DELLA family duplication events lead to different selective constraints in angiosperms

Texte intégral

(1)DELLA family duplication events lead to different selective constraints in angiosperms Jean Keller, Pauline Delcros, Cyril Libourel, Francisco Cabello-Hurtado, Abdelkader Aïnouche. To cite this version: Jean Keller, Pauline Delcros, Cyril Libourel, Francisco Cabello-Hurtado, Abdelkader Aïnouche. DELLA family duplication events lead to different selective constraints in angiosperms. Genetica, Springer Verlag, 2020, 148 (5-6), pp.243-251. �10.1007/s10709-020-00102-6�. �hal-02929503�. HAL Id: hal-02929503 https://hal.archives-ouvertes.fr/hal-02929503 Submitted on 9 Nov 2020. HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés..

(2) 1. DELLA family duplication events lead to different selective constraints in angiosperms.. 2. Authors: Keller J.1,2, Delcros P.1, Libourel C.2, Cabello-Hurtado F.1, Aïnouche A.1. 3. Affiliation : 1UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l’Univers de. 4. Rennes), Université de Rennes 1, 35042 Rennes, France ; 2LRSV, Université de Toulouse,. 5. CNRS, UPS, Castanet-Tolosan, France. 6. Corresponding author: [email protected]. 7 8. Acknowledgement. 9. We acknowledge the GenOuest bioinformatics core facility (https://www.genouest.org) for providing the. 10. computing infrastructure. Part of this work was conducted at the LRSV laboratory, which belongs to the. 11. TULIP Laboratoire d’Excellence (LABEX) (ANR-10-LABX-41). JK and CL were supported by the research. 12 13. project Engineering Nitrogen Symbiosis for Africa (ENSA), which is funded through a grant to the University. 14. research grant from the University of Rennes 1 – French Ministry of Higher Education and Research. This. 15. work benefited from the International Associated Laboratory “Ecological Genomics of Polyploidy”. 16. supported by CNRS (INEE, UMR CNRS 6553 Ecobio), University of Rennes 1, Iowa State University. 17. (Ames, USA).. of Cambridge by the Bill & Melinda Gates Foundation (OPP1172165). JK was supported by a doctoral. 18 19 20. 1.

(3) 21. Abstract. 22. Gibberellic acid (GA) is a major plant hormone involved in several biological processes from the flowering. 23. to the symbiosis with microorganisms. Thus, the GA regulation is crucial for plant biology. This regulation. 24. occurs via the DELLA proteins that belong to the GRAS transcription factor family. DELLA proteins are. 25. characterised by a DELLA N-terminal and a GRAS C-terminal domains. It is well known that DELLA. 26. activity appears after the bryophytes divergence and then evolved in the vascular plant lineages. Here we. 27 28. present the phylogeny of DELLA across 75 species belonging to various lineages from algae, liverworts and. 29. divergence and the other specific to the eudicots lineage. Comparative analysis of DELLA subclades in. 30. angiosperms revealed the loss in Poaceae and strong alteration in other species of the DELLA functional. 31. domain in the DELLA2 clade. In addition, molecular evolution analysis suggests that each of the clades. 32. (named DELLA1.1, DELLA1.2 and DELLA2) evolved differently but copies of each subclade are under. 33. strong purifying selection. This also suggests that, although the DELLA functional domain is altered in. 34. DELLA2, DELLA2 orthologs are still functional and operate in a different way compared to DELLA1 copies.. 35. In angiosperms, additional duplication events occurred and led to duplicate copies in species, genus or family. 36. such as in the Fabaceae subfamily Papilionoideae. This duplication led to the formation of additional paralogs. 37. in the DELLA1.2 subclade (DELLA1.2.1 and DELLA1.2.2). Interestingly, both copies appeared to be under. 38. relaxing selection revealing different evolutionary fate of the DELLA duplicated copies.. angiosperms. Our study confirmed two main duplication events, the first occurring before the angiosperms. 39 40. Keywords: DELLA, evolutionary history, gene duplication, Fabaceae, molecular evolution. 41 42. 2.

(4) 43. Introduction. 44. Plant hormones play essential roles in various plant biology aspects and a huge diversity of these compounds. 45. have been identified so far (Davies 2010). Among them, the gibberellic acid (GA), a tetracyclic di-terpenoid. 46. molecule, is particularly involved in plant growth, seed germination and development or flowering (Gupta. 47. and Chakrabarty 2013). It also plays a role in responses to environmental stimuli (water, light, temperature). 48. as well as during the symbiosis with microorganisms such as nitrogen-fixing bacteria and arbuscular. 49 50. mycorrhizal fungi (Lievens et al. 2005; Davies 2010; Ferguson et al. 2011; Gupta and Chakrabarty 2013;. 51. Regulation of GA acitvity occurs through the DELLA (named according the conserved DELLA amino acid. 52. motif at the protein N-terminal domain) proteins which form a complex with the GA receptor GID1. 53. repressing the expression of downstream GA-responsive genes (Ueguchi-Tanaka et al. 2007; Sun 2011).. 54. Fixation of GA to the GID1 receptor induces the proteasome-dependant degradation of DELLA proteins (Fu. 55. et al. 2002). This GA-GID1-DELLA complex has been observed in all the investigated vascular plants but is. 56. absent from bryophytes although a DELLA copy has been identified in Physcomitrella patens (Yasumura et. 57. al. 2007; Sun 2011). This suggests that the DELLA repressor activity of GA evolved after the bryophyte. 58. divergence (~430 MYA) and there is no functional data about the DELLA role and mode of action prior this. 59. divergence (Kenrick and Crane 1997; Yasumura et al. 2007; Wang and Deng 2014). In angiosperms, DELLA. 60. proteins are known to be involved in the regulation of a wide range of fundamental biological processes such. 61. as flowering, root and shoot development, and is a key regulator of both arbuscular mycorrhizal and root. 62. nodule nitrogen-fixing symbiosis (Maekawa et al. 2009; Ferguson et al. 2011; Floss et al. 2013; Yu et al.. 63. 2014; Pimprikar et al. 2016). For example, during nitrogen-fixing symbiosis, DELLA is thought to be a key. 64. regulator of the infection process by directly regulating symbiotic genes NSP1/NSP2, ERN1, NF-YA1 and by. 65. enhancing the binding between CYCLOPS and NSP2 (Fonouni-Farde et al. 2016; Jin et al. 2016). Recently,. 66. DELLA proteins were also shown to be likely involved in the cytokinin genetic pathway regulation during. 67. nodulation in Pisum sativum L. (Dolgikh et al. 2019).. 68. DELLA constitutes a subfamily of the GRAS transcription factors superfamily and DELLA proteins are. 69. characterised by a N- and C- terminal conserved DELLA and GRAS domains respectively (Pysh et al. 1999).. 70. The GRAS family encompasses between 8 and 13 subfamilies according to authors and surveyed species. 71. (Cenci and Rouard 2017). Although evolutionary history of the GRAS family has been recently investigated. 72. in representative angiosperms model species (Cenci and Rouard 2017), there is little data about the DELLAs. 73. evolution in angiosperms.. 74. In this context, we performed a large screening of DELLA proteins in more than 50 species representing all. 75. the main plant orders. This allowed us to confirm different duplication events in the DELLA subfamily, with. 76 77. a subclade exhibiting severe alterations of the DELLA functional domain. In addition, molecular evolution. 78. with currently a strong purifying constraint within each of these subclades.. Takeda et al. 2015; Martín-Rodríguez et al. 2016). analysis revealed that the angiosperms DELLA subclades have experienced different selective constraints. 3.

