American Journal of Botany

Volume 105, Issue 3 p. 291-301

Research Article

Open Access

Plastid phylogenomic analysis of green plants: A billion years of evolutionary history

Matthew A. Gitzendanner,

Corresponding Author

Matthew A. Gitzendanner

[email protected]

orcid.org/0000-0002-7078-4336

Department of Biology, University of Florida, Gainesville, FL, 32611 USA

Genetics Institute, University of Florida, Gainesville, FL, 32610 USA

Author for correspondence (e-mail: [email protected])Search for more papers by this author

Pamela S. Soltis,

Pamela S. Soltis

Florida Museum of Natural History, University of Florida, Gainesville, FL, 32611 USA

Genetics Institute, University of Florida, Gainesville, FL, 32610 USA

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Gane K.-S. Wong,

Gane K.-S. Wong

orcid.org/0000-0001-6108-5560

Department of Biological Sciences, University of Alberta, Edmonton AB, T6G 2E9 Canada

Department of Medicine, University of Alberta, Edmonton AB, T6G 2E1 Canada

BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083 China

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Brad R. Ruhfel,

Brad R. Ruhfel

Department of Biological Sciences, Eastern Kentucky University, Richmond, KY, 40475 USA

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Douglas E. Soltis,

Douglas E. Soltis

Department of Biology, University of Florida, Gainesville, FL, 32611 USA

Florida Museum of Natural History, University of Florida, Gainesville, FL, 32611 USA

Genetics Institute, University of Florida, Gainesville, FL, 32610 USA

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Matthew A. Gitzendanner,

Corresponding Author

Matthew A. Gitzendanner

[email protected]

orcid.org/0000-0002-7078-4336

Department of Biology, University of Florida, Gainesville, FL, 32611 USA

Genetics Institute, University of Florida, Gainesville, FL, 32610 USA

Author for correspondence (e-mail: [email protected])Search for more papers by this author

Pamela S. Soltis,

Pamela S. Soltis

Florida Museum of Natural History, University of Florida, Gainesville, FL, 32611 USA

Genetics Institute, University of Florida, Gainesville, FL, 32610 USA

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Gane K.-S. Wong,

Gane K.-S. Wong

orcid.org/0000-0001-6108-5560

Department of Biological Sciences, University of Alberta, Edmonton AB, T6G 2E9 Canada

Department of Medicine, University of Alberta, Edmonton AB, T6G 2E1 Canada

BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083 China

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Brad R. Ruhfel,

Brad R. Ruhfel

Department of Biological Sciences, Eastern Kentucky University, Richmond, KY, 40475 USA

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Douglas E. Soltis,

Douglas E. Soltis

Department of Biology, University of Florida, Gainesville, FL, 32611 USA

Florida Museum of Natural History, University of Florida, Gainesville, FL, 32611 USA

Genetics Institute, University of Florida, Gainesville, FL, 32610 USA

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First published: 30 March 2018

https://doi.org/10.1002/ajb2.1048

Citations: 194

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Abstract

Premise of the Study

For the past one billion years, green plants (Viridiplantae) have dominated global ecosystems, yet many key branches in their evolutionary history remain poorly resolved. Using the largest analysis of Viridiplantae based on plastid genome sequences to date, we examined the phylogeny and implications for morphological evolution at key nodes.

Methods

We analyzed amino acid sequences from protein-coding genes from complete (or nearly complete) plastomes for 1879 taxa, including representatives across all major clades of Viridiplantae. Much of the data used was derived from transcriptomes from the One Thousand Plants Project (1KP); other data were taken from GenBank.

Key Results

Our results largely agree with previous plastid-based analyses. Noteworthy results include (1) the position of Zygnematophyceae as sister to land plants (Embryophyta), (2) a bryophyte clade (hornworts, mosses + liverworts), (3) Equisetum + Psilotaceae as sister to Marattiales + leptosporangiate ferns, (4) cycads + Ginkgo as sister to the remaining extant gymnosperms, within which Gnetophyta are placed within conifers as sister to non-Pinaceae (Gne-Cup hypothesis), and (5) Amborella, followed by water lilies (Nymphaeales), as successive sisters to all other extant angiosperms. Within angiosperms, there is support for Mesangiospermae, a clade that comprises magnoliids, Chloranthales, monocots, Ceratophyllum, and eudicots. The placements of Ceratophyllum and Dilleniaceae remain problematic. Within Pentapetalae, two major clades (superasterids and superrosids) are recovered.

Conclusions

This plastid data set provides an important resource for elucidating morphological evolution, dating divergence times in Viridiplantae, comparisons with emerging nuclear phylogenies, and analyses of molecular evolutionary patterns and dynamics of the plastid genome.

