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.

Exploring virus-host-environment interactions in a chemotrophic-based underground estuary.

BACKGROUND: Viruses play important roles in modulating microbial communities and influencing global biogeochemistry. There is now growing interest in characterising their ecological roles across diverse biomes. However, little is known about viral ecology in low-nutrient, chemotrophic-based environments. In such ecosystems, virus-driven manipulation of nutrient cycles might have profound impacts across trophic levels. In particular, anchialine environments, which are low-energy underground estuaries sustained by chemotrophic processes, represent ideal model systems to study novel virus-host-environment interactions. RESULTS: Here, we employ metagenomic sequencing to investigate the viral community in Bundera Sinkhole, an anchialine ecosystem rich in endemic species supported by microbial chemosynthesis. We find that the viruses are highly novel, with less than 2% representing described viruses, and are hugely abundant, making up as much as 12% of microbial intracellular DNA. These highly abundant viruses largely infect important prokaryotic taxa that drive key metabolic processes in the sinkhole. Further, the abundance of viral auxiliary metabolic genes (AMGs) involved in nucleotide and protein synthesis was strongly correlated with declines in environmental phosphate and sulphate concentrations. These AMGs encoded key enzymes needed to produce sulphur-containing amino acids, and phosphorus metabolic enzymes involved in purine and pyrimidine nucleotide synthesis. We hypothesise that this correlation is either due to selection of these AMGs under low phosphate and sulphate concentrations, highlighting the dynamic interactions between viruses, their hosts, and the environment; or, that these AMGs are driving increased viral nucleotide and protein synthesis via manipulation of host phosphorus and sulphur metabolism, consequently driving nutrient depletion in the surrounding water. CONCLUSION: This study represents the first metagenomic investigation of viruses in anchialine ecosystems, and provides new hypotheses and insights into virus-host-environment interactions in such 'dark', low-energy environments. This is particularly important since anchialine ecosystems are characterised by diverse endemic species, both in their microbial and faunal assemblages, which are primarily supported by microbial chemosynthesis. Thus, virus-host-environment interactions could have profound effects cascading through all trophic levels.

Evolution of endoglucanase genes in subterranean and surface isopod crustaceans from Central Western Australia.

Recent studies have identified a significant number of endogenous cellulase genes in various arthropods, including isopods, allowing them to process hydrocarbons efficiently as a food source. While this research has provided insight into underlying gene-level processes in cellulose decomposition by arthropods, little is known about the existence and expression of cellulase genes in species from cave environments where carbohydrates are sparse. To investigate whether endogenous cellulase genes are maintained in subterranean species, we sequenced the transcriptomes of two subterranean paraplatyarthrid isopod species from calcrete (carbonate) aquifers of central Western Australia and a related surface isopod species. Seven protein-coding open-reading frames associated with endoglucanase genes were identified in all species. Orthology inference analyses, using a wide range of cellulase sequences from available databases, supported the endogenous origin of the putative endoglucanase genes. Selection analyses revealed that these genes are primarily subject to purifying selection in most of the sites for both surface and subterranean isopod species, indicating that they are likely to encode functional peptides. Furthermore, evolutionary branch models supported the hypothesis of an adaptive shift in selective pressure acting on the subterranean lineages compared with the ancestral lineage and surface species. Branch-site models also revealed a few amino acid sites on the subterranean branches to be under positive selection, suggesting the acquisition of novel adaptations to the subterranean environments. These findings also imply that hydrocarbons exist in subsurface aquifers, albeit at reduced levels, and have been utilized by subterranean isopods as a source of energy for millions of years.

Sensitivity of northwest Australian tropical cyclone activity to ITCZ migration since 500 CE.

Tropical cyclones (TCs) regularly form in association with the intertropical convergence zone (ITCZ), and thus, its positioning has implications for global TC activity. While the poleward extent of the ITCZ has varied markedly over past centuries, the sensitivity with which TCs responded remains poorly understood from the proxy record, particularly in the Southern Hemisphere. Here, we present a high-resolution, composite stalagmite record of ITCZ migrations over tropical Australia for the past 1500 years. When integrated with a TC reconstruction from the Australian subtropics, this time series, along with downscaled climate model simulations, provides an unprecedented examination of the dependence of subtropical TC activity on meridional shifts in the ITCZ. TCs tracked the ITCZ at multidecadal to centennial scales, with a more southward position enhancing TC-derived rainfall in the subtropics. TCs may play an increasingly important role in Western Australia's moisture budgets as subtropical aridity increases due to anthropogenic warming.

Differential transcriptomic responses to heat stress in surface and subterranean diving beetles.

