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.

Aquatic environmental DNA: A review of the macro-organismal biomonitoring revolution.

Environmental DNA (eDNA) is the fastest growing biomonitoring tool fuelled by two key features: time efficiency and sensitivity. Technological advancements allow rapid biodiversity detection at both species and community levels with increasing accuracy. Concurrently, there has been a global demand to standardise eDNA methods, but this is only possible with an in-depth overview of the technological advancements and a discussion of the pros and cons of available methods. We therefore conducted a systematic literature review of 407 peer-reviewed papers on aquatic eDNA published between 2012 and 2021. We observed a gradual increase in the annual number of publications from four (2012) to 28 (2018), followed by a rapid growth to 124 publications in 2021. This was mirrored by a tremendous diversification of methods in all aspects of the eDNA workflow. For example, in 2012 only freezing was applied to preserve filter samples, whereas we recorded 12 different preservation methods in the 2021 literature. Despite an ongoing standardisation debate in the eDNA community, the field is seemingly moving fast in the opposite direction and we discuss the reasons and implications. Moreover, by compiling the largest PCR-primer database to date, we provide information on 522 and 141 published species-specific and metabarcoding primers targeting a wide range of aquatic organisms. This works as a user-friendly 'distillation' of primer information that was hitherto scattered across hundreds of papers, but the list also reflects which taxa are commonly studied with eDNA technology in aquatic environments such as fish and amphibians, and reveals that groups such as corals, plankton and algae are under-studied. Efforts to improve sampling and extraction methods, primer specificity and reference databases are crucial to capture these ecologically important taxa in future eDNA biomonitoring surveys. In a rapidly diversifying field, this review synthetises aquatic eDNA procedures and can guide eDNA users towards best practice.

Subterranean carbon flows from source to stygofauna: a case study on the atyid shrimp Stygiocaris stylifera (Holthuis, 1960) from Barrow Island (WA).

Groundwater biota are crucial for the ecological functioning of subterranean ecosystems. However, while knowledge of the taxonomic diversity of groundwater invertebrates (stygofauna) is increasing, functional ecological information is still limited. Here, we investigate seldom empirically tested assumptions around stygofaunal trophic plasticity in coping with oligotrophic habitats. We focus on Barrow Island (Western Australia), an ideal natural laboratory due to the occurrence of natural oil seeps in association with aquifers. The trophic position and food source use of the endemic atyid shrimp Stygiocaris stylifera (Holthuis, 1960) were assessed via δ13C and δ15N stable isotope analysis (SIA). Background information on the environmental conditions was gathered through hydrochemical data and δ13C SIA combined with 14C data from dissolved inorganic/organic carbon and particulate organic carbon from groundwater samples. Our results indicate carbon enrichment in proximity to the natural oil seepage coupled with changes in trophic positions of S. stylifera from higher consumers/predators to biofilm grazers/decomposers. These results are consistent with an increased involvement of hydrocarbon seeps and associated microbial communities in the carbon flows and confirm potential for the trophic flexibility in stygofauna. Further investigations involving other trophic groups will help elucidate the functioning of the ecosystems at a community level.

eDNA in subterranean ecosystems: Applications, technical aspects, and future prospects.

Monitoring of biota is pivotal for the assessment and conservation of ecosystems. Environments worldwide are being continuously and increasingly exposed to multiple adverse impacts, and the accuracy and reliability of the biomonitoring tools that can be employed shape not only the present, but more importantly, the future of entire habitats. The analysis of environmental DNA (eDNA) metabarcoding data provides a quick, affordable, and reliable molecular approach for biodiversity assessments. However, while extensively employed in aquatic and terrestrial surface environments, eDNA-based studies targeting subterranean ecosystems are still uncommon due to the lack of accessibility and the cryptic nature of these environments and their species. Recent advances in genetic and genomic analyses have established a promising framework for shedding new light on subterranean biodiversity and ecology. To address current knowledge and the future use of eDNA methods in groundwaters and caves, this review explores conceptual and technical aspects of the application and its potential in subterranean systems. We briefly introduce subterranean biota and describe the most used traditional sampling techniques. Next, eDNA characteristics, application, and limitations in the subsurface environment are outlined. Last, we provide suggestions on how to overcome caveats and delineate some of the research avenues that will likely shape this field in the near future. We advocate that eDNA analyses, when carefully conducted and ideally combined with conventional sampling techniques, will substantially increase understanding and enable crucial expansion of subterranean community characterisation. Given the importance of groundwater and cave ecosystems for nature and humans, eDNA can bring to the surface essential insights, such as study of ecosystem assemblages and rare species detection, which are critical for the preservation of life below, as well as above, the ground.

