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.

Detection of vertebrates from natural and artificial inland water bodies in a semi-arid habitat using eDNA from filtered, swept, and sediment samples.

Biomonitoring is vital for establishing baseline data that is needed to identify and quantify ecological change and to inform management and conservation activities. However, biomonitoring and biodiversity assessment in arid environments, which are predicted to cover 56% of the Earth's land surface by 2100, can be prohibitively time consuming, expensive, and logistically challenging due to their often remote and inhospitable nature. Sampling of environmental DNA (eDNA) coupled with high-throughput sequencing is an emerging biodiversity assessment method. Here we explore the application of eDNA metabarcoding and various sampling approaches to estimate vertebrate richness and assemblage at human-constructed and natural water sources in a semi-arid region of Western Australia. Three sampling methods: sediment samples, filtering through a membrane with a pump, and membrane sweeping in the water body, were compared using two eDNA metabarcoding assays, 12S-V5 and 16smam, for 120 eDNA samples collected from four gnammas (gnamma: Australian Indigenous Noongar language term-granite rock pools) and four cattle troughs in the Great Western Woodlands, Western Australia. We detected higher vertebrate richness in samples from cattle troughs and found differences between assemblages detected in gnammas (more birds and amphibians) and cattle troughs (more mammals, including feral taxa). Total vertebrate richness was not different between swept and filtered samples, but all sampling methods yielded different assemblages. Our findings indicate that eDNA surveys in arid lands will benefit from collecting multiple samples at multiple water sources to avoid underestimating vertebrate richness. The high concentration of eDNA in small, isolated water bodies permits the use of sweep sampling that simplifies sample collection, processing, and storage, particularly when assessing vertebrate biodiversity across large spatial scales.

Taking eDNA underground: Factors affecting eDNA detection of subterranean fauna in groundwater.

Stygofauna are aquatic fauna that have evolved to live underground. The impacts of anthropogenic climate change, extraction and pollution on groundwater pose major threats to groundwater health, prompting the need for efficient and reliable means to detect and monitor stygofaunal communities. Conventional survey techniques for these species rely on morphological identification and can be biased, labour-intensive and often indeterminate to lower taxonomic levels. By contrast, environmental DNA (eDNA)-based methods have the potential to dramatically improve on existing stygofaunal survey methods in a large range of habitats and for all life stages, reducing the need for the destructive manual collection of often critically endangered species or for specialized taxonomic expertise. We compared eDNA and haul-net samples collected in 2020 and 2021 from 19 groundwater bores and a cave on Barrow Island, northwest Western Australia, and assessed how sampling factors influenced the quality of eDNA detection of stygofauna. The two detection methods were complementary; eDNA metabarcoding was able to detect soft-bodied taxa and fish often missed by nets, but only detected seven of the nine stygofaunal crustacean orders identified from haul-net specimens. Our results also indicated that eDNA metabarcoding could detect 54%-100% of stygofauna from shallow-water samples and 82%-90% from sediment samples. However, there was significant variation in stygofaunal diversity between sample years and sampling types. The findings of this study demonstrate that haul-net sampling has a tendency to underestimate stygofaunal diversity and that eDNA metabarcoding of groundwater can substantially improve the efficiency of stygofaunal surveys.

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.

Pilbarana, a new subterranean amphipod genus (Hadzioidea: Eriopisidae) of environmental assessment importance from the Pilbara, Western Australia.

The Pilbara and nearby regions in north-western Western Australia have an exceptionally high diversity of short-range endemic invertebrates inhabiting threatened groundwater-dependent habitats. Amphipod crustaceans, in particular, are dominant in these communities, but are poorly understood taxonomically, with many undescribed species. Recent molecular phylogenetic analyses of Pilbara eriopisid amphipods have, nonetheless, uncovered a previously unknown biodiversity. In this study, we formally establish a new genus, Pilbarana Stringer & King gen. nov., and describe two new species, P. grandis Stringer & King sp. nov. from Cane River Conservation Park and P. lowryi Stringer & King sp. nov. from the Fortescue River Basin near the Hamersley Range, using a combination of molecular and morphological data. The new genus is similar morphologically to the two additional Western Australian eriopisid genera, Nedsia Barnard & Williams, 1995 and Norcapensis Bradbury & Williams, 1997, but represents a genetically divergent, reciprocally monophyletic lineage, which can be differentiated by its vermiform body shape, the presence of an antennal sinus, and by the length and form of the antennae and uropods. This research signifies an important contribution to knowledge of Pilbara subterranean communities and has critical implications for future environmental impact assessments and conservation management.

Development and evaluation of a custom bait design based on 469 single-copy protein-coding genes for exon capture of isopods (Philosciidae: Haloniscus).

