Article Platyzoan Paraphyly Based on Phylogenomic Data Supports a Noncoelomate Ancestry of ...

17 Pages·2014·0.79 MB·English

Platyzoan Paraphyly Based on Phylogenomic Data Supports
a Noncoelomate Ancestry of Spiralia
Torsten H. Struck,*,1,2 Alexandra R. Wey-Fabrizius,3 Anja Golombek,1 Lars Hering,4 Anne Weigert,5
Christoph Bleidorn,5 Sabrina Klebow,3 Nataliia Iakovenko,6,7 Bernhard Hausdorf,8 Malte Petersen,1
Patrick Ku¨ck,1 Holger Herlyn,9 and Thomas Hankeln3
1ZoologicalResearchMuseumAlexanderKoenig,Bonn,Germany
2UniversityofOsnabru¨ck,FB05Biology/Chemistry,AGZoology,Osnabru¨ck,Germany
3InstituteofMolecularGenetics,BiosafetyResearchandConsulting,JohannesGutenbergUniversity,Mainz,Germany
4AnimalEvolutionandDevelopment,InstituteofBiologyII,UniversityofLeipzig,Leipzig,Germany
5MolecularEvolutionandSystematicsofAnimals,InstituteofBiology,UniversityofLeipzig,Leipzig,Germany
6DepartmentofBiologyandEcology,OstravianUniversityinOstrava,Ostrava,CzechRepublic
7DepartmentofInvertebrateFaunaandSystematics,SchmalhausenInstituteofZoologyNASofUkraine,Kyiv,Ukraine
8ZoologicalMuseum,UniversityofHamburg,Hamburg,Germany
9InstituteofAnthropology,JohannesGutenbergUniversity,Mainz,Germany
*Correspondingauthor:E-mail:[email protected].
Associateeditor:GregoryWray
Abstract
Based on molecular data three major clades have been recognized within Bilateria: Deuterostomia, Ecdysozoa, and
Spiralia.WithinSpiralia,small-sizedandsimplyorganizedanimalssuchasflatworms,gastrotrichs,andgnathostomulids
have recently been grouped together as Platyzoa. However, the representation of putative platyzoans was low in the
respectivemolecularphylogeneticstudies,intermsofboth,taxonnumberandsequencedata.Furthermore,increased
substitutionratesinplatyzoantaxaraisedthepossibilitythatmonophyleticPlatyzoarepresentsanartifactduetolong-
branchattraction.Inordertoovercomesuchproblems,weemployedaphylogenomicapproach,therebysubstantially
increasing1)thenumberofsampledspecieswithinPlatyzoaand2)species-specificsequencecoverageindatasetsofup
to82,162aminoacidpositions.Usingestablishedandnewmeasures(long-branchscore),wedisentangledphylogenetic
signalfrommisleadingeffectssuchaslong-branchattraction.Indoingso,ourphylogenomicanalysesdidnotrecovera
monophyletic origin of platyzoan taxa that, instead, appeared paraphyletic with respect to the other spiralians.
Platyhelminthes and Gastrotricha formed a monophylum, which we name Rouphozoa. To the exclusion of
Gnathifera, Rouphozoa and all other spiralians represent a monophyletic group, which we name Platytrochozoa.
PlatyzoanparaphylysuggeststhatthelastcommonancestorofSpiraliawasasimple-bodiedorganismlackingcoelomic
A
cavities,segmentation,andcomplexbrainstructures,andthatmorecomplexanimalssuchasannelidsevolvedfromsuch
r
a simply organized ancestor. This conclusion contradicts alternative evolutionary scenarios proposing an annelid-like t
i
ancestorofBilateriaandSpiraliaandseveralindependenteventsofsecondaryreduction. c
l
e
Introduction
ancestorofDeuterostomia,EcdysozoaandSpiraliahadaseg-
Molecular data have profoundly changed the view of the mentedandcoelomatebodyorganizationresemblingthatof
bilaterian tree of life by recognizing three major clades: anannelid,andthatmorphologicallymoresimplyorganized
Deuterostomia, Ecdysozoa, and Spiralia (Halanych 2004; taxa such as nematodes or flatworms (Platyhelminthes)
Edgecombe et al. 2011). The term Spiralia is occasionally evolved by secondary reductions (Brinkman and Philippe
used as a synonym for Lophotrochozoa (Halanych 2004). 2008; De Robertis 2008; Couso 2009; Tomer et al. 2010;
However, the term Lophotrochozoa is actually reserved for Chesebro et al. 2013). This is in stark contrast to the tradi-
all descendants of the last common ancestor of Annelida, tional“acoeloid–planuloid” hypothesisfavoringevolutionof
Mollusca, and the three lophophorate taxa (Halanych Bilateriafromasimplebodyorganizationtowardmorecom-
2004), whereas the more comprehensive taxon Spiralia in- plex forms with a last common ancestor resembling a flat-
cludes all animals with spiral cleavage and, hence, also worm without segmentation and coelomic cavities (Hyman
Platyhelminthes (Edgecombe et al. 2011). Herein, we use 1951;Halanych2004;Hejnoletal.2009).Unravelingthephy-
Spiraliainthetermsofthemoreinclusivedefinition. logeneticrelationshipswithinBilateriaiscrucialtoresolvethis
Previous results of the molecular phylogenetic analyses controversy(Halanych2004;Edgecombeetal.2011).
