*4.8. Multiple Alignments of Mitochondrial Gene Sequences and Selection of SNPs Specific for Pre-Defined Taxa*

SNPs specific for selected taxa were identified based on the newly developed software SNPtax which uses a set of custom scripts based on BioPerl [91] and PRANK v.170427 [92] as alignment tool. By extracting the taxonomic information together with the genic sequences from different mitochondrial genomes in GenBank format and then generating multiple alignments for each gene—at every position in the alignment—the taxon can be determined for which a certain base is characteristic. The resulting output can then be screened for selected taxa of interest to retrieve taxon-specific SNPs.

In total, the complete mitochondrial genome sequence of 13 tree species (see Section 2.2) were included in the analysis presented in this study. Because the *Betula pendula* genome (LT855379) was only available without annotation, the sequence was submitted to the GeSeq server [86,93] for gene calling and functional annotation with NC\_028096 as a reference. The resulting GenBank file was used as input for the SNPtax analysis beside the GenBank files of the other 12 species.