Transcriptome-based exon capture approaches, along with next-generation sequencing, are allowing for the rapid and cost-effective production of extensive and informative phylogenomic datasets from non-model organisms for phylogenetics and population genetics research. These approaches generally employ a reference genome to infer the intron-exon structure of targeted loci and preferentially select longer exons. However, in the absence of an existing and well-annotated genome, we applied this exon capture method directly, without initially identifying intron-exon boundaries for bait design, to a group of highly diverse Haloniscus (Philosciidae), paraplatyarthrid and armadillid isopods, and examined the performance of our methods and bait design for phylogenetic inference. Here, we identified an isopod-specific set of single-copy protein-coding loci, and a custom bait design to capture targeted regions from 469 genes, and analysed the resulting sequence data with a mapping approach and newly-created post-processing scripts. We effectively recovered a large and informative dataset comprising both short (<100 bp) and longer (>300 bp) exons, with high uniformity in sequencing depth. We were also able to successfully capture exon data from up to 16-year-old museum specimens along with more distantly related outgroup taxa, and efficiently pool multiple samples prior to capture. Our well-resolved phylogenies highlight the overall utility of this methodological approach and custom bait design, which offer enormous potential for application to future isopod, as well as broader crustacean, molecular studies.

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.

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.

Desert springs: deep phylogeographic structure in an ancient endemic crustacean (Phreatomerus latipes).

Desert mound springs of the Great Artesian Basin in central Australia maintain an endemic fauna that have historically been considered ubiquitous throughout all of the springs. Recent studies, however, have shown that several endemic invertebrate species are genetically highly structured and contain previously unrecognised species, suggesting that individuals may be geographically 'stranded in desert islands'. Here we further tested the generality of this hypothesis by conducting genetic analyses of the obligate aquatic phreatoicid isopod Phreatomerus latipes. Phylogenetic and phylogeographic relationships amongst P. latipes individuals were examined using a multilocus approach comprising allozymes and mtDNA sequence data. From the Lake Eyre region in South Australia we collected data for 476 individuals from 69 springs for the mtDNA gene COI; in addition, allozyme electrophoresis was conducted on 331 individuals from 19 sites for 25 putative loci. Phylogenetic and population genetic analyses showed three major clades in both allozyme and mtDNA data, with a further nine mtDNA sub-clades, largely supported by the allozymes. Generally, each of these sub-clades was concordant with a traditional geographic grouping known as spring complexes. We observed a coalescent time between ∼2-15 million years ago for haplotypes within each of the nine mtDNA sub-clades, whilst an older total time to coalescence (>15 mya) was observed for the three major clades. Overall we observed that multiple layers of phylogeographic history are exemplified by Phreatomerus, suggesting that major climate events and their impact on the landscape have shaped the observed high levels of diversity and endemism. Our results show that this genus reflects a diverse fauna that existed during the early Miocene and appears to have been regionally restricted. Subsequent aridification events have led to substantial contraction of the original habitat, possibly over repeated Pleistocene ice age cycles, with P. latipes populations becoming restricted in the distribution to desert springs.

Evolution in caves: Darwin's 'wrecks of ancient life' in the molecular era.

Cave animals have historically attracted the attention of evolutionary biologists because of their bizarre 'regressive' characters and convergent evolution. However, understanding of their biogeographic and evolutionary history, including mechanisms of speciation, has remained elusive. In the last decade, molecular data have been obtained for subterranean taxa and their surface relatives, which have allowed some of the classical debates on the evolution of cave fauna to be revisited. Here, we review some of the major studies, focusing on the contribution of phylogeography in the following areas: biogeographic history and the relative roles of dispersal and vicariance, colonization history, cryptic species diversity and modes of speciation of cave animals. We further consider the limitations of current research and prospects for the future. Phylogeographic studies have confirmed that cave species are often cryptic, with highly restricted distributions, but have also shown that their divergence and potential speciation may occur despite the presence of gene flow from surface populations. Significantly, phylogeographic studies have provided evidence for speciation and adaptive evolution within the confines of cave environments, questioning the assumption that cave species evolved directly from surface ancestors. Recent technical developments involving 'next generation' DNA sequencing and theoretical developments in coalescent and population modelling are likely to revolutionize the field further, particularly in the study of speciation and the genetic basis of adaptation and convergent evolution within subterranean habitats. In summary, phylogeographic studies have provided an unprecedented insight into the evolution of these unique fauna, and the future of the field should be inspiring and data rich.

Molecular phylogeny of the benthic shallow-water octopuses (Cephalopoda: Octopodinae).

Octopus has been regarded as a "catch all" genus, yet its monophyly is questionable and has been untested. We inferred a broad-scale phylogeny of the benthic shallow-water octopuses (subfamily Octopodinae) using amino acid sequences of two mitochondrial DNA genes: Cytochrome oxidase subunit III and Cytochrome b apoenzyme, and the nuclear DNA gene Elongation Factor-1alpha. Sequence data were obtained from 26 Octopus species and from four related genera. Maximum likelihood and Bayesian approaches were implemented to estimate the phylogeny, and non-parametric bootstrapping was used to verify confidence for Bayesian topologies. Phylogenetic relationships between closely related species were generally well resolved, and groups delineated, but the genes did not resolve deep divergences well. The phylogenies indicated strongly that Octopus is not monophyletic, but several monophyletic groups were identified within the genus. It is therefore clear that octopodid systematics requires major revision.