Collodictyon--An Ancient Lineage in the Tree of Eukaryotes.

0.92 MB·English

by  Zhao, Sen

Collodictyon—An Ancient Lineage in the Tree of Eukaryotes
Sen Zhao,(cid:1),1 Fabien Burki,(cid:1),2 Jon Bra˚te,1 Patrick J. Keeling,2 Dag Klaveness,1 and
Kamran Shalchian-Tabrizi*,1
1Microbial Evolution Research Group, Department of Biology, University of Oslo, Oslo, Norway
2Canadian Institute for Advanced Research, Botany Department, University of British Columbia, Vancouver, British Columbia,
Canada
(cid:1)These authors contributed equally to this work.
*Corresponding author: E-mail: [email protected].
Associate editor: Herve´ Philippe
Abstract
Thecurrentconsensusfortheeukaryotetreeoflifeconsistsofseverallargeassemblages(supergroups)thatarehypothesizedto
R
describe the existing diversity. Phylogenomic analyses have shed light on the evolutionary relationships within and between
e
supergroupsaswellasplacednewlysequencedenigmaticspeciesclosetoknownlineages.Yet,afeweukaryotespeciesremainof s
e
unknown origin and could represent key evolutionary forms for inferring ancient genomic and cellular characteristics of
a
eukaryotes. Here, we investigate the evolutionary origin of the poorly studied protist Collodictyon (subphylum Diphyllatia) by r
c
sequencingacDNAlibraryaswellasthe18Sand28SribosomalDNA(rDNA)genes.Phylogenomictreesinferredfrom124genes
h
placed Collodictyon close to the bifurcation of the ‘‘unikont’’ and ‘‘bikont’’ groups, either alone or as sister to the potentially
a
contentiousexcavateMalawimonas.PhylogeniesbasedonrDNAgenesconfirmedthatCollodictyoniscloselyrelatedtoanother
r
genus,Diphylleia,andrevealedaverylowdiversityinenvironmentalDNAsamples.TheearlyanddistinctoriginofCollodictyon t
i
suggests that it constitutes a new lineage in the global eukaryote phylogeny. Collodictyon shares cellular characteristics with c
l
ExcavataandAmoebozoa,suchasventralfeedinggroovesupportedbymicrotubularstructuresandtheabilitytoformthinand e
broad pseudopods. These may therefore be ancient morphological features among eukaryotes. Overall, this shows that
Collodictyon is a key lineage to understand early eukaryote evolution.
Key words: 18S and 28S rDNA, Collodictyon, Diphyllatia, tree of life, phylogenomics, cDNA, pyrosequencing.
Introduction and complex genome histories (Simpson and Roger
2004; Parfrey et al. 2006; Roger and Simpson 2009).
Overthelastfewyears,molecularsequencedatahavead-
Identification of sister lineages to these supergroups is
dressed some of the most intriguing questions about the
crucialforresolvingtheeukaryotetreeandunderstanding
eukaryote tree of life. Phylogenomic analyses have con-
the early history of eukaryotes. If these key lineages exist,
firmed the existence of several major eukaryote groups
theymaybefoundamongthefewspeciesthatharbordis-
(supergroups)aswellasshownvariouslevelsofevidences
tinct morphological features but are of unknown evolu-
fortherelationshipsamongthem(Burkietal.2007;Parfrey
tionary origin in single-gene phylogenies (Patterson 1999;
et al. 2010). Recently, two new large assemblages, SAR
Shalchian-Tabrizi et al. 2006; Kim et al. 2011). Indications
(Stramenopila,Alveolata,andRhizaria)andCCTH(Crypto-
thatsuchenigmaticspeciescanbeplacedintheeukaryote
phyta, Centrohelida, Telonemia, and Haptophyta), were
tree come from recent phylogenomic analyses. For in-
proposed to encompass a large fraction of the eukaryote
diversity,togetherwiththeothersupergroupsOpisthokon- stance, Ministeria (Opisthokonta), Breviata (Amoebozoa)
ta,Amoebozoa,Archaeplastida,andExcavata(Patronetal. and Telonemia, Centroheliozoa, and Picobiliphyta have
2007; Burki et al. 2009). Solid phylogenomic evidence been shown to constitute deep lineages within their re-
supports the monophyly of Amoebozoa, Opisthokonta, spective supergroups (Shalchian-Tabrizi, Minge, et al.
Archaeplastida, and SAR (Rodriguez-Ezpeleta et al. 2007; 2008;Burkietal.2009;Mingeetal.2009;Yoonetal.2011).
Burki et al. 2009; Minge et al. 2009), but the monophyly Here,weinvestigateamemberofsuchakeylineage,Col-
of Excavata and CCTH (also called Hacrobia; Okamoto lodictyon, which was first described in 1865 (Carter 1865),
et al. 2009) remains controversial, often dependent on butits cellularstructure andouter morphologywere ana-
the selection of taxa and gene data set (Burki et al. lyzedonlyrecently(Klaveness1995;Brugerolleetal.2002).
2009;Hampletal.2009;Baurainetal.2010).Despiteseveral Collodictyonwasoriginallyproposedtobecloselyrelatedto
attempts, the evolutionary relationships between these Diphylleia and Sulcomonas and classified in the family
supergroups are still uncertain because of the ancient Diphylleidae(Cavalier-Smith1993;thesynonymousfamily
©TheAuthor(s)2012.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense
(http://creativecommons.org/licenses/by-nc/3.0),whichpermitsunrestrictednon-commercialuse,distribution,and Open Access
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Mol. Biol. Evol. 29(6):1557–1568. 2012 doi:10.1093/molbev/mss001 Advance Access publication January 6, 2012 1557
MBE
Zhao et al. · doi:10.1093/molbev/mss001
CollodictyonidaeinBrugerolleetal.2002)andsubphylum 250–600 bp was eluted from a preparative agarose gel
Diphyllatia(Cavalier-Smith2003).Collodictyonisanomniv- and sequenced by the Norwegian ultra-high throughput
orousamoeba-flagellatewithamixofcellularfeaturesthat sequencing service unit at the University of Oslo and
makesituniqueamongeukaryotes.Thecellhasanegg-or Macrogen Inc (South Korea) yielding a total of 300,000
heart-like outline without walls or any other external sequence reads.
