Groundwater is a hidden global keystone ecosystem.

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

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

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

Temperature variation in caves and its significance for subterranean ecosystems.

Climate change affects all ecosystems, but subterranean ecosystems are repeatedly neglected from political and public agendas. Cave habitats are home to unknown and endangered species, with low trait variability and intrinsic vulnerability to recover from human-induced disturbances. We studied the annual variability and cyclicity of temperatures in caves vis-à-vis surface in different climatic areas. We hypothesize that cave temperatures follow the average temperature pattern at the surface for each location with a slight delay in the signal, but we found three different thermal patterns occurring in caves: (1) high positive correlation and a similar thermal pattern to the surface, (2) low correlation and a slight thermal delay of the signal from the surface, and (3) high negative correlation with an extreme delay from the surface. We found daily thermal cycles in some caves, which may potentially control the circadian rhythms of cave organisms. Our results show that caves had lower thermal amplitude than the surface, and that thermal averages within caves approximately correspond to the to the annual average of surface temperature. Caves buffer external temperature and act as refugia for biota in extreme climatic events. Likewise, temperature increases at surface will lead to increment in caves, threatening subterranean biota and ecosystem services.

Acetaminophen induced antioxidant and detoxification responses in a stygobitic crustacean.

A variety of veterinary and human medicinal products (VHMPs) are found in groundwater, an often-neglected habitat inhabited by species with unique traits, stygobitic species. It is crucial to understand the effect of VHMPs on stygobitic species because they may respond differently to stressors than surface species. Our hypothesis is that groundwater species may be more susceptible to environmental contaminants due to less plasticity in their detoxification response and acquisition of energy because subterranean habitats are more stable and isolated from anthropogenic activities. We performed a battery of biomarkers associated with important physiological functions on the stygobitic asellid crustacean Proasellus lusitanicus, after a 14-day exposure to acetaminophen, a commonly used pharmaceutical and pollutant of groundwaters. Our results show a decrease in total glutathione levels and an increase in glutathione S-transferase activity, suggesting a successful detoxification response. This helps explaining why acetaminophen did not cause oxidative damage, as well as had no effect on cholinesterase activity nor in aerobic production of energy. This study shows the remarkable capacity of P. lusitanicus to tolerate sublethal concentrations of VHMP acetaminophen. Most ecotoxicological studies on stygobitic species focused on the lethal effects of these compounds. The present study focuses on consequences at sublethal concentrations. Future studies should assess the stress levels induced to better predict and estimate the impacts of contaminants on groundwater ecosystems.

Cave-adapted millipedes from Portugal: species conservation profiles.

Background: Amongst the cave-dwelling millipedes (Diplopoda), there are several endemic species in Portugal with a very small geographical distribution. These species play an important role in the decomposition of organic matter in subterranean ecosystems and are vulnerable to disturbance from human activities, such as habitat destruction, pollution infiltrating from the surface and cave tourism. New information: We present the IUCN Red List profiles for cave-adapted millipedes (Diplopoda) from Portugal and propose conservation measures to prevent extinction. Overall, cave-adapted millipedes from Portugal represent an endemic part of the country's biodiversity and conservation efforts will help maintain the delicate ecological balance of subterranean ecosystems.

Thermal acclimation and metabolic scaling of a groundwater asellid in the climate change scenario.

Metabolic rate has long been used in animal adaptation and performance studies, and individual oxygen consumption is used as proxy of metabolic rate. Stygofauna are organisms adapted to groundwater with presumably lower metabolic rates than their surface relatives. How stygofauna will cope with global temperature increase remains unpredictable. We studied the thermal acclimation and metabolic scaling with body mass of a stygobitic crustacean, Proasellus lusitanicus, in the climate change scenario. We measured oxygen consumption rates in a thermal ramp-up experiment over four assay temperatures and tested two hypotheses: (i) P. lusitanicus exhibits narrow thermal plasticity, inadequate for coping with a fast-increasing thermal regime; and (ii) oxygen consumption rates scale with the body mass by a factor close to 0.75, as commonly observed in other animals. Our results show that P. lusitanicus has low thermal plasticity in a fast-increasing thermal regime. Our data also suggest that oxygen consumption rates of this species do not follow mass-dependent scaling, potentially representing a new trait of metabolic optimization in groundwater habitats, which are often limited in food and oxygen. Species with limited dispersal capacities and rigid metabolic guilds face extinction risk due to climate change and omitting groundwater ecosystems from climate change agendas emphasizes the unprotected status of stygofauna.

Species conservation profiles of cave-adapted terrestrial isopods from Portugal.

