bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Comparative analyses of 3654 chloroplast genomes unraveled new insights into the evolutionary mechanism of green plants

Ting Yang1,2,4, Xuezhu Liao1,2, Lingxiao Yang3, Yang Liu1,2, Weixue Mu1,2, Sunil Kumar Sahu1,2,5, Xin Liu1,2,5, Mikael Lenz Strube4, Bojian Zhong3*, Huan Liu1,2,5*

1BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China 2China National GeneBank, Jinsha Road, Dapeng New District, Shenzhen 518120, China 3College of Life Sciences, Nanjing Normal University, Nanjing, China 4Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800, Kgs. Lyngby, Denmark 5State Key Laboratory of Agricultural Genomics, BGI-Shenzhen, Shenzhen 518083, China

*Corresponding authors Huan Liu Email: [email protected]

Bojian Zhong E-mail: [email protected]

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Abstract Background Chloroplast are believed to arise from a cyanobacterium through endosymbiosis and they played vital roles in photosynthesis, oxygen release and metabolites synthesis for the plant. With the advent of next-generation sequencing technologies, until December 2018, about 3,654 complete chloroplast genome sequences have been made available. It is possible to compare the chloroplast genome structure to elucidate the evolutionary history of the green plants. Results We compared the 3654 chloroplast genomes of the green plants and found extreme conservation of gene orders and gene blocks in the green plant such as ATP synthase cluster, Phytosystem, Cytochrome cluster, and Ribosomal cluster. For the chloroplast-based phylogenomics, we used three different data sets to recover the relationships within green plants which accounted for biased GC content and could mitigate the bias in molecular data sets by increasing taxon sampling. The main topology results include: I) Chlorokybales + Mesostigmatales as the earliest-branching lineage and a clade comprising Zygnematales+ Desmidiales formed a grade as the sister group to the land plants, II) Based on matrix AA data, Bryophytes was strongly supported as monophyletic but for matrix nt123 data, hornworts, mosses and liverworts were placed as successive sister lineages of Tracheophytes with strong support, III) Magnoliids were placed in the outside of Monocots using the matrix nt123 data and the matrix AA data, IV) Ceratophyllales + Chloranthales as sister to the Eudicots using matrix nt123 data, but when using matrix nt12 data and AA data, only Ceratophyllales sister to the Eudicots.

Conclusion We present the first of its kind large scale comparative analyses of the chloroplast coding gene constitution for 3654 green plants. Some important genes likely showed co-occurrence and formed gene cluster and gene blocks in Streptophyta. We found a

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clear expansion of IRs (Inverted Repeats) among seed plants. The comprehensive taxon sampling and different data sets recovered a strong relationship for green plants.

Keywords: Chloroplast genome; Phylogenetics; Evolution; Viridiplantae; Inverted Repeats; Gene expansion

Introduction Chloroplast are one of the most important organelle of land plants and green algae which are essential for plant growth and development, and are involved in photosynthesis, lipid metabolism, and other cellular processes. According to endosymbiotic theory, chloroplast originated from a cyanobacterial endosymbiont into a eukaryotic host, and the photosynthetic eukaryotes were endosymbionts of non-photosynthetic eukaryote hosts to form secondary chloroplast, then the cellular chimaera subsequently diversified into glaucophytes, red algae, and green plant/algae. However, it is unclear whether the chloroplast of red algae, green algae, and green plants were from a single origin or multiple origins [1]. As the transfer of the chloroplast genes to the nucleus was an ongoing process, the phylogenetic tree based on some chloroplast genes may be complex, if sequences involved in the analyses are from different origins. However, chloroplast genomic DNA (cpDNA) are conserved in gene content, the similar set of genes in cpDNA could be explained in terms of large-scale gene transfer in an ancestral lineage and could help us to understand chloroplast origin and evolution. For instance, the presence of gene clusters like psbB/T/N/H could be considered as an indication of monophyly [2, 3]. cpDNA of green plants (Viridiplantae) normally exhibit a conserved genome structure which contains two copies of an inverted repeat (IR) separating the small single-copy region (SSC) and the large single-copy region (LSC). The chloroplast genome size of green plants normally ranges from 107 kb (Cathaya argyrophylla, Pinaceae family) [4] to 218kb (Pelargonium, Geraniaceae family) [5]. However, some angiosperm lineages may have extreme variations in their genome size, for

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instance, Cutinus (Cytinaceae) chloroplast genome is around 20 kb, while Chlorophyta (i.e., Floydiella, Chaetopeltidaceae) chloroplast genomes have been reported to host an unexpected large size of 520 kb [6]. The size of cpDNA has been compared within many clades [7, 8], and many factors could explain these chloroplast genome size variation, like (a) variations of intergenic regions, intron lengths, etc. [9,

10]；(b) IR region variation [5, 11]; (c) gene loss [12].

For cpDNA of green plants, hotspots for structural variation include the IRs, gene loss, gene transfer, and gene arrangement. For the IR variation, the lengths of IRs are likely to be expanded, contracted or to be completely lost. The IR analyses of all green plants showed that short IRs are frequently found in Bryophyta followed by Chlorophyta, the lowest among Polypodiopsida followed by basal Magnoliophyta, Magnoliidae, Commelinids [11], and in Papilionoideae, Pinaceae, cupressophytes, IRs are nearly lost or missing [8, 13, 14]. Regarding the gene variation, the cpDNA of green plants are normally conserved, but gene losses are widely seen especially in parasitic plants such as Cuscuta and Epifagus, which have partially or completely lost the photosynthetic ability [15]. To understand the origin and relationships of green plants, the phylogenetic analyses have been widely performed based on nuclear, mitochondrial [16], and chloroplast loci [17, 18]. The phylogenetic relationship among Chlorophyta has been reviewed recently [19-22] and the branching orders of the prasinophyte lineages, the relationships among core chlorophyte clades (Chlorodendrophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae) required further deep analyses. Meanwhile, regarding the ferns [23, 24], and Bryophytes [25, 26], transcriptome sequencing data was used to resolve the debated topologies within the ferns and Bryophytes. For the gymnosperm group, Lu et al. (2014) used two nuclear genes and performed near complete sampling of extant gymnosperms genera, and found that the cycads are the basal-most lineage of gymnosperms rather than a sister to Ginkgoaceae, a sister relationship between Podocarpaceae and Araucariaceae [27]. For seed plants, Burleigh et al. used four nuclear loci, five chloroplast loci and four mitochondrial loci

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from 31 genera to resolve the seed plant tree of life [17]. For basal angiosperms, Moore et al. used 61 chloroplast genes from 45 taxa to reconstruct the phylogenetic order among basal angiosperms [28]. Likewise, the largest chloroplast phylogenetic study has been performed across green plants by using a nearly complete set of protein-coding sequences, based on 360 species of the green plants, and 1879 taxa representing all the major subclades [29, 30]. With the advent of next-generation sequencing technologies, enormous efforts have been made to sequence the whole chloroplast genomes of plants. Until December 2018, over 3,000 complete chloroplast genome sequences have been made available in the National Center for Biotechnology Information (NCBI) organelle genome database. This large amount of complete cpDNA sequences could be effectively utilized to understand the evolution of the chloroplast genomes and phylogenetic relationships among plants. With so many chloroplast genomes, we tried to answer three main questions from this study: i) After the split of Streptophyta and Chlorophyta, how the evolution shaped Streptophyta and what were the similarities the in the genome? ii) IRs degenerated widely in red algal and have uneven size distribution in Viridiplantae, what is the formation mechanism behind IRs? iii) does increasing taxon sampling would help to resolve phylogenetic questions of relationships in Viridiplantae? In the present study, we comprehensively analyzed the available chloroplast genomes of Viridiplantae comprising 3,654 taxa, 298 families, and 111 orders. We compared the genomic organizations in their cpDNAs between major clades, including gene gain/loss, gene copy number, GC content, gene cluster, and gene blocks. We also covered a wide range of green plants species to construct the chloroplast-based phylogenetic trees. Increasing taxon sampling together with the whole coding genes of chloroplast helped us to resolve phylogenetic questions of relationships in Viridiplantae.

Results General Characteristics of the Genomes

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The genome size and gene organization in chloroplast genomes In this study, the complete chloroplast genomes (cpDNA) of 3,654 taxa representing 298 families, and 111 orders were selected. The size of cpDNA ranged from 71,666 bp to 521,168 bp. Liverworts, mosses, and gymnosperm displayed the smallest average genome size, while Chlorophyta had the largest genome size variation. Even though the chloroplast genome size showed large variation, but there were 120–130 conserved genes as well. We recovered 79 protein-coding genes from all the sequenced cpDNA, but seven genes: ndhF, psaA, psaB, rpoB, rpoC1, rpoC2, ycf2 had no information regarding gene annotation (Liu et al 2019) (see methods), so we only investigated 72 protein-coding genes in the follow-up analysis. To investigate the gene content similarity in the green plants, we calculated the average gene number in every order and the overview of the genes are presented in Fig 1. According to the copy number of the gene, samples were divided into three main clades, the first clade comprised some of Chlorophyte, Charophyta, moss, liverworts, fern-and-horsetails, and the second contained most of Chlorophyta, Genetal and Pinales of Gymnosperm, Santalales of Eudicots which possessed no ndh family. The third is Eudicots and Monocots which contained two copies of rpl23, rpl2, ndhB, rps7, and rps12. We also compared the gene content in the green plants by using the Spearman correlation (Fig S1). Some genes appeared to co-occur, the NADH dehydrogenase (ndh) genes showed a strong positive correlation with other genes within the family (r>0.7) except ndhB (r ~0.5), however, ndhB showed a strong positive correlation with rpl2, rpl23, rps12, rps7, and ycf1. Ndh family showed a negative correlation with both clpP and infA genes. We also found that some families were closely correlated such as psb family (psbE, psbF, and psbH) had a positive correlation to pet family (petA, petB, petD, petG, and petL). Similarly, the introns in land-plant chloroplast genomes are generally conserved. The Chlorophyta and Charophyta possess the least intron number. Most of the genes lacked introns with the exception among several ribosomal proteins and

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photosynthesis genes such as atpF, ndhA, petD, rpl16 and rps12 which possess at least one intron (in Streptophyta). The intron number of clpP gene showed a high divergence, 2327 species showed the presence of two introns, and more than 100 species owned 3-4 introns. Moreover, most of the clpP gene without introns were found among Chlorophyta, gymnosperms and Poaceae (Fig S2, Table S1).

