*2.3. 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. domestica*, *P. betulifolia*), and two Rosoideae 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. domestica* 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

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. The enlarged phylogenetic tree with bootstrap values is shown in Supplementary Figure S2.


**Table 2.** Numbers of complete TPSs with both domains from Rosaceae species that were used for the phylogenetic tree of Figure 3.