(5) 79. Material & Methods. 80. Data mining. 81. Proteomes of 75 plant species covering the main Angiosperms lineages (list available in Supplementary Table. 82. S1) were screened against the Pfam database (Finn et al. 2016) and hits with an e-value lower than 0.01 and. 83. both GRAS (PF12041.8) and DELLA (PF03514.14) domains were retained. To complement this search and. 84. take into account potential missing domains (in cases of losses or miss-annotated proteins), published and. 85 86. well characterized DELLA protein from Arabidopsis thaliana, Oryza sativa, Lupinus angustifolius,. 87. were used as queries for a tBLASTn v2.7.1+ search against the 75 plant species with default parameters. 88. (Camacho et al. 2009) and an e-value threshold of 1-60. Finally, protein sequences shorter than 200 amino. 89. acids were removed from the dataset to eliminate partial assembly and putative remnants (retained and. 90. removed sequences are available in Supplementary Data S1 and S2 respectively).. 91. In order to explore the absence of DELLA proteins in algae, the Algae-PrAS database containing 31 genomes. 92. (http://alga-pras.riken.jp/) was also screened with the reference DELLA sequences from A. thaliana and O.. 93. sativa as queries using the BLASTp v2.7.1+ algorithm and an e-value threshold of 0.01. Until recently, scarce. 94. data were available for Zygnematophyceae algae, the likely closest lineage of land plants. Two. 95. Zygnematophyceae genomes (Mesotaenium endlicheranium and Spirogloea muscicola, this latter belonging. 96. to a new subclass likely to be the closest from the Zygnematophyceae/embryophytes separation) have been. 97. published and were screened for DELLA homologs (Cheng et al. 2019).. 98. Phylogenetic analysis. 99. Retained protein sequences were aligned using MUSCLE (Edgar 2004) with default parameters. Alignment. 100. was subjected to maximum likelihood analysis using IQ-TREE v1.5.5 (Nguyen et al. 2015). The best-fitted. 101. evolutionary model was determined using ModelFinder as implemented in IQ-TREE (Kalyaanamoorthy et. 102. al. 2017). Maximum likelihood analysis was conducted with 10,000 replicates Ultra Fast Bootstraps (Hoang. 103. et al. 2018). Trees were visualized and annotated uing the Interactive Tree of Life platform (Letunic and Bork. 104. 2016).. 105. Molecular evolution analyses. 106. For each clade of interest identified through the phylogenetic analysis, sequence logos were generated from. 107. protein alignments using the WebLogo v3.5 platform with default parameters (Crooks 2004). In addition, to. 108. compare signature of selection among DELLA homolog clades identified in phylogeny, we estimated dN/dS. 109. ratios from all pairwise comparison of protein-coding sequences using yn00 module from the PAML package. 110. v4.9g (Yang and Nielsen 2000; Yang 2007). We also used the RELAX hypothesis testing framework from. 111. HyPhy (Pond et al. 2005; Wertheim et al. 2015) to look for relaxation (K<1) or intensification (K>1) of the. 112 113. signature of selection. To estimate signature of selection, codons were aligned according to the protein. Medicago truncatula and Amborella trichopoda (Sato et al. 2014; Cenci and Rouard 2017; Hane et al. 2017). alignment using the pal2nal v14 Perl script (Suyama et al. 2006) and all gapped positions were removed. We. 4.

(6) 114. used the following command line for each clade tested (SupplementaryTable2): hyphy relax CPU=1 --. 115. alignment input_file.fna --code Universal --type Nucleotide --keyword value Foreground.. 116 117. Results and discussion. 118. DELLA family emerged with land plants and experienced multiple duplication events. 119. To understand the origin of the DELLA family, the Algae-PrAS database that contains more than 30 genomes. 120 121. of non-land plants species (green and red algae, Glaucophyceae) and other organisms such as Oomycetes and. 122. confirmed the absence of DELLA orthologs in these species. In the two Zygnematophyceae genomes, no. 123. DELLA were detected but other GRAS family members were identified and are likely resulting from. 124. horizontal gene transfer from bacteria (Cheng et al. 2019). These findings are consistent with results from. 125. Yasamura et al. (2007) who suggested an emergence of DELLA proteins along with the land colonization by. 126. plants. To improve our understanding of the evolutionary dynamics of DELLA family, 75 land plants. 127. genomes were investigated and 293 DELLA orthologs were identified by the phylogenetic analysis. 128. (excluding putative remnants and misassembled sequences). Among them, one sequence with a DELLA. 129. functional domain was identified in the liverwort Marchantia polymorpha (Marpol_Mapoly0058s0044.1). 130. consistently with the hypothesis that the DELLA family originated at least in the common ancestor of the. 131. vascular and non-vascular plants (Hernández-García et al. 2019). Previous findings revealed that the GID1-. 132. DELLA complex does not exist in the bryophytes lineage (Yasumura et al. 2007; Hirano et al. 2007). In. 133 134. bryophytes, DELLA has been recently proposed as transcriptional co-activator that could have been exapted. 135. putative DELLA’s interactors in Physcomitrella patens (no DELLA-GID1 interaction), Solanum. 136. lycopersicum and Arabidopsis thaliana suggest that DELLA evolution is correlated with an increasing. 137. coordination of transcriptional programs between bryophytes and vascular plants (Briones-Moreno et al.. 138. 2017).. 139. Phylogenetic analysis shows that DELLA family underwent multiple duplication events at different points. 140. of the land plant evolution as recently shown by Hernández-Garcia and colleagues (2019). Indeed, results. 141. revealed a first duplication event that occurred before the divergence of angiosperms (Fig.1, with branch. 142. support values to be retrieved in the Supplementary Data S3) leading to the formation of two main clades,. 143. called DELLA2 (blue clade in Fig.1; corresponding to DELLA3 in Hernández-Garcia et al. 2019) and. 144. DELLA1 (orange, green and red clades) accordingly to Cenci and Rouard (2017). DELLA sequences from. 145. Marchantia polymorpha and the lycophyte Selaginella moellendorffii seem to belong to the DELLA2 clade;. 146. however, these branches are not supported (UFB value < 50%) and these sequences are likely at the base of. 147 148. the whole DELLA family. As demonstrated in recent phylogeny and in this study the DELLA1/DELLA2. 149. seed plants (c.a 319 MYA) (Jiao et al. 2011; Hernández-García et al. 2019). Another level of duplication has. diatoms was mined using DELLA protein sequences from Arabidopsis thaliana and Oryza sativa. Results. in vascular plants (Hernández-García et al. 2019). Previously published results based on RNAseq data and. clades emergence is probably due to the Whole Genome Duplication (WGD) event that occurred prior the. 5.

(7) 150. been identified in the DELLA1 clade. Indeed, this latter, with the basal Angiosperms Amborella trichopoda. 151. at its base, can be divided into three main subclades (Fig.1). The first, in green, is exclusively composed by. 152. Monocot species whereas the two others only contain Eudicot species. These two Eudicot subclades emerged. 153. from the core Eudicot WGD event that occurred approximately 125 MYA (Van de Peer et al. 2017) and were. 154. arbitrarily named DELLA1.1 (orange clade in Fig.1) and DELLA1.2 (red clade). Finally, additional. 155. duplication events limited to species or subclades were identified such as in Brassicaceaee DELLA1.1 or the. 156. DELLA1.2 of Papilionoideae (WGD of the Papilionoideae common ancestor, around 60 MYA, see below).. 157. For each sequence, gene structure was extrapolated from the annotation file, when available, and results were. 158. mapped on the phylogeny (Fig. 1; outer circle). Excepting few sequences, most of the genes present a non-. 159. intronic structure and a length ranked between 1000 and 2000 bp.. 160. Analysis of the functional domains revealed alterations in DELLA2. 161. DELLA proteins are characterised by the presence of two functional domains. The first (DELLA) is in the. 162 163. N-terminal region of the protein and is specific to the DELLA family whereas the second (GRAS) is shared. 164. Arabidopsis thaliana characterised two motifs required for the interaction with GID1 (Murase et al. 2008):. 165. DELLA§LXYXVXXXXMAXVAXXLEXLEX§ and TVHYNPXXLXXWXXXM (where X and § stand for. 166. any and non-polar amino acids respectively).. 167. DELLA and GRAS functional domains identified using the Pfam search were mapped on the phylogenetic. 168. tree (Fig.1, second outer circle) and revealed that most of the sequences possess both domains with few. 169. exceptions that can be explained by assembly and annotation issues or putative pseudogenes. However, it. 170. appears that all the six Poaceae from the DELLA2 clade have lost the DELLA functional domain (absence. 171. of red right pointing pentagram in Fig.1, and Supplementary Figure 1). The loss of the DELLA domain has. 172. been previously reported in rice (Itoh et al. 2005; Cenci and Rouard 2017) and our results indicate that this. 173. loss is likely to have occurred before diversification of the Poaceae from their common ancestor. Indeed,. 174. Monocots from other orders such as Spirodela polyrhiza, Zostera marina (two Alismatales) and Musa. 175 176. accuminata (Zingiberales) retained both DELLA and GRAS domains (Supplementary Figure 1). At the. 177. two DELLA2 copies of rice (also called SLRL1 and SLRL2 for SLR-like1 and 2) revealed that SLRL1 is. 178. positively affected by GA treatment whereas SLRL2 is not. In addition, trans-complementation of slr1. 179. (DELLA1) mutant by SLRL1 rescued the phenotype (Itoh et al. 2005). Since the N-terminal DELLA domain. 180. is missing in SLRL1 and 2, proteins were not degraded following the GA treatment but also exhibit a lower. 181. activity against GA than a SLR1 truncated copy missing the DELLA domain (Itoh et al. 2005). Itoh and. 182. colleagues suggest that SLR1 and SLRL1 can function together and are required for a fine-tuning of GA. 183. signalling.. 184. In a second approach, the focus has been made on the DELLA functional domain extracted from the protein. 185. alignment and amino acids conservation represented using the WebLogo tool (Crooks 2004) for each main. with other members of the GRAS superfamily. Functional analysis of the DELLA domain in the model plant. opposite, all Monocots DELLA1 proteins have a full DELLA domain. Previous functional analysis of the. 6.