Gene gain/loss in chloroplast genomes Although the genetic content and number of protein-coding genes are generally conserved in the chloroplast genomes, the gene gain and loss have been reported. A total of 72 protein-coding genes were investigated from 3654 species (Table S1). For gene gain, we found that, from Nymphaeales, almost all the flowering plants have two copies of ndhB, rpl2, rpl12, rpl23, rps12, and rps7 genes which correlated with IRs expansion, especially rps12 with four copes. In Campanulaceae, Ericaceae and Fabaceae, ndh family genes were duplicated. For gene loss, we found some genes were more likely lost in the green plant. The chloroplast translation initiation factor 1 (infA) and the ribosomal protein L22 (rpl22) are the two housekeeping genes. We found that infA was absent in 1825 taxa, and it was more frequently observed among angiosperms, especially in Eudicots. On the other hand, rpl22 was missing in 474 taxa mainly in Chlorophyta and legumes, suggesting that both genes were possibly transferred from chloroplast to the nucleus during evolution, as reported in an earlier study [31]. The ndh genes are related to the cyclic electron in the photosystem I complex, and has been thought to be lost in the higher plants [32]. In our study, ndh genes were found to be lost in at least 300 species, mainly in Chlorophyta, Pinaceae, Ephedrales, Welwitschiales, Gnetales and some species of Orchidaceae. At the same time, except ndh gene family, petN, rpl22, rpl33, rps15, rps16 were lost in Chlorophyta, and rps16 were lost in Gymnosperm and Bryophytes. In addition, we also observed the loss of accD and ycf1 genes together in more than 800 species (almost are Poales). Ycf1 is thought to have a

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functional role of assembling the ACCase holoenzyme [33, 34], and both these two genes have been related to fatty acid synthesis.

Gene conservation and rearrangement It is well known that the structure of chloroplast genomes conserved and the order of genes is relatively consistent in land plants. This opens up the possibility of reconstructing insertions, deletions and inversions during the evolution of green plant. In this study, 72 protein-coding genes were ordered according to the annotated position. In Arabidopsis thaliana, cluster analysis has been done based on chloroplast transcriptomes expression and finally chloroplast genes divided into eight sub-clusters [35]. To calculate the blocks frequency in Streptophyta, we first removed the samples in the order which have the same gene content, and finally obtained 1517 cpDNA and the blocks frequency are listed in Table S2. Based on the functional categories, there are three major gene clusters. The frequency of ATP synthase cluster: atpA-atpF-atpH-atpI was 74%, atpE-atpB was 82%, Phytosystem and Cytochrome cluster: petA-psbJ-psbL-psbF-psbE-petL-petG was 80% psbB-psbT-psbN-psbH-petB-petD was 85%. Ribosomal cluster: rps8-rpl14-rpl16-rps3 was 83%, rpl33-rps18-rpl20 was82% and rpoA_rps11_rpl36 was 85%. In Monocots and Eudicots, we observed three photosystem gene clusters with high frequency: psbM/D/C/Z [60%], psbJ/L/F/E [85%] and psbB/T/N/H [88%]. PsbJ/L/F/E and psbB/T/N/H nearly conserved in all the green plants and liked to form blocks: psbB/T/N/H-petB-petD-rpoA-rps11-rpl36 [78%], psbJ/L/F/E-petL-petG-psaJ-rpl33-rps18-rpl20 [76%] in Streptophyta. But psbM/D/C/Z block showed the highest variability in the green plant. PsbD and psbC genes encode the D2 and CP43 proteins of the photosystem II complex, and they are generally co-transcribed [36]. Similarly, psbM is highly light-sensitive and plays important roles in such conditions, in fact, the knock-out of psbM leads to a

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significant decrease in the activity of photosystem II [37]. In Chlorophyta, psbD/C/Z, psbZ/M, and psbD/C were found to be widely distributed, but in the Charophyta branch, only psbD/C/Z block exists. Later in Bryophytes, psbZ/C/D and psbM were connected by ATP synthase: atpA/F/H/I. From ferns and horsetails clade, psbM/D/C/Z was formed. In Cycadales which likely represents all the seed plant, showed the presence of complete psbM/D/C/Z blocks, but in Pinales, psbM and psbD/C/Z were separated. In Poaceae atpA/F/H/I-rps2-petN-psbM was especially inverted leading to the production of larger block psbK/I/M/D/C/Z. Except gene cluster, there are still some large blocks which contained the more than one functional category gene. The largest block: (atpA-atpF-atpH-atpI) (1) - (rps2-petN-psbM) (2) -(psbD-psbC-psbZ) (3) - (rps14-ycf3-rps4) (4) [51%]- (ndbJ-ndhK-ndhC-atpE-atpB-rbcL) (5) [70%] -accD-psaI-(ycf4-cemA-petA-psbJ-psbL-psbF-psbE-petL-petG-psaJ-rpl33-rps18-rpl2 0) [69%]- (psbB-psbT-psbN-psbH-petB-petD-rpoA-rps11-rpl36) [78%] was found with high frequency in Streptophyta, and numbers in [] are the block frequency. In Poaceae, (1), (2) and (3) block were with complicated situation which experienced several rounds of recombination and in some genus of Fabaceae, (3) -(4) and (5) were cross interchanged. In streptophytes, the cpDNA reserved a large gene block:(psbB-psbT-psbN-psbH-petB-petD) [85%]- (rpoA-rps11-rpl36) [85%]-infA-(rps8-rpl14-rpl16-rps3-rpl22-rps19-rps2-rps23) [61%] which included S10-spc regions and connected directly with IRb (Table S3). IRs normally contain tRNAs and rRNAs, but in this study, we didn’t annotate tRNA and rRNAs, we focused on coding genes: rps19-rpl2-rpl23-ndhB-rps7-rps12 which were newly acquired in IRs for angiosperms [8] (Fig 2). The rps19-rpl2-rpl23 were conserved in the green plants, but ndhB-rps7-rps12 showed great variation. The ndbB were found in some green algae such as Prasinococcales and Palmophyllales, then from Charophyta, ndhB-rps7-rps12 and rps19-rpl2-rpl23-ndhB-rps7 blocks were formed. In the Bryophytes and Lycophytes, ndhB-rps7-rps12 widely existed and Dendrocerotaceae have a complete block of rps19-rpl2-rpl23-ndhB-rps7-rps12. In

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ferns and horsetails, except rps19-rpl2-rpl23-ndhB-rps7-rps12 block in Marattiales, most orders have ndhB-rps12-rps7-psbA-ycf1 block which is near the IRs regions. In Amborella and Nymphaeales, IRs contained rps19-rps2-rps23-ndhB-rps7-rps12 which is similar to all other angiosperms but Trithuria filamentosa and Cabomba caroliniana had the largest IRs which contained psbB-psbT-psbN-psbH-petB-petD-rpoA-rps11- rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22-rps19-rps2-rps23-ndhB-rps7-rps12.

GC bias The GC content is deemed to be in connection with the amino acid composition based on former research[29]. In this study, the GC content at different codon positions of all the 72 protein-coding genes including the first, second, and third codon positions, together with ntNo3rd and ntAll data sets were used for the analysis. The average GC content of ntNo3rd matrix ranged from 36.3% to 58.2%, while the ntAll data set had a slightly lower GC content of 38.2%, varying from 29.0% to 55.4% (Table S1). The third codon position of all 14 clades owned the lowest GC content compared to other position, and there was an obvious difference between different clades. Therefore, the One-Way ANOVA was carried out using SPSS to test whether there was any significance between different clades, and the result revealed that the GC characters were the same in Chlorophyta and Charophyta, and there was a non-significant difference between angiosperms. Furthermore, the GC content of Lycophytes and fern were significantly higher than all the other clades (p < 0.01), and the GC content in the clades after Lycophytes and fern were higher than that of Bryophytes and algae (Fig S3).