ornamentation in spite of a highly vacuolated cytoplasm
(Rhodes 1917; Klaveness 1995). It possesses four equally Sequence Analysis
long flagella and mitochondria with unconventional Allthe454pyrosequencingreadswereassembledintocon-
tubular-shaped cristae. An important character of Collo- tigsusingNewblerv2.5(Marguliesetal.2005)withdefault
dictyon is a broad ventral feeding groove dividing the cell parameters. We retrieved contigs larger than 200 bp with
longitudinally. This groove is supported by both left and significantsimilaritytogenesrecentlyusedinamultigene
rightmicrotubularrootsalongtheentirelengthofthelips, phylogeny (Burki et al. 2010). The translated contigs were
similar to comparable structures in other eukaryotes such screened by BlastP using our single-gene sequences as
as in Excavata (Simpson 2003). It also forms pseudopods queries, and the homologous copies (e value , 1 (cid:1) 10(cid:2)20)
typical of Amoebozoa at the base of the groove, which wereaddedtothesingle-genedataset.Thesenewsequen-
are actively used for catching prey. ces were automatically aligned by Mafft with the linsi
Despiteitsinterestingmorphologicalfeatures,itremains algorithm(Katohetal.2002),andambiguouslyalignedpo-
unclear whether Collodictyon is closely related to either sitionswereremovedusingGblocks(Castresana2000)with
ExcavataorAmoebozoaortoanyoftheothersupergroups halfofthegappedpositionsallowed,theminimumnumber
becausenomoleculardataareavailable.Furthermore,the ofsequencesforaconservedandaflankpositionsetto50%
position of the closely related Diphylleia is totally unre- of the number of taxa, the maximum of contiguous non-
solvedin18SribosomalDNA(rDNA)phylogenies(Bruger- conservedpositionssetto12,andtheminimumlengthof
olle et al. 2002; Shalchian-Tabrizi et al. 2006). In order to ablocksetto5.Theorthologyandpossiblecontamination
exploretheoriginofCollodictyon,weestablishedaculture in eachsingle-gene alignment were assessedby maximum
of Collodictyon triciliatum, sequenced the 18S and 28S likelihood (ML) reconstructions with 100 bootstrap repli-
rDNA genes, and carried out a deep survey of a cDNA li- catesusingRAxMLv7.2.6underthePROTCATLGFsubsti-
brary with 454 pyrosequencing. About 300,000 sequence tution model (Stamatakis 2006), followed by visual
reads were generated and used to assemble an alignment evaluationoftheresultingindividualtrees.Forseveralsin-
of 124 genes (27,638 amino acid characters) that covered gle genes (i.e., prmt8, tubb, rpsa, suclg1, tcp1-beta, hsp90,
ataxon-richsamplingofeukaryotes(79species).Tofurther ubc, and crfg), the PROTGAMMALGF model was used in
understandtheevolutionaryhistoryofthislineage,wealso additiontothePROTCATLGFmodelforbetteridentifica-
screenedthecDNAlibraryforthedihydrofolatereductase tionoftheorthology.Weusedpublishedglobaleukaryotic
(DHFR)andthymidylatesynthase(TS)genesandextended trees such as in Rodriguez-Ezpeleta et al. (2007) and
the DHFR gene by 3# Rapid Amplification of cDNA Ends Burki et al. (2009) as framework to identify and remove
(RACE) and polymerase chain reaction (PCR). thesequencesthatshowedunexpectedgroupingandwere
supported with more than 70% bootstrap in the single
genestrees.Inordertoidentifyhiddenparalogsinthedata,
Materials and Methods
we added more taxa in the single-gene phylogenetic
Culturing,Harvesting,andcDNALibraryConstruction analyses than in analyses of the supermatrix. Deletion of
Collodictyon triciliatum was isolated from Lake A˚rungen, long-branch taxa (i.e., Trichomonas, Giardia, and Spironu-
Norway,andculturedonamodifiedGuillardandLorenzen cleus) was done in a subsample of the single-gene align-
medium(GuillardandLorenzen1972).Collodictyontricilia- ments, but it did not change the phylogeny or the
tum was inoculated in a culture of the cryptomonad bootstrap values significantly. Hence, although inclusion
Plagioselmis nannoplanktica (Klaveness 1995; Shalchian- offast-evolvingspeciescouldpotentiallyintroducesystem-
Tabrizi,Bra˚te,etal.2008).cDNAlibrarieswereconstructed atic errors in the trees, these types of taxa seemed not to
byVertisBiotechnologyAG(Freising,Germany)according stronglyimpactourparalogidentification.Importantly,we
to their random-primed cDNA protocol: Total RNA was includedgenesequencesfromthecryptomonadGuillardia
extractedwithmirVanaRNAisolationkit(Ambion,Austin, theta in all alignments in order to phylogenetically distin-
TX),andpoly(A)þRNAwasisolatedfromthetotalRNA. guish sequences from Collodictyon and its prey (P. nanno-
First-strand cDNA synthesis was performed with random- planktica). This left in total 124 single-gene alignments
ized primers, and second-strand cDNA was synthesized containing Collodictyon sequences that were used for fur-
usingGublerandHoffmanprotocol(GublerandHoffman ther analyses. The concatenation of the 124 single genes
1983). Double-stranded DNA (dsDNA) was blunted, and was done by Scafos (Roure et al. 2007) and amounted
454 GSFLX adapters A and B were ligated to its 5# to 27,638 amino acid positions with average missing
and 3# ends. dsDNA carrying both adapters was selected characters 34.4% (For detail, see supplementary table S2,
and amplified with PCR (24 cycles). Differently expressed SupplementaryMaterialonline).Thesequencesgenerated
genes were normalized with a method developed by here were submitted to GenBank with accession number
Vertis Biotechnology AG. cDNA in the size range of JN618831–JN618979.Thesingle-genetreesandalignments
1558
MBE
Phylogenomics of Collodictyon · doi:10.1093/molbev/mss001
