The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte

16 Pages·2017·2.84 MB·English

by  Mark N. Puttick

Article
The Interrelationships of Land Plants and the Nature
of the Ancestral Embryophyte
Graphical Abstract Authors
MarkN.Puttick,JenniferL.Morris,
TomA.Williams,...,HaraldSchneider,
green algae ggrreeeeeennn aallggaaee
DavidePisani,PhilipC.J.Donoghue
lhmivooersnrwswoorrtt “bryophytes” lhmmhmlmiivvooooeerrssnnnrrwwwsswwwooooorrrrtttt bryophytes“bryophytes” Correspondence
Tracheophyta TTrraaaccchhheeoopphhyyta [email protected](H.S.),
[email protected](D.P.),
[email protected](P.C.J.D.)
ggrreeeeeenn aallggaaee green algae ggrreeeeeennn aallggaaee In Brief
lmlhlhmiiivvvooooeerrsssnnrrwwssswwwoooorrrrtttt ypy“bryophytes” llhmiivvooeersnrrrwwswooorrrttt Bryophyta lhmhhmlhhiivvooooooeerrssnnrrwwwssswwooooorrrrtttt bryophytes“bryophytes” Pusunupitttpiincogkrtleivftoearrlwb.rroeyrsotsoplhavynetdeamm‘‘Sooesntsoaeppshhayyntlyad.’’Tchlaedire
TTrrraaaccchhheeeoopphhyyta Tracheoppphhyyttaa TTrraaccchhheeeoopphhyytta
resultsindicatethattheancestralland
plantwasmorecomplexthanhasbeen
envisagedbasedonphylogenies
green algae ggrreeeeennn aallggaaee recognizingliverwortsasthesister
lhmivooersnrwswoorrtt “bryophytes” hmhmhhllliiivvvoooooeeerrrssnnrrrwwwssswwooooorrrrrttttt “bryophytes”“bryophytes” lineagetoallotherembryophytes.
Tracheophyta TTrraaaccchhheeeooopphhyyttaa
Highlights
d Earlylandplantrelationshipsareextremelyuncertain
d Weresolvethe‘‘Setaphyta’’cladeofliverwortsplusmosses
d Thesimplebodyplanofliverwortsresultsfromlossof
ancestralcharacters
d Theancestrallandplantwasmorecomplex
Putticketal.,2018,CurrentBiology28,1–13
March5,2018ª2018TheAuthor(s).PublishedbyElsevierLtd.
https://doi.org/10.1016/j.cub.2018.01.063
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Current Biology
Article
The Interrelationships of Land Plants
and the Nature of the Ancestral Embryophyte
MarkN.Puttick,1,2,3,9JenniferL.Morris,1,4,9TomA.Williams,2CymonJ.Cox,5DianneEdwards,4PaulKenrick,6
SilviaPressel,3CharlesH.Wellman,7HaraldSchneider,3,8,*DavidePisani,1,3,*andPhilipC.J.Donoghue1,10,*
1SchoolofEarthSciences,UniversityofBristol,LifeSciencesBuilding,TyndallAvenue,BristolBS81TQ,UK
2SchoolofBiologicalSciences,UniversityofBristol,LifeSciencesBuilding,TyndallAvenue,BristolBS81TQ,UK
3DepartmentofLifeSciences,TheNaturalHistoryMuseum,CromwellRoad,LondonSW75BD,UK
4SchoolofEarthandOceanSciences,CardiffUniversity,MainBuilding,ParkPlace,CardiffCF103AT,UK
5CentrodeCieˆnciasdoMar,UniversidadedoAlgarve,Gambelas,8005-319Faro,Portugal
6DepartmentofEarthSciences,TheNaturalHistoryMuseum,CromwellRoad,LondonSW75BD,UK
7DepartmentofAnimalandPlantSciences,UniversityofSheffield,AlfredDennyBuilding,WesternBank,SheffieldS102TN,UK
8CenterofIntegrativeConservation,XishuangbannaTropicalBotanicalGarden,ChineseAcademyofSciences,Menglun,Yunnan,China
9Theseauthorscontributedequally
10LeadContact
*Correspondence:[email protected](H.S.),[email protected](D.P.),[email protected](P.C.J.D.)
https://doi.org/10.1016/j.cub.2018.01.063
SUMMARY and the fossil record, and prompts re-evaluation
of the phylogenetic affinity of early land plant
The evolutionary emergence of land plant body fossils, the majority of which are considered stem
plans transformed the planet. However, our under- tracheophytes.
standing of this formative episode is mired in the
uncertainty associated with the phylogenetic rela-
INTRODUCTION
tionships among bryophytes (hornworts, liverworts,
and mosses) and tracheophytes (vascular plants).
Theevolutionaryemergenceoflandplantbodyplansisoneof
Hereweattempttoclarifythisproblembyanalyzing
the most formative episodes in the evolution of our planet
alargetranscriptomicdatasetwithmodelsthatallow
[1, 2]. Land plant innovations, including stomata, vascular and
for compositional heterogeneity between sites.
rootingsystems,symbioseswithfungi,and,eventually,leaves,
Zygnematophyceae is resolved as sister to land
expanded the sequestration of CO through photosynthesis
2
plants, but we obtain several distinct relationships and silicate weathering [1]. These weathering effects resulted
between bryophytes and tracheophytes. Concate- in changes in atmospheric CO over long timescales [1, 3, 4],
2
natedsequenceanalysesthatcanexplicitlyaccom- aswellaskeychangestotheenvironmentsuchasthedevelop-
modate site-specific compositional heterogeneity ment of soils [5]. Early land plants have even been invoked in
give more support for a mosses-liverworts clade, shapingterrestriallandscapesbyconstrainingsedimentological
‘‘Setaphyta,’’ as the sister to all other land plants, processes [6]. Unfortunately, a detailed understanding of this
episodeisobscuredbyuncertaintyassociatedwiththephyloge-
and weak support for hornworts as the sister to all
netic relationships among bryophytes (hornworts, liverworts,
other land plants. Bryophyte monophyly is sup-
and mosses) and tracheophytes (vascular plants), for which
ported by gene concatenation analyses using
almosteverypossiblesolutionhasbeenproposed(e.g.,[7];Fig-
models explicitly accommodating lineage-specific
ures1A–1G).Intheabsenceofphylogeneticresolution,itisnot
compositional heterogeneity and analyses of gene
possibletoestablishthesequenceinwhichembryophyte,bryo-
trees. Both maximum-likelihood analyses that phyte,andtracheophytebodyplancharacterswereassembled.
