Stage 1: Karyotype Stage 2: Gene content & order Step 3

Transcription

1 Supplementary Figure Method used for ancestral genome reconstruction. MRCA (Most Recent Common Ancestor), AMK (Ancestral Monocot Karyotype), AEK (Ancestral Eudicot Karyotype), AGK (Ancestral Grass Karyotype) were reconstructed according to a two-stage procedure. While the ancestral karyotype is reconstructed in the first stage, the ancestral gene content of such karyotypes, as the gene order, are inferred in the second stage. Schematic representation below of the two-steps method involving: () karyotyping (based on core-ppgs), yielding the ancestral protochromosomes, and (2) gene order enrichment (based on ppgs) defining the complete set of opgs on the protochromosomes, using CIP/CALP Blast parameters as well as DRIMM-Synteny, MGRA tools as detailed in the Methods section of the manuscript. Step Genome alignment (ppg) Blast - CIP/CALP PROTOGENES Step 2 ppg conserved in all species (core-ppg). Stage : Karyotype Stage 2: Gene content & order Step 3 Step 4 Step 5 Syntenic Blocks with ppg (DRIMM Synteny) Enrichment of ancestral karyotype with ppg Step 9 Step 6 Syntenic Blocks (SB) (DRIMM Synteny) SB filtering (<5 core-ppg) SB merging (MGRA) Ancestral chromosome number Step Step 8 Ancestral gene order (opg) CHROMOSOMES GENE ORDER ANCESTRAL GENOME Nature Genetics: doi:0.038/ng.383

2 CAR 5 (A5 grasses) CAR /5 paralogy CAR (A grasses) Ancestor Ancestor Ancestor b2 Supplementary Figure 2 Schematic representation of ancestral genome reconstruction. The ancestral genome reconstruction procedure is illustrated for AGK (Ancestral Grass Karyotype chromosome, right) deriving from the orthologous chromosomes r (rice), s3 (sorghum), b2 (Brachypodium with telomere, tel.) defining A (cf dotplots and chromosome schema entitled CAR, left top) and orthologous chromosomes r5, s9, b2 (centromere, cent.) defining A5 (cf dotplots and chromosome schema entitled CAR5, left bottom), based on ppg and core-pg defining opg (color code). The post-duplication A5 and A protochromosomes are ultimately fused into a pre-duplication AGK protochromosome based on the paralogy observed between r-r5, s3-s9, b2 tel-cent (cf dotplots entitled CAR /5 paralogy, middle left), based on ppg and core-pg defining opg (cf color code) as defined in the Methods section of the manuscript. Modern synteny/paralogy Ancestors post-wgd Ancestor pre-wgd r s3 b2 (tel.) r s3 b2 (tel.) opg r r5 r s3 b2 (tel.) s3 s9 r5 s9 b2 (cent.) T C T T C T b2 ppg core-ppg A ppg core-ppg A5 Ancestor pre-wgd AGK core-ppg ppg opg opg r r5 s3 s9 b2 (tel. / cent.) r5 s9 b2 (cent.) r5 s9 b2 (cent.) 2 Nature Genetics: doi:0.038/ng.383