initiatedastillon-goingdebateabouttheevolutionofcom- Whilerecentphylogenomicstudiesrecoveredmostofthe
plexity in Bilateria. It was proposed that the last common relations of the major branches within Deuterostomia and
(cid:2)TheAuthor2014.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.Allrightsreserved.Forpermissions,please
e-mail:[email protected]
Mol.Biol.Evol.31(7):1833–1849 doi:10.1093/molbev/msu143 AdvanceAccesspublicationApril18,2014 1833
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Strucketal. . doi:10.1093/molbev/msu143
Ecdysozoa, the internal phylogeny of Spiralia is still unclear worms)andGastrotrichaeven5-foldincomparisontopre-
(Edgecombe et al. 2011). Indeed, spiralian animals exhibit a vious large-scale analyses of spiralian relationships (Dunn
widevarietyandplasticityindevelopmentandmorphology et al. 2008; Hejnol et al. 2009). After orthology assignment
includingbodyorganization(Nielsen2012)whichgaveriseto (Ebersbergeretal. 2009),thedatawerefurtherscreenedfor
the distinction of two major taxa: Lophotrochozoa and sequence redundancy (Kvist and Siddall 2013), potentially
Platyzoa (Halanych 2004; Edgecombe et al. 2011). As men- paralogous sequences (Struck 2013) and contamination
tioned above, Lophotrochozoa comprises at least annelids (Struck2013)resultinginapruningofabout7%ofsequence
(ringed worms), lophophorates, and mollusks (Halanych data (supplementary tables S3–S8, Supplementary Material
2004)andhenceanimalswithamorecomplexmorphology. online).
In contrast, Platyzoa subsumes more simple appearing taxa
suchasflatworms,hairybacks(Gastrotricha),wheelanimals Brute-Force Approach: More Taxa and Data
(classicalRotifera),thorny-headedworms(Acanthocephala),
Phylogenetic reconstructions based on the largest data sets
and jaw worms (Gnathostomulida) (Cavalier-Smith 1998).
d01 with 82,162 amino acid positions and 38.3% sequence
Although some authors regard Platyzoa as sister to
coverage (supplementary table S9, Supplementary Material
Lophotrochozoa (Edgecombe et al. 2011), others place
online) recovered monophyly of both Platyzoa and
Platyzoa within Lophotrochozoa, thus rendering Spiralia
Lophotrochozoa with strong bootstrap support (BS) of
synonymous with Lophotrochozoa (Halanych 2004).
99 for both (fig. 1). Within Platyzoa, monophyly
Importantly, unique morphological autapomorphies sup-
of Platyhelminthes, of Syndermata, as well as of
porting the monophyly of Platyzoa are lacking (Giribet
Gnathostomulidawasmaximallysupported,whereasmono-
2008)andphylogeneticanalysesofnuclearandmitochondrial
phyly of Gastrotricha was not recovered. The chaetonotid
datafailedtoresolvethequestionaswell(Papsetal.2009a,
gastrotrich Lepidodermella squamata appeared as sister to
2009b; Bernt et al. 2013). Nevertheless, there seems to be a
Platyhelminthes(BS46),whereasthemacrodasyidangastro-
tendency for a weakly supported monophylum Platyzoa as
trichs formed a monophylum (BS 74) as sister to all other
long as larger data sets were analyzed (Halanych 2004;
platyzoan taxa (BS 68). Finally, Gnathostomulida was sister
Hausdorf et al. 2007; Struck and Fisse 2008; Hejnol et al.
to Syndermata (BS 61) consistent with the Gnathifera
2009; Paps et al. 2009a; Witek et al. 2009). However, across
hypothesis(Ahlrichs1997;HerlynandEhlers1997).
all these analyses placement of platyzoan taxa appeared
Tostudytheinfluenceofunstabletaxa,leafstabilityanal-
unstable, probably due to low data and taxa coverage
yseswereperformed.Withaleafstabilityindexof0.876,the
(Edgecombeetal.2011).Moreover,parallelevolutionofchar-
gastrotrich Lep. squamata was the most unstable species
acterstatesonlongbranches(alsoknownasLBA)mightalso
withinthesampledplatyzoans,followedbythetwognathos-
haveconfoundedtheseanalyses(Edgecombeetal.2011).In
tomulid species (0.941) and the macrodasyidan gastrotrich
summary,monophylyofPlatyzoaandthephylogeneticposi-
Dactylopodola baltica (0.969) (fig. 2 and supplementary
tionsoftheplatyzoantaxawithinSpiraliaarestillcontentious
table S10, Supplementary Material online). Excluding these
althoughtheirpositionshavemajorimplicationsforbilaterian
four platyzoan taxa from data set d01 and conducting a
evolution.Inparticular,monophylyof Platyzoaandaplace-
new phylogenetic reconstruction did not influence the
ment within Lophotrochozoa would be in line with the
remaining topology, but led to an increased BS value of
theory of a more complex ancestry (Brinkman and Philippe
99 for a clade uniting Platyhelminthes and Syndermata and
2008), whereas paraphyletic Platyzoa with respect to
decreased values for the monophyly of both Platyzoa and
Lophotrochozoa would support the “acoeloid–planuloid”
Lophotrochozoa (BS 82 and 86, table 1). Thus, the four un-
hypothesis.
stable taxa showed some influence on support for the phy-
Results and Discussion logenetic placement of other platyzoan taxa. Therefore, we
excludedthesefour taxa from thefollowing analyses, which
Toaddressthemajoroutstandingissuesofbilaterianphylog-
addressedthepotentialroleofLBAonplatyzoanphylogeny
enywithrespecttospiralianandmorespecificallyplatyzoan
inmoredetail.
