Diet of a reintroduced marsupial 757
Table 1), as well as the bait used in traps (apple, peanut butter and oats). Leaf samples were freeze-dried for 2 days prior to analysis. The dual locus barcoding approach of Wilkinson et al. (2017) was used to develop the local refer- ence library for the rbcL (Kress & Erickson, 2007) and ndhJ (Schmitz-Linneweber et al., 2001) barcodes and sequenced on the MiSeq platform (Illumina, San Diego, USA) at the University of Adelaide.
Scat analysis: plants
FIG. 1 Sites where brushtail possum Trichosurus vulpecula scats were collected within Ikara-Flinders Ranges National Park (I-FRNP), the known area of occupancy (95 and 100% minimum convex polygons) for possums during the study period, and the location of the National Park within South Australia.
Plant samples were also collected for a DNA reference library. The availability of perennial species was averaged across the year as differences betweenmonthswere probably a result of the varied locations of survey sites. The per cent of canopy that was new growth was not accounted for. The availability of annuals was calculated monthly. Where therewas any doubt about the life history of a species, it was considered annual. Because the vegetation survey method used resulted in recording eucalypts (used as shelter sites) in all surveys, the availability of eucalypts was assessed using transect data from another study conducted simulta- neously (Moseby et al., 2020); the proportion of belt transect segments (100 m long, 20 m wide, location randomly selected, stratified by habitat type) containing eucalypts was calculated, based on 27.5 km worth of transects surveyed within possum habitat.
Local plant DNA-barcode reference library
We developed a plant DNA reference library for the study site, which included 165 plant species (Supplementary
We used amodification of the two-stepPCRstrategy described by Bell (2011) for the amplification of rbcL and ndhJ bar- codes from scat samples. Scats (2–3 per sample) were freeze- dried and homogenized with tungsten-carbide beads in a TissueLyser (Qiagen, Melbourne, Australia) and DNA was extracted using an ISOLATE II Plant DNA Kit (Bioline, London, UK) according to the manufacturer’s instructions. The first amplification was performed in 20 μl reaction volume consisting of 1 × MyFi Buffer (Bioline, Sydney, Australia), 0.2 nM of each forward and reverse primer, 1.6U MyFi Polymerase (Bioline) and 20 ng of DNA. PCRs were performed on a RotorGene RG-6000 machine (Corbett Life Science, Melbourne, Australia) using the following thermocycling conditions: for rbcL, 95 °C for 1 minute fol- lowed by 35 cycles of 95 °C for 15 seconds, 55 °C for 15 sec- onds, 72 °C for 15 seconds; and for ndhJ, 95 °C for 1 minute followed by 35 cycles of 95 °C for 15 seconds, 50 °C for 15 seconds, 72 °C for 15
seconds.Amplification products were then purified using the Agencourt AMPure XP system (Beckman Coulter, Sydney, Australia) at a ratio of 0.8× beads to PCR product. The second PCR was performed using Nextera 96 index
adapter sequences (Illumina, San Diego, USA) to add iden- tifying sequences to the amplification products from the first PCR. This was achieved by adding the following into a 12.5 μL reaction volume: 1 × MyFi Buffer (Bioline), 1.6 U MyFi Polymerase (Bioline), 0.4 nM of paired Nextera 96 Index Sequences and 4 μL of purified PCR product. The amplification conditions consisted of 95 °C for 1 minute followed by 5 cycles of 95 °C for 5 seconds, 55 °C for 10 seconds and 72 °C for 10 seconds. Amplified products were then performed using the Agencourt AMPure XP PCR Purification beads at a ratio of 0.6 × beads to PCR product and quantified by qPCR with reference to known PhiX standards(Illumina)using theSYBRFASTqPCRKit (Kapa Biosystems, Wilmington, USA) on a RotorGene RG-6000 machine (Corbett Life Science, Melbourne, Australia). The pooled library was then diluted and a 16pM aliquot
was paired-end sequenced on a MiSeq V3 sequencer, using a 600-cycle Version 3 kit (Illumina) according to the man- ufacturer’s instructions. The MiSeq Bcl output files were demultiplexed and converted to fastq files using MiSeq Reporter 2.6 (Illumina).
Oryx, 2021, 55(5), 755–764 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International doi:10.1017/S0030605319000991
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