## 2 Results and Discussion
### Multivariate Analysis for Leaf Volatiles among Nine Cultivars  
Multivariate analysis is one of the most appropriate approaches to evaluate variations or diversity of leaf volatiles during leaf development from a range of cultivars. In the present study, the complete data set was selected for principle component analysis (PCA) and hierarchical cluster analysis (HCA) and was performed on average contents of all compounds from each cultivar regardless of developmental stage. The PCA horizontal and vertical axis explained 35.93% and 24.43% of total variance, respectively, and suggested the existence of six clusters from nine cultivars (Figure 4). The dendrogram based on Euclidean distance between cultivars performed on leaf volatiles showed four major groups, with subgroups in  
Group I included volatiles from young and mature leaves of pomelos, with E-ocimene and \(\beta\)-pinene as the major components (Figure 5), which is consistent with the data from PCA analysis (Figure 4). Group II contained two subgroups, with subgroup II-A represented by young leaves of Liubencheng and Qingjia and the mature leaf of Ponkan, comprised of linalool and substantial amounts of \(\beta\)-terpinene, and subgroup II-B, which exhibited a high percentage of linalool in young leaves of Ponkan and Hongshigan only (Figure 5). It was also noted that linalool, the main component of group-II, was present at \(<\)2% in all samples of group-I (Table 2). Group III, a \(\gamma\)-terpinene/\(\beta\)-pinene group, was dominated by a high amount of \(\gamma\)-terpinene in young leaves of Satsuma and in Huyou, or \(\beta\)-pinene in mature leaves of Hongshigan (Figure 5). Group IV could be divided into two subgroups, as well, with subgroup IV-A represented by volatiles of mature leaves of Satsuma, Qingjia and Liubencheng, characterized by a high amount of \(\alpha\)-citral and \(\beta\)-citral, and subgroup IV-B represented by young and mature leaves of Eureka lemon, with a high percentage of limonene as the major volatile (Figure 5). Notably, the separation of subgroup IV-B (Figure 5) was also observed from PCA analysis (Figure 4).  
These results revealed that the concentration of leaf volatiles is variable among citrus types, although an underlying intraspecific similarity and interspecific chemical polymorphism in leaf volatiles was found. A similar finding has been previously reported for the chemical polymorphism of citrus essential oils [22,51]. The segregation of cultivars based on leaf volatile profile is in agreement with phylogenic studies based on morphological and biochemical characteristics (DNA markers) with citron (_C. medica_), mandarin (_C. reticulata_) and pomelo (_C. grandis_), identified as the only true  
Figure 5: Hierarchical cluster analysis based on Euclidean distance performed on young (Y) and mature (M) leaf volatiles from nine cultivars. Coding names of cultivars: see Table 1.  
biotypes [52, 53]. A chemical variability was observed for Ponkan (_C. reticulata_) and Satsuma mandarin (_C. unshiiu_), and similar results have been previously reported [21, 22], which emphasized the high chemical polymorphism in mandarins in the Tanaka system [54], which classified mandarins into a different species. Our results, in association with data from previous studies, provide a new insight into leaf volatile variability among cultivars. These results can be helpful for characterization of citrus cultivars based not only on DNA fingerprints, but leaf volatile profiles, as well.