Viridiplantae (green plants), comprising perhaps 500,000 species (Govaerts, 2003; Guiry, 2012; Judd et al., 2016), dominate terrestrial and many aquatic environments. Species of Viridiplantae fall into two major clades: Chlorophyta and Streptophyta. Chlorophyta (chlorophytes) include approximately 4300 species of marine, freshwater, terrestrial, and symbiotic “algae”. The remaining green plants, Streptophyta (streptophytes), include freshwater “algae” (Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Charophyceae, Coleochaetophyceae, and Zygnematophyceae); hornworts, mosses, and liverworts (together “bryophytes”, comprising 16,000 species); Lycopodiophyta (lycophytes; 1200 species); Monilophyta (monilophytes; ferns; 10,000 species); and Spermatophyta (seed plants; gymnosperms and angiosperms; with the angiosperms having perhaps 350,000–400,000 species); Lycopodiophyta, Monilophyta, and Spermatophyta collectively make up Tracheophyta (tracheophytes; vascular plants). (Note that throughout this paper, italicized names of larger groups correspond to clade names for which phylogenetic definitions have been provided, e.g., Cantino et al. 2007 and Podani 2015; non-italicized names reflect traditional Linnaean taxonomy.)

Despite the huge ecological and economic importance of Viridiplantae, many fundamental relationships remain unclear. Relationships within the Viridiplantae branch of the tree of life have been difficult to reconstruct for a combination of reasons, including the enormous age of the clade, significant extinction of major lineages, as well as extensive heterogeneity in the rates of molecular evolution (Soltis et al., 2002; Smith and Donoghue, 2008; Smith, 2009; Zhong et al., 2011; Rothfels et al., 2012; Ruhfel et al., 2014). Nonetheless, enormous progress has been made in recent years in clarifying our understanding of the evolution of Viridiplantae based on both molecular and paleobotanical research (reviewed by Ruhfel et al., 2014; Wickett et al., 2014; Chen et al., 2016; Hall and McCourt, 2016; Soltis et al., 2017).

Early phylogenetic analyses of Viridiplantae relied heavily on 18S-26S rDNA sequences (Chaw et al., 1997; Soltis et al., 1999b; Nickrent et al., 2000), with occasional use of mtDNA genes (Duff and Nickrent, 1999; Qiu et al., 1999, 2007), but by far most analyses have focused on plastid genes (e.g., Qiu and Palmer, 1999; Magallón and Sanderson, 2002; Qiu, 2008; Smith et al., 2009; Ruhfel et al., 2014). Importantly, regardless of genes or genome, most analyses have also been severely limited in terms of taxonomic sampling (but see Källersjö et al., 1998; Smith et al., 2009; Zanne et al., 2014 for increasingly large numbers of species but limited numbers of plastid genes)—broad comprehensive, phylogenomic analyses have not been conducted. The largest plastid phylogenomic study to date sampled across Viridiplantae, but the 360 species included in that study represent <0.1% of Viridiplantae species diversity (Ruhfel et al., 2014). The nuclear phylogenomic study of Wickett et al. (2014) sampled 852 genes but for only 103 species. Broad mitochondrial phylogenies remain problematic in Viridiplantae in part because of frequent horizontal gene transfer.

As a result of several decades of phylogenetic study, many of the major clades of Viridiplantae are now well defined. Nonetheless, major questions remain regarding the relationships among these major clades of green life. For example, the closest relatives of Embryophyta remain enigmatic (Nickrent et al., 2000; Karol et al., 2001; Qiu et al., 2006; Lemieux et al., 2007, 2014; Turmel et al., 2006, 2007). Similarly, the relationships among the three bryophyte lineages (mosses, liverworts, and hornworts) have varied greatly among analyses, with all possible relationships recovered (e.g., Nickrent et al., 2000; Shaw and Renzaglia, 2004; Renzaglia et al., 2007; Ruhfel et al., 2014; Wickett et al., 2014). Relationships among seed plant lineages are also contentious (reviewed by Soltis et al., 2017; Bowe et al., 2000; Chaw et al., 2000; Mathews, 2009; Finet et al., 2012; Zhong et al., 2010; Ruhfel et al., 2014).

Here we provide the largest phylogenomic analysis of the Viridiplantae clade yet conducted, based on 1879 taxa with representatives across all major subclades of Viridiplantae using complete (or nearly complete) plastid protein-coding sequences. This comprehensive phylogenetic framework for Viridiplantae reflects a wealth of new sequence data. We compare our results to recent plastid and nuclear phylogenomic analyses of Viridiplantae based on far fewer species (Ruhfel et al., 2014; Wickett et al., 2014), highlight differences in nuclear and plastid topologies, discuss implications for morphological innovation, and illustrate areas for future research. We hope that the broad phylogenetic framework we provide for Viridiplantae will be of enormous value to evolutionary biologists, ecologists, molecular biologists, and genomicists.

