doi: 10.1038/s41477-022-01129-7. Epub 2022 Apr 18.
The Cycas genome and the early evolution of seed plants
Yang Liu # 1 2, Sibo Wang # 3, Linzhou Li # 3, Ting Yang # 3, Shanshan Dong # 4, Tong Wei # 3, Shengdan Wu # 5, Yongbo Liu # 6, Yiqing Gong 4, Xiuyan Feng 7, Jianchao Ma 8, Guanxiao Chang 8, Jinling Huang 7 8 9, Yong Yang 10, Hongli Wang 3 11, Min Liu 3, Yan Xu 3 11, Hongping Liang 3 11, Jin Yu 3 11, Yuqing Cai 3 11, Zhaowu Zhang 3 11, Yannan Fan 3, Weixue Mu 3, Sunil Kumar Sahu 3, Shuchun Liu 4, Xiaoan Lang 4 12, Leilei Yang 4, Na Li 4, Sadaf Habib 4 13, Yongqiong Yang 14, Anders J Lindstrom 15, Pei Liang 16, Bernard Goffinet 17, Sumaira Zaman 17, Jill L Wegrzyn 17, Dexiang Li 12, Jian Liu 7, Jie Cui 18, Eva C Sonnenschein 19, Xiaobo Wang 20, Jue Ruan 20, Jia-Yu Xue 21, Zhu-Qing Shao 22, Chi Song 23, Guangyi Fan 3, Zhen Li 24, Liangsheng Zhang 25 26, Jianquan Liu 27, Zhong-Jian Liu 28, Yuannian Jiao 29, Xiao-Quan Wang 29, Hong Wu 30, Ertao Wang 31, Michael Lisby 32, Huanming Yang 3, Jian Wang 3, Xin Liu 3, Xun Xu 3, Nan Li 4, Pamela S Soltis 33, Yves Van de Peer 34 35 36, Douglas E Soltis 37 38, Xun Gong 39, Huan Liu 40, Shouzhou Zhang 41
Affiliations
- PMID:35437001
- PMCID: PMC9023351
- DOI: 10.1038/s41477-022-01129-7
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The Cycas genome and the early evolution of seed plants
Yang Liu et al. Nat Plants.2022 Apr.
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Abstract
Cycads represent one of the most ancient lineages of living seed plants. Identifying genomic features uniquely shared by cycads and other extant seed plants, but not non-seed-producing plants, may shed light on the origin of key innovations, as well as the early diversification of seed plants. Here, we report the 10.5-Gb reference genome of Cycas panzhihuaensis, complemented by the transcriptomes of 339 cycad species. Nuclear and plastid phylogenomic analyses strongly suggest that cycads and Ginkgo form a clade sister to all other living gymnosperms, in contrast to mitochondrial data, which place cycads alone in this position. We found evidence for an ancient whole-genome duplication in the common ancestor of extant gymnosperms. The Cycas genome contains four homologues of the fitD gene family that were likely acquired via horizontal gene transfer from fungi, and these genes confer herbivore resistance in cycads. The male-specific region of the Y chromosome of C. panzhihuaensis contains a MADS-box transcription factor expressed exclusively in male cones that is similar to a system reported in Ginkgo, suggesting that a sex determination mechanism controlled by MADS-box genes may have originated in the common ancestor of cycads and Ginkgo. The C. panzhihuaensis genome provides an important new resource of broad utility for biologists.
© 2022. The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
a, Illustration ofCycas panzhihuaensis.b, Chronogram of seed plants on the basis of the SSCG-NT12 dataset inferred using MCMCTree. All branches are maximally supported by bootstrap values (ML) and posterior probabilities (ASTRAL). I, II, III, VI, V and VI indicate internal branches for which the pie charts depicting gene tree incongruence are complemented by histograms (lower panel) showing quartet support for the main topology (q1), the first alternative topology (q2) and the second alternative topology (q3). O, Ordovician; S, Silurian; D, Devonian; C, Carboniferous; P, Permian; T, Triassic; J, Jurassic; K, Cretaceous; Pg, Palaeogene; N, Neogene; Q, Quaternary; Ma, million years ago.c, DiscoVista species tree analysis: rows correspond to the nine hypothetical groups tested (see Supplementary Note 5 for details) and columns correspond to the results derived from the use of different datasets and methods. SSCG, single-copy genes; LCG, low-copy genes; MT, mitochondrial genes; PT, plastid genes; AA, amino acid sequences; NT, nucleotide sequences; NT12, codon 1st + 2nd positions; ASTRAL, coalescent tree inference method using ASTRAL; CONCAT, maximum likelihood tree inferred with IQ-TREE based on concatenated datasets; STAG, species tree inference using software STAG with low-copy genes (one to four copies); Original, original organellar nucleotide sequences; RNA Editing, organellar genes with RNA editing site modified. Strong support, the clade is reconstructed with a support value >95%. Weak support, the clade is reconstructed with support value <95%. Weak rejection, the clade is not recovered, but the alternative topology is not conflict if poorly supported branches (<85%) are collapsed. Strong rejection, the clade is not recovered, and the alternative topology is conflict even when poorly supported branches (<85%) are collapsed.d, Diversification of Cycadales. The chronogram of 339 cycad species was inferred with MCMCTree based on 100 nuclear single-copy genes with concordant evolutionary histories. All illustrations are specifically created for this study (a high-resolution version is available athttps://db.cngb.org/codeplot/datasets/public_dataset?id=PwRftGHfPs5qG3gE ).
