The introduction of non-native species into new environments can cause significant ecological harm, and is considered a major conservation threat. As populations of invasive species continue to establish and increase across the globe, novel methods can provide new insights into their biology, and potentially aid in management. In this study, we examined the diet of non-native chital deer (Axis axis) in a tropical savanna environment in northern Australia. Using DNA metabarcoding of faecal samples, we described the dietary items consumed by 149 individuals over a two-year sampling period and associated each item with individual body condition. The DNA metabarcoding method detected significantly more dietary items consumed by individual chital deer at each of the taxonomic levels (family, genus, and species) when compared with previous analyses. We observed marked differences in diet composition across multiple seasons and sites. Significantly more sequences from the genera Terminalia, Diospyros, Jasminum, and Hakea were detected in samples collected from individuals in poor condition during the dry season, suggesting that a different suite of food resources is being consumed by a subset of individuals during periods when forage quantity and quality is low. Most notably, our results indicated that chital are consuming a browse-dominated diet throughout the year, differing from previous macroscopy analyses which suggested chital are predominantly grazers during the wet season in northern Australia. Our findings give support for the use of DNA metabarcoding to qualitatively assess diet composition compared to macroscopic analysis and suggest that the restricted availability of food during the dry season may result in the consumption of poor quality and detrimental dietary items.

This study was conducted across two cattle stations in the Einasleigh Uplands bioregion of northern Australia, approximately 110 km north of Charters Towers (20° 4' 35.1'' S, 146° 15' 24.6'' E). Faecal samples were collected from both stations during sampling programs in the October 2014 and 2015 dry seasons, and the March 2015 and 2016 wet seasons. A total of 67 males and 85 females were necropsied across each of the four sampling events. For each of the sampled deer, faecal samples were removed from the rectum, placed in separate field collection bags, and frozen at -4 °C. Samples were removed directly from culled individuals, thereby reducing the likelihood of contamination by pollen, seeds, or plant material not consumed by deer.

Faecal samples for the DNA extraction protocol were prepared by homogenising the collected pellets from each individual deer within their separate field collection bags. 200 mg from each homogenised faecal sample was transferred into a 2 mL microcentrifuge tube, and DNA extractions were performed using a QIAmp Fast DNA Stool Mini Kit (Qiagen), according to the manufacturer’s instructions. All extractions were undertaken in a Polymerase Chain Reaction (PCR) free room, treated with 15 μL of Proteinase K, and eluted in a final volume of 200 μL of Buffer ATE. Throughout the process, six extractions containing no faecal material were performed to monitor potential contamination and serve as negative controls. DNA extraction concentrations were quantified with a NanoDrop Spectrophotometer and stored at 4 °C.

An established primer pair for metabarcoding plants was used to amplify segments of the chloroplast trnL intron P6 loop gene region (Taberlet et al. 2007). An Illumina nextera adapter sequence followed by the trnL gene specific primer sequence was used in the first round of PCR. Each PCR mixture comprised 15.5 μL of nuclease-free H₂O, 0.5 μL of MyTaq™ HS DNA Polymerase (Bioline), 5 μL of 5x MyTaq Reaction Buffer (containing dNTPs, MgCl₂, and enhancers at optimal concentrations), 0.5 μL of each forward and reverse primer (10 μM), and 3 μL of DNA template for a 25 μL reaction. The PCR conditions contained an initial denaturation step at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 10 s, followed by a final extension step at 72°C for 2 min. In cases where samples failed to amplify, DNA samples were diluted at 1/10 and 1/100 rates with nuclease-free H₂O until amplification was successful. PCRs included both negative extraction and nuclease-free H₂O controls to test for contamination. 162 PCR products were sent to the Australian Genome Research Facility (AGRF), where amplicons underwent final purification, indexing, and normalisation steps to ensure equal DNA concentrations across all samples. Sequencing of amplicons was performed using a MiSeq reagent V3 kit (150 cycles) on an Illumina Miseq High-Throughput sequencer.