MATERIALS AND METHODS

The One Thousand Plants Project (1KP project; onekp.com) used various Illumina sequencing technologies to generate approximately 2 Gbp of sequence from each of approximately 1400 RNA-seq libraries built in most cases from vegetative samples (leaves for vascular plants or leaves and stems for tracheophytes with microphyllous leaves) collected from taxonomically diverse Viridiplantae. These data were filtered and assembled with SOAP-denovo-Trans (Wickett et al., 2014), and these assemblies were the starting point of the present study.

To obtain the plastid gene sequences from the SOAP-denovo-Trans transcriptome assemblies, we developed a new pipeline. We first used BLASTX (v. 2.2.29, Altschul et al., 1990) searches against each of the 78 protein-coding genes over 100 bp long typical of plastid genomes from the data set of Ruhfel et al. (2014). BLAST hits having e-values <10 × e⁻²⁵ were used to extract the matching gene regions from scaffolds. Any fragment that had BLAST hits to multiple plastid genes was discarded. Because the plastid data were found to contain many transcripts that did not correspond to the correct taxon (likely due to either misassembly or contamination), we performed a BLASTN search, initially against the Ruhfel et al. (2014) data set. Samples were divided into 13 broad groups (“algae”, nonseed land plants, gymnosperms, basal angiosperms, monocots, basal eudicots, Saxifragales, campanulids, Caryophyllales, Cornales and Ericales, malvids, fabids, and lamiids), and if the best BLAST hit was to a sample outside these groups, the fragment was discarded. To take advantage of the increased coverage provided by the 1KP data added in the initial round, a second round of BLASTN searches and filtering was conducted, using the Ruhfel et al. (2014) data, plus the new fragments that had passed the initial filter above. This approach was used to increase sampling in the BLAST database across the phylogenetic breadth and reduce the chances of missing true matches where the initial data set may have had limited samples to match in some under-represented groups. All fragments that passed this second BLAST filter were retained. As many samples had multiple fragments for each gene, these were aligned to the Ruhfel et al. (2014) data for the corresponding gene using the –add option of Mafft (v. 7.125, Katoh and Standley, 2013). A single sequence for each taxon was obtained by joining non-overlapping fragments with gap characters and computing the consensus of overlapping portions of fragments. Fragments with greater than 5% sequence divergence in regions of overlap were discarded.

Data from all complete plastid genomes in GenBank as of 27 December 2014 were downloaded and parsed into gene files based on their annotations. Our data set thus consisted of the original Ruhfel et al. (2014) samples (360), additional genomes in GenBank (380), and 1KP samples (1139) (Appendix S1, see the Supplemental Data with this article). We used 52 red and brown algal samples to root the tree, leaving 1827 representatives of Viridiplantae.

Ruhfel et al. (2014), as well as initial analyses of our own data, found that shifts in GC content over the tree result in unconventional placement of some groups (e.g., Lycopodiophyta) in analyses of DNA sequences (see also Smith, 2009); thus, we restricted our analyses to an amino acid data set and did not conduct analyses removing third positions, or RY-coding as was done in Ruhfel et al. (2014). All gene sequences for all samples were therefore translated to amino acids using transeq (EMBOSS v. 6.5.7.0, Rice et al., 2000).

The amino acid data sets for each gene were aligned using Mafft. Each gene alignment was then processed with TrimAL (v. v1.4.rev15, Capella-Gutiérrez et al., 2009) using the gappyout option to remove poorly aligned positions. The individual gene alignments were concatenated, and samples with a concatenated total of fewer than 1000 amino acids were removed from the data set.

The resulting data set was analyzed in EXaML (v. 3.0.14, Kozlov et al., 2015). For the maximum likelihood (ML) analysis, 10 random and 10 parsimony starting trees were used. The per-site rate category model (PSR) was used. One hundred bootstrap (BS) data sets were analyzed; each bootstrap replicate used a single, randomly selected starting tree from the 10 random trees used for the ML search.

RESULTS

Data set properties

We recovered 78 protein-coding genes across the data set (Table 1). The concatenated alignment for the 78 genes from 1879 taxa consisted of 18,328 amino acid positions. The trimmed data set had approximately 30% missing data. In some cases, entire genes were missing from many samples (e.g., petN was recovered from only 650 taxa, as compared to psbA, recovered from 1829 taxa; see Table 1). Figure 1 summarizes data set completeness across the genes and taxa.