3 Supplementary Figure 3 AGK (pre-ρ / post-ρ) reconstruction. AGK was reconstructed in comparing sorghum, rice and Brachypodium, following the strategy detailed in the Methods section of the manuscript and delivering a post-ρ AGK with 2 chromosomes and 424 genes and pre-ρ AGK with chromosomes and 00 genes. a. Detailed procedure for AGK reconstruction from modern genomes (top) and the identification of ppgs, core-ppgs and opgs following karyotyping and enrichment procedures for pre-ρ and post-ρ ancestors. These newly defined AGKs (pre-ρ / post-ρ) clearly refine previously reported grass ancestors based on solely 6464 ppgs by Murat et al. (Ref 25, cf manuscript), corresponding to 84 % of the 9430 ppgs defined here for the new 2-protochromosomes AGK and 6246 opgs by Murat et al. (Ref 25, cf manuscript), corresponding to 89 % of the 00 opgs defined here for the new -protochromosomes AGK. b. Dotplot representation of the comparison between AGK ancestors (n=, n=2; y-axis) and modern species (x-axis) with a five-colors code illuminating AMK chromosomes. The dotplot representation of post-ρ AGK and pre-ρ AGK against the sorghum-rice-brachypodium genomes demonstrated that the reconstructed AGKs are robust and accurate with all chromosomes from either the modern species or from the ρ-block pairs entirely covered by the inferred karyotypes (i.e. 00% modern genomes/chromosomes integrated into the post-ρ and preρ AGK protochromosomes). c. The inferred n= AGKs (pre-ρ, AGK-) defines precise and exhaustive syntenic chromosome relationships between the modern grasses as detailed in the table. Following the proposed method applied on rice, Brachypodium and sorghum, we inferred a post-ρ AGK consisting of 2 protochromosomes containing 424 ordered protogenes (opgs). Alignment of the two subgenomes generated by ρ made it possible to reconstruct a pre-ρ AGK, consisting of 00 ordered protogenes (opgs) on protochromosomes. The transition from the pre-ρ to post-ρ AGK involved seven known paralogous ancestral chromosome pairs (using the rice chromosome nomenclature): A-A5, A2-A4, A2-A6, A3-A, A3-A0, A8-A9, A-A2. a Oryza sativa v.0 2 chromosomes genes Ancestor Post-ρ Karyotyping Step I.9430 Potential protogenes (ppg) Step II. 6 ppg conserved in the three species (Core-pPG). Step III. 33 ppg In Syntenic Blocks (SB). Step IV. 93 ppg after filtering Step V. 93 protogenes (opg) in 2 Protochromosomes (SB). Brachypodium distachyon v2. 5 chromosomes genes Sorghum bicolor v.0 0 chromosomes 242 genes Ancestor Pre-ρ Karyotyping Step II. 835 ppg (duplicated) Step III. 94 ppg / 88 SB. Step IV. 55 ppg / 68 SB. Step V. 55 opg / 68 SB Protochromosomes. Enrichment Step VI ppg non core-ppg. Step VII. 522 ppg In Syntenic Blocks. Step VIII. 424 opg in 2 Protochromosomes. Enrichment Step VI. 60 opg / 68 groups Protochromosomes. Step VII. 00 opg in Protochromosomes. 3 Nature Genetics: doi:0.038/ng.383

4 Ancestor Post-ρ Ancestor Pre-ρ b Ancestor Post-ρ 2 Rice 2 Brachy 5 Sorghum 0 2 Rice 2 Brachy 5 Sorghum 0 c AGK Pre-ρ AGK Post-ρ Rice Brachypodium Sorghum AGK AGK AGK AGK AGK AGK AGK Nature Genetics: doi:0.038/ng.383

5 Supplementary Figure 4 Oil palm (pre-p / pre-τ) ancestor reconstruction. The oil palm paleohistory was reconstructed following the strategy detailed in the Methods section of the manuscript and delivering a pre-p oil palm ancestor of 0 chromosomes with 396 genes and a pre-τ oil palm (or AMK) of 5 chromosomes with 60 genes. a. Detailed procedure for AMK (pre-p / pre-τ) reconstruction from the oil palm genome, leading to the identification of ppgs, core-ppgs and opgs following the karyotyping and enrichment procedures for pre-τ and post-τ ancestors. The analysis unveiled the p duplication (p-p6, p3-p, p4-p, p2-p6, p5-p4, p2-p0, p5-p0, p3-p5, p8-p0, p2-p8, p2-p9, p-p8, p3-p0, p3-p paralogous blocks) as well as the τ duplication (p-p6/p3-p, p2-p8-p0/p2-p-p8-p9, p4-p/p2-p6, p5-p4/p2-p5-p0, p3-p-p0/p3-p5 paralogous blocks) b. Dotplot representation of the comparison between oil palm (6 chromosomes), oil palm pre-p (n=0), AEK (pre-γ n=, post-γ n=2) and AGK (pre-ρ n=, post- ρ n=2) with a five-colors code illuminating AMK chromosomes. The observed synteny and paralogy are entirely integrated in 5 CARs (diagonal color code) delivering a pre-p oil palm ancestor of 0 chromosomes (396 ordered protogenes, opgs) and a pre-τ AMK of 5 chromosomes (60 ordered protogenes, opgs). a Elaeis guinensis (20965 genes). Ancestor Pre-p Karyotyping Step I.2998 Potential proto-genes (ppg) Ancestor Pre-τ Karyotyping Step I.064 Potential proto-genes (ppg) Step II ppg Step II. 064 ppg Step III ppg In Syntenic Blocks (SB). Step III. 30 ppg In Syntenic Blocks. Step IV. 286 ppg after filtering. Step IV. 30 ppg after filtering. Step V opg in 0 Protochromosomes. Step V. 30 opg in 5 Protochromosomes. Enrichment Step VI. 24 opg (duplicated) Step VII. 396 opg in 0 ProtoChromosomes Enrichment Step VI. 30 opg (duplicated) Step VII. 60 opg in 5 Protochromosomes 5 Nature Genetics: doi:0.038/ng.383