(8) 186. clade of DELLA (Fig.2). Results revealed a high conservation of amino acids required for interaction with. 187. GID1 across the different DELLA1 subclades (DELLA1.1, DELLA1.2 and DELLA1 from Monocots).. 188. However, severe disruption of the DELLA motifs can be observed in the DELLA2 clade (Fig.2; red arrows).. 189. Indeed, the methionine from the MAXVA motif and at the end of the second DELLA subdomains is not. 190. conserved anymore among the DELLA2 sequences. Other disruptions of the DELLA domain are also. 191. characteristic of the DELLA2 clade such as the low conservation of the first leucine in the DELLA first motif. 192. or the alteration of the TV motif in the second DELLA motif (Fig.2). This low-conservation pattern observed. 193. in the DELLA2 clade could be explained by different hypothesis such as the pseudogenisation of the. 194. DELLA2 copies, a new function of these proteins, or another way for these proteins to interact with GID1.. 195. To test whether the DELLA2 clade is under relaxed selective constraints, different evolutionary tests were. 196. applied. To this aim, only sequences with entire DELLA domains were retained and evolutionary tests. 197. performed on the domain alignment. The analysis of non-synonymous versus synonymous (dN/dS) revealed. 198 199. a higher dN/dS ratio when comparing DELLA2 to the other DELLA1 subclades, whereas these latter had. 200. (RELAX) was applied with the DELLA2 clade marked as foreground branch (Supplementary Table 2).. 201. According to pairwise comparisons, estimating the selective intensity parameter (K) suggests that the. 202. DELLA2 clade is subjected to an ongoing process of relaxing selection (K=0.81, p-val≈0.00074, LR=11.39).. 203. Conversely, the higher value found for DELLA1 (K=1.28, p-val≈0.00038, LR=12.61) suggests. 204. intensification of the purifying selection (K>1) to maintain the proper role of DELLA1. In addition, logo and. 205. amino acid frequency for each position of the DELLA motifs (Fig.2 and Supplementary Table S3) show that. 206. mutations of essential amino acid in DELLA2 clade are shared by most of the sequences in this clade. For. 207. example, methionine from MAXVA stretch is replaced by leucine in 100% of sequences. Similarly, almost. 208. 90% of DELLA2 sequences share a glycine instead of a glutamate normally expected at the second position. 209. of the DELLA first motif. Altogether, these analyses suggest that modification of DELLA2 sequences. 210. occurred in the DELLA2 ancestor and then, have been maintained. These results are in line with a previous. 211 212. report suggesting that positive selection acted on the DELLA family during its evolution (Chen et al. 2013).. 213. similarly to Poaceae DELLA2, these proteins function along with their DELLA1 counterparts and act as. 214. weaker GA signalling repressors without being degraded. Interestingly, it has been shown that the RGL1. 215. protein of Arabidopsis thaliana (DELLA1.2 subclade) works without being degraded upon GA application. 216. (Wen and Chang 2002). All together, these results suggest that the multiple rounds of duplication in the. 217. DELLA family led to a slight subfunctionalization of DELLA copies, all involved in the GA regulation. 218. pathway. In their work, Yasumura and colleagues (2007), proposed a stepwise evolution of the DELLA-GA. 219. complex in plants with the GID1-DELLA interaction evolving in vascular plants. Here, we show that the. 220. DELLA family pursued its evolution in Angiosperms by experiencing several rounds of duplication, leading. 221. to sub-clades under different selective constraint. In addition, our results revealed that the loss of DELLA. 222. domain is shared among the DELLA2 proteins of Poaceae, supporting the hypothesis of a different. 223. mechanism (degradation-independent for example) for DELLA in this family (Itoh et al. 2005).. similar dN/dS ratios (Fig.3). To determine if relaxed selection is acting on the DELLA2 clade, a branch model. To date, there is no detailed studies of DELLA2 proteins from Eudicot lineage. However, it is possible that,. 7.

(9) 224. DELLAs from the Fabaceae show similar evolutionary pattern compared to that of the DELLA family. 225. DELLA proteins play major role in various biological aspects of plant life. In legumes, they also have a. 226. critical role for the establishment of the root nodule nitrogen-fixing symbiosis. Recently, DELLA proteins. 227. have been proposed to be also involved in the regulation of cytokinin genetic pathway through interaction. 228. with KNOX and BELL transcription factors (Dolgikh et al. 2019). Thus, a focused analysis on the DELLA. 229. sequences from the Fabaceae family has been performed and showed similar evolutionary pattern compared. 230. to results discussed above.. 231. First, the evolutionary pattern of DELLAs confirmed that the three main DELLA clades (DELLA2,. 232. DELLA1.1 and DELLA1.2) are conserved among the Fabaceae (Fig.4A). DELLA1 and DELLA2 copies. 233. both present mutations in amino acids involved in the interaction with GID1 (Fig.4B). The branch model. 234. analysis on both clades showed that DELLA1 and DELLA2 copies have a similar pattern of selection with. 235. no sign of relaxation/intensification (Supplementary Table S2). However, no change in the selective. 236 237. constraint was detected among the different clades suggesting that, also in Papilionoideae, there is no positive. 238. Second, in the DELLA1.2 subclade, an additional duplication event occurred leading to the formation of two. 239. subclades arbitrarily called DELLA1.2.1 and DELLA1.2.2. These two subclades likely originated from the. 240. Papilionoideae WGD that occurred 60MYA. Analysis of the selective constraints strongly suggests that both. 241. paralogs (K=0.25, p-val=7.3E-08, LR=28.98 and K=0.26, p-val=7.2E-08, LR=29 for DELLA1.2.1 and. 242. DELLA1.2.2 respectively) are under pseudogenization process. This is also supported by the apparent loss. 243. of DELLA1.2.1 copy in seven out of the 15 legume samples investigated.. selection acting on them.. 244 245. Conclusion. 246. DELLA proteins have a critical role in numerous aspects of plant life, from development to flowering but. 247. also in specific aspects such as in the nitrogen-fixing symbiosis. Phylogenetic analysis of DELLA family. 248. including more than 70 species confirmed its emergence in land plants along with its absence in algae. Results. 249 250. of phylogenetic analysis of the DELLA family is consistent with previous reports and show the existence of. 251. from a duplication event that occurred before the Angiosperms divergence and two additional DELLA1. 252. subclades (DELLA1.1 and DELLA1.2) emerged following the Eudicots whole genome duplication. A. 253. detailed analysis of the evolution of the three DELLA clades revealed that they are all under purifying. 254. selection although their ancestors seem to have been subjected to a relaxed selective constraint. In the. 255. DELLA2 clade, our results strongly support the loss of the DELLA domain before diversification of Poaceae. 256. and severe alterations of this motif in other DELLA2 proteins suggesting that DELLA2 protein could function. 257. in a degradation-independent manner. Finally, DELLA proteins from the Fabaceae family revealed a similar. 258. evolutionary history with additional duplication due to the Whole Genome Duplication of the Papilionoideae. two main duplication events leading to the formation of three clades: DELLA1 and DELLA2 clades emerged. 8.

(10) 259. ancestor leading to additional DELLA1.2.1 and DELLA1.2.2 clades. Both clades are under relaxing selection. 260. suggesting they are likely to be pseudogenized or neofunctionalized.. 261. All together this study supports the hypothesis of a complex evolutionary history of DELLA proteins in. 262. Angiosperms with multiple changes in the selective constraint. To extend these results, functional data on. 263. the different DELLA copies from both Monocots and Eudicots would be of a great interest to better. 264. understand the biological consequences of these differential selective constraints.. 265. Conflict of interest. 266. The authors declare that they have no conflict of interest.. 267 268. References. 269 270 271. Briones-Moreno A, Hernández-García J, Vargas-Chávez C, et al (2017) Evolutionary Analysis of DELLA-Associated Transcriptional Networks. Front Plant Sci 8:626. https://doi.org/10.3389/fpls.2017.00626. 272 273. Camacho C, Coulouris G, Avagyan V, et al (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421. https://doi.org/10.1186/1471-2105-10-421. 274 275. Cenci A, Rouard M (2017) Evolutionary Analyses of GRAS Transcription Factors in Angiosperms. Front Plant Sci 8:. https://doi.org/10.3389/fpls.2017.00273. 276 277. Chen J, Cheng T, Wang P, et al (2013) Genome-wide bioinformatics analysis of DELLA-family proteins from plants. Plant Omics Journal, POJ 6(3):201–207. 278 279 280. Cheng S, Xian W, Fu Y, et al (2019) Genomes of Subaerial Zygnematophyceae Provide Insights into Land Plant Evolution. Cell 179:1057-1067.e14. https://doi.org/10.1016/j.cell.2019.10.019. 281 282. Crooks GE (2004) WebLogo: A Sequence Logo Generator. Genome Res 14:1188–1190. https://doi.org/10.1101/gr.849004. 283. Davies PJ (ed) (2010) Plant Hormones. Springer Netherlands, Dordrecht. 284 285 286. Dolgikh AV, Kirienko AN, Tikhonovich IA, et al (2019) The DELLA Proteins Influence the Expression of Cytokinin Biosynthesis and Response Genes During Nodulation. Front Plant Sci 10: 432. https://doi.org/10.3389/fpls.2019.00432. 287 288. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340. 289 290 291. Ferguson BJ, Foo E, Ross JJ, Reid JB (2011) Relationship between gibberellin, ethylene and nodulation in Pisum sativum. New Phytol 189:829–842. https://doi.org/10.1111/j.14698137.2010.03542.x. 9.