compare the fit of each gene tree to proposed spe- This is a prerequisite for determining their intrinsic molecular
ciestreesandBayesiansupertreeestimationbased developmental causes and extrinsic environmental conse-
ongenetreessupportbryophytemonophyly.Ofthe quences,thephylogeneticinterpretationoffossilembryophytes,
15 distinct rooted relationships for embryophytes, and,consequently,establishingthetimescaleoverwhichthese
we reject all but three hypotheses, which differ only characters evolved—facilitating tests of hypotheses on their
roleintransformingtheplanet.
in the position of hornworts. Our results imply that
Although the monophyly of Embryophyta (land plants) and
the ancestral embryophyte was more complex than
Tracheophyta(vascularplants)isuniversallyaccepted,various
has been envisaged based on topologies recog-
hypothesesontheinterrelationshipsofbryophytes(hornworts,
nizing liverworts as the sister lineage to all other
liverworts,andmosses)andtracheophyteshavegainedsupport
embryophytes.Thisrequiresmanyphenotypicchar-
inthelast30years.Thefundamentaldistinctionisbetweenbryo-
acterlossesandtransformationsintheliverwortline- phyte monophyly (e.g., [7–11]; Figure 1A) and paraphyly (e.g.,
age, diminishes inconsistency between phylogeny [12–17]; Figures 1B–1G). Indeed, at least seven alternative
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green algae hornwort Bryoliverwortphyta moss Tracheophyta green algae hornwort“bryoliverwortphytes”moss Tracheophyta green algae hornwort“bryoliverwortphytes”moss Tracheophyta green algae liverwort“bryomossphytes”hornwort Tracheophyta
A B C D
Bryophyta Hornwort-sister Hornworts-sister Liverworts-Mosses sister
(i) Bayesian & mul(cid:2)-species coalescent supertrees (i) Bayesian supertree*
(iv) CAT-GTR (3/30 jack-knife) (iv) CAT-GTR (10/30 jack-knife) (iv) CAT-GTR (10/30 jack-knife)
(v) NDCH2 across-branch
composi(cid:2)onal heterogeneity
green algae liverwort“bryomossphytes”hornwort Tracheophyta green algae liverwort“bryohornwortphytes”moss Tracheophyta green algae liverwort“bryohornwortphytes”moss Tracheophyta
E F G
Liverworts-Mosses-Hornworts Liverworts-Hornworts-Mosses Liverworts plus Hornworts-Mosses
Figure1. TheSevenCompetingTopologiesIdentifiedbetweenthePrincipalEmbryophyteLineagesandTracheophytes,IncludingaSum-
maryoftheSupportfortheThreeMainTopologiesIdentifiedintheAnalyses
Thesevenprincipalcompetinghypothesesofbryophyteandtracheophyterelationships:(A)bryophytemonophyly;(B)hornwortssistertoacladeofmossesplus
liverworts,itselfsistertotracheophytes;(C)mosses,liverworts,andhornwortsaresuccessivesisterlineagesoftracheophytes;(D)acladeofliverwortsand
mossesasasisterlineageofhornwortsplustracheophytes;(E)hornworts,mosses,andliverwortsaresuccessivesisterlineagesoftracheophytes;(F)mosses,
hornworts,andliverwortsaresuccessivesisterlineagesoftracheophytes;and(G)hornwortsplusmossescompriseaclade,sistertotracheophytes,and
liverwortsareanoutgrouptoallthree.ThereissignificantsupportformonophyleticbryophytesfromanalysesemployingBayesiansupertreeestimationfrom
genetrees(i),significanttestsofthemaximum-likelihoodfitofgenetrees(ii)andsequencedata(iii)tothetopologies,andCAT-GTRanalysesinPhylobayes(iv).
However,bothBayesiansupertreeestimation(i)andmaximum-likelihoodfitofgenetrees(ii)significantlyrejecthornwortssisterandmosses-liverwortssister,but
bothofthesetopologiesarefoundmoreconsistentlyinjack-knifeCAT-GTRanalysesthanmonophyly(iv).
topologieshavebeenproposedfrommorphologicalandmolec- [11] and the use of overly simplistic models with assump-
ular analyses (Figures 1A–1G; Table 1), leading to the current tions—stationarity, reversibility, and homogeneity—that are
consensus of a polytomy between mosses, liverworts, horn- violated in real sequence data [22]. Therefore, any attempt to
worts,andtracheophytes[18].Theidentityoftheembryophyte understand these early relationships requires substitution
outgroupremainsequallyuncertain,witholderstudiessupport- models and analytic methods that can incorporate these
ing the morphologically complex stoneworts (Charales) as the complexities.