Background: Terrestrial isopods (Oniscidea) are the most diverse group of troglobionts in caves of continental Portugal. They occur in all karst regions of Portugal, play a major role in decomposition of organic matter in caves and may act as umbrella species for the conservation of all other cave-adapted invertebrates. New information: We present the IUCN Red List profiles for the cave-adapted terrestrial isopods from continental Portugal, based on recent distribution data from caves.

Penetrative and non-penetrative interaction between Laboulbeniales fungi and their arthropod hosts.

Laboulbeniales are a highly specialized group of fungi living only on arthropods. They have high host specificity and spend their entire life-cycle on an arthropod host. Taxonomic characters of Laboulbeniales are based on the architecture of the cells of the parenchymal thallus, i.e. the visible part of the fungus outside the host. The extent of the fungus spreading inside the host-the haustorium-remains largely unknown. The attachment to the arthropod host is fundamental to understand the fungus-animal interaction, but how this truly occurs is unclear. Recent evidences question the strictly parasitic life-style of Laboulbeniales. We used micro-computed tomography (µCT) and 3D reconstructions to visualize, for the first time, the complete structure of Laboulbeniales species in situ on their hosts. We compared the haustoriate species, Arthrorhynchus nycteribiae on an insect host to the non-haustoriate species, Rickia gigas on a millipede host. Our results confirm that some Laboulbeniales species are ectoparasitic and have a haustorial structure that penetrates the host's cuticle, while others are ectobionts and are only firmly attached to the host's cuticle without penetrating it. The presence and the morphology of the haustorium are important traits for Laboulbeniales evolution, and key factors for future understanding of host dependence and specificity.

Cave-adapted beetles from continental Portugal.

BACKGROUND: The cave biodiversity of continental Portugal faces tremendous conservation challenges, mostly linked to their direct destruction and contamination infiltrating from the surface. Beetles are the most diverse insects and one of the most diverse arthropod groups in caves of Portugal. NEW INFORMATION: We present the IUCN Red List profiles for the cave-adapted beetles from continental Portugal, all endemic to their respective geological units and massifs. Ground beetles (Carabidae) are the most diverse family of cave-adapted beetles in continental Portugal, followed by rove beetles (Staphylinidae). Beetles in caves of Portugal are mostly terrestrial and only one species is known to have evolved to live in groundwater. Trechus is the most diverse genus with four species, followed by Domene with three species and by Speonemadus and Iberoporus, both with one cave-adapted species. The aim of this contribution is to assess all endemic cave-adapted species of beetles from continental Portugal and to support their specific protection, to promote adequate management of surface habitats and the establishment of priority areas for conservation. The main biodiversity erosion drivers that are impacting the conservation of the studied species are pollution infiltrating from the surface, urbaniation, modifications of the natural habitat for touristic purposes and mining, quarrying and energy production infrastructures.This document can be used in spatial planning and territory management in karst, based on the current scientific knowledge.

Sensitivity of a widespread groundwater copepod to different contaminants.

Groundwater is an indispensable resource for humankind and sustainable biomes functioning. Anthropogenic disturbance threatens groundwater ecosystems globally, but to which extent groundwater organisms respond to stressors remains poorly understood. Groundwater animals are rare, with small populations, difficult to find and to breed in the lab, which poses a main challenge to the assessment of their responses to pollutants. Despite the difficulties, assessing the toxicity of a large spectrum of stressors to groundwater organisms is a priority to inform towards appropriate environmental protection of these ecosystems. We tested the sensitivity to CuSO4, diclofenac, and NaCl of a groundwater population of the copepod Diacyclops crassicaudis crassicaudis and compared its sensitivity with the model organism Daphnia magna. We ranked its sensitivity using a species sensitivity distribution (SSD) approach using the feasible data available for groundwater and surface crustaceans. Our results show that the most toxic compound was CuSO4 for which higher amount of data was recorded and wider variability in response was observed. It was followed by diclofenac, largely lacking data for groundwater-adapted organisms, and the least toxic compound was NaCl. The differential sensitivity between D. crassicaudis and D. magna was contaminant-dependent. As a general trend D. crassicaudis was always distributed in the upper part of the SSD curves together with other groundwater-adapted organisms. Our results highlight that the widespread groundwater populations of the D. crassicaudis species complex, which can be successfully breed in the lab, may provide a reasonable approach to assess the ecological effects of anthropogenic stressors in groundwater ecosystems.

Fundamental research questions in subterranean biology.

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.

Salinity and temperature increase impact groundwater crustaceans.

