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.
Citation: Lu Y, Ran J-H, Guo D-M, Yang Z-Y, Wang X-Q (2014) Phylogeny and Divergence Times of Gymnosperms Inferred from Single-Copy Nuclear Genes. PLoS ONE 9(9): e107679. doi:10.1371/journal.pone.0107679
Editor: Sven Buerki, Royal Botanic Gardens, Kew, United Kingdom
Received: March 7, 2014; Accepted: August 19, 2014; Published: September 15, 2014
Copyright: © 2014 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All gene sequences are deposited in NCBI and the GenBank accession numbers are shown in Table S1. All LFY and NLY gene sequences determined in this study are deposited in NCBI under GenBank accession numbers KF377856-KF377901, KF377904-KF377918 and KF377921-KF377963, and the trees and alignments are deposited in TreeBase (number S16207).
Funding: This work was supported by the National Natural Science Foundation of China (Grant Nos. 31170197, 31330008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
A solid organismal phylogeny is fundamental to study evolutionary biology and historical biogeography. In recent years, the angiosperm phylogeny group (APG) III system has provided an evolutionary framework for studying angiosperms . However, phylogenetic relationships of the main lineages of gymnosperms, either classified into four subclasses (Cycadidae, Ginkgoidae, Gnetidae and Pinidae) by Christenhusz et al.  or into the widely accepted five clades (cycads, ginkgos, cupressophytes, Pinaceae and gnetophytes), are still in hot debate. Gymnosperms, which have been resolved as the sister group of angiosperms by increasing evidence from morphological, molecular phylogenetic and evolutionary developmental studies –, bear important information of seed-plant evolution, and represent an important link in the evolution of many gene families and biological pathways. Therefore, a better understanding of evolutionary relationships within gymnosperms can also help us to interpret the evolution of seed plants, and even molecular evolution in land plants.
Gymnosperms have a rich fossil record that is very useful for phylogenetic reconstruction, but this group suffered a dramatic extinction in the Cenozoic  and currently comprises 12 families, 83 genera, and only a little more than 1,000 species , which makes it difficult to resolve some interfamilial and intergeneric relationships (see review by Wang and Ran ). The early molecular phylogenetic studies of gymnosperms only sampled a small part of the recognized genera , , –, and in particular most published molecular phylogenies were constructed based on uniparentally inherited cytoplasmic DNA markers and/or the multi-copy nuclear ribosomal DNA , , –. Despite that 53 genera representing all extant main lineages of gymnosperms were studied in Ran et al. , the main focus of the study was the fast evolution of the mitochondrial gene rps3 in Conifer II (cupressophytes) and the underlying mechanisms. Some other studies of gymnosperms mainly focused on individual families or clades, such as conifers , , Cupressaceae –, Pinaceae  and cycads , . Although great progress has been made on understanding the phylogeny of gymnosperms in recent years, more interesting phylogenetic hypotheses have been proposed and hotly debated (see review by Wang and Ran ), like the phylogenetic position of Gnetales and the relationship between cycads and ginkgos , , , . Till now, there is not a molecular phylogeny of gymnosperms that is reconstructed based on a complete sampling of all extant genera, although this ancient and widespread plant group has huge ecological and economic value. Also, it would be interesting to know whether the phylogenetic relationships of gymnosperms inferred from cytoplasmic DNA are supported by evidence from the nuclear genome, given the complex inheritance patterns of organellar genes in this group . Moreover, phylogenetic relationships within some lineages, such as Pinaceae , , Podocarpaceae  and Zamiaceae , , , need to be further resolved.
Due to the fast development of genome sequencing technologies, phylogenomic analyses have been increasingly used in reconstructing the tree of life, and the efficiency of using multiple single- or low-copy nuclear genes for phylogenetic analysis has been widely recognized . However, this is still difficult for gymnosperms with large and complex nuclear genomes characterized by long introns and numerous gene-like fragments . For example, based on ESTs, Lee et al.  analyzed millions of amino acid sites from 150 species across land plants, and placed Gnetales as sister to the rest of the gymnosperms, but their dataset suffered greatly from missing data and poor alignment (our unpublished analysis). Nevertheless, Yang et al.  successfully used two sister nuclear genes LEAFY (LFY) and NEEDLY (NLY), which originated from an ancient gene duplication in the common ancestor of seed plants and encode transcription factors regulating the development of reproductive structures in gymnosperms , , to reconstruct the phylogeny of Cupressaceae comprising all its 32 genera. They also confirmed that both LFY and NLY exist as single copy in gymnosperms, even in the polyploid species, and are excellent markers for studying the phylogeny and evolution of gymnosperms .
In this study, on the basis of Yang et al. , we use LFY and NLY gene sequences to reconstruct the phylogeny of gymnosperms based on a complete sampling of extant genera, in effort to provide an evolutionary framework for future studies on this important group. In addition, some controversial interfamilial and intergeneric relationships are resolved and discussed. Moreover, benefiting from the rich fossil record, we estimate the divergence times of different lineages, which would further help us understand the diversification history of gymnosperms.
Materials and Methods
Ninety species representing all recognized genera of extant gymnosperms were sampled. Most genera were represented by one species each, since the coding sequences of LFY and NLY used to reconstruct the phylogeny of gymnosperms are very conserved among congeneric species. If using introns of the two genes, the sequences are unalignable between the main clades of gymnosperms , and most congeneric species do not form monophyletic groups, respectively, due to the wide interspecific sharing of alleles as reported in Pinus . Therefore, the addition of more congeneric species can not significantly improve the resolution of intergeneric relationships of gymnosperms when using single-copy nuclear genes like LFY and NLY. Nevertheless, we sampled two species of Pinus to represent its two subgenera with an ancient divergence, and more species from the Juniperus-Cupressus- Callitropsis-Xanthocyparis-Hesperocyparis clade, in which the generic division is controversial . The origins of materials, including the data downloaded from NCBI, are shown in Table S1.
DNA and RNA extraction, PCR and RT-PCR amplification, cloning and sequencing
Total DNA was extracted from silica-gel dried leaves using either the modified CTAB method  or the DNAsecure Plant Kit (Tiangen, Beijing, China). Young leaves and reproductive organs of Ephedra equisetina were collected for total RNA extraction, which followed the modified Trizol method (Tiangen). The first-strand cDNA was produced using the 5′ RACE system (Invitrogen) and the 3′ RACE kit (Tiangen). Polymerase chain reaction (PCR) was conducted in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA) or an Eppendorf Mastercycler (Eppendorf Scintific, Westbury, NY, USA), in a volume of 25 µl containing 50–200 ng of DNA or cDNA template, 6.25 pmol of each primer, 0.2 mM of each dNTP, 2 mM MgCl2, and 0.75 U of ExTaq DNA polymerase (Takara Biotechnology, CO., Ltd. Dalian, China). PCR cycles were as follows: one cycle of 4 min at 94°C, four cycles of 1 min at 94°C, 30 s at 55–58°C, and 1.5–6.0 min at 72°C, followed by 32 cycles of 30 s at 94°C, 30 s at 53–55°C and 1.5–6.0 min at 72°C, with a final extension step for 10 min at 72°C.
After separation by 1.5% agarose gel electrophoresis, the PCR products were purified using the TIANgel Midi Purification Kit (Tiangen) and identified by direct sequencing with the PCR primers. Then, the correct PCR products were cloned with the pGEM-T Easy Vector System II (Promega, Madison, USA). Ten clones with the correct insertion, confirmed by EcoR I digestion, were picked for each species and screened for variation by sequencing with T7 primer. All distinct clones were further sequenced using SP6 and internal primers. Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit., and the sequencing products were separated on a 96-capillary 3730XL DNA analyzer (Applied Biosystems). All newly sequenced LFY and NLY genes, totaling 104 sequences, are deposited in NCBI under GenBank accession numbers KF377856-KF377901, KF377904-KF377918 and KF377921-KF377963 (Table S1). The primers used for amplifying and sequencing the LFY and NLY genes are shown in Table S2.