Phylogenetic analysis To conduct the phylogenetic analysis, the concatenated alignment of three data sets

for the 72 genes from 3654 species were used with 6 Rhodophyta as outgroups，which

consisted of 4,724 amino acid positions (AA). A total of 44,187 positions for the

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matrix containing all codon positions (nt123) and 29,458 positions for the matrix containing all but the third codon positions (nt12). We used two programs: IQ-TREE and RAxML to construct the phylogenetic tree, but they both produced nearly the same topology, so we only used IQ-TREE to illustrate our results. The topology is summarized in Fig 3-4 and the details of the phylogenetic trees are provided in supplemental materials (Fig S4-S8). For some debated clades, the summary of the similarities and conflicts in topologies derived from these three data sets are presented in Table 1. All phyla of green plants except Charophyta are recovered as monophyletic. Within Chlorophyte, both matrix nt12, nt123 and the matrix AA supported that Palmophyllales and Prasinococcales are both the earliest-diverging lineage of the Chlorophyta (SH-alrt == 33%, UFboot = 100%). Chlorophyceae is strongly supported as monophyletic, with Chlamydomonadales + Sphaeropleales sister to a clade of Chaetophorales and Chaetopeltidales. The relationship of Chlorophyceae and Ulvophyceae (non-monophyletic) is uncertain. Within Streptophyta, Charophyta lineages formed a paraphyletic assemblage with the land plants. Among the Charophyta groups, Chlorokybales + Mesostigmatales are the earliest-branching lineage and a clade of Zygnematales+ Desmidiales formed a grade as the sister group to the land plants. Within land plants, Bryophytes were weakly supported as monophyletic based on matrix nt12 (SH-alrt == 33%, UFboot = 100%), while matrix AA, strongly supported Bryophytes as monophyletic (SH-alrt == 100%, UFboot = 100%), both matrix nt12 and matrix AA supported liverworts and mosses are uniting and sister to hornworts. For matrix nt123 data, hornworts, mosses and liverworts were found to be successive sister lineages of Tracheophytes (SH-alrt == 100%, UFboot = 100%). Within Euphyllophyta in the matrix nt12 and nt123, a well-supported Monilophyta is sister to Spermatophyta (UFboot = 100%), but the matrix aa indicated that Monilophyta is sister to Bryophytes (SH-alrt = 90.9%, UFboot = 100%). Within Monilophyta, matrix nt12 supported Ophioglossales as the earliest-diverging lineage (UFboot =100%), but matrix nt123 supported Equisetales as the earliest branch

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(UFboot =100%). Within Spermatophyta, gymnosperms were designated as sister to angiosperms. Within gymnosperms, the subclades were well supported in three data sets, the clade of Cycadales + Ginkgoales is sister to the rest of gymnosperms. While the Gnetales, Welwitschiales along with Ephedrales, formed a clade (UFboot = 100%), which are sister to the clade comprising Cupressales and Araucariales. Within angiosperms, in matrix nt12 and nt123, the Amborellales is recovered as the sister to all other angiosperms, followed by Nymphaeales, nevertheless, the placement of Nymphaeales is outermost in angiosperms in the matrix AA with weakly support (SH-alrt =34.9%, UFboot = 100%). The placement of Ceratophyllales is certain in the outside of Eudicots in the three data sets, while the Magnoliids is placed in the outside of monocots in matrix nt123 (SH-alrt =100%, UFboot = 100%), and nested between monophyletic monocot and eudicot in matrix nt12 (SH-alrt =64.9%, UFboot = 59%) and matrix AA (SH-alrt =90.8%, UFboot = 35%). While checking the amino acid composition in Magnoliids, we found the composition deviated significantly from the ‘master’ distribution using the chi2 test. The relationship between COM clade supported Oxalidales is sister to Celastrales + Malpighiales. The major subclades were typically well supported in Monocots and Eudicots, but the position of Vitales, Gentianales, Petrosaviales and Arecales remained problematic. To further verify the phylogenetic analysis, the data of amino acids from the former research were added into the tree construction [30], and the results showed that the data of the same orders are clustered together, and the topology of the major clade is consistent with the matrix nt12 (Fig S9).

Selective pressure of chloroplast genes By dissecting the chloroplast genomes (cpDNA), we learned the special gene gain/loss, gene copy number, gene cluster order, and GC content along the evolution of the green plants, we also tried to understood which clades and cpDNA genes were under purifying selection, nonsynonymous substitution (dN), synonymous substitution (dS) and the dN/dS ratio were also calculated. The ndh subunits were

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thought to be related to the synthesis of photosystem I complex and involved in the adjustment of the redox level of the cyclic photosynthetic electron transporters [38]. Almost all the photosynthetic land plants have the ndh genes except in Chlorophyta, gymnosperms and some species of Orchidaceae (Table S1). To investigate the selective pressure of photosynthesis-related genes influenced by the absence of ndh subunits, 37 species in all 14 clades (10 of 37 species lost all ndh genes) were selected and the sequence of 14 photosynthesis genes existing in all the 37 species were chosen (Fig 5, Table S3). The results of the dN/dS analysis of 37 species revealed that the species which lost ndh genes might have a higher dN and dS, especially in Eudicots and gymnosperms. The dN/dS was extremely high in Chlorophyta, implying the occurrence of positive selection and dN/dS of all Streptophyta species except Downingia cuspidata is less than 1, suggesting that chloroplast genes are under purifying selection, even the rate was higher in the species without ndh genes. In addition, the independent analysis of 14 photosynthesis genes suggested that dS and dN in all genes for angiosperms have a relatively higher dN without ndh genes, except psbK (Fig S10, Fig S11).

Discussion The green plant's chloroplast genome The chloroplast gene content in the green plants are conserved and plasmid genome architectures have been discussed based on recombination events of rDNA operons [39], but how similarity of the genome in Streptophyta is unknown. In our study, we found the same function class likely to form gene cluster, ATP synthase, Phytosystem and Cytochrome, Ribosomal cluster appeared more than one with high frequency. In Streptophyta, a block: (psbB-psbT-psbN-psbH-petB-petD) [85%] -(rpoA-rps11-rpl36) [85%]-infA-(rps8-rpl14-rpl16-rps3-rpl22-rps19-rps2-rps23) [61%] widely existed and was located nearly IRs regions, parts of them are the S10–spc–alpha operon locus which first appeared in eubacteria and archaebacteria. Euglena and glaucophyte plasmids in the S10-spc regions contained rpl23-rpl2-rps19-rpl22-rps3-rpl16-rps17-

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rpl14-rpl5-rps8[40] which were identical to that in the E.coli operons[41], even in prokaryotic genomes [42], this location in cpDNA may come from archaebacteria to the green plant. What is more interested, in Arabidopsis thaliana, psbB-psbT-psbN-psbH-petB-petD and rps3-rpl22-rps19-rps2-rps23 have the same transcriptomes expression pattern which was exactly different from rpoA-rps11-rpl36-rps8-rpl14-rpl16 under various biological conditions[a1]. Near the S10–spc–alpha operon locus, there existed another larger block: atpA-atpF-atpH-atpI- rps2-petN-psbM-psbD-psbC-psbZ-rps14-ycf3-rps4- ndbJ-ndhK-ndhC-atpE-atpB-rbcL-accD-psaI-ycf4-cemA-petA-psbJ-psbL-psbF-psbE- petL-petG-psaJ-rpl33-rps18-rpl20-psbB-psbT-psbN-psbH-petB-petD-rpoA-rps11-rpl 36 which are ATP synthase, Phytosystem II, Cytochrome and Ribosomal genes.

Dynamic Evolution of IR in green plant

IR in green algal showed large fluctuation in size from 6.8 kb to 45.5kb, and sustained losses in major groups of green algal but in the green lineage, IR underwent expansion [43, 44]. When compared the IRs regions in green plants, not only for Eudicots but also all angiosperm, chloroplast have experienced an expansion at the end of IRs. For angiosperm, rps19-rpl2-rpl23-ndhB-rps7-rps12 gene copies were newly acquired in IRs, and there is always a big block connected with IRb. In the fern and horsetails, ndhB-rps12-rps7 appeared but not in IRs by rearrangement. In Gymnosperm except for Pinales, ndhB-rps7-rps12 block was copied and inverted to form rps19-rpl2-rpl23-ndhB-rps7-rps12, but we cannot tell whether the ndhB-rps7-ycf12 block was gained by fusion or rearrangement. From Amborellales and Nymphaeales, rps19-rpl2-rpl23-ndhB-rps7-rps12 gene copies were found in IRs, especially in Nymphaeales which still contains the largest IRs block.

Congruence and conflict in phylogenetic trees with other studies There are two previous chloroplast’s phylogenetic analysis of Ruhfel et al. (2014) [28] and Gitzendanner, M.A., et al (2017) [29] where they used 360 and 1879 taxa to study the green plants respectively. Most topologies of our phylogenetic tree were 14 bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

consistent, however, there were some differences between our results and those of the two studies. Based on the AA analysis of Gitzendanner, M.A., et al (2017) recovered Bryophyte clade as monophyletic, which is similar to our results but with an uncertain relationship to Lycophytes and fern. In our matrix AA analysis, we found Bryophyte + Lycophytes are sister to ferns and horsetails (UFboot = 100%). With matrix nt123, hornworts, mosses, and liverworts were identified as successive sister lineages of Tracheophytes (UFboot = 100%). Both these two topologies were well supported by previous research [18, 45]. In our study, Magnoliids were placed at the outside of Monocots in matrix nt123 (UFboot = 100%), but nested between monophyletic Monocot and Eudicot in matrix nt12 (UFboot = 59%) and matrix AA (UFboot = 35%). But, from the gene constitution in Magnoliids, they are similar to both Monocot and Eudicot. In Matthew et al. (2017) analysis, Magnoliids and Chloranthales form a weakly supported clade (BS = 61%) that is sister to a clade of Monocots (BS = 100%) and Eudicots. When we combined the dataset from Gitzendanner, M.A., et al (2017) with our AA sequences and re-analyzed, Magnoliids moved outside of the Monocots (SH-alrt =97.5%, UFboot = 95%). Ruhfel et al. (2014) recovered Ceratophyllales as sister to the Monocots using matrix nt12 with low support (BS = 52%) and when using matrix nt123, Ceratophyllales was placed between the Monocots and Eudicots (BS = 52%). We recovered Ceratophyllales + Chloranthales as sister to the Eudicots using nt123 data (UFboot=73%, UFboot=99%), but when using matrix nt12 and AA data, only Ceratophyllales sister to the Eudicots (UFboot=100%).

