## 3 Discussion
### The Plant Basis Difference between B. marginatum var. stenophyllum and Other Four Species  
This study presents, for the first time, the differences in volatile oil contents among medicinal species/varieties of the genus _Bupleurum_. _B. scorzonerifolium_ [PERSON]. showed the highest volatile oil content, followed by _B. smithii_ [PERSON] and _B. marginatum_ var. _stenophyllum. B. chinense_ DC. and _B. marginatum_ Wall. ex DC. showed the lowest volatile oil contents, which was less than 1/5 of _B. scorzonerifolium_ [PERSON]. We speculated that the reason for these significant differences is related to the geographical location of the plant habitat. Volatile oil is also an important secondary metabolite product that both the above- and below-ground parts of the plant generate in response to external stimuli [37]. Recently, extensive studies have revealed its role in plant abiotic stress resistance, especially in cold resistance. It has been shown that volatile compounds could ensure the survival of plants under severe cold conditions by maintaining membrane integrity and fluidity through the accelerated synthesis of abundant unsaturated fat [38]. Studies in several medicinal plants, such as _Litsea cubba_, _Camellia sinensis_, and _Astragalus mongholicus_, have shown that the levels of their volatile oil are most positively correlated with higher latitude or lower annual average temperature [37; 39; 40; 41]. The evolution research of the genus _Iuglans_ through pan-genome analyses further revealed that in eastern black walnut, with the sharp reduction of the effective population during the glacial climate, there was a large number of InDel sites in the genes involved in fatty acid synthesis [42]. Together, all these pieces evidence confirmed that volatile components such as volatile oil are indispensable for the response of plants to cold. In this study, the volatile oil contents exhibited significant positive correlation with latitude; that is, _B. scorzonerifolium_ Wild., with the highest volatile oil content, is located in the region with the highest altitude and latitude (1800 m, 35\({}^{\circ}\)40\({}^{\prime}\) N), _B. marginatum_ Wall. ex DC., with the lowest volatile oil content, is located in the region with the low altitude and latitude (350 m, 29\({}^{\circ}\)45\({}^{\prime}\) N), and so on (Table 2). The ranking of the annual average temperature of the above regions was also similar as that of altitude or content. Thus, the temperature difference based on altitude and latitude might be the important reason underlying the differences in volatile oil content among different _Bupleurum_ species/varieties. Moreover, we demonstrated that for medicinal plants, in addition to the widely studied volatile oils in the above-ground parts, the volatile oils in the roots are also highly sensitive to environmental temperature. Interestingly, in contrast to the ranking of volatile oil content, the odor analysis of dried samples using e-nose technology showed that the strongest response on the volatile organic compound sensor (S8 in Figure 1B) was from _B. marginatum_ Wall. ex DC., which come from the region with the lowest latitude, while the weakest response was from _B. scorzonerifolium_ Wild., which is located in the region with the highest latitude. This result is consistent with the fact that aromatic plants are more common in subtropical and tropical regions as they need to attract insect pollination over a longer reproductive period. The above results indicated that there are significant differences in the volatile compounds of the _Bupleurum_ species/varieties, which might be related to the environmental temperature. As shown by our metabolomic analysis (Figure 3D), there are sharp differences between _B. marginatum_ var. _stenophyllum_ and other species in the contents of many volatile oil components. To explore the underlying molecular mechanism, transcriptome sequencing was conducted, and numerous genes involved in the biosynthesis of volatile components were found to be only differentially expressed in _B.