Supplementary Methods
Mitochondrial data. A 356-bp fragment of the mtDNA control region (CR) was
analyzed for 246 harbour porpoises including: 148 sequences of animals from the
northern Bay of Biscay (49 from Tolley & Rosel (2006) and 99 from Walton (1997)),
30 from the Iberian coast (13 from Tolley & Rosel (2006) and 17 newly sequenced in
this study), and 72 from the Black Sea (8 sequences from Rosel et al. (1995), 2 from
Tolley & Rosel (2006), and 62 new sequences generated in this study) (see table
S1).
The 79 new sequences were obtained as follows: genomic DNA was extracted from
soft tissue using the Qiagen DNeasy Tissue kit. The complete mtDNA CR was
amplified by polymerase chain reactions (PCR) using newly designed forward and
reverse primers located in the flanking regions (respectively: PPL1: 5’-AAA TAC CTC
GGT CTT GTA AAC C-3’ and PPS1: 5’-AAG TTT AAG CTA CAT TAA CGT GTG G3’). PCR was conducted in a 25 µl reaction volume containing 50 ng of genomic
DNA, 0.4 U Taq DNA polymerase, thermophilic DNA polymerase buffer (1X), 2 mM
MgCl2, 0.5 µM of each primer, and 400 µM of dNTPs. The PCR conditions started
with an initial denaturation step at 95 °C for 2 min, followed by 35 cycles of
denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min, and an extension
step at 72°C for 1 min (30 min for final extension). The sequencing reactions were
conducted
on
a
96-capillary
MegaBACE-1000
DNA
Analyzer
(Amersham
Biosciences) using the manufacturer’s protocol and three sequencing primers: PPL1,
L15825, and H16265 previously designed (Rosel et al. 1999). Sequences were
edited using Sequence navigator (ABI), aligned primarily using Clustal X and then
checked visually.
1
IM analyses using microsatellite and mtDNA D-loop. We conducted the IM
simulation contrasting the harbour porpoise population from the Black Sea with those
in the Atlantic. We first applied the analysis to the mtDNA CR dataset alone, and then
pooled all loci together (i.e., the 10 microsatellite loci and the 356 bps mtDNA CR).
We used a similar procedure for the mtDNA CR data set as described for
microsatellite loci (see the core ms), with a Hasegawa-Kishino-Yano (HKY) mutation
model (Hasegawa et al. 1985) for the mtDNA CR, and 5 to 15 independent
Metropolis coupled chains. Preliminary runs showed that migration rate posterior
density distributions were not different from zero. We therefore simplified the model
by assuming equal migration rates between populations after their divergence. We
also applied a refined version of the IM model that allows for population size change
by adding a supplemental parameter to estimate the proportion (s and (1-s)) of
individuals from the ancestral population that founded the first and the second
descendent populations, respectively (Hey 2005). With this additional complexity in
the parameter space of the model, MCMC convergence was unachievable for our
complex multilocus microsatellite dataset. We were therefore only able to apply the
seven parameters scenario to the mtDNA dataset.
To convert parameter estimates into demographic units, we used a plausible range of
mutation rates between 3.3 x 10-8 bp-1 year-1 (µmtL) and 4.3 x 10-8 bp-1 year-1 (µmtH)
(i.e. respectively 1.17 x 10-5 and 1.53 x 10-5 year-1 for a sequence of 356 bps), as
suggested in Tolley & Rosel (2006). Estimates of  were converted into effective
population sizes using this range of mutation rate and the same range of generation
time as describe above in the microsatellite analysis.
2
Finally, we ran IM simulations pooling all loci together following a similar procedure
as that described for microsatellite loci, using a SMM for microsatellite evolution and
a HKY substitution model for mtDNA evolution.
Supplementary Figures and Tables legends
Table S1
MtDNA control region haplotypes used in the IM analyses comparing Atlantic (AT) vs.
Black Sea (BS) porpoises, count of each haplotype, and Genebank accession
number
Table S2
Details of the IM runs’ conditions for the three longest runs and Effective Sample
Size at the end of each run
Figure S1
Comparison of marginal posterior probability densities for each parameter of the IM
model obtained from the replicated runs conducted on the microsatellite data set
comparing Iberian (IB) vs. northern Bay of Biscay (NBB) porpoises.
Figure S2
Same as figure S2, but for the microsatellite data set comparing Atlantic (AT) vs.
Black Sea (BS) porpoises.
Figure S3
Comparison of marginal posterior probability densities for each parameter of the IM
model comparing Atlantic (AT) vs. Black Sea (BS) porpoises using mtDNA data set,
microsatellite data, and all loci combined together (see supplementary methods for
3
IM runs condition with mtDNA). S refers to the model including (or not) population
size change during the divergence.
Supplementary References
Hasegawa, M., Kishino, H. & Yano, T. 1985 Dating of the human-ape splitting by a molecular
clock of mitochondrial DNA. J Mol Evol 22, 160-74.
Hey, J. 2005 On the number of the New World founders: a population genetic portrait of the
poepling of the Amercias. PLoS Biol 3, e193.
Hey, J. & Nielsen, R. 2004 Multilocus methods for estimating population sizes, migration
rates and divergence time, with applications to divergence of Drosophila
pseudoobscura and D. persimilis. Genetics 167, 747-760.
Rosel, P., Dizon, A. E. & Haygood, M. G. 1995 Variability of the mitochondrial control region
in populations of the harbour porpoise, Phocoena phocoena, on interoceanic and
regional scales. Can J Fish Aquat Sci 52, 1210-1219.
Rosel, P. E., France, S. C., Wang, J. Y. & Kocher, T. D. 1999 Genetic structure of harbour
porpoise Phocoena phocoena populations in the northwest Atlantic based on
mitochondrial and nuclear markers. Mol Ecol 8, S41-S54.
Tolley, K. A. & Rosel, P. E. 2006 Population structure and historical demography of eastern
North Atlantic harbour porpoises inferred through mtDNA sequences. Mar Ecol Progr
Ser 327, 297-308.
Walton, M. J. 1997 Population structure of harbour porpoise Phocoena phocoena in seas
around the UK and adjacent waters. Proc R Soc B-Biol Sci 264, 84-94.
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