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The metacommunity concept provides a theoretical framework that aims at explaining organism distributions by a combination of environmental filtering, dispersal, and drift. However, few works have attempted a multitaxon approach and even fewer have compared two distant biogeographical regions using the same methodology. We tested the expectation that temperate (mediterranean-climate) pond metacommunities would be more influenced by environmental and spatial processes than tropical ones, because of stronger environmental gradients and a greater isolation of waterbodies. However, the pattern should be different among groups of organisms depending on their dispersal abilities. We surveyed 30 tropical and 32 mediterranean temporary ponds from Costa Rica and Spain, respectively, and obtained data on 49 environmental variables. We characterized the biological communities of bacteria and archaea (from the water column and the sediments), phytoplankton, zooplankton, benthic invertebrates, amphibians and birds, and estimated the relative role of space and environment on metacommunity organization for each group and region, by means of variation partitioning using generalized additive models. Purely environmental effects were important in both tropical and mediterranean ponds, but stronger in the latter, probably due to their larger limnological heterogeneity. Spatially correlated environment and pure spatial effects were greater in the tropics, related to higher climatic heterogeneity and dispersal processes (e.g., restriction, surplus) acting at different scales. The variability between taxonomic groups in the contribution of spatial and environmental factors to metacommunity variation was very wide, but higher in active, compared with passive, dispersers. Higher environmental effects were observed in mediterranean passive dispersers, and higher spatial effects in tropical passive dispersers. The unexplained variation was larger in the tropical setting, suggesting a higher role for stochastic processes, unmeasured environmental factors, or biotic interactions in the tropics, although this difference affected some actively dispersing groups (insects and birds) more than passive dispersers. These results, despite our limitations in comparing only two regions, provide support, for a wide variety of aquatic organisms, for the classic view of stronger abiotic niche constraints in temperate areas compared with the tropics. The heterogeneous response of taxonomic groups between regions also points to a stronger influence of regional context than organism adaptations on metacommunity organization.

Movile Cave, located in southeastern Romania close to the Black Sea, is one of the most remarkable diversity hotspots worldwid with 52 species of invertebrates of which 37 are endemic (Brad et al. 2021). Due to the high concentration of hydrogen sulfide in its water, the primary production of organic matter in Movile Cave relies almost entirely on the chemoautotrophic activity of microorganisms, notably sulfur-oxidizing gammaproteobacteria belonging to the genus Thiothrix. In the presence of oxygen, these filamentous bacteria can oxidize hydrogen sulfide and reduce it into various sulfidic compounds, generating energy in the process. In sulfidic ecosystems, Thiothrix bacteria are frequently found free-living but also as epibionts or ectosymbionts growing on other organisms, such as amphipods (Flot et al. 2014). However, it is unclear whether Thiothrix bacteria also grow on microcrustaceans such as copepods or ostracods, of which several species are known from the sulfidic mesothermal aquifer of Mangalia, where Movile Cave is located. To find it out, we combined DNA sequencing using the reference bacterial 16S ribosomal RNA gene with morphological observations (including fluorescence microscopy). Our results reveal that Thiothrix bacteria are indeed present on microcrustaceans from Movile Cave and surrounding wells in the Mangalia region, highlighting the versatility of Thiothrix-crustacean associations in sulfidic ecosystems. This is the first report of an association between Thiothrix and groundwater microcrustaceans, and the second report of an association between Thiothrix and a nonmarine ostracod (Khalzov et al. 2021).

Located in central Greece, Melissotrypa Cave is a hypogenic cave that contains a sulfidic lake. Biological investigations in this cave led to the discovery of several endemic troglobitic invertebrates, some of which occur in high densities of up to 200 specimens per square meter. The subterranean aquatic community appears to rely on a rich autochthonous food-base provided by chemoautotrophic sulfur-oxidizing microorganisms that form microbial biofilms floating on the surface of the sulfidic lake. Several terrestrial species inhabiting the upper sections of the cave rely on allochthonous food brought in from the surface such as vegetal detritus falling into the cave’s entrance shaft, and bat guano. Melissotrypa Cave allows the comparative study of chemoautotrophically and photoautotrophically based subterranean communities thriving within the same cave.

Caves with permanent and temporary ice formations exists at mid-to-high northern latitudes, at elevations between 0 and >3000 meters above sea level. In such caves, rare and endemic species or glacial relicts strictly tied to cold microclimates habitat conditions have evolved and the negative temperatures have contributed significantly in shaping the structural pattern of both terrestrial and aquatic communities. Aquatic dwellers inhabiting ice caves are likely to show resistance and have special physiological adaptation to cope with constantly low air and water temperatures. Ghețarul de la Vârtop cave is a short (340 m) cave located in the Apuseni Natural Park (northwest Romania) that hosts temporary ice formations near its entrance. In 2021, we have initiated a study aiming to understand how low temperatures in the cave are shaping the structure of underground fauna, along a temperature gradient through the cave. The sampling design imply monthly monitoring of air temperature (hourly measurements using data loggers), water physical and chemical characteristics and the structure of aquatic fauna communities. In this paper we present preliminary data on the environmental conditions and aquatic invertebrate communities present in percolation water and associated gours from the cave. Aquatic fauna is represented by nematodes, oligochaetes and several crustacean species the majority stygobites, of which at least three are potentially new to science. The surprising presence of Acanthocyclops reductus, considered a Tertiary relict living in warm water raises discussions on its tolerance to highly variable temperatures but also on the colonisation history of the caves on a regional scale. Investigations of cave aquatic fauna in caves with permanent and temporary ice offer hints to understand the ecology of the fauna, and also to further assess the mechanisms involved in adaptations of species to cope with constantly low-water temperatures.