DNA sequence analysis
Sequence alignments were generated with CLUSTAL X  and manually refined. The variable sites and variability of conspecific clones were calculated using MEGA5  and BioEdit v7.2.0 , respectively. Introns of the two nuclear genes could not be reliably aligned among distantly related gymnospermous families, and thus were excluded when constructing the entire phylogeny of gymnosperms. However, some intron regions are relatively conserved and alignable within cycads and Podocarpaceae, respectively, and thus were included in the alignments to infer the intergeneric relationships of these groups. The aligned sequences were further trimmed using the Gblocks server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html).
We used the software DAMBE  to test substitution saturation for the two datasets LFY and NLY, and the results showed that none of them was substitutionally saturated. To determine whether the two gene datasets can be combined, we checked variation of clones in each species, and found that many species did not show clone polymorphism of LFY and NLY and no more than two distinct clones occurred in the same individual. In particular, the conspecific clones showed a high sequence similarity of over 95%. Then, we tried to conduct separate phylogenetic analyses for LFY and NLY that included all distinct clones, and the results showed that conspecific clones grouped together except two LFY clones from the tetraploid species Fitzroya cupressoides that were discussed in Yang et al. . Therefore, we randomly selected one clone from each species for the further analyses. The incongruence length difference test (ILD) , implemented in PAUP* 4.0b10 , CONCATERPILLAR (a hierarchical likelihood ratio test) , and CADM (a test of congruence among distance matrices)  were performed to assess congruence between different datasets. According to the three tests, no significant incongruence existed between LFY and NLY (Table 1), so we combined the two genes for phylogenetic analysis.
Initially, we used the LFY + NLY coding sequences (CDS) and the 1st+2nd codon positions, respectively, to reconstruct the phylogeny of all sampled gymnosperms. The fern Angiopteris lygodiifolia was used as outgroup for two reasons. First, as mentioned in the introduction, the two nuclear genes of gymnosperms originated from a duplication event in the common ancestor of seed plants, and the NLY gene was lost in angiosperms. Thus, the LFY gene of ferns may represent an ancestral state of the two genes. Second, the LFY gene sequence cannot be reliably aligned between gymnosperms and angiosperms, although a sister relationship between the two groups is supported by most recent studies (see review by Wang and Ran ). The results showed that the phylogenetic trees generated from different methods all supported cycads as a monophyletic group and the basal-most clade of gymnosperms (Fig. S1). To avoid long-branch attraction (LBA) artifacts, the phylogeny of gymnosperms was further reconstructed using cycads as functional outgroups. In addition, to better resolve the intergeneric relationships within cycads and Podocarpaceae that were controversial, we conducted separate phylogenetic analyses for the two lineages with combined LFY + NLY sequences, and compared gene trees generated from CDS and CDS+intron, respectively. The sister groups were chosen as outgroups, including Ginkgo biloba for cycads , as well as Araucaria heterophylla and Agathis robusa for Podocarpaceae . When introns were included, the sequences could not be aligned among different gymnospermous families, and therefore the generated trees were not rooted or rooted with a functional outgroup, such as Cycas for cycads. The details of all datasets used for phylogenetic analyses are shown in Table 2. The trees and alignments are deposited in TreeBase (number S16207).
Phylogenetic relationships were reconstructed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI), respectively. The MP analyses were implemented in PAUP* 4.0b10 , using heuristic searches with 1000 random addition sequence replicates, starting trees obtained via stepwise addition, tree-bisection-reconnection (TBR) branch swapping, MulTrees and Collapse options in effect, and a maximum of 2000 trees saved for each replicate. Robustness of the nodes (50% majority-rule consensus) was tested by the bootstrap analysis  using 1000 replicates with the same settings as above. The evolutionary models for the ML and BI analyses were optimized in jModeltest 2.0  and MrModeltest 2.3 , using Akaike Information Criterion (AIC), respectively. The best models for analyses are shown in Table 2. The ML analyses were carried out in PHYML version 2.4.4  with a BIONJ tree as a starting point, and support values for the nodes were calculated based on 100 bootstrap replicates. The Bayesian inference was performed with MrBayes 3.1.2 . One cold and three heated Markov chain Monte Carlo chains were run for 10,000,000 generations with random initial trees, and every 1000 generations were sampled. The first 20% of the samples were discarded as burn-in and a 50% majority-rule consensus tree was generated based on the trees sampled after generation 2,000,000.
The Shimodaira-Hasegawa test (SH test)  and the Kishino-Hasegawa test (KH test) , implemented in PAUP* 4.0b10, were used to test alternative phylogenetic hypotheses for the deep lineages with controversial phylogenetic positions. The different positions of three taxa, including Ginkgoaceae (sister to cycads or conifers + Gnetales), Gnetales (sister to conifers, Conifer II, Pinaceae, or other gymnosperms), and Sciadopityaceae (sister to Cupressaceae + Taxaceae + Cephalotaxaceae or Araucariaceae + Podocarpaceae), were compared. Alternative tree topologies were generated in PhyML 2.4.4 , and the tree files were run in PAUP to calculate the p-value for each topology.
Divergence time estimation
Based on the LFY + NLY coding sequences, the divergence times of gymnosperms were estimated using the Markov chain Monte Carlo (MCMC) method, which was implemented in BEAST v1.7.5 , under an uncorrelated lognormal-relaxed clock model of rate variation among lineages. The topology was constrained to reflect the ML tree, and a GTR+I+G substitution model was used. Mean substitution rates were allowed to vary. Sauquet et al.  suggested that more age constraints could lead to improved time estimates, but risky age constraints might strongly influence estimated ages. Hence, we incorporated 11 fossil constraints that were widely recognized and used in previous molecular dating of gymnosperms or seed plants , , , and nearly each main lineage of gymnosperms was calibrated by at least one fossil record (For details, see Table S3).