6 AEK (post-γ) AGK (post-ρ) Oil palm pre-p Oil palm Oil palm pre-p b opg opg opg 2 0 AEK (pre-γ) Oil palm pre-p AGK (pre-ρ) 6284 opg 00 opg 2 AEK (pre-γ) AGK (pre-ρ). 6 Nature Genetics: doi:0.038/ng.383

7 Oil Palm Oil Palm Supplementary Figure 5 Oil palm (pre-p) and AMK (pre-τ) ancestors. The oil palm genome allowed the characterization of the shared ancestral τ duplication through a mixed approach including Ks and synteny inference of paralogs for the lineage-specific p duplication (see also previous Supplementary Figure 4) as well as the monocot τ duplication. a. Schematic representation of Ks distribution separating two WGDs (recent p WGD in red and ancient τ WGD in blue, top illustration) in the oil palm genome; and the corresponding gene pairs defining paralogous blocks (diagonals) on the oil palm-oil palm dotplot comparison (bottom illustration) with p duplication in red (p-p6, p3-p, p4-p, p2-p6, p5-p4, p2-p0, p5-p0, p3-p5, p8- p0, p2-p8, p2-p9, p-p8, p3-p0, p3-p paralogous blocks) and τ duplication in blue (p-p6/p3-p, p2-p8-p0/p2-p-p8-p9, p4- p/p2-p6, p5-p4/p2-p5-p0, p3-p-p0/p3-p5 paralogous blocks). b. Schematic representation of the deduced most parsimonious (reduced number of fusion and fission events) evolutionary model from a pre-τ AMK structured in 5 chromosomes (with 60 genes) deriving a n=0 post-τ AMK (396 genes) followed by a chromosomal fusion (between A-A9) to derive a n=9 intermediate. The second WGD (p) derives a n=8 (2x9) intermediate followed by 5 chromosomal fissions (breaks) and fusions to reach the modern oil palm genome (n=6), top illustration. Dotplot diagonals correspond to the p and τ WGDs from the oil palm-oil palm comparison with a colour code highlighting the five AMK (oil palm pre- τ) protochromosomes (right), and entirely covering the modern oil pam genome (with pre-τ AMK ancestor as color code at the center). a b #Paralogous pairs ks Oil Palm Oil Palm Nature Genetics: doi:0.038/ng.383

