*2.2. Identification of Potentially Taxon-Specific SNPs in Mitochondrial Genes*

In this study, the software SNPtax was developed allowing for the identification of potentially taxon-specific SNPs in mitochondrial genes using GenBank files of related species and outgroup species of interest as input. The software identifies SNPs specific for a pre-defined taxon based on multiple alignments of genic sequences extracted from the GenBank files together with related taxonomic information. SNPtax is freely available on https://github.com/tsciow/SNPtax.

In the search for SNPs that are potentially specific for different taxa within the Fagales, the GenBank files of the following 13 tree species were used as an input for SNPtax analysis: three Fagales species, i.e., *F. sylvatica* (GenBank Acc. MT446430; mitochondrial genome assembled and annotated in this study), *Quercus variabilis* (MN199236), and *Betula pendula* (LT855379; draft annotation in this study, see Materials and Methods) as well as 10 non-Fagales species of deciduous trees and conifers, i.e., *Bombax ceiba* (NC\_038052), *Eucalyptus grandis* (NC\_040010), *Lagerstroemia indica* (NC\_035616), *Populus alba* (NC\_041085), *Populus davidiana* (NC\_035157), *Populus tremula* (NC\_028096), *Populus tremula* x *Populus alba* (NC\_028329), *Liriodendron tulipifera* (NC\_021152), *Ginkgo biloba* (NC\_027976), and *Pinus taeda* (NC\_039746).

In total, 18 protein-coding genes were identified that are annotated in all 13 species. These genes were considered for the identification of potential taxon-specific SNPs. Only SNPs in conserved regions were taken into account. This way, we could identify 30 SNPs in 11 genes potentially specific for *F. sylvatica*, 29 SNPs in nine genes for Fagaceae (potential Fagaceae-specific allele occurred only in the sequences of *F. sylvatica* and *Quercus variabilis*; see above), and 27 SNPs in nine genes for Fagales (specific allele only in *F. sylvatica, Quercus variabilis* and *Betula pendula*). All SNPs potentially specific for *F. sylvatica*, Fagaceae, and Fagales are summarized in Table S3. The SNPs are located in 13 different mitochondrial genes.
