# Molecular and Functional Evolution of the Spermat Sesquiterpene Synthases
## 2 Results
### The Phylogeny of Spermat STSs in the TPS-a Subfamily  
TPS-a is the angiosperm-specific subfamily, which dominates the STS family in spermatophyte. The expansion of TPS-a subfamily may occur after the split of the monocot and dicot lineages by gene duplication and divergence, because of the obvious species-clustered phenomenon. The TPS-a clade is comprised of 282 representative STSs (Figure 7), in which monocotyled and dicotyledon STSs clustered separately, indicating the common ancestor. In the monocot clade (red), STSs from _Zingiberales_ and _Poles_ are clustered separately (Figure 7), suggesting large-scale gene duplications which probably occurred not long ago. STSs are obviously functionally clustered in the _Poles_ clade, including the 1,6-cyclized product branch (black) of representative bisabolene and 1,11-cyclized product branch (blue) of representative (E)-\(\beta\)-caryophyllene. All functionally characterized dicotyledon STSs in the TPS-a subfamily are from core eudicots, including _Rosanae_ (blue) and _Asterids_ (yellow). The state of clustered STSs in angiosperms is consistent with plant species classification, which indicates that STSs are developing with species evolution.  
Figure 6: (**a**) The phylogeny of spermatophyte STSs in the TPS-g subfamily. STSs with diiterpene synthases function in vitro are shown by red branches and uniformly distributed in the phylogenetic tree. (**b**) The phylogeny of spermatophyte STSs in the TPS-b subfamily. STSs with monoterpene synthases function in vitro are shown by red branches. STSs producing 1,6-cyclized sesquiterpenes and 1,10/1,11-cyclized sesquiterpenes are marked with grey dots and red dots, respectively. Products of STSs marked with black rectangles are acyclic farnesene or nerolidol.  
TPS-a contains two major clades, among which, Clade II has more members: members in the A1 and A2 clades are all from _Asterales_ and members in the R1 clade are mainly from _Rosanae_. In the A1 clade, STSs catalyzing initial 1,6-ring closures are mainly distributed in _Artemisia_ (grey dot), and the other STSs mainly catalysis initial 1,10-ring closures. A few of 1,6-cyclized STSs (grey dots) are distributed in the A2 and R1 clades, and the others are 1,10/1,11-cyclized STSs, among which some neighbor STSs produce 1,10- or 1,11-cyclized products, respectively (marked with black rectangles), suggesting that conversion of these two structures are common in STS evolution. Although Clade I has fewer members, it contains all orders of _Asterids_ and the most important fabids and malvids of the _Rosanae_. It can be considered that their ancestral proteins appeared early before the large-scale differentiation of dicotyledons. Interestingly, almost all germacrene A synthases (GASs) fell into this clade. Germacrene A is an intermediate catalyzed by several STSs to form endesmane-type sesquiterpene [31], the characteristic products of _Asteraceae_ and _Celstrates_. Germacrene A and (E)-\(\beta\)-farnesene are both aphid alarm pheromones and probable ancestral forms of plant sesquiterpenes, which naturally dovetails with the function-driven hypothesis.  
Figure 7: The phylogeny of spermatophyte STSs in the TPS-a subfamily. Blue shadow represents the _Rosanae_ STSs, yellow shadow represents the _Asterids_ STSs, red shadow represents the _monocot_ STSs, and orange shadow represents the _magnoliid_ STSs. Red branches show the STSs with identical products, blue branches show the STSs with 1,11-cyclized functions and green branches show the probable HGT members from _Santalum album_. STSs producing 1,6-cyclized sesquiterpenes are marked with grey dots, and the neighbor STSs producing 1,10- or 1,11-cyclized products, respectively, are marked with black rectangles.