*2.3. Sequence Alignment and Phylogenetic Analysis*

The protein sequences of *Arabidopsis thaliana*, *Oryza sativa*, *Brachypodium distachyon*, and *Sorghum bicolor* were aligned with *Zea mays* HSFs through Clustal W [53]. Phylogenetic analysis was performed with the neighbor-joining method implemented in MEGA7.0 and tests were carried out with 1000 bootstrap replicates [54].

#### *2.4. Gene Duplication and Evolutionary Analysis*

Gene duplication events were investigated by following two parameters: (1) the length of an alignable sequence covers > 80% of the longer gene; and (2) the similarity of the aligned regions > 70% [55,56]. To analyze the molecular evolutionary rates of duplicated gene pairs, the non-synonymous substitution (Ka) and synonymous substitution (Ks) ratio were calculated using Ka/Ks calculation tool (http://services.cbu.uib.no/tools/kaks, accessed on 1 August 2021). In principle, the value of Ka/Ks < 1 signifies the purifying selection (negative selection), Ka/Ks > 1 signifies positive selection, and Ka/Ks = 1 means neutral selection [57]. Based on a rate of 6.1 × <sup>10</sup>−<sup>9</sup> substitutions per site per year, the divergence time (T) was calculated as T = Ks/(2 × 6.1 × <sup>10</sup>−9) × <sup>10</sup>−<sup>6</sup> million years ago (Mya) for HSF genes [57]. The duplicated HSF genes were connected using Tbtools [58].
