| Summary: | (1) Background: The purpose of this study was to compare the free volatile compounds of 18 <i>Veronica</i> species (Plantaginaceae), as previously analyzed by gas chromatography coupled with mass spectrometry, with their DNA sequences for internal transcribed spacers ITS2 and ITS1-5.8S-ITS2 of the nuclear ribosomal DNA. (2) Methods: Two sets of DNA sequence data were generated and used for phylogenetic analysis: ITS2 sequences (~360 bp) obtained by next-generation sequencing and ITS1-5.8S-ITS2 sequences (~580 bp) sequenced by the Sanger sequencing method. Clustering from previously analyzed free volatile compounds was performed by Ward’s method. (3) Results: Both sets of DNA sequence data showed that the 18 analyzed <i>Veronica</i> species were grouped into eight main groups corresponding to the following subgenera: <i>Pentasepalae</i>, <i>Pocilla</i>, <i>Chamaedrys</i>, <i>Veronica</i>, <i>Beccabunga</i>, <i>Cochlidiosperma</i>, <i>Stenocarpon</i> and <i>Pseudolysimachium</i>. Results of the clustering analysis of free volatile compounds showed better clustering when using microwave-extracted volatiles. Three clusters were detected with the following main compounds: hexahydrofarnesyl acetone, hexadecanoic acid, phytol, caryophyllene oxide and <i>(E)</i>-caryophyllene. (4) Conclusion: The phylogenetic analysis of ITS2 data obtained by NGS technology and ITS1-5.8S-ITS2 data obtained by Sanger sequencing resulted in the grouping of 18 <i>Veronica</i> species into eight subgenera, which is in accordance with the existing classification. Statistical testing showed that there was no correlation between such clustering of <i>Veronica</i> species and clustering that was based on free volatile compounds. The achieved results can be viewed in the light of parallel evolution among some of the species of the <i>Veronica</i> genus as well as the fact that volatile compound composition can be influenced by environmental factors or epigenetic modifications.
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