Both Conifer II and Gnetales are characterized by a high frequency of ancient mitochondrial gene transfer to the nuclear genome.

BACKGROUND: Mitochondrial gene transfer/loss is common in land plants, and therefore the fate of missing mitochondrial genes has attracted more and more attention. The gene content of gymnosperm mitochondria varies greatly, supplying a system for studying the evolutionary fate of missing mitochondrial genes. RESULTS: Here, we studied the tempo and pattern of mitochondrial gene transfer/loss in gymnosperms represented by all 13 families, using high-throughput sequencing of both DNA and cDNA. All 41 mitochondrial protein-coding genes were found in cycads, Ginkgo and Pinaceae, whereas multiple mitochondrial genes were absent in Conifer II and Gnetales. In Conifer II, gene transfer from mitochondria to the nucleus followed by loss of the mitochondrial copy was common, but complete loss of a gene in both mitochondrial and nuclear genomes was rare. In contrast, both gene transfer and loss were commonly found in Gnetales. Notably, in Conifer II and Gnetales, the same five mitochondrial genes were transferred to the nuclear genome, and these gene transfer events occurred, respectively, in ancestors of the two lineages. A two-step transfer mechanism (retroprocessing and subsequent DNA-mediated gene transfer) may be responsible for mitochondrial gene transfer in Conifer II and Gnetales. Moreover, the mitochondrial gene content variation is correlated with gene length, GC content, hydrophobicity, and nucleotide substitution rates in land plants. CONCLUSIONS: This study reveals a complete evolutionary scenario for variations of mitochondrial gene transferring in gymnosperms, and the factors responsible for mitochondrial gene content variation in land plants.

A Comprehensive Evolutionary Study of Chloroplast RNA Editing in Gymnosperms: A Novel Type of G-to-A RNA Editing Is Common in Gymnosperms.

Although more than 9100 plant plastomes have been sequenced, RNA editing sites of the whole plastome have been experimentally verified in only approximately 21 species, which seriously hampers the comprehensive evolutionary study of chloroplast RNA editing. We investigated the evolutionary pattern of chloroplast RNA editing sites in 19 species from all 13 families of gymnosperms based on a combination of genomic and transcriptomic data. We found that the chloroplast C-to-U RNA editing sites of gymnosperms shared many common characteristics with those of other land plants, but also exhibited many unique characteristics. In contrast to that noted in angiosperms, the density of RNA editing sites in ndh genes was not the highest in the sampled gymnosperms, and both loss and gain events at editing sites occurred frequently during the evolution of gymnosperms. In addition, GC content and plastomic size were positively correlated with the number of chloroplast RNA editing sites in gymnosperms, suggesting that the increase in GC content could provide more materials for RNA editing and facilitate the evolution of RNA editing in land plants or vice versa. Interestingly, novel G-to-A RNA editing events were commonly found in all sampled gymnosperm species, and G-to-A RNA editing exhibits many different characteristics from C-to-U RNA editing in gymnosperms. This study revealed a comprehensive evolutionary scenario for chloroplast RNA editing sites in gymnosperms, and reported that a novel type of G-to-A RNA editing is prevalent in gymnosperms.

Phylotranscriptomics reveals the complex evolutionary and biogeographic history of the genus Tsuga with an East Asian-North American disjunct distribution.

