Sexual dimorphism in subterranean amphipod crustaceans covaries with subterranean habitat type.

Sexual dimorphism can evolve in response to sex-specific selection pressures that vary across habitats. We studied sexual differences in subterranean amphipods Niphargus living in shallow subterranean habitats (close to the surface), cave streams (intermediate), and cave lakes (deepest and most isolated). These three habitats differ because at greater depths there is lower food availability, reduced predation, and weaker seasonality. Additionally, species near the surface have a near-even adult sex ratio (ASR), whereas species from cave lakes have a female-biased ASR. We hypothesized (a) a decrease in sexual dimorphism from shallow subterranean habitats to cave lake species because of weaker sexual selection derived from changes in the ASR and (b) an increase in female body size in cave lakes because of stronger fecundity selection on account of oligotrophy, reduced predation, and weaker seasonality. We measured body size and two sexually dimorphic abdominal appendages for all 31 species and several behaviours related to male competition (activity, risk-taking, exploration) for 12 species. Species with an equal ASR that live close to the surface exhibited sexual dimorphism in all three morphological traits, but not in behaviour. The body size of females increased from the surface to cave lakes, but no such trend was observed in males. In cave lake species, males and females differed neither morphologically nor behaviourally. Our results are consistent with the possibility that sexual and fecundity selection covary across the three habitats, which indirectly and directly, respectively, shape the degree of sexual dimorphism in Niphargus species.

No room for males in caves: Female-biased sex ratio in subterranean amphipods of the genus Niphargus.

Sex allocation theory predicts that the proportion of daughters to sons will evolve in response to ecological conditions that determine the costs and benefits of producing each sex. All else being equal, the adult sex ratio (ASR) should also vary with ecological conditions. Many studies of subterranean species reported female-biased ASR, but no systematic study has yet been conducted. We test the hypothesis that the ASR becomes more female-biased with increased isolation from the surface. We compiled a data set of ASRs of 35 species in the subterranean amphipod Niphargus, each living in one of three distinct habitats (surface-subterranean boundary, cave streams, phreatic lakes) representing an environmental gradient of increased isolation underground. The ASR was female-biased in 27 of 35 species; the bias was statistically significant in 12 species. We found a significant difference in the ASR among habitats after correction for phylogeny. It is most weakly female-biased at the surface-subterranean boundary and most strongly female-biased in phreatic lakes. Additional modelling suggests that the ASR has evolved towards a single value for both surface-subterranean boundary and cave stream-dwelling species, and another value for 9 of 11 phreatic lake dwellers. We suggest that a history of inbreeding in subterranean populations might lower inbreeding depression such that kin selection favours mating with siblings. This could select for a female-biased offspring sex ratio due to local mate competition among brothers. The observed patterns in sex ratios in subterranean species make them a group worthy of more attention from those interested in sex allocation theory.

The late blooming amphipods: Global change promoted post-Jurassic ecological radiation despite Palaeozoic origin.

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 previously 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.

Is subterranean lifestyle reversible? Independent and recent large-scale dispersal into surface waters by two species of the groundwater amphipod genus Niphargus.

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.

Epigean gammarids survived millions of years of severe climatic fluctuations in high latitude refugia throughout the Western Carpathians.

Isolated glacial refugia have been documented in Central Europe for a number of taxa, but conclusive evidence for epigean aquatic species has remained elusive. Using molecular data (mitochondrial and nuclear markers), we compared the spatial patterns of lineage diversity of the widely distributed Gammarus fossarum species complex between two adjacent biogeographically and geomorphologically distinct Central European regions: the Bohemian Massif and the Western Carpathians. We investigated if the observed patterns of spatial diversity are more likely to stem from historical or present-day factors. Phylogenetic and phylogeographic analyses revealed eight phylogenetically diverse lineages: two exhibiting local signatures of recent demographic expansion inhabit both regions, while the other six display a relict distributional pattern and are found only in the Western Carpathians. Molecular dating indicates that these lineages are old and probably diverged throughout the Miocene (7-18Ma). Furthermore, their distribution does not seem to be constrained by the present boundaries of river catchments or topography. The contrasting spatial patterns of diversity observed between the two regions thus more likely result from historical rather than contemporaneous or recent factors. Our results indicate that despite the high latitude and proximity to the Pleistocene ice sheets, the Western Carpathians functioned as long-term glacial refugia for permanent freshwater fauna, allowing the uninterrupted survival of ancient lineages through millions of years of drastic climatic fluctuations.

