RESEARCH ARTICLE Open Access From algae to angiosperms

26 Pages·2014·1.94 MB·English

Ruhfeletal.BMCEvolutionaryBiology2014,14:23
http://www.biomedcentral.com/1471-2148/14/23
RESEARCH ARTICLE Open Access
–
From algae to angiosperms inferring the
phylogeny of green plants (Viridiplantae) from
360 plastid genomes
Brad R Ruhfel1*, Matthew A Gitzendanner2,3,4, Pamela S Soltis3,4, Douglas E Soltis2,3,4 and J Gordon Burleigh2,4
Abstract
Background: Next-generation sequencing has provided a wealth of plastid genome sequence data from an
increasingly diverse set of green plants (Viridiplantae).Although these data have helped resolve the phylogeny of
numerous clades (e.g., green algae, angiosperms, and gymnosperms), theirutility for inferring relationships across all
green plants is uncertain. Viridiplantae originated700-1500 million years ago and may comprise as many as 500,000
species. Thisclade represents a major source ofphotosynthetic carbon and contains animmensediversity oflife
forms, including some ofthe smallest and largest eukaryotes. Here weexplore the limits and challenges of inferring
acomprehensivegreenplantphylogenyfromavailablecompleteornearlycompleteplastidgenomesequencedata.
Results:Weassembledprotein-codingsequencedatafor78genesfrom360diversegreenplanttaxawithcomplete
ornearlycompleteplastidgenomesequencesavailablefromGenBank.Phylogeneticanalysesoftheplastiddatarecovered
well-supportedbackbonerelationshipsandstrongsupportforrelationshipsthatwerenot observed in previous analyses
of major subclades within Viridiplantae. However, there also is evidence of systematic error in some analyses. In
several instances we obtained strongly supported but conflicting topologies from analyses of nucleotides versus
amino acid characters, and the considerable variation in GC content among lineages and within single genomes
affected the phylogenetic placement of several taxa.
Conclusions: Analysesoftheplastidsequencedatarecoveredastronglysupportedframeworkofrelationshipsfor
greenplants.Thisframeworkincludes:i)theplacementofZygnematophyceaceassistertolandplants(Embryophyta),ii)
acladeofextantgymnosperms(Acrogymnospermae)withcycads+Ginkgosistertoremainingextantgymnosperms
andwithgnetophytes(Gnetophyta)sistertonon-Pinaceaeconifers(Gnecuptrees),andiii)withinthemonilophyteclade
(Monilophyta),Equisetales+PsilotalesaresistertoMarattiales+leptosporangiateferns.Ouranalysesalsohighlightthe
challengesofusingplastidgenomesequencesindeep-levelphylogenomicanalyses,andweprovidesuggestionsfor
futureanalysesthatwilllikelyincorporateplastidgenomesequencedataforthousandsofspecies.Weparticularly
emphasizetheimportanceofexploringtheeffectsofdifferentpartitioningandcharactercodingstrategies.
Keywords:Compositionbias,Phylogenomics,Plastidgenomesequences,Plastomes,RY-coding,Viridiplantae
Background than one billion years old [11-14]. Reconstructing the
Viridiplantae, or green plants, are a clade of perhaps phylogenetic relationships across green plants is challen-
500,000species[1-6]thatexhibit anastoundingdiversity ging because of the age of the clade, the extinction of
of life forms, including some of the smallest and largest major lineages [15-17], and extreme molecular rate and
eukaryotes [3,7]. Fossil evidence suggests the clade is at compositional heterogeneity [18-22]. Most phylogenetic
least 750 million years old [8-10], while divergence time analyses of Viridiplantae have recovered two well-
estimates from molecular data suggest it may be more supported subclades, Chlorophyta and Streptophyta
[23,24]. Chlorophyta contain most of the traditionally
*Correspondence:[e-mail] recognized “green algae,” and Streptophyta contain the
1DepartmentofBiologicalSciences,EasternKentuckyUniversity,Richmond,
land plants (Embryophyta), as well as several other
KY40475,USA
Fulllistofauthorinformationisavailableattheendofthearticle lineages also considered “green algae”. Land plants
©2014Ruhfeletal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycredited.
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include the seed plants (gymnosperms and angiosperms; We assembled available plastid genome sequences to
Spermatophyta), which consist of ~270,000 to ~450,000 build a phylogenetic framework for Viridiplantae that
species[1,3]. reflects the wealth of new plastid genome sequence data.