relationships,weappliedaphylogenomicapproach,generat-
ing transcriptome sequence data for 10 putative platyzoan
LBA Accounts for Monophyly of Platyzoa
and two nemertean species using second-generation se-
quencing technology and a modified RNA amplification Monophyletic Platyzoa as sister to Lophotrochozoa gained
method,whichallowedthegenerationofsequencinglibraries strong support in the analyses described above. However,
from as few as 10 specimens of microscopic species of thoroughinspectionofthetopology(fig.1)revealedconsid-
Gnathostomulida,Gastrotricha,andclassicalRotifera(supple- erablebranchlengthheterogeneity,withlongbranchesinthe
mentarytableS1,SupplementaryMaterialonline).Thesedata analyzed platyzoan lineages and rather short branches in
werecomplementedwithtranscriptomicorgenomicdataof lophotrochozoan and ecdysozoan lineages. Hence, the ob-
53 other spiralian and ecdysozoan species, including addi- servedstrongsupportformonophyleticPlatyzoamightorig-
tional representatives of Platyzoa (supplementary table S2, inatefromartificialratherthanphylogeneticsignal(Bergsten
Supplementary Material online). Hereby, the taxon cover- 2005; Edgecombe et al. 2011; Ku¨ck et al. 2012). For the tree
ageofPlatyzoaincreased3.5-foldandforindividualplatyzoan derivedfromdatasetd01andshowninfigure1,theLBscores
taxasuchasSyndermata(wheelanimalsandthorny-headed showedabimodaldistributionwithaminimumbetweenthe
1834
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* Opisthorchis
* Clonorchis
* Fasciola
* Schistosoma
Schistosoma
* * Echinococcus
* Echinococcus
62 * Taenia Platyhelminthes
* Moniezia
Spirometra
* Nematoplana
99 * * DuDguesgieasia
Schmidtea
* * Stylochoplana P
Paraplanocera
Macrostomum Gastrotricha la
Lepidodermella
* Pomphorhynchus ty
* Echinorhynchus
* Paratenuisentis z
68 92 Macracanthorhynchus o
* Philodina a
* Adineta Syndermata
Rotaria
* * * BrBacrahciohniounsus
99 Lecane
Seison
61 * Gnathostomula Gnathostomulida
Gnathostomula
* Turbanella
* Macrodasys Gastrotricha
Megadasys
74 Dactylopodola
* Biomphalaria
* Aplysia
* Lottia
* * * CrassoMstryetialus Mollusca
* Idiosepius L
Euprymna
90 Neomenia o
Chaetopleura p
* Lumbricus h
* Tubifex
* Helobdella Annelida o
Capitella t
73 9*0 TuCbAeulpvlahinnaeulolsltahrix Nemertea roc
99 * Bugula h
* Flustra o
93 Alcyonidium Ectoprocta z
* Tubulipora o
Cristatella a
* * PPeeddiciceelllilninaa Entoprocta/Cycliophora
Symbion
Lingula Brachiopoda
Terebratalia
* Priapulus
Echinoderes Ecdysozoa
* Daphnia
Apis
0.2
FIG.1. Maximum-likelihood(ML)treeobtainedbyanalysisofdatasetd01with65taxaand82,162aminoacidpositions.OnlyBSvalues(cid:2)50areshown
atthebranches.*Maximalsupportof100.Highertaxonomicunitsareindicated.
twohighestoptimaatanLBscorevalueof0(fig.2B).Putative clade was sister to monophyletic Lophotrochozoa (BS 95;
platyzoan species had generally higher LB score values than table1),whereasSyndermatawassistertoallotherspiralian
lophotrochozoanandecdysozoanspecies(fig.2andsupple- taxa.Thus,exclusionoflong-branchedspecieshadatremen-
mentarytableS11,SupplementaryMaterialonline).Onlythe dous effect on the analyses rendering a strongly supported
LBscoresinferredforStylochoplanaandParaplanocerawithin monophyly of Platyzoa with BS values >95 into a
Platyhelminthes, the two Brachionus species and Lecane in paraphyletic assemblage, in which the clade consisting
Syndermata,andMegadasysandMacrodasysinGastrotricha of Gastrotricha + Platyhelminthes and Lophotrochozoa
approximated those of most lophotrochozoans and ecdy- obtainedstrongsupportwithaBSvalueof95.
sozoans. On the other hand, Symbion (Cycliophora),
Alcyonidium, and Tubulipora (Ectoprocta) showed values Biases Causing Monophyletic Platyzoa
>0,resemblingthoseofmostoftheplatyzoanspeciessam- Togainfurtherinsightsintotheissueofmono-versuspara-
pled(fig.2). phyletic Platyzoa, we analyzed the data with respect to the
To assess the effect of long branches on tree reconstruc- differentpropertiesofindividualgenes.Indetail,westudied
tion, all species with LB scores above 0 were excluded from theeffectofgene-specificproportionsofhydrophobicamino
data set d01 (82,162 positions). Interestingly, monophyly of acidsandmissingdata,basecompositionandbranchlength
Platyzoa was no longer recovered (fig. 3). Gastrotricha now heterogeneity,andevolutionaryratesontreereconstruction.
emergedassistertoPlatyhelminthes(BS96;table1),andthis Acommonprocedureistochooseoneofthesepropertiesas
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themostinfluentialoneeitherbasedapriorionliteratureora branch length heterogeneity (Brinkman and Philippe 2008),
posterioriontheobtainedresults(e.g.,BrinkmanandPhilippe didnotcorrelatewithactualmeasurementsofbranchlength
2008; Simmons 2012b; Nesnidal et al. 2013; Nosenko et al. heterogeneity(R2=0.0324and0.0635;supplementaryfig.S4,
2013;Roureetal.2013;SalichosandRokas2013).Herein,we SupplementaryMaterialonline).
usedanotherprocedurebasedonthevariabilityexhibitedin AswewantedtotestforLBA,weusedthedirectmeasure-
thedataitselfpriortoanalysesofalternativedatasetsreflect- mentofbranchlengthheterogeneityinsteadofevolutionary
ingdifferentdegreesofdatareduction.Accordingtotheprin- rate. Thus, we generated data sets with either different
cipalcomponentanalysis(Alexeetal.2008),thefirstprincipal degrees of missing data (d02–d06), proportion of low base
component explained 31.0% of the variance between the composition heterogeneity (d07), or low branch length
different genes. It was mainly derived from the proportion heterogeneity (d08) as well as genes being part of the 70%
of missing data and base composition heterogeneity with or95%confidenceintervalsofthefirsttwoprincipalcompo-
eigenvectors pointing into opposite directions (supplemen- nents (d09 and d10) (supplementary tables S9 and S13,
tary fig. S3 and table S12, Supplementary Material online). Supplementary Materialonline).Basedon the resultsof the
Branchlengthheterogeneityandevolutionaryratewerethe principalcomponentanalysis,wepresentindetailtheresults
largest factors in the second component, which explained ofthreedatasetsd07(lowbasecompositionheterogeneity),
26.8% of the variance. Correlation analyses showed that in d08 (low branch length heterogeneity), and d02. The latter
ourcaseevolutionaryrate,whichisoftenusedasaproxyfor combinesalowdegreeofmissingdatawithahighnumberof
positions.