Members of the Eucyclops graeteri species complex are widely distributed in European groundwaters and share some morphocharacters such as: a long aesthetasc on the ninth segment of female antennula, short or very reduced spinule row ("serra") on furcal rami, lateral hairiness of 5th thoracic somite strongly reduced, and few or no hairs on the distal margin of the intercoxal sclerite of 4th legs pair. a long aesthetasc on the ninth segment of female antennula, short or very reduced spinule row ("serra") on furcal rami, lateral hairiness of 5th thoracic somite strongly reduced, and few or no hairs on the distal margin of the intercoxal sclerite of 4th legs pair. All these characters are subject to strong selective pressure in the underground environment; therefore, the study of molecular markers is needed to better define the taxonomy and phylogeny of this species group. The Southern Dobrogea region in Romania conceals dark, isolated, and sulfide-rich aquifers, accessible through artificial wells, springs, and the Movile Cave. The chemoautotrophically-based Movile Cave hosts a member of the E. graeteri complex, described several years ago as a subspecies (E. graeteri scythicus Plesa, 1989). The morphological and molecular analyses performed on specimens of this species complex collected from Southern Dobrogea revealed new records for E. graeteri scythicus both in sulfidic and non-sulfidic artificial wells dug near the cave as well as a species putatively new to science collected in three sulfidic wells from Mangalia. Mitochondrial markers allocated the Movile Cave population in a distinct clade within the genus Eucyclops, Eucyclops serrulatus s.l. being the closest taxon. The putative new species belongs to a different clade within the subfamily Eucyclopinae, raising doubts on the monophyly of this species complex. Furthermore, our investigations revealed the occurrence of ectosymbiont sulfur-oxidizing Thiothrix bacteria associated with E. graeteri scythicus suggesting that this putative symbiosis could allow this taxon to better cope with sulphidic-rich environments. These unique sulfidic groundwater environments therefore provide an interesting model system in which to study the taxonomy of copepods and their adaptation to very harsh environmental conditions.

Sulphidic cave ecosystems are remarkable evolutionary hotspots that have witnessed adaptive radiation of their fauna represented by extremophile species having particular traits. Ostracods, a very old group of crustaceans, exhibit specific morphological and ecophysiological features that enable them to thrive in groundwater sulphidic environments. Herein, we report a peculiar new ostracod species Pseudocandona movilaensis sp. nov. thriving in the chemoautotrophic sulphidic groundwater ecosystem of Movile Cave (Romania). The new species displays a set of homoplastic features specific for unrelated stygobitic species, for e.g., triangular carapace in lateral view with reduced postero–dorsal part and simplification of limb chaetotaxy (i.e., loss of some claws and reduction of secondary male sex characteristics), driven by a convergent or parallel evolution during or after colonization of the groundwater realm. P. movilaensis sp. nov. thrives exclusively in sulphidic meso-thermal waters (21°C) with high concentrations of sulphides, methane, and ammonium. Based on the geometric morphometrics-based study of the carapace shape and molecular phylogenetic analyses based on the COI marker (mtDNA), we discuss the phylogenetic relationship and evolutionary implication for the new species to thrive in groundwater sulphidic groundwater environments.

The subfamily Candoninae Kaufmann, 1900a is one of the most taxonomically difficult and species-rich lineages of non-marine Ostracoda. It is widely distributed, inhabiting all kinds of surface and subterranean waters, but its taxonomy remains unclear and generic relationships ambiguous due to a number of homoplasies. To verify the morphology-based taxonomy and monophyly of the European morphogenera of Candoninae we propose the analysis of the mitochondrial fragment encoding cytochrome c oxidase subunit I (COI). We investigated 75 individuals of 25 species representing eight of 13 extant genera of Candoninae known from Europe. The phylogenetic analyses revealed a complex pattern of relationships and a noteworthy incongruence with the currently used taxonomic system. The monophyletic status of the morphogenera CandonaBaird, 1845, FabaeformiscandonaKrstić, 1972, and PseudocandonaKaufmann, 1900a appeared questionable, but instead monophyly of some species groups distinguished morphologically within these genera seemed to be confirmed in our study. The phylogenetic position of some other morphogenera remained unresolved. Our analysis, focusing on well-supported nodes, nevertheless revealed a handful of salient points on the evolution of Candoninae that deserve further investigation.

Survey of the aquatic subterranean fauna from caves, springs, interstitial habitat, wells in deserts, artificial tunnels (Khanas) of five countries of the former URSS (Kazakhstan, Kyrgyzstan, Tadjikistan, Turkmenistan, Uzbekistan) located far east the Caspian Sea. The cave fauna present some originalities: - the rich fauna of foraminiferida in the wells of the Kara-Kum desert (Turkmenistan); - the cave fish Paracobitis starostini from the Provull gypsum Cave (Turkmenistan); - the presence of a rich stygobitic fauna in the wells of the Kyzyl-Kum desert (Uzbekistan); - the rich stygobitic fauna from the hyporheic of streams and wells around the tectonic Issyk-Kul Lake (Kyrgyzstan); - the eastern limit of the European genus Niphargus from the sub-lacustrin springs on the eastern shore of the Caspian Sea (Kazakhstan); - the presence of cave fauna of marine origin. Approximately 96 stygobionts, 9 stygobionts/stygophiles and 3 troglobionts are recorded.

