*4.10. Validation of the CAPS Markers*

The Fagales-specific marker 5\_Fagales\_*matR* was tested for "Fagales-specificity" using four families with ten genera, 41 species, and 75 individuals within the order Fagales, and additionally, nine other orders including 11 families, 17 genera, and 25 species with one individual each (Table S5). For validation of the Fagaceae-specificity of the marker 4\_Fagaceae\_*nad7*, three genera, and 23 species with 57 individuals from the family Fagaceae, and three further families within the order Fagales using seven genera and 15 species were used. Additionally, outside the Fagales, nine orders including 11 families, 17 genera, and 26 species and individuals were tested for validation of this family-specific marker. For validation of both *Fagus*-specific markers—3\_Fagus\_*matR* and 3\_Fagus\_*ccmFc*—58 or 60 *Fagus* individuals were used, respectively. Outside from the genus *Fagus*, further 76/79 individuals from 55/57 species in 25 genera, 13 families, and eight orders were tested (Table S5).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2223-7747/9/10/1274/s1, Figure S1: validation of the mitochondrial genome sequence of *F. sylvatica* (MT446430) with long Nanopore MinION reads by mapping of corrected reads (>10,000 bp) to the final assembly (Fasyl\_mt: mtDNA of *F. sylvatica*; mapped reads in green: forward reads; mapped reads in read: reverse reads). Figure S2: creation of start codons (related triplet underlined) by RNA editing in transcripts of *nad4L, cox1*, and *nad1* detected in mappings of RNA-Seq data from *F. sylvatica* to the mitochondrial genome (MT446430). Figure S3: comparison of the gene order in the mitochondrial genomes of *Liriodendron tulipifera* (NC 021152), *F. sylvatica* (MT446430), and *Quercus variabilis* (MN199236) using geneCo ([89,90]; only potential protein-coding genes without trans-splicing were compared). Figure S4: The *ccmB*/*rpl*10-gene cluster in the mtDNA of three Fagales species (detail enlargement of the visualization of GenBank files using CLC-GWB; *F. sylvatica*, MT446430; *Quercus variabilis,* MN199236, and draft annotation of *Betula pendula*, LT855379.1 in File S4; \*: reverse complement of the annotated *Betula pendula* mtDNA sequence is shown to obtain a uniform presentation of the gene cluster). Table S1: lists of interspersed repeats in the mtDNA sequences of *F. sylvatica* (MT446430), *Quercus variabilis* (MN199236), and *Betula pendula* (LT855379.1) identified using the tool "ROUSFinde1\_1" [26]. Table S2: summary of results of BlastN analyses of the DNA sequences of all *F. sylvatica* interspersed repeats versus *Quercus variabilis* or *Betula pendula* repeats, respectively (BlastN details in Files S3 and S4; detailed information to the repeats in Table S1). Table S3: mitochondrial SNPs with target alleles that are potentially specific for *F. sylvatica*, Fagaceae, or Fagales, respectively (SNPs used for CAPS markers highlighted in yellow; based on multiple alignments of DNA sequences of 18 mitochondrial genes from 13 species of deciduous trees and conifers; SNP positions related to *Populus tremula* genes; GenBank NC\_028096). Table S4: primer sequences for mitochondrial CAPS markers developed in this study (see Table 1). Table S5: details on individuals used for validation of the four mitochondrial CAPS markers developed in this study (see Table 1). File S1: result of BlastN analysis of the DNA sequence of the largest repeat from *Quercus variabilis* to the mtDNA sequence of *F. sylvatica* (MT446430). File S2: result of BlastN analysis of the DNA sequence of the largest repeat from *Quercus variabilis* to the mtDNA sequence of *Betula pendula* (LT855379.1). File S3: results of BlastN analyses of the DNA sequences of all *F. sylvatica* repeats versus *Quercus variabilis* repeats. File S4: results of BlastN analyses of the DNA sequences of all *F. sylvatica* repeats versus *Betula pendula* repeats. File S5: draft—GenBank file of the *Betula pendula* mitochondrial genome created by draft annotation of the mtDNA sequence LT855379.1 using GeSeq [86,93].

**Author Contributions:** Conceptualization, B.K. and H.S.; methodology, M.M., H.S., T.S., K.S.-S., and B.K.; software, M.M., T.S., and K.S.-S.; validation, H.S. and B.K.; formal analysis, M.M., T.S., K.S.-S., H.S., and B.K.; investigation, M.M., H.S., B.K., T.S., K.S.-S., A.P.L.M., H.L., M.L., and B.F.; resources, H.L. and M.L.; data curation, M.M., T.S., K.S.-S., and A.P.L.M.; writing—original draft preparation, B.K, M.M., H.S., T.S., A.P.L.M., and B.F.; writing—review and editing, all authors; visualization, B.K. and H.S.; supervision, B.K., H.S., H.L., M.L., and B.F.; project administration, B.K., H.S., H.L., and B.F.; funding acquisition, B.K., H.S., B.F., and H.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the German Federal Environmental Foundation, grant number 33949/01 (project "Wood DNA barcoding"); by the FEDERAL Ministry of Food and Agriculture, and the Federal Ministry for the Environment, Nature Conservation and Nuclear Safety in the research program "Waldklimafonds", grant number 28W-C-4-092-10 (project "GenMon"); by the BAVARIAN MINISTRY FOR FOOD, AGRICULTURE AND FORESTRY, grant number P31; and by core funding of the THÜNEN INSTITUTE.

**Acknowledgments:** We are very grateful to Stefanie Palczewski and Marie-Fee August for technical assistance. We also thank the Morton Arboretum and the North Carolina State University for providing samples of North American oaks. For the Asian oak species, we thank the Bashkirian State Agrarian University and the Korea National Arboretum. Furthermore, we thank the Botanical Gardens of Bayreuth, Bochum, Eberswalde, Goettingen, Marburg, and our former colleague Lasse Schindler who provided us with material of different species.

**Conflicts of Interest:** The authors declare no conflict of interest.