aswellastheconcatenatedalignmentareavailableathttp:// Testing Robustness of Trees by Removal of
www.mn.uio.no/bio/english/people/aca/kamran/. Fast-Evolving Sites
WeappliedtheAIRpackage(Kumaretal.2009;Yang2007)
Phylogeny of rDNA and Multigene Alignments to estimate evolutionary rates of sites under the Whelan
ReconstructionsofMLphylogeniesfrom18Sand28SrDNA and Goldman þ GAMMA model. The ML topology con-
sequence alignments were done using RAxML v7.2.6. The structedfromasampleof76taxa(i.e.,removaloftwoMa-
besttreewasdeterminedafter100heuristicsearchesstart- lawimonas species and Collodictyon) was used as starting
ingfromdifferentrandomtreesunderthegeneraltimere- treefortheestimateofsiterates.Therationaleforchoosing
versible (GTR) þ GAMMA þ I model. Bootstrap analyses thistopologywastoensurethatthesiterateswerecalcu-
wereperformedwith100pseudoreplicatesusingthesame lated independently of the evolutionary affinity between
model as in the initial tree search. Bayesian analyses were thesetwolineagesandtheirpositionsinthetree.Thesites
done with MrBayes v3.1.2 (Huelsenbeck and Ronquist werethenremovedin5%intervals(i.e.,removalofthe5%,
2001)undertheGTRþGAMMAþIþCOVevolutionary 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50% fastest
model that accounts for covarion substitution pattern evolvingsites)fromafullalignmentthatcontainedthetwo
acrossthesequences.Twoindependentruns,eachstarting MalawimonasspeciesandCollodictyon(i.e.,79taxa)andan
from a random tree for Markov chain Monte Carlo alignmentwhereonlythetwoMalawimonasspecieswere
(MCMC) chains, were run for 6,000,000 (18S rDNA) and removed (i.e., 77 taxa). The bootstrap values (BP) for the
4,000,000 (18S þ 28S rDNA) generations and sampled ev- nodesdefiningthesupergroupsaswellasfortheposition
ery 100 generations. Posterior probabilities and average ofCollodictyonandMalawimonaswereinferredfromeach
branchlengthswerecalculatedfromtheconsensusoftrees oftheseprocessedalignmentsbyRAxMLv7.2.6underthe
sampled after burn-in set to 3,000,000 (18S rDNA) and PROTCATLGF model (with 100 bootstrap replicates).
1,000,000(18Sþ28SrDNA)generations.Chainswerecon- Thesetrimmedalignmentswerethenusedfortheestima-
sideredtobeconvergentwhentheaveragesplitfrequency tionofaminoacidcomposition(seesupplementarymate-
was lower than 0.01. rials and methods, Supplementary Material online). All
Severalconcatenatedproteinalignmentswithdifferent bioinformatics analyses were done on the Bioportal at
taxonomiccompositionswereconstructedtoinvestigate the University of Oslo (www.bioportal.uio.no; Kumar
theinfluenceofspeciessamplingandmissingdataonthe et al. 2009).
phylogeny of Collodictyon. Phylogenies were inferred by
ML and Bayesian approaches, as implemented in RAxML Topology Comparisons
v7.2.6 and Phylobayes v3.2 (Lartillot and Philippe 2004), Topologytestingwasperformedusingtheapproximately
respectively.FollowingboththeAkaikeinformationcrite- unbiased (AU) test (Shimodaira 2002). For each tested
rionandthelikelihoodratiotestcomputedwithProtTest tree, site likelihoods were calculated using RAxML
3.0 (Darriba et al. 2011), the optimal model LG þ v7.2.6withthePROTGAMMALGFmodel,andtheAUtest
GAMMA þ F available in RAxML v.7.2.6 was chosen to wasperformedusingCONSEL(ShimodairaandHasegawa
infer ML trees. The best ML topology was determined 2001).
in heuristic searches from ten random starting trees.
Due to computational burden, statistical support was 3# RACE and Sequencing of the DHFR-TS Genes
evaluatedwith100bootstrapreplicatesunderthePROT- All assembled contigs were used as queries in BLAST
CATLGF model that approximates the gamma distribu- searchagainstthenonredundantproteinsequencesdata-
tion for site-rate variation (Stamatakis et al. 2008). base available at NCBI. Three contigs (contig15348,
Bayesian inferences were done with the CAT site- contig15349, and contig06264) showed a significant sim-
heterogeneousmixturemodel.TwoindependentMCMC ilaritytotheDHFRgene(evalue,1(cid:1)10(cid:2)10).Inorderto
chainsinPhyloBayesstartingfromrandomtreeswererun verify that these contigs belong to Collodictyon and not
for 24,000 cycles with trees being sampled every cycle. the prey, we designed forward and reverse primers, then
Consensustopologyandposteriorprobability(PP)values different combinations of primers were used to amplify
were calculated from saved trees after burn-in. Conver- genomic DNA from three cultures: 1) P. nannoplanktica
gence between the two chains was ascertained by exam- (PN),2)P.nannoplankticaþC.triciliatum(PNþCT),and
ining the difference in frequency for all their bipartitions 3)ChlorellapyreuoidosaþC.triciliatum(CPþCT).Bands
(maxdiff , 0.15). In addition, a bootstrap analysis under were observed on the agarose gel solely when using for-
the CAT model was performed on 100 pseudoreplicates ward primer in contig15348 and reverse primer in con-
generatedbySeqboot(Phylippackage;Felsenstein2001). tig15349 for PCR amplification from PN þ CT and CP
For each replicate, two Phylobayes MCMC chains were þ CT cultures. Both sequences were identical and
run for 5,000 cycles with a conservative burn-in of matched the 3#-end region of contig15348 and the 5#-
2,000cycles.Manualverificationof10%randomlychosen endregionofcontig15349.Sinceidenticalsequenceswere
replicates showed that the burn-in was optimal between only obtained in the cultures containing Collodictyon, it
1,000 and 2,000 cycles. Consense (Phylip package) was confirmed that these two contigs corresponded to the
used to calculate the bootstrap support based on these Collodictyon gene, not the Plagioselmis or Chlorella one.