The metacommunity concept provides a theoretical framework that aims at explaining organism distributions by a combination of environmental filtering, dispersal and drift. With the development of statistical tools to quantify and partially isolate the role of each of these processes, empirical metacommunity studies have multiplied worldwide. However, few works attempt a multi-taxon approach and even fewer compare two distant biogeographical regions using the same methodology. Under this framework, we tested the expectation that temperate (mediterranean-climate) pond metacommunities would be more influenced by environmental and spatial processes than tropical ones, because of stronger environmental gradients and greater isolation of waterbodies.We surveyed 30 tropical and 32 mediterranean temporary ponds from Costa Rica and Spain, respectively, and obtained data on 49 environmental variables (including limnological, hydrogeomorphological, biotic, climatic, and landscape variables). We characterized the biological communities of Bacteria and Archaea (from both the water column and the sediments), phytoplankton, zooplankton, benthic invertebrates, amphibians and birds, and estimated the relative role of space and environment on metacommunity organization for each group and region, by means of variation partitioning using Generalized Additive Models (GAMs).Environmental selection was important in both tropical and mediterranean ponds, but markedly stronger in the latter, probably due to their larger limnological heterogeneity. Spatialized environment and pure spatial effects were greater in the tropics, related to higher climatic heterogeneity and dispersal processes (e.g. restriction, surplus) acting at different scales. The variability between taxonomic groups in spatial and environmental contributions was very wide. Effects on passive and active dispersers were similar within regions but different across regions, with higher environmental effects in mediterranean active dispersers. The residual (unexplained) variation was larger in tropical pond metacommunities, suggesting a higher role for stochastic processes and/or effects of biotic interactions in the tropics. Overall, these results provide support, for a wide variety of organisms related to aquatic habitats, for the classical view of stronger abiotic niche constraints in temperate areas compared to the tropics.

Movile Cave hosts one of the world’s most diverse subsurface invertebrate communities. In the absence of matter and energy input from the surface, this ecosystem relies entirely on in situ primary productivity by chemoautotrophic microorganisms. The energy source for these microorganisms is the oxidation of hydrogen sulfide provided continuously from the deep thermomineral aquifer, alongside methane, and ammonium. The microbial biofilms that cover the water surface, the cave walls, and the sediments, along with the free-swimming microorganisms, represent the food that protists, rotifers, nematodes, gastropods, and crustacean rely on. Voracious water-scorpions, leeches, and planarians form the peak of the aquatic food web in Movile Cave. The terrestrial community is even more diverse. It is composed of various species of worms, isopods, pseudoscorpions, spiders, centipedes, millipedes, springtails, diplurans, and beetles. An updated list of invertebrate species thriving in Movile Cave is provided herein. With 52 invertebrate species (21 aquatic and 31 terrestrial), of which 37 are endemic for this unusual, but fascinating environment, Movile Cave is the first known chemosynthesis-based groundwater ecosystem. Therefore, Movile Cave deserves stringent attention and protection.

The increasing human impact in Romanian caves raises the urgency of publishing a correct database of the strictly-adapted cave fauna. Previous attempts at indexing cave fauna and classifying caves by using their fauna opened many questions regarding the use of an incomplete list of cave species and mixed lists of troglobionts/stygobionts with troglophiles/stygophiles for ranking caves with priority for protection. It has also become obvious that there is a need to publish a list of Romanian cave species that are under threat. Cave species in Romania (and elsewhere) are endemic on small ranges, are unique and must be considered as important units for conservation. A cave must be equally protected if it has one or more rare and strictly endemic cave species. Although not exhaustive, we here provide the first checklist of Romanian troglobionts/stygobionts developed in the framework of the DARKFOOD and GROUNDWATERISK projects, coordinated by the “Emil Racovita” Institute of Speleology, Cluj-Napoca, Romania. The GIS application was used to complement the checklist of cave species with data on caves and surface environments above the caves. Until complete data on species diversity and population sizes are made available for each cave, measures of conservation can be implemented, based on the presence/absence of cave species, while classifications of caves for protection, based on the number of species, must be avoided. We also propose a list of Romanian caves with fauna that are under threat and a tentative Red List of Romanian troglobiont/stygobionts. This is the first database with identified troglobiont and stygobiont species of Romania, with a critical analysis of their distribution inside the country. A list of caves that need protection for their rare and unique species and a tentative Red List of Romanian cave fauna are also added. A total of 173 species were identified, of which 77 troglobionts and 96 stygobionts are currently registered in 366 caves. The database is divided into two parts, one part with a list of troglobionts, their revised systematic position, cave name, cave code and geographic region; and the second part with the same information on stygobionts. The database represents the contribution of many active researchers, who are the authors of this paper and of review publications of many other authors of the "Emil Racoviță" Institute of Speleology.

