# Molecular and Functional Evolution of the Spermat Sesquiterpene Synthases
## 2 Results
### The Phylogeny of Spermat STSs in the TPS-b/g Subfamily  
Based on current knowledge, the TPS-b and TPS-g clades are the angiosperm-specific subfamilies [16]. They are closely related to the TPS-a clade and have substantially diverged from the other TPS clades (Figure 3). The TPS-g clade is comprised of 37 members producing acyclic products (Figure 6a), which are almost all bi- or tri-functional TPSs [21]. The STSs that can produce diterpenes in vitro (red branches in Figure 6a) are evenly placed in the TPS-g clade, indicating that the TPS-g ancestor might be a multifunctional diterpene synthase derived from TPS-f proteins. The TPS evolution involves not only a change in substrate specificity but also in subcellular localization. A relative recent example of such an event may exist in the _Antifrntinum majus_ bifunctional TPS _AmNES/LIS-2_ sharing 95% amino acid sequence identity with _AmNES/LIS-1_, which has an additional 30 amino acids in the N-terminus, leading to its localization in plastids and accounting for linalool formation, whereas _AmNES/LIS-1_ is localized in cytosol and is responsible for nerolidol biosynthesis [27]. Another good example of neofunctionalization of duplicated STS genes involving a change in subcellular localization is the pair of _CsLIS/NES-1_ and _CsLIS/NES-2_[28]. In contrast to the cytosolic STSs, most mono- and diterpene synthases have obvious N-terminus plastid transit peptides. The phylogeny of STSs in the TPS-g subfamily indicates that ancestral diterpene synthase gradually evolved to form monoterpene synthase and then to form acyclic STSs.  
The embryonic form of STSs was evolved by the expansion of the substrate spectrum. Then, STSs having a broad substrate spectrum undergo variable splicing or exon loss resulted in losing N-terminus plastid transit peptides, and gradually formed monofunctional STS under the restriction of FPP substrate pool.  
Three multifunctional TPSs of _PITPS2_, _PITPS3_, and _PITPS4_ from _Phaseolus lunatus_ phylogenetically clustered into the TPS-g subfamily. They are likely derived from a duplication of an ancestral gene followed by subfunctionalization. _PITPS3_ can convert FPP to (E)-nerolidol, which is the precursor of the homotrepene (_E_)-4,8-dimethyl-1,3,7-nonatriene (DMNT) [29]. _PITPS2_ and _PITPS4_ can produce (_E,E_)-geranyllinalool from GGPP. Homotrepene (_E,E_)-4,8,12-trimethyl-1,3,7,11-tridecatriene (TMTT) is then produced from (_E,E_)-geranyllinalool [29]. DMNT and TMTT are herbivore-induced plant volatiles, which enables the plants to attract natural enemies of herbivores [30]. Sesquiterpenes probably evolved gradually during the plants coping with insects and other environmental pressures. The phylogenetic position and physiological function of TPS-g STSs may suggest that (E)-nerolidol is the early form of plant sesquiterpenes.  
TPS-b (Figure 6b) is the sister group of TPS-g with more complicated evolutionary history, fortunately displays clustering characteristic of product structures. The outside Farnesene-branch contains 7 farresene synthases from different sources, and most of the other STSs produce 1,6-cyclized sesquiterpenes (members in bisabolyl cation-branch and marked with grey dots). Three 1,10- or 1,11-cyclized STSs are sporadically distributed in  
Figure 5: The phylogeny of spermatophyte STSs in the TPS-d subfamily.  
the phylogenetic tree (red dot). Meanwhile, many TPS-b members have the activity of monoterpene synthase in vitro (red branches in Figure 6b), leading the TPS-b subfamily to present a transitional state of different product types of monoterpenes and sesquiterpenes. Therefore, we speculated that the possible evolutionary process of TPS-b members was similar to that of TPS-d members, that is, the chain products gradually evolved to form small rings (C6-C1 closure) and then to form large rings (C10-C1 and C11-C1 closure).