## 2 Results
### Phylogenetic Analysis of DoTPS Genes in D. officinalae  
To gain further insight into the evolutionary relationships among the TPS subfamilies, an unrooted phylogenetic tree was constructed using the neighbor-joining (NJ) method implemented in MEGA 7.0 with the Jones-Taylor-Thornton (JTT) model based on multiple sequence alignment of TPS members from _Abies grandis_, _A. thaliana_, _Apostasia shenzhenica_, _D. officinalae_, _Oryza sativa_, _Phalaenopsis equsstris_, _Populus trichocarpa_, _Selaginella moellendorffii_, _Solanum lycopeneicum_, and _Sorghum bicolor_ (Figure 3, Table S6). The phylogenetic tree demonstrates that TPS proteins were clustered into seven subfamilies (TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h) according to the published report [1; 15]. Thirty-four DoTPS proteins appeared in only four groups (TPS-a, TPS-b, TPS-c, and TPS-e/f, 14, 16, 1, and 3, respectively), and 88.2% of them belonged to TPS-a or TPS-b subgroups. Similarly, there were 20 and 9 TPS proteins present in _P. equestris_ and _A. shenzhenica_, the amount of TPS-a, and TPS-b accounted for 12/20, and 7/9, respectively (Figure 3). This phenomenon was similar to that in _A. thaliana_ and _O. sativa_[1; 10]. Remarkably, in the TPS-a group, dicotyledonous and monocotyledonous plants formed distinct subgroups, which were observed previously [1; 15] and termed them as TPS-a1 (dicots) and TPS-a2 (monocots), suggesting that the _TPS-a_ genes evolved independently. Similar to the TPS-a group, the TPS-c group was further divided into dicot and monocot subclades. Consistent with a previous report, the TPS-d subfamily was specific to gymnosperms, and the TPS-h subfamily was only observed in _S. moellendorffii_[1; 11; 12; 17], inferring that they might play a particular role in these species.  
Multiple sequence alignment of DoTPS proteins was further analyzed. As illustrated in Figure 4, the arginine-tryptophan motif, R(R)X\({}_{8}\)W, was found in all the DoTPS-a and DoTPS-b proteins, except DoTPS8, at the \(N\)-terminus. It plays a role in initiation of the isomerization cyclization reaction [1; 10]. However, the arginine-tryptophan motif, R(R)X\({}_{8}\)W, varied or was even absent in TPS-c and TPS-e/f proteins. Two aspartate-rich motifs, DDxxD and NSE/DTE, are essential for cleaving prenyl diphosphate substrate by chelating a trio of Mg\({}^{2+}\) or Mn\({}^{2+}\) at the C-terminus [9; 11]. The DDxxD motif was conserved in almost all the DoTPS proteins, except three DoTPS proteins (DoTPS1, DoTPS8 of the TPS-a group, and DoTPS31 of the TPS-c group). DoTPS31 was the only TPS-c member in _D. officinalae_. The TPS-c subfamily is mainly found in land plants and its prenyl diphosphate unit is not cleaved [1]. The NSE/DTE motif was absent in DoTPS8, -11, and -12 of the TPS-a group, DoTPS1, -19, and -24 of the TPS-b group, and DoTPS32 of the TPS-e/f group (Figure 4). Taken together, gene structure and amino acid alignment were consistent with the phylogenetic analysis. The functions of DoTPS proteins in the same group could be inferred from known TPS proteins, according to their phylogenetic relationships.  
Figure 3: Phylogenetic analysis of TPS proteins in ten higher plant species. A phylogenetic tree was constructed by the neighbor-joining method with the Jones–Taylor–Thornton model and pairwise deletion option using MEGA 7.0 with 1000 bootstrap replicates. Tree visualization and labeling was performed on FigTree v1.4.4 ([[http://tree.bio.ed.ac.uk/software/figtree/](http://tree.bio.ed.ac.uk/software/figtree/)]([http://tree.bio.ed.ac.uk/software/figtree/](http://tree.bio.ed.ac.uk/software/figtree/))). The TPS family was divided into seven subfamilies as previously reported [1,15]: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h. Circles represented monocotyledonous plants, squares represented dicotyledonous plants, the cyan diamond indicates _Abies grandis_, and yellow triangle indicates _Selaginella moellendorffii_.