# Molecular and Functional Evolution of the Spermat Sesquiterpene Synthases
## 3 Discussion
For cyclic sesquiterpenes, they can be formed by typically catalyzing reaction cascades with additional steps, such as the isomerization of carbon-carbon double bond in the initial cation to allow alternate ring closures or additional cyclization [9]. Successive gene duplications and the subsequent accumulation of mutations led to the multitude of STSs, many of which catalyze more complex reactions than the ancestor. Although it is universally accepted that evolution of natural product biosynthesis has led to the formation of more and more complex structures, this process has rarely been documented at the level of a specific enzyme and plant group [45]. Overall, we speculated the early possible evolutionary process of spermatophyte STSs is from acyclic sesquiterpenes to cyclic sesquiterpenes, and the C6-C1 closure sesquiterpenes(small rings) may have formed earlier than C10-C1/C11-C1 closure sesquiterpenenes. Interestingly, in some specific STSs, evolution may stop at a certain stage to form a series of characteristic metabolites under certain selective pressures. For example, _Artemisia_ species' STSs are clustered in the A1 clade of the TPS-a subfamily (Figure 7) and obviously originated from a common ancestor dedicated to producing 1,6-cyclized sesquiterpenes. Among these, AaADS from _Artemisia annua_ produces the artemisinin specific intermediate amorpha-4,11-diene (C6-C1 cyclized bicyclic sesquiterpenes) [46]; however, other STSs highly homologous to ADS from _Artemisia_ species cyclize FPP to (+)-a-bisabolol (C6-C1 monocyclic sesquiterpenes) [47]. Systematic study on the evolutionary changes of STS structure is an effective way to elucidate its function. Over the last three decades, high-resolution crystal structures have become available for STSs, and the enzymatic structure-function relationships have revealed the evolutionary relationships of STSs [1; 12]. It was recognized early that the structure of STS products depending on the initial substrate conformation imposed by the enzymatic active site cavity [48]. For example, _SaSQS2_ is the representative of C6-C1 cyclized STSs, and the shape of FPP conformation is close to natural straight chain in its active site cavity [49]. For C10-C1 cyclized STSs of _TEAS_[50] and _XC1_[51], the shapes of FPP are obviously curved. For the formation of acyclic sesquiterpene, we choose the medium/long-chain-length prenyl pyrophosphate synthase as the representative because there is no crystal structure of acyclic STSs, in which the FPP conformation is almost natural straight chain [52] (Figure S2). Overall, in the evolution of STSs, the change of residues in the active site cavity made the straight chain FPP gradually bend, which made the C1 carbocation gradually approach the intramolecular double bond, and endowed STSs with the ability to form small ring and even large ring sesquiterpenes. Horizontal gene transfer (HGT) also plays an important role in the evolution of STSs [53]. Sixteen _Santalum_ STSs have been functionally charactered (Table S3), among which, 13 STSs are clustered in TPS-b and their products are mainly C6-C1 cyclized sesquiterpene \(\beta\)-bisabolene (Figure 6b), while three STSs are clustered in the R1 clade of the TPS-a subfamily and their products are mainly C10-C1 cyclized sesquiterpenes germacrene D-4-ol, hedycayol and C11-C1 cyclized sesquiterpene a-humulene (Figure 7). This indicates that the evolutionary sources of STSs in these two parts are completely different. Sandalwood is a semi-parasitic plant, whose survival is inseparable from the host plant. Some studies have shown that sandalwood prefers to parasitize on nitrogen fixing woody plants [54], and HGT events between sandalwood and host plants have also been reported [55; 56], which may indicate that STSs in sandalwood come from HGT. These parasitic plants are likely to obtain the synthesis ability of terpenenes and other secondary metabolites from the host through HGT, so as to better adapt to the environment or communicate with the host.