100 Bayesian consensus trees. Total RNA was isolated from PN þ CT cultures with
1559
MBE
Zhao et al. · doi:10.1093/molbev/mss001
Ammonia sp.
Reticulomyxa filosa
Allogromia sp.
Minchinia teredinis
Urosporidium crescens
Gromia oviformis
1.0/77 Phagomyxa oLdEoMnDte0ll5a2e
Plasmodiophora brassicae
1.0/62 Bodomorpha RmTin5iimina20
-/84 LKM30
BOLA383
Lecythium sp.
LKM48
1.0/63 Gymnophrys cometa
1.0/63 RT5iin4
N-Por
Acanthometra sp.
Arthracanthida sp.
Chaunacanthida sp.
0.84/- SyDmHp1h4y7aEcKanDt1h7id a
1.0/56 AT4.94 OLI11032 SAR
Acrosphaera sp. CR6A
Sphaerozoum punctatum
0.87/- AT8.54
Bacillaria paxillifer
0.63/- 0.83/- RT7iinO2LIC1i1li0o2p5h rys infusionum
0.99/- Achlya bisexualis
BOLA515
0.69/- DH148EKD53
BAQA072
OLI51105
0.88/- CCaSfe.Ete0r4ia5 roenbergensis
OLI11066
OLI11150
0.78/59 0.79/75 RT 5iin25 CS.E036
Diplophry s sp.
0.66/61 Labyrinthuloides minutaUlkenia profunda
Gonyaulax spinifera
0.88/- 1.0/- ColpNoodcetillluac pao snctiicnatillans
Loxophyllum utriculare
Oxytricha nova
0.55/- Cryptosporidium parvum
OLI11056
Emiliania hOuLxIl1e1y0i72 Haptophyta
OLI11007
-/- Pavlova salina
Telonema subtilis RCC404.5
0.73/- RA001219.10
0.63/- TReAlo0n1e0m41a2 s.u1b7tilis RCC358.7 Telonemia
RA000412.136
0.81/- BL010625.25
Telonema antarcticum
0.79/- AT4.11 Amastigomonas debruynei Apusozoa
-/- AT4.50
Apusomonas proboscidea
0.98/- Chlamydaster sterni Raphidiophrys ambigua Centrohelida
TCS 2002
0.70/- GlaucCocyyasntoisp tnyocshteo cghloineeoacryusmtis Glaucophyta
0.81/- Cyanophora paradoxa Goniomonas truncata Cryptophyta
1.0/74 GuillarCdoiam tphseotap ogon coeruleus Rhodophyta
Glaucosphaera vacuolata
0.84/- RT1n14cul
0.51/- SRchTe5riifnfVe2lo ialv doux bciaarteri Viridiplantae
OLI11059
OLI11305
RT5iin8
Helianthus annuus LKM101
Trypanosoma cruzi
AT4.56 AT1.3
-/- 0.69/57 Ichthyobodo necator
AT 4.96
DH148EKB1
Diplonema ambulator
0.79/- Euglena gracilis
RT8n7
Paravahlkampfia ustiana
RT5in38
0.96/- BOLA212 Naegleria gruberi
BOLA458
C2.E026
0.59/- C3.E012 CS.R003
0.87/- DH148EKD18
0.99/- BOLA048
DH145EKD11
0.68/- 1.0/- CS.E022 Jakoba liberaReclinomonas americana Excavata
0.99/68 UJankcoubltuar iendc aeruckeararytaote clone BOLA187
Uncultured eukaryote clone BOLA366
Breviata anathema
Malawimonas jakobiformis
0.89/- OStxryembloonmaass stipx. strix
0.40/- 0.89/- Trimastix pyrifTorrmimisastix marina
C1.E027
0.53/76 DUipnhcyullletuiare rdo tCaonlslodictyonidae partial Retortamonas sp. Diphyllatia
Collodictyon triciliatum
Amoeba proteus
0.53/- Leptomyxa retiBcuOlaLtAa868
Hartmannella vermiformis
LKM74
0.95/- Mayorella sp. Amoebozoa
Acanthamoeba castellanii
1.0/78 Platyamoeba stenopodia RT5iin44 LEMD267
Filamoeba nolandi
0.99/- Phalansterium solitarium
0.63/- Filobasidiella neoformans
Saccharomyces cerevisiae
0.57/50 Monosiga brevicollis Opisthokonta
Nuclearia simplex
0.99/68 Lumbricus rubellus
Podocoryne carnea
0.2
FIG.1.18SrDNAphylogenyoftheDiphyllatiaspeciesCollodictyontriciliatum(highlightedbyblackbox)andDiphylleiarotans.Thetopology
wasreconstructedbyMrBayesv3.1.2undertheGTRþGAMMAþIþcovarionmodel.Posteriorprobabilities(PP)andMLbootstrapsupports
(BP,inferredbyRAxMLv7.1.2underGTRþGAMMAþImodel)areshownatthenodes.ThicklinesindicatePP.0.90andBP.80%.Dashes
‘‘-’’ indicate PP, 0.5 orBP , 50%. Afew long branches are shortened by50% (/) or 75%(//).
1560
MBE
Phylogenomics of Collodictyon · doi:10.1093/molbev/mss001
Bigelowiella sp.
Thaumatomonas sp.