(11) 292 293 294. Finn RD, Coggill P, Eberhardt RY, et al (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–D285. https://doi.org/10.1093/nar/gkv1344. 295 296 297. Floss DS, Levy JG, Levesque-Tremblay V, et al (2013) DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci 110:E5025–E5034. https://doi.org/10.1073/pnas.1308973110. 298 299 300. Fonouni-Farde C, Tan S, Baudin M, et al (2016) DELLA-mediated gibberellin signalling regulates Nod factor signalling and rhizobial infection. Nat Commun 7:. https://doi.org/10.1038/ncomms12636. 301 302 303. Fu X, Richards DE, Ait-ali T, et al (2002) Gibberellin-Mediated Proteasome-Dependent Degradation of the Barley DELLA Protein SLN1 Repressor. Plant Cell 14:3191–3200. https://doi.org/10.1105/tpc.006197. 304 305. Gupta R, Chakrabarty SK (2013) Gibberellic acid in plant: Still a mystery unresolved. Plant Signal Behav 8:e25504. https://doi.org/10.4161/psb.25504. 306 307 308 309. Hane JK, Ming Y, Kamphuis LG, et al (2017) A comprehensive draft genome sequence for lupin ( Lupinus angustifolius ), an emerging health food: insights into plant-microbe interactions and legume evolution. Plant Biotechnol J 15:318–330. https://doi.org/10.1111/pbi.12615. 310 311 312 313. Hernández-García J, Briones-Moreno A, Dumas R, Blázquez MA (2019) Origin of GibberellinDependent Transcriptional Regulation by Molecular Exploitation of a Transactivation Domain in DELLA Proteins. Mol Biol Evol 36:908–918. https://doi.org/10.1093/molbev/msz009. 314 315 316 317. Hirano K, Nakajima M, Asano K, et al (2007) The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens. Plant Cell 19:3058–3079. https://doi.org/10.1105/tpc.107.051524. 318 319 320. Hoang DT, Chernomor O, von Haeseler A, et al (2018) UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol Biol Evol 35:518–522. https://doi.org/10.1093/molbev/msx281. 321 322 323. Itoh H, Shimada A, Ueguchi-Tanaka M, et al (2005) Overexpression of a GRAS protein lacking the DELLA domain confers altered gibberellin responses in rice: Rice SLR1-like genes. Plant J 44:669–679. https://doi.org/10.1111/j.1365-313X.2005.02562.x. 324 325. Jiao Y, Wickett NJ, Saravanaraj A, et al (2011) Data from: Ancestral polyploidy in seed plants and angiosperms. Nature 473 (7345): 97 100. https://doi.org/10.1038/nature09916.. 326 327 328. Jin Y, Liu H, Luo D, et al (2016) DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways. Nat Commun 7:(1). https://doi.org/10.1038/ncomms12433. 329. 10.

(12) 330 331 332. Kalyaanamoorthy S, Minh BQ, Wong TKF, et al (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14:587–589. https://doi.org/10.1038/nmeth.4285. 333 334. Kenrick P, Crane PR (1997) The origin and early evolution of plants on land. Nature 389 (6646): 33 39Nature 389:33–39. https://doi.org/10.1038/37918. 335 336 337. Letunic I, Bork P (2016) Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res 44:W242–W245. https://doi.org/10.1093/nar/gkw290. 338 339 340. Lievens S, Goormachtig S, Den Herder J, et al (2005) Gibberellins Are Involved in Nodulation of Sesbania rostrata. Plant Physiol 139:1366–1379. https://doi.org/10.1104/pp.105.066944. 341 342 343. Maekawa T, Maekawa-Yoshikawa M, Takeda N, et al (2009) Gibberellin controls the nodulation signaling pathway in Lotus japonicus. Plant J 58:183–194. https://doi.org/10.1111/j.1365-313X.2008.03774.x. 344 345 346. Martín-Rodríguez JA, Huertas R, Ho-Plágaro T, et al (2016) Gibberellin–Abscisic Acid Balances during Arbuscular Mycorrhiza Formation in Tomato. Front Plant Sci 7:. https://doi.org/10.3389/fpls.2016.01273. 347 348 349. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol Biol Evol 32:268–274. https://doi.org/10.1093/molbev/msu300. 350 351 352. Pimprikar P, Carbonnel S, Paries M, et al (2016) A CCaMK-CYCLOPS-DELLA Complex Activates Transcription of RAM1 to Regulate Arbuscule Branching. Curr Biol 26:987– 998. https://doi.org/10.1016/j.cub.2016.01.069. 353 354. Pond SLK, Frost SDW, Muse SV (2005) HyPhy: hypothesis testing using phylogenies. Bioinformatics 21:676–679. https://doi.org/10.1093/bioinformatics/bti079. 355 356 357. Pysh LD, Wysocka-Diller JW, Camilleri C, et al (1999) The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. Plant J 18:111–119. https://doi.org/10.1046/j.1365-313X.1999.00431.x. 358 359 360. Sato T, Miyanoiri Y, Takeda M, et al (2014) Expression and purification of a GRAS domain of SLR1, the rice DELLA protein. Protein Expr Purif 95:248–258. https://doi.org/10.1016/j.pep.2014.01.006. 361 362. Sun T (2011) The Molecular Mechanism and Evolution of the GA–GID1–DELLA Signaling Module in Plants. Curr Biol 21:R338–R345. https://doi.org/10.1016/j.cub.2011.02.036. 363 364 365. Suyama M, Torrents D, Bork P (2006) PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res 34:W609– W612. https://doi.org/10.1093/nar/gkl315. 366 367 368. Takeda N, Handa Y, Tsuzuki S, et al (2015) Gibberellin regulates infection and colonization of host roots by arbuscular mycorrhizal fungi. Plant Signal Behav 10:e1028706. https://doi.org/10.1080/15592324.2015.1028706 11.

(13) 369 370 371. Ueguchi-Tanaka M, Nakajima M, Motoyuki A, Matsuoka M (2007) Gibberellin Receptor and Its Role in Gibberellin Signaling in Plants. Annu Rev Plant Biol 58:183–198. https://doi.org/10.1146/annurev.arplant.58.032806.103830. 372 373. Van de Peer Y, Mizrachi E, Marchal K (2017) The evolutionary significance of polyploidy. Nat Rev Genet 18:411–424. https://doi.org/10.1038/nrg.2017.26. 374 375 376. Wang Y, Deng D (2014) Molecular basis and evolutionary pattern of GA–GID1–DELLA regulatory module. Mol Genet Genomics 289:1–9. https://doi.org/10.1007/s00438-0130797-x. 377 378. Wen C-K, Chang C (2002) Arabidopsis RGL1 Encodes a Negative Regulator of Gibberellin Responses. Plant Cell 14:87–100. https://doi.org/10.1105/tpc.010325. 379 380 381. Wertheim JO, Murrell B, Smith MD, et al (2015) RELAX: Detecting Relaxed Selection in a Phylogenetic Framework. Mol Biol Evol 32:820–832. https://doi.org/10.1093/molbev/msu400. 382 383. Yang Z (2007) PAML 4: Phylogenetic Analysis by Maximum Likelihood. Mol Biol Evol 24:1586–1591. https://doi.org/10.1093/molbev/msm088. 384 385 386. Yang Z, Nielsen R (2000) Estimating Synonymous and Nonsynonymous Substitution Rates Under Realistic Evolutionary Models. Mol Biol Evol 17:32–43. https://doi.org/10.1093/oxfordjournals.molbev.a026236. 387 388 389. Yasumura Y, Crumpton-Taylor M, Fuentes S, Harberd NP (2007) Step-by-Step Acquisition of the Gibberellin-DELLA Growth-Regulatory Mechanism during Land-Plant Evolution. Curr Biol 17:1225–1230. https://doi.org/10.1016/j.cub.2007.06.037. 390 391 392. Yu N, Luo D, Zhang X, et al (2014) A DELLA protein complex controls the arbuscular mycorrhizal symbiosis in plants. Cell Res 24:130–133. https://doi.org/10.1038/cr.2013.167. 393 394. Figure legends. 395. Fig. 1: Maximum likelihood tree of DELLA proteins (model JTT+F+R10). Tree was rooted using three. 396. SCARECROW (SCR, belonging to another subfamily of GRAS transcription factor) sequences from. 397. liverworts Marchantia polymorpha and are collapsed in the “outgroup” clade. DELLA2, Monocots. 398. DELLA1, Eudicots DELLA1.1 and DELLA1.2 are indicated by coloured branches as follow: blue, green,. 399. orange, red, respectively. The first outer circle indicates the main Angiosperm lineages as described below. 400. the tree. The second outer circle represents the presence/absence and the position of DELLA (red right. 401. pointing pentagram) and GRAS (dark blue rectangle) domains. Size and position of symbols is proportional. 402 403. to their length and position in the protein. The third outer circle illustrates the gene structure, extrapolated. 404. proportional to their length and positions in the gene. Branch support was tested using the UFB2 method and. from the annotation file when available. Blue squares represent gene exons, their size and position are. 12.

