Investigating the internal and external ecology of six subterranean diving beetle species from the Yilgarn region of Central Australia

Abstract

The ecology of subterranean ecosystems and stygofauna (subterranean aquatic animals) has largely been unexplored in an Australian context. The Yilgarn region of Western Australia is known as a biodiversity hotspot in relation to stygofauna from isolated calcrete aquifers, and it is home to the most diverse assemblage of subterranean, predatory diving beetles in the world. This study used extensive grids of boreholes to access calcrete aquifers at Sturt Meadows and Laverton Downs pastoral stations to investigate how subterranean species interact with their external and internal environment, focusing on six subterranean beetle species. A mix of traditional ecological monitoring and next-generation sequencing methods were employed to examine the following specific questions: What are the types of prey available in these calcrete systems and how do they change in abundance over time? What are the natural gut microbial communities associated with these predatory beetle species? Moreover, can metagenomic analyses be used to identify trophic differences among species, including adults and larvae, and determine whether beetle species eat other beetle species? Ecological monitoring over an 11-year period identified that rainfall and, in particular, major recharge events are important for the diversity and distribution of stygofauna within the calcrete at Sturt Meadows. Average taxon richness was highest shortly after periods of high rainfall, and when dominant taxa (i.e. amphipods and copepods) were excluded, evenness decreased after both high and low rainfall suggesting that dominant taxa are an important factor driving the system. Common taxa (i.e. amphipods and copepods) within the calcrete had broad distributions and high abundance levels, while rare taxa (oligochaete worms) had restricted distributions and low abundances. All taxon groups had lower abundances and narrower distributions after periods of intermediate and low rainfall. Over the 11-year period, the majority of boreholes sampled did not show changes in evenness, suggesting that the Sturt Meadows calcrete is a reasonably stable ecosystem with episodic fluctuations, most likely attributed to rainfall events. The gut microbiome was investigated in six beetle species from two separate aquifers using random shotgun sequencing (metagenomic analyses). The bacterial and viral communities were investigated separately, but the investigation showed similar results as follows: In both the viral and bacterial analyses the microbial communities varied greatly by location and there was a distinct signature in the microbial communities depending on whether samples were collected from aquifers or laboratory aquaria. There were also distinct differences among the beetle species and their stage of development (adult versus larvae), which are most likely accounted for by trophic differences among the beetles. In both the bacterial and viral analyses a large number of the sequences were novel and unable to be identified, suggesting major differences in their microbiome compared to previously studied invertebrates. The unknown sequences will once identified, provide further insights into the microbial communities of these subterranean environments. Like the bacteria, viral sequences provided evidence that calcretes had been influenced by anthropogenic activities on the surface, with a large number of vertebrate viruses infiltrating the calcrete system. The mitochondrial (mt) genomes of four of the beetle species, Limbodessus palmulaoides and P. macrosturtensis, P. mesosturtensis and P. microsturtensis, were characterised to provide a framework for future trophic analyses of the beetle gut contents and as a basis for further assessment of the molecular evolution of mtDNA genes associated with evolution underground. The mt genomes were all consistent with both previously sequenced dytiscid beetle mt genomes and the inferred ancestral insect mt genome. All four mt genomes were circular, contained the expected 37 genes and ranged from 16,504 to 16,868 bp. The overall structure (gene number, orientation and order) was consistent with the ancestral insect mt genome, and the genome size variation resulting from length variation of intergenic regions and the CR is consistent with other surface dytiscid species sequenced. These ecological and molecular analyses show a complex interconnected system between the surface and subterranean environments. They also demonstrate that metagenomics research can be used effectively for investigating the trophic ecology of species, particularly in taxa where traditional methods are ineffective or difficult to undertake. Preliminary analyses of the beetle metagenomes suggested that the beetle species at Sturt Meadows are not only eating other invertebrates from the calcrete but are also eating the other beetle species. This result may have been difficult to elucidate using traditional methods (e.g. metabarcoding) given how closely related the beetle species are to each other. This research also highlights that the subterranean beetles provide an excellent model system, not only for future microbiome work but also for investigating the adaptive and regressive evolution of the genome associated with moving from surface to underground habitats.