For the most recent common ancestor (MRCA) of gymnosperms (A), a minimum age of 306.2 Ma was set based on Cordaixylon iowensis, the oldest cordaitean coniferophyte found in the Laddsdale Coals (Cherokee Group, Desmoinesian Series; 307.2±1.0 Ma) near What Cheer of Iowa, and a maximum age of 366.8 Ma was set based on the well-documented first appearance of seeds (in the form of preovules) in the Upper Fammenian (Upper Devonian) VCo Spore Biozone –. In cycads, the stem age of Lepidozamia (B) was constrained to a minimum age of 33.9 Ma based on the fossil of Lepidozamia leaves from the Eocene of Australia, which possesses cuticular characters that are unique to Lepidozamia . In the family Pinaceae, the stem age of Picea (C) was constrained by Picea burtonii from the Apple Bay locality, Vancouver Island, British Columbia, dated to the Valanginian Stage of the Early Cretaceous (≥133 Ma). This seed cone fossil shares multiple morphological and anatomical characteristics with extant Picea, especially in the distribution and branching pattern of resin canals in the ovule scale . For Gnetales, its crown node (D) was calibrated based on Eoantha zherikhinii (≥125 Ma), which is a reproductive organ with whorls of scales and is considered closely related to Gnetum and Welwitschia , . In Conifer II, we set a minimum age of 172 Ma for the Araucariaceae-Podocarpaceae split (E) based on the first appearance of Araucarites phillipsii-Brachyphyllum mammilare from the Aalenian (172–176 Ma) , and 28 Ma for the Podocarpus-Retrophyllum split (F) based on Retrophyllum australe from the West Dale Flora of southwestern Australia (dated to 28–48 Ma) . The two calibrations were also used in Leslie et al. . Araucarites phillipsii, with seed cones similar to those in Araucariaceae, was considered as the first unambiguous evidence for the stem or crown of the plant family, and Brachyphyllum mammilare was found to have pollen cones that produced relatively large, non-saccate pollen comparable to modern Araucaria and foliages that contained oval sclereids similar to those in extant Araucaria cunninghamii. In addition, Retrophyllum australe had distinctive heterofacially flattened foliage similar to Nageia and Afrocarpus. For the split of Taxaceae-Cupressaceae (G), a minimum age of 197 Ma was set based on Palaeotaxus rediviva from the Skromberga Colliery in Scania, Sweden (dated to 197–201 Ma) , which showed an axillary short shoot that terminated in a single ovule and bore helically arranged sterile scales on seed cones identical to extant Austrotaxus and Taxus. The remaining fossil constraints were used to set a minimum age for four nodes in Cupressaceae s.l., as in Yang et al. , including the MRCAs of Sequoia-Metasequoia-Sequoiadendron (H, 140 Ma, Sequoia in early Cretaceous) , , Glyptostrobus-Taxodium (I, 99 Ma, Glyptostrobus in Cretaceous) , , Diselma-Fitzroya-Widdringtonia (J, 95 Ma, Widdringtonia in Cretaceous) , and Juniperus-Cupressus-Hesperocyparis (K, 33.9 Ma, Juniperus in the Eocene/Oligocene boundary) .
Since the age estimates by BEAST are usually older than those by PL (penalized likelihood) and the ages estimated with lognormal priors are slightly younger than those estimated with either uniform or exponential priors, Sauquet et al.  suggested that using lognormal priors can decrease the uncertainty in age estimates. Therefore, in this study, all fossil constraints were given lognormal prior distributions in the BEAST estimate. For the root constraint, we used a stdev of 0.5, a prior mean of 3.6, and an offset of 290.7 Ma. For a better comparability of our results with previous divergence time estimates of gymnosperms, the other constraints were set following Leslie et al.  and Yang et al. . The minimum age was set by the age of fossil, with a 95% confidence interval of the probability distribution extending 20 or 40 million years earlier than this minimum age, since the test by Leslie et al.  found that the fossil calibrations associated with the two prior age distributions led to very similar divergence time estimates. We ran four independent MCMC runs of 100 million generations, sampling every 2,500 generations. Tracer v1.5 was used to check convergence of the chains to the stationary distribution, ensuring the Effective Sample Size (ESS) >200. The first 20% of the generations were discarded as burn-in and trees were summarized with TreeAnnotator. The final tree and divergence times were visualized using FigTree v1.4.0.
In this study, we cloned and sequenced the LFY and NLY genes from 41 genera of 7 families (Table S1). These new data combined with the sequences downloaded from GenBank (mostly reported in Yang et al. ) completely represented all extant genera of gymnosperms. In Parasitaxus usta, the only parasitic conifer, we only got a pseudogene of NLY, in which several indels in the second exon led to an ORF shift. It is interesting that, by RT-PCR, we obtained cDNA sequences of both LFY and NLY genes from Ephedra equisetina and the LFY gene had two clone types that differed by a 9-bp deletion.
Both LFY and NLY sequences amplified from genomic DNA comprised three exons and two introns, and almost covered the full length of the two genes. The exon length was conserved, totally about 1000 bp, but the intron length varied greatly among different groups. A long repeat occurred in the first intron of the NLY gene of four Taxaceae genera (Pseudotaxus, Austrotaxus, Amentotaxus and Torreya), making it difficult to sequence the full length of the gene. Also, the first NLY intron of Cathaya and Pseudotsuga, two genera of the pine family, was difficult to sequence due to long length or complex structures. The detailed information of the sequence alignments for phylogenetic analyses, including sequence lengths and numbers of variable and parsimony-informative sites, is shown in Table 2.
Since the MP analysis is more easily affected by long branch attraction (LBA) than the ML and BI analyses –, we did not show the MP trees in this study. As mentioned earlier, when Angiopteris lygodiifolia was used as outgroup, all phylogenetic trees generated supported cycads as a monophyletic and basal-most group of gymnosperms, followed by Ginkgo (see the ML and BI trees in Fig. S1). When cycads were used as functional outgroups, the ML and BI trees generated from combined LFY and NLY CDS were topologically identical to each other, except for some branches with low statistical support. In the ML tree (Fig. 1), Ginkgoaceae was sister to the remaining gymnosperms excluding cycads, and Pinaceae was sister to a clade that was further divided into two sister subclades, i.e., Gnetales and conifer II (Cupressophytes). The conifer II was split into two lineages. One consisted of Sciadopityaceae, Podocarpaceae and Araucariaceae, and Sciadopityaceae was weakly supported to be sister to Podocarpaceae-Araucariaceae. Within the other lineage, Cephalotaxaceae was embedded in Taxaceae, and the two families formed a monophyletic group sister to Cupressaceae. In addition, this nuclear gene tree provided a relatively good resolution for intergeneric relationships in some families such as Pinaceae and Cupressaceae.
Numbers associated with branches are bootstrap percentages higher than 50%. The cycads were used as functional outgroups.
Although the trees generated with different rooting or from different codon positions (all CDS vs. 1st+2nd codons) were similar in topology, they differed in the positions of Gnetales and Sciadopityaceae (Fig. 2). For instance, in the phylogenetic tree generated from the first and second codon positions and rooted with cycads (Fig. 2D), Gnetales was weakly supported to be sister to Pinaceae, and Sciadopityaceae sister to a well-supported clade containing Cupressaceae and Taxaceae-Cephalotaxaceae. Moreover, many intra-familial relationships were poorly resolved (tree not shown), perhaps due to the declined phylogenetic signals caused by the removal of the third codon positions.
A and C, All three codon positions were used; B and D, 1st and 2nd codon positions were used. A and B, Angiopteris lygodiifolia was used as outgroup; C and D, The cycads were used as functional outgroups. Numbers associated with branches are bootstrap percentages of ML higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively.
Since the phylogenetic positions of some genera of cycads and Podocarpaceae were controversial in previous studies , , –, –, here we reconstructed internal relationships of the two groups, respectively. When only the LFY and NLY CDS was used, the phylogenetic signals were insufficient to resolve some intergeneric relationships (Figs. 3, 4), therefore we added the conserved intron regions of the two genes into analysis. For cycads, the addition of introns neither changed the tree topology nor greatly improved the resolution (Fig. 3), and the generated trees suggested a basal position of Dioon in Zamiaceae, a sister relationship between Zamia and Microcycas, and a close relationship among Encephalartos, Lepidozamia and Macrozamia. However, the resolution of internal relationships of Podocarpaceae was improved by adding intron sequences, with high support values for most nodes (Fig. 4). A large clade was strongly supported and well resolved, containing Microcachrys, Saxegothaea, Pherosphaera, Acmopyle, Dacrycarpus, Dacrydium, Falcatifolium, Afrocarpus, Podocarpus, Nageia and Retrophyllum. Within the clade, there existed two monophyletic sister groups. One was the ‘dacrydioid’ group comprising Dacrycarpus, Dacrydium and Falcatifolium, and the other was the ‘podocarpoid’ group including the Retrophyllum-Nageia subclade and the Afrocarpus-Podocarpus subclade. In addition, a close relationship among Manoao, Lagarostrobos and Parasitaxus was revealed (Fig. 4).