a, Inference of the number of gene families with duplicated genes surviving after WGD events mapped on a phylogenetic tree depicting the relationships among 16 vascular plants included in this study. The number of gene families with retained gene duplicates reconciled on a particular branch of the species tree are shown above the branch across the phylogeny (Methods). Numbers in square brackets denote the number of gene families with duplicated genes also supported by synteny evidence.b, Evolutionary analyses and phylogenetic profiles depicting the gains (light green), losses (light red), expansions (light yellow) and contractions (light blue) of orthogroups, according to the reconstruction of the ancestral gene content at key nodes and the dynamic changes of the lineage-specific gene characteristics.
a, Heatmap showing relative expression of genes in 11 co-expression modules by WGCNA across 4 developmental stages of the seed: S1, unpollinated ovule; S2, early stage of pollinated ovule; S3, late stage of pollinated ovule; and S4, fertilized ovule.b, Quantification of eight plant phytohormone amounts in the same four developmental stages of theCycas seed as above. The grey histogram represents the amount of hormone (n = 2 biologically independent experiments) and the error bar represents the standard error.c, Phylogeny of SSPs in some representative species in land plants. The SSPs analysed include germin-like protein (GLP), legumin-like SSP (l-SSP), vicilin-like SSP (v-SSP) and v-AMP. A maximum likelihood tree with 500 bootstrap replicates was constructed using RAxML. Bootstrap values (≥50%) for each major clade (highlighted in colour) and the relationships among them are provided. TheCycas sequences are highlighted in red.d, Expression levels of SSP in different tissues ofC. panzhihuaensis.
a, Manhattan plot of GWAS analysis of sex differentiation in 31 male and 31 femaleCycas samples. The red horizontal dashed line represents the Bonferroni-corrected threshold for genome-wide significance (α = 0.05).P values were calculated from a mixed linear model association of SNPs. Association analyses were performed once with a population of 31 male and 31 female individuals.b, Ratio ofπ,FST and difference of pooled heterozygosity (ΔHp) within a 100-kb sliding window between the female and male sequences. Colour represents values from low (blue) to high (red).c, Genome alignment of the MSY scaffolds with the corresponding female-specific region on chromosome 8. Scaffolds are separated by grey dashed lines. Red lines represent alignments >5 kb on the forward strand, and blue lines represent those on the reverse strand. Pink boxes ina–c represent the most differentiated regions between the sex chromosomes.d, Photographs of microsporophyll and megasporophyll ofC. panzhihuaensis. Bar, 1 cm.e, Sex-specific expression ofMADS-Y (CYCAS_034085) andCYCAS_010388 in male and female reproductive organs. Microsporophyll tissues were collected before meiosis (BFm), during prophase (Prophase), after meiosis (AFm) and before pollination (BFp); female tissues were collected at 0, 7, 11 and 21 days post-pollination.f, Phylogeny ofMADS-Y homologues across land plants. Genes from MSY and autosomes are marked on the right, and those fromSelaginella andPhyscomitrium are used as outgroups. Numbers above branches represent bootstrap scores from IQ-TREE.g, Molecular genotyping of male and female cycad samples fromCycas debaoensis,Macrozamia lucida andZamia furfuracea using primers specific to homologues ofMADS-Y andCYCAS_010388. Source data
a, Phylogenetic analysis of the TcdA/TcdB pore-forming domain containing proteins shows that the genes encoding four cytotoxin proteins ofCycas were likely acquired from fungi through an ancient horizontal gene transfer event. The maximum likelihood tree was generated by RAxML with the PROTCATGTR model and 1,000 bootstrap replicates. The numbers above the branches are bootstrap support values.b, The expression level of four cytotoxin proteins in different tissues ofC. panzhihuaensis. The digital expression values were normalized using the TPM method.c,d, Mortalities ofPlutella xylostella (c) andHelicoverpa armigera (d) after treatment with phosphate buffered saline (PBS) and cytotoxin. The asterisk indicates a significant difference (two-sided Student’st-test,P < 0.05,n = 3 biologically independent experiments), whereas the error bar represents the standard error.e,f, Morphologies ofPlutella xylostella (e) andHelicoverpa armigera (f) after receiving PBS and cytotoxin treatments.
