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Gymnosperms are seed-bearing vascular plants, such as cycads, ginkgo, yews and conifers, in which the ovules or seeds are not enclosed in an ovary.The word "gymnosperm" comes from the Greek word gymnospermos, meaning "naked seeds".Gymnosperm seeds develop either on the surface of scale or leaf-like appendages of cones, or at the end of short stalks.
The emergence of seed plants consisting of angiosperms and gymnosperms marked a momentous event in the evolution of land plants and the change of earth environments. Angiosperms and gymnosperms diverged in the Lower Mississippian1, followed by rapid radiation of flowering plants resulting in approximately 352,000 extant species on Earth compared to only 1000 species of gymnosperms. There is a variety of morphological/anatomical diversity and metabolic versatility between angiosperms and gymnosperms, but the underlying genetic and biochemical mechanisms are largely elusive2,3.
Genome sequences are key to addressing critical questions of plant evolution. Analyses of genomes of representative gymnosperms have shown unique aspects of gene and genome evolution that distinguish themselves from flowering plants13,14,15,16,17,18,19. However, understanding the biological and evolutionary mechanisms of phenotypic diversity between flowering and non-flowering plants is still challenging, partly due to the limited availability of genomic resources of gymnosperms.
Whole-genome duplications (WGDs) have occurred across the breadth of eukaryote phylogeny27. In gymnosperms, several WGDs have been recognized although some of them remain in controversy16,17,18,19,28. The Ks distribution of 3859 paralogue groups within T. grandis indicated the absence of recent WGDs. However, we observed a peak of Ks ranging from 1 to 2 and a summit at 1.4, representing a potential ancient WGD that occurred in the common ancestor of conifers and ginkgophytes, a lineage diverged from gnetophytes (Fig. 1d). We then used a tree-based approach29, which calculates the frequency of gene duplication on every branch of a phylogeny by reconciliation of gene tree and species tree, to cross validate the WGD event. Analysis of 19,649 gene trees from eight selected species led to the discovery of three ancient WGD signals including two (zeta and omega) reported previously17,28 and one that was consistent with the Ks analysis (Supplementary Fig. 5). Whole-genome comparison showed high collinearity among genomes of T. grandis and two evolutionarily distant gymnosperms, Sequoiadendron giganteum and Ginkgo biloba (Supplementary Fig. 6), and also revealed traces of collinear blocks that were duplicated in both T. grandis and G. biloba but not in Gnetum montanum, agreeing with the timing at which the newly discovered WGD occurred (Fig. 1e).
a Gene family expansion and contraction during the evolution of green plants. The maximum likelihood phylogeny was built with 219 low-copy orthologous groups. Gene family analysis was started with 10,345 orthologous groups that were shared by the most recent common ancestor (MRCA) of green plants. Numbers on branches are the sizes of expanded (blue) and contracted (red) gene families at each node. Colored pies on the right represent the sizes of expanded/contracted gene families as well as gained/lost genes for each leaf node of the tree. b Expression of MIKCC type MADS-box genes in vegetative and reproductive tissues of T. grandis. c Proposed reproductive organ identity genes in T. grandis. The AP3/PI-like genes (TG7g01668 and TG7g01669) and TG8g01565 were predominantly expressed in male and female cones, respectively. The AG-like (TG10g01848), AGL6-like (TG2g00325) and AP1/SEP-like (TG4g01441) genes were expressed in both female and male cones, with the first two showing a pattern biased to female cones. d Maximum likelihood phylogeny showing the bacterial origin of T. grandis genes. Blue pies indicate bootstrap support greater than 80% at the corresponding branches. e Expression of putative horizontally transferred genes in different tissues. Source data are provided as a Source Data file.
Gymnosperms have unenclosed or naked seeds on the surface of scales or leaves, while flowers and fruits are angiosperm innovations. Phylogeny-based homolog search using well-studied flower development genes32 showed sporadic distribution of these homologs in gymnosperms and non-seed plants (Supplementary Data 6), indicating the stepwise emergence accompanied with secondary loss of flower development genes during the evolution of land plants, as exemplified by NOP10 (required for female gametophyte formation in flowers)33 and WUS (required for shoot and floral meristem integrity)34 genes that emerged early in land plants and were subsequently lost in both T. grandis and T. wallichiana (Supplementary Data 6).
Lignin is a major component of plant secondary cell wall and is derived from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monolignols. S-lignin is restricted to flowering plants and some lycophytes, whereas G- and H-lignin are fundamental to all vascular plants2. Consistently, two key genes for S-lignin biosynthesis, F5H and COMT, were found only in angiosperms but not in gymnosperms. Unlike angiosperms in which vessels comprise major water-conducting elements in xylem43, gymnosperm woods are mainly composed of tracheids2. Vessel differentiation is regulated by VASCULAR-RELATED MAC-DOMAIN (VND) proteins44, while fiber development is associated with NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST)/SECONDARY WALL-ASSOCIATED NAC DOMAIN (SND) proteins45. The T. grandis genome encoded genes homologous to VND4/5/6, but lacked homologs of VND1/2/3, NST and SND1 (Supplementary Fig. 11), which, combined with the finding of divergent regulatory networks of VND/NST homologs in conifer and flowering plants during wood formation46, suggests a close link between vessel formation and the emergence of master NAC transcription factors as well as their regulatory networks in angiosperms.
