Getting to the Root of Organic Inputs in Groundwaters: Stygofaunal Plant Consumption in a Calcrete Aquifer

Abstract

Groundwater environments interact with and support subterranean biota as well as superficial aquatic and terrestrial ecosystems. However, knowledge of subterranean energy flows remains incomplete. Cross-boundary investigations are needed to better understand the trophic structures of groundwater ecosystems and their reliance on carbon inputs from aboveground. In this study we used carbon and nitrogen stable isotope analyses combined with radiocarbon fingerprints to characterise organic flows in groundwater ecosystems. We coupled these data with DNA metabarcoding of the gut contents of stygofauna to further elucidate organic matter (OM) sources and shifts in diet preferences. Samples were collected from the arid zone Sturt Meadows calcrete aquifer under low rainfall (LR) and high rainfall (HR) conditions. Bayesian modelling of Δ14C, δ13C, and δ15N data indicated that primary consumers (copepods) incorporated mainly particulate organic carbon (POC) under LR but during HR shifted to root derived material (either exudates or direct root grazing). By contrast, diets of secondary consumers (amphipods) were dominated by root material under both LR and HR. Our DNA metabarcoding-based results indicate that amphipods relied primarily on root inputs from perennial trees (likely Eucalyptus and Callitris) during the dry season (LR). Under HR, diets of both amphipods and copepods also included organic material derived from a broad range of more shallow rooted shrubs, and ephemeral herbs and grasses. Our findings illustrate the complexity of functional linkages between groundwater biota and surface terrestrial ecosystems in environments where aboveground productivity, diversity and OM flux to groundwater are intimately linked to often episodic rainfall.

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

“…As such, it is intended as the first step in developing a robust protocol for effective eDNA‐based stygofaunal detection and monitoring. Environmental impact assessment and monitoring for subterranean fauna is currently conducted using traditional approaches such as haul‐net sampling, but, as with most sampling protocols, these methods have inherent biases (Saccò, Campbell, et al, 2022; Saccò, Guzik, et al, 2022). We show that broad eukaryote 18S metabarcoding assays were more successful than haul‐net sampling in detecting the breadth of major stygofaunal taxon groups known from groundwater on Barrow Island, but that key taxa were notably absent from metabarcoding results compared to those collected using haul nets and identified morphologically.…”

“…In particular, they feed on protozoan and metazoan prey and bacterial biomass which contribute to groundwater purification and nutrient cycling, important for ecosystem functioning (Cunillera‐Montcusí et al, 2022; Herrmann et al, 2020). Platyhelminthes (Catenulida) and copepods (e.g., Cyclopoida) are considered high‐level predators in some groundwater systems (Herrmann et al, 2020), although recent studies suggest they may also be feeding on roots (Saccò, Campbell, et al, 2022) and bacteria (Saccò et al, 2021). These organisms are themselves preyed on by stygobiont teleosts, if present (Humphreys & Feinbergz, 1995), and are important for the food web structure and trophic dynamics of groundwater systems.…”

“…Whole specimens used to assess a baseline for morphological species diversity were collected using haul‐nets (similar to plankton‐nets—see Saccò, Campbell, et al, 2022; Saccò, Guzik, et al, 2022), which were 45 or 95 mm in diameter, depending on the size of the PVC casing. Five (Allford et al, 2008) to six (EPA guidance) haul‐net samples were taken at each bore, half with 150‐μm mesh and half with 50‐μm mesh, alternating mesh size between hauls.…”

“…Improvements have already been observed; recent studies have successfully used single‐species assays to detect rare and endangered subterranean species (Boyd et al, 2020; Gorički et al, 2017; Niemiller, Porter, et al, 2018; Vörös et al, 2017) and investigate genetic differences (haplotypes) between populations (White et al, 2020). Additionally, Saccò, Blyth, Humphreys, Karasiewicz, et al (2020), Saccò, Guzik, et al, 2022) have highlighted the potential value of eDNA for community‐level detection of subterranean fauna and their diets (Korbel et al, 2017; Saccò, Campbell, et al, 2022; West et al, 2020) in a large range of habitats and for all life stages, thereby avoiding the need for: (i) specialized taxonomic expertise (Bohmann et al, 2014; Thomsen & Willerslev, 2015); and (ii) destructive sampling for identification. However, before an eDNA metabarcoding approach can be used as a standard method for monitoring and assessment of groundwater communities, it is essential that the appropriate testing and development of the methods are undertaken to assess the efficacy, accuracy and speed of species detection.…”

Groundwater is a hidden global keystone ecosystem

“…Since then, some evidence for trophic specialization within trophic levels has been identified (Ercoli et al, 2019;Francois et al, 2016Francois et al, , 2020Saccò, Blyth, Humphreys, Karasiewicz, et al, 2020;, including the discovery of autotrophic systems based on chemoautotrophic bacteria that serve as primary producers (Sarbu et al, 1996). These aspects, together with existing multiple trophic levels within species-rich groundwater communities (Hutchins et al, 2016;Premate et al, 2021;Saccò, Blyth, Humphreys, et al, 2019;Saccò, Blyth, Humphreys, Cooper, et al, 2020) and the frequent dependency on surface carbon sources in biodiverse shallow groundwater ecosystems Saccò, Campbell, et al, 2022;Simon et al, 2003), make it difficult to identify suitable keystone species in most cases.…”