While many of the major green plant clades are well Furthermore, we highlight analytical challenges for re-
defined, questions remain regarding the relationships solving the green plant tree of life with this type of data.
among them. For example, the closest relatives of land We performed phylogenetic analyses of protein-coding
plants have varied among analyses [23,25-29], as have data on 78 genes from 360 taxa, exploring the effects of
the relationships among the three bryophyte lineages different partitioning and character-coding protocols for
(mosses, liverworts, and hornworts) [29-35]. The rela- the entire data set as well as subsets of the data. While
tionships among extant gymnosperms also remain con- our analyses recover many well-supported relationships
tentious, particularly with respect to the placement of and reveal strong support for some contentious relation-
Gnetophyta[20,36-43]. ships, several factors, including base composition biases,
Most broad analyses of green plant relationships based can affect the results. We also highlight the challenges
on nuclear gene sequence data have relied largely on of using plastid genomedata indeep-level phylogenomic
18S/26S rDNA sequences [30,37,44,45], although recent analyses and provide suggestions for future analyses that
analyses have employed numerous nuclear genes [40,46]. will incorporate plastid genome data for thousands of
Some studies have used mitochondrial gene sequence species.
data, often in combination with other data [29,47,48].
However, investigations of green plant phylogeny typic- Results
ally have either largely or exclusively employed chloro- Dataset
plast genes (e.g., [29,49-52]). Sequence data from the We assembled plastid protein-coding sequences from 360
plastid genome have transformed plant systematics and species (Additional file 1) for which complete or nearly
contributed greatly to the current view of plant relation- completeplastidgenomesequenceswereavailableonGen-
ships. With the plastid genome present in high copy Bank. Of the 360 species, there were 258 angiosperms
numbers in each cell in most plants, and with relatively (Angiospermae), 53 gymnosperms (Acrogymnospermae, in-
little variation in gene content and order [53], as well as cluding three Gnetophyta), seven monilophytes (Monilo-
few reported instances of gene duplication or horizontal phyta), four lycophytes (Lycopodiophyta), three liverworts
gene transfer [54,55], the plastid genome provides a (Marchantiophyta), one hornwort(Anthocerotophyta), two
wealth of phylogenetically informative data that are rela- mosses(Bryophyta),sixtaxafromtheparaphyleticstrepto-
tively easy to obtain and use [56,57]. Although early phyticalgae, and26 chlorophyticalgae(Chlorophyta). The
phylogenetic studies using one or a few chloroplast loci phylogenetic character matrices contained sequences from
provided fundamental insights into relationships within 78genesandthefollowingnumberofalignmentpositions:
and among green plant clades, these analyses failed to 58,347bpforthematrixcontainingallnucleotidepositions
resolve some backbone relationships [56-59]. These (ntAll) and the RY-coded (RY) version of the ntAll matrix;
remainingenigmaticportionsofthegreenplanttreeoflife 38,898 bp in the matrix containing only the first and sec-
ultimately motivated the use of entire, or nearly entire, ond codon positions (ntNo3rd), and 19,449 amino acids
plastidgenomesequencesforphylogeneticinference. (AA).The number of genespresent per taxon varied from
Complete sequencing of the relatively small (~150 kb) 18to78(mean=70),whilethenumberoftaxapresentper
plastid genome has been technically feasible since the gene ranged from 228 to 356 (mean=322; see Additional
mid-1980s [60,61], although few plastid genomes were file 2). Taxa with few genes present, such as Helicospori-
sequenced prior to 2000 (see [62,63]). Next-generation dium (18 genes) and Rhizanthella (19 genes), represent
sequencing (NGS) technologies, such as 454 [62] and highly modified complete plastid genomes of non-
Illumina [64-67], greatly reduced the cost and difficulty photosynthetic species[70,71].Thepercentageofmissing
of sequencing plastid genomes, and consequently, the data (gaps and ambiguous characters) was ~15.6% for
number of plastid genomes available on GenBank in- eachofthefourdatasets.Thepatternofdataacrosseach
creased nearly six-fold from 2006 to 2012 [68]. Phylogen- of the four matrices is decisive, meaning that it can
eticanalysesbasedoncompleteplastidgenomesequences uniquely define a single tree for all taxa [72]. The data
have provided valuable insights into relationships among contain 100% of all possible triplets of taxa, and are de-
andwithinsubcladesacrossthegreenplanttreeoflife(re- cisive for 100% of all possible trees. All alignments have
cently reviewed in [26,35,68,69]). Still, studies employing beendepositedintheDryadDataRepository[73].
complete plastid genomes generally have either focused
on subclades of green plants or have had relatively low GCbias
taxon sampling. Thus, they have not addressed the major GC content varied considerably both among lineages
relationshipsacrossallgreenplantssimultaneously. and also within single genomes, and chi-square tests
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rejected the null hypothesis of homogeneous base fre- AA (Additional file 10). Average support values among
quencies (Table 1). The average GC content in the ntAll all internal nodes in the ML trees were slightly higher
matrix was 38.9%, and it ranged from 54.3% in Selagin- in the ntAll phylogeny (~94% bootstrap support [BS];
ella uncinata to 27.5% in Helicosporidium sp. (Figure 1, Additional file 7) compared to the other data sets
Additional file 3). Also, the average GC content varied (~90-91% BS; Additional files 8, 9, and 10). The ntAll
among first, second, and third codon positions, with by phylogeny also had the most clades resolved with ≥
far the most variation among lineages at the third codon 70% BS (92%; 327 bipartitions resolved out of 357 pos-
position (Figure 1, Additional file 3). Although there was sible) while the ntNo3rd, RY, and AA data sets had 87%,
extensive heterogeneity in GC content across all species, 87%, and 86% of the possible bipartitions resolved at ≥
there was relatively little variation among the seed plant 70% BS, respectively. All resulting trees have been depos-
taxa (Figure 2). There also was significant correlation itedintheDryadDataRepository[73].