The southwestern Carpathians as an ancient centre of diversity of freshwater gammarid amphipods: insights from the Gammarus fossarum species complex.

Gammarus fossarum is a diverse species complex of epigean freshwater amphipods throughout Europe. Due to their poor dispersal capabilities and ubiquity, these crustaceans may serve as a model for investigating the influence of historical factors on the contemporary distribution and diversity patterns of freshwater macrozoobenthos. Here, we investigate the fine-scale phylogeographic structure of this complex across its range in the southwestern Carpathian Mountains, which comprises two areas that are geographically isolated from its main European distribution area as well as from each other. Given the Tertiary age of many freshwater Gammarus species, we hypothesize that the southwestern Carpathian populations reflect a relict distribution pattern. We used two mitochondrial and three nuclear markers from 32 localities to reconstruct phylogenetic relationships and estimate the timings of divergence among southwestern Carpathian and non-Carpathian lineages. Cryptic diversity was evaluated from mitochondrial markers by employing phylogenetic and distance-based methods. We distinguished at least 16 cryptic microendemic taxa, some of them coexisting, distributed in the southwestern Carpathians in a mosaic-like pattern. These lineages form a monophyletic group together with several lineages from southeastern Europe. Estimated divergence times indicate a Middle Miocene origin of this clade, with many deep splits dating back to more than 10 Ma. This time frame corresponds with a period of intense geological subsidence in the region that gave birth to the Pannonian Basin. We conclude that subsidence could have been an important driver of diversification in freshwater Gammarus and that the southwestern Carpathians represent an ancient centre of diversity for these crustaceans.

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.

The interplay between habitat use, morphology and locomotion in subterranean crustaceans of the genus Niphargus.

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.

Continental-scale patterns of hyper-cryptic diversity within the freshwater model taxon Gammarus fossarum (Crustacea, Amphipoda).

Traditional morphological diagnoses of taxonomic status remain widely used while an increasing number of studies show that one morphospecies might hide cryptic diversity, i.e. lineages with unexpectedly high molecular divergence. This hidden diversity can reach even tens of lineages, i.e. hyper cryptic diversity. Even well-studied model-organisms may exhibit overlooked cryptic diversity. Such is the case of the freshwater crustacean amphipod model taxon Gammarus fossarum. It is extensively used in both applied and basic types of research, including biodiversity assessments, ecotoxicology and evolutionary ecology. Based on COI barcodes of 4926 individuals from 498 sampling sites in 19 European countries, the present paper shows (1) hyper cryptic diversity, ranging from 84 to 152 Molecular Operational Taxonomic Units, (2) ancient diversification starting already 26 Mya in the Oligocene, and (3) high level of lineage syntopy. Even if hyper cryptic diversity was already documented in G. fossarum, the present study increases its extent fourfold, providing a first continental-scale insight into its geographical distribution and establishes several diversification hotspots, notably south-eastern and central Europe. The challenges of recording hyper cryptic diversity in the future are also discussed.

Contrasting life history strategies in a phylogenetically diverse community of freshwater amphipods (Crustacea: Malacostraca).

Differences in life histories are commonly exhibited within ecological communities, especially among species that display increased variations in body size and morphology and are phylogenetically distant. To examine the relationship between morphological dissimilarity and life history divergence, we investigated three morphologically distinct and distantly related species of freshwater amphipods that co-occur throughout the Danube lowlands - Gammarus balcanicus dacicus, Niphargus valachicus and Synurella ambulans - by collecting monthly samples during a one-year period. Results revealed that the studied species differ significantly with respect to fecundity, size at maturity, number of generations per year, duration and timing of the reproductive period and egg volume. Despite some overlap, each species possesses a unique combination of traits, supporting the hypothesis that life history variation within freshwater amphipod communities can reflect dissimilarities regarding body size, morphology and evolutionary relationships. However, it is not yet clear which of these factors has the most significant contribution to life history divergence.