Journal of Biogeography
SUPPORTING INFORMATION
Evolutionary and contemporary responses to habitat fragmentation detected in a
mesic zone marsupial, the long-nosed potoroo (Potorous tridactylus) in southeastern
Australia
Greta. J. Frankham, Kathrine A. Handasyde and Mark D.B. Eldridge
Appendix S2 - Supplementary Methods and Results
METHODS
Mitochondrial DNA analyses.
A ~630 base pair (bp) fragment of the tRNA proline end of Control Region (CR) was
amplified from 347 individuals using primers L15999M and H16498M (Fumagalli et al.,
1997). Polymerase Chain Reactions (PCRs) were carried out as described in Frankham et
al., (2014). Sequencing was resolved on an Applied Biosystems 3730xl sequencer at
Australian Genome Research Facility (AGRF) Sydney.
Standard population diversity indices (h, haplotype diversity; π, nucleotide diversity) and
pairwise differentiation (ΦST) amongst populations were estimated using ARLEQUIN
3.5.1.2 (Excoffier & Lischer, 2010).
Isolation by distance (IBD) was tested for using Mantel tests in ISOLATION BY DISTANCE
WEB SERVICE (IBDWS) 3.22 (Jensen et al., 2005). The natural logarithm of ΦST/(1- ΦST)
(Rousset, 1997) and geographic distance was used, due to the large variance in both data
sets. Pairwise geographic distances were calculated in GENALEX 6.0 (Peakall & Smouse,
2006). IBD tests were run separately for the southern mainland (including and excluding
French Island) and the Tasmanian lineages. The northern mainland lineage was not
Frankham et al. Biogeography of Potorous tridactylus
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analysed due to small sample sizes. Significance of the correlation between genetic and
geographic distances was tested with 10,000 permutations of the data.
A hierarchical Analysis of Molecular Variance (AMOVA) (Excoffier et al. 1992) was
performed in ARLEQUIN to compare the (a priori) taxonomic classification of Johnston
and Sharman 1977 (Tasmanian and mainland subspecies separated by Bass Strait), with
the genetic lineages/subspecies (a posterior) identified by Frankham et al., (2012).
10,000 permutations were carried out to test significance of values estimated, and
corrected using the Bonferroni method (Rice 1989)
Past population changes were investigated via mismatch analysis (ARLEQUIN) and
DNASP 5.10.01 (Librado & Rozas, 2009). Mismatch analysis was conducted on the
southern mainland lineage (including and excluding French Island) and the Tasmanian
lineage. Two models of expansion were tested; a demographic or sudden expansion
model (Rogers & Harpending, 1992) and a spatial range expansion (Excoffier, 2004). The
raggedness index (r) was used to determine the goodness-of-fit statistic for the observed
and expected mismatch distributions, with significance estimated using the parametric
bootstrapping procedure (10,000 replicates). A non-significant r is indicative of
population expansion.
DNASP 5.10.01 (Librado & Rozas, 2009) was also used to investigate past population
changes by calculating Fs (Fu, 1997) and R2 (Ramos-Onsins & Rozas, 2002).
Significance values were evaluated by coalescent simulations (2000 replicates). If
population expansion has occurred a significantly negative Fs value and a significantly
small R2 value were expected.
Nuclear DNA (microsatellite) analysis
Ten microsatellite loci were amplified across all 354 individuals, (conditions previously
described in Frankham et al., 2013; 2014). Deviations from Hardy-Weinberg Equilibrium
(HWE) and genotypic Linkage Disequilibrium (LD) were tested for using GENEPOP web
3.4 (Raymond & Rousset, 1995) with 1000 dememorizations, 500 batches and 5000
iterations per batch, P-values were adjusted using Bonferroni corrections (Rice 1989).
Frankham et al. Biogeography of Potorous tridactylus
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Observed heterozygosity (HO) and expected heterozygosity (HE) were calculated in
GENALEX 6.0 (Peakall & Smouse, 2006) and private alleles (unique to single populations)
and rare alleles (occurring at a frequency  0.05 within a population) were identified.