The disjunct distribution between East Asia and North America is one of the best established biogeographic patterns. A robust phylogeny is fundamental for understanding the biogeographic histories of taxa with this distribution pattern. Tsuga (hemlock) is a genus of Pinaceae with a typical intercontinental disjunct distribution in East Asia and eastern and western North America, and its phylogeny has not been completely reconstructed in previous studies. In this study, we reconstructed a highly resolved phylogeny of Tsuga using 881 nuclear genes, 60 chloroplast genes and 23 mitochondrial genes and explored its biogeographic and reticulate evolutionary history. The results of phylogenetic analysis, molecular dating and ancestral area reconstruction indicate that Tsuga very likely originated from North America in the late Oligocene and dispersed from America to East Asia via the Bering Land Bridge during the middle Miocene. In particular, we found complex reticulate evolutionary pattern among the East Asian hemlock species. T. sieboldii possibly originated from hybridization with the ancestor of T. chinensis from mainland China and T. forrestii as the paternal donor and the ancestor of T. diversifolia and T. ulleungensis as the maternal donor. T. chinensis (Taiwan) could have originated by hybridization together with T. sieboldii and then evolved independently after dispersal to the Taiwan Island, subsequently experiencing mitochondrial DNA introgression with T. chinensis from mainland China. Moreover, our study found that T. chinensis from western China is more closely related to T. forrestii than to T. chinensis from eastern China. The nonmonophyletic T. chinensis needs taxonomic reconsideration.

The flattened and needlelike leaves of the pine family (Pinaceae) share a conserved genetic network for adaxial-abaxial polarity but have diverged for photosynthetic adaptation.

BACKGROUND: Leaves have highly diverse morphologies. However, with an evolutionary history of approximately 200 million years, leaves of the pine family are relatively monotonous and often collectively called "needles", although they vary in length, width and cross-section shapes. It would be of great interest to determine whether Pinaceae leaves share similar morpho-physiological features and even consistent developmental and adaptive mechanisms. RESULTS: Based on a detailed morpho-anatomical study of leaves from all 11 Pinaceae genera, we particularly investigated the expression patterns of adaxial-abaxial polarity genes in two types of leaves (needlelike and flattened) and compared their photosynthetic capacities. We found that the two types of leaves share conserved spatial patterning of vasculatures and genetic networks for adaxial-abaxial polarity, although they display different anatomical structures in the mesophyll tissue differentiation and distribution direction. In addition, the species with needlelike leaves exhibited better photosynthetic capacity than the species with flattened leaves. CONCLUSIONS: Our study provides the first evidence for the existence of a conserved genetic module controlling adaxial-abaxial polarity in the development of different Pinaceae leaves.

The complete mitochondrial genome of Taxus cuspidata (Taxaceae): eight protein-coding genes have transferred to the nuclear genome.

BACKGROUND: Gymnosperms represent five of the six lineages of seed plants. However, most sequenced plant mitochondrial genomes (mitogenomes) have been generated for angiosperms, whereas mitogenomic sequences have been generated for only six gymnosperms. In particular, complete mitogenomes are available for all major seed plant lineages except Conifer II (non-Pinaceae conifers or Cupressophyta), an important lineage including six families, which impedes a comprehensive understanding of the mitogenomic diversity and evolution in gymnosperms. RESULTS: Here, we report the complete mitogenome of Taxus cuspidata in Conifer II. In comparison with previously released gymnosperm mitogenomes, we found that the mitogenomes of Taxus and Welwitschia have lost many genes individually, whereas all genes were identified in the mitogenomes of Cycas, Ginkgo and Pinaceae. Multiple tRNA genes and introns also have been lost in some lineages of gymnosperms, similar to the pattern observed in angiosperms. In general, gene clusters could be less conserved in gymnosperms than in angiosperms. Moreover, fewer RNA editing sites were identified in the Taxus and Welwitschia mitogenomes than in other mitogenomes, which could be correlated with fewer introns and frequent gene losses in these two species. CONCLUSIONS: We have sequenced the Taxus cuspidata mitogenome, and compared it with mitogenomes from the other four gymnosperm lineages. The results revealed the diversity in size, structure, gene and intron contents, foreign sequences, and mutation rates of gymnosperm mitogenomes, which are different from angiosperm mitogenomes.

Phylogenomics disentangles the evolutionary history of spruces (Picea) in the Qinghai-Tibetan Plateau: Implications for the design of population genetic studies and species delimitation of conifers.