between nucleotide composition and amino acid com- The monophyly of Chlorophyta receives 100% BS in all
position. Plastid genomes that are GC-rich had a signifi- analyses. Prasinophyceae are consistently not monophy-
cantly higher percentage (Figure 3; p<0.001) of amino letic.Instead,theprasinophyteNephroselmisissistertoall
acids that are encoded by GC-rich codons (i.e., G, A, R, other Chlorophyta (Figure 9; Additional files 4, 5, and 6),
and P). Similarly, GC-rich plastid genomes had a signifi- while remaining Prasinophyceae form a clade that is vari-
cantlylowerpercentage(Figure4;p<0.001)ofaminoacids ously supported (ntAll 97% BS, ntNo3rd 78% BS, RY 93%
thatarecodedbyAT-richcodons(i.e.,F,Y,M,I,N,andK). BS, and AA 68% BS) and is sister to a clade of the
remaining Chlorophyta. Chlorophyceae are monophyletic
Phylogeneticanalyses (100%BSinallanalyses),butTrebouxiophyceaeandUlvo-
Inthephylogeneticanalysesofalldatasetsandpartitioning phyceae are not monophyletic, and the relationship of
schemes, the partitioning strategy with the most partitions Chlorophyceaetotheselineagesisunresolved.
consistently fit the data best based on the AICc (Table 2). We consistently recovered a single set of relationships
These best-fit models partitioned the AA matrix by gene among the streptophytic algae subtending the land plant
(78 partitions) and the nucleotide (ntAll, ntNo3rd) and RY clade. Zygnematophyceae are sister to land plants,Coleo-
matricesbycodonpositionandgene(234partitions).Alla chaetophyceae are sister to Zygnematophyceae+Embryo-
posterioribootstoppinganalysesindicatedthatconvergence phyta, Charophyceae are sister to Coleochaetophyceae+
of support values had been reached after 100 replicates, (Zygnematophyceae+Embryophyta), and a clade of
and thus our choice of 200 replicates was more than suffi- Mesostigmatophyceae+Chlorokybophyceae is sister to all
cienttoobtainreliablebootstrapvalues. other Streptophyta. Each of these relationships has ≥86%
We will focus on reporting the relationships of major BSsupport(Figures5,6,7,and8).
clades of Viridiplantae shown in the 50% maximum like- The branching order of the non-vascular land plant
lihood (ML) majority-rule bootstrap consensus summary lineages differs among analyses. In analyses of the ntAll
treesforeachdataset:ntAll(Figure5),ntNo3rd(Figure6), and RY data sets, Marchantiophyta (liverworts), followed
RY (Figure 7), and AA (Figure 8). These summary trees by Bryophyta (mosses), and then Anthocerotophyta
collapse some clades for ease of viewing the major rela- (hornworts) are the earliest-branching land plant
tionships within Viridiplantae. A summary of important lineages, with Anthocerotophyta the immediate sister
resultsandconflictsamongthesefourdatasetsisgivenin to the vascular plants (Tracheophyta; Figures 5 and 7).
Table 3. We provide full majority-rule bootstrap consen- In the ntAll andRYanalyses,theserelationshipshad≥89%
sus trees for the ntAll (Figures 9, 10, 11, 12, 13, and 14), BS support except for the Bryophyta+(Anthocerophyta+
ntNo3rd(Additionalfile4),RY(Additionalfile5),andAA Tracheophyta) relationship in the ntAll analysis, which
(Additionalfile6)data sets.MLtreeswithbranchlengths receivedonly69%BS(Figure5).Incontrast,inthentNo3rd
andBSvaluesarealsoprovided:ntAll(Additional file 7), andAAanalyses,BryophytaandMarchantiophytaformeda
ntNo3rd (Additional file 8), RY (Additional file 9), and clade (78% BS [Figure 6] and 99% BS [Figure 8], respect-
ively),followedbyAnthocerophytaassistertoTracheophyta
Table1Chi-squaretestsofnucleotidecomposition (94%[Figure6]and53%BS[Figure8],respectively).