(14) 405. were masked from the tree for readability reason. Support values can be retrieved in the Supplementary Data. 406. S3 corresponding to the rooted tree shown here in Newick format.. 407. Fig. 2: Logos of functional DELLA domains generated for each phylogenetic clade. For DELLA2, strongly. 408. affected sites are indicated by red arrows. Size of letter is proportional to the amino acid conservation at a. 409. given site. Blank in the logo represents highly gapped sites in the alignment. Theoretical functional models. 410. are written in black above logos and essential amino acids for the interaction with GID1, as defined by Murase. 411. et al. (2008), are indicated in bold.. 412. Fig. 3: Boxplot representing pairwise dN/dS comparisons among the DELLA clades. All pairwise. 413. comparison between all DELLA sequences were calculated. For each clade (X-axis), comparison with other. 414. clades and itself (white waves) are shown separately. The dN/dS ratios were estimated using the Yang &. 415. Nielsen method (Yang and Nielsen 2000).. 416. Fig. 4: Maximum likelihood tree of DELLA proteins (model JTT+ R5) in the Papilionoideae clade (A).. 417 418. Alignment of DELLA functional domains mapped on the phylogenetic tree. Blue background is proportional to identity level for each site. Functional DELLA subdomains are indicated by red rectangles (B).. 419 420. Supplementary Data. 421. Supplementary Data S1: Protein sequences used for phylogenetic analysis. 422. Supplementary Data S2: Protein sequences discarded prior the phylogenetic analysis (less than 200 amino. 423. acids). 424. Supplementary Data S3: Rooted tree presented in Figure 1 in Newick format.. 425. Supplementary Table S1: Summary table of DELLA sequences repartition. For each species, sequence ID is. 426. indicated for each DELLA clade. Absence of sequence for a given species/clade is indicated by empty cells.. 427. In addition, order, family, reference and abbreviation used for each species are indicated in the last four. 428. columns. For Vitis vinifera, the DELLA2 sequence is indicated but not retained in the analysis since it seems. 429. that this sequence is missassembly.. 430 431. Supplementary Table S2: Results of the branch analysis for signature of selection acting on the different. 432. Supplementary Table S3: Amino acid frequency matrices for each DELLA copy. Numbers represent. 433. percentage of each amino acid at each position in the DELLA functional domain. First column indicates the. 434. consensus of DELLA domain.. DELLA clades.. 435. 13.

(15) 436. Supplementary Figure 1: Alignment of DELLA domains in DELLA2 Monocots and Medicago truncatula as. 437. reference. Blue-coloured names represent sequences with a predicted DELLA domain by Pfam. The DELLA. 438. functional domains are indicated by black squares with the theoretical sequence indicated below. Musa. 439. acuminate, Siprodela polyrhiza and Zostera marina belong to diverse lineages of Monocots outside the. 440. Poaceae clade.. 441. 14.