A laborious and difficult task in current tree of life reconstruction is to resolve evolutionary relationships of closely related congeneric species that originated from recent radiations. This is particularly difficult for forest species with long generation times and large effective population sizes such as conifers. The Qinghai-Tibetan Plateau (QTP) and adjacent areas are considered a species diversity center of Picea, harboring 11 species (including 5 varieties) of this genus, but evolutionary relationships of these species are far from being resolved due to recent radiations, morphological convergence, and frequent interspecific gene flow. In this study, we use these spruce species to test whether phylotranscriptomic analysis, combined with population genetic analysis, can disentangle their evolutionary relationships, and to explore whether reticulate evolution has occurred among them. Phylogenomic analyses indicate that all spruce species in the QTP and neighboring areas, except P. asperata and P. crassifolia, cluster together, and in particular, nearly all taxa (including varieties) reflect reciprocally monophyletic lineages, although the two species P. likiangensis and P. brachytyla are not monophyletic. We found that, compared to herbaceous plants, many more genes (a minimum of 600 OGs for Picea) are required to resolve interspecific relationships of conifers. Contrary to previous studies, our data do not support a hybrid origin of P. purpurea, but suggests a hybrid origin for P. brachytyla var. brachytyla and P. likiangensis var. rubescens. We emphasize that the species or species complex used for population genetic and phylogeographical studies should be monophyletic.

Phylotranscriptomics resolves interspecific relationships and indicates multiple historical out-of-North America dispersals through the Bering Land Bridge for the genus Picea (Pinaceae).

A robust phylogeny is prerequisite to understand the evolution and biogeography of organisms. However, ancient and recent evolutionary radiations occurred in many plant lineages, which pose great challenges for phylogenetic analysis, especially for conifers characterized by large effective population sizes and long generation times. Picea is an important component of the dark coniferous forests in the Northern Hemisphere. Previous studies improved our understanding of its evolutionary history, but its interspecific relationships and biogeographic history remain largely unresolved. In the present study, we reconstructed a well-resolved phylogeny of Picea by comparative transcriptomic analysis based on a complete species sampling. The phylogenetic analysis, together with molecular dating and ancestral area reconstruction, further supports the North American origin hypothesis for Picea, and indicates that this genus experienced multiple out-of-North America dispersals by the Bering Land Bridge. We also found that spruces in the Japanese Archipelago have multiple origins, and P. morrisonicola from the Taiwan Island has a close relationship with species from the Qinghai-Tibetan Plateau and adjacent regions. Our study provides the first complete phylogeny of Picea at the genomic level, which is important for future studies of this genus.

Phylogenomics resolves the deep phylogeny of seed plants and indicates partial convergent or homoplastic evolution between Gnetales and angiosperms.

After decades of molecular phylogenetic studies, the deep phylogeny of gymnosperms has not been resolved, and the phylogenetic placement of Gnetales remains one of the most controversial issues in seed plant evolution. To resolve the deep phylogeny of seed plants and to address the sources of phylogenetic conflict, we conducted a phylotranscriptomic study with a sampling of all 13 families of gymnosperms and main lineages of angiosperms. Multiple datasets containing up to 1 296 042 sites across 1308 loci were analysed, using concatenation and coalescence approaches. Our study generated a consistent and well-resolved phylogeny of seed plants, which places Gnetales as sister to Pinaceae and thus supports the Gnepine hypothesis. Cycads plus Ginkgo is sister to the remaining gymnosperms. We also found that Gnetales and angiosperms have similar molecular evolutionary rates, which are much higher than those of other gymnosperms. This implies that Gnetales and angiosperms might have experienced similar selective pressures in evolutionary histories. Convergent molecular evolution or homoplasy is partially responsible for the phylogenetic conflicts in seed plants. Our study provides a robustly reconstructed backbone phylogeny that is important for future molecular and morphological studies of seed plants, in particular gymnosperms, in the light of evolution.

Phylogeny and evolutionary history of Pinaceae updated by transcriptomic analysis.