homogeneityamonglineages
Within Tracheophyta, the ntNo3rd, RY, and AA data
Data χ2 df p sets all place Lycopodiophyta sister to a Euphyllophyta
ntAll 31350.257185 1077 <0.0001 clade(Monilophyta+Spermatophyta;≥89%BS,Figures6,
ntNo3rd 11968.002464 1077 <0.0001 7, and 8). However, the analysis of the ntAll data set
places Monilophyta sister to aclade of Lycopodiophyta+
ntAll(Position1) 8366.331439 1077 <0.0001
Spermatophyta(75% BS,Figures 5,6,7,8,9,and10).
ntAll(Position2) 6003.338041 1077 <0.0001
Our analyses of Monilophyta generally reveal strong
ntAll(Position3) 46288.248785 1077 <0.0001
support for a clade of Equisetales+Psilotales as sister
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0
7
0
6
0
5
ntent 40
o
C
C
G
nt
e
erc 30
P
0
2
0
1
0
ntAll ntNo3rd 1st codon 2nd codon 3rd codon
position position position
Figure1BoxplotsofpercentGCcontentinthentAllandntNo3rddatasetsaswellasinthefirst,second,andthirdcodonpositions
ofthentAlldataset.
to Marattiales+leptosporangiate ferns (represented by Nymphaeales+(Austrobaileyales+Mesangiospermae) re-
CyathealesandPolypodiales).Thelowest supportobtained ceives81%BS(Figure6)inthentNo3rd analysesand 70%
was for Equisetales+Psilotales in the ntNo3rd analysis BS (Figure 8) in the AA analyses. The remaining angio-
(84% BS; Figure 6) and ntAll (89% BS; Figure 5); all other sperms (Mesangiospermae) receive 100% BS in all ana-
nodesinallanalysesreceived>90%BS,withMarattiales+ lyses. Within Mesangiospermae, the relationships among
leptosporangiatefernsreceiving≥99%BS. Monocotyledoneae, Magnoliidae, Eudicotyledoneae, and
Within Spermatophyta, all analyses place the extant Ceratophyllum (Ceratophyllales) are not well supported
gymnosperms (Acrogymnospermae) sister to Angiosper- andvarydependingontheanalysis.Thestrongest support
mae with 100% BS. Within extant gymnosperms, Cyca- for the placement of Ceratophyllales is75% BS as sister to
dales and Ginkgoales form a clade (≥ 98% BS in ntAll, EudicotyledoneaeintheRYanalysis(Figure7).
ntNo3rd, and AA; 51% BS in RY) that is sister to a clade Chloranthales receive 61-69% BS as sister to the well-
inwhichGnetophyta(100%BSinallanalyses)arenested supported (100% BS in ntAll, RY; 83% BS in ntNo3rd)
within the paraphyletic conifers. There is generally high Magnoliidae. However, Magnoliidae are not monophy-
support(100%BSinntAll[Figure5],ntNo3rd[Figure6], letic in the AA analyses, where Piperales are sister to
and AA [Figure 7]; 87% BS [Figure 8] in RY) placing Ceratophyllales(67% BS;Figure8).
Gnetophyta as sister to a clade of Araucariales+Cupres- Withinthemonocotclade(Monocotyledoneae),Acorales,
sales. This “Gnecup” clade [sensu 16, 30, 41] is then sis- followed by Alismatales, have 100% BS in all analyses as
tertoPinales,whichhas100%BSinallanalyses. subsequent sisters to the remaining monocots. In three of
In all analyses, Angiospermae receive 100% BS, and our analyses (ntAll, ntNo3rd, and AA), a variously sup-
Amborella(Amborellales)issistertoallotherangiosperms, ported clade (72%, 69%, and 80% BS, respectively) of
followedbyNymphaeales,andthenAustrobaileyales.These Liliales+(Pandanales+Dioscoreales) is sister to a clade
relationships are mostly supported by 100% BS. However, (>95% BS in these three analyses) of the remaining
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0
7
0
6
0
5
nt
onte 40
C
C
G
nt
erce 30
P
0
2
0
1
0
ntAll ntNo3rd 1st codon 2nd codon 3rd codon
position position position
Figure2BoxplotsofpercentGCcontentinseedplants(Spermatophyta;onleft)andthedatasetasawhole(Viridiplantae;onright)in
thentAllandntNo3rddatasetsaswellasthefirst,second,andthirdcodonpositionsofthentAlldataset.Foreachpairofboxplots,
valuesforseedplants(Spermatophyta)areontheleft,andvaluesforallgreenplanttaxa(Viridiplantae)areontheright.
0
5
0
4
s
d
ci
a
no 30
mi
a
P
R
A
G
ent 20
c
er
P
y = 0.41x + 8.83
0 r2 = 0.494
1
p < 0.001
0
0 10 20 30 40 50 60
Percent GC content
Figure3CorrelationbetweenpercentGCnucleotidecontentinthentAllmatrixandpercentofaminoacidsintheAAmatrixthatare
codedforbyGC-richcodons(G,A,R,andP).