Analysesofthesethreedatasetsexcludingthefourabove-
mentioned unstable platyzoan taxa consistently resulted in
1.0 A
Ecdysozoa paraphyletic Platyzoa (fig. 4and table 1) as observed before
Pa.
bility index EActonpNnreoeMcmtleiardotealalusca SBtr..Me.LMe.a.Tu.Al.DSya.. GEnatsoPtrploartotrycichtahe/alCmyincltihoepshora Syndermata wd(B0hS1e.7n6O,en8x4cce,luamnddion7rge1,l,oRPnloagut-ybphrhaeonlmzcohianetidnhettasaxbwaleaf1sro)smaisntdetrhLetooplahGrogaetsrtodrcoahttraoiczshoeaat
Leaf sta 0.2G5 nathostomulida wThaesrecclaodveereodf aGsaastmrootnriochpah/yPlelatticyhgerlomuipnt(hBeSs98w,a4s7,saisntder95to).
y Lophotrochozoa (BS 72, 86, and 75, table 1) and
0.9 Brachiopoda frequenc00..0155 STyhnudse,remithatear wbyasinscisrteearsintogtthheeaclolvoetrhaegres(pdi0ra2l)iaonrtdaexcareaagsaining.
base composition or branch length heterogeneity (d07
Lep. B -25 0 LB score 50 and d08) paraphyletic Platyzoa was recovered (table 1), as
-30 0 60 a clade comprising Gastrotricha, Platyhelminthes and
LB score
Lophotrochozoa gained strong branch support exceeding
FIG.2. LeafstabilityindicesandLBscoresbasedontheMLanalysisof valuesof70.
datasetd01with65taxaand82,162aminoacidpositions.(A)Plotof
Additional exclusion of long-branched species (figs. 2
leafstabilityindicesagainstLBscores.(B)DistributionofLBscores.The
and 3) reproduced paraphyly of Platyzoa in all analyses,
dashedlineinAindicatesLBscore=0.Shadesdistinguishecdysozoan
even with maximum BS in some analyses. Again
andlophotrochozoanspecies(lightgray)fromputativeplatyzoanspe-
cies (dark gray). Some species are also labeled: Pa, Paraplanocera; St, Platyhelminthes was sister to Gastrotricha (BS 93, 97, and
Stylochoplana; Br, Brachionus; Le, Lecane; Me, Megadasys; Ma, 54,fig.5,Rouphozoaintable1)andbothweremoreclosely
Macrodasys; Da, Dactylopodola; Lep, Lepidodermella; Sy, Symbion; Tu, related to the lophotrochozoan taxa than to Syndermata
Tubulipora;Al,Alcyonidium. (BS 100, 100, and 50, fig. 5, Platyzoa Para. in table 1).
Table 1. Bootstrap Support (BS) for Monophyly and Paraphyly of Platyzoa as well as Monophyly of Rouphozoa.
Data Set Excl. Taxa # Pos. # Taxa Platyzoa Rouphozoa
Mono. Para. Mono.
d01 (all data) None 82,162 65 99a 0 3
Unstable 82,162 61 82b 1 1
LB 82,162 34 3 95a 96a
d02 (high coverage) Unstable 36,513 61 3 86b 84b
LB 36,513 34 0 100a 93b
d07 (low base frequency heterogeneity) Unstable 37,907 61 19 75b 71b
LB 37,907 34 0 100a 97a
d08 (low branch length heterogeneity) Unstable 29,133 61 18 72b 76b
LB 29,133 34 10 50 54
Excl.,excluded;#pos.,numberofpositions;#taxa,numberoftaxa;LB,long-branchedtaxa.
aSupportvaluesarepartofthe95%confidenceset.
bSupportvaluesarepartofthe70%confidenceset.
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PlatyzoansandSpiralia . doi:10.1093/molbev/msu143
* Biomphalaria
* Aplysia
* Lottia
* Crassostrea
* Mytilus Mollusca
* Idiosepius
L
Euprymna
Neomenia o
73 Chaetopleura p
99 Tubifex h
* Lumbricus o
* Helobdella Annelida
t
Capitella r
76 73 Alvinella o
60 * Tubulanus Nemertea c
Cephalothrix h
* * BuguFlalustra Ectoprocta oz
95 * CristatellPaedicellina Entoprocta oa
Pedicellina
86 Terebratalia Brachiopoda
Lingula
* Stylochoplana
96 Paraplanocera P
* Macrodasys G la
Megadasys
t
* * BraLcheBciroaanncuehsionus Sy ast yh
63 Echinoderes n r e
Priapulus E d o lm
* DaApphinsia cd er tri in
y m c
0.2 s h th
oz ata a es
o
a
FIG.3. MLtreeobtainedbyanalysisofdatasetd01with34taxaand82,162aminoacidpositions.AlltaxaexceedingLBscores>0intreeoffigure1
wereexcluded.OnlyBS(cid:2)50areshownatthebranches.*Maximalsupportof100.Highertaxonomicunitsareindicated.