Table 1. List of the 78 protein-coding plastid genes used in the analysis and alignment properties. AA = amino acids

Gene	Number of taxa with sequences	Maximum ungapped length (AA)	Aligned length, pre-trimAL (AA)	Aligned length, post-trimAL (AA)	Average pairwise %-identity (AA)
accD	1216	1017	4376	235	79.3
atpA	1616	556	629	488	89.7
atpB	1589	577	731	498	92.1
atpE	1651	149	185	128	74.4
atpF	1597	230	395	180	70.6
atpH	1536	130	147	79	96.1
atpI	1621	313	448	246	88.0
ccsA	1476	447	1112	316	66.1
cemA	1514	582	1207	229	67.0
clpP	1538	556	1097	195	71.8
infA	1090	116	159	61	75.6
matK	1257	546	1057	364	64.9
ndhA	1443	385	649	362	79.7
ndhB	1304	545	786	518	88.0
ndhC	1389	135	170	119	86.4
ndhD	1403	531	681	495	80.7
ndhE	1301	111	129	99	84.1
ndhF	1327	791	1186	778	68.0
ndhG	1349	222	271	174	75.3
ndhH	1459	506	668	388	87.4
ndhI	1401	189	233	179	86.4
ndhJ	1447	174	228	155	85.8
ndhK	1403	318	520	285	83.7
petA	1711	394	548	320	84.1
petB	1751	235	264	213	95.5
petD	1718	224	307	155	93.3
petG	717	50	55	36	90.5
petL	671	86	119	30	79.4
petN	650	237	238	28	94.9
psaA	1750	769	944	745	92.7
psaB	1746	748	863	727	92.7
psaC	1717	103	110	74	95.4
psaI	669	226	275	33	78.3
psaJ	731	77	89	40	82.7
psbA	1837	359	417	350	96.5
psbB	1789	510	645	505	91.7
psbC	1787	487	570	449	94.6
psbD	1823	373	425	351	96.1
psbE	1748	490	497	81	90.6
psbF	718	49	56	38	94.5
psbH	1577	94	120	71	81.1
psbI	697	230	254	34	95.3
psbJ	717	50	62	40	87.8
psbK	733	91	117	42	83.4
psbL	679	43	43	37	93.9
psbM	709	97	135	33	87.2
psbN	851	97	122	42	87.7
psbT	717	508	508	32	93.0
psbZ	1196	99	123	60	82.5
rbcL	1774	490	548	474	92.2
rpl2	1713	314	491	263	80.1
rpl14	1732	147	195	121	81.6
rpl16	1716	171	260	130	81.4
rpl20	1618	157	290	112	66.8
rpl22	1407	194	335	184	66.9
rpl23	1432	112	201	89	73.3
rpl32	662	88	169	41	70.9
rpl33	1479	76	112	65	76.4
rpl36	729	196	196	35	84.8
rpoA	1565	858	2417	325	67.5
rpoB	1345	2143	4523	1069	72.3
rpoC1	1351	2348	5920	682	72.9
rpoC2	1264	3629	9252	1355	63.3
rps2	1658	951	1131	235	75.9
rps3	1690	745	1392	202	69.6
rps4	1637	257	538	200	76.5
rps7	1666	279	439	154	79.4
rps8	1650	177	339	129	72.7
rps11	1686	161	258	135	73.9
rps12	1511	165	324	103	87.8
rps14	1664	116	134	98	78.8
rps15	1400	116	168	39	77.2
rps16	1151	99	123	77	80.8
rps18	1566	251	596	98	76.0
rps19	1699	215	371	91	76.0
ycf2	657	2481	6703	335	76.9
ycf3	1614	201	330	167	85.7
ycf4	1,570	209	400	183	75.1

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Heatmap showing presence of sequence data for each gene and taxon across the phylogeny. Branches on the phylogeny are colored green to indicate 1KP transcriptome data and black to indicate data from NCBI or Ruhfel et al. (2014; mostly NCBI and genomic data). Genes are arranged alphabetically, as in Table 1 with *accD* on the inside and *ycf4* on the outside of the arcs; one or more letters are placed where the letter of the gene name changes, e.g., “i” indicates the position of *infA*, “*psb*” indicates where *psa* genes end and *psb* genes begin, etc. Heatmap colors: black, no sequence data for gene; blue, 0–25%; white, 25–50%; pink, 50–75%; red, 75–100% of full-length gene.

Missing genes may be due to lack of expression at the time of sampling or true loss from the plastomes of some species. Gene recovery was low in the outgroups, possibly due to the lack of a close reference genome for the BLAST searches among these taxa. Within Chlorophyta, the ndh genes are missing in both transcriptome and genome sequences, consistent with previous reports (Martín and Sabater, 2010; Wicke et al., 2011). Similarly, in the gymnosperms, Pinaceae and Gnetophyta lack the ndh genes. Poales have lost accD (Wicke et al., 2011). The eurosids also show a loss of infA in both genome sequences and transcriptomes, a result consistent with Millen et al. (2001). In contrast, many genes not recovered from transcriptomes are found in related genome sequences; for example, recovery of psbF through psbZ genes was low from transcriptomes, although these genes are generally present in genome sequences.