immediatesistercladeandmorerecentstudiessupportingthe Hereweanalyzealargetranscriptomicaminoacidalignment
‘‘pondscum’’Zygnematophyceaeasthelandplantsistergroup from103speciesofalgae(ChlorophytaandStreptophyta)and
(e.g.,[7,19,20]). Embryophyta (mosses, hornworts, liverworts, and tracheo-
The failure of different studies to reach congruence on the phytes)fromWickettetal.[7]usingamodel(CAT-GTR+G[23])
fundamental relationships among embryophytes is most likely thataccountsforcompositionalheterogeneityacrosssites[23]
duetoinadequatephylogeneticmodelsratherthaninsufficient andadata-recodingstrategy(Dayhoff-6)thatreduceslineage-
data [11, 21, 22]. For example, the lack of consensus at the specific compositional heterogeneity; the same recoded data-
rootofembryophyteshasbeenattributedtodirectionalevolu- sets were also analyzed using the node-discrete composition
tion in nucleotide sequences leading to compositional biases heterogeneity (NCDH) model [24] that explicitly accounts for
2 CurrentBiology28,1–13,March5,2018
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Table1. SummaryoftheTopologiesIdentifiedbetweenthePrincipalEmbryophyteLineages,aswellastheAlgalOutgroupto
EmbryophytaandtheDataTypeAnalyzed
EmbryophyteSisterGroupa Data Reference
BryophyteMonophyly
Charales nuclearrDNAb Horietal.[56]
Charales nuclearrDNAb Steeleetal.[57]
Coleochaetales/Charales spermultrastructure Garbaryetal.[8]
Coleochaetales spermultrastructure Mishleretal.[14]
Charales spermultrastructure Renzagliaetal.[58]
charophytes/chlorophytes chloroplastaminoacids Nishiyamaetal.[9]
Chlorophyta/Coleochaetales chloroplastnucleotidesb Quandtetal.[59]
Coleochaetales chloroplastnucleotidesc GoremykinandHellwig[10]
Coleochaetales chloroplastaminoacidsb Rodrı´guez-Ezpeletaetal.[60]
Zygnematales chloroplastaminoacidsandnucleotidesb Turmeletal.[61]
Zygnematales chloroplastaminoacids Lemieuxetal.[62]
Zygnematales chloroplastaminoacids Coxetal.[11]
Zygnematales chloroplastaminoacids Civanetal.[63]
Zygnematales nuclearaminoacids Wickettetal.[7]
(Hornwort,((Liverwort,Moss),Tracheophyta))
Coleochaetales nuclearrDNA Heddersonetal.[38]
Coleochatales/Klebsormidiales nuclearrDNA Heddersonetal.[64]
Coleochaetales spermultrastructure GarbaryandRenzaglia[40]
Chlorophytes mitochondrialrDNA DuffandNickrent[65]
Chlorophyta/Coleochaetales nuclearandchloroplastnucleotidesc NishiyamaandKato[66]
Charales/Zygnematales nuclear,mitochondrial,chloroplastnucleotidesand Nickrentetal.[67]
nuclearrDNAc
Coleochaetales morphology,nuclearandmitochondrialnucleotides Renzagliaetal.[58]
Zygnematales nuclearnucleotidesc Wickettetal.[7]
(Hornwort,(Liverwort,(Moss,Tracheophyta)))
Charales morphology Bremeretal.[68]
Coleochaetales morphology,nuclearrDNA Mishleretal.[14]
((Liverwort,Moss),(Hornwort,Tracheophyta))
Coleochaetales chloroplastnucleotidesd Nishiyamaetal.[9]
Zygnematales chloroplastnucleotides Turmeletal.[61]
Zygnematales chloroplastnucleotides Lemieuxetal.[62]
Zygnematales chloroplastnucleotidesc Qiuetal.[17]
Zygnematales chloroplastnucleotidesc Gaoetal.[20]
Zygnematales chloroplastnucleotides Karoletal.[69]
Zygnematales chloroplastaminoacids Ruhfeletal.[19]
Zygnematales chloroplastaminoacids Lemieuxetal.[70]
(Liverwort,(Moss,(Hornwort,Tracheophyta)))
‘‘Charophyceae’’ morphology Parenti[71]
Coleochaetes/Charales chloroplastnucleotides Lewisetal.[72]
Charales chloroplastnucleotides Delwicheetal.[73]
Coleochaetales chloroplastgenomestructure Kelchetal.[74]
Coleochaetales chloroplastnucleotidesc Nishiyamaetal.[9]
Coleochaetales chloroplastnucleotides Wolfetal.[75]
Charales/Coleochaetales mitochondrialnucleotides Groth-Maloneketal.[76]
CharalesorZygnematales chloroplastnuclecotideandrDNA,andmitochondrial Qiuetal.[17]
andnuclearrDNA
Charales chloroplastandmitochondrialnucleotides,andnuclear, Qiuetal.[77]
chloroplast,andmitochondrialrDNA
(Continuedonnextpage)
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Table1. Continued
EmbryophyteSisterGroupa Data Reference
Charales chloroplastnucleotides Smithetal.[27]
Zygnematales chloroplastnucleotides Gaoetal.[20]
Zygnematales chloroplastnucleotides ChangandGraham[28]
Chlorophytes/charophytes chloroplastnucleotidesandnuclearrDNA Fiz-Palaciosetal.[78]
Coleochaetales chloroplastnucleotides Magallo´netal.[79]
CharalesorZygnematales mitochondrialnucleotides Turmeletal.[80]
Charales/Coleochaetales chloroplastnucleotides Kimetal.[81]
Charales mitochondrialnucleotidesandaminoacids Liuetal.[21]
Zygnematales chloroplastnucleotides Ruhfeletal.[19]
Zygnematales chloroplastnucleotides Zhongetal.[29]
(Liverwort(Hornwort(Moss,Tracheophyta)))
‘‘charophytes’’ morphology MishlerandChurchill[13]
Charales/Coleochaetales morphology Bremer[31]
Coleochaetales morphology Bremeretal.[68]
Coleochaetales morphologyandchloroplastandnuclearrRNA Mishleretal.[14]
Charales/Coleochaetales morphology KenrickandCrane[15]
Charales chloroplastandmitochondrialnucleotides,nuclearrDNA Karoletal.[45]
(Liverwort((Moss,Hornwort),Tracheophyta))
Klebsormidiales/Coeleochaetales nuclearrRNA Watersetal.[12]
Klebsormidiales chloroplastandnuclearrDNA Mishleretal.[14]
Chlorophytes mitochondrialrDNA DuffandNickrent[65]
(Moss,(Liverwort,(Hornwort,Tracheophyta)))
Charales mitochondrialnucleotides Liuetal.[21]
Coleochaetales chloroplastaminoacids Lemieuxetal.[82]
(Moss,(Hornwort,(Liverwort,Tracheophyta)))
Charales nuclearaminoacids Floydetal.[83]
aEitherthedesignatedoutgroupsor,ifidentified,themostcloselyrelatedtaxontolandplants.
bNotethatalthoughbryophytesweremonophyletic,theywereembeddedinaparaphyletictracheophyte.
cProteincoding-genes1+2codonpositionsonly[7,9,10,20,67].
dExcludingthenucleotidesitescodingleucineandthirdcodonpositionsandfourfolddegeneratesites.
