The genome of giant waterlily provides insights into the origin of angiosperms, leaf gigantism, and stamen function innovation

Abstract

As some of the earliest evolving flowering plants, waterlilies offer unique insights into angiosperm evolution. Giant Amazonian waterlilies (genus Victoria) are of particular interest due to their production of the world's largest floating leaves and gigantic flowers that entrap pollinating beetles. Here, we report chromosome-level genome assemblies of Victoria cruziana and three related waterlilies: Euryale ferox, Nymphaea mexicana, and Brasenia schreberi. We found an ancient whole-genome duplication event specific to the Nymphaeales. We reveal major gene duplication and loss events throughout the evolution of angiosperms, with substantial implications for flower development and the biosynthesis of floral volatile organic compounds (FVOCs) in waterlilies. Importantly, we report a unique division of labor in the stamen function of V. cruziana linked to beetle attraction by FVOCs. This is related to the ultra-high expression of VicSABATHa along with Vicchitinase, possibly linked to protection from damage by trapped beetles. Overexpression of VicSABATHa in tobacco leaves reveals a capacity to produce volatile fatty acids, confirming its role in their catalytic synthesis. Overall, these findings provide novel insights into the evolution and adaptations of waterlilies and flowering plants in general.

Keywords: early angiosperms; floral scent biosynthesis; leaf gigantism; stamen innovation; waterlily genomes.

Figure 1

Phylogenetic relationships of waterlilies with other plants and genome assemblies of V. cruziana, E. ferox, N. mexicana, and B. schreberi in the present study. (A) Phylogeny of 29 representative angiosperms, including six waterlily species: V. cruziana, E. ferox, N. mexicana, B. schreberi, N. thermarum, and N. colorata. (B) Morphology of leaves and flowers in V. cruziana, E. ferox, B. schreberi, and N. mexicana. (C) BUSCO assessment of six waterlily genomes.

Figure 2

WGT and WGD events in Nymphaeales. (A) Density distribution of estimated synonymous substitution rates (Ks) of syntelog pairs from intragenomic comparisons of V. cruziana, B. schreberi, E. ferox, N. mexicana, and N. colorata, and an intergenomic comparison between V. cruziana and B. schreberi. Labeled peaks indicate potential WGD events. (B) Syntenic dot plots for V. cruziana and B. schreberi. Red rectangles represent collinear blocks in B. schreberi; blue rectangles represent collinear blocks in V. cruziana. (C) Phylogenetic framework and genome evolution history, including WGTs and WGDs, in waterlilies. WGD, whole genome duplication; WGT, whole genome triplication.

Figure 3

Gene duplication and loss events in the evolutionary history of angiosperms. (A) Venn diagram showing the numbers of specific and common gene families in Nymphaeales, monocots, magnoliids, and eudicots. (B) Simplified phylogenetic relationships of major angiosperm clades, highlighting gene family number variation and key genes involved in molecular regulatory mechanisms (Supplemental Figures 7–25). Genes in solid red boxes represent genes gained in the lineage; genes in dashed blue boxes represent genes lost in the lineage.

Figure 4

Developmental mechanisms underlying leaf gigantism. (A) Heatmap showing relative expression profiles of genes in 12 co-expression modules inV. cruziana leaves and petals, generated by WGCNA. The 12 modules were divided into three groups corresponding to three stages of leaf development. Genes in solid boxes in Figure 2A are correlated with the turquoise module shown in Supplemental Figure 24. Genes in dashed boxes correspond to the magenta, yellow, and green modules in Supplemental Figure 24. (B) Heatmap showing the expression profiles of key genes involved in early-stage leaf and petal growth. (C) Histogram showing the expression profiles of putative functional miRNAs involved in leaf size regulation in V. cruziana. (D) Proposed molecular mechanism underlying leaf gigantism in V. cruziana.

Figure 5

Molecular regulatory mechanisms of flower development, with emphasis on stamen development, in V. cruziana. (A) Heatmap of ABC(D)E gene expression. Scale bars: 1 mm. Se, sepal; Pe, petal; St, stamen; Ca, carpel; w, whorl. (B) Heatmap showing gene expression in 16 co-expression modules across various floral whorls and stamen developmental stages. (C) Volcano plot showing differentially expressed genes between stamens from day 1 and day 2 flowers. (D) Key regulatory genes involved in the development of different stamen whorls in V. cruziana.

Figure 6

Floral Volatile Organic Compounds (FVOCs) and pollination syndrome in V. cruziana. (A) GC-MS peak plot showing FVOCs emitted from whole flowers of V. cruziana during 2 consecutive days of blooming. The x-axis represents retention time; the y-axis represents relative abundance. (B) GC-MS peak plot of volatile compounds emitted from the inner two whorls of V. cruziana (mainly comprising stamens). The x-axis represents retention time; the y-axis represents relative abundance. (C) GC-MS peak plot showing emissions of methyl esters and benzenoids from V. cruziana flowers during 2 consecutive days of blooming. The x-axis represents retention time; the y-axis represents relative abundance. (D) Methyl ester biosynthesis pathway in V. cruziana. Ado, adenosine; Hcy, homocysteine. Genes expressed on the first and second blooming days are displayed in rows. (E) Functional validation of VicSABATHa and NcSABATH1 through GC-MS analysis of volatiles in N. benthamiana overexpression lines. The x-axis represents retention time; the y-axis represents relative abundance.