(16) 0. 10. 00. 10. Pruper Prupe.7G236100.1. Maldom MDP0000825698. HO. 0. ul. rv. 50. ry. M. us. 0. Am. 10. bt. ri. ev. m. 27. .T. U.. rp. rp. ol. ol. 1.. po. po. Se lm. oe. tgr. Arah. Ara al A. ly0. lm. Se. Ou. 0. Ma. Ma. G. SM. ipo. ar. Tr v. rb. Sp. sm. Am. Ma. Ma. u. Zo. Zo. lS. sc. old. ly0. 00. 02. 12. 24. 2.. 0. 45. 86. 34. 72. 01. 00. 71. 70. 1g. 24. 23. 55. 72. s0. G. 3G. 16. 03. 12. O. .5. 34. G0. g0. 44. 71. 95. 6P. 08. 00. 3s. 13. hr. C. ita. 00. o0. 8s. Se. c.. Ac. 00. 05. oe. bi. 00. 0.. 1. .1. .1. 1. 00. .1. .1. .1. 06. 41. p. lyr A. L3G. 131 20.t 1 14s0 007 a .1 AT3G Capr 0345 ub C arub 0.1 .000 Capg 3s02 ra Ca 59.1 gra.2 0 9 Boes 8s00 tr Bos 43.1 tr.190 46s00 61.1 Brarap Brara.E 03510 .1 Braole cap Bo l02877 Eutsal T 7 hhalv10 020431 m Caprub C arub.000 6s1572.1 Capgra C agra.6170s 0019.1 Boestr Bostr .26527s0463 .1 Aratha AT5G17 490.1 raha. Arath. 69. DELLA2. .109. DELLA1. Aralyr AL6G28580.t1 Brarap Brara.J01802.1. Braolecap Bol019798. 1 G 0. r1 99 4H 49 U .1 g 1 3 00 0. RV s0 90 30 O O 2 H 4 20 1 C l 1 56 u G O v 9 1G 019 5 t L ita. or 0 a a 0 H ys 95 Se bic. 08 r 04 O o 00 a0 ita t 0 8 S A 00 Se rbic -R Zm 00 97 ay A So 73 m Zm -R .1 1 a 1 y 9 a 00 Ze NU 039 m 84 a N 41 0.1 U Ze nuc G 90 l NN oe3 03 c 0 Ne c G lnu Aq e3 1 Ne coe co 7.1 u.t u Aq 37 rya 4 Aq coe 3 0 1 a 1 u 0 x.t Dc 44 Aq uq ar A 0h 61 ac v 0 i -R 0 D 3 lB 23 12 95 tvu A 3 p e 0 B 1-R 62 eS 19 iol 01 UR 14 A 0 Sp i 2 100 u 6 eq 117 UR 0 A 1 Ch 0 ui 11 IVT eq SV Ch 073 R0 nG 1 i A v 36 t C 680 Vi 302 rD 056 R0 uca 17g 91 r CA 0 h Da D 2 r QC 21 uca XR 8g0 Da hr0 Han 901 C 3 n RQ an 036 Hel 12g anX r H h C nn RQ Hela anX 45.1 nn H 004 E la . t e u H ig M 96.1 gut 003 Mim ut.L .1 Mig .1 t u g 0250 0820 Mim ut.L0 0017 v2 0 t Mig 3 .1 u 7 0 g 8 7 988 Mim X38 0002 RAE 3 v2 xc F 7 e 8 a r 8 3 F AEX .2 xc FR 4052 Frae eu01 O l y rs u e 5 le 0 0 40 O 92.m0 m 296 0.1 Ricco 17700 G i.002 i Gora ra s o 6 G 9 5t1 EG010 Thecc1 Thecac 041.t1 Jugreg g1 A-1 3672.t1 ne-0.25-mRN Jugreg g1 06-snap-ge -scaffold010 er ak m ol Casm P0409830.2 Querob Qrob 40 del.supercontig 2.2 .mo Carpap evm 1 cds XP 010554410.1 2856 Tarhas lcl NW 010966881.1 4543 87.1 0105277 Tarhas lcl NW 010961290.1 cds XP Tarhas lcl NW 010960187.1 cds XP 010553798.1 3192. Eutsal Thhalv10015535m Tarhas lcl. DELLA1.1. NW 010966372.1 cds XP 010549753.1 24315. .1 .1. .1. .1 .1. 70. .1. .1. 70. .1. Monocots Basal Eudicots Eudicots. 85. 4.2 .su .1 009 per 290 con com .1 t i g 286 116 cac 77. .43 The m0 Gos cc1 000 rai E 55 G0 Gor 1 ai.0 916 Cits 01G 5t1 in o 089 rang 400 e1.1 .1 C g00 itcle Ros 815 Cicle chi R 3m v10 chiO 011 BHm 367 Hum Chr3 m lup H g04 L.SW 930 .v1.0 11 .G01 5633 Morn .1 ot L4 84 0 Mald 2720 om M 1 DP00 0066 Maldo 9451 m MD P000 Pyrbre 09503 lcl NW Pyrco 87 00898 m PC 8416.1 P0196 cds XP 49.1 00933 4448.1 31731 Maldom MDP00 005605 49 Pyrcom Pyrbre lcl PCP0102 NW 0089 14.1 88216.1 cd s XP 0185 05947.1 22 014 Maldom MD P000018148 Prumum lcl NC 2 024129.1 cds XP 008229195.1 13 596 Pruavi Pav sc0000464 .1 g350.1.mk. 59. Jugreg g1321.t1. Ric. The. Pruper Prupe.3G162500.1. rpa. pe. vm. .mo. del. Lag. sic. Lsi. 09G. 97. 14. r5.. Ch. 22. el.. C0. O3. EL. vm te. el M. cm. Cu. csa. Ca. Basal Angiosperms. Jugreg g16324.t1. First outer circle legend. Querob Qrob P0505290.2. Cu. 66. .1 30. od. .m. Cit. lan. Cla. 01. 02. 29. 70. .1. 30 99. 39 02. 4G h0 Cm. aC. h0. 4G. 76. 30 74. 00. g1. 5G. 01. LG. 4.1. oC. Cm. os. ax. Cu. cm. 37. 40. 52. g0 13. 00. 5G h1. oC. h1 aC. Cm. ax. Cu. Cu. cp. ep. Cp. Cp. Cm os. cm. cm Cu. cm. 69. 40. 00. 1G. h0 4.1. LG. aC Cu. Cu. cp. ep. ax cm. 00. 1G. h0. Cm. os. Cu. 39. g1 02. G. 1L. Cm. oC. 4.. Cp. 00. 82. 00. .1. 30. 83. 00. 4G. h1. aC. cm. Cu. Cu. cp. ep. ax cm. Cu. 00. 4G. h1. oC. Cm. p. pe. os. uc. cm Cu. C. Cm. C. p4. .1. LG. 03. g0. 60. 90. .1. DELLA1.2. Eutsal Thhalv10007323m Boestr Bostr.7128s0107.1 Capgra Cagra.3501 s0003.1 Caprub Carub .0001s1470.1 Aratha AT1G 14920.1 Arahal Ar aha.1686 4s0002.1 Aralyr A L1G2697 0.t1 Boestr Bostr.7 903s00 21.1 Aratha AT2G0 1570.1 Aralyr AL5G 10630 .t1 Capg ra Ca gra.2 235s0 Capru 013.1 b Ca rub.0 Euts 005s al Th 0051 halv1 .1 Brao 0005 leca 482m p Bo Brar l018 ap B 868 rara Bra .I02 olec 029 ap B .1 Bra ol00 rap 466 Bra 7 Citc ra.F le C 034 72.1 icle Cits v10 in o 017 r a 466 Lup nge m ang 1.1 g04 Lup Lup 313 007 ang 1 m 545 Lup Lot .1 jap 009 Lj4 138 Cic g . 3v2 ari 1 Ca 436 Tri 16 su 020 49 bl .1 Me 1.1 cl D dtr F9 uM 74 Tri 05 pra tru 0.1 nA Tp Ca 17 cd 57 jca sG Ch 57 jC Gl AU r8g 7T ym ca 44 0 G j 37 an ax 52 Gl AC 63 1.1 22 ym Gl v 8 2 y 96 1 33 ax Vi m m 9 57 a.0 gr RN Gl 9 a 5G ym A9 dV Ph 90 14 a.0 av ra 2 04 di0 Cu ul 8G 00 Ph 27 c 09 .1 Cu ma v 1s 58 x C ul.0 00 00 cm 03 Cu 05 .1 m o G 0 s a c . Ch 29 pe 1 Cm Cu 15 04 p oC Cp 00 Cu cpe G0 h0 .1 p 4. 2 c 4 12 Cp m 1L C G0 uc ax 10 G 4. 22 00 m .1 20 os Cm 1LG g0 0. a 01 41 C Ch 1 m g1 90 15 oC .1 84 G h1 40 5G 009 .1 59 01 0. 00 1 00 .1. .1 10 1 36 2. g0 8. 13 13 29 19 LG 05 5. 0 .1 hr 2 3C p4 .C 93 O C el 23 p .1 EL od .1 pe l M 94 .1 46 .m e 12 uc 32 660 m 54 C 03 39 ev 89 6 cm 0 t 5 G . 