lineage-specific compositional heterogeneity. Finally, we CompositionalHeterogeneityamongSites
exploretherelativefitamongsequencedataandgenetreesto The position of hornworts is unresolved in a focal analysis of
seven proposed hypotheses for the relationships of early land genespresentinatleast95%oftaxa(20,512aminoacids).Anal-
plants (Figures 1A–1G). Analyses under the CAT-GTR model ysisofthesedatawiththeCAT-GTRmodelyieldsaphylogenyin
are not conclusive, supporting several topologies. However, whichtherelationshipsbetweenthebryophytephylaandthetra-
bryophytemonophylyissignificantlyfavoredoverallalternative cheophytes are unresolved (Figures 3 and 4). Using posterior
topologies (1) in superalignment analyses using the NDCH2 predictive tests to estimate the number of Dayhoff-recoded
model,(2)whencomparingthelikelihoodfitofgenetreesandse- aminoacidsateachsiteinthealignment,thereisasignificant
quences to different hypotheses, and (3) through Bayesian difference between the empirical data and those estimated
supertreeanalysesofgenetrees. from the CAT-GTR model (Z score: 3.85), but this model fit is
betterthanthepredictionfromtheGTRmodelwithnocomposi-
RESULTS tionalheterogeneityandtheempiricaldata(Zscore:27.24).An-
alyses without the hornworts did not change the relationships
Theresultsofourphylogeneticanalysesoverwhelminglysupport amongtheremainingbryophyteandtracheophyteclades.
the clade uniting liverworts and mosses that we name ‘‘Seta- Fivefullyresolvedalternativetopologiesaresupportedbythe
phyta.’’Toalesserdegree,wefindsupportforasisterrelation- jack-knifeanalysesofthefulldataset(Figure4).Thejointmost
ship between Setaphyta and hornworts and, hence, the commonlysampledconsensustree(10/30replicates)fromthe
monophylyofthebryophytes(Figures2,3,and4).Supportfor jack-knifeanalysesshowshornwortsassistertotracheophytes,
the monophyly of the three bryophyte groups is mainly from withliverwortsandmossescomprisingasistercladeatthebase
the use of hypothesis tests using prior topologies, but also ofembryophytes(Figure4B).Thepositionofhornwortschanges
supertreeestimationfromgenetrees. in the remaining phylogenies to be either at the base of
4 CurrentBiology28,1–13,March5,2018
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Pyramimonas parkeae C
NUMreoopnnhoermmosaaes lstmipx iso ppiysrtihfoorsmtigisma hlorop
Mesostigma viride Ch
CSphilroortoakeynbiuas m aitnmuotaphyticus hkby.
Entransia fimbriata K
Klebsormidium subtile lb
Chara vulgaris C
Chaetosphaeridium globosum C
Coleochaete irregularis o
SCpoilreoogcyhraae stpe scutata Zle.
Mesotaenium endlicherianum y
Cylindrocystis brebissonii gn
Cylindrocystis cushleckae e
Mougeotia sp m
Netrium digitus a
Roya obtusa to
Penium margaritaceum ph
Cosmarium ochthodes .
Nothoceros aenigmaticus H
Nothoceros vincentianus o
Bazzania trilobata L
Metzgeria crassipilis iv
Sphaerocarpos texanus e
Ricciocarpos natans rw
SMMpaahrrccahhgaannnuttmiiaa lpeeomslcyaumrrgioiinrpahtaa ort Sebryo
BRCPPorhoeylysryuasutmtrcloiaoc dbahmorruyigntmure epm nclulta oercp mufpu mcamraeteupunnislselare Mos taphytaphytes
Hedwigia ciliata se
Leucodon brachypus s
Thuidium delicatulum
Rhynchostegium serrulatum
Anomodon attenuatus
Selaginella moellendorffii 1kp L
Selaginella moellendorffii genome y
Huperzia squarrosa co
Pseudolycopodiella caroliniana p
Dendrolycopodium obscurum h.
Equisetum diffusum
Pteridium aquilinum
Alsophila spinulosa P
Angiopteris evecta te S
GPOsipnihlkoigotuogm lbo isnlosubudamum petiolatum rid. trep
Zamia vazquezii to
CCyyccaass mruimchpohliiitzii Gy ph
Cedrus libani m y
EPipnhuesd traae sdianica no ta
s
Gnetum montanum p
PWreulmwnitsocphitiyas m ainradbinilais erm
Sciadopitys verticillata a
Taxus baccata e
Juniperus scopulorum
Cunninghamia lanceolata
Amborella trichopoda T
Nuphar advena r
AKcaodrsuusr aa mheetreicroacnluitas achEm
Dioscorea villosa eb
OBZSSYSCueoramoracyarblcicgczl aahmhhaalxi yu cabfs pmiubyelaoamosrt dmmibnv iiaauaecuunmondtoltau ooxdnmrsiasantaaclehyon SpermatopEupophyrtaryophyta
HSPLSieaaorirrruoscutdeatmueanyan dan drhmiaraeo egnncrlro aiytcrubiadlrinaapatiafera Angioshytahyllop
PAEoqscduhoilespcghhiyaoll ulfzomiram pcoeaslltiaafotrunmica perm hyta
a
Vitis vinifera e
Larrea tridentata
Medicago truncatula
Boehmeria nivea
Populus trichocarpa
Hibiscus cannabinus
Arabidopsis thaliana
Carica papaya
Kochia scoparia
Diospyros malabarica
Tanacetum parthenium
Inula helenium
Rosmarinus officinalis
Ipomoea purpurea
Catharanthus roseus
Allamanda cathartica
Figure2. TheMainTopologySupportedbyAnalysesRecoversaMonophyleticBryophyta
Abbreviations: Chloro, Chlorophyta; Chkb, Chlorokybophyceae; Klb, Klebsormidiophyceae; C, Charophyceae; Cole, Coleochaetophyceae; Zygnemato,
Zygnematophyceae;Ho,hornworts;Lycophy,Lycophyta;Pterid.,Pteridophyta.ImagesofKlebsormidium,Chara,andSpirogyraarefromPhylopic.