Paracercomonas marina
Aureococcus anophagefferens
0.97/57 Pelagomonas calceolata
Apedinella radians
0.81/- Rhizochromulina cf. marina
Dictyocha speculum
0.98/- Glossomastix chrysoplasta
Pinguiococcus pyrenoidosus
1.00/68 Tribonema aequale
0.75/85 Dictyota dichotoma
0.63/- Fucus gardneri
Laminaria angustata
Scytosiphon lomentaria SAR
Choristocarpus tenellus
0.84/59 Skeletonema pseudocostatum
Cylindrotheca closterium
0.97/63 Rhizosolenia setigera
Ochromonas danica
Chrysolepidomonas dendrolepidota
Synura sphagnicola
Mallomonas rasilis
Hyphochytrium catenoides
0.90/- Phytophthora megasperma
Cryptosporidium parvum
Toxoplasma gondii
0.83/- CochPleordkininiusums paotllaynkrtiickuosides
Paramecium tetraurelia
Phaeocystis antarctica Haptophyta
Centroheliozoa sp. Prymnesium patelliferum Centrohelida
0.97/- Cyanophora paradoxa Glaucophyta
0.84/- Glaucocystis nostochinearum
Roombia truncata
Goniomonas sp. Goniomonas truncata Cryptophyta
Guillardia theta
0.80/- Cryptomonas paramecium
Bangia fuscopurpurea Rhodophyta
Cyanidioschyzon merolae
0.76/- Chlamydomonas pulsatilla
0.99/- 0.80/- 0.91/68 PeOdoiagsatrmuomc hdluapmleyxs zimbabwiensis
Chlorella vulgaris Viridiplantae
Pseudochlorella sp.
0.98/74 Arabidopsis thaliana
0.97/- Marchantia polymorpha
Closterium selenastrum
0.90/0.60 Closterium ehrenbergii
Chara globularis
Histiona aroides Excavata
0.99/45 Reclinomonas americana
Trimastix pyriformis Diphyllatia
Collodictyon triciliatum
1.00/56 RhizamoebaA scaaxnothnaicmaoeba castellanii Amoebozoa
Hartmannella vermiformis
Apusomonas proboscidea
Amastigomonas bermudensis Apusozoa
Ancyromonas sigmoides
Planomonas micra
0.85/- Filobasidiella neoformans
Amanita bisporigera
1.00/79 Mucor racemosus
Saccharomyces cerevisiae
1.00/- 0.65/61 Aspergillus oryzae
Schizosaccharomyces pombe
Pneumocystis carinii
Nuclearia simplex Glomus mosseae Opisthokonta
0.66/- Antipathes galapagensis
0.90/68 Chironex fleckeri
Beroe ovata
Leucosolenia sp.
Suberites ficus
0.96/61 Monosiga brevicollis
Capsaspora owczarzaki
0.53/61 Ichthyophonus hoferi
0.03
FIG.2.18Sþ28SrDNAphylogenyofCollodictyontriciliatum(highlightedbyblackbox)reconstructedwithMrBayesv3.1.2undertheGTRþ
GAMMAþIþcovarionmodel.NumbersatnodesarePPandMLbootstrapvalues(BP,inferredbyRAxMLv7.2.6undertheGTRþGAMMAþ
I model). Thick lines show PP . 0.9 and BP . 80%. Nodes marked with symbol ‘‘-’’ indicate BP , 50% or PP , 0.5. Some branches are
shortened byhalf in ordertosave space(marked with‘‘/’’).
theRNAqueous-MicroKit(Ambion,Austin,TX)following Results and Discussion
thestandardprotocol.The3#RACEsystemfromInvitro-
Collodictyon Is an Ancient and Distinct Eukaryote
gen (Carlsbad, CA) was performed to obtain the full-
length 3#-end of the DHFR cDNA. Two specific forward Lineage
primers (DHFR1F: 5#-CGAGTGCGTTGAATGATTCGT- InordertoclarifytheoriginofCollodictyon,wefirstobtained
CAAA-3# and DHFR2F: 5#-CTCAATGTTATTGTCAG- the18SrDNAsequenceforC.triciliatum.Phylogeneticanal-
CAGCACT-3#), together with a universal reverse ysisrecoveredmostoftheeukaryotesupergroupsasmono-
primer (AUAP: 5#-GGCCACGCGTCGACTAGTAC-3#), phyleticclades,exceptCCTHandArchaeplastida,congruent
were used in a two-step protocol to improve the speci- with several recent reports (fig. 1; Burki et al. 2007, 2008;
ficityoftheamplificationprocess.ThePCRproductswere Yoonetal.2008;Hampletal.2009).Moreinterestingly,this
sequencedtovalidatewhethertheDHFRgeneandtheTS phylogenyrobustlysupportedCollodictyonandDiphylleiaas
gene were fused or not (GenBank accession number: sister lineages with 100% bootstrap support (BP) and 1.00
JN618830). posterior probabilities (PP), confirming that these two
1561
MBE
Zhao et al. · doi:10.1093/molbev/mss001
Table 1.Maximum likelihood bootstrapvalues (ML)andbayesian posteriorprobabilities (Bayes) ofthe EukaryoteSupergroups in the
Phylogenomic Trees.
79Taxa 74Taxaa 77Taxab 72Taxac
20% 20%
AllSites Removedd AllSites Removedd
AllSites AllSites
Nodee Groups ML Bayes ML Bayes ML ML Bayes ML Bayes ML
A Opisthokonta 100 1.00 100 1.00 100 100 1.00 100 1.00 100
B Unikonts 79 0.99 99 1.00 87 57 0.99 96 1.00 58
C Amoebozoa 86 1.00f 100 1.00 100 84 1.00f 100 1.00 100
D Collodictyon1Malawimonas 86 0.79 98 0.63 94 NA NA NA NA NA
E Excavata 100 1.00 100 1.00 100 100 1.00 100 1.00 100
F Bikonts 98 1.00 98 1.00 95 98 1.00 100 1.00 93
G Archaeplastida - 0.98 63 0.84 - - 0.99 71 0.95 -
H Archaeplastida1CCTH1SAR - 1.00 81 1.00 - - 1.00 88 1.00 -
I CCTH - * 54 * - 50 * 60 * -
J SAR 98 1.00 100 1.00 96 99 1.00 100 1.00 96
NOTE.—‘‘-’’indicatebootstrapvalues,50%orPP,0.5;‘‘*’’indicatethatCCTH(Cryptophyta,Centrohelida,Telonemia,andHaptophyta)isnotmonophyletic.
aFivetaxa(Leishmania,Trypanosoma,Sawyeria,Entamoeba,andBreviata)wereremoved.
bTwoMalawimonastaxawereremoved.