The plastid topology

The topology we obtained is summarized in Fig. 2, with the full tree with species names provided in Appendices S2 (cladogram with BS values) and S3 (phylogram). Viridiplantae are recovered as monophyletic (BS = 99%), within which Chlorophyta and Streptophyta (each with BS = 97%) are sisters (BS = 54%), with a small clade (BS = 100%) of Mesostigma, Chlorokybus, and the enigmatic Spirotaenia (Gontcharov and Melkonian, 2004) sister to this larger clade. The glaucophytes are sister to Viridiplantae (BS = 99%).

Within Chlorophyta, Prasinophyceae, Trebouxiophyceae, and Ulvophyceae are each nonmonophyletic, and the relationship of Chlorophyceae (monophyletic with BS = 99%) to these lineages is uncertain.

Within Streptophyta, Klebsormidiophyceae, along with Entransia and Interfilum, form a clade (BS = 100%) that is sister to the remaining streptophytes, which form a grade of Charales, Coleochaetales (BS = 100%), and a clade of Zygnematophyceae (BS = 96%) and embryophytes (BS = 97%); this clade of Zygnematophyceae and embyrophytes has BS support of 93%.

Within embryophytes, bryophytes are strongly supported as monophyletic (BS = 95%), with hornworts (BS = 100%) sister to a clade (BS = 93%) of liverworts and mosses, each with BS = 100%. The monophyly of Tracheophyta is poorly supported (BS = 54%); within Tracheophyta, a well-supported (BS = 100%) Lycopodiophyta is sister to a weakly supported (BS = 60%) Euphyllophyta clade comprising Monilophyta (ferns; BS = 100%) + Spermatophyta (BS = 97%).

Within Monilophyta, a well-supported clade (BS = 100%) of Equisetum + Psilotaceae is sister to Marattiales + leptosporangiate ferns (Polypodiidae sensu the Pteridophyte Phylogeny Group: PPG I, 2016) (BS = 100%). Within Spermatophyta, extant gymnosperms (Acrogymnospermae; BS = 100%) are sister to Angiospermae (BS = 97%). Within gymnosperms, Cycadophyta (cycads; BS = 100%) and Ginkgo form a clade (BS = 98%) that is sister to a clade in which Gnetophyta are nested within conifers (BS = 100%). We find strong evidence for the “Gne-Cup” hypothesis, with Gnetophyta forming a well-supported clade (BS = 98%) with Cupressophyta sensu Cantino et al. (2007), the latter comprising a clade (BS = 100%) of Araucariaceae (BS = 100%), Podocarpaceae (BS = 100%), Sciadopitys, Cephalotaxaceae (BS = 100%), Taxaceae (BS = 100%), and Cupressaceae (BS = 100%). This clade of Gnetophyta + Cupressophyta is sister to Pinaceae (BS = 100%).

The monophyly of angiosperms receives 97% BS support, with Amborella sister to all other extant angiosperms (BS = 80%); Nymphaeales (BS = 100%) and Austrobaileyales (BS = 100%) are successive sisters (BS = 80%) to all other angiosperms (Mesangiospermae; BS = 97%), comprising Magnoliophyta (magnoliids), Chloranthales, monocots, Ceratophyllum, and eudicots. Magnoliids (BS = 95%) and Chloranthales (BS = 100%) form a weakly supported clade (BS = 61%) that is sister to a clade (BS = 56%) of monocots (BS = 100%) and eudicots (BS = 97%) + Ceratophyllum (41%).

The placement of Ceratophyllum remains problematic, with only 41% bootstrap support for its placement as sister to the eudicots. Ranunculales are sister to the remaining eudicots (BS = 97%), followed by a grade of Proteales (BS = 100%), Meliosma, and Trochodendrales + Buxales (forming a clade with BS = 52%). Gunnerales are sister to Pentapetalae (97%), and Dillenia is sister to the remaining eudicots (BS = 97%), which comprise a clade of superrosids (BS = 99%) and superasterids (BS = 48%).

Within the superrosids, Vitales are sister to the remainder (BS = 61%), which comprise Saxifragales (BS = 100%) sister to the core rosids/eurosids (BS = 99%); the core rosids comprise the malvid and fabid clades, although each is weakly supported with only BS = 61% and 62%, respectively. The COM clade (Celastrales, Oxalidales, Malpighiales), long a problematic region of angiosperm phylogeny (e.g., Wurdack and Davis, 2009; Sun et al., 2015), is strongly supported as a clade (BS = 90%) and placed within the fabids as sister (BS = 74%) to Zygophyllales. Within the COM clade, there is weak support (BS = 59%) for (Celastrales + Oxalidales), which are in turn sister to Malpighiales (BS = 100%).

Within the superasterid clade, Santalales (BS = 100%) are sister to the rest of the clade (BS = 63%), with Berberidopsidales sister to a clade (BS = 94%) of Caryophyllales (BS = 100%) and asterids (BS = 98%). The asterids comprise a clade of [Cornales (BS = 100%) sister to a clade of Ericales (BS = 100%) + (campanulids (BS = 98%) + lamiids (BS = 91%))].