8 Supplementary Figure 6 Dotplot-based deconvolution of the AMK (pre-τ), AGK (pre-ρ) and oil palm genomes into 5 CARs. The complete dotplot-based comparative genomics deconvolution into reconstructed CARs of the observed synteny and paralogy between the oil palm (pre-p with 0 chromosomes and 396 genes), AMK (pre-τ with 5 chromosomes and 60 genes) and AGK (preρ with chromosomes and 00 genes) genomes validates the five proposed protochromosomes as the origin of monocots, i.e. AMK. AGK-AMK (right), AGK-oil palm (left) and oil palm-amk (centre) dotplot comparisons illustrate the complete deconvolution of synteny/paralogy relationships (diagonals) into 5 CARs (color code), as illuminated as a case example for AK (external red arrows) deriving orthologous/paralogous blocks on AGK (chromosomes A-4-6) and oil palm (chromosomes -3-6-). Oil pam (6 chromosomes, z-axis) and AGK ( ancestral chromosomes from rice, Brachypodium and sorghum comparison, y-axis) genomes are entirely covered with 5 independent ancestral chromosome (AMK CARs, x-axis) that do not share any orthologous or paralogous relationship. AMK AK AK2 AK3 AK4 AK5 AGK AMK Oil Palm 8 Nature Genetics: doi:0.038/ng.383

9 AMK Supplementary Figure Dotplot-based deconvolution of the AMK (pre-τ), pineapple genomes into 5 CARs. The dotplot-based comparative genomics of the pineapple (25 chromosomes and genes) genome and the pre-τ AMK (5 chromosomes and 60 genes) genome revealed the precise nature of the σ event as a hexaploidization event, with a clear -to-6 (two regions inherited from τ and three from σ) chromosomal relationships between AMK and pineapple genomes. Dotplot (top) comparison of AMK (post-τ, y-axis) and pineapple (x-axis) illustrating a -to-6 relationship (horizontal) observed between the 5 AMK protochromosomes (or CARs, diagonal color code) and pineapple chromosomes. As a case example for AK (bottom), the number of identified conserved genes (y-axis) is shown as a distribution of 6 pineapple orthologous chromosomes (x-axis). The -to-6 relationship observed between AMK and pineapple involves τ (reported as a tetraploidization) and σ, a paleohexaploidization event. The observed difference in ancestral gene retention between the triplicated regions inherited from σ in pineapple (chromosomes -2-6 and chromosomes -5-0 as case example) is in favour of a post-polyploidy subgenome dominance with chromosomes 0- as the least fractionated (LF), chromosomes 5-6 as the medium fractionated (MF) and chromosomes -2 as the most fractionated (MF2) compartments. The comparison of the pineapple genome and AMK (pre-τ, 5 protochromosomes with 60 ordered protogenes) allowed us to unveil the precise nature of the σ event as a hexaploidization with a clear -to-6 (two regions inherited from τ and three from σ) chromosome relationships between the investigated genomes, then deriving the AMK pre-σ with 9 chromosomes (from the fusion of two ancestral A-A9 post-τ chromosomes) and the AMK post-σ with 2 chromosomes. The σ hexaploidization event shows the characteristic of the subgenome dominance phenomenon following hexaploidization involving MF2 (most fractionated), MF (medium fractionated) and LF (least fractionated) compartments as reported in the case of the ancestral hexaploidization in rosids by Murat et al. (Ref. 24, cf manuscript) and Brassicacae by Murat et al. (Ref. 32, cf manuscript). 5 4 τ 3 LF MF 2 σ Pine Apple LF MF MF2 LF MF MF2 PA PA5 PA0 PA PA2 PA6 PA PA6 PA2 PA0 PA5 PA 9 Nature Genetics: doi:0.038/ng.383

