Appendix S2 – Case study genetic methods
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Sample collection and preparation
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Leaf material was collected from individuals of each species in the state of South Australia
5
(SA) during August 2007-November 2008 and placed into bags of silica-gel in the field. For
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the Allocasuarina robusta study, samples were collected from populations of congeners A.
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mackliniana (Fleurieu Peninsula (FP) n=63, South East (SE) n=13), A. pusilla (FP n=20, SE
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n=12) and A. paludosa (SE n=20) to confirm the genetic distinctiveness of A. robusta. For A.
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robusta itself we collected samples from four broad areas within its distribution which is
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centred around the township of Mt Compass on the Fleurieu Peninsula (North
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(Dingabledinga) n=10; Central (Mt Compass, Yundi, Tooperang) n=63; Southwest
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(Myponga, Hindmarsh Tiers) n=25; South (Hindmarsh Valley) n=25). Samples were
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collected from each of the two known remnant populations of Prostanthera eurybioides, at
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Monarto (n=20) and Mt Monster (n=20), and from re-stocked plants at each location
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(Monarto, n=20; Mt Monster, n=20). In all cases we sampled plants that were as widely-
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spaced as possible to avoid sampling related individuals if genetic structure exists in the
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populations.
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Genomic DNA was subsequently extracted from dried leaf material using a commercial
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service (Australian Genome Research Facility, Adelaide). We conducted amplified fragment
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length polymorphism (AFLP) analysis following the protocol of Vos et al. (1995) with a few
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minor modifications. We used EcoRI and MseI as rare and frequent cutter enzymes,
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respectively, and approximately 100 ng DNA per reaction. Pre-selective amplification was
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performed using adapter primers with a single selective base at the 3’ end (EcoRI-A, MseI-
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C). Selective amplification was performed with three primer combinations, with each of the
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EcoRI and MseI primers having three selective nucleotides attached to the 3’ end (Table S1).
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Each of the EcoRI-selective primers was fluorescently-labelled (FAM, VIC, PET or NED)
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and fragments were analysed by capillary separation on an ABI 3730 (Applied Biosystems),
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using a commercial service (Australian Genome Research Facility, Adelaide). Fragments
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were sized using the GS-LIZ500 standard and scored as present (1) or absent (0) using the
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GENEMAPPER software (Applied Biosystems). A peak scoring threshold of 400 rfu was used
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for FAM-, NED- and VIC-labelled fragments and 200 rfu for PET-labelled fragments as
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fluorescence was weaker for this dye. All automatic scoring in GENEMAPPER was manually
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checked and adjusted. We assessed a putative locus as being present if a band was present in
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two or more individuals, and polymorphic if it was absent in two or more individuals. We
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assessed genotyping error by PCR amplifying approximately 5% of samples twice. Error
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rates were calculated as the proportion of loci mismatching between repeated samples.
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Genetic data analysis
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The genetic structure of sampled populations was initially visualised using Principal
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Coordinates analysis of inter-individual genetic distances (Euclidian distances based on
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diploid binary AFLP data), calculated and plotted in GENALEX v6.5 (Peakall & Smouse
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2012) to confirm our a priori identification of populations. We quantified genetic
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differentiation between populations using FST as calculated in the program AFLP-SURV
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(Vekemans 2002). In AFLP-SURV, allele frequencies were estimated using the Bayesian
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method with non-uniform prior distribution and assuming populations were not in Hardy-
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Weinberg equilibrium, in which case we entered values of FIS (inbreeding coefficient, gained
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from analysis described below) as follows: Allocasuarina robusta FIS=0.05; Prostanthera
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eurybioides FIS=0.2. Standard errors for FST were estimated from 10 000 permutations. We
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used average heterozygosity (HE) at AFLP loci as our measure of genetic diversity, calculated
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in the program AFLP-SURV using allele frequencies as above (Vekemans 2002). The
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inbreeding coefficient, FIS, was calculated in the program I4F (Chybicki et al. 2011), which
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uses Markov Chain Monte Carlo to obtain posterior distributions of FIS using a model of
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identity-by-descent. To run the simulations, we set α and β parameters to 1 (uniform priors)
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for both inbreeding and allele frequencies and ran the Gibbs sampling with 60 000 steps with
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a ‘burn-in’ of 10 000 steps.
