological groups and whether ecologically or phylogenetically distinct species are more likely to co-occur. We found that 68% of species co-occurrences were not different from random expectation, indicating that most species had access to most sites within each region. The remaining 32% co-occurred either significantly more or less often than expected by chance. Cluster analysis of functional morphological characters showed that interstitial species belong to two feeding types, micro- and macrofeeders, likely representing two peaks of the interstitial adaptive landscape, and hinting that niche divergence, as a mechanism allowing coexistence, is favoured. Finally, we found that the number of co-occurrences increases with increasing differentiation of functional morphology, but not phylogenetic differences. We conclude that ecological differentiation may be important in shaping such interstitial communities.

Knowledge on the diversity and distribution of subterranean organisms is still scattered, even in faunistically relatively well-researched countries such as Switzerland. This is mostly due to the restricted access to these subterranean habitats. Better knowledge on these organisms is needed, because they contribute substantially to overall biodiversity of a region, often contain unique elements of biodiversity, and can potentially be indicative of the ecological status of subterranean ecosystems that are providing important ecosystem services such as drinking water. Past research on subterranean organisms has often used highly specialised sampling techniques and expert knowledge. Here, we show that inclusion of non-professionals can be an alternative and highly promising sampling strategy. We retrieved citizen science-based samples from municipal groundwater wells across Switzerland, mainly from the Swiss Plateau. Opportunistic samples from 313 sites revealed a previously undocumented groundwater fauna including organisms from different major invertebrate groups, with a dominance of crustaceans. Here, we studied amphipods of the genus Niphargus. Among all 363 individuals sampled, we found in total eight nominal species. Two of them, namely N. fontanus and N. kieferi, are reported for Switzerland for the first time. We also found four further phylogenetic lineages that are potentially new species to science. One of them is here formally described as Niphargus arolaensissp. nov. The description is based on molecular and morphometric data. Our study proves the suitability of citizen science to document subterranean diversity, supports groundwater conservation efforts with data, and raises awareness for the relevance and biodiversity of groundwater amphipods among stakeholders.

Adaptive radiations are bursts of evolutionary species diversification that have contributed to much of the species diversity on Earth. An exception is modern Europe, where descendants of ancient adaptive radiations went extinct, and extant adaptive radiations are small, recent and narrowly confined. However, not all legacy of old radiations has been lost. Subterranean environments, which are dark and food-deprived, yet buffered from climate change, have preserved ancient lineages. Here we provide evidence of an entirely subterranean adaptive radiation of the amphipod genus Niphargus, counting hundreds of species. Our modelling of lineage diversification and evolution of morphological and ecological traits using a time-calibrated multilocus phylogeny suggests a major adaptive radiation, comprised of multiple subordinate adaptive radiations. Their spatio-temporal origin coincides with the uplift of carbonate massifs in South-Eastern Europe 15 million years ago. Emerging subterranean environments likely provided unoccupied, predator-free space, constituting ecological opportunity, a key trigger of adaptive radiation. This discovery sheds new light on the biodiversity of Europe., There are relatively few known extant adaptive radiations in Europe that predate the Pleistocene. Here, Borko et al. characterize the diversity and diversification of the subterranean amphipod genus Niphargus, showing evidence for a large adaptive radiation associated with massif uplift 15 million years ago.

Five decades ago, a landmark paper in Science titled The Cave Environment heralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field. S.M acknowledge support by the CAWEB project “Testing macroecological theory using simplified systems”, funded by the European Commission through Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) individual fellowships (Grant no. 882221). I.R.A. was supported by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Norma Transitória – DL57/2016/ CP1375/CT0003. D.E. was supported by IFREMER and by the CERES “Climate change and European Aquatic Resources” project funded by European Commission through Horizon 2020 research and innovation programme (Grant no. 678193). C.F., Z.F., and M.Z. were supported by the Slovenian Research Agency (program P1-0184, project N1-0069). J.E.K. was funded by NSF awards DEB1754231 and IOS1933428, and EDGE award 1923372. F.M. was supported by the French National Research Agency projects CONVERGENOMICS (ANR-15-CE32-0005) and EUR H2O’Lyon (ANR-17-EURE-0018). A.M. was supported by the ANCAVE project “Anchialine caves to understand evolutionary processes”, funded by the European Commission through Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) individual fellowships (Grant no. 745530). P.A.V.B. was supported by the project AZORESBIOPORTAL – PORBIOTA (ACORES-01-0145-FEDER-000072), financed by FEDER in 85% and by Azorean Public funds by 15% through Operational Program Azores 2020. A.S.P.S.R. was supported by VILLUM FONDEN (Grant no. 15471) and by a Carlsbergfondet grant (CF19-0609). T.G.P. was supported by Vale S.A. and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for individual fellowship (RDP 00092-18). T.P. was supported by the Karst Research Programme P6-0119, LifeWatch ERIC, RI-SI LifeWatch, and EU H2020 project eLTER. info:eu-repo/semantics/publishedVersion