Pinaceae comprises 11 genera, and represents the largest family of conifers with an extensive wild distribution in the Northern Hemisphere. Intergeneric relationships of Pinaceae have been investigated using many morphological characters and molecular markers, but phylogenetic positions of four genera, including Cathaya, Cedrus, Nothotsuga and Pseudolarix, remain controversial or have not been completely resolved. To completely resolve the intergeneric relationships of Pinaceae, we conducted a comparative transcriptomic study of 14 species representing all Pinaceae genera. Multiple data sets, containing up to 6,369,681 sites across 4676 loci, were analyzed using concatenation and coalescent methods. Our study generated a robust topology, which divides Pinaceae into two clades, one (pinoid) including Cathaya, Larix, Picea, Pinus, and Pseudotsuga, and the other (abietoid) including Abies, Cedrus, Keteleeria, Nothotsuga, Pseudolarix, and Tsuga. Cathaya and Pinus form a clade sister to Picea; Cedrus is sister to the remaining abietoid genera, and the two genera Nothotsuga and Tsuga form a clade sister to Pseudolarix. The discordant positions of Cathaya, Cedrus and Pseudolarix in different gene trees could be explained by ancient radiation and/or molecular homoplastic evolution. The hybrid origin hypothesis of Nothotsuga is not supported. Based on molecular dating, extant Pinaceae genera diverged since about 206 Mya, earlier than the break-up of Pangea, and the divergence among the pinoid genera occurred earlier than the split among the abietoid genera. Moreover, our study indicates that two radiation events occurred in the evolution of Pinaceae genera, and some important morphological characters evolved multiple times based on ancestral state reconstruction.

A high frequency of allopolyploid speciation in the gymnospermous genus Ephedra and its possible association with some biological and ecological features.

The origin and evolution of polyploids have been studied extensively in angiosperms and ferns but very rarely in gymnosperms. With the exception of three species of conifers, all natural polyploid species of gymnosperms belong to Ephedra, in which more than half of the species show polyploid cytotypes. Here, we investigated the origin and evolution of polyploids of Ephedra distributed in the Qinghai-Tibetan Plateau (QTP) and neighbouring areas. Flow cytometry (FCM) was used to measure the ploidy levels of the sampled species that are represented by multiple individuals from different populations, and then, two single-copy nuclear genes (LFY and DDB2) and two chloroplast DNA fragments were used to unravel the possible origins and maternal donors of the polyploids. The results indicate that the studied polyploid species are allopolyploids, and suggest that allotetraploidy is a dominant mode of speciation in Ephedra. The high percentage of polyploids in the genus could be related to some of its biological attributes such as vegetative propagation, a relatively high rate of unreduced gamete formation, and a small genome size relative to most other gymnosperms. Significant ecological divergences between allotetraploids and their putative progenitors were detected by PCAs and anova and Tukey's tests, with the exception of E. saxatilis. The overlap of geographical distributions and ecological niches of some diploid species could have provided opportunities for interspecific hybridization and allopolyploid speciation.

Mitochondrial introgression and complex biogeographic history of the genus Picea.

Biogeographic history of plants is much more complex in the Northern Hemisphere than in the Southern Hemisphere due to that both the Bering and the North Atlantic land bridges contributed to floristic exchanges in the Cenozoic, which led to hybridization between congeneric species from different continents. It would be interesting to know how intercontinental gene flow and introgression have affected plant phylogenetic reconstruction and biogeographic inference. In this study, we reinvestigated the phylogenetic and biogeographic history of Picea, a main component of the Northern Hemisphere forest with many species that originated from recent radiation, using two chloroplast (cp), one mitochondrial (mt) and three single-copy nuclear gene markers. The generated gene trees are topologically highly discordant and the geographically closely related species generally show a close affinity of mtDNA rather than cp- or nuclear DNA, suggesting that inter- and intra-continental gene flow and mtDNA introgression might have occurred commonly. However, all gene trees resolved Picea breweriana as the basal-most lineage, which, together with fossil evidence, supports the North American origin hypothesis for the genus. Both dispersal and vicariance have played important roles in the evolution of Picea, and the Bering Land Bridge could have mediated the "North America to Eurasia" dispersal at least two times during the Miocene and Pliocene. Our study again demonstrates the importance of applying data from three genomes for a clear understanding of evolutionary histories in the pine family. Any markers from a single genome alone will not reveal a clear picture of the phylogenetic relationships among closely related congeneric species. In particular, mtDNA markers should be cautiously used, considering that introgression of the maternally inherited mtDNA with a lower rate of gene flow (by seeds) could have occurred much more frequently than that of the paternally inherited cpDNA with a higher rate of gene flow (by pollen) in Pinaceae.