Phylogenetic analysis of desaturases in green plants (Viridiplantae) showed that TgDES1 clustered with desaturases exclusively from non-angiosperm organisms, and this monophyletic clade was close to the family containing sphingolipid desaturases including AtSLDs from Arabidopsis (Fig. 4a). Interestingly, the TgDES1 clade clearly separated from the group harboring AL10 and AL21, two proteins that were found to be responsible for SCA biosynthesis in the basal eudicot Anemone leveillei48. Structure modeling of TgDES1, AtSLD2 and AL21 showed overall similar structures between TgDES1 and AtSLD2, particularly in the region where the active center was formed, whereas structure of AL21 was relatively diverged from TgDES1 (Fig. 4b). Since flowering plants rarely synthesize SCA, our phylogenetic and structural evidence suggested that this is possibly due to the loss of TgDES1 clade desaturases, while the ability of SCA biosynthesis in particular species of eudicots was largely attributed to the secondary gain of the Δ5-desaturase activity of evolutionarily independent counterparts. Similarly, close homologs of TgELO1 were not found in flowering plants but present in early land plants and algae, suggesting the co-evolution of Δ5-desaturase and Δ9-elongase in plants (Supplementary Fig. 13).
Characterization of protein sequences revealed the conservation of an N-terminal cytochrome b5-like domain and three histidine-rich boxes of TgDES1 clade desaturases (clade 1) and their two closely related groups (group 1 and group 2 of clade 2), whereas striking variation was observed in the first two histidine-rich boxes among different groups (Fig. 4c). A previous study reported that site-directed substitution of histidine boxes could influence substrate chain-length specificity and selectivity50. The single amino acid substitution likely directs the outcome of the desaturation reaction by modulating the distance between substrate fatty acyl carbon atoms and active center metal ions51. To test whether sequence variation of histidine-rich domains determined substrate specificity that led to the success of SCA biosynthesis, we replaced the histidine-rich domain of Arabidopsis desaturase AtSLD2 with that of TgDES1, and transiently expressed the construct in N. benthamiana leaves. We noted that TgELO1 was not coexpressed with the engineered desaturase gene because 20:2Δ11,14-PC, the product of Δ9-elongase catalysis, could be detected in leaves of the wild-type tobacco. SCA was undetectable in N. benthamiana leaves expressing wild-type AtSLD2; however, switch of either of the two histidine-rich boxes from TgDES1 was sufficient to synthesize SCA in N. benthamiana leaves (Fig. 4d). Taken together, our data suggest that mutations in these two histidine-rich motifs of desaturases have led to the alternation of substrate specificity and consequently the evolution of specific clade for SCA biosynthesis, loss of which marks the significant metabolic diversity between gymnosperms and angiosperms.
Seed development in gymnosperms is a long process spanning multiple years3. To understand whether and how DNA methylation participates in seed development of T. grandis, as is evident in flowering plants52, we profiled seed methylomes at three developmental stages (Fig. 5a; Supplementary Data 9). Genes involved in DNA methylation of all three cytosine contexts (CG, CHG, CHH) were identified in the T. grandis genome (Supplementary Data 3). The global average methylation levels of mCG, mCHG, mCHH in T. grandis seed genome were 83%, 69% and 4%, respectively. Both mCG and mCHG methylation levels were higher than those in most of previously studied angiosperms53, coinciding with the proposal of positive correlation between genome sizes and mCG/mCHG methylation levels54. mC of all sequence contexts was enriched at centromeric and peri-centromeric regions, despite that both mCG and mCHG were also broadly distributed in chromosome arms (Fig. 1b). In flowering plants, exons of genes are sometimes enriched with mCG but depleted with both mCHG and mCHH, which is referred to as gene body methylation (gbM)55. We observed the enrichment of mCG and depletion of mCHH in T. grandis genes; however, enrichment of mCHG was also found in transcribed regions (Fig. 5b and Supplementary Fig. 14a, b), which is similar to the pattern found in conifers56. GbM has been proposed to regulate gene transcription55. We observed a clear enrichment of mCG instead of mCHG/mCHH on moderately expressed genes, for which the expression was positively correlated with methylation levels (Fig. 5c and Supplementary Fig. 14c), indicating functional conservation of gbM in the sister lineage of angiosperms. Evolution of gbM is hypothesized to be associated with DNA methylation silencing of TEs in proximity of genes55. Consistently, we found that LTR-RTs, which were the major component of TEs in gene regions, were highly methylated (Fig. 5d), and that genes with TE insertions had both higher expression and CG methylation than those without TEs (Supplementary Fig. 15). 781b155fdc