A, CDS; B, CDS+Intron. Numbers associated with branches are bootstrap percentages of ML and MP higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively. Ginkgo biloba was used as outgroup in Fig. 3A.
A, CDS; B, CDS+Intron. Numbers associated with branches are bootstrap percentages of ML and MP higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively. Araucaria heterophylla and Agathis robusa were used as outgroups in Fig. 4A.
The results of the SH and KH tests are shown in Table S4. The trees placing Ginkgoaceae with conifers + Gnetales were better than the trees placing the family sister to cycads, but the trees placing Sciadopityaceae with Podocarpaceae + Araucariaceae were not significantly different from the trees placing Sciadopityaceae sister to Cupressaceae + Taxaceae + Cephalotaxaceae. The sister relationship between Gnetales and the other gymnosperms was rejected by both SH and KH tests for the CDS dataset, and by the KH test for the dataset of the 1st+2nd codon positions. In addition, the topology placing Gnetales sister to conifers was rejected by the KH test for the CDS dataset. There was not significant difference in ln score between the other two topologies (Gnetales sister to Conifer II or Pinaceae).
Divergence time estimation
The divergence time estimation based on combined LFY and NLY CDS suggested a Triassic-Jurassic origin of the crown group for most families (Fig. 5). The mean ages and 95% HPDs are shown in Table S5. The most recent common ancestor (MRCA) of cycads was dated to the Middle Jurassic (158.1 Ma), and that of Pinaceae to the Lower Triassic (198.4 Ma). The divergence time between Cupressaceae and Taxaceae s.l. was close to that between Podocarpaceae and Araucariaceae, i.e., in the Late Triassic to the Early Jurassic. Most gymnosperm genera originated in the Cretaceous to the Cenozoic (Fig. 5).
A time scale is shown at the bottom. A–K indicate fossil calibration points. 1–10, A, D, E and G indicate some nodes of interest. Median ages of nodes are shown, with horizontal bars indicating the 95% highest posterior density intervals.
Evolution and phylogenetic utility of the LFY and NLY genes in gymnosperms
Our study indicates that both LFY and NLY genes occur in all extant genera of gymnosperms except that NLY has not been found in Gnetum (Table S1). Frohlich and Parker  found that the LFY-NLY gene pair originated from a duplication event in the common ancestor of seed plants, and then both paralogous genes were remained in gymnosperms while NLY was lost in angiosperms. Using this information of gene evolution as strong evidence, they also proposed the mostly male theory of flower origin given the important role of LFY in flower development, although this theory is not supported by the study of Vazquez-Lobo et al. . The study of Frohlich and Parker  only sampled a few species from gymnosperms, and supposed the existence of NLY in Gnetum. Frohlich  further mentioned the occurrence of both LFY and NLY in Ephedra (his unpublished observations), a close relative of Gnetum. Our present study has covered all extant gymnospermous genera, and the results suggest that each studied species harbors both LFY and NLY genes, except that NLY is still not found in Gnetum. Therefore, our study further supports that the LFY-NLY gene pair originated from an ancient gene duplication, at least before the divergence of gymnosperms. In addition, we have successfully obtained the LFY and NLY genes of Ephedra by RT-PCR and RACE. Moreover, the selection test suggests that both LFY and NLY genes have experienced strong purifying selection in gymnosperms (our unpublished data), implying their conserved functions.
Currently, functions of LFY and NLY in gymnosperms are still not very clear , –, but it is clear that both of them exist as single-copy genes suitable for phylogenetic reconstruction in gymnosperms based on the present study and Yang et al. . No more than two distinct clones were found in the same individual. Although the NLY sequence obtained from the parasitic Parasitaxus usta represents a pseudogene, its exon region shares a high similarity (over 90%) with that of other Podocarpaceae species, and thus still could be used in phylogenetic analysis. Actually, the LFY gene has been successfully utilized in phylogenetic and biogeographic studies of several gymnosperm groups, including Gnetum , Thuja , and Pseudotsuga . In particular, the intergeneric relationships of Cupressaceae s.l. have been well resolved by the LFY and NLY genes .
Interfamilial relationships of gymnosperms
Our study provides the first molecular phylogeny of gymnosperms covering all extant families and genera, and the phylogeny is based on two single-copy nuclear genes LFY and NLY (Fig. 1). This nuclear gene phylogeny is topologically largely consistent with most previous phylogenies of gymnosperms constructed based on cytoplasmic and/or nuclear ribosomal DNA , , . That is, the cycads diverged first, followed by Ginkgoaceae, and then conifers plus Gnetales. Within conifers, Podocarpaceae is sister to Araucariaceae, and Cephalotaxaceae-Taxaceae sister to Cupressaceae (Fig. 2). However, we did not found a sister relationship between cycads and Ginkgoaceae as suggested by chloroplast phylogenomic analyses ,  as well as genome-scale nuclear and plastid data .
The present study seems to supports the monophyly of Taxaceae s.l. that includes Cephalotaxus (Figs. 1, S1), which is consistent with the study of Leslie et al.  based on rbcL, matK, 18S and PHYP. To confirm whether the topology is really constant, we further conducted phylogenetic analyses for the Cephalotaxaceae-Taxaceae lineage using two species (Taiwania cryptomerioides and Cunninghamia lanceolata) of its sister group Cupressaceae as outgroups. The results indicate that Cephalotaxus is strongly supported to be sister to Taxaceae based on either LFY or LFY + NLY CDS, but is nested within Taxaceae with a weak support based on NLY (Fig. 6). The inconsistent positions of Cephalotaxaceae in different analyses could be caused by LBA artifacts or insufficient resolution of the markers. Actually, the evolutionary relationship of Cephalotaxaceae and Taxaceae has been controversial for a long time. All molecular studies based on chloroplast and/or nuclear ribosomal DNA suggested a sister relationship between the two families –, but many morphological studies supported the merge of them (see review by Ghimire and Heo ). A more broadly defined Taxaceae including Cephalotaxaceae has been suggested by Quinn et al.  based on rbcL and matK sequence analyses, and by Ghimire and Heo  based on a cladistic analysis of morphological characters. Also, in the new gymnosperm classification scheme of Christenhusz et al. , Cephalotaxaceae was merged into Taxaceae, and this taxonomic treatment has been adopted by Lang et al.  in the revision of Cephalotaxus. As discussed above, more studies are still needed to resolve the relationship between Cephalotaxaceae and Taxaceae.
A, combined LFY and NLY; B, LFY; C, NLY. Numbers associated with branches are bootstrap percentages of ML and MP higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively. Taiwania cryptomerioides and Cunninghamia lanceolata were used as outgroups.
The systematic position of Gnetales has been debated for several decades, which involves six main hypotheses (see reviews by Braukmann et al.  and Wang and Ran ), i.e., anthophyte, gnetales-other seed plants, gnetales-other gymnosperms, Gnetifer, Gnecup and Gnepine , , , , , –. The last three hypotheses all support a close relationship between Gnetales and conifers. In particular, the Gnepine hypothesis (Gnetales sister to Pinaceae) is supported by more and more molecular phylogenetic studies after eliminating bias in data analyses (see review by Wang and Ran ), despite the fact that the Gnecup hypothesis (Gnetales sister to conifer II or cupressophytes) is still supported by a couple of recent phylogenomic studies using all chloroplast genes , . According to the present study, the Gnetales has a close relationship with conifers, although it has not been resolved whether the Gnecup or Gnepine hypothesis is correct. In the trees generated from combined LFY and NLY CDS, Gnetales is strongly supported as sister to conifer II (Fig. 2A and 2C), which is corroborated by the SH and KH tests (Table S4). However, when excluding the third codon positions and using cycads as functional outgroups, the most popular Gnepine hypothesis is recovered with low support (Fig. 2D, Table S4). A similar phenomenon is also observed in Sciadopityaceae. When all CDS sequences are used, this family is moderately supported as sister to the Podocarpaceae-Araucariaceae clade (Fig. 2A and 2C), but when excluding the third codon positions it is revealed as sister to the Taxaceae-Cephalotaxaceae-Cupressaceae clade (Fig. 2B and 2D) as found in most previous studies , –, . The topological conflicts on phylogenetic positions of Gnetales and Sciadopityaceae may be attributed to LBA artifacts that could occur when the fast-evolving third codon positions are included in analyses. Zhong et al.  also found that the LBA artifacts and parallel changes could mislead the phylogenetic placement of Gnetales when using chloroplast genome data, and the removal of fast-evolving genes can effectively alleviate the LBA artifacts, thereby recovering a sister relationship between Gnetales and Pinaceae.