Evolution and biogeography of gymnosperms.

Living gymnosperms comprise only a little more than 1000 species, but represent four of the five main lineages of seed plants, including cycads, ginkgos, gnetophytes and conifers. This group has huge ecological and economic value, and has drawn great interest from the scientific community. Here we review recent advances in our understanding of gymnosperm evolution and biogeography, including phylogenetic relationships at different taxonomic levels, patterns of species diversification, roles of vicariance and dispersal in development of intercontinental disjunctions, modes of molecular evolution in different genomes and lineages, and mechanisms underlying the formation of large nuclear genomes. It is particularly interesting that increasing evidence supports a sister relationship between Gnetales and Pinaceae (the Gnepine hypothesis) and the contribution of recent radiations to present species diversity, and that expansion of retrotransposons is responsible for the large and complex nuclear genome of gymnosperms. In addition, multiple coniferous genera such as Picea very likely originated in North America and migrated into the Old World, further indicating that the center of diversity is not necessarily the place of origin. The Bering Land Bridge acted as an important pathway for dispersal of gymnosperms in the Northern Hemisphere. Moreover, the genome sequences of conifers provide an unprecedented opportunity and an important platform for the evolutionary studies of gymnosperms, and will also shed new light on evolution of many important gene families and biological pathways in seed plants.

Reciprocal expression of MADS-box genes and DNA methylation reconfiguration initiate bisexual cones in spruce.

The naturally occurring bisexual cone of gymnosperms has long been considered a possible intermediate stage in the origin of flowers, but the mechanisms governing bisexual cone formation remain largely elusive. Here, we employed transcriptomic and DNA methylomic analyses, together with hormone measurement, to investigate the molecular mechanisms underlying bisexual cone development in the conifer Picea crassifolia. Our study reveals a "bisexual" expression profile in bisexual cones, especially in expression patterns of B-class, C-class and LEAFY genes, supporting the out of male model. GGM7 could be essential for initiating bisexual cones. DNA methylation reconfiguration in bisexual cones affects the expression of key genes in cone development, including PcDAL12, PcDAL10, PcNEEDLY, and PcHDG5. Auxin likely plays an important role in the development of female structures of bisexual cones. This study unveils the potential mechanisms responsible for bisexual cone formation in conifers and may shed light on the evolution of bisexuality.

Three genome-based phylogeny of Cupressaceae s.l.: further evidence for the evolution of gymnosperms and Southern Hemisphere biogeography.

Phylogenetic information is essential to interpret the evolution of species. While DNA sequences from different genomes have been widely utilized in phylogenetic reconstruction, it is still difficult to use nuclear genes to reconstruct phylogenies of plant groups with large genomes and complex gene families, such as gymnosperms. Here, we use two single-copy nuclear genes, together with chloroplast and mitochondrial genes, to reconstruct the phylogeny of the ecologically-important conifer family Cupressaceae s.l., based on a complete sampling of its 32 genera. The different gene trees generated are highly congruent in topology, supporting the basal position of Cunninghamia and the seven-subfamily classification, and the estimated divergence times based on different datasets correspond well with each other and with the oldest fossil record. These results imply that we have obtained the species phylogeny of Cupressaceae s.l. In addition, possible origins of all three polyploid conifers were investigated, and a hybrid origin was suggested for Cupressus, Fitzroya and Sequoia. Moreover, we found that the biogeographic history of Cupressaceae s.l. is associated with the separation between Laurasia and Gondwana and the further break-up of the latter. Our study also provides new evidence for the gymnosperm phylogeny.