Considering the progressive depletion of groundwater quantity and the alteration of superficial and groundwater quality in the Madrid region, a highly populated area with extended urbanizations in the north and agricultural activities in the south, we conducted a monitoring study over a period of 2.5 years (2015–2017). The study was conducted in the Jarama-Tajuna shallow alluvial aquifer system located southeast of Madrid, where the exploitation of the aquifer and the Jarama river have increased exponentially in the last decade being affected by both urban and agricultural activities. Our aims were to provide: i) a geochemical characterization of the surface and groundwater properties; ii) identify the process responsible for the geochemical evolution and mineralization of the waters; iii) assess the water quality (i.e. nitrates, ammonia, sodium and chloride as potential contaminants) and the water suitability for irrigation (SAR, Wilcox, KI and MH indexes); and iv) identify the main sources of contamination in the area. A set of plots, ion ratios, correlation coefficients, multivariate statistical analyses and indexes were performed. Results indicated that rock weathering largely controls the hydrogeochemistry of the system and that wastewater treatment plant discharges and agricultural practices significantly affect the composition of the water, causing an important decline of both surface and groundwater chemical quality. Nonetheless, water suitability for irrigation is admissible. Thus, taking additional measures to increase its quality are not necessary. With this study we aimed to establish a base line to evaluate future changes in the groundwater properties from the Madrid region enabling the planners and policy makers to develop a strategy to mitigate the impact of the exponential increase of urban and agriculture activities on groundwater resources.

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.

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. Fil: Di Lorenzo, Tiziana. Consiglio Nazionale delle Ricerche; Italia. Istituto di Ricerca sugli Ecosistemi Terrestri; Italia Fil: Castaño Sánchez, Andrea. Universidad de Alcalá; España Fil: Di Marzio, Walter Dario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina Fil: García Doncel, Patricia. Universidad de Alcalá; España Fil: Nozal Martínez, Leonor. Universidad de Alcalá; España Fil: Galassi, Diana Maria Paola. Universita degli Studi dell'Aquila; Italia Fil: Iepure, Sanda. Universidad de Alcalá; España

Chile has large extensions of arid and semi-arid regions throughout the whole country, where the intensive demands and use of water resources, especially groundwater for irrigations and mining activities, increased dramatically over the last decades. The aquifer depletions due to water abstraction for irrigation and nutrient loads, exert major alterations of water quality, groundwater recharge and the natural renewal rate. All these factors diminish the aquifer value for the users and contribute to the degradation of groundwater as environment and habitat for fauna. This intensive use of groundwater resources in Chile brought to significant social and economic benefits, but their inadequate management resulted in negative environmental, legal and socioeconomic consequences. In this study, we aimed at providing a first assessment of environmental alterations of groundwater ecosystems from agricultural watersheds in northern Chile by specifically evaluating the effects of nitrogen and pesticide loads on groundwater communities and identifing the ecosystem service alterations due to agricultural activities. The study has been performed in a glacial aquifer from Coquimbo region; 250 km north of Santiago de Chile, the floodplain of which is dominated by agriculture (fruits tress, vineyards). Due to low regional precipitations (100-240 mm/year) the aquifer is primarily recharged by snowmelt from the Andean chain and surface runoff. The relative groundwater levels, groundwater temperature, chemical analysis of nitrogen and total phosphorus and pesticide concentrations were examined, along with the evaluation of crustacean biodiversity and spatial distribution pattern. Stygofauna taxonomic richness and the presence of stygobites have been related more to groundwater level stability than to chemical water parameters indicating that over-exploitation has a negative impact on habitat suitability for groundwater invertebrates. Groundwater biota assessment is essential in understanding the impact produced by agriculture activities on groundwater as a resource and as ecosystem, a nexus that becomes more and more widely recognized. The rationale and the preliminary results of this study are summarized in the Suppl. material 1.

Ice caves are extreme environments where low temperatures significantly contribute to shaping the structural pattern of terrestrial and aquatic biocoenosis. Extreme ice cave inhabitants range from troglobiotic beetles to stygobites, crustaceans, insects, and Acarina that show resistance to low temperatures. Many species are rare and endemic and are strictly tied to cold, microclimate habitat conditions. Here we provide a review of the environmental settings, habitats, and recent and extant fauna in ice caves in Europe, Canada, and North America.

On the catchment spatial scale, rivers can be considered a patchy discontinuity from headwaters to mouth with significant implications on the structure and functioning of the hyporheic zone (HZ) biota. The present study aims to determine the spatial variation of ostracod assemblages from a fluvial HZ of two groundwater-fed rivers of the Jarama basin (Central Spain). We hypothesised that ostracod abundance and composition are subject to changes in water condition, substratum type geology and surface water/groundwater exchanges along the hyporheic flow path. Individual measures of 29 physico-chemical parameters, water discharge, sediment size and sediment organic matter (as loss of ignition) were measured at a depth of 20–40 cm at 20 hyporheic sites of the Henares and Tajuna Rivers. The results show that factors like elevation, carbonate water type, high permeable riverbed substratum and groundwater upwelling at headwaters contribute to shape a diverse, mixed assemblage formed by stygophyle and stygoxene ostracods (14 species). Conversely, low-mineralised waters, siliceous riverbeds with poor permeability, and relatively low surface/ ground water exchanges in the middle and lower sectors, limit species diversity and ostracod abundance to exclusively stygoxene elements (nine species). Ostracod abundance correlates positively with fine sands (0.125–0.25 mm) (Spearman, r¼0.70), and negatively with temperature (r¼�0.40), SO4 2� (r¼�0.45) and Mg2þ (r¼�0.40). Trace metal (Cu, Mn, Ni) accumulation in the intermediate and lower sectors of both rivers appears harmful for Ilyocypris brady (r¼�0.46) and Pryonocypris zenkeri (r¼�0.40), whereas As relates negatively to the abundance of Candona candida (r¼�0.51), Pryonocypris zenkeri (r¼�0.60) and Herpetocypris brevicaudata (r¼�0.72). The results highlight the significance of recent ostracods as a proxy to assess the water conditions in a HZ and to provide forthcoming approaches to depict surface-subsurface hydrological exchanges.