Intergeneric relationships within gymnospermous families
The combined LFY and NLY CDS phylogeny provides a good resolution for intergeneric relationships within four families including Cupressaceae, Pinaceae, Taxaceae and Araucariaceae (Figs. 1, S1). The LFY + NLY phylogeny of Cupressaceae has been discussed in detail by Yang et al. . For Pinaceae, all of the eleven genera form two strongly supported clades. One clade comprises Cedrus, Pseudolarix, and two pairs of sister genera, i.e., Nothotsuga-Tsuga and Keteleeria-Abies, while the other clade includes the sister genera Pseudotsuga and Larix, and the three closely related genera Pinus, Cathaya and Picea (Figs. 1, S1). The revealed intergeneric relationships are largely congruent with the finding of Wang et al. , and are generally consistent with the results of morphological and anatomical analyses (see review by Farjón ). However, Wang et al.  did not completely resolve the systematic position of Cedrus. According to the present study, Cedrus is sister to the Nothotsuga-Tsuga-Pseudolarix-Keteleeria-Abies clade (Figs. 1, S1), which is consistent with most recent molecular phylogenetic studies , . Moreover, like Wang et al. , our study supports the monotypic genus Cathaya as sister to Picea (Figs. 1, S1), rather than to Pinus as suggested by Lin et al. . In Taxaceae, Torreya is sister to Amentotaxus, and Austrotaxus is closely related to the sister genera Pseudotaxus and Taxus (Figs. 1, 6, S1), corroborating previous studies , –. For Araucariaceae, the previous rbcL gene analysis suggested a basal position of Wollemia in the family . However, the present study supports Wollemia as sister to Agathis (Figs. 1, S1), consistent with more recent studies , , , .
The concatenated LFY and NLY CDS can not resolve some intergeneric relationships of cycads and Podocarpaceae very well (Figs. 1, S1). It is interesting that the addition of intron sequences can improve the resolution in Podocarpaceae but not in cycads (Figs. 3, 4), although divergence times of the cycad genera are similar to or longer than those of the Podocarpaceae genera (Fig. 5) , . Consistent with most previous studies , , , , the phylogeny of cycads inferred from either CDS or CDS+Intron sequences of LFY and NLY supports the genus Dioon from tropical America as the basal-most lineage in Zamiaceae (Fig. 3), rather than sister to the Bowenia-Ceratozamia-Stangeria-Microcycas-Zamia clade in the PHYP tree constructed by Nagalingum et al. . Actually, despite low support values in some clades, the present LFY + NLY gene tree is topologically very similar to the recently reconstructed phylogeny of cycads based on five single-copy nuclear genes . For instance, the two genera Zamia and Microcycas, also from tropical America, have a sister relationship and form a clade sister to Stangeria, while the African Encephalartos and the Australian Lepidozamia form a clade sister to Macrozamia from Australia (Fig. 3). Moreover, our study also does not support the establishment of the family Boweniaceae or Stangeriaceae that was based on morphological analyses , , since the two genera Bowenia and Stangeria are nested within Zamiaceae and do not form a monophyletic clade (Fig. 3), as found in most previous molecular phylogenetic analyses , , , .
Compared to the CDS dataset, the CDS+Intron dataset provides a much better resolution for intergeneric relationships of Podocarpaceae (Fig. 4), a large family comprising 19 genera with a wide distribution in the tropics, especially in the Southern Hemisphere , . Our study strongly supports a large clade comprising 11 genera, of which the Australian Microcachrys and the South American Saxegothaea diverged first, followed by the two Australian genera Pherosphaera and Acmopyle, and then the three genera Dacrycarpus, Dacrydium and Falcatifolium (all distributed in Asia and Australia) forming the ‘dacrydioid’ group sister to the ‘podocarpoid’ group that include Retrophyllum, Nageia, Afrocarpus and Podocarpus. In addition, we found a close relationship among the three Australian genera Manoao, Lagarostrobos and Parasitaxus and a sister relationship between Prumnopitys and Sundacarpus (Fig. 4B). This phylogeny of Podocarpaceae constructed from nuclear genes is topologically highly consistent with those inferred from plastid DNA fragments  and from a combined analysis of nrITS1, NLY intron 2 and rbcL sequences as well as anatomical and morphological data . However, our nuclear gene phylogeny strongly supports two pairs of sister genera Retrophyllum-Nageia and Afrocarpus-Podocarpus (Fig. 4B). The genus Phyllocladus is nested within Podocarpaceae (Fig. 4), and thus the family status of Phyllocladaceae is not supported.
Divergence times of gymnosperms
The divergence time estimation is very helpful to interpret the temporal evolution of organisms. Previous studies have provided divergence time estimates for different gymnospermous groups, such as Pinaceae , cycads , Podocarpaceae , Cupressaceae , , and conifers . However, only Crisp and Cook  estimated divergence times of gymnosperms as a whole using molecular clock, and in their study many extant genera were not sampled.
Our present study provides divergence time estimates for gymnosperms based on a sampling of all extant families and genera (Fig. 5). The estimated crown ages of some groups such as Pinaceae, cycads and Podocarpaceae are approaching to those reported in previous studies , , . However, the estimated crown age of Cupressaceae and divergence times of most genera of this family are a little younger than those reported in Yang et al.  and Mao et al. . This could be attributed to the discrepancy of different dating methods and delineation of different fossil calibrations.
Based on the molecular dating analysis, all of the extant five lineages of gymnosperms (cycads, ginkgos, cupressophytes, Pinaceae and gnetophytes) originated at least before 300 Ma (in the Carboniferous), but the crown ages of all families except Ginkgoaceae and Sciadopityaceae are younger than 200 Ma (Fig. 5), indicating that drastic extinctions occurred in the early evolution of gymnosperms, which might be caused by the two extreme cooling events in the Carboniferous and Triassic . After 200 Ma, the divergence speed of genera is moderate when extinction is not considered (Fig. 7), although recent studies showed that the pulse of extinction and speciation in the Cenozoic, even in the late Tertiary, shaped today's species diversity of gymnosperms , . Leslie et al.  found that lineages of conifers that diversified mainly in the Southern Hemisphere show a significantly older distribution of divergence ages than their counterparts in the Northern Hemisphere. However, interestingly, we found that extant coniferous genera in the Northern Hemisphere are older than those in the Southern Hemisphere on average (Fig. 8A). In fact, if excluding the several genera that originated before 150 Ma, the distribution of divergence ages of the remaining genera is very similar between the two hemispheres (Fig. 8B). Of great interest is to investigate why more ancient genera survive in the Northern Hemisphere than in the Southern Hemisphere. Moreover, to get a more accurate estimation of the divergence times and a solid reconstruction of the evolutionary dynamics of gymnosperms, more nuclear genes or genome sequences should be used in future studies, and more reliable fossils are needed to be found.