Allelic richness (A) and FST were determined in FSTAT 2.9.3 (Goudet, 1995) and
corrected for sample size (Rice, 1989).
Principal Components Analysis (PCA) was also used to visualise relationships amongst
population using GENALEX.
Mantel tests were conducted to test for isolation by distances (IBD) patterns using
IBDWS (Jensen et al., 2005), using the natural logarithm FST/(1-FST) (Rousset, 1997) and
the natural logarithm of the geographic data. IBD tests were run separately for the
southern mainland lineage and the Tasmanian lineage. Significance of the correlation
between genetic and geographic distances was tested with 10,000 permutations of the
data.
RESULTS
Mitochondrial DNA
Haplotype diversity (h) ranged from 0.0 (Lake Tyres [16]) to 0.89 (East Gippsland - East
site [15]). Overall nucleotide diversity (π) was low to moderate, ranging from 0% (Lake
Tyres [16]) to 1.79% (Budderoo [9]) (Table 1).
Within lineage population differentiation was high with the most ΦST values > 0.9. Lower
(some non-significant) values were found between populations in close geographic
proximity (8 km apart), for example French Island East [19] and West [20] populations;
0.021 (Appendix S1; Table S3).
AMOVA analyses indicated that the majority of genetic variation in P. tridactylus was
found between populations. The taxonomic classification of two sub-species (a priori),
with Bass Strait as the major biogeographic barrier, was found to explain 72.49% of the
variance. Whereas a posterior grouping of populations into the three genetic lineages
Frankham et al. Biogeography of Potorous tridactylus
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identified in this study (separated by Bass Strait and the Sydney Basin) explained 87.43%
of the variation (Appendix S1; Table S4).
IBD was non-significant across the southern mainland lineage (p = 0.16, r2 = 0.02), but
was highly significant across the Tasmanian lineage (p = 0.006, r2 = 0.45) (Appendix S1;
Fig.S1a, b).
Nuclear DNA (microsatellite)
354 individuals were successfully genotyped at 10 loci. Loci in some populations
deviated significantly from HWE, however this was not consistent across loci or
populations, thus all 10 loci were retained in the analysis. LD was not consistently
identified for any loci or populations.
All 10 loci were polymorphic, with the number of alleles ranging from 5 (G26.4) to 42
(Y151), averaging 20.4 alleles per loci (Table 1). Mean allelic richness (A) (n = 7)
averaged 4.86, ranging from 2.72 (French Island – West [20]) to 6.29 (East Gippsland –
West [14]); 7.68 was recorded in the pooled northern mainland lineage. HE averaged
0.67, ranging from 0.47 (French Island – West [20]) to 0.80 (East Gippsland – East [15])
(Table 1).
Estimates of pairwise FST were significant between all pairs of populations after
correction for multiple comparisons (Rice, 1989) (Appendix S1; Table S3). FST values
between populations in close geographic proximity (5–10 km) were low to moderate (e.g.
0.03 between two East Gippsland sites [14,15]; 0.07 between French Island East [19] and
West [20]), as were those between Tasmanian populations (0.06–0.17). The highest
differentiation was consistently recorded in comparisons that included the insular French
Island populations (East [19] and West [20]) (0.29–0.42).
PCA analysis also identified significant structuring within the microsatellite data
(Appendix S1; Fig.2.). The first axis accounted for (37.0%) of the variation and the
second (21.4%). Three distinct population clusters were identified; the mainland
Frankham et al. Biogeography of Potorous tridactylus
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populations, Tasmanian populations and French Island populations. The inclusion of a
third axis accounted for 71.7% of the total variation and separated the previous three
groups as well as another comprising the isolated Mt Clay + Western Victoria [23, 24,
25] and Lake Tyres [16] populations. Excluding the French Island populations [19, 20]
did not reveal any additional structuring
IBD was significant across the southern mainland lineage (p = 0.04, r2 = 0.09), as well as
highly significant across Tasmania (p = 0.0002, r2 = 0.45) (Appendix S1; Fig.S1c, d).
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