Salt to conserve: a review on the ecology and preservation of hypersaline ecosystems.

When it comes to the investigation of key ecosystems in the world, we often omit salt from the ecological recipe. In fact, despite occupying almost half of the volume of inland waters and providing crucial services to humanity and nature, inland saline ecosystems are often overlooked in discussions regarding the preservation of global aquatic resources of our planet. As a result, our knowledge of the biological and geochemical dynamics shaping these environments remains incomplete and we are hesitant in framing effective protective strategies against the increasing natural and anthropogenic threats faced by such habitats. Hypersaline lakes, water bodies where the concentration of salt exceeds 35 g/l, occur mainly in arid and semiarid areas resulting from hydrological imbalances triggering the accumulation of salts over time. Often considered the 'exotic siblings' within the family of inland waters, these ecosystems host some of the most extremophile communities worldwide and provide essential habitats for waterbirds and many other organisms in already water-stressed regions. These systems are often highlighted as natural laboratories, ideal for addressing central ecological questions due to their relatively low complexity and simple food web structures. However, recent studies on the biogeochemical mechanisms framing hypersaline communities have challenged this archetype, arguing that newly discovered highly diverse communities are characterised by specific trophic interactions shaped by high levels of specialisation. The main goal of this review is to explore our current understanding of the ecological dynamics of hypersaline ecosystems by addressing four main research questions: (i) why are hypersaline lakes unique from a biological and geochemical perspective; (ii) which biota inhabit these ecosystems and how have they adapted to the high salt conditions; (iii) how do we protect biodiversity from increasing natural and anthropogenic threats; and (iv) which scientific tools will help us preserve hypersaline ecosystems in the future? First, we focus on the ecological characterisation of hypersaline ecosystems, illustrate hydrogeochemical dynamics regulating such environments, and outline key ecoregions supporting hypersaline systems across the globe. Second, we depict the diversity and functional aspects of key taxa found in hypersaline lakes, from microorganisms to plants, invertebrates, waterbirds and upper trophic levels. Next, we describe ecosystem services and discuss possible conservation guidelines. Finally, we outline how cutting-edge technologies can provide new insights into the study of hypersaline ecology. Overall, this review sheds further light onto these understudied ecosystems, largely unrecognised as important sources of unique biological and functional diversity. We provide perspectives for key future research avenues, and advocate that the conservation of hypersaline lakes should not be taken with 'a grain of salt'.

Rainfall as a trigger of ecological cascade effects in an Australian groundwater ecosystem.

Groundwaters host vital resources playing a key role in the near future. Subterranean fauna and microbes are crucial in regulating organic cycles in environments characterized by low energy and scarce carbon availability. However, our knowledge about the functioning of groundwater ecosystems is limited, despite being increasingly exposed to anthropic impacts and climate change-related processes. In this work we apply novel biochemical and genetic techniques to investigate the ecological dynamics of an Australian calcrete under two contrasting rainfall periods (LR-low rainfall and HR-high rainfall). Our results indicate that the microbial gut community of copepods and amphipods experienced a shift in taxonomic diversity and predicted organic functional metabolic pathways during HR. The HR regime triggered a cascade effect driven by microbes (OM processors) and exploited by copepods and amphipods (primary and secondary consumers), which was finally transferred to the aquatic beetles (top predators). Our findings highlight that rainfall triggers ecological shifts towards more deterministic dynamics, revealing a complex web of interactions in seemingly simple environmental settings. Here we show how a combined isotopic-molecular approach can untangle the mechanisms shaping a calcrete community. This design will help manage and preserve one of the most vital but underrated ecosystems worldwide.

The critical thermal maximum of diving beetles (Coleoptera: Dytiscidae): a comparison of subterranean and surface-dwelling species.