Conclusion The structure of chloroplast genomes is mostly consistent in green plants and formed several gene clusters and gene block except in Chlorophyta. This structural conservatism might be a result of the common genes between cyanobacteria or the same function categories are more likely to form a gene cluster. Topologies of phylogenetic tree of green plants, more extensive taxon indeed increased the phylogenetic resolution for some controversial clades. Matrix nt12 data produced

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similar results with matrix AA data, matrix nt123 data affected the position of Bryophyte and Magnoliids. In general, for some controversial clades that are deep within green plants, such as, Bryophyte, dense taxon sampling did not improve phylogenetic accuracy anymore, data set will effort the topologies. Thus, resolving the controversial deep-level clades, simply increasing taxon sampling may not be necessary. In addition, chloroplast genome analysis alone seems unlikely to solve the relationship of these controversial clades. Using large numbers of nuclear genes or selecting the nuclear genes with stronger phylogenetic signals may help to solve these deep-level questions.

Methods Taxon and Gene Sampling The complete or nearly complete chloroplast genomes of 3246 species in GenBank (as of May 18, 2018) and 731 species of Ruili Botanical Garden were retrieved and used as the raw data (Liu et al 2019). The duplicated samples and the species with incorrect annotation or significant gene losses were excluded from the analysis. Six problematic species (Monoraphidium neglectum, CM002678; Nothoceros aenigmaticus, NC-020259; Nymphaea ampla, NC-035680; Allium sativum, NC-031829; Bambusa oldhamii, NC-012927 and Potentilla micrantha, HG931056) were subjected to re-annotation with GeneWise v2.4.1 (Birney et al. 2000). In addition, 50% missing genes of a species and 50% missing species of a gene was also set as the filter elements, and a total of 3654 species with 72 conserved protein-coding genes were obtained for the further analysis. The 298 families from 111 orders of Chlorophyta, Charophyta, Bryophytes, Pteridophyta, Bryophytes, Gymnosperm and Angiosperms were used to represent most major lineages of green plants, and 6 species of Rhodophyta as outgroups were used. The details (species name, family names, order names, genome size, and accession numbers) of 3654 chloroplast genomes are listed in Supplementary Table S1.

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Sequence Alignment DNA sequences of 72 protein-coding genes were extracted from each genome according to the annotation files. A total of three data sets were made, which included the matrix of all nucleotide positions (nt123), the matrix containing only the first and second codon positions (nt12), and the matrix of amino acids (AA). The nucleotide sequence of each coding gene was processed individually with TranslatorX using MAFFT v7.017 (Katoh et al. 2013) to align the aa sequences, and then the TrimAL software was used to trim the poorly aligned positions with the gappyout option. The genes with 50% missing data were also discarded on the basis of the sequence alignment for further filtration, and the final alignments of each gene were obtained by repeating the aforementioned steps. The nucleotide and amino acid sequence alignments of 72 protein-coding genes were connected subsequently, and a total of 44,187 sites of the ntAll matrix, 29,458 sites of nt12 matrix and 14,724 amino acid positions of AA matrix were used for further analysis.

Phylogenetic analyses Three datasets containing 72 protein-coding genes of 3654 species with no partitioning strategies were used to reconstruct the phylogenetic tree based on IQ-TREE with 5000 ultrafast bootstrap replicates and 1000 bootstrap replicates for SH-aLRT, together with GTR+F+R10 model for nucleotide sequences and JTT+F+R10 model for amino acids sequences. ML analysis was also conducted using RAxML v8.2.4 (Stamatakis 2014) under the GTRCAT model for nucleotide and PROTGAMMAWAG model for amino acids. The 100 bootstrap replicates were set to test the reliability of each node for ML analysis. The optimal partitioning scheme was referred according to Brad et. (2014. For the AA data, we partitioned the data sets by gene (72 partitions). For the nucleotide (nt123), we used program PartitionFinder (ref) to find the best partitioning strategies by each codon position within each gene, to reduce the computational burden, only 731 species of Ruili Botanical Garden were used for PartitionFinder. The 148 partitions

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were selected with lnL: -3013791.4635925293, AICc: 6034073.71459. Partitioning strategies for both AA data and nt123 to construct phylogenetic tree were unable to complete due to time limitations resulting from the large samples of our data. To verify the topologies of the phylogenetic tree, the amino acids sequences of 72 genes of 1901 samples in former research (Gitzendanner et al. 2018) were downloaded to analyze along with our data using the IQ-TREE. The Tree-doctor was used to obtain the simplified trees in order levels. The species of Rhodophyta was set as outgroups to re-root the result, and the iTOL (https://itol.embl.de/) was used for data visualization.

Evolutionary Rate Estimation. According to the gene losses of ndh subunits, 37 species in all 14 clades (10 of 37 species lost all ndh genes) were selected as the representative species, and the sequence of 14 photosynthesis genes existing in all 37 species was selected. The alignments and the topology derived from IQ-TREE of all nucleotide positions (nt12) of each gene and the gene set in the selected species were used to perform the evolutionary rate analysis. The codeml of PAML was used to estimate the ratio of nonsynonymous to synonymous nucleotide substitutions (dN/dS) for each branch of 14 genes corresponding to photosynthesis.

Competing interests

The authors declare that they have no competing interests.

Data availability The data that support the findings of this study have been deposited in the CNSA (https://db.cngb.org/cnsa/) of CNGBdb with accession code CNPhis0000538.

Acknowledgements

18 bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

This work was supported by the grants of Basic Research Program, Shenzhen Municipal Government of China (No. JCYJ20160331150739027 and No. JCYJ20150831201123287), and the Guangdong Provincial Key Laboratory of Genome Read and Write (Grant No. 2017B030301011). Table legends Table 1. Summary of consensus topologies derived from the three data sets.

Figure legends Fig 1. The gene constitution in the green plants is displayed as a heatmap. In heat map, the data is displayed in a grid where each row represents order and each column represent average gene number in the order. Fig 2. Coding genes in IRs in Streptophyta. Coding genes in IRs and upstream are shown. Fig 3. Chloroplast phylogenomic tree based on the matrix nt12 of 72 protein-coding genes of 3654 green plants and six Rhodophyta using IQTREE. The colors in the internal circle indicate different families while the colors in the external circle indicate different orders. The green branches represent the branch with UFboot more than 95%. Fig 4. Summary of the phylogenomic tree based on three data sets of 72 protein-coding genes of 3654 green plants and six Rhodophyta using IQTREE. Fig 5. Phylogenomic tree of selective pressure for 37 species in all 14 clades. The red branch stands for the length of the branch which was changed to 1. “*” indicated the species without ndh genes.

Supporting information Fig S1. Gene content in the green plants, the correlation of the genes was calculated by the Spearman method. Fig S2. Characters of the chloroplast genome in the green plant. The box plots represent the median (central line), first and third quartiles (box), and outliers (dot).

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Fig S3. GC content of different codon positions in green plant. The One-Way ANOVA and other statistical analyses were performed in SPSS software, and the lowercase represents the significance between 14 clades (p < 0.01). Fig S4. Chloroplast phylogenomic tree based on the matrix nt123 of 72 protein-coding genes of 3654 green plants and six Rhodophyta using IQTREE. The colors on the internal circle indicate different families, while the colors on the external circle indicate different orders. Fig S5. Chloroplast phylogenomic tree based on the matrix aa of 72 protein-coding genes of 3654 green plants and six Rhodophyta using IQTREE. The colors on the internal circle indicate different families while the colors on the external circle indicate different orders. Fig S6. Chloroplast phylogenomic tree based on the matrix nt12 of 72 protein-coding genes of 3654 green plants using RaXML. The colors on the internal circle indicate different families while the colors on the external circle indicate different orders. Fig S7. Chloroplast phylogenomic tree based on the matrix nt123 of 72 protein-coding genes of 3654 green plants using RaXML. The colors in the internal circle indicate different families while the colors in the external circle indicate different orders. Fig S8. Chloroplast phylogenomic tree based on the matrix aa of 72 protein-coding genes of 3654 green plants and 1901 species in the former research using IQTREE. The colors on the internal circle indicate different families while the colors on the external circle indicate different orders. Fig S9. Summary of the phylogenomic tree based on matrix aa of 72 protein-coding genes of 3654 green plants and 1901 species obtained from earlier reports using IQTREE. Fig S10. Phylogenomic tree of dN for 14 photosynthesis genes in 37 species. “*” indicated the species without ndh genes.

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Fig S11. Phylogenomic tree of dS for 14 photosynthesis genes in 37 species. The red branch stands for the length of the branch which was changed to 10. “*” indicated the species without ndh genes.

Table S1. The gene blocks frequency in Streptophyta Table S2. The detailed information and characters of the species used in this study. Table S3. The gene blocks frequency in Streptophyta Table S4. The hexadecimal colors used in this study. Table S5. The 37 selected species for the analysis of dN/dS.