10 Supplementary Figure 8 Dotplot-based deconvolution of the AMK (pre-τ and post-σ) and oil palm (pre-p) genomes into 5 CARs. The complete dotplot-based comparative genomics deconvolution of the observed synteny between the oil palm (pre-p, 0 chromosomes and 396 genes), AMK pre-τ (5 chromosomes and 60 genes), and AMK post-σ (2 chromosomes and 208 genes) genomes into five independent CARs validated the origin of monocots from the five proposed protochromosomes, the tetraploid and hexaploid nature of respectively the τ (referenced as x2) and σ (referenced as x3) polyploidization events. a. AMK (pre-τ, 5 chromosomes)/amk (post-σ, 2 chromosomes) (right), AMK (post-σ, 2 chromosomes)/oil palm (pre-p, 0 chromosomes) (left) and AMK (pre-τ, 5 chromosomes)/ oil palm (pre-p, 0 chromosomes) (centre) dotplot comparisons illustrate the complete deconvolution of synteny/paralogy relationships (diagonals) into 5 CARs (color code, bottom tight). b. AMK pre-τ, AMK post-σ and oil palm pre-p, are entirely covered with 5 independent ancestral chromosomes (AMK CARs) defining a -to-6 (τ(x2) and σ(x3)) chromosomal relationship between AMK pre-τ / AMK post-σ, a -to-6 (τ(x2) and σ(x3)) chromosomal relationship between AMK post-σ / oil palm pre-p and a -to- 2 (τ(x2)) chromosomal relationship between AMK pre-τ / oil palm pre-p as AMK post-σ / AMK post-σ and detailed in the table with the ancestral chromosomes referred to as AK X and modern chromosome as X'. a AMK post-σ x6 = τ(x2) + σ(x3) Pre-P x6 = τ(x2) + σ(x3) AMK AMK AK AK2 AK3 AK4 AK5 b AMK Pre-τ AMK Post-τ/Pre-p (x2) Oil palm (x4) AMK Post-σ (x6) AGK Pre-ρ (oil palm) (x6) Pineapple (x6) AK AK AK AK AK Nature Genetics: doi:0.038/ng.383

11 Supplementary Figure 9 AEK (pre-γ, post- γ) reconstruction. AEK was reconstructed in comparing cocoa, grape and peach, following the strategy detailed in the Methods section of the manuscript, delivering a post-γ AEK with 2 chromosomes with 9022 genes and pre-γ AEK with chromosomes with 6284 genes. a. Detailed procedure for AEK reconstruction from modern genomes (top) and the identification of ppgs, core-ppgs and opgs following the karyotyping and enrichment procedures for pre-γ and post-γ AEK ancestors. The proposed pre-γ and post-γ AEK ancestors considerably refine previous reported AEK based on solely 02 ppgs by Murat et al. (Ref. 24, cf manuscript), corresponding to 48 % of the 430 newly defined ppgs for the 2-protochromosome AEK, and 626 opgs by Murat et al. (Ref. 24, cf manuscript), corresponding to 0 % of the 6284 newly defined opgs for the -protochromosomes AEK b. Dotplot representation of the comparison between AEK ancestors (n=, n=2; y-axis) and modern species (x-axis) with a five-colors code illuminating the AMK chromosomes. The accuracy of the reconstructed post-γ AEK and pre-γ AEK is validated by the dotplot comparisons, which unveil 00% coverage of all chromosomes either from modern species or from the γ-block triplets into the reconstructed n= and n=2 AEK protochromosomes. c. The inferred n= AEKs (pre- γ, AEK-) defines exhaustive and accurate syntenic chromosome relationships between the modern eudicots as detailed in the table. The transition from the pre-γ AEK to the post-γ AEK involved seven known paralogous ancestral chromosome triplets (using the grape chromosome nomenclature): A-A4-A, A2-A5-A2-A6, A3-A4-A-A8, A4-A9-A, A5-A- A4, A6-A8-A3, A0-A2-A9. a Theobroma cacao CocoaGen DB 0 chromosomes genes Ancestor Post- γ Karyotyping Step I. 430 Potential protogenes (ppg) Prunus persica v.0 8 chromosomes 2256 genes Vitis vinifera Genoscope 2X 9 chromosomes 2364 genes Ancestor Pre- γ Karyotyping Step II. 500 ppg conserved in the three species (core-ppg). Step III. 269 ppg in Syntenic Blocks (SB). Step IV. 580 ppg after filtering. Step V. 520 protogenes (opg) in 2 Protochromosomes. Enrichment Step VI ppg non core-ppg. Step VII. 30 ppg In Syntenic Blocks. Step VIII opg in 2 Protochromosomes. Step II. 499 ppg (duplicated) Step III. 36 ppg / 42 SB. Step IV. 36 ppg / 42 SB. Step V. 352 opg / 4 SB Protochromosomes. Enrichment Step VI. 335 opg / 4 groups Protochromosomes. Step VII opg in Protochromosomes. Nature Genetics: doi:0.038/ng.383