Anthropogenic impacts in groundwater ecosystems remain poorly known. Climate change is omnipresent, while groundwater salinization poses serious long-term environmental problems in arid and semi-arid regions, and is exacerbated by global warming. Both are present threats to the conservation of groundwater ecosystems, which harbour highly specialized species, with peculiar traits and limited geographic distributions. We tested the temperature and salinity tolerance of groundwater-adapted invertebrates to understand the effect of global warming and salinization in groundwater ecosystems. We used species representative of groundwater-adapted crustaceans: two copepods (harpacticoid and cyclopoid) and one syncarid, endemic to Australia. Our results show that 50% of the populations died at salt concentrations between 2.84 to 7.35 g NaCl/L after 96 h, and at 6.9 °C above the ambient aquifer temperature for copepods and more than 10 °C for syncarids. Both copepods were more sensitive to temperature and NaCl than the syncarid. We calculated a salinity risk quotient of 9.7 and predicted the risk of loss of 10% of syncarid and 20% of copepod population abundances under a worst-case scenario of global warming predictions for 2070. These results highlight that both salinity and temperature increases pose a risk to the ecological integrity of groundwater ecosystems.

Ecotoxicological effects of anthropogenic stressors in subterranean organisms: A review.

How anthropogenic stressors affect biodiversity is a central question in a changing world. Subterranean ecosystems and their biodiversity are particularly vulnerable to change, yet, these species are frequently neglected in analyses of global biodiversity and assessments of ecological status and risk. Are these hidden species affected by anthropogenic stressors? Do they survive outside of the current thermal limits of their ecosystems? These and other important questions can be addressed with ecotoxicological testing, relating contaminants and temperature resistance of species with measured environmental concentrations and climatic data. Ecotoxicological knowledge specific to subterranean ecosystems is crucial for establishing thresholds for their protection, but such data are both scarce and scattered. Here, we review the existing ecotoxicological studies of these impacts to subterranean-adapted species. An effort that includes 167 measured endpoints and presents a database containing experimentally derived species' tolerance data for 28 contaminants and temperature, for 46 terrestrial and groundwater species, including fungi and animals. The lack of standard data among the studies is currently the major impediment to evaluate how stressors affect subterranean-adapted species and how differently they respond from their relatives at surface. Improving understanding of ecotoxicological effects on subterranean-adapted species will require extensive analysis of physiological responses to a wide range of untested stressors, standardization of testing protocols and evaluation of exposures under realistic scenarios.

Hyperparasitism in caves: Bats, bat flies and ectoparasitic fungus interaction.

Bat flies (Diptera: Nycteribiinae) are highly specialized bloodsucking bat ectoparasites. Some of the ectoparasitic bat flies are themselves parasitized with an ectoparasitic fungus of the genus Arthrorhynchus (Laboulbeniales). Ascospores of the fungus attach to the cuticle of a bat fly and develop a haustorium that penetrates the host cuticle. This interaction defines the fungus as a hyperparasite. Both the fly and the fungus are obligate parasites and this peculiar case of hyperparasitism has remained largely unstudied. We studied the prevalence of Laboulbeniales, genus Arthrorhynchus, in natural populations of bat flies infesting the bat species Miniopterus schreibersii, Myotis bechsteinii, My. blythii, My. daubentonii, My. escalerai and My. myotis in Portuguese caves. Laboulbeniales were found infecting 10 of the 428 screened bat flies (2.3%) in natural populations, with fewer infections in winter. Images obtained with transmission electron microscopy show the fungal haustorium within the bat fly host tissue, from where it extracts nutrition.

Studies of Laboulbeniales on Myrmica ants (IV): host-related diversity and thallus distribution patterns of Rickia wasmannii.

Fungal species identities are often based on morphological features, but current molecular phylogenetic and other approaches almost always lead to the discovery of multiple species in single morpho-species. According to the morphological species concept, the ant-parasitic fungus Rickia wasmannii (Ascomycota, Laboulbeniales) is a single species with pan-European distribution and a wide host range. Since its description, it has been reported from ten species of Myrmica (Hymenoptera, Formicidae), of which two belong to the rubra-group and the other eight to the phylogenetically distinct scabrinodis-group. We found evidence for R. wasmannii being a single phylogenetic species using sequence data from two loci. Apparently, the original morphological description (dating back to 1899) represents a single phylogenetic species. Furthermore, the biology and host-parasite interactions of R. wasmannii are not likely to be affected by genetic divergence among different populations of the fungus, implying comparability among studies conducted on members of different ant populations. We found no differences in total thallus number on workers between Myrmica species, but we did observe differences in the pattern of thallus distribution over the body. The locus of infection is the frontal side of the head in Myrmica rubra and M. sabuleti whereas in M. scabrinodis the locus of infection differs between worker ants from Hungary (gaster tergites) and the Netherlands (frontal head). Possible explanations for these observations are differences among host species and among populations of the same species in (i) how ant workers come into contact with the fungus, (ii) grooming efficacy, and (iii) cuticle surface characteristics.