The topology for monocots generally agrees with recent analyses (with the exception of the placement of Petrosaviales; see below) (Chase et al., 2006; Graham et al., 2006; Givnish et al., 2010; Soltis et al., 2011; Hertweck et al., 2015). We found that Acoraceae (Acorales), followed by Alismatales, are each well supported (BS = 100%) and are subsequent sisters to all other monocots, which form a well-supported clade (BS = 100%). Within this remaining large clade of monocots, the major subclades are typically well supported: Commelinidae (Arecales, Commelinales, Poales, and Zingiberales), Dioscoreales, Petrosaviales, Pandanales, Liliales, and Asparagales. After Acoraceae and Alismatales, we recover Pandanales as sister to all remaining monocots, a placement that is weakly supported (remaining monocots have BS = 76%). Following Pandanales, Liliales and then Asparagales are sister to the remaining monocots. We then recover Petrosaviales as sister to the weakly supported (BS = 62%) remaining monocots consisting of Arecales + Commelinidae (BS = 83%). This placement of Petrosaviales differs from previous analyses in which following Acorales and Alismatales, Petrosaviales have been sister to all other monocots (Chase et al., 2006; Graham et al., 2006; see APG, 2016). Relationships obtained here within the larger clades of monocots are in general agreement with other recent analyses (see Chase et al., 2006; Graham et al., 2006; Givnish et al., 2010; Soltis et al., 2011; Hertweck et al., 2015). For example, within Commelinidae, we recovered Arecales as sister to Poales + (Commelinales + Zingiberales).

DISCUSSION

Low bootstrap support for some nodes

Several historically well-supported nodes in the Viridiplantae phylogeny received relatively weak support in our analyses (e.g., monophyly of vascular plants [BS = 54%]; monophyly of superasterids [BS = 48%]). While this could certainly be a sign of low support in this data set for these relationships, we believe most cases are due to the limited analyses we were able to conduct on each of the bootstrap data sets (a single analysis from one random starting tree). For the vascular plants, for example, about 40% of topologies found a large group of monilophytes sister to the remaining land plants (including the bryophytes). We reanalyzed six of these bootstrap data sets using a different random starting tree and found that all six found a tree consistent with the monophyletic vascular plant topology presented in Fig. 2. Thus, it appears that this reduced effort in analyzing bootstrap data sets generated trees that may have been stuck on local optima or suboptimal parts of tree space; these suboptimal trees varied in topology, yielding lower support for the monophyly of certain clades (see Mort et al., 2000). However, given that each bootstrap analysis uses about 300 CPU hours, using the same analytical rigor used for the maximum likelihood tree search (20 starting trees) would use over 600,000 CPU hours for the bootstrap analysis. Such an analysis was beyond the scope of our current study, although as data sets continue to grow, we encourage further research into the effects of limited analysis of bootstrap datasets (Mort et al., 2000) and new methods of inferring support for relationships (see Smith et al., 2018). It should also be acknowledged that it is possible that reduced rigor in analyses of bootstrap data sets may lead to higher bootstrap values, and support values should therefore be interpreted with caution.

Comparison to other plastid trees

Our results are generally congruent with the previous plastid genome analysis of Ruhfel et al. (2014); here we focus primarily on the amino acid analysis of that study. However, the current study includes five times as many taxa (1879) and provides much better coverage of all lineages of Viridiplantae. There are differences, however, between our results and those of Ruhfel et al. (2014). We recovered a well-supported (BS = 95%) bryophyte clade, but Ruhfel et al. (2014) found various relationships among hornworts, liverworts, and mosses, with the amino acid analysis supporting mosses and liverworts together (BS = 99%) as found here, but as sister to a clade of the single hornwort sample plus the remaining Embryophyta (BS = 53%). Indeed, none of the several different analyses of Ruhfel et al. (2014) recovered the bryophytes as monophyletic. Ruhfel et al. (2014) recovered Ceratophyllum and Piperales as sister to the monocots, although with low support (BS = 56%); we recovered Piperales as sister to Laurales (BS = 95%) and Ceratophyllum as sister to the eudicots, but with very low support (BS = 41%). The placement of Piperales in Ruhfel et al. (2014) disagrees with recent analyses and likely reflects limited taxon sampling in the magnoliids. Ceratophyllum has often been placed as sister to eudicots (e.g., Soltis et al., 2011; Sun et al. 2016; summarized by APG, 2016), but its position as sister to monocots has also been reported repeatedly (e.g., Zanis et al., 2002). The placement here of Dilleniaceae as sister to all core eudicots except Gunnera (i.e., to superrosids + superasterids) also conflicts with its position in Ruhfel et al. (2014), where it was sister to the superasterids (BS = 70%). The placement of Dilleniaceae has been problematic in all phylogenetic analyses of angiosperms (e.g., Soltis et al., 1999a, 2011).