One of the most challenging questions in the assessment of groundwater ecosystem health is whether assemblages of groundwater biota can be used reliably as biomarkers. In the present study, we aimed to: (i) explore the diversity, distribution and ecological composition of groundwater Crustacea Copepoda and Ostracoda communities from six aquifers in central Spain; and (ii) relate the structure of the species assemblages to the aquifers’ hydrogeological conditions, habitat features and hydrochemistry. The data sets were simultaneously collected from 24 boreholes drilled in six unconfined aquifers from the upper part of the Tajo catchment. Our results show that the assemblages are primarily influenced by the specific hydrogeological conditions of the aquifer, determined by groundwater flow, supply of organic matter from the surface and habitat features, whereas the aquifer's hydrochemistry has only a marginal effect. The assemblages from the Tertiary detrital aquifer were the most diverse, containing a mixture of stygobiont and non-stygobiont species distributed preferentially in the recharge area of the aquifer, characterized by medium hydraulic conductivity, high surface input and relatively high water renewal. The Quaternary alluvial aquifer type was found to have a moderate richness with several hyporheic dwellers, indicating an active surface/groundwater exchange with the adjacent stream channel. In contrast, the carbonate aquifer type was less diverse, but markedly distinct owing to the high occurrence of stygobionts. Our results provide a step forward in exploring the groundwater biodiversity of aquifers in Spain and towards incorporating an ecological perspective into overall assessments of groundwater status.

Rich, diverse assemblages comprising a total (live + dead) of twenty-one ostracod species belonging to fifteen genera were recovered from phreatic waters of the hypogenic Frasassi Cave system and the adjacent Frasassi sulfidic spring and Sentino River in the Marche region of the northeastern Apennines of Italy. Specimens were recovered from ten sites, eight of which were in the phreatic waters of the cave system and sampled at different times of the year over a period of five years. Approximately 6900 specimens were recovered, the vast majority of which were disarticulated valves; live ostracods were also collected. The most abundant species in the sulfidic spring and Sentino River were Prionocypris zenkeri, Herpetocypris chevreuxi, and Cypridopsis vidua, while the phreatic waters of the cave system were dominated by two putatively new stygobitic species of Mixtacandona and Pseudolimnocythere and a species that was also abundant in the sulfidic spring, Fabaeformiscandona ex gr. F. fabaeformis. Pseudocandona ex gr. P. eremita, likely another new stygobitic species, is recorded for the first time in Italy. The relatively high diversity of the ostracod assemblages at Frasassi could be attributed to the heterogeneity of groundwater and associated habitats or to niche partitioning promoted by the creation of a chemoautotrophic ecosystem based on sulfur-oxidizing bacteria. Other possible factors are the geologic age and hydrologic conditions of the cave and karst aquifer system that possibly originated in the early– middle Pleistocene when topographic uplift and incision enabled deep sulfidic waters to reach the local carbonate aquifer. Flooding or active migration would have introduced the invertebrates that now inhabit the Frasassi Cave system.

The hyporheic zone of stream ecosystems, located at the transition between surface channel and ground waters, exhibits patchy discontinuity on a longitudinal transect and induces changes in the structure and functioning of biotic communities. Nevertheless, the factors that influence biotic communities along spatial and temporal gradients remain poorly understood. This study aimed to characterise the diversity and spatial pattern distribution of Cyclopoida and Ostracoda from the hyporheic zone in relation to the riverbed sediment structure and the quantity of organic matter in two second-order rivers in central Spain, whose alluvium is derived from materials of contrasting geology (siliceous vs. carbonates). Both streams were found to be characterised by marked habitat heterogeneity along the hyporheic flow path, as reflected by the different lithology, riverbed permeability and distinct hyporheic biota assemblages. The results indicate that the alluvium riverbeds dominated by carbonates rocks and associated with high permeable riverbeds had slightly diverse hyporheic assemblages (25 species in all) with species of mixed ecology (stygobites / stygophiles / stygoxenes). Conversely, the siliceous alluvium riverbeds with low mineralised waters, associated with reduced permeability and relatively less active surface/ground water exchanges, displayed slightly lower diversity (23 species) with assemblages exclusively composed of stygophiles/stygoxenes. The results highlight the role of the riverbed substratum for the hyporheic biota and provide forthcoming approaches for depicting surface-subsurface hydrological exchanges.

It is assumed that the south-eastern Europe and especially the Romanian Carpathians were important regions for surface and underground invertebrates survival during glacial periods and acted as a source of post-glacial colonization processes. We analyzed data from 233 georeferenced records for 164 species of groundwater copepods and ostracods from Romania and used a comparative approach to recognize the determinants of the regional-scale richness, endemism, and distribution patterns, with a primary focus on species from the Carpathian Mountains. In addition, we examined the driving forces for the observed pattern of distribution and richness linked to contemporary (groundwater habitat fragmentation and heterogeneity, climate, vegetation) and historical (past climate and vegetation) environmental conditions. Our analyses showed that: (1) species richness was high, irrespective of habitat heterogeneity, in karst and non-karst areas; (2) the main driver accounting for high species richness in the karst landscape was the rainfall regime (> 1350 mm per year), whereas, in non-karst areas, it was woodland vegetation; and (3) there was significant species richness and richness of phyletic lineages in hypothetical forest glacial refugia of the Carpathian arc. The combination of the distribution pattern, diversification, and evolution of stygobite lineages provides reliable evidence for species persistence in the Romanian groundwater during Pleistocene.