00 Cu csa 01 a0 .0 9G Cl .v1 XP i0 n 21 Cu 1 ds 36 tla c Ls L.SW 197 i 44 1c Ci 1 . H 0 8 gs g0 r4 68 84 La lup 9 98 2 m Ch L4 62 0 m 1 ot Hu C . 2.1 n 0 N k BH 1 42 or m .1 lcl iO 41 21 M 0 .1. h 3 0 82 juj 10 4g Rc 96 00 g2 Ziz chi 32 H4 .1 XP s Fv .1G 21 ds e 2 Ro es 1c up 00 . av Pr 00 27 Fr sc er 41 4 939 02 up av 03 P Pr .1 4 40 NC vi 6 505 lcl ua 00 m Pr 351 00 1 . u P 2 009 um 69 8 MD XP Pr 27 m 797 ds P0 ldo 023 .1 c PC Ma m 000 058 DP rco 988 Py mM 008 1 W ldo 38. lN Ma e lc 210 P0 rbr 76 Py PC 141 1.1 R0 com 60. CA Pyr 112 rD 0 uca .1.1 11g Da 370 olyc 086 yc S 10g .1.1 Soll 380 olyc 086 yc S 10g Soll olyc 66.1 022 yc S Soll ut.H Mig 894.1 gut K00 Mim igut. 0.1 gut M 265.1 6153 Mim u009 0002 l Oe 3 v2 ursy 60.1 3887 Olee 1143 AEX 2 000 xc FR 873 v Frae EX38 c FRA 632.1 Fraex eu059 rsyl O 01 258 Oleeu 07350 93.1 36 T0103 158716 s XP 0 GSVIV Vitvin 98.1 cd 36741 154558 2232.1 l NW 0 P 01587 Zizjuj lc .1 cds X 8 704 545663 191.1 36 l NW 01 XP 015872 Zizjuj lc 569.1 cds 36740 W 015456 5872233.1 Zizjuj lcl N 1 cds XP 01 015456638. Zizjuj lcl NW 694.1 36257 cds XP 015871 015455898.1 Zizjuj lcl NW 03 015872192.1 367 456569.1 cds XP Zizjuj lcl NW 015 83-snap-gene-0.8-mRNA-1 Casmol maker-scaffold062. 24314 2.1 cds XP 010549754.1 32551 .1 681 558 010 967707.1 cds XP Tarhas lcl NW 010 02s1238.1 .00 rub Ca b Capru 002.1 gra.0463s0 Capgra Ca 350..1 66 1G AT Aratha m 018436 hhalv10 Eutsal T 655.1 1 0 .B Brara Brarap 1 4080.t AL2G2 Aralyr 4.1 8 2 0 s 6959 Bostr.2 256 Boestr 0.1 5 2854 5 0 01 983 P 2 X 7.1 cds 5179 290.1 0105 50.1 0961 9 P 1 7 0 X 0 s W 3g cd s lcl N 87.1 radi0 1 0.1 V 0 d 6 Tarha 7 00 6 109 Vigra 121s NW 0 s lcl 00.1 ang0 a 5 h 0 .V r a 3 n T 2 iga 01G 0 ng v l. a 00.1 u ig V hv 158 1g2 vul P .1 0 a n h P 000 igu 040 ng V 0.1 18G . Vigu 0 a 5 m 6 Gly G21 x 71 1 a 1 . m a 335 Gly lym 3.1 515 ax G 451 4 4 m 1 U A Gly 1 GA RN s m 78. d c 2 152 9.1 Cv a 4 A 71 0 C 9 G 4 0 ari 7T F97 011 3 Cic 757 3g lcl D 58 5 r p 9 b h u n1 ra T 7C Tris 0.1 aja A1 Trip 8 c n 8 u 33 K aj C Mtr 4 c j 0 u 7T v Ca dtr g3 .KK 3.1 Me Lj6 aip 7 r p 8 A a tj a 23 .1 Lo p0 80 aip Ar Lu 54 0.1 g0 ng 6 a 73 .1 p i0 05 Lu ad 0 r g 1 V 50 g0 45 00.1 rad an g1 V . Vig 8 2 55 n n0 52 23 iga igu 05 0 8G gv V n 0 n D7 0 a ng aja XU ul. .1 c Vig v gu . i C h u 45 1 V aj lP . ad 294 c r u j A 50 8 av Ca p0 ur 36 2 Ph Lu ad 00 132 r g A 62 n 5G la0 0 pa 20 .1 i u C 3. L Ls r .2 n h ic 06 tla .C gs Ci el 02 d 2 La o 0 .m 3C m LO ev E t a M cs el m Cu uc C. Tarhas lcl NW 01096637. Tree scale: 1. .1. 30. 00. O. a. So. pip. a2. aff. tL. tit. ic. UA. sm. ou. Se. So. ac. sa. Maldom MDP00001 34341 Pyrbre lcl NW 008988236.1 cds XP 009371 410.1 23202 Pyrcom PC P004331.1 Cucpep C p4.1LG04 g09740.1 Cucmax CmaCh1 1G0057 80.1 Cucmo s CmoC h11G00 Cucma 5830.1 x Cma Ch10G Cucpe 00586 0.1 p Cp4 .1LG1 Lagsic 8g045 70.1 Lsi06 G 0091 Citlan 60.1 Cla0 1230 Cucm 2 el M ELO Cuc 3C02 sat e 5288 vm.m .2.1 Araip odel. a Ar Chr1 a ip.51 .194 Lotja 8 Q21 p Lj6 g3v Cic 095 ari C 947 a 01 0.1 Me 703 dtru .1 Mtr Trip u nA1 ra T 7C p57 Tris hr2 577 g03 ub TG 066 l c l DF Gly AC 41 ma 973 v 2 xG mR 226 Gly NA lym . 1 ma 319 c a.0 ds xG 39 Gly 4G GA lym 150 ma U2 a 1 5 x . 281 06G Gly 00. Gly 1 .1 1 ma 213 ma 033 xG Ph .10 100 9 av lym G1 .1 ul 90 a.2 Vig Ph 2 00 0G un vu .1 20 gV l.L Vig 05 00 igu a 00 20 n0 .1 Vig ng v 44 7g .1 iga rad 1 8 n Am 12 .Va Vr 00 ad btr ng .1 Zo i08 ie 06 s vm g0 g1 96 Zo mar 53 2 7.T 40 50 s Zo .4 .1 Sp mar U. s Am ipo Zo ma1 M Tr 06 sm lS us v1 g0 pip a1 ac M .0 00 3 o us u sc 30 GS 15G 5g0 ac aff M . 1 0 us old u 00 M 38 G U ac M 00 27 0. S A us 1 u 01 80 M A Br G ac 0.1 ch 0 SM UA ad u r1 06 A G P is UA ch 2 S 13 Br r2 M A P2 ad 00 UA ch i1 25 r8 00 Ac g1 P1 10 1 hr 10 82 00 10 90 50 1 P .1 19 00 65 1 0 00 1. 00 10. 0 50. Pruavi Pav sc0000704.1 g110.1.mk. Prumum lcl NC 024133.1 cds XP 008242703.1 26299. Pyrcom PCP026914.1 018506851.1 25641 988278.1 cds XP Pyrbre lcl NW 008 000662303 Maldom MDP0 .1 8.1.g00001 iscf0030207 Fraana FAN 001.1 2914.1.g00 N iscf0009 080.1 Fraana FA vH4 1g04 Fraves F 9571 r2g008 Hm Ch RchiOB 180.2 Roschi P0282 b Qrob NA-1 Quero .1-mR ene-0 7 binit-g 3700 1833-a 527.1 2 ffold0 1 1587 0 d-sca 387.t s XP maske g g43 4.1 cd ustus Jugre 5719 75t1 ol aug 0154 Casm 0379 lcl NW 1EG 00.1 Zizjuj hecc ac T 0670 Thec .1 010G 700 orai. 021 rai G 09G Gos 0 482 orai. 000 rai G 07.m Gos 5.88 298 om tig 7 n Ricc erco 07m sup 096 del. 1g0 .mo 25m e1. evm 108 ang r 0 pap in o v10 881 Car icle Cits 316 4 le C 0g0 922 Citc hr1 0 C R0 .1 RQ CA 100 anX rD nH 205 uca RA lan Da e7G He 05 63 A qco eA U2 7-R 56 NN uco A 21 Aq uc -R n 5 l NU 05 Ne A 11 cN -R 20 lnu 33 R6 Ne 34 .t1 3 e AU 20 zp ui R6 0 e hg.t1 eq 16 AU Ch iq .1 ui 98 0 1 eq 00 p1 54 4 Ch 84 eS 19 83 16 iol v8 30 8G Sp .1 B 0 0 l .00 08a 20 tvu 1 bic 37 00 0. Be So 93 G1 m0 2 ic . 9 3 Z . rb 00 2 g4 ita ay So 05 79 Se 0. am Os i1g4 07 Ze .1 tita C 0 32 ad Se LO 1 80 1g Br 0. at di 90 is 0 a 0 ys 1 .1 ad Br Or 1G 58 117 s Br i 0 Hr 5 G ad U1 r1 2g4 Br 3H RV di VU Bra HO R is ad Br. ul. Ho. rv. Ho. O.

