Journal of Biogeography
SUPPORTING INFORMATION
The contribution of Anatolia to European phylogeography:
the centre of origin of the meadow grasshopper, Chorthippus parallelus
E. Mahir Korkmaz, David H. Lunt, Battal Çıplak, Naci Değerli and Hasan H. Başıbüyük
Appendix S3 Results of demographic history and IMA2 analyses
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

The findings from neutrality and mismatch distribution analyses with possible
expansion time estimates based on cpnl-1 sequences for all clusters detected in the
result of DAPC (Table S1 & Fig. S1),
A more detailed explanation of the isolation-with-migration analysis (IMA2) using
COI–tRNALeu–COII and cpnl-1 regions to investigate the barrier position of the
Turkish Straits system,
The network analyses and demographic statistics of Thracian and north-west
Anatolian groups to test the barrier position of the Turkish Straits system (Table S1,
Figs S2 & S3).
The demographic history of all clusters
The results of the neutrality tests and the output from the mismatch distribution analysis including expansion time estimates for all clusters of Chorthippus parallelus are provided in Table S1. Fu’s Fs test yielded significant negative values in clusters B (−21.507,
P < 0.01) and D (−145.815, P < 0.01), suggesting an excess of unique or rare haplotypes
over what would be expected under neutrality with a recent sudden expansion event
(Table S1). Values of Fu’s Fs were, however, non-significant for cluster A and cluster C
(P > 0.5), indicating a past demographic constriction or bottleneck event. Likewise, Tajima’s D test rejected a scenario of selective neutrality and population equilibrium for
each clusters (Table S1, P > 0.05) except cluster D, which had a negative and significant
D value (−2.260, P < 0.01). The value of Tajima’s D for cluster D also suggested an excess
of high-frequency haplotypes, indicating a historically continuous gene-flow pattern
among populations (Oleksyk et al., 2010). This finding was also supported by the individual assignment plots of DAPC (Fig. 4).
The demographic dynamics of the four clusters were also inferred from mismatch distributions. The results showed that the mismatch distributions fitted unimodal curves for all clusters except cluster C, which exhibited a multimodal mismatch
distribution with a smaller effective sample size than expected (Fig. S1). The Harpending raggedness index (r) and variance (SSD) also indicated that the curves did not differ significantly from the expected distributions under the model of population expansion for clusters A and D (Table S1). On the other hand, significant values for r and/or
SSD were observed in cluster B and cluster C, suggesting a demographic history further
from the neutral expectations.
The barrier position of the Turkish Straits system
The sequences of both COI–tRNALeu–COII and cpnl-1 were used for IMA2 analysis.
Cpnl-1 is known to exhibit no (or limited) recombination (Ibrahim et al., 2002). IMA2
was used to generate: (1) marginal posterior probability densities of the population
parameters of divergence time, τ, where τ = Tµ, and where T is the time in years and µ is
the mutation rate; (2) population differentiation indices of the ancestral population and
the two extant lineages, θA, θ1 and θ2, respectively, where θ = Neµ for uniparentally
inherited loci or θ = 4Neµ for diploid autosomal loci, where Ne is the effective population
size; and (3) the migration rate for each population, m1 and m2, where m is the migration rate per mutation (m / µ), and where the population migration rate is M = 2Nem =
θm. We used an HKY substitution model, a locus inheritance scalar of 0.25 to account for
the uniparental inheritance, and a mean mutation rate per base of 0.0177 per million
years, with an interval set from 0.00475 to 0.025. This mutation rate was chosen from a
recent calibrated rate for partitioned mitochondrial genome in insects (Papadopoulou
et al., 2010) with the lowest and highest mutation rates (Percy et al., 2004; Shapiro et
al., 2006). Initially, we followed the procedure described by the IMA2 manual for setting
upper bounds of each parameter [θ (θ1 = θ2 = θA) = 10; τ = 10; m1 = 10; m2 = 10]. We
applied multiple runs using different random number of seeds to obtain appropriate
starting values for the bounds. To identify scales in parameter estimation, we then carried out additional runs with more acceptable upper bounds [finding consistent marginal peak locations (parameter estimates) with unimodal likelihood curves approaching
zero on both ends; see Hey & Nielsen, 2007, for details]. The final analysis running with
prior estimating parameters was as follows: θ = 200; τ = 2; m1 = 10; and m2 = 10. Our
analyses were carried out over two independent MCMC runs with 100,000 burn-in
generations and 2.5 million sampling generations, sampling every 100th generation. We
repeated the analyses three times using different random seed values.