In general, the increased taxon sampling in our analysis relative to Ruhfel et al. (2014) did not change the topology greatly or add bootstrap support to the nodes that were previously problematic. These problematic nodes may represent hard polytomies that result from rapid radiations (as proposed for mesangiosperms; Moore et al., 2010). Comparison of the plastid tree with a robust nuclear phylogeny may help reveal the causes of these remaining areas of poor resolution and support.

Discordance with other studies

In general, there is strong agreement between the plastid tree we report here and nuclear topologies based on 852 nuclear genes for 103 species reported by Wickett et al. (2014). However, several areas of discordance deserve discussion. We stress that most differences are weakly supported in either the plastid or nuclear trees (or both) and also conflict between concatenated vs. gene-tree-based nuclear trees (Wickett et al., 2014). Furthermore, some of the more noteworthy differences are represented by short branches in the ASTRAL nuclear tree. These branch lengths are proportional to coalescent units, and we infer that these problematic relationships reflect “non-tree-like” events, including ILS and hybridization. Perhaps the most noteworthy differences between plastid and nuclear topologies involve the relationships among bryophytes, among gymnosperms, and of magnoliids to either monocots or eudicots. These differences could be due to differences in taxon sampling between Wickett et al. (2014) and the current study; it will be important to rule out this issue through a comparable analysis of plastid and nuclear genes from the same large set of species (Green Plant Consortium, unpublished manuscript).

The position of Mesostigma + Chlorokybus has fluctuated in different analyses, with Lemiuex et al. (2007) finding this clade either sister to Streptophyta or Streptophyta + Chlorophyta. The nuclear analyses of Wickett et al. (2014) and the Green Plant Consortium (unpublished manuscript) both found Mesostigma + Chlorokybus sister to Streptophyta.

Our analyses of plastid data provide strong support (BS = 95%) for a bryophyte clade with hornworts sister to mosses + liverworts. In contrast, the nuclear tree of Wickett et al. (2014) based on concatenated genes provides 100% bootstrap support for mosses + liverworts, with hornworts sister to the tracheophytes; however, the ASTRAL analyses of nuclear gene trees recovered a bryophyte clade with 97% bootstrap support (Wickett et al., 2014).

Within gymnosperms, our plastid tree and that of Ruhfel et al. (2014) place the enigmatic Gnetophyta within conifers as sister to non-Pinaceae (the Gne-Cup hypothesis) with strong support (BS = 98%). In contrast, the concatenated nuclear tree of Wickett et al. (2014) placed Gnetophyta sister to Pinaceae with strong support (Gne-pine hypothesis; BS = 100%), while the ASTRAL tree found Gnetophyta sister to the conifers (Gnetifer hypothesis; BS = 89%). These topological differences in gymnosperm relationships have been noted previously (e.g., Bowe et al., 2000; Chaw et al., 1997; Lee et al., 2011; reviewed by Soltis et al., 2017).

Both concatenated and ASTRAL nuclear trees place magnoliids + Chloranthales sister to the eudicots with 100% bootstrap support (Wickett et al., 2014), while, in contrast, our analyses place magnoliids + Chloranthales sister to a clade of monocots and eudicots + Ceratophyllum. However, bootstrap support for these relationships is weak.

Another noteworthy discrepancy between plastid and nuclear trees is the placement of the COM clade (Celastrales–Oxalidales–Malpighiales) in differing positions within rosids. Previous analyses of nuclear genes (Wickett et al., 2014) have placed the clade in Malvidae, whereas plastid data place the clade within Fabidae, as seen here (reviewed by Sun et al., 2015).

Implications for Embryophyta evolution

Our results have major implications for resolving the sister group of Embryophyta. Despite many studies having found either Charophyceae or Coleochaetophyceae as sister to embryophytes (reviewed by Ruhfel et al. 2014; Wickett et al., 2014), recent analyses have converged with Zygnematophyceae as sister to all Embryophyta (BS = 93% in this study) (Timme et al., 2012; Ruhfel et al., 2014; Wickett et al., 2014). Thus, both plastid and nuclear genes support this placement. The data indicate that the complex morphological characters found in Zygnematophyceae, such as branching, parental retention of the egg, and plasmodesmata, either originated once in the common ancestor of Charophyceae, Coleochaetophyceae, Zygnematophyceae, and Embryophyta and were then lost in most lineages of Zygnematophyceae or these traits originated independently in Charophyceae, Coleochaetophyceae, and Embryophyta (reviewed by Judd et al., 2016).