Niphargus is one of the major freshwater amphipod genera in the world, distributed in groundwater in the Western Palearctic. Iran presents the eastern-most boundary of its range. Recent surveys using molecular markers unveiled that many populations from Western Iran deserve species status. In this study, we morphologically analyzed, described and illustrated four new species: N. persicus sp. nov., N. hosseiniei sp. nov., N. ilamensis sp. nov. and N. sohrevardensis sp. nov. All species were found in the west and north-west of Iran in Elburz and Zagros Mountain ranges. Therefore, the number of Niphargus species from Iran has been raised to 15, and we expect more species will be discovered in the future. In order to facilitate and further stimulate the taxonomy of the genus in the region, we constructed a database in the taxonomic program DELTA. The database describes 31 the most important morphological characters for 29 Asian Niphargus species, and is freely distributed via the World Amphipoda Database. In order to stimulate research on groundwater amphipods in the Middle East, we present how further taxonomy can benefit from this web-available data on morphology.

Amphipods from the genus Niphargus represent an important part of the Western Palearctic subterranean fauna. The genus is morphologically diverse, comprising several distinct ecomorphs bound to microhabitats in the subterranean environment. The most impressive among them are “lake giants,” a series of massive, large-bodied species. These range from morphologically distinct to morphologically cryptic taxa. We analysed the taxonomic structure of the Niphargus arbiter–Niphargus salonitanus species complex, belonging to “lake giants” from the Dinaric Karst (West Balkans), and assessed their phylogenetic, morphological and ecological diversity. Multilocus phylogeny suggested that the complex is monophyletic and nested within other cave lake ecomorphs. Unilocus and multilocus coalescence species delimitations indicated that the complex totals nine species. These species substantially overlap in morphology and cannot be unambiguously told apart without the use of molecular markers. An analysis of splitting events within a palaeogeological context, and modelling of environmental characteristics on the phylogeny unveiled a complex history of diversification. Part of this diversification might have been influenced by ecological divergence along the altitudinal gradient reaching from the Adriatic coast to inland Dinaric mountain chains and Poljes. Other splits coincide with the marine regression–transgression cycles during Pliocene. We describe Niphargus alpheus sp. n., Niphargus anchialinus sp. n., Niphargus antipodes sp. n., Niphargus arethusa sp. n., Niphargus doli sp. n., Niphargus fjakae sp. n. and Niphargus pincinovae sp. n., and by doing so hope to prompt their further research.

Ecologists increasingly rely on molecular delimitation methods (MMs) to identify species boundaries, thereby potentially increasing the number of putative species because of the presence of morphologically cryptic species. It has been argued that cryptic species could challenge our understanding of what determine large-scale biodiversity patterns which have traditionally been documented from morphology alone. Here, we used morphology and three MMs to derive four different sets of putative species among the European groundwater crustaceans. Then, we used regression models to compare the relative importance of spatial heterogeneity, productivity and historical climates, in shaping species richness and range size patterns across sets of putative species. We tested three predictions. First, MMs would yield many more putative species than morphology because groundwater is a constraining environment allowing little morphological changes. Second, for species richness, MMs would increase the importance of spatial heterogeneity because cryptic species are more likely along physical barriers separating ecologically similar regions than along resource gradients promoting ecologically-based divergent selection. Third, for range size, MMs would increase the importance of historical climates because of reduced and asymmetrical fragmentation of large morphological species ranges at northern latitudes. MMs yielded twice more putative species than morphology and decreased by 10-fold the average species range size. Yet, MMs strengthened the mid-latitude ridge of high species richness and the Rapoport effect of increasing range size at higher latitudes. Species richness predictors did not vary between morphology and MMs but the latter increased the proportion of variance in range size explained by historical climates. These findings demonstrate that our knowledge of groundwater biodiversity determinants is robust to overlooked cryptic species because the latter are homogeneously distributed along environmental gradients. Yet, our findings call for incorporating multiple species delimitation methods into the analysis of large-scale biodiversity patterns across a range of taxa and ecosystems., SCOPUS: ar.j, info:eu-repo/semantics/published

