DOI: 10.5061/dryad.d51c5b00s
title: Under the karst: detecting hidden subterranean assemblages using eDNA metabarcoding in the caves of Christmas Island, Australia

description: Subterranean ecosystems are understudied and challenging to conventionally
 survey given the inaccessibility of underground voids and networks. In
 this study, we conducted a eukaryotic environmental (eDNA) metabarcoding
 survey across the karst landscape of Christmas Island, (Indian Ocean,
 Australia) to evaluate the utility of this non-invasive technique to
 detect subterranean aquatic ‘stygofauna’ assemblages. Three metabarcoding
 assays targeting the mitochondrial 16S rRNA and nuclear 18S genes were
 applied to 159 water and sediment samples collected from 23 caves and
 springs across the island. Taken together, our assays detected a wide
 diversity of chordates, cnidarians, porifera, arthropods, molluscs,
 annelids and bryozoans from 71 families across 60 orders. We report a high
 level of variation between cave and spring subterranean community
 compositions which are significantly influenced by varying levels of
 salinity. Additionally, we show that dissolved oxygen and longitudinal
 gradients significantly affect biotic assemblages within cave communities.
 Lastly, we combined eDNA-derived community composition and environmental
 (water quality) data to predict potential underground interconnectivity
 across Christmas Island. We identified three cave and spring groups that
 showed a high degree of biotic and abiotic similarity indicating likely
 local connectivity. This study demonstrates the applicability of eDNA
 metabarcoding to detect subterranean eukaryotic communities and explore
 underground interconnectivity.
description: Six one-litre water replicates and one 50-ml sediment sample were
 collected from 23 cave and spring sites across Christmas Island in October
 2018, totaling 159 samples across a 110km<sup>2</sup> area.
 DNA was extracted from half of each filter membrane and 250 mg of each
 sediment sample using a DNeasy PowerLyzer PowerSoil Kit (Qiagen; Venlo,
 Netherlands) following the manufacturer’s instructions. DNA was amplified
 using three previously published PCR assays (16S and 18S) to largely
 target bony fish, molluscs and arthropods (such as crustaceans
 and insects) from our mixed environmental samples (see Scientific Reports
 publication for more information).  Libraries were sequenced on
 either a 300 cycle (for unidirectional sequencing of the 16S amplicons) or
 500 cycle (for paired-end sequencing of the 18S amplicons)
 MiSeq® V2 Standard Flow Cell on an
 Illumina MiSeq platform (Illumina, San Diego, USA), housed in the TrEnD
 Laboratory at Curtin University, Western
 Australia. Unidirectional and unmerged
 paired-end sequencing reads were demultiplexed using OBITools
 (v1.2.9) and the insect
 package in RStudio
 (v1.1.423), respectively.
description: We have uploaded demultiplexed (unfiltered) data for public use.
 This is in a fastq format and corresponds to sample IDs (see Scientific
 Reports publication for more information). We have also uploaded a
 taxonomic (read abundance) matrix that has gone through our quality
 filtering (DADA2) pipeline and has been blasted against NCBI's
 GenBank (2019). This can be directly used for multivariate statistical
 analyses.