A new stygobite species Acanthocyclops transylvanicus sp. nov. is described from the north-western Romanian Carpathians (Apuseni Mountains) and is accommodated in the subterranean kieferi-group. Acanthocyclops transylvanicus sp. nov. resembles closely A. biarticulatus Monchenko, 1972 in the segmentation pattern of the swimming legs (3.2/3.2/3.2/3.2), but differs by the following characters: antennary second endopodite segment with 7 setae; distal articles of endopodites of P1 and P3 with 4 and 5 setae, respectively; absence of coxopodite ornamentation pattern in leg 4; leg 4 endopodite with 3-segmented appearance, but lacking a functional articulation between the second and third segment. We assume that the 2-segmented nature of the leg 4 endopodite as observed in both species is a convergent acquisition. The adult shape of the leg 4 endopodite in A. transylvanicus sp. nov. results from the simple suppression of an arthrodial membrane formation. A. transylvanicus is the ninth representative know...

The purpose of this study was to examine the sensitivity, in a field situation, of the hyporheic fauna to pollution by heavy metals and also to test the use of oxidative stress enzymes produced by this fauna as a sensitive indicator of oxidative stress generated by chemical contamination. This was done by surveying the patterns of distribution, structure, and composition of hyporheic invertebrate communities in one of the most polluted rivers in Romania. Twelve permanent sampling stations with differing water qualities were established along a 180 km transect of the Aries River. Data on hyporheic invertebrate abundance and richness, chemistry of the surface and hyporheic water and interstitial suspended particles were analyzed via multifactorial analyses. In the downstream, more polluted stations, epigean species were less abundant and hyporheic communities, especially macrocrustaceans and oligochetes, became dominant. The higher levels of hyporheic invertebrate biodiversity in the moderately polluted stations compared to highly polluted, and the increase of the number of some hyporheos (especially macrocrustaceans) in the moderately polluted stations, suggested that the hyporheic fauna was more tolerant of heavy metal pollution than the surface water fauna of the area. However, the different richness and abundance of hyporheic fauna in sites of similar water chemistry suggested that additional factors, such as sediment structure are shaping the spatial distribution of hyporheic fauna. Strong correlations between superoxide dismutase (SOD) activity in pooled tissues extracts and some chemical parameters suggest that oxidative stress enzymes may prove to be sensitive indicators of chemical pollution in hyporheic zones.

Recent studies substantiate the importance of the unsaturated zone in ground-water biodiversity of karst areas. Few investigations, however, have addressed the temporal changes in community composition in relation to water physico-chemical features. We provide information on the distribution pattern of the fauna in dripping water at spatial and temporal scales. This is related to variation in water chemistry and other environmental features in five caves within two hydrographic basins of the Pa˘ durea Craiului Mountains (northwestern Romania). The analysis revealed no major pollution in the dripping water. The physico- chemical parameters varied within and between caves over one year. The dripping water fauna is hetero- geneously distributed within and between the caves, containing a mixture of epigean and hypogean species. This emphasizes high microhabitat partition and also underlines the influence of physico-chemical parameters. The species composition among the two hydrographic basins was different. Crustaceans are the best represented in terms of both abundance and species richness. Six out of 15 crustacean species are endemic to the Pa˘ durea Craiului Mountains and four are new to science. All these point to the importance of unsaturated karstic habitats as biodiversity hot spots in ground-water ecosystems.

Morphology of both the valves and limbs of several parthenogenetic populations of the widespread non-marine ostracod Cryptocandona vavrai Kaufmann (sensu lato) living in groundwater habitats of western (France) and south-eastern Europe (Romania) was examined for the extent of intra- and interpopulational variation. Geometric morphometrics and multivariate statistics were employed for the description of the valve morphological disparity whereas the discriminant analysis was performed for the comparative analysis of the limb chaetotaxy. The ranges of the most studied limb traits principally overlapped among the populations, however considerable differences in the carapace shape and size, reflecting microevolutionary changes at a macro-scale (inter-population) level, allowed distinguishing quantitatively some Romanian populations which deserve a separate specific status. Hence, a new species Cryptocandona racovitzai sp. nov. is described which differs from C. vavrai sensu stricto mainly in larger size, in more triangular valve shape as seen laterally and in less developed inner lamella of the valves. Finally, after examination of more material from Romanian groundwaters, we hypothesised the presence of more new species within the studied lineage and concluded that Cryptocandona vavrai sensu lato is a polymorphic lineage consisting of several geographically differentiated species rather than just only one widely dispersed species.