Unbiased Subgenome Evolution in Allotetraploid Species of Ephedra and Its Implications for the Evolution of Large Genomes in Gymnosperms.

The evolutionary dynamics of polyploid genomes and consequences of polyploidy have been studied extensively in angiosperms but very rarely in gymnosperms. The gymnospermous genus Ephedra is characterized by a high frequency of polyploidy, and thus provides an ideal system to investigate the evolutionary mode of allopolyploid genomes and test whether subgenome dominance has occurred in gymnosperms. Here, we sequenced transcriptomes of two allotetraploid species of Ephedra and their putative diploid progenitors, identified expressed homeologs, and analyzed alternative splicing and homeolog expression based on PacBio Iso-Seq and Illumina RNA-seq data. We found that the two subgenomes of the allotetraploids had similar numbers of expressed homeologs, similar percentages of homeologs with dominant expression, and approximately equal numbers of isoforms with alternative splicing, showing an unbiased subgenome evolution as in a few polyploid angiosperms, with a divergence of the two subgenomes at ∼8 Ma. In addition, the nuclear DNA content of the allotetraploid species is almost equal to the sum of two putative progenitors, suggesting limited genome restructuring after allotetraploid speciation. The allopolyploid species of Ephedra might have undergone slow diploidization, and the unbiased subgenome evolution implies that the formation of large genomes in gymnosperms could be attributed to even and slow fractionation following polyploidization.

Evolution of the Cinnamyl/Sinapyl Alcohol Dehydrogenase (CAD/SAD) gene family: the emergence of real lignin is associated with the origin of Bona Fide CAD.

Lignin plays a vital role in plant adaptation to terrestrial environments. The cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and might have contributed to the lignin diversity in plants. To investigate the evolutionary history and functional differentiation of the CAD gene family, we made a comprehensive evolutionary analysis of this gene family from 52 species, including bacteria, early eukaryotes and green plants. The phylogenetic analysis, together with gene structure and function, indicates that all members of land plants, except two of moss, could be divided into three classes. Members of Class I (bona fide CAD), generally accepted as the primary genes involved in the monolignol biosynthesis, are all from vascular plants, and form a robustly supported monophyletic group with the lycophyte CADs at the basal position. This class is also conserved in the predicted three-dimensional structure and the residues constituting the substrate-binding pocket of the proteins. Given that Selaginella has real lignin, the above evidence strongly suggests that the earliest occurrence of the bona fide CAD in the lycophyte could be directly correlated with the origin of lignin. Class II comprises members more similar to the aspen sinapyl alcohol dehydrogenase gene, and includes three groups corresponding to lycophyte, gymnosperm, and angiosperm. Class III is conserved in land plants. The three classes differ in patterns of evolution and expression, implying that functional divergence has occurred among them. Our study also supports the hypothesis of convergent evolution of lignin biosynthesis between red algae and vascular plants.

Fast evolution of the retroprocessed mitochondrial rps3 gene in Conifer II and further evidence for the phylogeny of gymnosperms.

The popular view that plant mitochondrial genome evolves slowly in sequence has been recently challenged by the extraordinarily high substitution rates of mtDNA documented mainly from several angiosperm genera, but high substitution rate acceleration accompanied with great length variation has been very rarely reported in plant mitochondrial genes. Here, we studied evolution of the mitochondrial rps3 gene that encodes the ribosomal small subunit protein 3 and found a dramatically high variation in both length and sequence of an exon region of it in Conifer II. A sequence comparison between cDNA and genomic DNA showed that there are no RNA editing sites in the Conifer II rps3 gene. Southern blotting analyses of the total DNA and mtDNA, together with the real-time PCR analysis, showed that rps3 exists as a single mitochondrial locus in gymnosperms. It is very likely that the Conifer II rps3 gene has experienced retroprocessing, i.e., the re-integration of its cDNA into the mitochondrial genome, followed by an evolutionary acceleration due to the intron loss. In addition, the phylogenetic analysis of rps3 supports the sister relationship between conifers and Gnetales. In particular, the monophyly of conifer II is strongly supported by the shared loss of two rps3 introns. Our results also indicate that the mitochondrial gene tree would be affected in topology when the "edited" paralogs are analyzed together with their genomic sequences.