References

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Density 0 5 10 15 20 3 2 1 0 and DensityPlot bioRxiv preprint Color Key Value doi: certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. https://doi.org/10.1101/655241

atpB atpI atpA rpl36 atpF rps11 rps18 rpl20 rbcL atpE rps14 psbF psbJ rps2 rps8 ;

rpl16 this versionpostedJune3,2019. rpl14 rps3 psaJ psbC psbE psbK ycf3 petA clpP atpH petG psbI petB psbB psbL psbD rps4 petD ycf4 psbN psbH psbT psaC The copyrightholderforthispreprint(whichwasnot psbZ psaI rps19 petL cemA psbM rpoA ccsA rpl32 psbA ycf1 ndhC ndhK ndhI ndhH ndhE ndhG ndhD ndhA ndhJ petN rpl22 accD rps15 rpl33 matK rps16 infA rpl23 rpl2 ndhB rps7 rps12 mosses_Takakiales fer Monocots_Petrosaviales Chlorophyta_orderNA5 Chlorophyta_orderNA4 Chlorophyta_Pyramimonadales Chlorophyta_Prasinococcales Chlorophyta_Palmophyllales Lycophytes_Selaginellales ferns_and_horsetails_Cyatheales ferns_and_horsetails_Polypodiales Gymnosperm_Ginkgoales liverworts_Metzgeriales Eudicots_Gentianales ANA_Grade_Austrobaileyales ferns_and_horsetails_Marattiales Gymnosperm_Cycadales ferns_and_horsetails_Psilotales Lycophytes_Isoetales Lycophytes_Lycopodiales hornworts_Anthocerotales Charophyta_Charales Charophyta_Chlorokybales hornworts_Dendrocerotales Eudicots_Vitales Eudicots_Ericales Eudicots_Saxifragales Eudicots_Proteales Eudicots_Cornales Eudicots_Apiales Eudicots_Lamiales Monocots_Alismatales Monocots_Liliales Eudicots_Ranunculales Magnoliids_Magnoliales Monocots_Dioscoreales Eudicots_Huerteales ANA_Grade_Nymphaeales Magnoliids_Piperales Monocots_Acorales Magnoliids_Laurales Eudicots_Dipsacales Eudicots_Caryophyllales Eudicots_Asterales Chloranthales_Chloranthales Charophyta_Mesostigmatales liverworts_Marchantiales Magnoliids_Canellales Ceratophyllales_Ceratophyllales Eudicots_Icacinales Eudicots_Aquifoliales Eudicots_Garryales Eudicots_Malvales Eudicots_Fagales fer ferns_and_horsetails_Equisetales Gymnosperm_Cupressales Gymnosperm_Araucariales Charophyta_Zygnematales Charophyta_Desmidiales ferns_and_horsetails_Osmundales mosses_Funariales mosses_Hypnales mosses_Pottiales mosses_Sphagnales mosses_Orthotrichales Charophyta_Coleochaetales liverworts_Ptilidiales Eudicots_Cucurbitales Eudicots_Myrtales Eudicots_Rosales Eudicots_Solanales Eudicots_Brassicales Eudicots_Malpighiales Eudicots_Fabales ferns_and_horsetails_Schizaeales Gymnosperm_Pinales Eudicots_Santalales Gymnosperm_Gnetales liverworts_Pelliales Monocots_Asparagales Monocots_Poales ANA_Grade_Amborellales Monocots_Pandanales Monocots_Commelinales Eudicots_Sapindales Eudicots_Celastrales Monocots_Arecales Monocots_Zingiberales Eudicots_Buxales Eudicots_Crossosomatales Eudicots_Boraginales Eudicots_Oxalidales Eudicots_Trochodendrales Eudicots_Geraniales ferns_and_horsetails_Salviniales Chlorophyta_orderNA1 Chlorophyta_orderNA9 Chlorophyta_Bryopsidales Chlorophyta_Ulvales Chlorophyta_Marsupiomonadales Chlorophyta_orderNA3 Chlorophyta_Pedinomonadales Chlorophyta_Chlamydomonadales Chlorophyta_Sphaeropleales Chlorophyta_orderNA8 Chlorophyta_Chaetopeltidales Chlorophyta_Chaetophorales Chlorophyta_Monomastigales Chlorophyta_orderNA6 Chlorophyta_Mamiellales Chlorophyta_Chlorodendrales Gymnosperm_Ephedrales Gymnosperm_Welwitschiales ns_and_horsetails_Ophioglossales ns_and_horsetails_Gleicheniales Charophyta psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 rps12 rps7 ndhB IRb IRa

Bryophytes psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 rps2 rps23 IRb IRa rps12 rps7 ndhB

Lycophytes psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 rps2 rps23 IRb IRa rps12 rps7 ndhB

Fern psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 rps19 rps2 rps23 IRb IRa rps12 rps7 ndhB bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

ycf2 ndhB rps7 rps12 rps12yfc1 rps7 ndhB ycf2 Gymnosperm psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 rps19 rps2 rps23 IRb IRa

rps19 rps2 rps23 ycf2 ndhB rps7rps7 rps12 rps12ps7ndhB rps7rps7 ndhBrps12 ycf2 rps23 rps2 Angiosperms Amborellales psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 IRb IRa

psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 rps19 rps2 rps23 ycf2ndhB ndhBrps7 rps7rps7 rps12ps7ndhByfc1 rps12ps7ndhB rps7rps7 ndhBrps12 ycf2 rps23 rps2 rps19 rpl22-rps3-rpl16-rpl14-rps8-infA-rpl36-rps11-rpoA psbB-psbT-psbN-psbH-petB-petD Nymphaeales IRb IRa

rps19 rps2 rps23 ycf2 ndhB rps7 rps12 rps12ps7ndhB rps7rps7 ndhBrps12 ycf2 rps23 rps2 rps19 Magnoliids psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 IRb IRa

rps19 rps2 rps23 ndhB rps7rps7rps7 rps12 rps12ps7ndhB rps7rps7 ndhBrps12 rps23 rps2 rps19 Monocots psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 IRb IRa

rps19 rps2 rps23 ndhBndhB rps7rps7 rps12 rps12ps7ndhB rps7rps7 ndhBrps12 rps23 rps2 rps19 Eudicots psbB-psbT-psbN-psbH-petB-petD rpoA-rps11-rpl36-infA-rps8-rpl14-rpl16-rps3-rpl22 IRb IRa bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Monocots

Austrobaileyales Dioscoreales Petrosaviales Pandanales Gymnosperm Nymphaeales Alismatales Liliales Amborellales

Acorales

Asparagales

Commelinales Zingiberales Pinales Cycadales,Ginkgoales Arecales Ferns & Horsetails Cupressales Araucariales