Groundwater belongs to the spatially most extensive, but least explored freshwater systems. On a global scale, the species richness of several subterranean invertebrate taxa parallels species richness found in surface waters, while on a local scale species richness hardly exceeds 20 species. This results in a high contribution of groundwater ecosystems to regional β- and γ-diversity, and to a smaller degree to α-diversity, and deserves focused attention. In general, more species are to be found in large cave systems. The second largest cave system in Europe is Holloch in Switzerland. In this paper we revised the taxonomic, phylogenetic and ecological diversity of the amphipod community in the Holloch cave system. While previous records listed five geographically widespread species of the genus Niphargus for this cave system, we could not confirm the presence of any of those species, but rather found three highly distinct species new to science. In this paper we describe Niphargus styx sp. nov., Niphargus ...

In light of recent alarming trends in human population growth, climate change, and other environmental modifications, a “Warning to humanity” manifesto was published in BioScience in 2017. This call reiterated most of the ideas originally expressed by the Union of Concerned Scientists in 1992, including the fear that we are “pushing Earth's ecosystems beyond their capacities to support the web of life.” As subterranean biologists, we take this opportunity to emphasize the global importance and the conservation challenges associated with subterranean ecosystems. They likely represent the most widespread nonmarine environments on Earth, but specialized subterranean organisms remain among the least documented and studied. Largely overlooked in conservation policies, subterranean habitats play a critical role in the function of the web of life and provide important ecosystem services. We highlight the main threats to subterranean ecosystems and propose a set of effective actions to protect this globally important natural heritage.

Subterranean animals are known for their highly evolved phenotypes. They are eyeless, depigmented and possess elongated appendages compared to their surface relatives. Increased antenna and leg length of cave species has traditionally been explained as a consequence of selection for non-visual senses and increased food-finding ability in an environment low in energy. Variation in appendage length between cave species is usually thought to result from differences in time since the colonization of the subterranean habitat. In this study, we analyzed appendage length variation in the Dinaric amphipod species Niphargus croaticus. Relative length of appendages varied substantially among populations. Using multilocus phylogenetic analysis, we showed that the species is nested within highly specialized N. steueri species complex and rejected the time hypothesis. Next, we explored the effects and strength of two environmental factors, water flow and presence of a competing species, N. subtypicus. Populations in caves with flowing water had shorter appendages than populations in cave lakes. Presence of the competing sister species did lead to longer appendages in stagnant water, but had no effect in flowing water. Abiotic factors had a stronger effect than biotic factors, but their relative strength differed among appendage pairs. High variation in appendage length between adjacent population shows that the morphology of cave arthropods is changing quickly and therefore cannot be used to predict species age. Rather than being a general adaptation to cave life, long appendages seem to be associated with the absence of water flow as well as character displacement when in sympatry with ecologically similar competing species.

Locomotion is an important, fitness-related functional trait. Environment selects for type of locomotion and shapes the morphology of locomotion-related traits such as body size and appendages. In subterranean aquatic arthropods, these traits are subjected to multiple, at times opposing selection pressures. Darkness selects for enhanced mechano- and chemosensory systems and hence elongation of appendages. Conversely, water currents have been shown to favor short appendages. However, no study has addressed the variation in locomotion of invertebrates inhabiting cave streams and cave lakes, or questioned the relationship between species’ morphology and locomotion. To fill this knowledge gap, we studied the interplay between habitat use, morphology and locomotion in amphipods of the subterranean genus Niphargus. Previous studies showed that lake and stream species differ in morphology. Namely, lake species are large, stout and long-legged, whereas stream species are small, slender and short-legged. We here compared locomotion mode and speed between three lake and five stream species. In addition, we tested whether morphology predicts locomotion. We found that the stream species lie on their body sides and move using slow crawling or tail-flipping. The species inhabiting lakes move comparably faster, and use a variety of locomotion modes. Noteworthy, one of the lake species almost exclusively moves in an upright or semi-upright position that resembles walking. Body size and relative length of appendages predict locomotion mode and speed in all species. We propose that integrating locomotion in the studies of subterranean species might improve our understanding of their morphological evolution.