A test of seven candidate barcode regions from the plastome in Picea (Pinaceae).

DNA barcoding, as a tool for species discrimination, has been used efficiently in animals, algae and fungi, but there are still debates on which DNA region(s) can be used as the standard barcode(s) for land plants. Gymnosperms, especially conifers, are important components of forests, and there is an urgent need for them to be identified through DNA barcoding because of their high frequency of collection in the field. However, the feasibility of DNA barcoding in gymnosperms has not been examined based on a dense species sampling. Here we selected seven candidate DNA barcodes from the plastome (matK, rbcL, rpoB, rpoC1, atpF-atpH, psbA-trnH, and psbK-psbI) to evaluate their suitability in Picea (spruce). The results showed that none of them or their different combinations has sufficient resolution for spruce species, although matK+rbcL might be used as a two-locus barcode. The low efficiency of these candidate barcodes in Picea might be caused by the paternal inheritance of the chloroplast genome, long generation time, recent radiation, and frequent inter-specific hybridization aided by wind pollination. Some of these factors could also be responsible for the difficulties in barcoding other plant groups. Furthermore, the potential of the nuclear LEAFY gene as a land plant barcode was discussed.

Evolutionary patterns of genic DNA methylation vary across land plants.

Little is known about patterns of genic DNA methylation across the plant kingdom or about the evolutionary processes that shape them. To characterize gene-body methylation (gbM) within exons, we have gathered single-base resolution methylome data that span the phylogenetic breadth of land plants. We find that a basal land plant, Marchantia polymorpha, lacks any evident signal of gbM within exons, but conifers have high levels of both CG and CHG (where H is A, C or T) methylation in expressed genes. To begin to understand the evolutionary forces that shape gbM, we first tested for correlations in methylation levels across orthologues(1,2). Genic CG methylation levels, but not CHG or CHH levels, are correlated across orthologues for species as distantly related as ferns and angiosperms. Hence, relative levels of CG methylation are a consistent property across genes, even for species that diverged ∼400 million years ago(3,4). In contrast, genic CHG methylation correlates with genome size, suggesting that the host epigenetic response to transposable elements also affects genes. Altogether, our data indicate that the evolutionary forces acting on DNA methylation vary substantially across species, genes and methylation contexts.

Phylogeny and divergence times of gymnosperms inferred from single-copy nuclear genes.

Phylogenetic reconstruction is fundamental to study evolutionary biology and historical biogeography. However, there was not a molecular phylogeny of gymnosperms represented by extensive sampling at the genus level, and most published phylogenies of this group were constructed based on cytoplasmic DNA markers and/or the multi-copy nuclear ribosomal DNA. In this study, we use LFY and NLY, two single-copy nuclear genes that originated from an ancient gene duplication in the ancestor of seed plants, to reconstruct the phylogeny and estimate divergence times of gymnosperms based on a complete sampling of extant genera. The results indicate that the combined LFY and NLY coding sequences can resolve interfamilial relationships of gymnosperms and intergeneric relationships of most families. Moreover, the addition of intron sequences can improve the resolution in Podocarpaceae but not in cycads, although divergence times of the cycad genera are similar to or longer than those of the Podocarpaceae genera. Our study strongly supports cycads as the basal-most lineage of gymnosperms rather than sister to Ginkgoaceae, and a sister relationship between Podocarpaceae and Araucariaceae and between Cephalotaxaceae-Taxaceae and Cupressaceae. In addition, intergeneric relationships of some families that were controversial, and the relationships between Taxaceae and Cephalotaxaceae and between conifers and Gnetales are discussed based on the nuclear gene evidence. The molecular dating analysis suggests that drastic extinctions occurred in the early evolution of gymnosperms, and extant coniferous genera in the Northern Hemisphere are older than those in the Southern Hemisphere on average. This study provides an evolutionary framework for future studies on gymnosperms.