Thermal tolerance limits in animals are often thought to be related to temperature and thermal variation in their environment. Recently, there has been a focus on studying upper thermal limits due to the likelihood for climate change to expose more animals to higher temperatures and potentially extinction. Organisms living in underground environments experience reduced temperatures and thermal variation in comparison to species living in surface habitats, but how these impact their thermal tolerance limits are unclear. In this study, we compare the thermal critical maximum (CTmax) of two subterranean diving beetles (Dytiscidae) to that of three related surface-dwelling species. Our results show that subterranean species have a lower CTmax (38.3-39.0°C) than surface species (42.0-44.5°C). The CTmax of subterranean species is ∼10°C higher than the highest temperature recorded within the aquifer. Groundwater temperature varied between 18.4°C and 28.8°C, and changes with time, depth and distance across the aquifer. Seasonal temperature fluctuations were 0.5°C at a single point, with the maximum heating rate being ∼1000x lower (0.008°C/hour) than that recorded in surface habitats (7.98°C/hour). For surface species, CTmax was 7-10°C higher than the maximum temperature in their habitats, with daily fluctuations from ∼1°C to 16°C and extremes of 6.9°C and 34.9°C. These findings suggest that subterranean dytiscid beetles are unlikely to reach their CTmax with a predicted warming of 1.3-5.1°C in the region by 2090. However, the impacts of long-term elevated temperatures on fitness, different life stages and other species in the beetle's trophic food web are unknown.

Refining trophic dynamics through multi-factor Bayesian mixing models: A case study of subterranean beetles.

Food web dynamics are vital in shaping the functional ecology of ecosystems. However, trophic ecology is still in its infancy in groundwater ecosystems due to the cryptic nature of these environments. To unravel trophic interactions between subterranean biota, we applied an interdisciplinary Bayesian mixing model design (multi-factor BMM) based on the integration of faunal C and N bulk tissue stable isotope data (δ13C and δ15N) with radiocarbon data (Δ14C), and prior information from metagenomic analyses. We further compared outcomes from multi-factor BMM with a conventional isotope double proxy mixing model (SIA BMM), triple proxy (δ13C, δ15N, and Δ14C, multi-proxy BMM), and double proxy combined with DNA prior information (SIA + DNA BMM) designs. Three species of subterranean beetles (Paroster macrosturtensis, Paroster mesosturtensis, and Paroster microsturtensis) and their main prey items Chiltoniidae amphipods (AM1: Scutachiltonia axfordi and AM2: Yilgarniella sturtensis), cyclopoids and harpacticoids from a calcrete in Western Australia were targeted. Diet estimations from stable isotope only models (SIA BMM) indicated homogeneous patterns with modest preferences for amphipods as prey items. Multi-proxy BMM suggested increased-and species-specific-predatory pressures on amphipods coupled with high rates of scavenging/predation on sister species. SIA + DNA BMM showed marked preferences for amphipods AM1 and AM2, and reduced interspecific scavenging/predation on Paroster species. Multi-factorial BMM revealed the most precise estimations (lower overall SD and very marginal beetles' interspecific interactions), indicating consistent preferences for amphipods AM1 in all the beetles' diets. Incorporation of genetic priors allowed crucial refining of the feeding preferences, while integration of more expensive radiocarbon data as a third proxy (when combined with genetic data) produced more precise outcomes but close dietary reconstruction to that from SIA + DNA BMM. Further multidisciplinary modeling from other groundwater environments will help elucidate the potential behind these designs and bring light to the feeding ecology of one the most vital ecosystems worldwide.

Tracking down carbon inputs underground from an arid zone Australian calcrete.

Freshwater ecosystems play a key role in shaping the global carbon cycle and maintaining the ecological balance that sustains biodiversity worldwide. Surficial water bodies are often interconnected with groundwater, forming a physical continuum, and their interaction has been reported as a crucial driver for organic matter (OM) inputs in groundwater systems. However, despite the growing concerns related to increasing anthropogenic pressure and effects of global change to groundwater environments, our understanding of the dynamics regulating subterranean carbon flows is still sparse. We traced carbon composition and transformations in an arid zone calcrete aquifer using a novel multidisciplinary approach that combined isotopic analyses of dissolved organic carbon (DOC) and inorganic carbon (DIC) (δ13CDOC, δ13CDIC, 14CDOC and 14CDIC) with fluorescence spectroscopy (Chromophoric Dissolved OM (CDOM) characterisation) and metabarcoding analyses (taxonomic and functional genomics on bacterial 16S rRNA). To compare dynamics linked to potential aquifer recharge processes, water samples were collected from two boreholes under contrasting rainfall: low rainfall ((LR), dry season) and high rainfall ((HR), wet season). Our isotopic results indicate limited changes and dominance of modern terrestrial carbon in the upper part (northeast) of the bore field, but correlation between HR and increased old and 13C-enriched DOC in the lower area (southwest). CDOM results show a shift from terrestrially to microbially derived compounds after rainfall in the same lower field bore, which was also sampled for microbial genetics. Functional genomic results showed increased genes coding for degradative pathways-dominated by those related to aromatic compound metabolisms-during HR. Our results indicate that rainfall leads to different responses in different parts of the bore field, with an increase in old carbon sources and microbial processing in the lower part of the field. We hypothesise that this may be due to increasing salinity, either due to mobilisation of Cl- from the soil, or infiltration from the downstream salt lake during HR. This study is the first to use a multi-technique assessment using stable and radioactive isotopes together with functional genomics to probe the principal organic biogeochemical pathways regulating an arid zone calcrete system. Further investigations involving extensive sampling from diverse groundwater ecosystems will allow better understanding of the microbiological pathways sustaining the ecological functioning of subterranean biota.