Gnetales

Lycophytes Poales

LiverwortsMosses CharophytaHornworts Chlorophyta

Rhodophyta

Magnoliids

Lamiales Chloranthales Piperales Canellales Laurales Magnoliales Ceratophyllales

Ranunculales

Proteales Buxales,Trochodendrales Gentianales Vitales Saxifragales

Geraniales

Myrtales

Solanales

Crossosomatales Boraginales Garryales Sapindales Icacinales

Huerteales

Malvales

Brassicales Superasterids

Asterales Superrosids Oxalidales Celastrales

Malpighiales

Cucurbitales Apiales Fagales

Dipsacales Rosales Aquifoliales

Ericales

Cornales

Santalales Fabales

Caryophyllales Eudicots-Fagales Eudicots-Cucurbitales Eudicots-Brassicales nt12 Eudicots-Cucurbitales nt123 Eudicots-Fagales aa Eudicots-Malvales Eudicots-Rosales Eudicots-Rosales Eudicots-Huerteales Eudicots-Fabales Eudicots-Fabales Eudicots-Sapindales 100/100 100/100 Eudicots-Celastrales Eudicots-Malpighiales 100/100 Eudicots-Crossosomatales Eudicots-Malpighiales Eudicots-Celastrales Eudicots-Myrtales Eudicots-Oxalidales Eudicots-Oxalidales Eudicots-Geraniales Eudicots-Brassicales Eudicots-Malvales Eudicots-Fagales 100/100 100/100 100/100 Eudicots-Malvales Eudicots-Brassicales Eudicots-Cucurbitales Eudicots-Huerteales Eudicots-Huerteales Eudicots-Rosales Eudicots-Sapindales Eudicots-Sapindales Eudicots-Fabales Eudicots-Crossosomatales Eudicots-Crossosomatales Eudicots-Malpighiales Eudicots-Myrtales Eudicots-Myrtales Eudicots-Celastrales Eudicots-Geraniales Eudicots-Geraniales Eudicots-Oxalidales Eudicots-Vitales Eudicots-Vitales Eudicots-Vitales 67.8/100 0/86 56.4/99 Eudicots-Saxifragales Eudicots-Saxifragales Eudicots-Saxifragales Eudicots-Gentianales Eudicots-Boraginales Eudicots-Gentianales Eudicots-Lamiales Eudicots-Solanales Eudicots-Boraginales Eudicots-Solanales Eudicots-Lamiales Eudicots-Solanales 100/100 Eudicots-Boraginales Eudicots-Gentianales Eudicots-Lamiales Eudicots-Garryales Eudicots-Garryales Eudicots-Garryales Eudicots-Icacinales Eudicots-Icacinales Eudicots-Icacinales Eudicots-Apiales Eudicots-Apiales Eudicots-Dipsacales Eudicots-Dipsacales Eudicots-Dipsacales Eudicots-Apiales Eudicots-Asterales Eudicots-Asterales Eudicots-Asterales Eudicots-Aquifoliales Eudicots-Aquifoliales Eudicots-Aquifoliales Eudicots-Ericales Eudicots-Ericales Eudicots-Ericales Eudicots-Cornales Eudicots-Cornales Eudicots-Cornales Eudicots-Caryophyllales Eudicots-Caryophyllales Eudicots-Caryophyllales Eudicots-Santalales Eudicots-Santalales Eudicots-Santalales Eudicots-Trochodendrales Eudicots-Trochodendrales Eudicots-Trochodendrales Eudicots-Buxales Eudicots-Buxales Eudicots-Buxales 100/100 100/100 100/100 Eudicots-Proteales Eudicots-Proteales Eudicots-Proteales 100/100 Eudicots-Ranunculales 99.4/99 Eudicots-Ranunculales 100/100 Eudicots-Ranunculales Ceratophyllales-Ceratophyllales Ceratophyllales-Ceratophyllales Ceratophyllales-Ceratophyllales 99.4/73 Magnoliids-Laurales Chloranthales-Chloranthales 99.4/71 Magnoliids-Magnoliales Magnoliids-Magnoliales Monocots-Zingiberales Magnoliids-Laurales Magnoliids-Canellales Monocots-Commelinales Magnoliids-Piperales Magnoliids-Piperales Monocots-Poales Magnoliids-Canellales 88.3/63 90.8/35 Chloranthales-Chloranthales Monocots-Arecales Chloranthales-Chloranthales Monocots-Poales Monocots-Asparagales Monocots-Commelinales Monocots-Arecales Monocots-Liliales Monocots-Zingiberales 69.4/59 Monocots-Zingiberales Monocots-Dioscoreales 100/100 Monocots-Poales Monocots-Commelinales Monocots-Pandanales Monocots-Arecales Monocots-Asparagales 13.5/51 Monocots-Petrosaviales Monocots-Asparagales Monocots-Liliales Monocots-Alismatales Monocots-Liliales Monocots-Dioscoreales Monocots-Acorales Monocots-Dioscoreales 100/100 100/100 Monocots-Pandanales 100/100 Magnoliids-Laurales Monocots-Pandanales Monocots-Petrosaviales Magnoliids-Magnoliales Monocots-Petrosaviales 81.4/99 Monocots-Alismatales Magnoliids-Piperales 99.9/100 Monocots-Alismatales 100/100 Monocots-Acorales Magnoliids-Canellales Monocots-Acorales 100/100 13/85 ANA_Grade-Austrobaileyales 99.8/100 ANA_Grade-Austrobaileyales ANA_Grade-Austrobaileyales ANA_Grade-Nymphaeales ANA_Grade-Nymphaeales ANA_Grade-Amborellales ANA_Grade-Amborellales ANA_Grade-Amborellales ANA_Grade-Nymphaeales Gymnosperm-Gnetales Gymnosperm-Gnetales Gymnosperm-Welwitschiales 100/100 100/100 Gymnosperm-Welwitschiales 100/100 Gymnosperm-Welwitschiales Gymnosperm-Gnetales 94.5/98 Gymnosperm-Ephedrales 94.6/100 Gymnosperm-Ephedrales 84.9/100 Gymnosperm-Ephedrales Gymnosperm-Cupressales Gymnosperm-Araucariales Gymnosperm-Araucariales Gymnosperm-Araucariales Gymnosperm-Cupressales Gymnosperm-Cupressales 100/100 Gymnosperm-Pinales 100/100 Gymnosperm-Pinales 100/100 Gymnosperm-Pinales Gymnosperm-Cycadales Gymnosperm-Cycadales Gymnosperm-Cycadales Gymnosperm-Ginkgoales Gymnosperm-Ginkgoales Gymnosperm-Ginkgoales 99.9/87 ferns_and_horsetails-Polypodiales 100/100 ferns_and_horsetails-Polypodiales mosses-Orthotrichales ferns_and_horsetails-Cyatheales ferns_and_horsetails-Cyatheales mosses-Hypnales ferns_and_horsetails-Salviniales ferns_and_horsetails-Salviniales mosses-Pottiales ferns_and_horsetails-Schizaeales ferns_and_horsetails-Schizaeales mosses-Funariales ferns_and_horsetails-Gleicheniales ferns_and_horsetails-Gleicheniales mosses-Tetraphidales ferns_and_horsetails-Osmundales ferns_and_horsetails-Osmundales 100/100 mosses-Sphagnales 88.2/86 ferns_and_horsetails-Marattiales 100/100 ferns_and_horsetails-Marattiales mosses-Takakiales ferns_and_horsetails-Equisetales 100/100 ferns_and_horsetails-Ophioglossales 99.7/100 liverworts-Ptilidiales ferns_and_horsetails-Ophioglossales ferns_and_horsetails-Psilotales liverworts-Metzgeriales ferns_and_horsetails-Psilotales ferns_and_horsetails-Equisetales liverworts-Pelliales 100/100 99/100 Lycophytes-Selaginellales Lycophytes-Isoetales liverworts-Marchantiales bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Lycophytes-Isoetales Lycophytes-Selaginellales hornworts-Dendrocerotales Lycophytes-Lycopodiales Lycophytes-Lycopodiales 100/100 hornworts-Anthocerotales mosses-Orthotrichales hornworts-Dendrocerotales Lycophytes-Selaginellales mosses-Hypnales hornworts-Anthocerotales Lycophytes-Isoetales 100/100 mosses-Pottiales mosses-Orthotrichales Lycophytes-Lycopodiales 100/100 mosses-Funariales mosses-Hypnales ferns_and_horsetails-Polypodiales mosses-Tetraphidales mosses-Pottiales ferns_and_horsetails-Cyatheales 90.9/100 mosses-Sphagnales mosses-Funariales ferns_and_horsetails-Salviniales mosses-Takakiales 100/100 mosses-Tetraphidales ferns_and_horsetails-Schizaeales 100/100 liverworts-Ptilidiales mosses-Takakiales ferns_and_horsetails-Gleicheniales liverworts-Metzgeriales mosses-Sphagnales ferns_and_horsetails-Osmundales 100/100 33/100 liverworts-Pelliales liverworts-Metzgeriales ferns_and_horsetails-Marattiales liverworts-Marchantiales 100/100 liverworts-Ptilidiales ferns_and_horsetails-Ophioglossales hornworts-Dendrocerotales liverworts-Pelliales ferns_and_horsetails-Psilotales hornworts-Anthocerotales liverworts-Marchantiales 100/100 ferns_and_horsetails-Equisetales Charophyta-Zygnematales Charophyta-Desmidiales Charophyta-Zygnematales Charophyta-Desmidiales Charophyta-Zygnematales Charophyta-Desmidiales Charophyta-Coleochaetales Charophyta-Coleochaetales 100/100 Charophyta-Coleochaetales 97.8/100 Charophyta-Charales 100/100 Charophyta-Charales Charophyta-Charales Charophyta-Mesostigmatales Charophyta-Chlorokybales Charophyta-Chlorokybales Charophyta-Chlorokybales Charophyta-Mesostigmatales Charophyta-Mesostigmatales Chlorophyta-Pedinomonadales Chlorophyta-Chlamydomonadales Chlorophyta-Marsupiomonadales Chlorophyta-Marsupiomonadales Chlorophyta-Sphaeropleales Chlorophyta-Pedinomonadales 100/100 Chlorophyta-Ulvales Chlorophyta-Chaetophorales Chlorophyta-Ulvales 100/96 Chlorophyta-Chlorodendrales Chlorophyta-Chaetopeltidales Chlorophyta-Chlorodendrales Chlorophyta-Bryopsidales Chlorophyta-Ulvales Chlorophyta-Bryopsidales Chlorophyta-Sphaeropleales Chlorophyta-Bryopsidales 100/100 Chlorophyta-Sphaeropleales Chlorophyta-Chlamydomonadales Chlorophyta-Chlorodendrales Chlorophyta-Chlamydomonadales 100/100 100/100 Chlorophyta-Chaetophorales Chlorophyta-Pedinomonadales 18/69 Chlorophyta-Chaetopeltidales Chlorophyta-Chaetopeltidales Chlorophyta-Marsupiomonadales Chlorophyta-Chaetophorales Chlorophyta-Monomastigales Chlorophyta-Monomastigales Chlorophyta-Monomastigales Chlorophyta-Mamiellales Chlorophyta-Mamiellales Chlorophyta-Mamiellales 100/100 99.9/100 Chlorophyta-Pyramimonadales Chlorophyta-Pyramimonadales Chlorophyta-Pyramimonadales Chlorophyta-Prasinococcales Chlorophyta-Palmophyllales Chlorophyta-Prasinococcales 100/100 100/100 100/100 Chlorophyta-Palmophyllales Chlorophyta-Prasinococcales Chlorophyta-Palmophyllales Rhodophyta Rhodophyta Rhodophyta

0.2 0.2 0.2 bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

dN dS dN/dS

Eudicots-Asterales-Asteraceae_NC374 Eudicots-Asterales-Campanulaceae_NC764* Eudicots-Lamiales-Lamiaceae_NC1381 Eudicots-Caryophyllales-Droseraceae_NC1044* Eudicots-Caryophyllales-Polygonaceae_NC2487 Eudicots-Santalales-Santalaceae_NC2680* Eudicots-Santalales-Olacaceae_NC1721 Eudicots-Fabales-Fabaceae_NC1179 Eudicots-Brassicales-Brassicaceae_NC641 Ceratophyllales-Ceratophyllales-Ceratophyllaceae_NC869 Magnoliids-Laurales-Lauraceae_NC1422 Magnoliids-Laurales-Lauraceae_NC1420* Magnoliids-Magnoliales-Magnoliaceae_NC1541 Chloranthales-Chloranthales-Chloranthaceae_NC870 Monocots-Asparagales-Orchidaceae_NC1856* Monocots-Asparagales-Orchidaceae_NC1788 Monocots-Poales-Poaceae_NC2327 Monocots-Poales-Poaceae_NC2473 ANA_Grade-Nymphaeales-Nymphaeaceae_NC1712 ANA_Grade-Amborellales-Amborellaceae_NC51 Gymnosperm-Pinales-Pinaceae_NC2023* Gymnosperm-Gnetales-Gnetaceae_NC1340* Gymnosperm-Cycadales-Cycadaceae_NC1016 Gymnosperm-Ginkgoales-Ginkgoaceae_NC1337 ferns_and_horsetails-Polypodiales-Athyriaceae_NC517 ferns_and_horsetails-Schizaeales-Schizaeaceae_NC2713* Lycophytes-Lycopodiales-Lycopodiaceae_NC1518 mosses-Sphagnales-Sphagnaceae_NC2781 mosses-Takakiales-Takakiaceae_NC2792 liverworts-Marchantiales-Marchantiaceae_NC1607 hornworts-Anthocerotales-Anthocerotaceae_NC63 Charophyta-Zygnematales-Zygnemataceae_NC3060 Charophyta-Mesostigmatales-Mesostigmataceae_NC2978 Chlorophyta-Prasinococcales-NA_NC2989* Chlorophyta-Prasinococcales-NA_NC2988 Chlorophyta-Palmophyllales-NA_NC2986 Chlorophyta-Mamiellales-Bathycoccaceae_NC2913*