The findings of network analyses and demographic history of the Thracian and north-west
Anatolian groups
The median-joining haplotype networks, using cpnl-1 and mtDNA haplotypes, separately yielded a star-like structure with the presence of mostly shared haplotypes among
both groups (Fig. S2a,b). The hypothesis of constant population size was rejected by
Fu’s Fs test (–76.586; P < 0.05 for cpnl-1; −30.746, P < 0.05 for COI–tRNALeu–COII; Table
S1). Moreover, Tajima’s D test showed a non-significantly negative value (−1.494 for
cpnl-1; –0.259 for COI–tRNALeu–COII, P > 0.05; Table S1). These results were also supported by the unimodal mismatch distribution (Fig. S3a,b), Harpending raggedness
index (r = 0.007 for cpnl-1; r = 0.019 for COI–tRNALeu–COII, P > 0.5; Table S1) and SSD
(0.001 for cpnl-1; 0.001 for COI–tRNALeu–COII, P > 0.05; Table S1). Values of τ for
Thrace and north-west Anatolian groups are presented with lower and upper intervals
in Table S1. Setting µ (for COI–tRNALeu–COII) to 0.0177, the putative expansion times
from τ using the combined mtDNA data were estimated at 0.017 Mya (0.011–0.023) for
both groups, a period just prior to the opening of Bosphorus around 6000 years ago
(Kerey et al., 2004). The BSP analysis (Drummond et al., 2005) also supports this expansion, which gradually became strong by the end of the Last Glacial Maximum (Fig. S3c).
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Table S1 Summary of demographic history statistics of the six clusters of Chorthippus parallelus obtained from DAPC including the groups analysed in IMA2.
Cluster A
Cluster B
Cluster C
Cluster D
cpnl-1
Mean (obs.)
τ
θ0
θ1
SSD
Raggedness
index (r)
t (Myr)
Fu’s Fs
Tajima’s D
0.547
0.908
(0.000–2.189)
0.000
0.900
0.005n.s.
0.169n.s.
0.041
(0.000–0.098)
−1.627n.s.
−0.312n.s.
2.840
5.766
(0.648–10.006)
0.000
3.840
0.007n.s.
0.022*
0.259
(0.029–0.450)
−21.507**
−0.417n.s.
Thrace and north-west Anatolian groups
COI–tRNALeu–COII
5.127
5.211
(2.896–7.250)
0.000
17.578
0.031*
2.807
1.482
(0.744–4.867)
1.350
n.a.
0.000n.s.
0.072n.s.
0.234
(0.130–0.326)
−3.299n.s.
−1.158n.s.
0.047n.s.
0.067
(0.033–0.219)
−145.815**
−2.260**
2.816
1.59
(1.09–2.22)
1.74
99.9
0.001n.s.
0.019n.s.
0.012
(0.004–0.057)
−30.746**
−0.259n.s.
cpnl-1
4.647
2.607
(0.965–8.523)
2522
35.42
0.001n.s.
0.007n.s.
—
−76.586**
−1.494n.s.
Mismatch analysis parameters by the clusters and the combined data of Thracian and north-west Anatolian groups: mean number of observed differences;
expansion parameter τ, (with lower and upper bounds at α = 0.05); θ is the substitution rate before (θ0) and after (θ1) the expansion; SSD tests the validity of a
stepwise expansion model based on the sum of squares deviations between the observed and expected mismatch, with probability values (n.s., P > 0.05; * P < 0.05;
** P < 0.01). Non-significant mismatch values suggest an expansion event. Harpending’s raggedness index is calculated similarly, and with probability values (* P <
0.05; ** P < 0.01). Non-significant raggedness values suggest an expansion event. Time since expansion (t) is calculated from τ = 2μt, where μ = 3.36% Myr−1 for
both gene region. Under a model of sudden population expansion, Fu’s FS and Tajima’s D are expected to be significantly negative.
Figure S1 Mismatch distribution analyses of the pairwise differences, observed and expected values (as
indicated) for the four clusters of Chorthippus parallelus.
Figure S2 Network graphs of cpnl-1 (A) and COI–tRNALeu–COII (B) haplotypes for Thracian (white) and
north-west Anatolian (black) groups of Chorthippus parallelus.
Figure S3 Mismatch distribution graphs (A, cpnl-1) and (B, COI–tRNALeu–COII), and Bayesian skyline plot
(C) for Thracian and north-west Anatolian groups. The Bayesian skyline plot (m = 5) is derived from the
COI–tRNALeu–COII dataset of Chorthippus parallelus. The median estimates are shown as a thick solid line,
and the 95% highest posterior density limits are shown by the grey area.