The 1st International Symposium on Ostracoda (ISO) was held in Naples (1963). The philosophy behind this symposium and the logical outcome of what is now known as the International Research Group on Ostracoda (IRGO) are here reviewed, namely ostracodology over the last 50 years is sociologically analysed. Three different and important historic moments for the scientific achievements of this domain are recognised. The first one, between about 1963 and 1983, is related to applied research for the oil industry as well as to the great interest in the better description of the marine environment by both zoologists and palaeontologists. Another important aspect during this period was the work by researchers dealing with Palaeozoic ostracods, who had their own discussion group, IRGPO. Gradually, the merger of this latter group with those dealing with post-Palaeozoic ostracods at various meetings improved the communication between the two groups of specialists. A second period was approximately delineated between 1983 and 2003. During this time-slice, more emphasis was addressed to environmental research with topics such as the study of global events and long-term climate change. Ostracodologists profited also from the research “politics” within national and international programmes. Large international research teams emerged using new research methods. During the third period (2003–2013), communication and collaborative research reached a global dimension. Amongst the topics of research we cite the reconstruction of palaeoclimate using transfer functions, the building of large datasets of ostracod distributions for regional and intercontinental studies, and the implementation of actions that should lead to taxonomic harmonisation. Projects within which molecular biological techniques are routinely used, combined with sophisticated morphological information, expanded now in their importance. The documentation of the ostracod description improved through new techniques to visualise morphological details, which stimulated also communication between ostracodologists. Efforts of making available ostracod information through newsletters and electronic media are evoked.

Teratocyclops cubensis Plesa, 1981 (Copepoda, Cyclopidae) is redescribed on the basis of specimens deposited in the collection of the Speleological Institute "Emil Racovitza", Cluj, Romania. The genus Teratocyclops Plesa, 1981 is redefined, mainly characterized by the following characters of the female: the general habitus of the body, the 11-segmented antennule, the presence of the vestigial exopodite on the antenna, the segmentation of the swimming legs, the structure of the distal segment of the endopodite of leg P4 with 1 spine and 3 setae, and the proximal segment of leg 5 fused to the somite. The relationship to the closely related genera Metacyclops Kiefer, 1927, Apocyclops Lindberg, 1942, and Goniocyclops Kiefer, 1955 are discussed. Teratocyclops cubensis Plesa, 1981 (Copepoda, Cyclopidae) est redecrit a partir des specimens deposes dans les collections de l'Institutul de Speologie "Emil Racovitza", de Cluj, Roumanie. Le genre Teratocyclops Plesa, 1981 est redefini. Il est principalement caracterise par les traits suivants de la femelle: la forme generale du corps, l'antennule a 11 segments, la presence de l'exopodite vestigial sur l'antenne, la segmentation des pattes natatoires et l'ornementation du dernier segment de l'endopodite de P4 comprenant 1 soie et 3 epines, le segment proximal de P5 soude au somite. Les affinites avec les genres voisins, notamment Metacyclops Kiefer, 1927, Apocyclops Lindberg, 1942 et Goniocyclops Kiefer, 1955 sont discutees.

In order to evaluate the water quality at the surface/groundwater interface (hyporheic zone), the pattern of microcrustacean assemblages in response to environmental stress caused by urban industrial contamination was studied in the Jarama River basin (central Spain) during high water discharges (March and April 2011). The clustering of biological variables and the concentration of urban contaminants in hyporheic waters showed that pristine hyporheic waters have moderate species diversity (two to seven species) and dominance of k strategist stygobites, whereas excessively contaminated sites are devoid by crustaceans. An intermediate level of disturbance in hyporheic waters is associated with a peak of species taxonomic diversity (four to nine species) and proliferation of r strategist more tolerant species. Typical species found in hyporheic zone, e.g., Paracyclops imminutus (Copepoda, Cyclopoida), Cryptocandona vavrai (Ostracoda) and Herpetocypris chevreuxi (Ostracoda), were good indicators of high concentrations of Cr, Mn, Ni, Cd, Pb and VOCs; whereas the stygobites do not show any significant correlation. The effectiveness of hyporheic crustaceans as efficient bioindicators for assessing the current ecological status of river ecosystems is emphasised.

Exceptionally well-preserved giant spermatozoa observed between abundant decalcified carapace valves of ostracods (Crustacea: Ostracoda) were found in Late Glacial to Holocene (14,400 to 10,000 cal years bp) lacustrine sediments in the southern Romanian Carpathians. Analysis by scanning electron microscopy and laser scanning confocal microscopy revealed good preservation of the appendages enabling specific identification as Cypria ophtalmica (Candonidae) and indication of the presence of both female and male specimens based on the sexual dimorphism of the second antenna. This record represents the oldest and richest direct evidence of virtually morphologically unaltered animal spermatozoa preserved in females after mating.

The female antennule of several cyclopid copepods of the genus Acanthocyclops Kiefer, 1927 is known to have a high degree of oligomerization in both segmentation and chaetotaxy. The present work provides information on the ontogenetic development of the antennule of three (sub-)species of Acanthocyclops accommodated in the kieferi-group, with an 11- segmented antennule in the adult female. These are, A. balcanicus bisetosus Iepure, 2001, A. milotai Iepure & Defaye, 2008, and Acanthocyclops n. sp. Iepure & Oarga (unpubl.). One developmental pattern has been observed that results in an 11-segmented antennule through several successive enlargements and the addition of segments in the proximal section of the limb as follows: the first segment adds a new segment distally and an arthrodial membrane proximally during the moult to CIII; and the second segment adds a new segment distally during the moult to CIV and proximally in the moult to CV. The same (neotenic) morphology of CV is retained in the adult. The addition of segments and the distribution of setae on each segment were compared and homologized with two subterranean representatives of the genus Diacyclops Kiefer, 1927 that share the same pattern in the adult female, i.e., the D. clandestinus (Kiefer, 1926) sensu lato and the D. languidoides (Lilljeborg, 1901) sensu lato. The position of marker elements (i.e., dorsal seta (ancestral segment XV), medial distal seta (ancestral segment XX), etc.) in various copepodid stages is used to identify homologous segments. The analyses of antennula development suggest that segment number and chaetotaxy during development are characters that are less informative for phylogenetic studies in the genera Acanthocyclops and Diacyclops.