Outer ring: The 11 chromosomes are labeled from Chr1 to Chr11. Inner rings 1-4 (from outside to inside): Repeat elements number shown in light purple. GC content colored indicated in light blue (y-axis min-max: 0.27–0.48). Expressed base percentage colored in light blue (y-axis min-max: 0–0.20). Gene numbers colored in light orange (y-axis min-max: 0-30). The sliding window of the inner rings 1-4 is 1 Mb. The inner ring 5 indicates the miRNA location over the genome. The blue lines inside represent the syntenic regions inCycas.
Extended Data Fig. 2. Comparative analysis of C. panzhihuaensis. (a) Comparison of the longest 10% of introns and gene in the representative land plants. The minimum, first quartile (Q1), median, third quartile (Q3), and maximum value was indicated in the box-plot by order after excluding the outliers. (b) Comparison of components of intron across the selected plants.
25 fossil calibrations and 2 secondary calibrations were used. Individual gene trees (1,569 NT tree) were mapped on the nuclear coalescent tree with Phyparts. The pie charts at each node show the proportion of genes in concordance (blue), conflict (green = a single dominant alternative; red = all other conflicting trees), and without enough information (gray). Quartet support for six internal branches I, II, III, IV, V, VI were indicated on the left panel as barcharts. Image courtesy of Zanqian Li and Xiaolian Zeng.
Example showing both the phylogenomic and syntenic evidence supporting an ancestral polyploidy event in extant gymnosperms. Four pairs of paralogous genes in OG0000093, OG0000255, OG00000276 and OG0000316 were duplicated before the divergence of gymnosperms and after the split of angiosperms and gymnosperms based on phylogenetic trees. These pairs of duplicated genes are located on the same syntenic block identified in theC. panzhihuaensis genome. The abbreviated name given before the protein ID represents species name: CYCAS:Cycas panzhihuaensis, Gb:Ginkgo biloba, ELO:Encephalartos longifolius, SEGI:Sequoiadendron giganteum, GMON:Gnetum montanum, PICABI:Picea abies, PITA:Pinus taeda.
(a) Phylogenetic tree of the NF-YB. The tree was constructed using the maximum likelihood method with 500 bootstrap replicates. The bootstrap values are shown on the branches. (b) Phylogenetic tree of the B3 domain containing the gene family ofC. panzhihuaensis. Bootstrap values are shown on the braches. (c) Transcript expression level is indicated by TPM during seed development. The phylogenetic trees were built using RAxML (estimating branch support values by bootstrap iterations with 500 replicates) with PROTGAMMAGTRX amino acid substitution model. The abbreviated name given before the protein ID represents species name: CYCAS:Cycas panzhihuaensis, Gb:Ginkgo biloba, SEGI:Sequoiadendron giganteum, GMON:Gnetum montanum, PICABI:Picea abies, PITA:Pinus taeda, ATH,Arabidopsis thaliana, DEBAO:Cycas debaoensis, AMTR:Amborella trichopoda, OS:Oryza sativa, AFILI:Azolla filiculoides, SACU:Salvinia cucullata, SELMO:Selaginella moellendorffii, PPATEH:Physcomitrella patens, MARPO:Marchantia polymorpha.
(a) Phylogenetic trees of CESA and CSL gene families. (b) Phylogenetic tree of CSLB and CSLH genes. (c) The phylogenetic tree of CSLE and CSLG genes. The CSLE/G from gymnosperm are the ancestral form of the angiosperm CSLE and CSLG. The phylogenetic trees were generated using RAxML with PROTCATGTR model and 500 bootstrap replicates. Bootstrap values ≥ 50% are shown. The abbreviated name given before the protein ID represents species name: CYCAS:Cycas panzhihuaensis, Gb:Ginkgo biloba, SEGI:Sequoiadendron giganteum, GMON:Gnetum montanum, PICABI:Picea abies, PITA:Pinus taeda, ATH,Arabidopsis thaliana, DEBAO:Cycas debaoensis, AMTR:Amborella trichopoda, OS:Oryza sativa.
(a) Sketch of the Cycas sperm. (b) Schematic diagram of flagellum loss events in green linage. (c) Distribution of outer dense fiber protein and other key flagellar proteins across representative embrophyta.
(a) Phylogenetic tree of the TPS gene family. The tree was constructed using RAxML (the maximum-likelihood method) with PROTCATGTR amino acid substitution model and 500 bootstrap replicates. The bootstrap values ≥ 50% are shown in the central branches. The red colors in the tree represent the cycas genes. (b) Heatmap of TPS gene family in different tissues ofC. panzhihuaensis. The * denotes theC.panzhihuaensis specific TPS genes.
(a) Heatmap of 1,971 genes differentially expressed in males and females’ organs. Arrows indicate CYCAS_034085 on the MSY and CYCAS_010388 on chromosome 2. (b) Expression of CYCAS_034085 on MSY and CYCAS_010388 on chromosome 2 in male microsporophyll and in the ovule.
Comment in
- Sexing cycads - a potential saviour.Clugston JAR, Kenicer GJ.Clugston JAR, et al.Nat Plants. 2022 Apr;8(4):326-327. doi: 10.1038/s41477-022-01133-x.Nat Plants. 2022.PMID:35437000No abstract available.
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