cTwoMalawimonastaxaandfivetaxa(Leishmania,Trypanosoma,Sawyeria,Entamoeba,andBreviata)wereremoved.
dRemovalofthe20%fastestevolvingsitesfromthealignment.
eThecapitalletterscorrespondtosupergroupsmarkedinfigure3.
fBreviataissistertoOpisthokonta(fig.3).
speciesindeedarecloselyrelated.Inanattempttoenrichthe CCTH, but these were instead placed as a sister to Opis-
speciesdiversityforthisgroupandestimatetheirpotential thokonta (0.75 PP) and SAR (0.91 PP). Of much interest,
abundance and diversity in nature, we searched for our analyses showed that Collodictyon branched outside
Collodictyon-like 18S rDNA sequences by blastn against any of the major lineages (fig. 3A and supplementary fig.
theenvironmentaldatabaseinNCBI.TwentyofthetopBlast S1A,SupplementaryMaterialonline),morespecificallyat
hits were used for phylogenetic analysis, but only a single the bifurcation of the so-called ‘‘unikonts’’ (Amoebozoa
partial sequence grouped with Diphylleia (results not and Opisthokonta) and ‘‘bikonts’’ (Archaeplastida, SAR,
shown),suggestingalowdiversityandabundanceoftheDi- Excavata,CCTH;thetermsunikontsandbikontsareused
phyllatia in the environment. This partial sequence was in- here for simplicity and do not refer to their original
cluded in the 18S phylogeny (fig. 1). description; Stechmann and Cavalier-Smith 2002; Roger
To improve the rDNA tree, we also sequenced the 28S and Simpson 2009). Although Collodictyon did not fall
rDNAgeneforCollodictyonandreconstructedacombined within any of the supergroups, an affinity to another
18S þ 28S rDNA phylogeny (fig. 2). This tree showed Col- enigmatic genus Malawimonas was recovered with 0.79
lodictyonasadeeplineagewithpossibleaffinitytoExcavata PP and 86% BP.
with 45% BP and 0.99 PP. Interestingly, our data did not To test whether the deep position of Collodictyonwas
show any affiliation to Apusozoa, even though this group stable or instead sensitive to taxonomic sampling, we
has been proposed to be closely related to Collodictyon performed several taxon removal experiments, but
(Cavalier-Smith 2003). Instead, the 18S þ 28S rDNA tree Collodictyon was consistently recovered in the same po-
suggested Apusomonas to be sister to Amoebozoa (56% sition. Most interestingly, the position of Collodictyon in
BP and 1.00 PP), although Ancyromonas grouped with the global eukaryote phylogeny remained identical when
the Opisthokonta (,50% BP and 1.00 PP). Malawimonas was removed from our alignment (fig. 3B
Because our 18S and 18S þ 28S rDNA trees suggested and supplementary fig. S1B, Supplementary Material on-
that Collodictyon might have diverged very early in eu- line).Itwasstillplacedclosetothesplitbetweenunikonts
karyote evolution and that these two genes alone were andbikonts,suggestingthatthispositionwasnotcaused
not sufficient to infer ancient relationships, we sought by erroneous attraction to Malawimonas or other Exca-
to increase the phylogenetic signal by constructing an vataspecies(i.e.,Trimastix;seesupplementaryfig.S2,Sup-
alignment of 124 protein-coding genes and 79 taxa. Phy- plementary Material online). The high statistical support
logenomic trees inferred with both Bayesian and ML forthebikontgrouprecoveredwiththisreduceddataset
methods consistently recovered most eukaryote super- stronglyexcludedCollodictyonfrombeingmemberofthis
groups as in recent studies (Rodriguez-Ezpeleta et al. assemblage (bikonts: BP 5 98% and PP 5 1.00). On the
2007; Burki et al. 2009; Hampl et al. 2009), generally with other hand, removing Malawimonas lowered the boot-
highstatisticalsupport(table1).Differingfrompublished strap support for the unikonts (BP 5 57% and PP 5
phylogenies (Burki et al. 2009; Minge et al. 2009), the 0.99; table 1), pointing to a possible attraction between
Bayesian inference (fig. 3A) did not recover Breviata as Collodictyonandthisothermajorgroup.Inordertoeval-
sistertoAmoebozoaandTelonemadidnotbranchwithin uatethepotentialimpactofmissingdataontheposition
1562
MBE
Phylogenomics of Collodictyon · doi:10.1093/molbev/mss001
A B
Missing
number Missing
of genes characters (%)
Homo Homo
Mus Mus
Gallus Gallus
Danio Danio
Drosophila Drosophila
Strongylocentrotus Strongylocentrotus
Nematostella Nematostella
Monosiga Monosiga Opisthokonta
A 0.99 CapSsapshpaoerraoforma A CapSspahspaoerraoforma
Ustilago Ustilago
Cryptococcus Cryptococcus
Schizosaccharomyces Schizosaccharomyces
Neurospora 0.93 Neurospora
B 0.75 PBhaytcraocmhyoccehsytrium B PBhaytcraocmhyoccehsytrium
* C 0.990.88 AcanthaHmaoretmbaanBnreelvlaiata C 0.990.92 AcanthaHmaortembaaBnrneevlliaata
0.86 PhysaruDMmicatsytoigsatemliouemba 0.89 PhysarDMumiacstytiogsatmeliouemba Amoebozoa
D CollodictyonEntamoeba Entamoeba
* 0.79 ‘Malawimonas californiana’ Collodictyon Diphyllatia
Malawimonas jakobiformis Histiona
* Histiona Reclinomonas
E ReclJinaokmoboanas E SecuJlaakmoobnaas
Seculamonas Stachyamoeba Excavata
* Stachyamoeba 0.98 Naegleria