0.1 1.0 0.5 bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

ycf4 ycf3 ycf1 rps8 rps7 1.0 rps4 rps3 rps2 rps19 rps18 rps16 rps15 rps14 0.8 rps12 rps11 rpoA rpl36 rpl33 rpl32 rpl23 rpl22 0.6 rpl20 rpl2 rpl16 rpl14 rbcL psbZ psbT psbN 0.4 psbM psbL psbK psbJ psbI psbH psbF psbE psbD 0.2 psbC psbB psbA psaJ psaI psaC petN petL 0.0 petG petD petB petA ndhK ndhJ ndhI ndhH −0.2 ndhG ndhE ndhD ndhC ndhB ndhA matK infA −0.4 clpP cemA ccsA atpI atpH atpF atpE atpB −0.6 atpA

accD

psbM rpl14

accD rpl16

rpl20

rpl22 rbcL

rpl23

rpl32

ndhC rpl33

psbN ndhD rpl36

ndhH

atpA

psbT atpB rpoA

psbZ atpE

rps11

rps12

rps14

ndhJ

rps15

rps16

rps18

rps19

petG

rpl2

ndhG

matK

ndhA ndhI

ndhB

ndhE

petL

psbL atpH

ndhK

atpF

psaC

psbC

psbD

psbH

petD

rps2

rps3

rps4 petN

rps7

rps8 psbF

psaJ

atpI

ccsA

psbJ

psaI

cemA

psbA psbI clpP

psbB

ycf1 psbE

ycf3 petA

ycf4 petB

psbK infA 5e+05

4e+05 200

3e+05 Genome size

2e+05 100 Number of protein-coding genes bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1e+05

0.4

1.6

0.3

1.4

0.2 Copy number Copy Intron number Intron

1.2 0.1

1.0 0.0

mosses mosses Eudicots liverworts Monocots Eudicots hornworts liverworts Magnoliids Monocots hornworts Magnoliids ChlorophytaCharophyta Lycophytes ANA_Grade ChlorophytaCharophyta Lycophytes ANA_Grade Gymnosperm Chloranthales Gymnosperm Chloranthales Ceratophyllales Ceratophyllales

ferns_and_horsetails ferns_and_horsetails bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Eudicots-Fagales-1kp Eudicots-Fagales Eudicots-Cucurbitales-1kp Eudicots-Cucurbitales Eudicots-Rosales-1kp Eudicots-Rosales Eudicots-Fabales Eudicots-Fabales-1kp Eudicots-Malpighiales-1kp Eudicots-Malpighiales Eudicots-Celastrales-1kp Eudicots-Celastrales Eudicots-Oxalidales Eudicots-Oxalidales-1kp Eudicots-Zygophyllales-1kp Eudicots-Brassicales-1kp Eudicots-Brassicales Eudicots-Malvales-1kp Eudicots-Malvales Eudicots-Huerteales Eudicots-Huerteales-1kp Eudicots-Sapindales Eudicots-Sapindales-1kp Eudicots-Crossosomatales Eudicots-Crossosomatales-1kp Eudicots-Myrtales Eudicots-Myrtales-1kp Eudicots-Geraniales-1kp Eudicots-Geraniales Eudicots-Vitales-1kp Eudicots-Vitales Eudicots-Saxifragales-1kp Eudicots-Saxifragales Eudicots-Gentianales-1kp Eudicots-Gentianales Eudicots-Boraginales-1kp Eudicots-Boraginales Eudicots-Solanales Eudicots-Solanales-1kp Eudicots-Lamiales Eudicots-Lamiales-1kp Eudicots-Garryales Eudicots-Garryales-1kp Eudicots-Icacinales Eudicots-Icacinales-1kp Eudicots-Asterales-1kp Eudicots-Asterales Eudicots-Dipsacales-1kp Eudicots-Escalloniales-1kp Eudicots-Apiales-1kp Eudicots-Apiales Eudicots-Dipsacales Eudicots-Aquifoliales-1kp Eudicots-Aquifoliales Eudicots-Ericales-1kp Eudicots-Ericales Eudicots-Cornales Eudicots-Cornales-1kp Eudicots-Caryophyllales Eudicots-Berberidopsidales-1kp Eudicots-Santalales-1kp Eudicots-Santalales Eudicots-Dilleniales-1kp Eudicots-Gunnerales-1kp Eudicots-Trochodendrales-1kp Eudicots-Trochodendrales Eudicots-Buxales Eudicots-Proteales-1kp Eudicots-Proteales Eudicots-Ranunculales Eudicots-Ranunculales-1kp Ceratophyllales-Ceratophyllales Ceratophyllales-Ceratophyllales-1kp Monocots-Zingiberales-1kp Monocots-Zingiberales Monocots-Commelinales-1kp Monocots-Commelinales Monocots-Poales Monocots-Poales-1kp Monocots-Arecales Monocots-Arecales-1kp Monocots-Asparagales-1kp Monocots-Asparagales Monocots-Dioscoreales-1kp Monocots-Dioscoreales Monocots-Pandanales-1kp Monocots-Pandanales Monocots-Liliales Monocots-Liliales-1kp Monocots-Petrosaviales Monocots-Petrosaviales-1kp Monocots-Alismatales-1kp Monocots-Alismatales Monocots-Acorales-1kp Monocots-Acorales Magnoliids-Laurales Magnoliids-Laurales-1kp Magnoliids-Magnoliales-1kp Magnoliids-Magnoliales Magnoliids-Piperales-1kp Magnoliids-Piperales Magnoliids-Canellales Chloranthales-Chloranthales Chloranthales-Chloranthales-1kp ANA_Grade-Austrobaileyales-1kp ANA_Grade-Austrobaileyales ANA_Grade-Nymphaeales-1kp bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not ANA_Grade-Nymphaeales ANA_Grade-Amborellales-1kp certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ANA_Grade-Amborellales Gymnosperm-Gnetales-1kp Gymnosperm-Gnetales Gymnosperm-Welwitschiales Gymnosperm-Welwitschiales-1kp Gymnosperm-Ephedrales Gymnosperm-Ephedrales-1kp Gymnosperm-Araucariales-1kp Gymnosperm-Araucariales Gymnosperm-Cupressales Gymnosperm-Cupressales-1kp Gymnosperm-Pinales Gymnosperm-Pinales-1kp Gymnosperm-Cycadales Gymnosperm-Cycadales-1kp Gymnosperm-Ginkgoales Gymnosperm-Ginkgoales-1kp mosses-Hypnales-1kp mosses-Hypnales mosses-Leucodontales-1kp mosses-Isobryales-1kp mosses-Bryales-1kp mosses-Orthotrichales mosses-Orthotrichales-1kp mosses-Grimmiales-1kp mosses-Dicranales-1kp mosses-Pottiales-1kp mosses-Pottiales mosses-Timmiales-1kp mosses-Funariales mosses-Funariales-1kp mosses-Diphysciales-1kp mosses-Buxbaumiales-1kp mosses-Tetraphidales-1kp mosses-Tetraphidales mosses-Polytrichales-1kp mosses-Andreaeales-1kp mosses-Takakiales-1kp mosses-Takakiales mosses-Sphagnales mosses-Sphagnales-1kp liverworts-Ptilidiales liverworts-Ptilidiales-1kp liverworts-Porellales-1kp liverworts-Jungermanniales-1kp liverworts-Metzgeriales-1kp liverworts-Metzgeriales liverworts-Pelliales liverworts-Pelliales-1kp liverworts-Pallaviciniales-1kp liverworts-Blasiales-1kp liverworts-Marchantiales liverworts-Marchantiales-1kp liverworts-Lunulariales-1kp liverworts-Sphaerocarpales-1kp hornworts-Dendrocerotales-1kp hornworts-Dendrocerotales hornworts-Notothyladales-1kp hornworts-Anthocerotales-1kp hornworts-Anthocerotales hornworts-Leiosporocerotales-1kp Lycophytes-Isoetales Lycophytes-Isoetales-1kp Lycophytes-Selaginellales Lycophytes-Selaginellales-1kp Lycophytes-Lycopodiales-1kp Lycophytes-Lycopodiales ferns_and_horsetails-Polypodiales-1kp ferns_and_horsetails-Polypodiales ferns_and_horsetails-Cyatheales-1kp ferns_and_horsetails-Cyatheales ferns_and_horsetails-Salviniales ferns_and_horsetails-Salviniales-1kp ferns_and_horsetails-Schizaeales ferns_and_horsetails-Schizaeales-1kp ferns_and_horsetails-Hymenophyllales-1kp ferns_and_horsetails-Gleicheniales-1kp ferns_and_horsetails-Gleicheniales ferns_and_horsetails-Osmundales ferns_and_horsetails-Osmundales-1kp ferns_and_horsetails-Marattiales ferns_and_horsetails-Marattiales-1kp ferns_and_horsetails-Ophioglossales ferns_and_horsetails-Ophioglossales-1kp ferns_and_horsetails-Psilotales ferns_and_horsetails-Psilotales-1kp ferns_and_horsetails-Equisetales ferns_and_horsetails-Equisetales-1kp Charophyta-Desmidiales-1kp Charophyta-Desmidiales Charophyta-Zygnematales Charophyta-Coleochaetales Charophyta-Coleochaetales-1kp Charophyta-Charales-1kp Charophyta-Charales Charophyta-Chlorokybales Charophyta-Chlorokybales-1kp Charophyta-Mesostigmatales Charophyta-Mesostigmatales-1kp Chlorophyta-Cladophorales-1kp Chlorophyta-Ulvales-1kp Chlorophyta-Chlorosarcinales-1kp Chlorophyta-Ulvales Chlorophyta-Ulotrichales-1kp Chlorophyta-Bryopsidales Chlorophyta-Bryopsidales-1kp Chlorophyta-Pedinomonadales Chlorophyta-Pedinomonadales-1kp Chlorophyta-Marsupiomonadales Chlorophyta-Chlorodendrales Chlorophyta-Chlorodendrales-1kp Chlorophyta-Chlorococcales-1kp Chlorophyta-Prasiolales-1kp Chlorophyta-Chlorellales-1kp Chlorophyta-Sphaeropleales-1kp Chlorophyta-Sphaeropleales Chlorophyta-Chlamydomonadales Chlorophyta-Microsporales-1kp Chlorophyta-Volvocales-1kp Chlorophyta-Chaetophorales Chlorophyta-Chaetophorales-1kp Chlorophyta-Chaetopeltidales-1kp Chlorophyta-Chaetopeltidales Chlorophyta-Oedogoniales-1kp Chlorophyta-Mamiellales-1kp Chlorophyta-Mamiellales Chlorophyta-Dolichomastigales-1kp Chlorophyta-Monomastigales Chlorophyta-Monomastigales-1kp Chlorophyta-Pyramimonadales-1kp Chlorophyta-Pyramimonadales Chlorophyta-Scourfieldiales-1kp Chlorophyta-Nephroselmidales-1kp Chlorophyta-Prasinococcales Chlorophyta-Prasinococcales-1kp Chlorophyta-Palmophyllales Chlorophyta-Trentepohliales-1kp Rhodophyta-Gigartinales-1kp Rhodophyta-Halymeniales-1kp Rhodophyta-Ceramiales-1kp Rhodophyta-Bangiales-1kp Rhodophyta-Porphyridiales-1kp Rhodophyta-Stylonematales-1kp Cryptophyta-Pyrenomonadales-1kp Cryptophyta-Cryptomonadales-1kp Heterokonta-Ectocarpales-1kp Ochrophyta-Chromulinales-1kp