[The Acanthocyclops kieferi (Chappuis, 1925) complex (Copepoda, Cyclopoida) includes exclusively stygobitic species, mainly widespread in the cave habitats of south-eastern Europe. A new species for the group, Acanthocyclops milotai n. sp., collected from pools supplied by percolation water in a cave in south-western Romania, is described. The new species is distinguished from its congeners by the following characters: the ornamentation pattern of the basis of the antenna with four spinules placed in an arch; the coxa of leg 4 with four spinules on its distal margin; furcal rami divergent, about 2.2 times as long as wide, with the lateralmost, terminal furcal seta 1.14 times as long as the medialmost one, and these both setiform. Inter-populational variability of some characters in the new species such as the shape of the anal operculum and the ornamentation of the coxa of leg P4 are discussed. Furthermore, the characters generally used to differentiate the taxa of the kieferi group are re-examined and discussed, and a brief description of the adaptive traits of these species to the groundwater is given. Biogeographical and ecological considerations about the kieferi species-complex in Romanian groundwater habitats are also mentioned. Le complexe Acanthocyclops kieferi (Chappuis, 1925) est compose d'especes stygobiontes largement repandues dans les habitats des grottes de l'Europe de l'Est. Une nouvelle espece de ce groupe, A. milotai n. sp., trouvee dans des bassins remplis d'eau de percolation d'une grotte du sud-ouest de la Roumanie, est decrite. La nouvelle espece se distingue de ses congeneres par les caracteres suivants: l'ornementation de l'antenne avec 3 spinules disposees en arc sur le basipodite et celle du coxopodite de P4, avec 4 spinules sur son extremite distale; les rames furcales divergentes, 2,2 fois plus longues que larges et la soie furcale terminale la plus laterale 1,14 fois plus longue que celle plus mediane, les deux setiformes. La variabilite intra-populationnelle de certains caracteres chez la nouvelle espece, comme la forme de l'opercule anal et l'ornementation du coxopodite de P4 est brievement discutee. Les caracteres generalement utilises pour differencier les taxons de ce groupe sont re-examines et discutes, et une courte description de leurs traits adaptatifs est donnee. Les caracteres biogeographiques et ecologiques de la nouvelle espece et du complexe A. kieferi dans les eaux souterraines de Roumanie sont brievement discutes., The Acanthocyclops kieferi (Chappuis, 1925) complex (Copepoda, Cyclopoida) includes exclusively stygobitic species, mainly widespread in the cave habitats of south-eastern Europe. A new species for the group, Acanthocyclops milotai n. sp., collected from pools supplied by percolation water in a cave in south-western Romania, is described. The new species is distinguished from its congeners by the following characters: the ornamentation pattern of the basis of the antenna with four spinules placed in an arch; the coxa of leg 4 with four spinules on its distal margin; furcal rami divergent, about 2.2 times as long as wide, with the lateralmost, terminal furcal seta 1.14 times as long as the medialmost one, and these both setiform. Inter-populational variability of some characters in the new species such as the shape of the anal operculum and the ornamentation of the coxa of leg P4 are discussed. Furthermore, the characters generally used to differentiate the taxa of the kieferi group are re-examined and discussed, and a brief description of the adaptive traits of these species to the groundwater is given. Biogeographical and ecological considerations about the kieferi species-complex in Romanian groundwater habitats are also mentioned. Le complexe Acanthocyclops kieferi (Chappuis, 1925) est compose d'especes stygobiontes largement repandues dans les habitats des grottes de l'Europe de l'Est. Une nouvelle espece de ce groupe, A. milotai n. sp., trouvee dans des bassins remplis d'eau de percolation d'une grotte du sud-ouest de la Roumanie, est decrite. La nouvelle espece se distingue de ses congeneres par les caracteres suivants: l'ornementation de l'antenne avec 3 spinules disposees en arc sur le basipodite et celle du coxopodite de P4, avec 4 spinules sur son extremite distale; les rames furcales divergentes, 2,2 fois plus longues que larges et la soie furcale terminale la plus laterale 1,14 fois plus longue que celle plus mediane, les deux setiformes. La variabilite intra-populationnelle de certains caracteres chez la nouvelle espece, comme la forme de l'opercule anal et l'ornementation du coxopodite de P4 est brievement discutee. Les caracteres generalement utilises pour differencier les taxons de ce groupe sont re-examines et discutes, et une courte description de leurs traits adaptatifs est donnee. Les caracteres biogeographiques et ecologiques de la nouvelle espece et du complexe A. kieferi dans les eaux souterraines de Roumanie sont brievement discutes.]

Biodiversity and ecology of fauna in percolating water from Slovenian and Romanian caves was studied. Research focused on unravelling the community structure of epikarst fauna, which is carried away by the trickles of percolating water from the epikarst and vadose zones. The major part of the fauna found in percolating water is represented by copepods. This fauna, originating in the epikarst, was analysed and by means of the systematic sampling and observation the same groups of animals were found in Slovenian and Romanian caves. Differences among caves and sampling points indicate that epikarst is a heterogenous habitat. Relationship between faunal richness and the physical characteristics of the water was found. Correlation between surface geomorphology and fauna in percolating water was statistically significant in the Postojna cave system.