# Molecular and Functional Evolution of the Spermat Sesquiterpene Synthases
## 2 Results  
### Dataset of Characterized Spermat STSs  
To obtain a comprehensive set of functional STSs, we searched for the public databases, a subset of UniProt, NCBI, and Database of Characterized Plant Sesquiterpene Syntheses ([[http://www.bioinformatics.nl/sesquiterpene/synthasedb/](http://www.bioinformatics.nl/sesquiterpene/synthasedb/)]([http://www.bioinformatics.nl/sesquiterpene/synthasedb/](http://www.bioinformatics.nl/sesquiterpene/synthasedb/))) in 10 January 2021, and manually reviewed literature linked to enzymes with the characteristic STS domain. Overall, we present a dataset of 474 manually curated characterized plant STSs. Among these, 421 STSs have sequence information, including 27 STSs from non-seed plants, 29 STSs from gymnosperm, and 365 STSs from angiosperm. The information of the 474 STSs, including gene name, species origin, GenBank, products (major and minor), and product type, is presented in Tables S1-S3. The dataset supports searching and sorting of all or subsets of the data. To investigate the functional evolution of plant STSs, our dataset covered not only typical plant STSs, but also monoterpene and diterpene synthases with STS ability. The phylogenetic tree of all 421 plant STSs (Figure 2) showed that non-seed plant STSs (green) clustered obviously, and they were relatively independent from other plant STSs. The gymnosperm STSs (blue) also clustered in a single clade, which showed the evolutionary difference between them and angiosperm STSs.  
We manually curated 447 characterized spermatophyte plant STSs, among which 394 STSs have sequence information (Tables S2 and S3). This is the largest and most diverse group of STSs currently known. The structure-based phylogenetic tree of all 394 spermatophyte STSs (Figure 3) showed that most angiosperm STSs belong to the TPS-a subfamily (red), while gymnosperm STSs belong to the TPS-d subfamily (green). In addition, a few STSs can be summarized as TPS-f (blue), TPS-g (pink), and TPS-b (light blue) subfamilies (Figure 3 outer ring). In order to show the evolutionary position of STSs in each clade more clearly, we selected 35 representative STSs (shaded in Tables S2 and S3) based on the clade location, species source, and product type to construct global phylogenetic tree (Figure 3, inner ring). The phylogeny of spermatophyte STSs is basically consistent with that of all plant TPSs [16]. The diterpene synthase clade (TPS-f) is the closest to the ancestral clade TPS-c, indicating that it might evolve from the ancestral bifunctional copalyl diphosphate synthase/_ent_-kaurene synthase (CPS/KS) [17]. Although gymnosperm STSs (the TPS-d clade) and angiosperm STSs derived from a common ancestor, they have an obvious evolutionary divergence, corresponding with the speculation that gymnosperms are monophyletic groups, which are far away from angiosperms [18]. The TPS-g clade contains many bifunctional monoterpene/sesquiterpene synthases, having many characteristics of the evolutionary transition state [16]. The neighbor clade TPS-b contains more actual monoterpene synthases, which indicates that most angiosperm STSs originated from a recent common ancestor and the ancestral protein gained the ability to produce sesquiterpenes through the continuous evolution from monoterpene synthases.