Appendix 1
Exclusion of PB7 and LS
Fig. S1 describes the results of D-statistic tests (Green et al. 2010; Durand et al. 2011) for
brown bear admixture into pairs of individual polar bears. Two outliers are observed: LS,
which has the most positive distribution of D-statistics (indicating that the LS polar bear
contains less admixture from brown bears than any other polar bear), and PB7, the only
polar bear for which the distribution of D-statistics does not include zero (suggesting that
PB7 may have bona fide admixture from brown bears). We suspect that both of these
results are artefacts resulting from errors in the sequencing libraries rather than admixture
with brown bears.
As reported previously (Cahill et al. 2013), the LS polar bear data contain DNA
damaged sites. This ancient DNA associated error biases the D-statistic away from zero.
The LS data were generated from a 40-year-old bone that is currently stored at the
National Museum of National History in Washington, DC (Cahill et al. 2013). These data
show nearly twice the number of C to T and G to A transitions when compared to the
polar bear reference genome than the other polar bears. This is most likely due to
miscoding lesions resulting from cytosine deamination to uracil, which is the most
common form of ancient DNA damage (Hofreiter 2001). Although damage does not
affect the D-statistic when the damaged individual is in the position of the potential
introgressor, it will bias the D-statistic when the damaged sample is included in either the
P1 or P2 positions (potential recipient of introgression). In this case, the effect is a false
positive match between the damaged individual and the outgroup, and consequent
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identification of any undamaged comparison individual as admixed, as seen for LS in
Fig. S1.
D-statistic results show that the PB7 polar bear has a stronger signal for brown
bear ancestry than any other polar bear (mean D= -0.033). However, D-statistic tests
involving PB7 and the Kenai brown bear (Ken) as potential introgressor are particularly
extreme, with an average D of -0.066 for D(PB7, other polar bear, Ken, black bear),
compared to -0.028 for D(PB7, other polar bear, other brown bear, black bear). Given
that the PB7 and Ken data were generated as part of the same project (Miller et al. 2012),
we explored the possibility that contamination between these data may exist.
We mapped the published genomic data from PB7 and Ken to a reference polar
bear mitochondrial genome (Delisle & Strobeck 2002) bwa (Li & Durbin 2010) and
samtools (Li et al. 2009) under the same parameters as the analyses described in the main
text (Methods). We then identified all positions in the mitochondrial genome where the
consensus mitochondrial genome sequences assembled for PB7 and Ken differed from
each other. At each of these positions, we then calculated the number of times the
consensus allele for Ken was observed in the PB7 data set, and vice versa. For most sites,
we observed zero reads in the Ken data set that matched the consensus assembly for PB7
(Fig. S2A). However, in the PB7 data set, 0.5-1.0% of PB7 reads match the Ken allele
(Fig. S2A). To determine whether this result could be due to differences in sequencing
depth, we down sampled the Ken and PB7 data sets to an equal number of reads, and
counted the number of sites in the down sampled data sets in which the consensus allele
for the opposite species was observed (Fig. S2B). We observed that as the number of
reads sampled increased so did the number of Ken alleles observed in the PB7 data set.
2
These results are most simply explained by small amounts of contamination of the PB7
data set with data from Ken. We therefore chose to exclude PB7 from further analysis.
Y-chromosome Analysis
Recent studies have shown that the Y-chromosome haplotypes found in polar
bears and form reciprocally monophyletic clades (Bidon et al. 2014). These findings
include the ABC islands brown bears which at the Y-chromosome fall within the brown
bear clade (Bidon et al. 2014). We assessed the same ~390 KB Y-chromosome scaffold
of the polar bear reference genome (Li et al. 2011) as Bidon et al (scaffold 297) and
calculated the sequence divergence between all male bears in our panel.
Using the pseudohaploidization method used elsewhere in this study (Methods)
we generated representative sequences for each individual at scaffold 297 and compared
pairwise differences. Within polar pairwise differences ranged from 1-4 differences per
10,000 sites. Polar bear to Baranof island brown bear differences range from 11-12.5
differences per 10,000 sties. Polar bear to American black bear differences range from
29-31 differences per 10,000 sites and the Baranof brown bear has 29 differences to the
American black bear per 10,000 sites.
Our analytical power in this analysis is limited by only having a single male
brown bear. However, insofar as our results are interpretable they show that the Baranof
island brown bear (a male ABC islands bear) possesses a Y-chromosome haplotype that
falls well outside the diversity of polar bear Y-chromosomes. Bidon et al observed
brown bear to polar bear divergence to be ~35% of brown/polar bear divergence to black
3
bear (Bidon et al. 2014). Our observation of ~39% (Figure S3) is qualitatively consistent
with previous results and probably indicative of minor filtering differences.
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Supplemental Figures
Figure S1 - D-statistic tests for brown bear admixture into individual polar bears
The boxplot shows the autosomal D-statistic for each polar bear with all possible
combinations of polar bear and brown bear introgressors. Negative values indicate that
the individual listed on the x-axis has more polar bear ancestry. Polar bears from the
Beaufort and Chukchi seas are depicted as red, those from Svalbard are blue, those from
Hudson Bay are yellow, and the Lancaster Sound bear is green.
Figure S2 – Tests for contamination of PB7 by Ken
The frequency of reads in the PB7 and Ken data sets that may be derived from
contamination by the other data set (A). To control for the detection of potential
contaminant reads based on differences in coverage, we sampled fixed numbers of reads
from each individual at variable sites. (B) The mean frequency of sites containing one or
more potentially contaminant reads of 20 random draws of N reads. Error bars equal two
standard deviations.
Figure S3 – Y-chromosome pairwise difference
The number of pairwise differences per site between male individuals in our panel of
bears at a ~390KB Y-chromosome scaffold. We find that the Baranof sample (the only
male brown bear in this study) falls outside the range of divergences observed between
polar bears. The level of divergence is consistent with previous studies analysing the
same scaffold with different individuals suggesting that brown bears and polar bears form
reciprocally monophyletic clades at the Y-chromosome (Bidon et al. 2014).
5
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