The ecological radiation of amphipods is striking among crustaceans. Despite high diversity, global distribution and key roles in all aquatic environments, little is known about their ecological transitions, evolutionary timescale and phylogenetic relationships. It has been proposed that the amphipod ecological diversification began in the Late Palaeozoic. By contrast, due to their affinity for cold/oxygenated water and absence of pre-Cenozoic fossils, we hypothesized that the ecological divergence of amphipods arose throughout the cool Late Mesozoic/Cenozoic. We tested our hypothesis by inferring a large-scale, time-calibrated, multilocus phylogeny, and reconstructed evolutionary patterns for major ecological traits. Although our results reveal a Late Palaeozoic amphipod origin, diversification and ecological divergence ensued only in the Late Mesozoic, overcoming a protracted stasis in marine littoral habitats. Multiple independent post-Jurassic radiations took place in deep-sea, freshwater, terrestrial, pelagic and symbiotic environments, usually postdating deep-sea faunal extinctions, and corresponding with significant climatic cooling, tectonic reconfiguration, continental flooding, and increased oceanic oxygenation. We conclude that the profound Late Mesozoic global changes triggered a tipping point in amphipod evolution by unlocking ecological opportunities that promoted radiation into many new niches. Our study also provides a solid, time-calibrated, evolutionary framework to accelerate research on this overlooked, yet globally important taxon.

Niphargidae is among the largest families of freshwater subterranean amphipods in the world, and one of the most important macroinvertebrate groups in European groundwaters. The knowledge of their species diversity on a country level is often incomplete due to demanding taxonomy. In this paper we critically evaluated species diversity of the family in Belgium, using published records and samples from our own field work. Morphology-based identifications confirmed the presence of genera Microniphargus and Niphargus, represented with one and eight species, respectively. Most of the species found in Belgium belonged to complexes of cryptic species, which are still awaiting their taxonomic epilogue. Four species belong to phylogenetic lineages distributed also across British Isles, whereas three species belong to lineages distributed in the west-continental Europe. Identity of one species was doubtful, and two populations, partially illustrated herein, showed distinct morphology and might belong to a new species. All together, Niphargidae were found at 172 localities, with 80% of sites harboring one species only. At 12 localities three to six species co-occurred. When mapped onto 10x10 km grid, up to seven different species were recorded per quadrate. Species richest quadrates were situated in the southeastern part of the country. Paucity of records from the northern part of the country imply lack of research, rather than absence of Niphargidae. Conservation status of Belgian Niphargidae has not been evaluated so far; assessment of this status would require further sampling and detailed taxonomic work.

The amphipod genusNiphargus(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 >425Niphargusspecies 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 ofNiphargusin Switzerland. First, we molecularly delimited, morphologically diagnosed, and formally described two new species, namelyNiphargusluchoffmannisp. n.andNiphargustonywhittenisp. n.Second, we updated and revised a checklist ofNiphargusin 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.

International audience; The species concept is the cornerstone of biodiversity science, and any paradigm shift in the delimitation of species affects many research fields. Many biologists now are embracing a new “species” paradigm as separately evolving populations using different delimitation criteria. Individual criteria can emerge during different periods of speciation; some may never evolve. As such, a paradigm shift in the species concept relates to this inherent heterogeneity in the speciation process and species category which is fundamentally overlooked in biodiversity research. Cryptic species fall within this paradigm shift: they are continuously being reported from diverse animal phyla but are poorly considered in current tests of ecological and evolutionary theory. The aim of this review is to integrate cryptic species in biodiversity science. In the first section, we address that the absence of morpho- logical diversification is an evolutionary phenomenon, a “process” counterpart to the long studied mechanisms of morphological diversification. In the next section regarding taxonomy, we show that molecular delimitation of cryptic species is heavily biased towards dis- tance-based methods. We also stress the importance of formally naming of cryptic species for better integration into research fields that use species as units of analysis. Finally, we show that incorporating cryptic species leads to novel insights regarding biodiversity pat- terns and processes, including large-scale biodiversity assessments, geographic variation in species distribution and species coexistence. It is time for incorporating multicriteria spe- cies approaches aiming to understand speciation across space and taxa, thus allowing inte- gration into biodiversity conservation while accommodating for species uncertainty.