CurrentBiology28,1–13,March5,2018 5
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A strict consensus B 50% majority−rule consensus
Monomastix opisthostigma Monomastix opisthostigma
Nephroselmis pyriformis C Nephroselmis pyriformis
Uronema sp h Uronema sp
Pyramimonas parkeae l Pyramimonas parkeae
MSCpheilsrooortosatkeigynbmiuaas m vaiitrnmiudoteaphyticus Ch ChlorokSyMbpeuirssoo taastetmignomiap ahm yvitniircuiduteas
Entransia fimbriata K Entransia fimbriata
Klebsormidium subtile l Klebsormidium subtile
Chara vulgaris C Chara vulgaris
Chaetosphaeridium globosum C Chaetosphaeridium globosum
Coleochaete irregularis o Coleochaete irregularis
Coleochaete scutata l Coleochaete scutata
SRNpoeiytrraoiu gomybr tadu issgapitus Zyg MesCotyaleinndiuromc yesntidSsl pibcirhreoebgriyisarsnaou snmpii
MPCeoenssimoutmaare imunmiaur mgoac erhintthadocliecdhueemsrianum nem CylindrocysNtiMest orciuuugmseh odletigciakit auseps
Cylindrocystis brebissonii Roya obtusa
Cylindrocystis cushleckae a Cosmarium ochthodes
Mougeotia sp t Penium margaritaceum
Nothoceros aenigmaticus H Nothoceros aenigmaticus
Nothoceros vincentianus Nothoceros vincentianus
Bazzania trilobata Bazzania trilobata
Metzgeria crassipilis L Metzgeria crassipilis
SRMMSppiaachhrrcccaaiohhgecaanraonnurcttmpiiaaao r lppese oomsnslcyaa umttreargioxniinarspanhtuaas iver Set bryo SMMpaahrRraccihSecharcpaonihnoctatciaaigaar pn rpepouomosmlys atm elrnegxoasiarntcpanauhuntraassii
Polytrichum commune a p Polytrichum commune
Ceratodon purpureus p h Physcomitrella patens
Physcomitrella patens m h y Ceratodon purpureus
HRBreoydsuuwmliag baiarryg cueimlniat etcauf mcapillare oss yta tes RosulaBbrryyHuuemmdw acifrg gciaea npctiiellliauarmtea
Anomodon attenuatus e Anomodon attenuatus
Leucodon brachypus s Leucodon brachypus
Rhynchostegium serrulatum Rhynchostegium serrulatum
Thuidium delicatulum Thuidium delicatulum
Selaginella moellendorffii 1kp Selaginella moellendorffii 1kp
Selaginella moellendorffii genome L Selaginella moellendorffii genome
PDseenuddroollyyccooppooddiiuemlla ocbasrcoulinruiamna yco DendrolycHouppoedriuzmia soqbusacurrrousma
Huperzia squarrosa S Pseudolycopodiella caroliniana
OAEqnpughiiisooepgttlueomrsiss ud emifvfeu pcsetuatmiolatum Pte tre EAAqlsunoigspieohtpiultame rs idpsi ifnefuuvesloucstmaa
APPlstseiolroipdtuhiumilma n saupqdinuuuimlilnousma rid pto OphioPgtleorsPisdsuiiulmomt up ameqt iunoiullaidntuuummm
GZainmkigao v baizloqbuaezii G p ZaGmiinak vgaoz bqiuloebzaii
Cycas micholitzii y Eh Cycas micholitzii
Cycas rumphii m my Cycas rumphii
PCiendursu sta leibdaani no bta CPeidnruuss tlaibeadnai
GEpnheetudmra msionnictaanum sp ry GnetEupmh emdoran tsainnuicma
PSWrceuialmwdniotsopcpithiytiysas vm aeinrrtadicbiniilllaiasta erm op ScWPiaerdulwompitnsitocyphsi itavye smr taiircnaidlblaiinltiaas
NATJCuamuunxnpbuipnhoseia nrbrergu alalhsacda c svtmarceitocinaahpa oulaplonorcdueamolata ae ETrachyta CunJAnuimnnigpbheoarrNeumTulsalipa axsh utclarasoircn p bhacuaoedlcopovcreolaaundttmaaaa
SKaardcsaunrad rhae gtelarborcalita Suphe KaSdasrucraan hderate grolacblirtaa
Houttuynia cordata pho Houttuynia cordata
PLSAieacriorrosurdeumeasan aa dhmmreoeennrrr iiytccuiaalinnpuaifsera ermyllophy LirAioPcdeoerrnsSuedsaar raou amnmm etauer lihricpiecaifannenruryaasi
Sabal bermudana apt Yucca filamentosa
EPADSCYOZSBueosqormioroacycadurlscicgczhl oiachlmhhaasepoxiyu ccgahfs rphmiubiyyelaaoaomoslat l dmiulbnf vzo ivmiaauaiecrialu−mmon l optncltu oooseadomsraslxlitisaaafnottaarucnlmehicyaon Angiosperm tophytahytaa BrEasPcCchohoydslpcoAScoShDphqSadimhcioubiooyuuliiaszrlllmmelgalOciu aghxbo m r da iuercaybZie umarsz poeafmttlaoeuni aa fb ruvolma cstmimiadrc−halnnltaoooyntauaiinolcssvoyomlaaeaaansxr
Kochia scoparia Kochia scoparia
DViitoiss pvyinroifes rmaalabarica ae DiospyrIonsu lma ahlealbeanriuicma
Inula helenium Tanacetum parthenium
Tanacetum parthenium Ipomoea purpurea
Rosmarinus officinalis Rosmarinus officinalis
Ipomoea purpurea Allamanda cathartica
Allamanda cathartica Catharanthus roseus
Catharanthus roseus Vitis vinifera
Boehmeria nivea Boehmeria nivea
Populus trichocarpa Larrea tridentata
Hibiscus cannabinus Medicago truncatula
Larrea tridentata Populus trichocarpa
Medicago truncatula Hibiscus cannabinus
Arabidopsis thaliana Arabidopsis thaliana
Carica papaya Carica papaya
Figure3. ResultofAnalysisof30Sampled20,000AminoAcid,Dayhoff-RecodedDatasetsAnalyzedundertheCAT-GTRModel
Strict(A)and50%majority-rule(B)consensus.Abbreviations:Chl,Chlorophyta;Ch,Chlorokybophyceae;Kl,Klebsormidiophyceae;C,Charophyceae;Col,
Coleochaetophyceae;Zygnemat,Zygnematophyceae;H,hornworts;Liv,liverworts;Lyco,Lycophyta;Pterid.,Pteridophyta.
embryophytes withthe moss-liverwort clade sisterto tracheo- consensusoftheresultsfromeachjack-knifeanalysisindicates
phytes (10/30 replicates; Figure 4) or within monophyletic a polytomy between the bryophyte phyla and tracheophytes
bryophytes at the base of Embryophyta (4/30 replicates; Fig- (Figure3A)andthe50%majority-ruleconsensusshowsasimilar
ure4C).Theremainingtopologiesshowvariationsofapolytomy polytomy, but with mosses and liverworts forming a clade
containing bryophytes at the base of embryophytes. A strict (Figure3B).