0.99 Naegleria Sawyeria Euglena Sawyeria
Euglena Trypanosoma
Trypanosoma Leishmania
Leishmania Arabidopsis
Arabidopsis Solanum
Solanum Oryza
Oryza Pinus
Pinus Physcomitrella
Physcomitrella Mesostigma
* Mesostigma Chlamydomonas
Chlamydomonas F Volvox
F Volvox Micromonas Archaeplastida
Micromonas 0.99 Ostreococcus
** G 0.99 GOrCashctrioleanordicaroucscus G PoGrCprahhcyoirlnaadriraus
* 0.98 0.99 PGoarlpdhieyrriaa Cyanidioschyzon 0.99 0.9C9yanophoGraaldieria Cyanidioschyzon
Cyanophora Glaucocystis
Glaucocystis Guillardia
* H 0.51 GPuRlialalagprihodisidaeiolmphisrys H 0.58 EmPiRlilaaangpiihaoisdeiolmphisrys
****** I 0.09.383 IPPmarIaEysvnmolmotconvihlnaeiarTisyCaniesuieailsmorcnBoePigmmheoaalonewaosdieallcatylum I 0.09.182 PPIrmyaImavslnonotcevoThasnCeriiuyaleosmrniBscePoigmmheaaoloenwoadiseallcatylum CCTH
0.91 AurTeohcaolacscsuiossira AurTehoacloacscsuiossira
* Laminaria Laminaria
* J PPhyythtoiupmhStchhoirzaochytrium J PPhyythtoiupmShtchhoirzaochytrium SAR
Alexandrium Alexandrium
* Karlodinium Karlodinium
* Oxyrrhis Oxyrrhis
Perkinsus Perkinsus
Toxoplasma Toxoplasma
Cryptosporidium Cryptosporidium
Tetrahymena Tetrahymena
Paramecium Paramecium
100 75 50 250255075100 0.1 0.1
FIG.3.PhylogenomicpositionofCollodictyoninferredfrom124genesundertheCATmixturemodelinPhyloBayesv3.2.Branchesthatreceived
1.00PParemarkedbyfilledcircles.ThebranchlengthofEntamoebaisshortenedby50%tosavespace.(A)Treetopologyconstructedwith79
taxafromthesaved18,000treesafterdiscardingthefirst6,000cyclesasburn-in(maxdiff50.137).Missingdataforeachtaxonisshownas
a color barplot (left bar: missing number of genes; right bar: missing percentage of characters). Bars marked by ‘‘ ’’ indicate the missing
*
percentageofcharactersisover60%ofthefull-lengthalignment.(B)Treetopologyconstructedwith77taxa(i.e.,twoMalawimonasexcluded)
from the saved 16,000 trees after discarding first 8,000 cycles as burn-in (maxdiff 5 0.083). CCTH is the abbreviation of Cryptophyta,
Centrohelida, Telonemia, and Haptophyta. Additional statistical support values for the main nodes in the tree marked by capital letters in
boxes arelisted intable1.
of Collodictyon, we removed taxa with more than 60% branching within unikonts or bikonts using similar taxo-
missingcharacters(fig.3A).Thephylogeniesinferredfrom nomic sampling as reported by Hampl et al. 2009 and
this data set showed Collodictyon in the same position, Rodriguez-Ezpeletaetal.2007(i.e.,Leishmania,Trypanosoma,
which indicated that taxa with low sequence coverage Sawyeria,Entamoeba,andBreviataremoved).Again,noal-
didnotaffecttheconstructionofCollodictyonphylogeny ternativepositionwasobservedforCollodictyon(seetable
(supplementary figs. S3 and S4, Supplementary Material 1 and supplementary fig. S5, Supplementary Material
online). Finally, we tested the possibility of Collodictyon online).
1563
MBE
Zhao et al. · doi:10.1093/molbev/mss001
est evolving sites showed strong evidence for excluding
Collodictyon from unikonts (PP 5 1.00; CAT-BP 5 93%)
or bikonts (PP 5 1.00; CAT-BP 5 100%) (fig. 5 and table
1). Cross-validation test showed that the CAT model fits
our data better than the LG model with a score averaged
over10replicatesof2451.36±132.9(allreplicatesfavored
the‘‘CAT’’model).Theglobalphylogenyinferredfromthe
CAT model should be favored, although both models re-
coveredthesamepositionofCollodictyon(fig.5Bandsup-
plementary fig. S6B, Supplementary Material online).
Hence, after the removal of the noisiest positions in our
alignment, Collodictyon was robustly placed close to the
bifurcation of unikonts and bikonts.
Consistent with the phylogenetic analyses mentioned
above, the AU test based on the data set without the
20% fastest evolving sites rejected topologies where
Collodictyon was placed within unikonts or bikonts. The
FIG. 4. Changes in bootstrap support for key nodes in the inferred sameresultsholdtrueforthebikontswhenthefull-length
treesasfast-evolvingsiteswereremoved.Siterateswereestimated
alignment was used, but the possibility of Collodictyon
from an alignment without two Malawimonas and Collodictyon
branching within unikonts, that is, sister to Amoebozoa
species (76 taxa). Sites were then removed in 5% increments from
alignments consisting of (A) 79 taxa (including Collodictyon and (P50.372)orOpisthokonta(P50.076),couldnotbedis-
Malawimonas) and (B) 77 taxa (including Collodictyon). ML carded at the 5% level of significance (table 2). These two
Bootstrap values (BP) for Collodictyon þ Malawimonas, unikonts, alternativetreeswereevaluatedbycomparingwiththeop-
bikonts, and Opisthokonta (used as a reference) were calculated timallikelihoodtopology(supplementaryfig.S1B,Supple-
underthePROTCATLGFmodelinRAxMLv7.2.6.BPvaluesshaded
mentary Material online) under a covarion model in
bygrayrectanglesarelistedintable1andsupplementaryfigureS6
ProCov (Wang et al. 2009). The alternative topologies ob-
(Supplementary Material online).
