*2.4. Phylogenetic Analysis of CDPK and CRK Gene Families*

## 2.4.1. In the Benincaseae Tribe

In the Benincaseae tribe, watermelon was predicted to diverge from its sister lineage bottle gourd around 10.4–14.6 million years ago (Mya) and from *Cucumis* 17.3–24.3 Mya [27]. To deeply analyze the phylogentic relationships of *CDPK* and *CRK* gene families in these four species, all full-length protein sequences of watermelon (18 CDPKs and six CRKs), melon (18 CDPKs and seven CRKs), cucumber (17 CDPKs and seven CRKs), and bottle gourd (17 CDPKs and seven CRKs) were aligned using the software MUSCLE, and were then used to construct an evolutionary tree using MEGA6.0 (Figure S4). Four CDPK groups (CDPK I, CDPK II, CDPK III, and CDPK IV) and one CRK group (CRK I) were observed in the distance tree. Combined with results from the integrated map, all groups (except for CDPK IV) were found to constitute homologs from at least four loci. Furthermore, most loci contained only one homolog from each individual species. Apparently, homologs from watermelon and bottle gourd were preferentially clustered together, compared to those from *Cucumis* species.

#### 2.4.2. In the Cucurbiteae Tribe

It has been reported that a whole genome duplication (WGD) event has occurred in the progenitor of the *Cucurbita* genus [26,27]. As shown in the phylogenetic tree constructed with all identified full-length protein sequences from *C. moschata* (30 CDPKs and 12 CRKs), *C. maxima* (31 CDPKs and 12 CRKs), and *C. pepo* (32 CDPKs and 12 CRKs), the majority of loci in five groups contained two copies of the homolog gene from each species (Figure S5). Generally, CDPKs or CRKs from the same sub-genome (A or B) of *C. moschata* and *C. maxima* grouped together in the distance tree. For example, both CmoCDPK7 and CmaCDPK8 from sub-genome B of two species clustered together in CDPK IV, while CmoCDPK14 and CmaCDPK15 from sub-genome A formed another sister clade. Based on these observations, origins of CDPKs on chromosome 4 in two *Cucurbita* species could be uncovered, although the boundary sites of three segments (two from sub-genome A and one from B) of chromosome 04 were ambiguous [26], e.g., CmoCDPK26 and CmaCDPK27 in Locus 15 (group CDPK I) were from sub-genome A (Table S3 and Figure S5), suggesting that the members (CmoCDPK9 and CmaCDPK10) in its sister clade originated from sub-genome B. Similarly, CmoCDPK8 and CmaCDPK9 were inferred to originate form sub-genome A, which formed the Locus 20 in group CDPK II. It is worthy to note that both loci 06 and 24 formed two independent clades in the phylogenetic tree, due to having far more CDPK members.

#### 2.4.3. In the Cucurbitaceae Family

To deeply analyze the evolutionary history of *CDPK* and *CRK* gene families in Cucurbitaceae, a total of 226 protein sequences were used to construct a phylogenetic tree (Figure 3). Similar to topological structures mentioned above, four CDPK and one CRK group could be further divided into 25 loci. Notably, six homologs (LsiCRK1, CsCDPK3, CsCDPK19, CpCRK1, CpCDPK1, and CpCDPK2), which failed to be mapped on the integrated map due to lack of enough flanking sequences, were clustered with their orthologs in the phylogenetic trees. CDPKs or CRKs from watermelon and bottle gourd still cluster together in most loci. Compared to sub-genome A, members from sub-genome B usually gathered together with those from the Benincaseae tribe, except for two loci in group CRK I (Figure S6), which is consistent with the evolutionary scenario of modern Cucurbitaceae genomes [27]. CDPK IV, as the smallest group in CDPK lineage, was clustered with CRK I rather than the other three CDPK groups in phylogenetic trees, indicating that groups CDPK IV and CRK I may have originated from a common ancestor [10,11,30].

**Figure 3.** Phylogenetic tree of *CDPK* and *CRK* genes from Cucurbitaceae species. Four CDPK groups and one CRK group can be found in the tree, which were further divided into 25 loci. Numbers on nodes represent bootstrap values, and values <65 are not shown. **Figure 3.** Phylogenetic tree of *CDPK* and *CRK* genes from Cucurbitaceae species. Four CDPK groups and one CRK group can be found in the tree, which were further divided into 25 loci. Numbers on nodes represent bootstrap values, and values <65 are not shown.

For the comparative evolutionary analysis of two gene families in plants, a phylogenetic tree was constructed using the 226 protein sequences in Cucurbitaceae, as well as that from *Arabidopsis*  (34 CDPKs and eight CRKs, Cruciferae), tomato (29 CDPKs and six CRKs, Solanaceae), pepper (31 CDPKs and five CRKs, Solanaceae), and rice (31 CDPKs and five CRKs, Poaceae) (Figure S7). There is no doubt that all groups contained homologs from these four families, suggesting that both gene families have conserved basal architectures in their evolutionary process. For the comparative evolutionary analysis of two gene families in plants, a phylogenetic tree was constructed using the 226 protein sequences in Cucurbitaceae, as well as that from *Arabidopsis*(34 CDPKs and eight CRKs, Cruciferae), tomato (29 CDPKs and six CRKs, Solanaceae), pepper (31 CDPKs and five CRKs, Solanaceae), and rice (31 CDPKs and five CRKs, Poaceae) (Figure S7). There is no doubt that all groups contained homologs from these four families, suggesting that both gene families have conserved basal architectures in their evolutionary process.