Subterranean habitats are generally very stable environments, and as such evolutionary transitions of organisms from surface to subterranean lifestyles may cause considerable shifts in physiology, particularly with respect to thermal tolerance. In this study we compared responses to heat shock at the molecular level in a geographically widespread, surface-dwelling water beetle to a congeneric subterranean species restricted to a single aquifer (Dytiscidae: Hydroporinae). The obligate subterranean beetle Paroster macrosturtensis is known to have a lower thermal tolerance compared to surface lineages (CTmax 38 °C cf. 42-46 °C), but the genetic basis of this physiological difference has not been characterized. We experimentally manipulated the thermal environment of 24 individuals to demonstrate that both species can mount a heat shock response at high temperatures (35 °C), as determined by comparative transcriptomics. However, genes involved in these responses differ between species and a far greater number were differentially expressed in the surface taxon, suggesting it can mount a more robust heat shock response; these data may underpin its higher thermal tolerance compared to subterranean relatives. In contrast, the subterranean species examined not only differentially expressed fewer genes in response to increasing temperatures, but also in the presence of the experimental setup employed here alone. Our results suggest P. macrosturtensis may be comparatively poorly equipped to respond to both thermally induced stress and environmental disturbances more broadly. The molecular findings presented here have conservation implications for P. macrosturtensis and contribute to a growing narrative concerning weakened thermal tolerances in obligate subterranean organisms at the molecular level.

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.

Parallel decay of vision genes in subterranean water beetles.

In the framework of neutral theory of molecular evolution, genes specific to the development and function of eyes in subterranean animals living in permanent darkness are expected to evolve by relaxed selection, ultimately becoming pseudogenes. However, definitive empirical evidence for the role of neutral processes in the loss of vision over evolutionary time remains controversial. In previous studies, we characterized an assemblage of independently-evolved water beetle (Dytiscidae) species from a subterranean archipelago in Western Australia, where parallel vision and eye loss have occurred. Using a combination of transcriptomics and exon capture, we present evidence of parallel coding sequence decay, resulting from the accumulation of frameshift mutations and premature stop codons, in eight phototransduction genes (arrestins, opsins, ninaC and transient receptor potential channel genes) in 32 subterranean species in contrast to surface species, where these genes have open reading frames. Our results provide strong evidence to support neutral evolutionary processes as a major contributing factor to the loss of phototransduction genes in subterranean animals, with the ultimate fate being the irreversible loss of a light detection system.

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.

Extreme genetic diversity among springtails (Collembola) in subterranean calcretes of arid Australia.

The subterranean islands hypothesis for calcretes of the Yilgarn region in Western Australia applies to many stygobitic (subterranean-aquatic) species that are "trapped" evolutionarily within isolated aquifers due to their aquatic lifestyles. In contrast, little is known about the distribution of terrestrial-subterranean invertebrates associated with the calcretes. We used subterranean Collembola from the Yilgarn calcretes to test the hypothesis that troglobitic species, those inhabiting the subterranean unsaturated (non-aquatic) zone of calcretes, are also restricted in their distribution and represent reciprocally monophyletic and endemic lineages. We used the barcoding fragment of the mtDNA cytochrome c oxidase subunit 1 (COI) gene from 183 individuals to reconstruct the phylogenetic history of the genus Pseudosinella Schäffer (Collembola, Lepidocyrtidae) from 10 calcretes in the Yilgarn. These calcretes represent less than 5% of the total possible calcretes in this region, yet we show that their diversity for subterranean Collembola comprises a minimum of 25 new species. Regionally, multiple levels of diversity exist in Pseudosinella, indicative of a complex evolutionary history for this genus in the Yilgarn. These species have probably been impacted by climatic oscillations, facilitating their dispersal across the landscape. The results represent a small proportion of the undiscovered diversity in Australia's arid zone.

Evidence for speciation underground in diving beetles (Dytiscidae) from a subterranean archipelago.

Most subterranean animals are assumed to have evolved from surface ancestors following colonization of a cave system; however, very few studies have raised the possibility of "subterranean speciation" in underground habitats (i.e., obligate cave-dwelling organisms [troglobionts] descended from troglobiotic ancestors). Numerous endemic subterranean diving beetle species from spatially discrete calcrete aquifers in Western Australia (stygobionts) have evolved independently from surface ancestors; however, several cases of sympatric sister species raise the possibility of subterranean speciation. We tested this hypothesis using vision (phototransduction) genes that are evolving under neutral processes in subterranean species and purifying selection in surface species. Using sequence data from 32 subterranean and five surface species in the genus Paroster (Dytiscidae), we identified deleterious mutations in long wavelength opsin (lwop), arrestin 1 (arr1), and arrestin 2 (arr2) shared by a sympatric sister-species triplet, arr1 shared by a sympatric sister-species pair, and lwop and arr2 shared among closely related species in adjacent calcrete aquifers. In all cases, a common ancestor possessed the function-altering mutations, implying they were already adapted to aphotic environments. Our study represents one of the first confirmed cases of subterranean speciation in cave insects. The assessment of genes undergoing pseudogenization provides a novel way of testing modes of speciation and the history of diversification in blind cave animals.

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.

Scratching the surface of subterranean biodiversity: Molecular analysis reveals a diverse and previously unknown fauna of Parabathynellidae (Crustacea: Bathynellacea) from the Pilbara, Western Australia.