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0
5
0
4
s
d
ci
a
mino 30
a
K
N
MI
Y
nt F 20
e
c
er
P
y = -0.53x + 49.00
0 r2 = 0.603
1
p < 0.001
0
0 10 20 30 40 50 60
Percent GC content
Figure4CorrelationbetweenpercentGCnucleotidecontentinthentAllmatrixandpercentofaminoacidsintheAAmatrixthatare
codedforbyAT-richcodons(F,Y,M,I,N,andK).
monocots (Asparagales+Commelinidae). However, in the remaining taxa. In the ntAll, ntNo3rd, RY, and AA ana-
RY-coded analysis, Pandanales+Dioscoreales(100% BS) is lyses, the clade of these remaining taxa receives 100%,
sistertoacladeofLiliales+(Asparagales+Commelinidae), 85%, 100%, and 62% BS, respectively. Relationships vary
which receives 69% BS (Figure 7). Here Asparagales+ among Sabiaceae, Proteales, and a clade ofthe remaining
Commelinidaeissupportedby80%BS. taxa,dependingontheanalysis.InthentAllandntNo3rd
Within the eudicots (Eudicotyledoneae), which receive analyses, Proteales+Sabiaceae are supported as a clade,
100% BS in all analyses, Ranunculales are sister to the although with only 63% and 60% BS, respectively.
Table2AICcscoresforeachofthephylogeneticmatrixpartitioningstrategies
Matrix Numberof Partitioningstrategy Numberof Log-likelihood AICc ΔAICc
characters partitions
ntAll 58,347 OnePart 1 −3135739.544116 6272952.811161 114533.884536
CodonPart 3 −3099273.099639 6200056.468462 41637.541838
GenePart 78 −3120195.077316 6243312.241766 84893.315142
CodonGenePart 234 −3076219.426792 6158418.926624 0
RY 58,347 OnePart 1 −1239354.453402 2480173.246480 21572.787069
CodonPart 3 −1235533.368070 2472537.854401 13937.394990
GenePart 78 −1234706.178899 2471197.311314 12596.851903
CodonGenePart 234 −1228081.159986 2458600.459411 0
ntNo3rd 38,898 OnePart 1 −1387913.034830 2777313.721117 30326.016847
CodonPart 2 −1385570.086154 2772645.570816 25657.866546
GenePart 78 −1376158.263023 2755293.787916 8306.083646
CodonGenePart 156 −1371218.716450 2746987.704270 0
AA 19,449 OnePart 1 −1418038.152084 2837614.101717 8353.616354
GenePart 78 −1413039.660496 2829260.485363 0
PartitioningstrategiesjudgedtobethebestbytheAICcareinbold.
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100 100 Brassicales
1010010010011000008110010107070 93 11111111900000000100000000 COSMFFCRGMMaaaruoxeaayobgpacsrrllsvptaaaiualnaiasidnllrlgdeeleolbeaeihaassssissltielloaaeesmllsseeasstalesFabidaeMalvidaeSuperrosidae
Celastrales P
100713001001009959918010010010010091090100609 75111111111100000000000000000000 VDGBDAGBZSCCEGSAALSayaoeroiapqsiiaoueatplimlgancrltrixurenrrnaaseabrynaotlintyinnaeialfgireofaeaarplaeieolaacsaiaalrpslnnhelreaisllellgacedieheasyaaslsesleaosyseclllleeaasllpeselsalseaseslieesdsales Lami.Campanuli.Superasteridae entapetalae EudicotyledoneaeMesangiospermaeAngiospermaeSperm
761001010010005921800100 810603072111111111000000000000000000 DPTBSCAAPRDZCPriausorarieaaonooeopnxabrnsmgscctaaadliyeuiechaabtmlrpaaoneosocaleeonlpscreedgersegauahaleasianolllyeeenlaelanleslsdlaselaserlscaeselsease Commelinidae Monocotyledon atophyta TracheophytaEmbryophyta Streptophyta
100100100 10099111000000 LLMAAailclaiisuaogmrlreanaalsoleetlasisaleless Magneae
100 68 85 PCiapneerallaleless oliid
100 100 CAuhslotrraonbtahialeleysales ae
Nymphaeales
10010071500 100100 11001001100010010900810011111010000000000000 EWCGGLAIPASCCPsypomreiyoyuninnaechalcleepyaaokbulaetwpttrhgglpocaaeeddoieoiaorlltnssreeadesaaadrseslsilcillealliaeeealaahlsllasessllleeieealssesssless Gnetophyta Acrogymnospermae MonLyco.
