# Diversity and Functional Evolution of Terpene Synthases in Rosaceae
## 2 Results
### Varied Family Number of TPSs among Different Clades in Rosaceae  
To explore the evolution of TPSs in Rosaceae, we chose two representative species from each subfamily in Rosaceae for phylogenetic tree construction, including two Prunoideae species (_P. persica, P. mume_), two Maloideae species (_M. domesticia, P. betulifolia_), and two Rosociaceae species (_F. vesca, R. chinensis_). The detailed distribution of family numbers of different TPS clades is listed in Table 2. The TPS-a clade is the major determinant of family size of individual species; it is the largest group with more TPS copies, followed by TPS-b and TPS-g, TPS-c/e/f have a relatively small family size. On the whole, Rosoideae species owned more TPS copies, followed by Maloideae species, and Prunoideae species. The family number of TPSs from _R. chinensis_ is up to 57, of which 37 are TPS-a members. The six Rosaceae species that we chose nearly contained all five TPS clades; however, no TPS-f member was detected in _F. vesca_. The family number distribution of different TPS clades also varied among three Rosaceae subfamilies. For example, more than two TPS-c gene copies existed in the Maloideae and Rosoideae subfamilies, while only one copy was detected in Prunoideae species. The diversity of family number of TPS-f is also obvious; both _M. domesticia_ and _P. betulifolia_ owned three copies, while for Rosoideae species, only one copy was encoded in _R. chinensis_. For Prunoideae species, one copy of TPSs from the TPS-f clade was detected in _P. mira_ and _P. mume_, while three copies were detected in _P. persica_, indicating the recent expansion of the TPS-f clade in modern peach. The phylogenetic relationship revealed that lineage-specific expansions of the  
Figure 2: The unrooted phylogenetic tree of TPSs and motifs comparison between different TPS clades. (**A**) The maximum-likelihood phylogenetic tree of the TPS proteins in three Rosaceae species (_P. persica, M. domesticia, F. vesca_). The TPS-a, TPS-g, TPS-b, TPS-c, TPS-e, and TPS-f clades are shaded in blue, green, yellow-green, red, benzo, purple, respectively. (**B**) The frequency of different motifs among different TPS clades. (**C**) The seqLog of ‘DDxxD’ motif in the C-terminal domain of different TPS clades, the bit score represents the information content for each position in the sequence. TPSs were widely observed within different TPS clades. These expansions are denoted in the phylogenetic tree (Figures 3 and S2), the clustering relationship indicated that many expansions occurred after the split of sister lineages, such as the split of Maloideae and Prunoideae. Two Rosoideae species (_F. vesca_, _R. chinensis_) owned more lineage-specific expansions compared with the other two Rosaceae subfamilies, especially in the branch of TPS-a clade. On the other hand, in the lineage-specific expansion branch, except that many copies were shared by sister species, species-specific expansions were also detected widely. _R. chinensis_ was also the most striking species with more copies of TPSs arising by species-specific expansion. Figure 3: Phylogenetic tree of complete TPSs from six representative Rosaceae species. The TPS members were identified from six species including two Prunoideae species (_P. persica_, _P. mume_), two Maloideae species (_M. domestica_, _P. betulifolia_), and two Rosoideae species (_F. vesca_, _R. chinensis_); only those TPSs containing both PF01397 and PF03936 domains were used. The phylogenetic tree from full-length amino acid sequences was constructed using the MEGA with maximum likelihood (ML) method. Representative sequences of TPSs from _Vitis vinifera_ were used as outgroups. The branches of TPS-a, TPS-g, TPS-b, TPS-c, TPS-e, and TPS-f clades are indicated by blue, green, yellow-green, red, benzo, and purple colors, respectively. The lineage-specific expansion of TPSs in Prunoideae, Maloideae, Rosoideae are indicated by yellow squares, grey diamonds, red triangles, respectively.