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 consumers to further elucidate organic matter 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 terrestrial surficial ecosystems in environments where aboveground productivity, diversity and organic matter flux to groundwater are intimately linked to often episodic rainfall.

Specimens of AM1, AM2, C and H were used for gut DNA metabarcoding analysis targeting plant material (primer trnL g-h). Prior to DNA extraction, stygofaunal animals (three to five individuals per pool; n = 84) were placed in petri dishes containing ultrapure water and UV sterilized in a UV oven for 10 min to eliminate any environmental DNA species that may be contained on the exoskeleton. Details on the DNA extraction, controls, sequencing and blasting for stygofaunal gut analysis are described in detail in Saccò et al. (2021a). A minimum threshold of five OTU counts was established for the presence-absence analysis. Only taxa known to occur in the Goldfields Region of Western Australia (Florabase; Western Australian Herbarium, 1988) were considered in this study.