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Sample sizes were roughly equivalent in each of the case studies (Table S3) with the
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exception of Allocasuarina robusta. For this species we re-ran all analyses using a modified
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dataset that reduced the size of the Central population to n=25 using random sampling to
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account for sample size effects on our genetic parameter estimates. The results from the
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modified data set varied only slightly for each parameter (± 0.01), therefore we present the
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full dataset results in the main text.
Table S2: Details of AFLP primer combinations used in each case study, number of polymorphic loci, average total heterozygosity (HT)
and genotyping error rate.
Species
Primer combination
No. loci scored
No. loci polymorphic
HT
(%)
Allocasuarina robusta
Prostanthera eurybioides
Error rate
(%)
eACG/mCAG (VIC)
21
13 (61.9)
0.229
3.2
eAGC/mCGA (FAM)
14
11 (78.6)
0.240
1.6
eAAC/mCCGT (NED)
14
13 (92.9)
0.235
2.9
eACG/mCAA (VIC)
60
41 (68.3)
0.259
2.3
eAGC/mCCG (NED)
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35 (77.8)
0.232
2.9
eAGC/mCAA (FAM)
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30 (71.4)
0.252
3.1
eAGC/mCAG (PET)
12
9 (75.0)
0.262
2.5
Table S3: Details of sample sizes and genetic diversity statistics for populations of each threatened species, Prostanthera eurybioides,
Cassinia tegulata, Acacia enterocarpa and Allocasuarina robusta. N = number of individuals genotyped, HE = mean expected
heterozygosity (± standard error), PLP = proportion of loci polymorphic and FIS = inbreeding coefficient (± standard error).
Species/Population
Location
N
HE
PLP
FIS
North
35°16’ S/138°38’ E
10
0.284 ± 0.026
81.6
not resolved
Central
35°21’ S/138°37’ E
61
0.219 ± 0.023
73.5
0.070 ± 0.032
Southwest
35°23’ S/138°31’ E
25
0.250 ± 0.020
81.6
0.032 ± 0.014
South
35°26’ S/138°35’ E
25
0.219 ± 0.024
63.3
0.050 ± 0.025
121
0.235 ± 0.021
75.5
0.108 ± 0.038
36
0.235 ± 0.013
74.1
0.106 ± 0.039
Allocasuarina robusta
TOTAL
Prostanthera eurybioides
Monarto (All)
139°9’ E/35°7’ S
-
Natural
20
0.264 ± 0.014
80.3
0.160 ± 0.057
-
Planted
16
0.205 ± 0.014
66.0
0.153 ± 0.061
39
0.234 ± 0.016
59.2
0.351 ± 0.069
Mt Monster (All)
140°19’ E/36°12’ S
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Natural
20
0.238 ± 0.016
61.2
0.292 ± 0.083
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Planted
19
0.228 ± 0.017
57.1
0.388 ± 0.097
75
0.249 ± 0.014
72.1
0.230 ± 0.044
TOTAL
1
References
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Chybicki IJ, Oleksa A, Burczyk J (2011) Increased inbreeding and strong kinship structure in
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4
Taxus baccata estimated from both AFLP and SSR data. Heredity, 107, 589–600.
Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic
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software for teaching and research - an update. Bioinformatics (Oxford, England), 28,
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2537–9.
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Vekemans X (2002) AFLP-SURV version 1.0. Laboratoire de Génétique et Ecologie
Végétale, Université Libre de Bruxelles, Belgium.
Vos P, Hogers R, Bleeker M et al. (1995) AFLP: a new technique for DNA fingerprinting.
Nucleic Acids Research, 23, 4407–4414.