12 Ancestor Post-γ Ancestor Pre-γ b 2 2 Ancestor Post-γ Grape 9 Peach 8 Cacao 0 c Grape 9 Peach 8 Cacao 0 AEK Pre-γ AEK Post-γ Grape Peach Cocoa AEK AEK AEK AEK AEK AEK AEK Nature Genetics: doi:0.038/ng.383

13 Supplementary Figure 0 Dotplot-based deconvolution of the AMK, AEK and Amborella genomes into 5 CARs. In order to identify the most parsimonious structure of the MRCA for eudicots/monocots from the comparison of AEK and AMK, an outgroup species is required. Amborella trichopoda is a basal angiosperm with no specific WGD event making it the most interesting candidate in reconstructing eudicots/monocots MRCA. Amborella genome contains 3 chromosomes for an estimated size of 80 Mb. The available genome is assembled into 545 scaffolds (06 Mb) and annotated genes. 38 scaffolds are assigned by FISH to 2 over the 3 chromosomes, representing 34% of the genome (i.e annotated genes, 204 Mb). The dotplot-based comparative genomics of the reconstructed AEK and AMK unveiled a synteny signal between these two principal families of flowering plants, in the form of 5 CARs recovered from 5 orthologs that appeared to display a higher degree of structural conservation (with one-to-one chromosome relationships on average, i.e.3) between the AMK and the Amborella genome, than between the AEK and Amborella (with one-to-two chromosome relationships on average, i.e.9). The complete deconvolution of the observed syntenies between the Amborella, AMK (pre-τ) and AEK (pre- γ) genomes into independent CARs ultimately validated the existence of a angiosperm progenitor with a minimum of fifteen proposed protochromosomes. a. AMK (pre-τ, 5 chromosomes)/aek (pre-γ, chromosomes) (right), AMK (pre-τ, 5 chromosomes)/amborella (3 chromosomes) (left) and AEK (pre-γ, chromosomes)/amborella (3 chromosomes) (centre) dotplot comparisons illustrate the complete deconvolution of synteny/paralogy relationships (diagonals, color code from the 5 AMK CARs, top) into 5 CARs (rectangles). 3-D dotplot comparison is provided at the centre with the associated 2-D dotplots provided for details. b. chromosome-to-chromosome synteny relationships (table lines corresponding to colored arrows on the panel A) illustrate a.3 ratio between Amborella and AMK chromosomes and a.9 ratio between Amborella and AEK chromosomes. c. Illustration of the close structural relationship at the chromosome level between Amborella and AMK compared to Amborella and AEK with Amborella chromosome 4, structurally conserved with AMK chromosome 4, whereas two orthologous regions are identified in AEK chromosomes 5 (CAR number 4 on the panel A) and 6 (CAR number 8 on the panel A). d. Illustration of the synteny relationships (5 CARs from the panel A and detailed in the table from panel B) as colored blocks (reflecting the 5 AMK CARs) between Amborella AEK and AMK, suggesting AMK as closest representative of the angiosperm MRCA of 5 CARs. The comparison of the reconstructed AEK (pre-γ with chromosomes and 6284 protogenes) and AMK (pre-τ with 5 chromosomes and 60 protogenes) deliver a syntenic signal between these two main families of flowering plants consisting in 5 CARs recovered from 5 orthologs. a AMK 5 AK AK2 AK3 AK4 AK AEK 3 0 Amborella trichopoda Nature Genetics: doi:0.038/ng.383

14 B b CARs AEK- AMK AEK chr (n=) AMK chr (n=5) Amborella trichopoda chr (n=3) Nb AEK regions Nb AMK regions NA NA NA NA c C AMK chr4 AEK chr5 Amborella chr4 chr6 CAR 4 CAR 8 D Average.9.3 Angiosperms d Angiosperms Basal angiosperms Eudicots 5 MRCA E-M Monocots Basal angiosperms Eudicots 5 MRCA Monocots x Amborella trichopoda AEK AMK x Amborella trichopoda AEK AMK 4 Nature Genetics: doi:0.038/ng.383