Moreover, comparing the trees without long-branched spe- Thus,combiningBayesianandmaximum-likelihoodanal-
cies(figs.3and5)totheonewithallspecies(fig.1)showsthat yses with different data and taxa exclusion strategies could
nowsimilarbranchlengthsleadtothe“platyzoan”andlopho- not recover monophyletic Platyzoa in contrast to analyses
trochozoanspecies(figs.3and5).Additionally,thestandard using only large numbers of data (figs. 1, 3–6 and table 1).
deviationofthespecies-specificLBscoresforthetreesshown Consideringall10datasets(i.e.,d01–d10),BSformonophy-
in figures 3 and 5 are 10.3 and 9.3, respectively, and, letic Platyzoa substantially increased with additional amino
hence, lower than the standard deviation of 15.4 for the acidpositions(darkgraylineinfig.7A),whereassupportfor
tree of figure 1. This means that the latter exhibits much paraphylydecreased(blacklineinfig.7A).Incontrast,support
stronger branch length heterogeneity across all taxa than formonophylyofLophotrochozoawasnotstronglyaffected
the former two. Similarly, the standard deviations for the bythenumberofpositionsanalyzed(lightgraylineinfig.7A).
classical tip-to-root distances are lower for the trees of It is a well-known phenomenon of LBA that it is positively
figures 3 and 5 with 0.054 and 0.143 than for the tree of misleading;thatis,withincreasingnumbersofpositionsthe
figure1with0.202. artificialgroupismorerobustlyrecovered(Felsenstein1978;
We also used a Bayesian approach with the GTR+CAT Huelsenbeck 1997; Bergsten 2005). On the other hand, ex-
model,asthisisknowntobemorerobusttowardLBAthan cludinglong-branchedspeciesfromanalysesdidnotleadto
classicalMLmodelssuchasLG(Lartillotetal.2007).Dueto suchcorrelations.Inparticular,supportforthemonophylyof
computational time restrictions and high memory require- Platyzoa remained low irrespective of the number of align-
ments,wewerenotabletousethelargedatasetd01(82,162 mentpositions(darkgraylineinfig.7B).
positions). Instead, we chose data set d02 (low to medium- Additionally,wedeterminedforeachdatasetthenumber
lowdegreeofmissingdata;36,513positions;46.1%coverage; of single-gene trees supporting monophyly or paraphyly of
supplementary table S9, Supplementary Material online) Platyzoa. Across all data sets the percentage of single genes
as the principal component analysis indicated coverage supporting platyzoan paraphyly ranged from 8.6% to 11.7%
as the most influential property in the first and,thus,washigherthanthepercentagesupportingmono-
component. Importantly, the Bayesian approach did not phyletic Platyzoa, ranging from 0.5% to 3.2% (table 2).
recover monophyletic Platyzoa, but instead a clade Interestingly, decreasing the degree of missing data (i.e.,
includingGastrotricha + Platyhelminthesandmonophyletic d01–d06) and, hence, increasing the number of taxa per
Lophotrochozoa(posteriorprobability[PP]=1.00,fig.6)and gene, the ratio of the percentage of trees supporting
again Gnathostomulida + Syndermata was sister to this paraphyly relative to the percentage of trees supporting
clade(PP=1.00,fig.6). monophyly strongly increased (black line in fig. 7C).
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* Aplysia
89 * Biomphalaria
Lottia
* Mytilus
97 Crassostrea Mollusca L
Neomenia
* Euprymna o
Idiosepius p
72 86 ChaetL.ingula Brachiopoda h
Terebratalia o
93 * 98 TuCbueTplauhnbauilfosetxhrix Nemertea tr
* Lumbricus o
* Helobdella Annelida c
98 Capitella h
62 Alv*inella Flustra o
92 * Bugula z
* Cristatella TubuliporaAlcyonidium Ectoprocta oa
93 * * PPeeddiicceelllliinnaa Entoprocta/Cycliophora
Symbion* Opisthorchis P
* Clonorchis l
* Fasciola a
86 * Schistosoma t
Schistosoma y
62 * * * * * EEcchThianineoonccioaoccccuussMoniezia hel
96 Spirometra m
Nematoplana
72 * Dugesia i
* Dugesia n
81 * * StylPocahraoppllaannoacera Schmidtea th
Macrostomum e
84 * 99 TuMrabcarnoedllaasys Gastrotricha s
Megadasys S
* * PoEmchpihnoorrhhyynncchhuuss y
* Paratenuisentis n
93 Macracanthorhynchus d
* Philodina
* Adineta e
Rotaria r
* * * BraBchraiocnhuiosnus m
Lecane a
Seison
* Apis t
Daphnia Ecdysozoa a
* Echinoderes
Priapulus
0.2
FIG.4. MLtreeobtainedbyanalysisofdatasetd02with61taxaand36,513aminoacidpositions.Onlypartitionswithlowtomediumuptolow
degreesofmissingdatawereincludedandthefourunstabletaxa(Lepidodermellasquamata,Dactylopodolabaltica,andthetwoGnathostomulida
species)wereexcluded.OnlyBS(cid:2)50areshownatthebranches.*Maximalsupportof100.Highertaxonomicunitsareindicated.
Directlyaddressingbiasesinthedatasuchasbaseorbranch figs. 4 and 8). Analysis of data set d07 excluding unstable
lengthheterogeneitydidnothavesuchaneffectontheratio. taxa except Gnathostomulida (i.e., Lepidodermella and
In the case of LBA only strategies as used herein, which are Dactylopodola) and of data set d02 excluding all long-
able to attenuate its misleading effect by excluding either branched taxa recovered Gnathostomulida as part of a
biased data or species or by increasing taxon coverage per clade with Gastrotricha and Platyhelminthes (table 3).
gene,canrevealwhetherornotanassemblyoflong-branched However, all other analyses placed Gnathostomulida as
taxa is artificially grouped together (Bergsten 2005). In con- sister to Syndermata with BS values of up to 91, even
clusion, our analyses support platyzoan paraphyly, whereas though overall BS remained low (fig. 8 and table 3).
recovery of monophyletic “Platyzoa” is most probably due Moreover, the Bayesian analysis also recovered a sister
toLBA. group-relationship of Gnathostomulida and Syndermata
with strong support (PP=0.98, fig. 6). This position of
Position of Gnathostomulida Gnathostomulida as sister to Syndermata is consistent with
In addition to LBA, the inference of a stable topology was the Gnathifera hypothesis (Ahlrichs 1997; Herlyn and Ehlers
hamperedbytheinclusionofGnathostomulidaandthetwo 1997).MonophylyofGnathiferahasalsobeenfoundinpre-
gastrotrichs Lepidodermella and Dactylopodola. In order to vious studies based on ribosomal protein data (Witek et al.