The divergence times was based on the median ages of the nodes from the BEAST analysis (see Fig. 5).
The ML and BI trees of gymnosperms constructed from combined LFY and NLY sequences. Numbers associated with branches are bootstrap percentages of ML higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively. A, ML tree from the CDS sequences with Angiopteris lygodiifolia as outgroup; B, BI tree from the CDS sequences with Angiopteris lygodiifolia as outgroup; C, ML tree from the 1st+2nd codon positions with Angiopteris lygodiifolia as outgroup; D, BI tree from the 1st+2nd codon positions with Angiopteris lygodiifolia as outgroup; E, BI tree from the CDS sequences with cycads as functional outgroups; F, ML tree from the 1st+2nd codon positions with cycads as functional outgroups; G, BI tree from the 1st+2nd codon positions with cycads as functional outgroups.
Sources of materials.
Primers used for the PCR amplification and sequencing.
The eleven calibration points used in divergence time estimation for gymnosperms. All constraints were given lognormal prior distributions, where the minimum age was set by the age of the fossil constraint and 95% confidence interval of the probability distribution extending 20 or 40 million years earlier than the minimum age.
Results of the Shimodaira-Hasegawa (SH) test and the Kishino-Hasegawa (KH) test.
Ages of selected clades.
We are indebted to Profs. Chad Husby (Montgomery Botanical Center, USA), Frank Van Caekenberghe (National Botanic Garden of Belgium), Peter Brownless and Peter Hollingsworth (Royal Botanic Garden Edinburgh), Christopher Quinn (Royal Botanic Gardens of Australia), Shou-Zhou Zhang (Shenzhen FairyLake Botanical Garden, China), and Zhiduan Chen (Institute of Botany, Chinese Academy of Sciences) for their kind help in sampling some genera of gymnosperms. We also thank Prof. Robert P. Adams (Baylor University, USA) and the Royal Botanic Garden, Kew (UK) for providing some DNA samples, Ms. Wan-Qing Jin for her assistance in DNA sequencing, and the two anonymous reviewers for their insightful comments and suggestions on the manuscript.
Conceived and designed the experiments: XQW. Performed the experiments: YL. Analyzed the data: JHR YL XQW DMG. Contributed reagents/materials/analysis tools: XQW YL ZYY JHR. Wrote the paper: XQW YL JHR.
- 1. The Angiosperm Phylogeny Group (2009) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Bot J Linn Soc 161: 105–121.
- 2. Christenhusz MJM, Reveal JL, Farjón A, Gardner MF, Mill RR, et al. (2011) A new classification and linear sequence of extant gymnosperms. Phytotaxa 19: 55–70.
- 3. Winter KU, Bec ker A, Munster T, Kim JT, Saedler H, et al. (1999) MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants. Proc Natl Acad Sci U S A 96: 7342–7347.
- 4. Chaw SM, Parkinson CL, Cheng YC, Vincent TM, Palmer JD (2000) Seed plant phylogeny inferred from all three plant genomes: Monophyly of extant gymnosperms and origin of Gnetales from conifers. Proc Natl Acad Sci U S A 97: 4086–4091.
- 5. Bowe LM, Coat G, dePamphilis CW (2000) Phylogeny of seed plants based on all three genomic compartments: Extant gymnosperms are monophyletic and Gnetales' closest relatives are conifers. Proc Natl Acad Sci U S A 97: 4092–4097.
- 6. Mundry M, Stutzel T (2004) Morphogenesis of the reproductive shoots of Welwitschia mirabilis and Ephedra distachya (Gnetales), and its evolutionary implications. Org Divers Evol 4: 91–108.
- 7. Hajibabaei M, Xia J, Drouin G (2006) Seed plant phylogeny: Gnetophytes are derived conifers and a sister group to Pinaceae. Mol Phylogenet Evol 40: 208–217.
- 8. Braukmann TWA, Kuzmina M, Stefanović S (2009) Loss of all plastid ndh genes in Gnetales and conifers: extent and evolutionary significance for the seed plant phylogeny. Curr Genet 55: 323–337.
- 9. Zhong B, Yonezawa T, Zhong Y, Hasegawa M (2010) The position of gnetales among seed plants: overcoming pitfalls of chloroplast phylogenomics. Mol Biol Evol 27: 2855–2863.
- 10. Wu CS, Lin CP, Hsu CY, Wang RJ, Chaw SM (2011) Comparative chloroplast genomes of Pinaceae: insights into the mechanism of diversified genomic organizations. Genome Biol Evol 3: 309–319.
- 11. Xi Z, Rest JS, Davis CC (2013) Phylogenomics and coalescent analyses resolve extant seed plant relationships. PLoS One 8: e80870.
- 12. Ruhfel B, Gitzendanner M, Soltis P, Soltis D, Burleigh J (2014) From algae to angiosperms-inferring the phylogeny of green plants (Viridiplantae) from 360 plastid genomes. BMC Evol Biol 14: 23.
- 13. Wang X-Q, Ran J-H (2014) Evolution and biogeography of gymnosperms. Mol Phylogenet Evol 75: 24–40.
- 14. Crisp MD, Cook LG (2011) Cenozoic extinctions account for the low diversity of extant gymnosperms compared with angiosperms. New Phytol 192: 997–1009.
- 15. Chaw SM, Zharkikh A, Sung HM, Lau TC, Li WH (1997) Molecular phylogeny of extant gymnosperms and seed plant evolution: analysis of nuclear 18S rRNA sequences. Mol Biol Evol 14: 56–68.
- 16. Ran J-H, Gao H, Wang X-Q (2010) Fast evolution of the retroprocessed mitochondrial rps3 gene in Conifer II and further evidence for the phylogeny of gymnosperms. Mol Phylogenet Evol 54: 136–149.
- 17. Rai HS, Reeves PA, Peakall R, Olmstead RG, Graham SW (2008) Inference of higher-order conifer relationships from a multi-locus plastid data set. Botany 86: 658–669.
- 18. Leslie AB, Beaulieu JM, Rai HS, Crane PR, Donoghue MJ, et al. (2012) Hemisphere-scale differences in conifer evolutionary dynamics. Proc Natl Acad Sci U S A 109: 16217–16221.
- 19. Gadek PA, Alpers DL, Heslewood MM, Quinn CJ (2000) Relationships within Cupressaceae sensu lato: A combined morphological and molecular approach. Am J Bot 87: 1044–1057.
- 20. Kusumi J, Tsumura Y, Yoshimaru H, Tachida H (2000) Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences. Am J Bot 87: 1480–1488.
- 21. Mao K, Milne RI, Zhang L, Peng Y, Liu J, et al. (2012) Distribution of living Cupressaceae reflects the breakup of Pangea. Proc Natl Acad Sci U S A 109: 7793–7798.
- 22. Yang Z-Y, Ran J-H, Wang X-Q (2012) Three genome-based phylogeny of Cupressaceae s.l.: Further evidence for the evolution of gymnosperms and Southern Hemisphere biogeography. Mol Phylogenet Evol 64: 452–470.
- 23. Wang X-Q, Tank DC, Sang T (2000) Phylogeny and divergence times in Pinaceae: evidence from three genomes. Mol Biol Evol 17: 773–781.
- 24. Zgurski JM, Rai HS, Fai QM, Bogler DJ, Francisco-Ortega J, et al. (2008) How well do we understand the overall backbone of cycad phylogeny? New insights from a large, multigene plastid data set. Mol Phylogenet Evol 47: 1232–1237.
- 25. Nagalingum NS, Marshall CR, Quental TB, Rai HS, Little DP, et al. (2011) Recent synchronous radiation of a living fossil. Science 334: 796–799.