Elucidating stygofaunal trophic web interactions via isotopic ecology.

Subterranean ecosystems host highly adapted aquatic invertebrate biota which play a key role in sustaining groundwater ecological functioning and hydrological dynamics. However, functional biodiversity studies in groundwater environments, the main source of unfrozen freshwater on Earth, are scarce, probably due to the cryptic nature of the systems. To address this, we investigate groundwater trophic ecology via stable isotope analysis, employing δ13C and δ15N in bulk tissues, and amino acids. Specimens were collected from a shallow calcrete aquifer in the arid Yilgarn region of Western Australia: a well-known hot-spot for stygofaunal biodiversity. Sampling campaigns were carried out during dry (low rainfall: LR) and the wet (high rainfall: HR) periods. δ13C values indicate that most of the stygofauna shifted towards more 13C-depleted carbon sources under HR, suggesting a preference for fresher organic matter. Conversion of δ15N values in glutamic acid and phenylalanine to a trophic index showed broadly stable trophic levels with organisms clustering as low-level secondary consumers. However, mixing models indicate that HR conditions trigger changes in dietary preferences, with increasing predation of amphipods by beetle larvae. Overall, stygofauna showed a tendency towards opportunistic and omnivorous habits-typical of an ecologically tolerant community-shaped by bottom-up controls linked with changes in carbon flows. This study provides baseline biochemical and ecological data for stygofaunal trophic interactions in calcretes. Further studies on the carbon inputs and taxa-specific physiology will help refine the interpretation of the energy flows shaping biodiversity in groundwaters. This will aid understanding of groundwater ecosystem functioning and allow modelling of the impact of future climate change factors such as aridification.

New light in the dark - a proposed multidisciplinary framework for studying functional ecology of groundwater fauna.

Groundwaters provide the vast majority of unfrozen freshwater resources on the planet, but our knowledge of subsurface ecosystems is surprisingly limited. Stygofauna, or stygobionts -subterranean obligate aquatic animals - provide ecosystem services such as grazing biofilms and maintaining water quality, but we know little about how their ecosystems function. The cryptic nature of groundwaters, together with the high degree of local endemism and stygofaunal site-specific adaptations, represent major obstacles for the field. To overcome these challenges, and integrate biodiversity and ecosystem function, requires a holistic design drawing on classical ecology, taxonomy, molecular ecology and geochemistry. This study presents an approach based on the integration of existing concepts in groundwater ecology with three more novel scientific techniques: compound specific stable isotope analysis (CSIA) of amino acids, radiocarbon analysis (14C) and DNA analyses of environmental samples, stygofauna and gut contents. The combination of these techniques allows elucidation of aspects of ecosystem function that are often obscured in small invertebrates and cryptic systems. Carbon (δ13C) and nitrogen (δ15N) CSIA provides a linkage between biogeochemical patterns and ecological dynamics. It allows the identification of stygofaunal food web structures and energy flows based on the metabolic pathway of specific amino groups. Concurrently, 14C provides complementary data on the carbon recycling and incorporation within the stygobiotic trophic webs. Changes in groundwater environmental conditions (e.g. aquifer recharge), and subsequent community adaptations, can be pinpointed via the measurementof the radiocarbon fingerprint of water, sediment and specimens. DNA analyses are a rapidly expanding approach in ecology. eDNA is mainly employed as a biomonitoring tool, while metabarcoding of individuals and/or gut contents provides insight into diet regimes. In all cases, the application of the approaches in combination provides more powerful data than any one alone. By combining quantitative (CSIA and 14C) and qualitative (eDNA and DNA metabarcoding) approaches via Bayesian Mixing Models (BMM), linkages can be made between community composition, energy and nutrient sources in the system, and trophic function. This suggested multidisciplinary design will contribute to a more thorough comprehension of the biogeochemical and ecological patterns within these undervalued but essential ecosystems.