100 Marattiales ilo
69 89 Equisetales ph
10097100 111000000 APMBrnsayitlrohocpothahcaleyentrsatoiotopphhytyata yta
100 Zygnematophyceae
Coleochaetophyceae
100 Charophyceae
100 Mesostigmatophyceae
100 Chlorokybophyceae
63 100 TCrhelbooroupxhioypcehayeceaep.p. C
10087100 95578186 PTUUTrrrllevveaoobbsppooinhhuuoyyxxpcciiohoeepypaachheeeyy apccpe.ee.pp aa.p.ee.p pp...pp.. hlorophyta
Prasinophyceae p.p.
Figure5Fiftypercentmaximumlikelihoodmajority-rulebootstrapconsensussummarytreeofViridiplantaeinferredfromtheall
nucleotidepositions(ntAll)analysis.Datasetderivedfrom78protein-codinggenesoftheplastidgenome(ntax=360;58,347bp;missingdata~15.6%).
Bootstrapsupportvalues≥50%areindicated.Terminalswithatrianglerepresentcollapsedcladeswith>2taxa.NotepositionofLycopodiophytaas
sistertoSpermatophytaislikelycausedbybasecompositionbias(seetext).SeeFigures9,10,11,12,13,and14forthecompletetreeand
Additionalfile1fortaxonomy.Lami.=Lamiidae;Campanuli.=Campanulidae;Lyco.=Lycopodiophyta.
However,intheRYanalysis,Protealesaresistertoaclade The placement of Dilleniaceae remains uncertain. The
containing Sabiaceae plus the remaining taxa, which has family is sister to Superrosidae in the ntAll (95% BS),
79% BS. In the AA analysis, relationships among these ntNo3rd (77% BS),and RY (57% BS) analyses, but appears
threecladesareunresolved. assistertoSuperasteridae(70%BS)intheAAanalysis.
Among the remaining eudicots, we consistently recov- Within Superrosidae, a clade of Vitales+Saxifragales
ered Trochodendrales as sister to Buxales+Pentapetalae is supported in the ntAll (75% BS), ntNo3rd (70% BS),
and Gunnerales as sister to the remaining lineages of Pen- and AA (78% BS) analyses. In the RYanalysis, the rela-
tapetalae: Dilleniaceae, Superrosidae, and Superasteridae. tionship among Saxifragales, Vitales, and remaining
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100 100 Brassicales
1001007089100 70100895592 111111110000000000000000 CSOMFFCRGMMaaarouxeaayopgbascrrllsvptaiaaaulnaisaidnlllrgdeleoelbeaeihaassssislstielolaaeesmsllseeasstalesFabidaeMalvidaeSuperrosidae
Celastrales
77 70100 ZSyagxoifprahgyallaleless E
10052541001010009611000088541007310099957591300991001011000001005532 52160006911111111111111111110000000000800000000000009000000000000000 PAADVSBLPDTBGABDGAAPRGDZCSCCEAALSiarrisrauoaolceiaoqpiirisalauoaeoanitpooloiesimlpaaonxrabnclrtiurensnmnrrgaassctcmaebrnyaarleladtliynuytinieneeachaiaalagbirefaeomrlaapaalaaeenioossecaolscaalliaearelespnnlolnelrtscareeieleeldslgaracldeisseehaegsaaaualsassleaellesiosaeecysonllllleeelneeaaspeelslsnalessdslasesasealserliesecadseslaealsees Lami.Campanuli.SuperasteridaeCommelinidaePentapetalae udicotyledoneaeMonocotyledoneae Mesangiospermae AngiospermaeSpermatophyta EuphyllophytaTracheophyta Embryophyta Streptophyta
81100 61839215020 CCLPCMaiahepaunlregoarearnrtaalololealnpelsilatehshlsyealsllaelses Magnoliidae
100 100 ANuysmtrpohbaaeilaelyeasles
100100919009140093100 101000 101090910110700101180000000011111111110800000000040000000000 ABSCAWGALEGIZPPMPCMCEsyyrrepnmoqisouyynianyaanecgihtlcaluepelyaroharuokblonacewtiarpttspghgolpcoteeaaheddtaoeihieocatornmlasltsreaeldieteysaaearadsenaslsilitcarelllaslliaaeetlealaaoeleihtsolalosselslpsleseeiepalpshesssshlheysyytsatcaeaeGnetophyta AcrogymnospermaeMonilophytaLycopodiophyta
Coleochaetophyceae
100 Charophyceae
100 Mesostigmatophyceae
59 100 CChhlloorroopkhyybcoepaheyceae
10078100 108672800 UTPTUTrrrrlleevevaoobbbsppoooinhhuuuoyyxxxpcciiioooheepppyaahchheeeyyy capccpee.ee.ppa aap.e.ee. p ppp....ppp... Chlorophyta
Prasinophyceae p.p.
