DOI: 10.5061/dryad.gqnk98sxd
title: DNA metabarcoding provides new insight into the diet of invasive chital deer (Axis axis) in a tropical savanna landscape

description: 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.
description: 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 <em>trnL </em>intron P6
 loop gene region (Taberlet et al. 2007). An Illumina nextera adapter
 sequence followed by the <em>trnL </em>gene specific primer
 sequence was used in the first round of PCR. Each PCR mixture comprised
 15.5 μL of nuclease-free H<sub>2</sub>O, 0.5 μL of MyTaq™ HS
 DNA Polymerase (Bioline), 5 μL of 5x MyTaq Reaction Buffer (containing
 dNTPs, MgCl<sub>2</sub>, 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 H<sub>2</sub>O until
 amplification was successful. PCRs included both negative extraction and
 nuclease-free H<sub>2</sub>O 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.
description: # DNA metabarcoding provides new insight into the diet of invasive chital
 deer (Axis axis) in a tropical savanna landscape
 [https://doi.org/10.5061/dryad.gqnk98sxd](https://doi.org/10.5061/dryad.gqnk98sxd) ## Description of the data and file structure The files in this repository are for 'DNA metabarcoding provides new insight into the diet of invasive chital deer (*Axis axis*) in a tropical savanna landscape' ### Files and variables #### File: MQuin\_-*Supplementary\_Material*-\_Taxonomy\_Table.xlsx **Description:**  The taxonomy table provides the full taxonomic classification for each Amplicon Sequence Variant (ASV) at the levels of Class, Order, Family, Genus, and Species. The growth form of each classified ASV is detailed in the 'Form' column, and whether an ASV is native or exotic is detailed in the 'Endemicity' column. The sum of the sequence reads for each ASV across all samples is included in the column 'Total Sequence Reads'. Lastly, the 'Frequency of Occurrence' column provides information on the number of samples that each ASV was detected in. Missing values are indicated by blank cells.  Specifically, 'Form' descriptions are concordant with the summaries of plant shape and structure provided by the AusTraits Plant Directory (APD) Ontology (Wenk et al. 2023). A description for each categorical value for 'Form' is as follows: Tree: A tall, woody, perennial plant, usually with a single main trunk, and its leaves predominantly elevated above the ground surface. Shrub: A woody plant that is distinguished by NOT being a tree, due to one or more of the following characteristics, 1) they are less than 8 metres in height; 2) if a plant community includes trees, the shrubs are shorter than the surrounding trees and not part of the canopy; 3) in comparison to trees, the leaves are more distributed along the entire trunk; 4) the plant has multiple relatively narrow stems arising at or near ground level. Herb: A seed-bearing plant which does not have a woody above-ground stem. Climber: Plant that climbs up another plant’s stem or branches, rather than being able to support itself. This includes climbing plants that are either herbaceous (a vine) or woody (a liana). Grass: Herbaceous plant with a grass-like morphology that is within the order Poales. Taxa both with and without a tussock form are mapped to this term.