Groundwater is an extreme environment due to its absence of light, resource scarcity and highly fragmentary nature. Successful groundwater colonizers underwent major evolutionary changes and exhibit eye and pigment loss (troglomorphies). Consequently, their chances of dispersal and survival in the well-connected surface waters are greatly decreased, resulting in significant endemism. The West Palaearctic subterranean amphipod genus Niphargus comprises hundreds of narrowly endemic and troglomorphic species. Nevertheless, a few are known to occur in surface waters, two of which, N. hrabei and N. valachicus, have extremely large ranges that even exceed those of many surface-water amphipods. We tested if this pattern results from a secondary colonization of the relatively well-connected epigean environment, and whether this ecological shift promoted the large-scale dispersal of these species. Results showed that despite their ecological and zoogeographic similarities, N. hrabei and N. valachicus are not closely related and independently colonized surface waters. Their phylogeographic patterns indicate Middle to Late Pleistocene dispersal episodes throughout the Danube lowlands, and relatively modest yet significant genetic differentiation among populations. Clustering based on morphology revealed that the two species are phenotypically closer to each other than they are to most other epigean congeners. We presume that the ecological shift to surface environments was facilitated by their ability to thrive in hypoxic waters where rheophilic competitors from the family Gammaridae cannot survive. In conclusion, our results indicate that adaptation to groundwater is not a one-way evolutionary path and that troglomorphic species can occasionally recolonize and widely disperse in surface waters.

Molecular taxonomy often uncovers cryptic species, reminding us that taxonomic incompleteness is even more severe than previous thought. The importance of cryptic species for conservation is poorly understood. Although some cryptic species may be seriously threatened or otherwise important, they are rarely included in conservation programs as most of them remain undescribed. We analysed the importance of cryptic species in conservation by scrutinizing the South European cryptic complex of the subterranean amphipod Niphargus stygius sensu lato. Using uni- and multilocus delineation methods we show that it consists of 15 parapatric and sympatric species, which we describe using molecular diagnoses. The new species are not mere “taxonomic inflation” as they originate from several distinct branches within the genus and coexist with no evidence of lineage sharing. They are as evolutionarily distinct as average nominal species of the same genus. Ignoring these cryptic species will underestimate the number of subterranean endemics in Slovenia by 12 and in Croatia by four species, although alpha diversity of single caves remains unchanged. The new taxonomy renders national Red Lists largely obsolete, as they list mostly large-ranged species but omit critically endangered single-site endemics. Formal naming of cryptic species is critical for them to be included in conservation policies and faunal listings.

Aim Three broad mechanisms have been proposed to explain geographic variation in species range size: habitat area/heterogeneity, climate seasonality and long-term climate variability. However, it has proved difficult to disentangle their relative role, particularly as temperature seasonality often covaries with the amplitude of long-term temperature oscillations. Here, we shed new light onto this debate by providing the first continental-scale analysis of range size and beta diversity in groundwater habitats, where taxa are not exposed to latitudinal variation in temperature seasonality. Location Europe. Methods We compiled and mapped occurrence data for 1570 groundwater crustacean species. Generalized regression models were used to test for latitudinal variation in geographic range size and to assess the relative role of the three broad mechanisms in shaping present-day patterns of range size. We partitioned beta diversity into its spatial turnover and nestedness components and analysed their latitudinal variation across Europe. Results Median range size increases with latitude above 43° N and the range size of individual species is positively correlated to latitude, even after accounting for phylogenetic effects. Long-term temperature variability accounted for a substantially higher variation in median range size of groundwater crustaceans across Europe than precipitation seasonality and habitat heterogeneity, including aquifer area, elevation range, climatic rarity and productive energy. Spatial turnover contributes significantly more to beta diversity in southern regions characterized by stable historic climates than it does in northern Europe. Main conclusions Our findings add support to the historic climate hypothesis which suggests that patterns of increasing range size and decreasing species turnover at higher latitudes in the Palaearctic region are primarily driven by long-term temperature oscillations rather than by climatic seasonality and the availability and heterogeneity of habitats.