Resolving relationships among the three lineages of bryophytes (hornworts, liverworts, and mosses) has been extremely difficult (reviewed by Ruhfel et al., 2014; Wickett et al., 2014; Judd et al., 2016). As multiple papers have indicated (Qiu et al., 1998; Nickrent et al., 2000; Renzagalia et al., 2000; Nishiyama et al., 2004; Chang and Graham, 2011; reviewed by Ruhfel et al., 2014; Wickett et al., 2014), every possible branching order of relationships among these three lineages has been obtained in analyses using diverse molecular and morphological data. Our result indicating a bryophyte clade mirrors the recent results of Wickett et al. (2014) using a large number of nuclear genes (but fewer taxa); however, as noted, this result for nuclear genes was obtained only from the coalescent analysis and not the concatenated analysis. Importantly, recent analyses of more taxa (but fewer nuclear genes than in Wickett et al. 2014) also reveals a bryophyte clade (Green Plant Consortium, unpublished manuscript).

Resolving the relationships among the three lineages of bryophytes has major implications for the origin and evolutionary transformation of the heteromorphic alternation of generations typical of all embryophytes. The traditional hypothesis, based on a grade of bryophytes, as often reported in previous studies, suggests that a gametophyte-dominant life cycle is ancestral in embryophytes (Ligrone et al., 2012; Judd et al., 2016) and the sporophyte-dominant life cycle of tracheophytes is derived. However, if bryophytes are indeed a clade, as our results indicate, then the alternation of generations of the common ancestor of all embryophytes cannot be unequivocally reconstructed, with gametophyte-dominant and sporophyte-dominant life cycles both plausible hypotheses of the ancestral state.

Future of plastid phylogenomics

We may be close to exhausting the power of the plastid genome for resolving deep-level phylogenetic questions of relationships in Viridiplantae (cf. Davis et al., 2014). Particularly enigmatic areas of the Viridiplantae tree of life first revealed with a few genes remain problematic with the entire protein-coding component of the plastid genome, despite dense taxon sampling in the problematic portions of the tree. Moreover, although some of the uncertain nodes are deep within Viridiplantae phylogeny, many others occur at more shallow levels, suggesting that perhaps different processes—extinction vs. recent radiations, for example—may be responsible at different points in the tree. Notable areas of uncertainty include the placement of Chloranthales and magnoliids, the positions of eudicot lineages, such as Dilleniaceae, and the placement of Ceratophyllaceae. Adding more taxa will continue to fill in the leaves of the tree, but will likely do little to improve support for deeper nodes. Filling missing genes may help with resolution and support, and adding noncoding regions will provide more characters (although such characters will also augment challenges of alignment and varying GC content). Thus, it seems unlikely that plastid genome analysis alone will recover a single, well-supported topology resolving all remaining deep-level questions.

Sequencing of the plastid genome has now become routine, and obtaining complete or nearly complete plastid genomes will soon be as commonplace as the sequencing of a few genes via PCR. Although current work suggests that plastid data will likely not resolve all long standing problems in Viridiplantae phylogeny, plastid genome sequencing has a very bright future and broad application as we move to the tips of the Viridiplantae tree of life, particularly within clades recognized as orders, families, and genera.

Future analyses should focus on elucidating the underlying causes responsible for the noteworthy discrepancies between plastid and nuclear phylogenetic trees and for regions of poor support. It is our hope that these studies will usher in a new era in molecular systematics that explicitly incorporates phylogenetic uncertainty and conflict in studying and using the tree of life. Despite its longstanding role in plant phylogenetics, the plastid genome provides only one perspective on plant evolutionary history, and a plastid-based tree should not be blindly accepted as the backbone tree for Viridiplantae. Incongruent placements between nuclear and plastid trees (e.g., the COM clade) are points of concern for reconstructing phylogeny, but opportunities for understanding the intricacies of plant evolution and should be the focus of intensive study.

ACKNOWLEDGEMENTS

The authors thank the many researchers across the world who supplied samples and, in many cases, extracted RNA for the 1KP project. Without these samples, this project would not have been possible. These major contributors are recognized as coauthors on the 1KP synthesis paper (Green Plant Consortium, unpublished manuscript). In addition, we thank University of Florida Research Computing for providing access to HiPerGator for the computational analyses conducted in this study. The authors also thank two anonymous reviewers and the editor for their helpful suggestions to improve the manuscript.

DATA ACCESSIBILITY

The assembled transcriptomes from the 1KP project used for this study are publicly available at http://www.onekp.com/public_data.html. All raw sequence data have been deposited in the SRA: see http://www.onekp.com/public_read_data.html for list of accession numbers. Scripts used to analyze the data as well as single-gene and concatenated amino acid alignments are available at https://github.com/magitz/1KP_Plastid.

Supporting Information

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Volume105, Issue3

Special Issue:Using and Navigating the Plant Tree of Life

March 2018

Pages 291-301

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Plastid phylogenomic analysis of green plants: A billion years of evolutionary history