Phylogenetic relationships
Maximum likelihood phylogenetic inference using whole mitochondrial genomes (15,268 nucleotides) showed that the Xerces Blue specimens form a monophyletic clade, as do the Silvery Blue specimens (Fig. 1A). We inferred a time-calibrated Bayesian phylogenetic tree from protein-coding genes (11,028 nucleotides) of the mitochondrial DNA genomes of Xerces Blue, Silvery Blue, and 12 related butterflies in Polyommatinae. The Xerces Blue and the Silvery Blue are recovered as sister taxa with high support (posterior probability=1). Because there are no known fossils to calibrate the time since divergence, we first used a molecular clock that spanned the range of rates frequently used for arthropod mitochondrial genes (1.5-2.3% divergence/Ma). This yielded an origin of this subgroup of Polyommatinae at 10.26 Ma (7.16-13.76 Ma 95% HPD interval) and divergence of the Xerces Blue from the Silvery Blue at 849,000 years ago (560,895-1,159,512 years 95% HPD interval, Fig. 1B). A second estimate based on larger-scale fossil-based calibrations (7) placed the origin of the subgroup at ca. 33 Ma (8) with the subsequent divergence of the Xerces Blue and Silvery Blue at 2.60 Ma (2.14-3.08 Ma 95% HPD interval, Figure 1B). The recent speciation of Xerces and Silvery blue is not obviously due to infection with theWolbachia , as no evidence of infection of the sampled specimens with this alpha-proteobacterium is detected in the raw read data.