758 H. Bannister et al. The ndhJ locus raw sequences were merged using


BBMerge Paired Read Merger 37.64 (Bushnell et al., 2017) and aligned to the local DNA reference database using a pairwise match of .99%in Geneious 11.1.5 identified to genus level. Sequences ,99% similar were identified to genus, but the confidence was lower. For the rbcL sequences, reads were trimmed and quality-filtered, and only reads of 300 bp with QF.30 were used for alignment to the rbcL reference database with a pairwise match of .99% (Bushnell et al., 2017).


Scat analysis: invertebrates and birds


We analysed possum scats for the presence of inverte- brate and bird DNA using a similar approach as described for plant DNA, using invertebrate (Hebert et al., 2004a; Foottit et al., 2008) and bird (Hebert et al., 2004b) specific COI primers. Amplification products were assessed by visu- alization following electrophoresis on a 1% agarose gel and products sequenced on the Illumina MiSeq platform using a 600-cycle v3 kit (Illumina), as described previously.


Data analysis


We conducted data analysis using R 3.5.0 (R Development Core Team, 2018). Genera making up ,1%of reads within a sample were removed from analyses to limit the inclusion of material resulting from incidental ingestion or environmen- tal contamination. Bait items were also omitted from results. The plant component of the diet of brushtail possums in the Ikara-Flinders Ranges National Park was investigated by calculating the frequency of occurrence in scats for each genus (the number of scats containing each genus), both monthly and pooled across the 12 months. We also calcu- lated relative abundance of genera within samples by mea- suring the proportion of each genera contained within each sample (i.e. the number of reads for a particular genus, di- vided by the total number of reads for that sample). We cal- culated an average pairwise Pianka’s niche overlap index to test for sex effects (Pianka, 1974). To investigate the influence of time since release on diet,


we calculated selectivity, frequency of occurrence in scats and proportions within scats monthly. Selectivity was calcu- lated using Jacob’s Index (Jacobs, 1974), which assesses plant preference or avoidance relative to environmental availabil- ity:


Jacob’s Index (D) = ri +pi−2ripi ri−pi


where ri = proportion of genera i within the diet (frequency of occurrence within scats), and pi = proportion that genus i is available (frequency of occurrence within the environ- ment). Maximum preference is a Jacob’s Index value of


+1,and maximum avoidance is −1. Genera present in ,5% of both scats and vegetation surveys were omitted from the


selectivity analysis to avoid skewing selectivity results because of low detectability. We created linear mixed-effects models using the pack-


age lme4 (Bates et al., 2015) to investigate the frequency of occurrence of plants in scats, proportions within scats, diet- ary richness (i.e. number of genera) and dietary diversity (using Shannon’s index of diversity) over time (Table 1). Model fit was assessed using Q-Q plots.A generalized linear model with a Poisson distribution was used to test whether the number of non-perennial genera available was related to rainfall (log-transformed), which could explain dietary changes. The height of plants (,0.5 m, 0.5–2.5mor.2.5 m) fall-


ing into each preference category (calculated overall rather than monthly) was calculated along with the overall fre- quency of occurrence in scats for plants in those height classes, to assess whether possums were selecting certain height classes.


Results


We collected a mean of 21.1 ± SE 2.8 scat samples monthly during the study period, from a total of 55 adult possums (26 female, 29 male) of known identity, totalling 253 sam- ples. A mean of 5.3 ± SE 0.7 and 4.0 ± SE 0.6 samples were collected from individual females and males, respectively, over the study period. Samples were collected from across the possums’ area of occupancy (Fig. 1). A total of 112 vege- tation surveys were conducted (mean number of surveys per month 9.3, range 7–12) during the same period, in both of the habitat types possums used for shelter; 57 in woodland habitat and 55 in creekline habitat. Ikara-Flinders Ranges National Park received 466 mm of rain during the study period, comparable to the 440 mm annual mean (Bureau of Meteorology, 2018). The number of annual genera available was not significantly related to rainfall in the preceding month (χ2


1 = 2.7,P = 0.098; Supplementary Fig. 1), although there was a positive trend.


Time since release Both dietary richness and diversity decreased with time since release (Fig. 2, Table 2). The frequency of occurrence in scats changed over time and with preference grouping, and the proportion of each genus within scats changed significantly over time based on preference grouping (Table 2).


General diet One hundred and one plant genera were detected in at least one sample, and 88 were present in at least five samples. Twelve genera were present in .20%of


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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