Amphipods are keystone species in many freshwater ecosystems. Understanding their distribution and diversity is crucial to ensure and preserve freshwater ecosystem functioning, particularly in the northern hemisphere. For the European Alps information on amphipods has been relatively limited until recently. We describe a new, widely distributed amphipod species, Gammarus alpinus sp. nov., found across the Alps and analyse its distribution, biogeography as well as genetic and morphological differentiation. Until now, this species has been reported as Gammarus lacustris. Based on genetic and morphometric measurements, we show that G. alpinus is highly divergent from G. lacustris. The latter has a circumpolar distribution, but may be absent from the Alps. The observed occurrence pattern of G. alpinus might be explained by a Pliocene range expansion and vicariance due to climate warming following the Quaternary glaciation. Historical drainage divides suggest a single recolonization route from a distinct southern refugium. While G. lacustris is widely distributed and not endangered at a global scale, G. alpinus is endemic to the Alps and its habitat is negatively affected by eutrophication, non-native species and possibly climate change.

The present study investigates morphologically similar species in the amphipod genus Niphargus, with special emphasis on three presumably closely-related species: N. fontanus, N. aquilex, and N. schellenbergi. The distribution ranges of these species overlap in Central Europe, and ambiguity of the current diagnostic characters has likely resulted in misidentifications. To address this issue, we carried out molecular-phylogenetic analyses of Cytochrome Oxidase Subunit 1 gene (COI) and 28S rDNA sequences for 34 individuals of the three species from 27 sites in Central Europe and Great Britain. We also included 14 GenBank sequences for two additional species, N. virei and N. rhenorhodanensis, to increase the resolution potential among our in-group taxa. In addition, about 100 specimens of the three species were examined for a reappraisal of diagnostic characters. Representative specimens taken from various subterranean habitat types were examined for each of the three species. A new morphological key, utilizing improved characters, is presented to clearly distinguish between N. fontanus, N. aquilex, and N. schellenbergi. Molecular-phylogenetic analysis using COI sequence data indicates geographically well-delimited clades for N. aquilex and N. fontanus. Our data suggest that some previously recorded niphargids from the Harz Mountains of Germany, initially identified as N. fontanus or N. aquilex, were all N. schellenbergi.

SUMMARY 1. Various groundwater habitats have exceptionally high levels of endemism caused by strong hydrographical isolation and low dispersal abilities of their inhabitants. More than 10% of macro-stygobiotic species nevertheless occupy relatively large ranges, measuring from some hundred to over 2000 km in length. These species represent a challenge because their distributions disregard hydrographical boundaries, and their means to disperse and maintain long-term gene flow are unknown. 2. Based on mitochondrial and nuclear gene sequences, we examined the phylogeographic structure of six formally recognised stygobiotic species (Niphargus virei, N. rhenorhodanensis, Troglocaris anophthalmus, T. hercegovinensis, Spelaeocaris pretneri, Proteus anguinus) and searched for cryptic lineage diversity in a genus-wide phylogeny of Niphargus. Using treebased criteria as well as comparative divergence measures, we identified cryptic lineages, which may tentatively be equated with cryptic species. 3. Fourteen analysed nominal stygobiotic species with large ranges emerged as highly diversified, splitting into 51 tentative cryptic lineages. The degree of divergence was within the range or larger than the divergence of other related pairs of sister species. A substantial part (94%) of the cryptic lineages had ranges

Boundaries of species distributions are the result of colonization-extinction processes. Survival on the boundary depends on how well individuals discriminate optimal from suboptimal habitat patches. Such behaviour is called habitat choice and was only rarely applied to macroecology, although it links species ecological niche and species distribution. Surface and subterranean aquatic species are spatially strongly segregated, even in the absence of physical barriers. We explored whether a behavioural response to light functions as a habitat choice mechanism that could explain species turnover between surface and subterranean aquatic ecosystems. In a controlled laboratory experiment, we studied the behavioural response to light of ten pairs of surface and subterranean amphipods that permanently co-occur in springs. Surface species showed a weak photophobic, photoneutral, and in one case, photophilic response, whereas all subterranean species showed a strong photophobic response. Eyeless subterranean but not eyed surface amphipods appear to orient themselves with light cues. On a local scale, this difference possibly diminishes harmful interactions between the co-occurring amphipods, whereas on a regional scale, photophobia could explain limited dispersal and a high degree of endemism observed among subterranean species.