6 CurrentBiology28,1–13,March5,2018
Pleasecitethisarticleinpressas:Putticketal.,TheInterrelationshipsofLandPlantsandtheNatureoftheAncestralEmbryophyte,CurrentBiology
(2018),https://doi.org/10.1016/j.cub.2018.01.063
M L H T H M L T M L H T M L H T M L H T L M H T M H L T Figure4. AssessingSupportforCompeting
Topologies
Thenumberoftimesseventopologieswerepro-
A B C D E F G
ducedfromthe30jack-knifeanalysesofDayhoff-
10 recodeddataundertheBayesianCAT-GTRmodel
(A–G)andthetopologyfromBayesiansupertree
c(10, 10, 3, 3, 2, 1, 1)fe replicatesjackkni468 e[lfif(eA2rgsoa5)tumsi]martbecaecasotalsiaumoecdmndhpemouooossanfifirtniit8lzoghiv5enete2sahrljewtglayhecomehnkreet-cestktoehntnarroieosfndeegedsneornsmuf(HuenSosos)tseuatsiersnneeldgdseaeesnoanpdpnseersnRtoahd(odIees)du.inrscT1ittge4hleydo8er:
lineage of hornworts plus tracheophytes; (B)
2
hornwortssistertoacladeofmossesplusliver-
worts,itselfsistertotracheophytes;(C)bryophyte
0
Index monophyly;(D)mosses,liverworts,andhornworts
are successive sister lineages of tracheophytes;
(E)mossesplusliverwortscompriseacladesister
852 ML gene trees 148 least heterogeneous gene trees
totracheophytes,thepositionofhornwortsunre-
Chlorophyta
solved;(F)hornworts,mosses,andliverwortsare
Chlorokybophyceae
Charales successivesisterlineagesoftracheophytes;and
Coleochaetales (G)thepositionofhornwortsandliverwortsunre-
Zygnematophyceae
solved, sister to tracheophytes, with mosses
hornworts
liverworts branchingearlier.TheBayesiansupertreeanalysis
0.96 of852genesrecoversabryophyteclade,including
mosses
thesetaphytecladeofliverwortsandmosses(H);
hornworts
however,bryophytesareparaphyleticinthetree
Lycophyta
Monilophyta 0.98 of 148 genes, with hornworts sister to tracheo-
phytes (I).Abbreviations: H, hornwort;M,moss;
Gymnospermae L,liverwort;T,tracheophyta.
Angiospermae 0.98 mainingembryophytesisrecovered(pos-
teriorprobability=0.98).Further,the148
least heterogeneous genes indicate that
H I
Charaissistertoembryophytes,whereas
all other estimated supertrees support a
Across-BranchCompositionalHeterogeneity Zygnematophyceae-Embryophyta relationship (Figure 4I). The
TheNDCH2analysisoftheDayhoff-recoded148leastheteroge- effective sample size (ESS) of all parameters was >200, and
neousgenesrecoveredmonophyleticbryophyteswithmaximal themedianestimateofthefreebetaparameterwas2.06.
(posteriorprobability[PP]=1.0)posteriorprobability.CAT-GTR We also estimated multi-species coalescent supertrees in
analysisofthesedatasupportsaliverwortplusmosscladesister Astral[26].Thesetreessupportbryophytemonophylywithlow
toahornwortsplustracheophytesclade,withmaximalposterior support(52.5)andalsosupportasister-grouprelationshipbe-
probability(PP=1.0). tweenCharaandtheembryophytes.
FitofSequenceAlignmentstoProposedTopologies FitofGeneTreestoProposedTopologies
Analyses of the sequence data rejected all topologies except Comparisonsofthelikelihoodofgenetreesandsevenproposed
monophyleticbryophytes(Table2).Noneofthefiveotheralter- embryophyte relationships support bryophyte monophyly
native topologies was sampled during the bootstrap analyses (Table 3). All alternative hypotheses can be rejected using the
(monophyletic bryophytes proportion = 1). Bryophyte mono- AUandKHtests,althoughthemoreconservativeSHtestcannot
phylyreceived thehighestsupportofsummedlikelihoodsand rejectthetopologyinwhichliverwortsandmossesarerecovered
issignificantlysupportedcomparedtoallalternativetopologies asthesistercladetotheremainingembryophytes(Table3,top).
using approximately unbiased (AU), Kishino-Hasegawa (KH), However,whenusingtheleastheterogeneous148genedataset,
andShimodaira-Hasegawa(SH)tests(Table2). itisnotpossibletorejectliverworts-mossessister,monophyletic
bryophytes,orliverwortsplushornworts-mossestopologiesus-
SupertreeEstimationusingGeneTrees ingtheAUtest(Table3,bottom).