elucidate the phylogenetic position of Gnathostomulida 2009;Hausdorfetal.2010)andisalsostronglysupportedby
within Spiralia, we reincluded the two formerly excluded thelikelyhomologyofgnathostomulidanjawsandrotiferan
gnathostomulidspeciesintodifferentdatasets.Importantly, trophi (Rieger and Tyler 1995; Haszprunar 1996; Ahlrichs
their inclusion did not alter the topology with respect to 1997;HerlynandEhlers1997;Jenner2004a).Forathorough
platyzoan paraphyly in any tree reconstruction (e.g., cf. analysisofthephylogeneticrelationswithinSyndermataand
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* Tubifex
63 * LumbricusHelobdella Annelida
* Alvinella
52 Capitella L
* Pedicellina Entoprocta
Pedicellina o
Terebratalia Brachiopoda p
Tubulanus Nemertea h
99 Cephalothrix o
* Biomphalaria t
* Aplysia r
* Lottia o
* Crassostrea c
* Mytilus Mollusca h
58 * Idiosepius o
Euprymna z
Chaetopleura o
84 Neomenia a
50 * Bugula
* Flustra Ectoprocta
Cristatella
Lingula Brachiopoda
76 54 * MegMaadcarsoydsasys G
* Paraplanocera P a
75 * Priapulus * BBraraLcchehicoiaonnnueusSstylocSynhoplana latyh strotr
Echinoderes Ecdysozoa d e i
* Apis e lm ch
Daphnia r
m i a
0.2 n
a t
t h
a
e
s
FIG.5. MLtreeobtainedbyanalysisofdatasetd08with34taxaand29,133aminoacidpositions.Onlypartitionswithlowdegreesofbranchlength
heterogeneitywereincludedandalltaxaexceedingLBscores>0intreeoffigure1wereexcluded.OnlyBS(cid:2)50areshownatthebranches.*Maximal
supportof100.Highertaxonomicunitsareindicated.
theimplicationfortheirevolution,werefertoarecenttran- common characteristic can nonetheless be utilized for
scriptome-basedstudy(Wey-Fabriziusetal.2014). naming the clade. We therefore suggest the name
Rouphozoa(derivedfromtheGreekwordrouphaoforingest-
ing by sucking) to define the last common ancestor of
A Novel View on Spiralian Phylogeny
Platyhelminthes and Gastrotricha and all its descendants.
In summary, our analyses support the monophyly of The clade of Rouphozoa + Lophotrochozoa can be named
LophotrochozoaandofacladecombiningGastrotrichaand Platytrochozoa, reflecting that it comprises Platyhelminthes
Platyhelminthes.Gnathiferaissistertoacladecomprisingthe andtaxawithatrochophorelarvaandallextantdescendants
aforementionedtaxa(fig.9).Nomorphologicalapomorphyis of the last common ancestor of Platyhelminthes and
known to date supporting either a monophyletic origin of Lophotrochozoa. Spiralia then comprises Gnathifera
Platyhelminthes and Gastrotricha or of Platyhelminthes, (Syndermata + Gnathostomulida)andPlatytrochozoa.
Gastrotricha,andLophotrochozoa(Jenner2004a;Rotheand
Implications for bilaterian evolution
Schmidt-Rhaesa2009)and,hence,couldbeusedfornaming
these two clades. However, whereas most of the other spir- TheparaphylyofPlatyzoawithrespecttoLophotrochozoais
alian taxa exhibit additional structures for food gathering in more in line with the traditional “acoeloid–planuloid” hy-
theirgroundpattern(e.g.,palpsinannelids,proboscisinne- pothesis than with the scenario of a last common ancestor
merteans, filter feeding apparatuses in lophophorates, ento- ofDeuterostomia,Ecdysozoa,andSpiraliawithasegmented
procts, and cycliophorans, as well as jaw-like elements and coelomate body organization resembling an annelid.
in rotifers, gnathostomulids, and mollusks), gastrotrichs Within Spiralia the non-coelomate, small-sized taxa succes-
and most flatworm species ingest food without such extra- sively branch off first (fig. 9). Both Gnathostomulida and
structures, just by dilating their rather simple pharynx. The Gastrotricha comprise small interstitial organisms with an
respective pharynx simplex is part of the ground pattern of acoelomate body organization and <4 or 2mm of length,
Platyhelminthesandenablestheswallowingofpreybyeither respectively (Nielsen 2012). Within Syndermata only the
sucking action or engulfment (Doe 1981). Gastrotricha pos- highly modified, parasitic Acanthocephala are larger than a
sess a Y-shaped or inverted Y-shaped sucking pharynx fewmillimetersandallexhibitapseudocoelomateorganiza-
(Kieneke et al.2008). Althoughgatheringfoodby sucking is tion (Herlyn and Ro¨hrig 2003; Nielsen 2012). Similarly, in
not necessarily an autapomorphy of these two taxa, this Platyhelminthes,theancestralconditionisalsoasmall-sized,
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* Biomphalaria
* Aplysia
* Lottia
* Mytilus
* Crassostrea Mollusca
* Idiosepius
* 0.86 Euprymna
0.99 Neomenia
Chaetopleura
* Terebaratalia Brachiopoda
Lingula
*0.94 ** TLuubmifebxricus Lophotrochozoa
Helobdella Annelida
* Alvinella
0.99 Capitella
* * TuCbuelpahnaulsothrix Nemertea
* Flustra
0.97 * Bugula
* Alcyonidium Ectoprocta
Tubulipora
Cristatella
* * * PPeeddiicceelllliinnaa Entoprocta/Cycliophora
Symbion
* Echinococcus
* Echinococcus
* Taenia
* * Moniezia
Spirometra
* * Opisthorchis
* Clonorchis
* Fasciola Platyhelminthes
* Schistosoma
* Schistosoma
* Dugesia
* Dugesia
* Schmidtea
Nematoplana
* * Stylochoplana
Paraplanocera
* 0.59 Macrostomum
* * TuMrbaacnreoldlaasys Gastrotricha
Megadasys S
* * EPchoimnpohrhoyrnhcyhnuchsus y
* Paratenuisentis n
* Macracanthorhynchus d
* * * PhiAloddinineata er
Rotaria
m
* * * BraBcrhaicohniuosnus
a
Lecane
0.98 Seison t
* Gnathostomula Gnathostomulida a
Gnathostomula
* Priapulus Ecdysozoa
Echinoderes
* Daphnia
Apis
0.2
FIG.6. BItreeobtainedbyanalysisofdatasetd02with63taxaand36,513aminoacidpositions.Onlypartitionswithlowtomediumuptolowdegrees
ofmissingdatawereincludedandonlythetwounstablegastrotrichtaxaLepidodermellasquamataandDactylopodolabalticawereexcluded.Only
PPs(cid:2)0.50areshownatthebranches.*Maximalsupportof1.00.Highertaxonomicunitsareindicated.
