Patterns of Genetic Variation of Nepeta nuda L. from the Central Balkans: Understanding Drivers of Chemical Diversity

Abstract

Nepeta nuda L., a notable medicinal species in the tradition of the Balkan region, is a rich source of bioactive iridoids and phenolics previously described as high-resolution taxonomical classifiers for the genus Nepeta. However, their potential in investigating intra-species differentiation is here described for the first time. The aim was to recognize the sources of natural chemical diversity and their association with the genetic variability both within and among N. nuda populations in the Central Balkans. Chemical diversity was assessed from methanol extracts and essential oils through untargeted and targeted metabolomics using state-of-the-art analytical tools, covering a broad spectrum of compounds that represent the N. nuda metabolome. We found that chemodiversity primarily resides within populations of N. nuda, and similar results were obtained at the DNA level using microsatellite markers. The low genetic and chemical differentiation of the studied N. nuda populations implies that their metabolomic profiles may be less influenced by geographic distance and variable environmental conditions within the Central Balkans, as they are under the pivotal control of their genetic backgrounds. Screening the distribution of the major bioactive compounds belonging to phenolics (phenolic acids and flavonoids) and iridoids (both aglycones and glycosylated forms), within and among N. nuda populations, is able to guarantee mass spectrometry-based tools for the selection of elite representative genotypes with practical importance. The knowledge acquired will allow us to delve deeper into the molecular background of N. nuda chemical diversity, which is the course of our further work.

Keywords: GC-MS; Lamiaceae; Nepeta nuda; UHPLC/QToF MS; chemodiversity; genetic variability; microsatellites; populations.

Figure 1

Map representing the populations of Nepeta nuda L. originating from the central Balkan Peninsula analyzed in the present study. For the coordinates, please refer to Table 4. Plant photos denote N. nuda individuals captured on site.

Figure 2

(A) Chemical structures of characteristic iridoid glycosides identified in Nepeta nuda methanol extracts (Glc—glucose or another hexose). (B) Heatmap of the scaled QToF MS data with the samples arranged according to the hierarchical cluster analysis (Spearman’s method of cluster agglomeration). (C) Principal component analysis (PCA) biplot constructed based on the QToF MS data, with the first two PCs explaining 49.04% of the total variance among N. nuda accessions. Participation of the variables (compounds) in the first two PCs is indicated by the corresponding loading plots.

Figure 3

(A) Representative TIC GC/MS chromatograms of 11 samples of EOs originating from Central Balkans populations of Nepeta nuda. (B) Heatmap of the scaled GC/MS data with the samples (both populations and compounds) arranged according to the hierarchical cluster analysis and adopting the Spearman’s method of cluster agglomeration. (C) Principal component analysis (PCA) biplot constructed based on the GC/MS data, with the first two PCs explaining 84.48% of the total variance among N. nuda accessions. Participation of the variables (compounds) in the first two PCs is indicated by the corresponding loading plots.

Figure 4

(A) UHPLC/(±)HESI-MS² quantitative data of targeted iridoids (green bars) and phenolics (orange bars) in methanol extracts of Nepeta nuda originating from 11 Central Balkans populations. Mean values ± SE are presented. Values labeled with different letters are significantly different (p < 0.05) according to post hoc Tukey’s test of one way ANOVA. (B) Principal component analysis (PCA) biplot constructed based on the quantitative data, with the first two PCs explaining 99.52% of the total variance among N. nuda accessions. For eleven N. nuda populations, 95% confidence ellipses are presented, colored according to the symbol legend. Participation of the variables (compounds) in the first two PCs is indicated by the corresponding loading plots. (C) Heatmap of the scaled UHPLC/(±)HESI-MS² quantitative data with populations and compounds arranged according to the hierarchical cluster analysis and adopting the Pearson’s method of cluster agglomeration.

Figure 5

Genetic diversity of Nepeta nuda populations from the Central Balkans, as estimated by EST-SSR and SSR markers. (A) Analysis of molecular variance. (B) Pairwise F_ST values of eleven populations of N. nuda. The green-to-orange color scale denotes low-to-high genetic differentiation. (C) PCoA biplot representing genetic clustering of individuals belonging to eleven populations of N. nuda. Cumulative variation explained by the two coordinates is 29.33% (19.34% COORD.1 + 9.99% COORD.2). (D) Individuals’ recruitment of the four genetic clusters inferred by STRUCTURE.