Whole-genome Duplication Reshaped Adaptive Evolution in A Relict Plant Species, Cyclocarya paliurus

Abstract

Cyclocarya paliurus is a relict plant species that survived the last glacial period and shows a population expansion recently. Its leaves have been traditionally used to treat obesity and diabetes with the well-known active ingredient cyclocaric acid B. Here, we presented three C. paliurus genomes from two diploids with different flower morphs and one haplotype-resolved tetraploid assembly. Comparative genomic analysis revealed two rounds of recent whole-genome duplication events and identified 691 genes with dosage effects that likely contribute to adaptive evolution through enhanced photosynthesis and increased accumulation of triterpenoids. Resequencing analysis of 45 C. paliurus individuals uncovered two bottlenecks, consistent with the known events of environmental changes, and many selectively swept genes involved in critical biological functions, including plant defense and secondary metabolite biosynthesis. We also proposed the biosynthesis pathway of cyclocaric acid B based on multi-omics data and identified key genes, in particular gibberellin-related genes, associated with the heterodichogamy in C. paliurus species. Our study sheds light on evolutionary history of C. paliurus and provides genomic resources to study the medicinal herbs.

Keywords: Cyclocarya paliurus; Genomics; Resequencing; Triterpenoid; Whole-genome duplication.

Figure 1

Morphology and genome duplications of C.paliurusA. Imparipinnate leaves. B. Mature fruits. C. Female and male flowers of PA-dip. D. Female and male flowers of PG-dip. E. Fruit wings that start to spread. F. Tetraploid plant. G. Genomic alignments between the basal angiosperm A. trichopoda and the basal eudicot V. vinifera, as well as PG-dip, PA-dip, and PA-tetra C. paliurus are shown. The conserved collinear blocks are shown as the gray lines in the background, and the green lines indicate cases in each round of WGD. C. paliurus, Cyclocarya paliurus; A. trichopoda, Amborella trichopoda; V. vinifera, Vitis vinifera; WGD, whole-genome duplication; PA-tetra, the protandrous tetraploid C. paliurus; Chr, chromosome; PG-dip, the protogynous diploid C. paliurus; PA-dip, the protandrous diploid C. paliurus.

Figure 2

Phylogenetic and comparative analyses of C. paliurus A. Phylogenetic relationship of C. paliurus, C. illinoinensis, J. nigra, P. stenoptera, A. thaliana, Z. jujuba, V. vinifera, P. trichocarpa, and O. sativa. The divergence time among different plant species is labeled at the bottom. B. Venn diagram of orthologous and species-specific gene families in different plant genomes. C. Evolutionary analysis of the diploid and tetraploid C. paliurus genomes with the distribution of Ks values of orthologs.D. Synteny analysis between PA-tetra and PA-dip genomes. “monoploid” indicates a reference genome assembly with only one representative haplotype retained, whereas “haplotype” indicates fully phased genome with all the four haplotypes. C. illinoinensis, Carya illinoinensis; J. nigra, Juglans nigra; P. stenoptera, Pterocarya stenoptera; A. thaliana, Arabidopsis thaliana; Z. jujuba, Ziziphus jujuba; P. trichocarpa, Populus trichocarpa; O. sativa, Oryza sativa; Ks, synonymous substitution rate; MYA, million years ago.

Figure 3

Dosageeffect contributes to increased growth adaptability and accumulation of terpenoids A. Scanning electron microscopy of stomata in diploid and tetraploid C. paliurus leaves. B. Comparison of chlorophyll content between diploid and tetraploid C. paliurus individuals. Statistical significance (n = 5) was determined using two-sided Student’s t-test. Error bars indicate mean ± SD of indicated replicates. **, P < 0.01. C. Comparison of Pn between diploid and tetraploid C. paliurus individuals. ***, P < 0.001. D. Heatmap showing the accumulation patterns of triterpenoids among the four samples. Sept_diploid indicates diploid samples collected in September; Sept_tetraploid indicates tetraploid samples collected in September; May_diploid indicates diploid samples collected in May; and May_tetraploid indicates tetraploid samples collected in May. E. Expression profiles of genes associated with cyclocaric acid B synthesis in C. paliurus tender and mature leaves for different ploidies. The scale ranging from blue (low) to red (high) indicates the expression magnitude of the FPKM values. The genes of CYP72A subfamily are shown in blue. The functions of the CYP716A14v2 and CYP716C subfamilies were identified by Miettinen and colleagues and Moses and colleagues , . SL, stomatal length; SA, stomatal aperture; SW, stomatal width; mag, magnification; WD, working distance; HV, high voltage; AM, ante meridiem; PM, post meridiem; SD, standard deviation; Pn, net photosynthetic rate; FPP, farnesyl pyrophosphate; SQS, squalene synthase; SQE, squalene epoxidase; bAS, β-amyrin synthesis; FPKM, fragments per kilobase per million.

Figure 4

Phylogenetic splits among C. paliurus populations A. Dispersion of 45 individuals sampled from 9 sites (a–i) across most of the geographic range of C. paliurus. Populations are plotted with dots color-coded based on dispersion by latitude and longitude. Yellow stars represent the co-presence of diploid and auto-tetraploid distributions, blue stars represent only auto-tetraploid distribution, and purple star represents the outgroup (Juglans regia). More details are shown in Table S14. The world map was constructed from http://bzdt.ch.mnr.gov.cn/index.html. B. A phylogeny for C. paliurus individuals estimated from SNPs in neutrally evolving sites. C. PCA showing clear separation between diploid and auto-tetraploid populations. D. Top: CV plot displaying CV error vs. K. K = 2 is the best fit. Bottom: ADMIXTURE plot for C. paliurus showing the distribution of genetic clusters (K = 2, 3, and 4). K = 2 (representing the divergence within diploid and auto-tetraploid clades) indicates the smallest CV error. SNP, single nucleotide polymorphism; PCA, principal component analysis; CV, cross-validation.