acoelomate organization as seen today in Catenulida and (forreview,seeKochetal.2014).Hence,segmentalcoelomsin
Macrostomorpha, which are <5mm in length (Nielsen annelids and arthropods are not necessarily homologous
2012).Within Spiralia,animalswithacoelomatebodyorga- structures(Kochetal.2014).Inaddition,thedevelopmental
nization are, according to our analyses, only found in origins of coelomic cavities in deuterostomes differ from
Lophotrochozoa (fig. 9). Thus, it is epistemologically more those in lophotrochozoans and panarthropods (Nielsen
parsimonious to assume that the last common ancestor of 2012). Considering these differences and our results, it is
Spiralia was an animal lacking a coelomic body cavity. moreprobablethatcoelomiccavitiesevolvedindependently
Although many relationships within Lophotrochozoa are withinthemajorbilateriancladesDeuterostomia,Ecdysozoa,
still unresolved in our study and warrant further investiga- andSpiralia.Clearly,furtheranalysesoftheunderlyinggenetic
tions,ouranalysessuggestthatwithinSpiraliacoelomiccav- regulatorynetworksincoelomformationacrossawidevari-
ities with a lining epithelium might have originated at the ety of coelomate and non-coelomate taxa are necessary to
earliestinthestemlineageofLophotrochozoa.Additionally, substantiateorrejectthisconclusion.
recentinvestigationsofdevelopmentandformationofcoe- ThepositionofcoelomateChaetognathawithinBilateriais
lomic cavities using a comparative anatomical approach alsoofinterestinthisaspect,butstillenigmaticbasedonboth
revealed considerable differences between Annelida and molecularandmorphologicaldata.Deuterostomeaswellas
Panarthropodaalreadyintheearlieststepsofcoelomogenesis protostomeaffinitiesincludingasistergrouprelationshipto
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typesofcoelomformation(Kapp2000;Perezetal.2014)and,
A100
hence,mightbeindicativeofaconvergentevolutionofcoe-
ort 80 lomiccavitiesinChaetognatha.However,ultrastructuralstud-
p ies of coelom formation are lacking at the moment (Perez
p
u60 etal.2014).
s
p Thealternativescenariowhereuponevolutionprogressed
a
r40 fromcomplextosimpleinBilateriaismainlybasedonsim-
t
ts ilarities in segmentation in vertebrates, arthropods, and an-
o
o20 nelids(DeRobertis2008;Couso2009;Chesebroetal.2013).
B
However,inouranalyses,Annelidawasalwaysdeeplynested
0
withinLophotrochozoa.Thus,similartotheevolutionofcoe-
B100 lomiccavities,asegmentedancestryofSpiraliawouldimply
t several independent losses of this organization, which we
r
o80 regard as less parsimonious. Moreover, Annelida and
p
p Arthropoda exhibit high plasticity in segmentation and, on
u
s60 the other hand, other spiralian andecdysozoan taxa exhibit
p
a varying degrees of repetitive organization in organ
r40
t systems. This includes Kinorhyncha, Monoplacophora and
s
t
o Polyplacophora, Eucestoda and other platyhelminths, some
o20
B nematodesandnematomorphs,andanemertean(Hannibal
0 and Patel 2013; Struck 2012). In addition, segmentation is
25 35 45 55 65 75 85 mostlyrestrictedtotissuederivedfromtheectoderminar-
Number of positions (*1000) thropods,fromthemesoderminvertebrates,andfromboth
C16 germlayersinannelids(Nielsen2012).Apossibleexplanation
forsimilaritiesinsegmentformationincludingdevelopmental
12 pathwayslikethenotchoscillationcouldbethatthesegene
regulatorynetworkshavebeenco-optedfromancestralnet-
o
ati 8 worksinvolvedintheorganizationofrepetitiveorgansystems
r (Davidson and Erwin 2006; Chipman 2010). However, this
hypothesis cannot be conclusively proven due to a current
4
lack of data on developmental gene pathways in taxa with
such repetitive organ systems (Chesebro et al. 2013).
0
Nonetheless,thespiralianphylogenyderivedhereinprovides
150 250 350 450 550
Number of genes additional support for the hypothesis that segmentation
evolved independently within Deuterostomia, Ecdysozoa,
FIG. 7. BS and the ratio of single-genes supporting paraphyly over
andSpiralia.
monophyly relative to the number of alignment positions or genes.