Figure6Fiftypercentmaximumlikelihoodmajority-rulebootstrapconsensussummarytreeofViridiplantaeinferredfromthefirstand
secondcodonpositions(ntNo3rd)analysis.Datasetderivedfrom78protein-codinggenesoftheplastidgenome(ntax=360;38,898bp;
missingdata~15.6%).Bootstrapsupportvalues≥50%areindicated.Terminalswithatrianglerepresentcollapsedcladeswith>2taxa.See
Additionalfile4forthecompletetreeandAdditionalfile1fortaxonomy.Lami.=Lamiidae;Campanuli.=Campanulidae.
Rosidae (Fabidae+Malvidae) is unresolved. Fabidae Figure 5) treesandformsatrichotomywithZygophyllales
and Malvidae are both recovered with≥99% BS in the andthecladeofFagales,Cucurbitales,Rosales,andFabales
ntAll and RY analyses. However, each clade receives in the ntNo3rd tree (70% BS; Figure 6). Zygophyllales are
only 70% BS in the ntNo3rd analysis. In the AA ana- sister to Geraniales (69% BS; Figure 8) in the AA tree and
lysis neither clade is monophyletic; Zygophyllales are sister to all other Fabidae in the ntAll and RYtrees (with
embedded (68% BS) within a clade of Malvidae taxa. 100%[Figure5]and99%BS[Figure7],respectively).
The COM clade (Celastrales, Oxalidales, Malpigh- Superasteridae (Santalales, Berberidopsidales, Caryo-
iales) is sister to a clade of Fagales, Cucurbitales, phyllales, and Asteridae) are recovered in all analyses.
Rosales, and Fabales in Fabidae in the AA (69% BS; This clade receives 100% BS in the ntAll and RY ana-
Figure 8), RY (82% BS; Figure 7), and ntAll (81% BS; lyses, 95% BS in the ntNo3rd analysis, and 66% BS in
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101000999150019090812001086207 7311111111190000000007000000000 SCOCFFCRMGMMBaaaerruoxeaayoapgblacsarrllssvptaiaaualnsasisaidnllrltgdeeiloerlcbeaeiahaassssaislstlielolealeaeemsslslsseeasstales MalvidaeFabidaeSuperrosidaeP
101001001000101010000698715100001007017190062100751009576517010001010010010050810053 160110009811111111111111111111111050000000000000000000000090000000000000000000000 AAANMAADPAGZADTBLVDCBSLGCPBCPCSDRGZCCEAPSLSaiaryisrauclooiasueaiapqorraheiiaiyaloauaoneipatpooleisoiulmpgoaatnxarcnbmsrlinlxrrsuenmrnnrgegaaeossccmabrtayrnaoarlaldtlytiieenyinutieeaernnachaaialafrrgbripoetfemaralapparlaaaaealooeaniooslesocloaalcsllaieaeaehprelasnllnohnneltrlpsbecelarelieellsgdeargaclesdeiisaslheaegysaaataeauhsaslsalaehslelseisaoeyeaelcsonllllsllyleieeeleneaaaalslelpeasslnaeellessslasdllslesaaseelealyseserlilesesacsadessesllsaeealsseesCampanuli. Lami.SuperasteridaeCommelinidaeentapetalae EudicotyledoneaeMonocotyledoneaeMagnoliidae Mesangiospermae AngiospermaeSpermatophyta EuphyllophytaTracheophyta Embryophyta Streptophyta
100861008991500101000 101000 101008101170001100050010011111111110900000000070000000000 MBPSCACPAWAGGMIEELCPsyproqnmeirsyoyunianaynaecichalutlepelyraoahrokbuolacwetiaprttsphgglopoctaaheeddtaoeiheoictaorlalnstsraeelidetseyaaeaadrsnaeslsilciterllalslliaaeetlaeleaaoeilhsolalsesslsltleseieeapalsesssslhleesyssta Gnetophyta AcrogymnospermaeMonilophytaLycopodiophyta
100 Zygnematophyceae
Coleochaetophyceae
100 Charophyceae
100 Mesostigmatophyceae
10092100 15999043780 CPUTUCPTrrrrhlhleevvaalloobboossppooriirnnoohhuuoopkyyxxpphycciioohhbyeeppycyoaaecchhpeeeeahyy aaepccpyee.ee.cpp aae.pp.eea..ppepp....pp.. Chlorophyta
Figure7Fiftypercentmaximumlikelihoodmajority-rulebootstrapconsensussummarytreeofViridiplantaeinferredfromtheRY-coded
(RY)analysis.Datasetderivedfrom78protein-codinggenesoftheplastidgenome(ntax=360;58,347bp;missingdata~15.6%).Bootstrapsupport
values≥50%areindicated.Terminalswithatrianglerepresentcollapsedcladeswith>2taxa.SeeAdditionalfile5forthecompletetreeand
Additionalfile1fortaxonomy.Lami.=Lamiidae;Campanuli.=Campanulidae.