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

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

Amblypygids of Timor-Leste: first records of the order from the country with the description of a remarkable new species of Sarax (Arachnida, Amblypygi, Charinidae).

The whip spider genus Sarax Simon, 1892 is widely distributed throughout Southeast Asia and part of the Indo-Malayan region. The genus is recorded from several Indonesian islands, but no species are known from inside the area that comprises the biogeographical region of Wallacea, despite being recorded from both sides of the area. An expedition to survey the biology of caves in Timor-Leste (formerly East-Timor) discovered populations of amblypygids living underground and including a remarkable new species of Sarax, S.timorensis sp. n., the first Amblypygi known from the island of Timor. The new species is here described bears the unique character state of two pairs of lateral eyes, instead of three or none as in all other living species of Amblypygi, and expands the biogeographic range of the genus. New records of amblypygids are given for two caves in Timor-Leste. A detailed description and a discussion of its distribution and the species characters are also provided.

The first stygobiont species of Coleoptera from Portugal, with a molecular phylogeny of the Siettitia group of genera (Dytiscidae, Hydroporinae, Hydroporini, Siettitiina).

Iberoporuspluto sp. n., the first stygobiont beetle from Portugal (Dytiscidae, Hydroporinae), is described from a single female from the cave Soprador do Carvalho (Coimbra). The species is highly troglomorphic, depigmented, blind, and with elongated appendages not adapted for swimming. A molecular phylogeny based on a combination of three mitochondrial and two nuclear genes showed the new species to be sister to I.cermenius Castro & Delgado, 2001 from Córdoba (south of Spain), within the subtribe Siettitiina of the tribe Hydroporini. Both species are included in a clade with Siettitiaavenionensis Guignot, 1925 (south of France) and Rhithrodytesagnus Foster, 1992 and R.argaensis Fery & Bilton, 1996 (north of Portugal), in turn sister to the rest of species of genus Rhithrodytes Bameul, 1989, in what is here considered the Siettitia group of genera. We resolve the paraphyly of Rhithrodytes by transferring the two Portuguese species to Iberoporus Castro & Delgado, 2001, I.agnus (Foster, 1992), comb. n. and I.argaensis (Fery & Bilton, 1996), comb. n.

Novelty upon novelty visualized by rotational scanning electron micrographs (rSEM): Laboulbeniales on the millipede order Chordeumatida.

Laboulbeniales are highly specific ectoparasitic fungi of arthropods (insects, millipedes, and arachnids). The first Laboulbeniales parasitizing the millipede order Chordeumatida (Diplopoda) were discovered and described as a new dioecious genus of Laboulbeniales, Thaxterimyces, to accommodate the new species T. baliensis. Also the millipede host is a new species and is described as Metopidiothrix sheari. This is the first time Laboulbeniales fungus and its millipede host are described as new together. Males of Metopidiothrix have the most extensive secondary sexual modifications in the entire class Diplopoda. Although nothing is known about the function of these modifications, the unique pattern of Laboulbeniales infection in the new millipede species is obviously related to host sexual behavior. Rotational Scanning Electron Micrographs (rSEM) are used to create a 3D comprehensive model to examine the fungal-host interaction, a more advanced visualization of the ectoparasitic fungus on its host. Laboulbeniales diversity on millipedes is still understudied, and a consistent effort is needed to unveil and understand the extent and diversity of this biological interaction. Due to their minute size and apparently non-detrimental effect on their hosts, Laboulbeniales in general have been largely ignored by mycologists and neglected by generations of entomologists. As a result a significant component of global biodiversity has been strongly underestimated, and a wealth of new discoveries is still to be made both in the field and in existing museum collections.

First continental troglobiont Cylindroiulus millipede (Diplopoda, Julida, Julidae).

The new species of millipede Cylindroiulusvillumi is described from a cave in the Estremenho karst massif in central Portugal. It is the first cave-adapted species of its genus with a strict subterranean life-style in continental Europe, and is the fifth blind species of the genus. The new species is illustrated with photographs and diagrammatic drawings. It is tentatively placed in the purely Iberian Cylindroiulusperforatus-group. The differences between the new species and its relatives are discussed, as well as its adaptations to a subterranean life-style.