Supportforacomplexbilaterianancestoralsoarosefrom
(AandB)BSformonophylyandparaphylyofPlatyzoaaswellasmono-
phyly of Lophotrochozoa relative to the number of positions. the observation of neuronal structures called mushroom
(A) Analyses based on 61 taxa, from which the four unstable taxa bodies that were consistently present in arthropods and
(Lepidodermella squamata, Dactylopodola baltica, and the two some annelids, as well as similar gene expression patterns
Gnathostomulida species) were excluded. (B) Analyses based on 34 noted in these bodies and in the vertebrate pallium (Heuer
taxa, from which all taxa exceeding LB scores >0 in tree of figure 1 et al. 2010; Tomer et al. 2010). However, within annelids,
were excluded. Light gray=monophyly of Lophotrochzoa, dark mushroom bodies occur exclusively in five families of the
gray=monophylyofPlatyzoa,black=paraphylyofPlatyzoa.Best-fitting
subgroup Errantia, which are all characterized by a high va-
trend lines generated by Excel are also shown in the same colors.
gility(Heueretal.2010;Strucketal.2011),whiletheyarenot
(C) Ratio ofthepercentageofsingle-genetreessupportingparaphyly
known for any other annelid or spiralian taxa (Rothe and
of Platyzoa to the percentage of single-gene trees supporting mono-
Schmidt-Rhaesa 2009; Heuer et al. 2010; Nielsen 2012;
phylyofPlatyzoarelativetothenumberofgenes.Diamonds=datasets
Loesel 2014). Thus, if such distinct higher brain centers are
d02andd03withreducedmissingdata;triangles=datasetsd01,d04–
d06 generated using MARE; circle=data set d07 with reduced base taken as an ancestral condition of a complex last common
heterogeneity;square=datasetd08withreducedbranchlengthhet- spiralian ancestor (Heuer et al. 2010), several losses within
erogeneity;crosses=datasetsd09andd10basedonconfidenceinter- Spiralia, including even several ones within Annelida, have
valsofPCA.Thebest-fittingtrendlinegeneratedbyExcelforthedata to be assumed. On the other hand, the gastrotrich nervous
setsd01–d06withdecreasingdegreesofmissingdataisshowninblack. systemconsistsof abrainwithasolidarch-likedorsalcom-
missurewithlaterallypositionedcellsomataandafineven-
Spiraliahavebeenproposed(Marletazetal.2006;Matusetal. tralcommissureaswellasapairoflongitudinal,lateroventral
2006; Dunn et al. 2008; Perez et al. 2014). Moreover, nerve cords joining posteriorly (Rothe and Schmidt-Rhaesa
Chaetognatha possess a unique type of coelom formation, 2009).Thisorganizationissimilartotheorganizationofthe
heterocoely,whichexhibitsnostrongsimilaritiestotheother nervous system of Acoelomorpha. Hence, in comparison to
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Table 2. Percentage of Single-Genes Supporting Monophyly or Paraphyly of Platyzoa.
Degree of Missing Data Heterogeneity PCA
Data Set d01 d02 d03 d04 d05 d06 d07 d08 d09 d10
# Genes 559 232 413 340 235 174 217 187 446 537
% Mono. 2.1 0.9 1.2 1.5 0.9 0.6 3.2 3.2 2.0 2.0
% Para. 9.7 8.6 9.4 9.1 9.4 8.6 11.5 11.8 10.1 9.9
% Lack 88.2 90.5 89.3 89.4 89.8 90.8 85.3 85.0 87.9 88.1
Para./Mono. 4.5 10 7.8 6.2 11 15 3.6 3.7 5 4.8
#Genes,numberofgenesindataset;%Mono.,percentageofsingle-genetreessupportingmonophylyofPlatyzoa;%Para.,percentageofsingle-genetreessupportingparaphyly
ofPlatyzoa;%Lack,percentageofsingle-genetreeslackingresolutionregardingthisquestion;Para./Mono.,ratioofthepercentageofsingle-genetreessupportingparaphylyof
Platyzoatothepercentageofsingle-genetreessupportingmonophylyofPlatyzoa;PCA,principalcomponentanalysis.
* Aplysia
85 * Biomphalaria
Lottia
98 * CrassoMstyrteialus Mollusca L
Neomenia o
* Idiosepius
Euprymna p
70 88 Chaetopleura h
TerebaLriantgaulilaa Brachiopoda o
93 * 99 TuCbueplaLhnuaumlosbthrricixus Nemertea tro
* * TubifexHelobdella Annelida c
99 Capitella h
56 Alvinella o
* Flustra
89 * Bugula z
Alcyonidium Ectoprocta o
* Tubulipora a
Cristatella
92 * * PPeeddiicceelllliinnaa Entoprocta/Cycliophora
Symbion P
* Echinococcus
* Echinococcus l
* Taenia a
62 * Moniezia t
Spirometra y
* * Clonorchis
66 * * OpiFsatosrcciohlias he
98 * SScchhisitsotososommaa lm
Nematoplana
66 * * DuDguegsieasia in
Schmidtea
* * Stylolochoplana t
Paraplanocera h
89 61 99 99 TMurabcarnoedlalasys GMacarosstotmrumotricha es Sy
Megadasys n
* Pomphorhynchus
* Echinorhynchus d
94 * Macracanthorhynchus Paratatenuisentis e
* Philodina r
* Adineta m
Rotaria
99 * Brachionus a
* Brachionus t
Lecane a
Seison
71 * Gnathostomula Gnathostomulida
99 Priapulus Gnathostomula
Echinoderes Ecdysozoa
* Apis
Daphnia
0.2
FIG.8. MLtreeobtainedbyanalysisofdatasetd02with63taxaand36,513aminoacidpositions.Onlypartitionswithlowtomediumuptolow
degreesofmissingdatawereincludedandonlythetwounstablegastrotrichtaxaLepidodermellasquamataandDactylopodolabalticawereexcluded.
OnlyBS(cid:2)50areshownatthebranches.*Maximalsupportof100.Highertaxonomicunitsareindicated.
thenet-likeplexuswithoutacerebralganglioninnon-bilater- ancestor of Spiralia showed that characteristic. Moreover,
iananimals,bothGastrotrichaandAcoelomorphaexpressa also, for example, platyhelminths, syndermatans, gnathosto-
certaindegreeofcondensationattheanteriorendtoforma mulids, or entoprocts show anterior condensations of the
more or less condensed commissural brain, but to a lesser central nervous system, but not to the same degree as in
degreethanotherbilateriantaxa(RotheandSchmidt-Rhaesa elaborate brains, which can be found in some mollusks or
2009).Thus,Gastrotrichamightstillexhibittheancestralbila- annelids(Northcutt2012;Loesel2014).Suchacondensation
terian condition indicative that also the last common is in general agreement with a small-sized, noncoelomate
1842