- 26. Wu CS, Chaw SM, Huang YY (2013) Chloroplast phylogenomics indicates that Ginkgo biloba is sister to cycads. Genome Biol Evol 5: 243–254.
- 27. Lee EK, Cibrian-Jaramillo A, Kolokotronis SO, Katari MS, Stamatakis A, et al. (2011) A functional phylogenomic view of the seed plants. PLoS Genet 7: e1002411.
- 28. Mogensen HL (1996) The hows and whys of cytoplasmic inheritance in seed plants. Am J Bot 83: 383–404.
- 29. Lin CP, Huang JP, Wu CS, Hsu CY, Chaw SM (2010) Comparative chloroplast genomics reveals the evolution of Pinaceae genera and subfamilies. Genome Biol Evol 2: 504–517.
- 30. Knopf P, Schulz C, Little DP, Stutzel T, Stevenson DW (2012) Relationships within Podocarpaceae based on DNA sequence, anatomical, morphological, and biogeographical data. Cladistics 28: 271–299.
- 31. Chaw SM, Walters TW, Chang CC, Hu SH, Chen SH (2005) A phylogeny of cycads (Cycadales) inferred from chloroplast matK gene, trnK intron, and nuclear rDNA ITS region. Mol Phylogenet Evol 37: 214–234.
- 32. Salas-Leiva DE, Meerow AW, Calonje M, Griffith MP, Francisco-Ortega J, et al. (2013) Phylogeny of the cycads based on multiple single-copy nuclear genes: congruence of concatenated parsimony, likelihood and species tree inference methods. Ann Bot 112: 1263–1278.
- 33. Zhang N, Zeng L, Shan H, Ma H (2012) Highly conserved low-copy nuclear genes as effective markers for phylogenetic analyses in angiosperms. New Phytol 195: 923–937.
- 34. Nystedt B, Street NR, Wetterbom A, Zuccolo A, Lin YC, et al. (2013) The Norway spruce genome sequence and conifer genome evolution. Nature 497: 579–584.
- 35. Vazquez-Lobo A, Carlsbecker A, Vergara-Silva F, Alvarez-Buylla ER, Pinero D, et al. (2007) Characterization of the expression patterns of LEAFY/FLORICAULA and NEEDLY orthologs in female and male cones of the conifer genera Picea, Podocarpus, and Taxus: implications for current evo-devo hypotheses for gymnosperms. Evol Dev 9: 446–459.
- 36. Moyroud E, Kusters E, Monniaux M, Koes R, Parcy F (2010) LEAFY blossoms. Trends Plant Sci 15: 346–352.
- 37. Syring J, Farrell K, Businský R, Cronn R, Liston A (2007) Widespread genealogical nonmonophyly in species of Pinus subgenus Strobus. Syst. Biol 56: 163–181.
- 38. Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant-tissues. Plant Mol Biol 5: 69–76.
- 39. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25: 4876–4882.
- 40. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
- 41. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 40: 95–98.
- 42. Xia X, Xie Z (2001) DAMBE: software package for data analysis in molecular biology and evolution. J Hered 92: 371–373.
- 43. Farris JS, Kallersjo M, Kluge AG, Bult C (1994) Testing significance of incongruence. Cladistics 10: 315–319.
- 44. Swofford DL (2002) Phylogenetic Analysis Using Parsimony (* and other methods), Version 4. Sinauer, Sunderland, MA.
- 45. Leigh JW, Susko E, Baumgartner M, Roger AJ (2008) Testing congruence in phylogenomic analysis. Syst Biol 57: 104–115.
- 46. Campbell V, Legendre P, Lapointe F-J (2009) Assessing congruence among ultrametric distance matrices. J Classif 26: 103–117.
- 47. Felsenstein J (1985) Confidence-limits on phylogenies - an approach using the Bootstrap. Evolution 39: 783–791.
- 48. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9: 772.
- 49. Nylander JAA (2004) MrModeltest v2. Program distributed by the author: Evolutionary Biology Centre, Uppsala Univ., Uppsala, Sweden.
- 50. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696–704.
- 51. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
- 52. Shimodaira H, Hasegawa M (1999) Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol 16: 1114–1116.
- 53. Kishino H, Hasegawa M (1989) Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in hominoidea. J Mol Evol 29: 170–179.
- 54. Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29: 1969–1973.
- 55. Sauquet H, Ho SY, Gandolfo MA, Jordan GJ, Wilf P, et al. (2012) Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales). Syst Biol 61: 289–313.
- 56. Magallón S, Hilu KW, Quandt D (2013) Land plant evolutionary timeline: Gene effects are secondary to fossil constraints in relaxed clock estimation of age and substitution rates. Am J Bot 100: 556–573.
- 57. Ravn RL, Swade JW, Howes RR, Gregory JL, Anderson RR, et al. (1984) Stratigraphy of the Cherokee Group and revision of Pennsylvanian stratigraphic nomenclature in Iowa. Iowa Geological Survey Technical Information Series 12: 1–76.
- 58. Rothwell GW, Scheckler SE, Gillespie WH (1989) Elkinsia gen. nov., a late Devonian gymnosperm with cupulate ovules. Bot Gaz 150: 170–189.
- 59. Peppers RA (1996) Palynological correlation of major Pennsylvanian (Middle and Upper Carboniferous) chronostratigraphic boundaries in the Illinois and other coal basins. Geol Soc Amer Mem 188: 1–111.
- 60. Hill RS (1980) Three new Eocene cycads from eastern Australia. Aust J Bot 28: 105.
- 61. Klymiuk AA, Stockey RA (2012) A Lower Cretaceous (Valanginian) seed cone provides the earliest fossil record for Picea (Pinaceae). Am J Bot 99: 1069–1082.
- 62. Krassilov VA (1982) Early Cretaceous flora of Mongolia. Palaeontolographica B 181: 1–43.
- 63. Krassilov VA (1986) New floral structure from the Lower Cretaceous of Lake Bakal area. Rev Paleobot Palinol 47: 9–16.
- 64. Harris TM (1979) The Yorkshire Jurassic Flora. V. Coniferales. British Museum of Natural History, London.
- 65. Hill RS, Merrifield HE (1993) An early Tertiary macroflora from West Dale, southwestern Australia. Alcheringa 17: 285–326.
- 66. Florin R (1958) On Jurassic taxads and conifers from north-western Europe and Eastern Greenland. Acta Hort Berg 17: 257–402.
- 67. Penny JS (1947) Studies on the conifers of the magothy flora. Am J Bot 34: 281–296.
- 68. Ma Q-W, Li F-L, Li C-S (2005) The coast redwoods (Sequoia, Taxodiaceae) from the Eocene of Heilongjiang and the Miocene of Yunnan, China. Rev Palaeobot Palynol 135: 117–129.
- 69. Aulenback KR, LePage BA (1998) Taxodium wallisii sp. nov.: first occurrence of Taxodium from the Upper Cretaceous. Int J Plant Sci 159: 367–390.
- 70. Miller CN (1977) Mesozoic conifers. Bot Rev 43: 217–280.
- 71. McIver EE (2001) Cretaceous Widdringtonia Endl. (Cupressaceae) from North America. Int J Plant Sci 162: 937–961.
- 72. Kvacek Z (2002) A new juniper from the Palaeogene of Central Europe. Fedd Repert 113: 492–502.
- 73. Felsenstein J (1978) Cases in which parsimony or compatibility methods will be positively misleading. Syst Zool 27: 401–410.
- 74. Hendy MD, Penny D (1989) A framework for the quantitative study of evolutionary trees. Syst Zool 38: 297–309.