Results from analyses of gene trees using Bayesian supertree
inference [25] strongly support monophyletic bryophytes. The DISCUSSION
posteriorprobabilityofthesplitsupportingmonophyleticbryo-
phytes was 1 across two independent chains (Figure 4H). Thephylogeneticrelationshipamongearlylandplantshasbeen
When the 148 least compositionally heterogeneous genes are oneofthemostrecalcitrantproblemsinphylogenetics.Wefind
analyzedalone(Figure4I),aliverwort-mosssistercladetothere- support for three topologies of bryophytes and tracheophytes
CurrentBiology28,1–13,March5,2018 7
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(2018),https://doi.org/10.1016/j.cub.2018.01.063
Table2. TheFitoftheWeightedAlignmenttotheSevenHypothesesofEarlyLandPlantTopologyUsingRELLBootstrapping
RELLBootstrap
Topology Likelihood DLikelihood Proportion AU SH KH
Monophyleticbryophytes (cid:2)19,802,012.115 0.000 1 1 1 1
Hornwortssister (cid:2)19,802,243.57 231.459 0 0 0.0013 0
Liverworts-mossessister (cid:2)19,802,248.226 236.111 0 0 0.0004 0
Liverworts-mosses-hornworts (cid:2)19,803,217.59 1,205.477 0 0.0002 0 0
Liverwortsplushornworts-mosses (cid:2)19,803,340.12 1,328.01 0 0.0019 0 0
Hornworts-liverworts-mosses (cid:2)19,803,399.22 1,387.107 0 0.0057 0 0
Liverworts-hornworts-mosses (cid:2)19,803,472.14 1,460.029 0 0 0 0
Thesummedlikelihoodandbootstrapproportionofalignmentssupportsthemonophyleticbryophytestopologyandsignificantlyrejectsallalternative
topologies.RELL,resamplingofestimatedloglikelihoods;AU,approximatelyunbiased;KH,Kishino-Hasegawa;SH,Shimodaira-Hasegawa.
fromanalysesusingmodelsthataccommodatesequencehet- uponmorphology(e.g.,[15])andtranscriptomes(e.g.,[7]).Our
erogeneity:(1)monophylyofthebryophytes,(2)hornwortsalone analyses suggest that this continuing controversy is due to a
earliest branching, and (3) liverworts plus mosses earliest combinationoffactors:embryophytesequencedataexhibitsig-
branching. Bryophyte monophyly is consistently supported by nificant compositional heterogeneity that is difficult to model
analysesthatcomparethefitofsequencedataandgenetrees even with the best current approaches [22]; there is a paucity
tohypothesizedrelationships,aswellasbyanalysesperformed ofsequencedataforkeyhornwortlineages;andbiological ef-
usingASTRAL.Supportforbryophytemonophylyisalsofoundin fects, such as incomplete lineage sorting, may be masking
theresultsofanalysesaccommodatingacross-branchcomposi- early-branchingrelationships.
tional heterogeneity. Overall, these results suggest bryophyte Inouranalyses,thechoice ofmodel didimpacttheinferred
monophyly, and support for alternative topologies might be a tree topology, and models accounting for across-site or
consequenceofincompletelineagesortingandlineagespecific across-tree heterogeneity did not agree with each other. Site
compositionalheterogeneityinthedata.Bryophytemonophyly specific heterogeneity is very high in this dataset and cannot
is congruent with recent studies that accommodate across- be accounted for in full also when applying Dayhoff recoding
branchcompositionalheterogeneity[11]butconflictwithresults (Z = j6j). In addition, it is evident from the results of the CAT-
fromalarge-scaleconcatenationanalysisusingsimplermodels GTR+G analyses, which resolve different tree topologies, that
[7](Figure2). theproblem mayalso befurthercomplicatedbythe presence
of incomplete lineage sorting in the data. However, both the
RejectionofProposedRelationships use of ASTRAL coalescent methods and the use of models
In the last 30 years, at least seven topologies have been pro- thataccommodatelineage-specificcompositionalheterogene-
posedforearlylandplants(Figures1A–1G;Table1).Ourana- ity support bryophyte monophyly. Thus, from a modeling
lysesallowustonarrowthistopologicaluncertaintydownsolely perspective, resolving the deepest relationships among land
tothepositionofhornworts:asthesistertoacladeofmosses plantsisachallengingproblem.Theapplicationofsite-hetero-
and liverworts in monophyletic bryophytes (Figure 1A), as the geneous models has resolved phylogenetic controversy else-
sisterlineagetootherembryophytes(Figure1B),orasthesister whereinthetreeoflife(e.g.,[32,33]),but,atpresent,itisunable
lineageoftracheophytes(Figure1D).Withallmethods,wecan to definitively resolve early land plant relationships (Figure 4).
reject previous hypotheses that do not find a moss-liverwort Furthermore,modelingofcompositionalheterogeneitybetween
clade(i.e.,Table1;Figures1Dand1E–1G),suchasthesucces- sitesmaynotbesufficientlycomplextoresolvelandplantrela-
sivebranchingofhornworts,liverworts,andmossessistertothe tionships, as it will not capture compositional heterogeneity
tracheophytes [13, 19–21, 27–31]. The three remaining topol- among lineages [24] or non-reversibility in sequence evolution
ogies represent a fundamental split in the topology witheither [34].Anadditionalfactoristhatthegenome-scaleaminoacidda-
monophyly or paraphyly of bryophytes (hornworts or liver- tasetsthatarenowavailableformanyembryophytelineagesare
worts-mosses as the sister lineage to all remaining embryo- too large to be easily modeled using the best-fitting models
phytes). Wickett et al. [7] found support for both bryophyte available. In particular, achieving convergence with the CAT-
paraphyly(hornwortssister—basedonconcatenationanalyses GTRmodelinPhylobayesortheNDCH2modelofP4becomes
of nucleotide data) and bryophyte monophyly (based upon a challengingbeyonddatasetsofaround20,000alignedpositions,
coalescentanalysisofareduceddatasetofaminoacids).Here evenwhenusingparallelization.
