## 2 Results and Discussion
### Statistical Analysis of the Obtained Results
Results consistent with PCA, HCA, and heatmap analysis were also obtained in supervised PLS-DA analysis. The PLS-DA scatter plot showed a clear differentiation among the two genetic groups of _C. suecica_, indicating that the groups differed in their composition and content of the detected volatile compounds (Figure 8). The model identified significant separation between the two groups (R\({}^{2}\)X = 0.98, Q\({}^{2}\) = 0.97, \(p\) < 0.001). PLS-DA analysis makes it possible to determine the importance of variables in projection (VIP), which are key to the separation of the tested samples into groups. Figure 9 presents 20 key VOCs (VIP > 1.40) differentiating C. _suecica_ samples belonging to different genetic groups. The higher the VIP result, the greater the contribution of the chemical compound to group separation. Among the chemical compounds indicated as the most important for distinguishing the studied groups, group 1, compared to group 2, was characterized by a reduced content of 14 compounds (**48**, **79**, **66**, **71**, **51**, **40**, **43**, **72**, **91**, **56**, **89**, **68**, **54**, and **70**) and an increased content of 6 compounds (**52**, **80**, **103**, **106**, **53**, and **61**). The importance of the above VOCs for the identification of _C. suecica_ groups was also confirmed by univariate analyses, such as fold change (FC), _t_-test, and volcano plot. According to the _t_-test (Table 2), the studied groups differed statistically significantly in the mean concentration of 78 chemical compounds, while the volcano plot, which combined the results of the fold change (FC) and _t_-test analyses into one single graph, showed significant differences for 64 VOCs: 38 sig. down, and 26 sig. up (Figures S6 and S7, Tables S7 and S8). The observed differences in the composition of volatile organic compounds in the two genetic groups of _C. suecica_[9] are so distinct that they can serve as a marker enabling the reliable identification of plants that cannot be recognized based on morphological features. Both groups of _C. suecica_ also differed clearly in their VOC composition from the other species of the genus _Calypogeia_ occurring in Europe analyzed so far [32; 33; 34; 36; 37]. The results of our study confirmed the high content of bisabola-2,10-diene [1,9]oxide (**80**) in _C. suecica_, a compound detected for the first time in this species by [PERSON] et al. [34]. It should be emphasized that bisabola-2,10-diene [1,9]oxide (**80**) occurred in both genetic  
Figure 5: Two-dimensional PCA scatter plot based on all 107 detected compounds in samples of _C. suecica_ collected in different years. The percentage of explained variance is 48.7% for PC1 and 14.3% for PC2. The year of sample collection is marked in different colors. Shaded ellipses indicate the 95% confidence regions. groups of _C. suecica_; however, the groups differed significantly in the average content of this compound. In group 1 it was 34.89%, while in group 2 it was several times lower, with an average of 3.20% (Tables 1 and 2). Group 2 _C. suecica_, unlike group 1, contained two unidentified compounds (RI = 1532, RI = 1594) in amounts of 11.64 and 16.17%, respectively. Both _C. suecica_ groups, similarly to _C. muelleriana_ and _C. azurea_, were found to include compounds of the azulene type [32; 36]. Azulenes are considered important chemical markers of _Calypogeia_ species [29]. According to previous studies, _C. fissa_ is dominated by acorane-type sesquiterpenes, which distinguishes this species from _C. suecica_[33]. In the case of _C. intefigristipula_, the dominant compounds are anastraptenes (15.61-25.26%) [37], which were also identified in _C. suecica_, but at a much lower level (4.69-7.13%). Bisabola-2,10-diene [1,9]oxide (**80**) is also present in _C. integristipula_, but in small amounts compared to _C. suecica_. The importance of the composition of volatile compounds as chemical markers has been proven in many studies of various liverwort species [16; 23; 24; 25; 26].