15 Supplementary Figure Gene Ontology enrichment comparing MRCA and extant genomes. We compared 3 plant species (3 monocots, 20 eudicots, one basal angiosperm (Amborella trichopoda), and three outgroup species (Picea abies as a representative of gymnosperms, Physcomitrella patens as a representative of mosses and Chlamydomonas reinhardtii as a representative of single-celled green algae)) to identify orthologous gene clusters representative of the angiosperm MRCA gene pool with 0263 gene groups specific to flowering plants (absent from the outgroup species). We performed a GO (Gene Ontology) enrichment analysis at the Biological process, Cellular component and Molecular function levels. Enriched GO terms (at the Biological process, Cellular component and Molecular function levels) in comparing 0263 angiosperm-specific gene clusters to the MRCA gene pool (a), in comparing the MRCA gene pool to that of modern species, considering Arabidopisis thaliana as a reference with the most robust GO terms and classification (b), and in comparing the modern genomes to the MRCA gene pool (c). GO analysis from the panel a delivers enriched terms in angiosperms compared to outgroups. GO analysis from the panel b delivers enriched terms in MRCA compared to extant species, i.e. basic functions or processes already present in the ancestor. Finally, GO analysis from panel c delivers enriched terms in extant species compared to MRCA, i.e. functions amplified during evolution. a 0263 angiosperm-specific genes compared to MRCA genes Biological process Cellular component Molecular function b MRCA genes compared to modern genomes (Arabidopsis) Biological process Cellular component Molecular function 5 Nature Genetics: doi:0.038/ng.383

16 c Modern genomes (Arabidopsis) compared to MRCA genes Biological process Cellular component Molecular function 6 Nature Genetics: doi:0.038/ng.383

17 Supplementary Figure 2 Genomic evolutionary plasticity of the AEK and AGK genomes. Comparisons of the genomes of the MRCA and extant modern species provided insight into the structural plasticity of angiosperm genomes and diversification in the response to polyploidy where: () post-duplication chromosomes have similar gene distributions, from the gene-rich telomeric regions to the gene-poor centromeric regions; (2) lineage-specific genes (i.e. not mapped onto the ancestral karyotypes and referred to as non-ak genes ) are preferentially located in pericentromeric and subtelomeric regions; (3) the ancestral gene pool is partitioned between paralogous blocks, forming the MF (most fractionated (MF) also described as sensitive (S)) and LF (least fractionated (LF) also described as dominant (D)) chromosomal compartments; (4) gene pairs evolved differentially between rapidly evolving and slowly evolving species (such as rice for the monocots and grape for the dicots); (5) ancestral genes tend to have larger numbers of exons than non-ancestral genes; (6) genes conserved in grasses have a higher GC content than those conserved in eudicots. Detailed analysis of the genomic features (gene number, AK genes, Ks, exon numbers, GC content) are reported in columns, for the monocot and eudicot species investigated (in rows). Statistically significant differences (t-test p<0.05) are indicated by red stars for each genomic feature tested (columns). Avg = average, SD = standard deviation, % = percentage, spe=specific, LF=least fractionated, MF=most fractionated. Clade Monocots Eudicots Species Partitionning Genes AK genes Ks (median) Exons number (avg) % GC content (avg) Rice LF ,4 5,8 5 9, MF ,8 5 9,8 Spe , Brachy LF , MF Spe 964 4, 56 9 Sorghum LF ,3 5,8 5 9,6 MF , 5 9, Spe ,5 5 0 Grape LF ,32,4 46 3,3 MF 2086,2 46 3,4 MF2 52,5 46 3, Spe 92 5, Cacao LF , MF 854 6, MF2 326, Spe ,3 4 5,5 Peach LF ,65 6, 46 3, MF 8 6,6 46 3, MF Spe ,4 45 4,8 % GC content (SD) P-value <0,05 Nature Genetics: doi:0.038/ng.383