- 75. Huelsenbeck JP (1995) Performance of phylogenetic methods in simulation. Syst Biol 44: 17–48.
- 76. Conran JG, Wood GM, Martin PG, Dowd JM, Quinn CJ, et al. (2000) Generic relationships within and between the gymnosperm families Podocarpaceae and Phyllocladaceae based on an analysis of the chloroplast gene rbcL. Aust J Bot 48: 715–724.
- 77. Kelch D (1998) Phylogeny of Podocarpaceae: comparison of evidence from morphology and 18S rDNA. Am J Bot 85: 986.
- 78. Hill KD, Chase MW, Stevenson DW, Hills HG, Schutzman B (2003) The families and genera of cycads: A molecular phylogenetic analysis of cycadophyta based on nuclear and plastid DNA sequences. Int J Plant Sci 164: 933–948.
- 79. Rai HS, O'Brien HE, Reeves PA, Olmstead RG, Graham SW (2003) Inference of higher-order relationships in the cycads from a large chloroplast data set. Mol Phylogenet Evol 29: 350–359.
- 80. Frohlich MW, Parker DS (2000) The mostly male theory of flower evolutionary origins: from genes to fossils. Syst Bot 25: 155–170.
- 81. Frohlich MW (2003) An evolutionary scenario for the origin of flowers. Nat Rev Genet 4: 559–566.
- 82. Mellerowicz EJ, Horgan K, Walden A, Coker A, Walter C (1998) PRFLL - a Pinus radiata homologue of FLORICAULA and LEAFY is expressed in buds containing vegetative shoot and undifferentiated male cone primordia. Planta 206: 619–629.
- 83. Mouradov A, Glassick T, Hamdorf B, Murphy L, Fowler B, et al. (1998) NEEDLY, a Pinus radiata ortholog of FLORICAULA/LEAFY genes, expressed in both reproductive and vegetative meristems. Proc Natl Acad Sci U S A 95: 6537–6542.
- 84. Shindo S, Sakakibara K, Sano R, Ueda K, Hasebe M (2001) Characterization of a FLORICAULA/LEAFY homologue of Gnetum parvifolium and its implications for the evolution of reproductive organs in seed plants. Int J Plant Sci 162: 1199–1209.
- 85. Carlsbecker A, Tandre K, Johanson U, Englund M, Engström P (2004) The MADS-box gene DAL1 is a potential mediator of the juvenile-to-adult transition in Norway spruce (Picea abies). Plant J 40: 546–557.
- 86. Dornelas MC, Rodriguez APM (2005) A FLORICAULA/LEAFY gene homolog is preferentially expressed in developing female cones of the tropical pine Pinus caribaea var. caribaea. Genet Mol Biol 28: 299–307.
- 87. Shiokawa T, Yamada S, Futamura N, Osanai K, Murasugi D, et al. (2008) Isolation and functional analysis of the CjNdly gene, a homolog in Cryptomeria japonica of FLORICAULA/LEAFY genes. Tree Physiol 28: 21–28.
- 88. Won H, Renner SS (2006) Dating dispersal and radiation in the gymnosperm Gnetum (Gnetales) - Clock calibration when outgroup relationships are uncertain. Syst Biol 55: 610–622.
- 89. Peng D, Wang X-Q (2008) Reticulate evolution in Thuja inferred from multiple gene sequences: implications for the study of biogeographical disjunction between eastern Asia and North America. Mol Phylogenet Evol 47: 1190–1202.
- 90. Wei X-X, Yang Z-Y, Li Y, Wang X-Q (2010) Molecular phylogeny and biogeography of Pseudotsuga (Pinaceae): insights into the floristic relationship between Taiwan and its adjacent areas. Mol Phylogenet Evol 55: 776–785.
- 91. Cheng Y, Nicolson RG, Tripp K, Chaw SM (2000) Phylogeny of Taxaceae and Cephalotaxaceae genera inferred from chloroplast matK gene and nuclear rDNA ITS region. Mol Phylogenet Evol 14: 353–365.
- 92. Wang X-Q, Shu Y-Q (2000) Chloroplast matK gene phylogeny of Taxaceae and Cephalotaxaceae, with additional reference to the systematic position of Nageia. Acta Phytotax Sin 38: 201–210.
- 93. Hao DC, Xiao PG, Huang BL, Ge GB, Yang L (2008) Interspecific relationships and origins of Taxaceae and Cephalotaxaceae revealed by partitioned Bayesian analyses of chloroplast and nuclear DNA sequences. Plant Syst Evol 276: 89–104.
- 94. Ghimire B, Heo K (2014) Cladistic analysis of Taxaceae s.l.. Plant Syst Evol 300: 217–223.
- 95. Quinn CJ, Price RA, Gadek PA (2002) Familial concepts and relationships in the conifers based on rbcL and matK sequence comparisons. Kew Bull 57 513–531.
- 96. Lang XD, Su JR, Lu SG, Zhang ZJ (2013) A taxonomic revision of the genus Cephalotaxus (Taxaceae). Phytotaxa 84: 1–24.
- 97. Donoghue MJ, Doyle JA (2000) Seed plant phylogeny: Demise of the anthophyte hypothesis? Curr Biol 10: R106–109.
- 98. Doyle JA, Donoghue MJ (1986) Seed plant phylogeny and the origin of angiosperms - an experimental cladistic approach. Bot Rev 52: 321–431.
- 99. Schmidt M, Schneider-Poetsch HA (2002) The evolution of gymnosperms redrawn by phytochrome genes: the Gnetatae appear at the base of the gymnosperms. J Mol Evol 54: 715–724.
- 100. Burleigh JG, Mathews S (2007) Assessing among-locus variation in the inference of seed plant phylogeny. Int J Plant Sci 168: 111–124.
- 101. Zhong B, Deusch O, Goremykin VV, Penny D, Biggs PJ, et al. (2011) Systematic error in seed plant phylogenomics. Genome Biol Evol 3: 1340–1348.
- 102. Farjón A (1990) Pinaceae: Drawings and Descriptions of the Genera Abies, Cedrus, Pseudolarix, Keteleeria, Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix and Picea. Königstein, Germany: Koeltz Scientific Books.
- 103. Setoguchi H, Osawa TA, Pintaud JC, Jaffre T, Veillon JM (1998) Phylogenetic relationships within Araucariaceae based on rbcL gene sequences. Am J Bot 85: 1507–1516.
- 104. Liu N, Zhu Y, Wei ZX, Chen J, Wang QB, et al. (2009) Phylogenetic relationships and divergence times of the family Araucariaceae based on the DNA sequences of eight genes. Chinese Sci Bull 54: 2648–2655.
- 105. Escapa IH, Catalano SA (2013) Phylogenetic analysis of Araucariaceae: Integrating molecules, morphology, and fossils. Int J Plant Sci 174: 1153–1170.
- 106. Stevenson DW (1992) A formal classification of the extant cycads. Brittonia 44: 220–223.
- 107. Stevenson DW (1990) Morphology and systematics of the Cycadales. Mem NY Bot Gard 57: 8–55.
- 108. Eckenwalder JE (2009) Conifers of the World: the Complete Reference. London: Timber Press.
- 109. Farjón A (2010) A Handbook of the World Conifers. Vol. 1, 2. Leiden: Brill Press.
- 110. Biffin E, Brodribb TJ, Hill RS, Thomas P, Lowe AJ (2012) Leaf evolution in Southern Hemisphere conifers tracks the angiosperm ecological radiation. Proc Roy Soc B-Biol Sci 279: 341–348.
- 111. Royer DL, Berner RA, Montañez IP, Tabor NJ, Beerling DJ (2004) CO2 as a primary driver of Phanerozoic climate. GSA Today 14: 4.