0.4 bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

atpA atpB atpE atpI petA psaC psbC psbE psbF psbJ psbK psbN psbT rbcL 1.0

Eudicots-Asterales-Asteraceae_NC374 Eudicots-Asterales-Campanulaceae_NC764* Eudicots-Lamiales-Lamiaceae_NC1381 Eudicots-Caryophyllales-Droseraceae_NC1044* Eudicots-Caryophyllales-Polygonaceae_NC2487 Eudicots-Santalales-Santalaceae_NC2680* Eudicots-Santalales-Olacaceae_NC1721 Eudicots-Fabales-Fabaceae_NC1179 Eudicots-Brassicales-Brassicaceae_NC641 Ceratophyllales-Ceratophyllales-Ceratophyllaceae_NC869 Magnoliids-Laurales-Lauraceae_NC1422 Magnoliids-Laurales-Lauraceae_NC1420* Magnoliids-Magnoliales-Magnoliaceae_NC1541 Chloranthales-Chloranthales-Chloranthaceae_NC870 Monocots-Asparagales-Orchidaceae_NC1856* Monocots-Asparagales-Orchidaceae_NC1788 Monocots-Poales-Poaceae_NC2327 Monocots-Poales-Poaceae_NC2473 ANA_Grade-Nymphaeales-Nymphaeaceae_NC1712 ANA_Grade-Amborellales-Amborellaceae_NC51 Gymnosperm-Pinales-Pinaceae_NC2023* Gymnosperm-Gnetales-Gnetaceae_NC1340* Gymnosperm-Cycadales-Cycadaceae_NC1016 Gymnosperm-Ginkgoales-Ginkgoaceae_NC1337 ferns_and_horsetails-Polypodiales-Athyriaceae_NC517 ferns_and_horsetails-Schizaeales-Schizaeaceae_NC2713* Lycophytes-Lycopodiales-Lycopodiaceae_NC1518 mosses-Sphagnales-Sphagnaceae_NC2781 mosses-Takakiales-Takakiaceae_NC2792 liverworts-Marchantiales-Marchantiaceae_NC1607 hornworts-Anthocerotales-Anthocerotaceae_NC63 Charophyta-Zygnematales-Zygnemataceae_NC3060 Charophyta-Mesostigmatales-Mesostigmataceae_NC2978 Chlorophyta-Prasinococcales-NA_NC2989* Chlorophyta-Prasinococcales-NA_NC2988 Chlorophyta-Palmophyllales-NA_NC2986 Chlorophyta-Mamiellales-Bathycoccaceae_NC2913* atpA atpB atpE atpI petA psaC psbC 10.0

Eudicots-Asterales-Asteraceae_NC374 Eudicots-Asterales-Campanulaceae_NC764* Eudicots-Lamiales-Lamiaceae_NC1381 Eudicots-Caryophyllales-Droseraceae_NC1044* Eudicots-Caryophyllales-Polygonaceae_NC2487 Eudicots-Santalales-Santalaceae_NC2680* Eudicots-Santalales-Olacaceae_NC1721 Eudicots-Fabales-Fabaceae_NC1179 Eudicots-Brassicales-Brassicaceae_NC641 Ceratophyllales-Ceratophyllales-Ceratophyllaceae_NC869 Magnoliids-Laurales-Lauraceae_NC1422 Magnoliids-Laurales-Lauraceae_NC1420* bioRxiv preprint doi: https://doi.org/10.1101/655241; this version posted June 3, 2019. The copyright holder for this preprint (which was not Magnoliids-Magnoliales-Magnoliaceae_NC1541 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Chloranthales-Chloranthales-Chloranthaceae_NC870 Monocots-Asparagales-Orchidaceae_NC1856* Monocots-Asparagales-Orchidaceae_NC1788 Monocots-Poales-Poaceae_NC2327 Monocots-Poales-Poaceae_NC2473 ANA_Grade-Nymphaeales-Nymphaeaceae_NC1712 ANA_Grade-Amborellales-Amborellaceae_NC51 Gymnosperm-Pinales-Pinaceae_NC2023* Gymnosperm-Gnetales-Gnetaceae_NC1340* Gymnosperm-Cycadales-Cycadaceae_NC1016 Gymnosperm-Ginkgoales-Ginkgoaceae_NC1337 ferns_and_horsetails-Polypodiales-Athyriaceae_NC517 ferns_and_horsetails-Schizaeales-Schizaeaceae_NC2713* Lycophytes-Lycopodiales-Lycopodiaceae_NC1518 mosses-Sphagnales-Sphagnaceae_NC2781 mosses-Takakiales-Takakiaceae_NC2792 liverworts-Marchantiales-Marchantiaceae_NC1607 hornworts-Anthocerotales-Anthocerotaceae_NC63 Charophyta-Zygnematales-Zygnemataceae_NC3060 Charophyta-Mesostigmatales-Mesostigmataceae_NC2978 Chlorophyta-Prasinococcales-NA_NC2989* Chlorophyta-Prasinococcales-NA_NC2988 Chlorophyta-Palmophyllales-NA_NC2986 Chlorophyta-Mamiellales-Bathycoccaceae_NC2913*

psbE psbF psbJ psbK psbN psbT rbcL

Eudicots-Asterales-Asteraceae_NC374 Eudicots-Asterales-Campanulaceae_NC764* Eudicots-Lamiales-Lamiaceae_NC1381 Eudicots-Caryophyllales-Droseraceae_NC1044* Eudicots-Caryophyllales-Polygonaceae_NC2487 Eudicots-Santalales-Santalaceae_NC2680* Eudicots-Santalales-Olacaceae_NC1721 Eudicots-Fabales-Fabaceae_NC1179 Eudicots-Brassicales-Brassicaceae_NC641 Ceratophyllales-Ceratophyllales-Ceratophyllaceae_NC869 Magnoliids-Laurales-Lauraceae_NC1422 Magnoliids-Laurales-Lauraceae_NC1420* Magnoliids-Magnoliales-Magnoliaceae_NC1541 Chloranthales-Chloranthales-Chloranthaceae_NC870 Monocots-Asparagales-Orchidaceae_NC1856* Monocots-Asparagales-Orchidaceae_NC1788 Monocots-Poales-Poaceae_NC2327 Monocots-Poales-Poaceae_NC2473 ANA_Grade-Nymphaeales-Nymphaeaceae_NC1712 ANA_Grade-Amborellales-Amborellaceae_NC51 Gymnosperm-Pinales-Pinaceae_NC2023* Gymnosperm-Gnetales-Gnetaceae_NC1340* Gymnosperm-Cycadales-Cycadaceae_NC1016 Gymnosperm-Ginkgoales-Ginkgoaceae_NC1337 ferns_and_horsetails-Polypodiales-Athyriaceae_NC517 ferns_and_horsetails-Schizaeales-Schizaeaceae_NC2713* Lycophytes-Lycopodiales-Lycopodiaceae_NC1518 mosses-Sphagnales-Sphagnaceae_NC2781 mosses-Takakiales-Takakiaceae_NC2792 liverworts-Marchantiales-Marchantiaceae_NC1607 hornworts-Anthocerotales-Anthocerotaceae_NC63 Charophyta-Zygnematales-Zygnemataceae_NC3060 Charophyta-Mesostigmatales-Mesostigmataceae_NC2978 Chlorophyta-Prasinococcales-NA_NC2989* Chlorophyta-Prasinococcales-NA_NC2988 Chlorophyta-Palmophyllales-NA_NC2986 Chlorophyta-Mamiellales-Bathycoccaceae_NC2913*