Like other crustacean families, the Parabathynellidae is a poorly studied subterranean and aquatic (stygobiontic) group in Australia, with many regions of available habitat having not yet been surveyed. Here we used a combined approach of molecular species delimitation methods, applied to mitochondrial and nuclear genetic data, to identify putative new species from material obtained from remote subterranean habitats in the Pilbara region of Western Australia. Based on collections from these new localities, we delineated a minimum of eight and up to 24 putative new species using a consensus from a range of molecular delineation methods and additional evidence. When we placed our new putative species into the broader phylogenetic framework of Australian Parabathynellidae, they grouped with two known genera and also within one new and distinct Pilbara-only clade. These new species significantly expand the known diversity of Parabathynellidae in that they represent a 22% increase to the 109 currently recognised species globally. Our investigations showed that sampling at new localities can yield extraordinary levels of new species diversity, with the majority of species showing likely restricted endemic geographical ranges. These findings represent only a small sample from a region comprising less than 2.5% of the Australian continent.

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.

Too hot to handle: Cenozoic aridification drives multiple independent incursions of Schizomida (Hubbardiidae) into hypogean environments.

The formation of the Australian arid zone, Australia's largest and youngest major biome, has been recognized as a major driver of rapid evolutionary radiations in terrestrial plants and animals. Here, we investigate the phylogenetic diversity and evolutionary history of subterranean short-tailed whip scorpions (Schizomida: Hubbardiidae), which are a significant faunal component of Western Australian hypogean ecosystems. We sequenced two mitochondrial (12S, COI) and three nuclear DNA markers (18S, 28S, ITS2) from ∼600 specimens, largely from the genera Draculoides and Paradraculoides, including 20 previously named species and an additional 56 newly identified operational taxonomic units (OTUs). Phylogenetic analyses revealed a large and rapid species radiation congruent with Cenozoic aridification of the continent, in addition to the identification of a new genus in Western Australia and the first epigean schizomid from the Pilbara. Here, we also synonymise Paradraculoides with Draculoides (new synonymy), due to paraphyly and a lack of reliable characters to define the two genera. Our results are consistent with multiple colonisations of the subterranean realm from epigean ancestors as their forest habitat fragmented and retracted, with ongoing fragmentation and diversification of lineages underground. These findings illustrate the remarkable diversity and high incidence of short-range endemism of Western Australia's subterranean fauna, which has important implications for identifying and managing short-range endemic subterranean fauna. They also highlight the advantages of including molecular data in subterranean fauna surveys as all specimens can be utilized, regardless of sex and life stage. Additionally, we have provided the first multi-gene phylogenetic framework for Australian schizomids, which will enable researchers and environmental consultants to identify new taxa or align them to existing lineages.

Shedding light on the hidden world of subterranean fauna: A transdisciplinary research approach.

Subterranean environments contain a diverse and unique obligate fauna: either aquatic living in the groundwater or terrestrial living in voids above the water table. In the arid region of the western part of the Australian continent, a particularly rich subterranean fauna coincides with a concentration of natural resource extraction operations. Since the inclusion of subterranean fauna in assessments of environmental impact in the mid-1990s, taxonomic research in Australia on this group of mainly invertebrates has grown exponentially. However, remaining knowledge gaps continue to frustrate both environmental regulators and development proponents due to high uncertainty in the decision-making process. In early 2017, the Western Australian Biodiversity Science Institute was tasked with leading the development of a research program to improve on the current state of knowledge of subterranean fauna. To balance the diverse environmental, economic and social needs of a range of stakeholders, transdisciplinary principles were applied to program development. A clear consensus on five broad focus areas to progress include: (1) data consolidation; (2) resilience to disturbance; (3) survey and sampling protocols; (4) abiotic and biotic habitat requirements; and (5) species delineation. In the context of these focus areas; we describe the research program development, reviewing the status of knowledge within each focus area, and the research initiatives to close the gaps in knowledge. We argue that, by adopting a transdisciplinary approach, the likelihood of success of the research program, as measured by the effective translation and adoption of research findings, will be maximized. This review is timely given the ever-increasing demand on groundwater systems for water extraction worldwide. A holistic understanding of the influence of anthropogenic activities on these ecosystems, and the functional role of organisms within them, will help to ensure that their health is not compromised.

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.

Sieve-type pore canals in the Timiriaseviinae-A contribution to the comparative morphology and the systematics of the Limnocytheridae (Ostracoda, Crustacea).

Examination of normal pore canals, especially sieve-type pore canals, in living and fossil representatives of ten genera of the family Limnocytheridae, subfamily Timiriaseviinae, has revealed important diversity of structure. These complex pore canals have been studied via high-resolution scanning electron microscopy (the Cartographic Method) and analysed via the application of newly devised indices to assess patterns of consistency and variation in both detailed structure of individual pores and of their distribution on the calcified valve. The timiriaseviine taxa are compared with species of the genera Limnocythere, sub-family Limnocytherinae and Cyprideis (family Cytherideidae). The relationship between the living animal and its aquatic environment is discussed in the light of previous studies and of new evidence herein. The importance of normal pore canals for systematics is highlighted by the recognition and definition of the new tribe Gomphodellini Danielopol, Cabral Lord nov. tribe, subfamily Timiriaseviinae, family Limnocytheridae.