the AA analysis. Santalales and Berberidopsidales are the ntAll tree, and sister to a weakly supported clade in-
strongly supported as subsequent sisters to Caryophyl- cluding Gentianales, Lamiales, and Solanales in the
lales+Asteridae. Within Asteridae, Cornales, followed ntNo3rd(Figure 6)andRY(Figure 7)trees.
by Ericales, are subsequent sisters to a strongly sup- Analysis of only the third codon positions (nt3rdOnly,
ported clade that comprises strongly supported Campa- Additional file 11) resulted in several very strong con-
nulidae and Lamiidae clades. Within Lamiidae, the flicts along the backbone of Viridiplantae when com-
placement of Boraginaceae is weak among the various pared to the topology from the ntNo3rd analyses. These
analyses.Boraginaceaearesister toGentianales(59% BS; conflictsincludethebackbonerelationshipswithinChloro-
Figure 8) in the AA tree, part of a trichotomy (100% BS; phyta, the placements of Cycadales and Lycopodiophyta,
Figure 5) with Lamiales and Solanales+Gentianales in the relationships of the three major bryophyte lineages,
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81801000 110000 SMBaraaplsvinsadilceaaslleess
9710068 619005538 695964 111111100000000000000 FCZMFCRGMayaruoeayoggbcsrrlspotaaauaasipnllrlgeleoebehihassssisytiloaaellmallseeleassstales Superrosidae
76 Oxalidales P
10063561001010009118000069231006210100070667992100831009190100010017080 5789801111111111111111111160000000000090000000000007000000004000000000 AAPRBDZCPLTCBBDVDCDPSGAGAASCCEAALGSSaiisrooraauaoralcepaoirsaqeniieiloauaeietopolioispmlaraaonclnxntbsmirlgrxurnerrnaaacasscemtbraanylaaryltldueiiineyitnnaeieacahaslgbfarimeateofrpalarlanaaaeoseiootaalealscocsielaaealrsrnpeolnenlclrtpesaeralgelisleedralegsacdieashgeesuaaaahllsslaaseeiselleaoscnoysaeelllllyeeleaenesaeselplanslsellessslldasseaeaslaessrlesilceadeseslasealsees Lami.Campanuli.SuperasteridaeCommelinidaeentapetalae MonocotyledoneaeEudicotyledoneae Mesangiospermae AngiospermaeSpermatophyta EuphyllophytaTracheophyta Embryophyta Streptophyta
100 71100 MPiapgenraoleliasles
70 6969 CLaaunreallleasles
Chloranthales
100 100 ANuysmtrpohbaaeilaelyeasles
10010093100539089100 100100 101090190110900019110000000011111111119000000000040000000000 AGCCPMILEPCWAGAZMBEPSsyyroqpsrnmieoyuyinananayecgialuchtlepelyroarhakuooblnactiweaprttsghpgolpctoaehaeeddtaoeioehaictorllamsntsreaelidteaesayearadsaneslsiilctalelrasllaileealtaeelaaoeihlstlolasoselssletlseeieopalpssesssphlheshyystycateaaeGnetophyta AcrogymnospermaeMonilophytaLycopodiophyta
Coleochaetophyceae
100 Charophyceae
100 Mesostigmatophyceae
69 100 CChhlloorroopkhybycoepaheyceae
10096100 168808770 UTTTUPrrrrlleevevaoobbbsppoooinhhuuuoyyxxxpcciiioooheepppyaahchheeeyyy cpapcce.e.eeppa aa..peee. p ppp....ppp... Chlorophyta
Prasinophyceae p.p.
Figure8Fiftypercentmaximumlikelihoodmajority-rulebootstrapconsensussummarytreeofViridiplantaeinferredfromtheamino
acid(AA)analysis.Datasetderivedfrom78protein-codinggenesoftheplastidgenome(ntax=360;19,449AAs;missingdata~15.6%).Bootstrap
supportvalues≥50%areindicated.Terminalswithatrianglerepresentcollapsedcladeswith>2taxa.SeeAdditionalfile6forthecompletetreeand
Additionalfile1fortaxonomy.Lami.=Lamiidae;Campanuli.=Campanulidae.
andbackbonerelationshipswithinPoales.Removaloffour they also may exacerbate the effects of systematic error,
taxa(Epifagus,Helicosporidium,Neottia,andRhizanthella) or error resulting from problems in the analysis, such as
with elevated rates of molecular evolution and few genes model inaccuracy. The high amount of agreement
present in the data sets did not significantly affect the among our various analyses and strong support for re-
resultingtopologies. sults generally consistent with previous studies (many of
which also used plastid genes) suggest that plastid gen-
Discussion ome sequence data hold much promise for resolving re-
While the enormous phylogenetic data sets that result lationships throughout the green plants. However,
from new genome or transcriptome sequencing efforts several areas of conflict between analyses using different
can ameliorate the effects of random or stochastic error, character-coding strategies demonstrate that plastid