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Raw reads were quality-trimmed by Trimmomatic48 with a sliding window option, minimum base quality of 20 and minimum read length of 25 bp. The assembled polar bear genome23 was used for reference mapping using BWA version 0.7.5a49 with the BWA-MEM algorithm on scaffolds larger than 1 Mb. Scaffolds shorter than 1 Mb in length were not involved in the mapping and analyses, due to potential assembly artefacts50 and for reducing the computational time in downstream analyses. Duplicate Illumina reads were marked by Picard tools version 1.106 (http://picard.sourceforge.net/) and the genome coverage was estimated from Samtools version: 0.1.1851.Freebayes version 0.9.14–1752 called Single Nucleotide Variants (SNVs) using the option of reporting the monomorphic sites with additional parameters as -min-mapping-quality 20, -min-alternate-count 4, -min-alternate-fraction 0.3 and -min-coverage 4 with insertion/deletion (indel) realignment. A custom Perl script created consensus sequences for each of the mapped bear individuals from the Variant Call Format (VCF) files, keeping the heterozygous sites and removing indels. In order to complete the taxon sampling of the ursine bears, reads from six previously published genomes (Supplementary Table 1) selected and on the basis of geographic distribution, availability and sequence depth and SNVs were called as described above. For the two high coverage ( > 30X) genomes, SNVs calling parameters (-min-coverage) were set as one-half of the average read depth after marking duplicates. Genome error rates53 were calculated on the largest scaffold (67 Mb) for all bear genomes, confirming a high quality of the consensus sequences. (Supplementary Methods and Supplementary Fig. 22).

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Data filtration, simulation of sequence length and topology testingThe next step was to create multi-species alignments for further phylogenetic analysis from all 13 bear individuals. In order to create a data set with reduced assembly and mapping artefacts, genome data was masked for TEs and simple repeats19 using the RepeatMasker54 output file of the polar bear reference genome available from http://gigadb.org/23. Since the polar bear reference genome RepeatMasker output file did not contain the simple repeat annotation, we repeatmasked the polar bear reference genome with the option (-int) to mask simple repeats. Next all bear genomes were masked with bedtools version 2.17.055 and custom Perl scripts. Non-overlapping, sliding window fragments of 100 kb were extracted using custom perl scripts together with the program splitter from the Emboss package56 (Supplementary Fig. 1), creating a dataset of 22,269 GFs from 13 bear individuals. Heterozygous sites, and repeat elements were all marked “N” and removed using custom Perl scripts. An evaluation of the minimum sequence length of GFs needed for phylogenetic analysis was done by estimating how much sequence data is needed to reject a phylogenetic tree topology using the approximate unbiased, AU test57. Only sufficiently long sequences can differentiate between alternative trees with statistical significance. The evaluation was done in two separate analyses: (a) with a simulated data set and (b) on a data set of 500 random GFs (Supplementary Methods).