International audience; The recognition of multi-causality and spatial non-stationarity in the determinants of large-scale biodiversity patterns requires to consider the role of multiple mechanisms, their interactions, and how these mechanisms vary in strength relative to each other across geographical space. Here, we challenge the view that historical climate stability primarily drives European patterns of groundwater crustacean diversity by testing also the role of spatial heterogeneity and productive energy. First, we predicted that the three mechanisms would be equally important at continental scale when analyzed separately, but that the importance of historical climate variability would weaken in joint analyses due to co-variation with the two other mechanisms. Second, we predicted that the three mechanisms would exhibit predictable latitudinal changes in their relative strength. To test these predictions, we selected predictors representing each mechanism and analyzed separately and jointly their effects and interactions using global regression models. We further mapped the independent and overlapping effects of mechanisms across Europe using partial geographically weighted regressions. When analyzed separately, the three mechanisms explained the same amount of variation in species richness, but in the joint analysis, the influence of historical climate stability became hidden in the variation shared with the other mechanisms. Topographic heterogeneity interacted synergistically with actual evapotranspiration and habitat heterogeneity on species richness. Spatial non-stationarity in the independent and overlapping effects of the three mechanisms was the most plausible expla- nation for the hump-shaped latitudinal pattern of crustacean species richness. Productive energy and spatial heterogeneity were important predictors at mid and southern latitudes, whereas historical climate stability overlapped with the two other mechanisms in northern Europe and productive energy in southern Europe. Multi-causality and spatial non-stationarity provide a broader perspective of groundwater biodiversity determinants that revives the importance of spatial heterogeneity and the strong dependence of subterranean communities on food supply from the surface.

24 páginas, 2 figuras, 1 tabla., The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EUWater Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel ecogenomic indices for routine application in biodiversity assessments of European fresh- and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments.

New species of the genus Niphargus is described and named as N. kermanshahi sp. nov. from a karst spring in west of Iran. This species is identified based on the analysis of morphological characters and 28S ribosomal DNA sequences. Taxonomic status and phylogenetic position of this species is discussed in comparison to other Iranian species of Niphargus genus.

Phenotypically similar species coexisting in extreme environments like sulfidic water are subject to two opposing eco-evolutionary processes: those favoring similarity of environment-specific traits, and those promoting differences of traits related to resource use. The former group of processes includes ecological filtering and convergent or parallel evolution, the latter competitive exclusion, character displacement and divergent evolution. We used a unique eco-evolutionary study system composed of two independent pairs of coexisting amphipod species (genus Niphargus) from the sulfidic caves Movile in Romania and Frasassi in Italy to study the relative contribution and interaction of both processes. We looked at the shape of the multifunctional ventral channel as a trait ostensibly related to oxygenation and sulfide detoxification, and at body size as a resource-related trait. Phylogenetic analysis suggests that the sulfidic caves were colonized separately by ancestors of each species. Species within pairs were more dissimilar in their morphology than expected according to a null model based on regional species pool. This might indicate competitive interactions shaping the morphology of these amphipod species. Moreover, our results suggest that the shape of the ventral channel is not subject to long-term convergent selection or to the process of environmental filtering, and as such probably does not play a role in sulfide tolerance. Nevertheless, the ancestral conditions reconstructed using the comparative method tended to be more similar than null-model expectations. This shift in patterns may reflect a temporal hierarchy of eco-evolutionary processes, in which initial environmental filtering became later on superseded by character displacement or other competition-driven divergent evolutionary processes., info:eu-repo/semantics/published

Recent studies indicate that morphologically cryptic species may be ecologically more different than would be predicted from their morphological similarity and phylogenetic relatedness. However, in biodiversity research it often remains unclear whether cryptic species should be treated as ecologically equivalent, or whether detected differences have ecological significance. In this study, we assessed the ecological equivalence of four morphologically cryptic species of the amphipod genus Niphargus. All species live in a small, isolated area on the Istrian Peninsula in the NW Balkans. The distributional ranges of the species are partially overlapping and all species are living in springs. We reconstructed their ecological niches using morphological traits related to feeding, bioclimatic niche envelope and species’ preference for epi-hypogean habitats. The ecological meaning of differences in niches was evaluated using distributional data and co-occurrence frequencies. We show that the species comprise two pairs of sister species. All species differ from each other and the degree of differentiation is not related to phylogenetic relatedness. Moreover, low co-occurrence frequencies in sympatric zones imply present or past interspecific competition. This pattern suggests that species are not differentiated enough to reduce interspecific competition, nor ecologically equivalent to co-exist via neutral dynamics. We tentatively conclude that the question of ecological equivalence relates to the scale of the study: at a fine scale, species’ differences may influence dynamics in a local community, whereas at the regional level these species likely play roughly similar ecological roles.