wefindequivocalsupportforthesehypothesesthroughestima- Asecondimportantfactorrelatestothesamplingofkeyline-
tionofphylogeniesbasedonCAT-GTR-model-basedanalyses ages.Despitethemodelingdifficultiesoutlinedabove,ourana-
ofaminoaciddata(Figures3and4). lysesenabledustoresolverobustlyallofthemainbranchesof
the land plant tree with the exception of the hornworts—the
DifficultiesintheResolutionofEarlyLandPlant most poorly sampled major lineage in our dataset. Hornworts
Phylogeny are currently represented by just two transcriptomes from
Despitethreedecadesofresearch,thephylogenyofearlyland congenericspecies(Nothoceros),leavingfourofthefiveorders
plants remains unresolved with large-scale analyses based unrepresented. Thus, improved genomic or transcriptomic
8 CurrentBiology28,1–13,March5,2018
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(2018),https://doi.org/10.1016/j.cub.2018.01.063
Table3. ComparisonoftheSumofApproximateLikelihoodsofGeneTrees,WhenFittoSevenAlternativeTopologiesofEarlyPlant
Relationships
MultiscaleBootstrap
Topology ApproximateLikelihood DLikelihood Proportion AU KH SH
852Maximum-LikelihoodEstimateTrees
Monophyleticbryophytes (cid:2)56,488.38 0 0.994 0.995 0.995 1
Liverworts-mossessister (cid:2)56,545.555 57.2 0.006 0.006 0.005 0.050
Hornwortsister (cid:2)56,563.42 75 9e(cid:2)5 7e(cid:2)5 5e(cid:2)5 0.023
Liverwortsplushornworts-mosses (cid:2)56,697.43 209.1 3e(cid:2)6 1e(cid:2)5 0 0
Hornworts-liverworts-mosses (cid:2)56,749.25 260.9 7e(cid:2)20 2e(cid:2)56 0 0
Liverworts-mosses-hornworts (cid:2)56,772.48 284.1 3e(cid:2)25 4e(cid:2)82 0 0
Liverworts-hornworts-mosses (cid:2)56,784.99 296.6 3e(cid:2)6 2e(cid:2)5 0 0
148LeastCompositionalHeterogeneousMaximum-LikelihoodEstimateTrees
Liverworts-mossessister (cid:2)9,816.57 0 0.823 0.969 0.937 0.996
Monophyleticbryophytes (cid:2)9,830.48 13.9 0.067 0.091 0.063 0.3
Liverwortsplushornworts-mosses (cid:2)9,833.95 17.4 0.128 0.079 0.077 0.179
Hornwortsister (cid:2)9,833.95 17.4 0.020 0.031 0.026 0.199
Liverworts-mosses-hornworts (cid:2)9,842.65 26.1 0.008 0.017 0.018 0.063
Hornworts-liverworts-mosses (cid:2)9,844.38 27.8 0.008 0.02 0.007 0.023
Liverworts-hornworts-mosses (cid:2)9,844.38 27.8 0.001 0.004 0.008 0.027
DatasetswereanalyzedusingthemethodofSteelandRodrigo[25].Top:forthe852genes,thebetavaluewassetto2.05(themedianvaluefromthe
P4analyses).Overallgenetrees,thesummedlikelihoodishighestforthemonophyleticbryophytestopology.Thereissignificantsupportformono-
phyleticbryophytesoverallotherpotentiallandplantrelationshipsusingtheAUandKH.Bottom:forthe148genetreeswiththeleastcompositional
heterogeneity,thebetavaluewassetto2.Analyseswererunwiththemaximum-likelihoodestimatetopologies.Theliverworts-mossessistertreewas
thetopologywiththehighestsupport,butbothmonophyleticbryophytesandliverwortsplushornwortsmossescouldnotberejected.
sampling of hornworts may help to place this final recalcitrant fit of sequence data to competing phylogenetic hypotheses
branch in the land plant tree. Liverwort diversity is also under (Tables2and3).Forexample,significanttestsoftherelativefit
represented, particularly for early-branching lineages. Thus, ofsequencedatatotopologiesrejecthornwortssister,andthe
thequalityofthedatasetcouldbeimprovedsignificantlythrough highest support and bootstrap proportion is for monophyletic
additionoftranscriptomedataforthehornwortsLeiosporoceros bryophytes(Table2).Ourresultsarecompatiblewithconcatena-
and/orAnthoceros,aswellastheearliest-diverginglineagesof tionprecludingresolutionofearlylandplantphylogeny,possibly
haplomitropsidliverworts,Haplomitriumand/orTreubia. due to incomplete lineage sorting (e.g., [41]). Furthermore, the
Finally, the current intractability of land plant relationships approach we apply here [37] is similar to the gene genealogy
mustbe,atleastinpart,biological.Partofthereasonmaybe interrogation(GGI)methodologyofArcilaetal.[42],whofound
horizontalgenetransfer,whichhasbeendocumentedbetween thattestingbetweenthefitofgenetreestohypothesizedspecies
hornwortsandpteridophytes[35],aswellasmossesandangio- treescouldbeusedtoresolvedifficultareasofthetreeoflife.Itis
sperms[36],potentiallybiasingtheestimationofphylogeniesus- possiblethegenetreeapproachherecanovercomeknowndif-
ingconcatenation.Theshortbranchesseparatingthefourmain ficulties in sequence evolution of early land plants [11, 22] to
lineagesoflandplantsmayindicatearapidadaptiveradiationre- resolve thesedifficult relationships whenconcatenation-based
sulting in conflicts among gene trees as a consequence of methodsstruggle.
incomplete lineage sorting of gene polymorphisms. Future WhilesupportforamonophyleticBryophytaisnotunequivo-
studiesmayaimtoidentifysetsofgenetreessupportingalterna- cal,wefindoverwhelmingsupportforacladeofmossesandliv-
tivetopologiesasaconsequenceofthesebiologicalprocesses. erwortsthatwename‘‘Setaphyta’’afterthesporophyteseta,a
stalk supporting the capsule in both liverworts and mosses.
SupportforMonophyleticBryophytesandtheLiverwort- However, we define Setaphyta phylogenetically, not based on
MossClade‘‘Setaphyta’’ anapomorphy,asthecladecomprisedoflivingmossesandliv-
Bayesiansupertreeestimation[25],genetreecoalescentanal- erworts,theirlastcommonancestorandallofitsdescendants.
ysis [26], and maximum-likelihood analyses [37] of gene trees
all support monophyletic bryophytes [7–11, 38–40]. Wickett ImplicationsforEarlyLandPlantEvolution
et al. [7] also found monophyletic bryophytes based on gene Mostrecentanalyses(e.g.,[19,21,28,29])havesupportedliver-
tree coalescence analyses of amino acids, and other studies wortsasthesistergrouptoallotherembryophytes.Thisresult
havesuggestedthatsupportforrelationshipsotherthanbryo- hasbeencompellingbecauseliverwortsaremissinganumber
phytemonophylyisartifactualinchloroplastdata[11].Wefind of characters that are otherwise shared between mosses and
the support for bryophyte monophyly based on analysis of vascular plants, e.g., stomata. As such, liverworts have been
genetrees,butthereisadditionalsupportfromanalysesofthe envisagedasanevolutionaryhalfwaystagebetweenprimitively
CurrentBiology28,1–13,March5,2018 9