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Key message The investigation provides initial knowledge on the distribution and evolution of the high and low methyl salicylate-producing trait in the    Betula    genus. Mislabelled birch species could be identified and removed.  The genus  Betula  is the largest group of ecologically and economically dominant perennial woody plants in subalpine forests. The taxonomy of  Betula  is complex due to an extensive history of hybridization and periodic introgression events among the species .  Although almost all land plants including birches produce methyl salicylate (MeSA) as a signaling molecule and in response to stress (“low MeSA producer”), some birch species produce high amounts of MeSA in the leaves and bark (“high MeSA producer”). Unfortunately, the evolution of high levels of MeSA production in the genus  Betula  remains unclear. The salicylic acid-binding protein 2 ( SABP2 ) and salicylic acid methyltransferase ( SAMT ) genes involved in MeSA biosynthesis were incorporated into this study to examine the interspecific relationship of high and low MeSA-producing birches. Additionally, eight chloroplast and three nuclear regions were included to evaluate their potential application in species determination. The analysis resulted in 25 and 61 nucleotide variations, respectively, which allowed for a visualization of the genetic architecture in the 18  Betula  species investigated. The high MeSA-producing  B. lenta, B. grossa,  and  B. alleghaniensis  formed the basal clade in the phylogenetic analysis, thus revealing their ancestral status, and the network analysis postulates that the diploid  B. lenta  is one of the ancestors of the genus  Betula . The results indicate that the ability to produce high levels of MeSA that were initially present in the genus has been lost several times during its evolution. Placing species of the subgenus  Acuminata  alongside the subgenus  Betula , together with a fragrance analysis, questions their ability to produce high levels of MeSA.

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Species determination and phylogenetic relationships of the genus Betula inferred from multiple chloroplast and nuclear regions reveal the high methyl salicylate-producing ability of the ancestor