(17) DELLA§LxYxVxxxxMAxVAxxLExLEx§ DELLA2. DELLA1 Monocots. DELLA1.1 Eudicots. DELLA1.2 Eudicots. TVHYNPxxLxxWxxxM.

(18) ●. 0.7. 0.6. dN/dS (Yang & Nielsen (2000) method). 0.5. ● ●. 0.3. ● ● ● ● ● ●. ● ● ●. 0.2. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ●. ●. ●. ●. ●. ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ●. ● ● ●. ● ● ●. ●. ● ● ● ● ●. ●. ● ● ● ●. ●. ●. ●. ● ● ● ●. ●. 0.4. ●. ●. ● ● ● ● ● ● ● ● ● ●. ●. ● ●. ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ●. ●. ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. DELLA clades DELLA1.1 ●. ●. ● ● ● ● ● ●. 0.1. 0.0. DELLA1.1. ● ● ●. ● ● ● ●. ● ●. ●. ● ●. ●. DELLA1.2. DELLA1 Monocots. DELLA clades. ● ● ● ●. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●. ● ● ● ●. DELLA1 Monocots. ● ●. ●. ● ● ●. DELLA1.2. DELLA2. ● ● ● ● ● ● ● ● ● ● ● ● ●. DELLA2.

(19) B. A. DELLA2. Glymax Glyma.10G190200.1 Glymax Glyma.20G200500.1. 100 87. Phavul Phvul.L002044.1 Vigung Vigun07g181200.1 Vigang vigan.Vang06g09640.4 Vigrad Vradi08g15350.1 Araipa Araip.51Q21 Lotjap Lj6g3v0959470.1 Cicari Ca 01703.1 Medtru MtrunA17 Chr2g0306641 Tripra Tp57577 TGAC v2 mRNA31939 Trisub lcl DF973226.1 cds GAU21281.1 10339 Lupang Lup007545.1. 98 100 97. 61. 64 72 93 94 100 100. DELLA1.1. 99. 100. 100 100 96 81 100 95 96. 100. DELLA1.2.1. 74 96 100 100. DELLA1.2. 100 99. 91. 90. DELLA1.2.2. 76. 74 100 81 100. 98. 88100. Papilionoideae WGD. 57 100. Tree scale: 0.1. 88 100. 20. 30. 40. 50. 60. 70. - GDL AGFGY - K VRSSE - LQHVAE NME - R LE NVMD I VNSS TNNN - - - ISQL AS - DT I F Y - NP SD I - - GSWIDT L DGDL AGFGY - K VRSSE - LQHVAENME - R LE NVMD I VNSS TNNN - - - ISQL AS - DT VF Y - NP SD I - - GSWVDT L DDHL AGLGY - K VRSSE - LCQVAANME - R LE NV I - - - - SS TD - - - - - LSQL AS - DT T L Y - DP SN IGLGSWVDT L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MS LP S - - - - HYHLSSDL - - - - - - - - -. Glymax Glyma.04G150500.1 Glymax Glyma.06G213100.1. 100. 10. DGQL AGLGY -KVRSSE - LCQVAENME -RLENA INTVNSSRD - - - - - ISQVVS -DAL L Y -DP SN IGLGSWVDTL. DGQL AGLGY - K VRSSE - LCQVAE NME - R LE NA INT VNSSRD - - - - - ISQVVS - DAL L Y - DP SN IGLGSWVDT L DGQL AGLGY - K VRSSE - LCQVAE NME - R LE NA INT VNSSRD - - - - - ISQVVS - DAL L Y - DP SN IGLGSWVDT L DGQL AGLGY - K VRSSE - LCQVAE NME - R LE NA INT VNSSRD - - - - - ISQVVS - DAL L Y - DP SN IGLGSWVDT L NE MVVDE GL - - - - - - - - L HD IML NSP - S L HHL P - - - - - - - - - - - - - QT THYN - YNYNY - NP F DDT T VSW IN - -. - - - - - - - - - -RVRP HR - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L L TLRS - - - - - - - - - - - - - - - - - - - - -. DGL L AGVGY - K VRSSE - LHQVAQN IE - R LE NV I - - VNSSSD - - - - - ISQ I AS - DT VHY - DP SD I - - GNWVDNL DGL L ANVGY - K VRSSE - LHQVAQNLE - R LE S A I - - VNSSSD - - - - - ISQF AS - DT VHY - DP SD I - - GNWVDNL DGL L ADVGY - K VKSSE - LHQVAQN IE - R LENV I - - VNSSD - - - - - - ISQF AS - DT IHY - DP SD I - - GNWVDNL DGL L ADVGY - K VKSSE - LHQVAQN IE - R LENV I - - VNSSSSD - - - - ISQF AS - DT VHY - DP SD I - - GNWVDNL DE L L AVVGY - K VRSSD - MAE VAQK IE - QLE E AM- - TNVE TN - - - - - ISS LSS - NT VHY - NP SD I - - S TWLESM Lupang Lup009138.1 DE L L AVVGY - K VRSSD - MAE VAQK IE - QLE E AM- - TNFDTG - - - - - ISS LSS - DT IHF - NP SD I - - S TWLESM Glymax Glyma.05G140400.1 DE L L AVVGY - K VRSSD - MAE VAQK LE - R LE E AM- - GNVQDD - - - - - L TDLSN - DAVHY - NP SD I - - SNWLQTM Glymax Glyma.08G095800.1 DE L L AVVGY - K VRSSD - MAE VAQK LE - R LE E AM- - GNVQDD - - - - - LP E ISN - DVVHY - NP SD I - - SNWLE TM Vigrad Vradi0271s00050.1 DE L L AVVGY - K VRSSD - MAE VAQK LE - R LE E AM- - GN IQDG - - - - - LKE LSD - GAAHY - NP SD I - - SNWLE TM Phavul Phvul.003G291500.1 DE L L AVVGY - K VRSSD - MAE VAQK LE - R LE E AM- - GNVQDG - - - - - LKE LSD - DAVHY - NP SD I - - SNWLQTM Lotjap Lj4g3v2436020.1 DE L L AVVGY - K VRSSD - MAE VAQK LE - QLE E AM- - - - - SDDT I - - - AHHLSS - DT IHF - NP AD I - - GTWLE TM Cicari Ca 16491.1 DE L L AVVGY - K VKSSD - MAE VAQK LE - QLEHAM- - GNFQDQDE A I I AQQLSN - DT VHY - NP SD I - - SNWLK TM Medtru MtrunA17 Chr8g0376381 DE L L AVVGY - K VKSSD - MAE VAQK LE - QLEQAMMGNNFHDHDE S T I AQHLSN - DT VHY - NP SD I - - SNWLQTM Tripra Tp57577 TGAC v2 mRNA9902 DE L L AVVGY - K VKSSD - MAE VAQK LE - QLEQAM- - GNFQDQDE S T I AQQLSN - DT VHY - NP SD I - - SNWLQTM Trisub lcl DF974050.1 cds GAU44521.1 33579 DE L L AVVGY - K VKSSD - MAE VAQK LE - QLEQAMNGGNFQDQDE S T I AQQLSN - DT VHY - NP SD I - - SNWLQTM Aradur Aradu.XUD70 DE L L AA IGYKK LRSKDNMADVGQK LDQQLE AVMMDNNVNARE DGTSSNVASDDAVVHYDDP NDV - - FPWEK TM Lupang Lup029445.1 DE L LE VLGY - K VRSSD - MADVAHK LE - K LE DV I - - VT TQQNG - - - - ISNL ATHHT VHY - NP TDL - - HGWVHNM Cajcaj Ccajan 05522 DK L L AGVGY - K VCSSD - MADVAQK LE - QLEMVM- - - - - DE DG - - - - IP HLP T - DT VHY - DP TDL - - YGWVQN I Phavul Phvul.008G235500.1 DR L L AGVGY - K VCSSN - MVDVAHK LD - QLE MVM- - - - - E GDG - - - - MSHL AN - NT VHY - DP TDL - - YGWVQN I Vigung Vigun08g145500.1 DK L L AGVGY - K VCSSD - MVDVAHK LD - QLE MVM- - - - - E E DG - - - - IP NL AS - DT VHY - DP TDL - - YGWVQN I Vigang vigan.Vang01g05730.1 DK L L AGVGY - K VCSSD - MVDVAHK LD - QLE MVM- - - - - E E DG - - - - ISHL AS - NT VHY - DP TDL - - YGWVQN I Vigrad Vradi06g05480.1 - - - - - - - - - - - - - - - - - MVDVAHK LD - QLE MVM- - - - - E E DG - - - - ISHL AS - NT VHY - DP TDL - - YGWVQN I Lupang Lup023873.1 DE L L AALGY - K VRSSD - MADVAQK LE - QLE MAM- - G I TQEDV - - - - ISHLSS - DT VHY - DP TDL - - YSWVQTM Araipa Araip.KK7TK DE L L AALGY - K VRNSD - MADVAQK LE - QLE MVM- - GT AQE DG - - - - ISHL AT - DT VHY - DP TDL - - YSWVQSM Lotjap Lj6g3v0433880.1 DE L L AALGY - K VRSSD - MADVAQKME - QLEMVM- - G I AQE DG - - - - ISHL AS - DT VHY - DP TDL - - YSWVQT I Medtru MtrunA17 Chr3g0110971 DE L L AALGY - K VRSSD - MADVAQK LE - QLE MVM- - GS AQEE G - - - - INHLSS - DT VHY - DP TDL - - YSWVQTM Cicari Ca 15278.1 DE F L AALGY - K VRSSD - MADVAQK LE - QLE MVM- - GC AQE DG - - - - INHLSS - DT VHY - DP ADL - - YSWVQTM Tripra Tp57577 TGAC v2 mRNA14515 DE L L AALGY - K VRSSD - MADVAQK LE - QLE MVM- - GS AQEE G - - - - INHLSS - DT VHY - DP TDL - - YSWVQTM Trisub lcl DF974049.1 cds GAU44513.1 33571 DE L L AALGY - K VRSSD - MADVAQK LE - QLE MVM- - GS AQEE G - - - - INHLSS - DT VHY - DP TDL - - YSWVQTM Cajcaj Ccajan 19583 DE L L AALGY - K VRSSD - MADVAQK LE - QLE MVM- - GS AQEE G - - - - ISHL AS - DT VHY - DP TDL - - YSWVQTM Glymax Glyma.11G216500.1 DE L L AALGY - K VR ASD - MADVAQK LE - QLE MVM- - GC AQE DG - - - - ISHL AS - DT VHY - DP TDL - - YSWVQSM Glymax Glyma.18G040000.1 DE L L AALGY - K VR ASD - MADVAQK LE - QLE MVM- - GC AQE EG - - - - ISHL AS - DT VHY - DP TDL - - YSWVQTM Phavul Phvul.001G230500.1 DE L L AALGY - K VR ASD - MADVAQK LE - QLE MVM- - GS AQEE G - - - - ISHLSS - Y T VHY - DP TDL - - HSWVQSM Vigung Vigun01g215800.1 DE L L AALGY - R VR ASD - MADVAQK LE - QLE MVM- - GS AQE DG - - - - ISHL AS - DT VHY - DP TDL - - HSWVQSM Vigang vigan.Vang0121s00760.1 DE L L AALGY - K VR ASD - MADVAQK LE - QLE MVM- - GS AQEE G - - - - ISHL AS - DT VHY - DP TDL - - HSWVQSM Vigrad Vradi03g07950.1 DE L L AALGY - K VR ASD - MADVAQK LE - QLE MVM- - GS AQEE G - - - - ISHL AS - DT VHY - DP TDL - - HSW- - - Consensus DE L L AAL GYKK VRSSDNMADVAQK L E QQL E MVMNTGNAQE DGE S T I ISHL AS +DT VHY +DP SD IGL YSWVQTM.

(20)