*3.2. Phylogenetic Analysis and Classification of Maize HSFs*

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In present study, the evolutionary relationship among *At*HSFs, *Os*HSFs, *Sb*HSFs, *Bd*HSFs, and *Zm*HSFs was explored. A total of 118 HSFs were divided into three classes and 15 sub-classes based on the phylogenetic tree (Figure 2; Table S2). Variation in HSF gene number was observed among different plant species and between sub-groups (Table 2). For example, *Arabidopsis thaliana* contains 21 HSFs (15 HSFAs, 5 HSFBs, and 1 HSFC), the *Oryza sativa* possess 25 HSFs (13 HSFAs, 8 HSFBs, and 4 HSFCs), *Zea mays* harbors 25 HSFs in its genome (15 HSFAs, 7 HSFBs, and 3 HSFCs), *Brachypodium distachyon* 24 HSFs (14 HSFAs, 7 HSFBs, and 4 HSFCs), while *Sorghum bicolor* contains 23 HSFs (12 HSFAs, 7 HSFBs, and 4 HSFCs) (Table 2). Results indicated that maize HSFs were close to sorghum HSFs. Similarly, HSFs of rice were closer to *Brachypodium* HSFs, which is in line with the botanical classification among monocots. In contrast to *Arabidopsis*, sub-class A1 contains fewer HSFs in monocots (Table 2). On the other hand, sub-class A2 appears to have expanded in monocots. There was no ortholog of *Arabidopsis* HSFA9, HSFB3 in monocots that might reflect the loss of these sub-groups in family Poaceae (Figure 2, Table 2). Contrarily, this could also signify the gain of these sub-groups in dicots. A higher number of class C HSFs was observed in monocots.

**Figure 2.** A neighbor-joining phylogenetic tree was constructed after aligning protein sequences of *Arabidopsis thaliana*, *Oryza sativa*, *Brachypodium distachyon*, *Sorghum bicolor*, and *Zea mays*. Overall, 21 AtHSFs (marron circle), 25 OsHSFs (turquoise rhombus), 23 SbHSFs (purple rectangular), 24 BdHSFs (yellow triangle), and 25 ZmHSFs (red square) were divided into three classes and further into 15 sub-classes.


**Table 2.** Distribution of HSF genes in different sub-classes in selected plant species.