18 Supplementary Figure 3 Dating the Angiosperm origin. 286 orthologous gene clusters, from the 5 angiosperm MRCA genes, containing single-gene copies from peach, grape, cocoa, Brachypodium, rice and sorghum were used to assess the age of angiosperm origin (considered here as the monocot/eudicot speciation date) using BEAUTi, BEAST and visualized in FigTree and DensiTree (see Methods section of the main manuscript) with a minimum age calibration from fossils of 65 mya for grasses and 25 mya for the eudicots. We inferred that angiosperms originated 24 (between ) mya. The figure illustrates the maximum clade credibility tree from divergence time estimates of angiosperms based on 286 ancestral genes conserved in peach, grape, cocoa, Brachypodium, rice and sorghum. The branch length illustrates the average substitutions per sites according to the time scale (in million years, my) associated with major geological epochs (bottom). The 95% highest posterior density (HPD) estimates for each well-supported speciation events are represented by horizontal blue bars and with age intervals mentioned in brackets. Red asterisks represent minimum age fossil calibrations with 65 my for grasses and 25 my for the eudicots. Dashed horizontal black lines represent age estimates of the angiosperm origin from the literature (references cited) and timetree ( Sorghum Smith et al. (200) [82-25] Seng et al. (204) [ ] Grasses [65-8] * 3 68 [60-5] Rice Angiosperms [90-238] 24 Brachy Magalloon and Sanderson (2005) [6-3] Timetree [68-94] Eudicots [8-09] 98 * 3 [64-8] Grape Peach Cocoa Million years CARBON. PERMIAN TRIASSIC JURASSIC CRETACEOUS TERTIARY QUAT Paleozoic Mesozoic Cenozoic 8 Nature Genetics: doi:0.038/ng.383

19 Supplementary Table 5 Synteny relationships between AEK, AGK, AMK based on MRCA. Chromosomal conservation (rows) between MRCA (CARs), AMK (AK-AK5), AEK (n= pre-γ and n=2 post-γ), AGK (n= pre-ρ and n=2 post-ρ) and the modern monocot (rice as representative, with chromosomes to 2) and eudicot (grape as representative, with chromosomes to 9) species (columns). MRCA Pre-γ (AEK n=) Post-γ (AEK n=2) Grape (reference) AMK (n=5) Pre-ρ (AGK n=) Post-ρ (AGK n=2) Rice (reference) CAR 4 Ch Ch-2-3 Ch2-5-6 Ch Ch-5 Ch-5 CAR 2 Ch2 Ch4-5-6 Ch4-9- AK Ch4 Ch2-6 Ch2-6 CAR Ch6 Ch6--8 Ch-4- Ch6 Ch8-9 Ch8-9 CAR 2 Ch Ch Ch0-2-9 Ch3 Ch3- Ch3- CAR 3 Ch Ch-2-3 Ch2-5-6 Ch2 Ch3-0 Ch3-0 CAR 0 Ch3 Ch-8-9 Ch5--4 AK2 CAR Ch4 Ch0--2 Ch6-8-3 Ch5 Ch2-4 Ch2-4 CAR 6 Ch5 Ch3-4-5 Ch Ch Ch-5 Ch-5 CAR 3 Ch3 Ch-8-9 Ch5--4 AK3 Ch3 Ch3- Ch3- CAR Ch4 Ch0--2 Ch6-8-3 Ch Ch-2 Ch3- Ch2 Ch3-0 Ch3-0 CAR 5 Ch2 Ch4-5-6 Ch4-9- CAR 4 Ch5 Ch3-4-5 Ch CAR 8 Ch6 Ch6--8 Ch-4- Ch3 Ch3- Ch3- AK4 Ch4 Ch2-6 Ch2-6 Ch5 Ch2-4 Ch2-4 Ch6 Ch8-9 Ch8-9 CAR 5 Ch2 Ch4-5-6 Ch4-9- CAR 9 Ch6 Ch6--8 Ch-4- AK5 Ch Ch-2 Ch-2 Ch4 Ch2-6 Ch2-6 Ch5 Ch2-4 Ch2-4 Ch6 Ch8-9 Ch8-9 Ch Ch-2 Ch-2 9 Nature Genetics: doi:0.038/ng.383