Electronic Supplementary Appendix S1. Method details
Taxon and data sampling
Individuals were captured by hand between 1996 and 2010 from almost all known populations of the
four populations (Table S2). DNA was extracted from one leg or part of the thorax by means of the
NucleoSpin® Tissue Kit (Macherey-Nagel). For PCR amplification the following primers were used:
Cytochrome Oxidase I (cox1, 657bp): HCO1490F and LCO2140R (1); a mitochondrial region
spanning the NADH subunit I (155bp), the tRNA-Leu gene (65bp) and the 16S rRNA gene
(107bp)(nadh1, 328bp): NDIF and ND1R (2); Cytochrome b (cytb, 435bp): CytbF and CytbR (2) and
enolase (448bp): EN37F and EN731R (3). For cad PCR primers had to be optimized. A first round of
PCR was performed with the primer combination CD439F – CD1098R (3), followed by a heminested PCR with the primers CalCAD688R: 5’-CCA AGT GGA TCC ACA TTT TCC (this study);
CalCAD630_F: 5’-GGC GGA CTT GGA TCT GGT TTT GC (this study) and CalCAD851_R: 5’GGA TCG AAT CCA ATT ACG TTT TCA TCT ACC and successfully amplified a 1284bp long
fragment. PCR conditions consisted of an initial denaturation step of 10 min followed by 39 cycles of
60 s at 95°C, annealing at 40 °C (cytb), 47 °C (cox1), 55-59°C (cad, cad nested) and 53 °C (nadh1,
enolase) for 45 s and an extension for 90 s at 72 °C. PCR products were purified and sequenced in
both directions with an ABI3130 (Applied Biosystems) automated sequencer. Haplotypes of each
individual are given in Table S1. The number of individuals that yielded unambiguous sequences per
population is given in Table S2.
Primer pairs to amplify the microsatellites are reported in (4) (CALO1, CALO3 and CALO4) and four
extra loci (CALO16, CALO21, CALO23, CALO24) that were developed in the same way by H.
Dhuyvetter (pers. comm.). Microsatellite amplification was performed using the QIAGEN ® Multiplex
PCR kit following the manufacturers protocol. Reactions contained ≤0,02µg DNA/µl, 1:1 ratio of
Multiplex PCR Master Mix to primer mix (0,2 µM of each primer). Two multiplex reactions,
containing the microsatellites CALO1, CALO3, CALO4, CALO16 and CALO21, CALO24, CALO23
were carried out with annealing temperature of 57°C. PCR amplifications included an initial HotStart
Taq activation step of 15 min at 95 °C, followed by 35 cycles of denaturing for 30 s at 94 °C,
annealing for 90 s at 57°C, extension for 60 s at 72 °C and a final extension step of 30 min at 60 °C.
Amplification products were run on an ABI3130 sequencer. Allele sizes were scored with GenScan TM
500LIZ® Size Standard (Applied Biosystems) and peak profiles were analyzed in GeneMapper v3.7
(Applied Biosystems).
Sequence data analysis
Sequences were aligned with ClustalW v1.83 (5) and nuclear haplotypes were reconstructed with the
PHASE algorithm (6) in DnaSP v5 (7). For the three mitochondrial genes, cox1, nadh1 and cytb, we
first tested for congruence based on the distance matrices by computing and testing the coefficient of
concordance (8) in the ape2.3 package in R2.14.2. This revealed that distance matrices were highly
congruent among the genes (W=0.89; P < 0.0001) and the three genes were therefore concatenated to
single mitochondrial haplotype sequences.
For phylogenetic inference, the best fitting nucleotide substitution model was, selected based on the
Bayesian Information Criterium (BIC) as implemented in jModelTest 0.1.1 (9), were GTR+I+G
(mitochondrial), HKY + G (cad) and TRN with equal base frequencies. No evidence for
recombination was found in any of the four sequences based on GARD analysis (10) as implemented
in the Datamonkey phylogenetic analysis suite (11).
Phylogenetic relationships among the unique haplotypes were reconstructed for each gene fragment
by BEAST v1.8.0 (12). For the BEAST analysis, we opted for a strict clock analysis. As our sample
composition consists of a dense sampling of Calosoma individuals from the Galapagos, defined as the
ingroup, and a five outgroup species from the mainland consisting of a single individual each, the
phylogenetic relationship among all haplotypes is unlikely to correspond to one of the tree priors
assumed in relaxed clock models, such as a constant population size coalescent, Yule or birth-death
tree prior. This might lead to biased results if weak priors are set simultaneously to the variation in
rates of nucleotide substitution among the branches. This was confirmed by some pilot runs of 10
million generations where a strong influence of the tree prior appeared under a model assuming
uncorrelated lognormal distributed rate variation among branches or a random local clock model (13).
These trees depicted a very deep basal split of the ingroup that was close to the split separating the
ingroup and outgroup C. scrutator and, because of the strong sequence divergence between the
ingroup and outgroup, very high substitution rates along the branch including the outgroup. However,
these pilot runs also revealed that there is little support for rate variation among branches within the
ingroup since the posterior distribution of rate variation among branches contains zero with high
probability and highest support was consequently given to a model with single estimated substitution
rate under a random local clock model for most genes. Based on these results, we imposed a strong
prior on rate variation among branches by assuming a strict molecular clock. Analyses were run by
default for 10 million generations of which the first 2 million generations were treated as burn-in and
discarded for the calculation of posterior probability estimates. We further specified a birth-death tree
prior and default prior distribution parameters for the substitution rates. Appropriateness of the burnin period, stationarity of the chain and effective sample sizes were checked with Tracer v1.4.1 (14).
Posterior probability values of the node heights and their 95% credibility values for the divergence
times were calculated with TreeAnnotator v.1.7.1 and visualized with FigTree v.1.2.2 . For indicative
purposes only, the ages of the branching events were obtained by calculating the average substitution
rate of the concantenated mtDNA. This was obtained by calculating a length weighted average of the
substitution rates of each mitochondrial gene fragment for beetles (15) and yielded an average rate of
0.04803 substitutions/site/MY.
Estimating introgression rates
A HKY and stepwise mutation model was used for the sequence and microsatellite data respectively.
With exception of the microsatellites, relative evolutionary rates and their 95% highest posterior
density (HPD) interval were specified for each locus to ease convergence. Given that these rates are
expressed in terms of the number of substitutions since divergence from the species C. scrutator from
the mainland, they only provide information on the relative rate and cannot be used for the estimation
of demographic parameters. For the concatenated mtDNA sequences, an inheritance scalar of 0.25
was specified. For θi , tij and mij a uniform prior with an upper bound of 50, 10 and 10 were specified
respectively.
Two independent analyses were run for 1,000,000 generations with different random seed values and
saving genealogies every 100’th step resulting in a total of 10,000 genealogies. The first 2,000
genealogies were treated as burn-in and discarded. To ensure sufficient exploration of the parameter
space, a geometric heating scheme with parameter values ha = 0.975 and hb = 0. 75 (see IMa2 manual
for details) was applied with one cold and 39 heated chains, resulting in an average swapping rate of
approximately 0.7 for the least heated chains. Statistical significance of introgression rates was tested
by means of a likelihood-ratio test as implemented in IMa2.
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