tained substantially lower likelihood values (DlnL 5 (cid:2)31
All phylogenetic analyses described above were done andDlnL5(cid:2)15)thantheoptimaltopology.Nevertheless,
based on a ‘‘concatenated model,’’ without considering inordertoexamineotherpossibleaffinitiesofCollodictyon
theevolutionarytempoandmodeofeachproteincompos- within Amoebozoa or Opisthokonta, 24 topologies where
ingtheconcatenatedalignment.Wethereforeassessedthe Collodictyonbranchedwithbasallineagesofunikontswere
impactofusinga‘‘separatemodel’’thattakesintoaccount compared.Strikingly,allofthemwererejected(P,0.05),
the evolutionary specificity of each gene (see supplemen- thus weakening the suspicion of a closer relationship be-
tary materials and methods, Supplementary Material on- tween Collodictyon and unikonts (supplementary fig. S7,
line). The topologies inferred from the separate model Supplementary Material online).
again recovered Collodictyon in the same position near
the bifurcation of unikonts and bikonts, either alone or Relationship between Collodictyon and
as sister to Malawimonas (supplementary fig. S1 and S5, Malawimonas
SupplementaryMaterialonline).Furthermore,theseparate Malawimonashasproventobeparticularlychallengingto
model generated similar bootstrap support values as the placeintheeukaryotetree,evenwithverylargealignments,
concatenatedmodel(seesupplementarytableS1,Supple- butithastypicallybeenassociatedwithExcavatabasedon
mentary Material online), altogether demonstrating that itsultrastructure(Simpson2003).Inouranalyses,Malawi-
thephylogeneticpositionofCollodictyonisnotanartifact monasgenerallybranchedoutsideofExcavata(fig.3A,sup-
caused by oversimplification of the concatenated model. plementary figs. S1A and S3A and S3C, Supplementary
To further investigate the evolutionary origin of Collo- Material online), in agreement with previous observations
dictyon,weattemptedtoincreasethephylogeneticversus (Rodriguez-Ezpeletaetal.2007;Hampletal.2009).Because
nonphylogeneticsignalratiobyremovingthefastestevolv- MalawimonasgroupedwithCollodictyonandnotwithEx-
ingsites,whichhavebeenshowntobearthehighestdegree cavatainourBayesianandMLtrees,wetookacloserlook
ofhomoplasy(BrinkmannandPhilippe1999).Becauseour at this relationship by applying several strategies. One
analyses suggested that Collodictyon is excluded from the modelviolationthatisknowntocausetreereconstruction
known eukaryote supergroups, we successively monitored artifactsisbiasintheaminoacid(AA)composition.Inter-
the statistical support for unikonts and bikonts. Most no- estingly, our heatmap analyses showed a weak deviation
tably,thebootstrapsupportfor unikontsincreasedas the fromaminoacidhomogeneitythatcouldpartiallyaccount
fastest evolvingsites wereremoved,reachingapeak value for the grouping of Collodictyon and Malawimonas, to-
of 96% after removing 20% of sites (table 1 and fig. 4B), gether with a few other taxa (supplementary fig. S8 and
whereas the bikonts remained highly supported (BP . table S3, Supplementary Material online). Removing up
95%)duringthisexperiment.Moreover,aBayesianphylog- to20%ofthefastestevolvingsitesseemednottoovercome
enyconstructedwiththealignmentremovingthe20%fast- theamino acidcompositional bias (supplementary fig.S8,
1564
MBE
Phylogenomics of Collodictyon · doi:10.1093/molbev/mss001
FIG. 5. Bayesian phylogeny of Collodictyon constructed from 124 genes after removal of the fastest evolving sites. The consensus topology was
calculatedundertheCATmodelfrom18,000savedtreesafterdiscardingthefirst6,000cyclesasburn-in.Branchesshowing1.00PParemarkedby
filledcircles.ThebranchlengthofEntamoebaisshortenedby50%tosavespace.(A)Treetopologyinferredfromthetrimmedalignmentwiththe20%
fastestevolvingsitesremoved(markedbygrayrectanglesinfig.4A).Chainswereconsideredtohaveconverged(maxdiff50.104).(B)Treetopology
inferredfromthetrimmedalignment(i.e.,twoMalawimonasexcluded)withthe20%fastestevolvingsitesremoved(markedbygrayrectanglesinfig.
4B).Chainswereconsideredtohaveconverged(maxdiff50.065).Numbersatthenodesin(B)indicatePP/bootstrapvaluescalculatedfromfrom100
pseudoreplicates with Phylobayes under CAT mixture model. Dashes ‘‘-’’ indicate bootstrap supports , 50%. CCTH is the abbreviation of
Cryptophyta,Centrohelida,Telonemia,andHaptophyta.Additionalstatisticalsupportvaluesforthesupergroupsareshownintable1.
Supplementary Material online). However, recoding the better fitted CAT model from the alignment after removing
amino acids into functional categories (Hrdy et al. 2004) the20%fastestevolvingsitesonlyweaklyrecoveredCollodictyon
still recovered the grouping of Malawimonas and Collo- andMalawimonasasagroup(PP50.63;fig.5Aandtable1).
dictyon(supplementaryfig.S9,SupplementaryMaterialon- Moreover, when Collodictyon and five other taxa (i.e., Leish-
line), suggesting that the bias may not significantly affect mania,Trypanosoma,Sawyeria,Entamoeba,andBreviata)were
the phylogeny. removedfromthedataset,Malawimonasgroupedassisterto
Despitethisapparentcloserelationshipbetweenthem,itis ExcavatainourMLtree(BP5 60%;supplementaryfig.S5B,
important to note that the Bayesian tree inferred under the Supplementary Material online), in agreement with recent
1565
MBE
Zhao et al. · doi:10.1093/molbev/mss001
Table 2.AU Test ofTreeTopologies.
PValueb
Rank TreeTopologyBasedonaSampleof79Taxaa Allc 20%d
1 (((Opst,Amoe),(Mala,Coll)),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.982 0.995
2 (((Opst,Amoe),Coll),((Exca,Mala),(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.064 0.045
3 (((Amoe,(Mala,Coll)),Opst),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.067 0.006
4 (((Opst,(Mala,Coll)),Amoe),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.009 0.005
5 (((Opst,Mala),(Coll,Amoe)),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.007 0.010
6 (((Opst,Coll),(Mala,Amoe)),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.006 0.006
7 ((((Opst,Amoe),Coll),Mala),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.005 0.014
8 (((Opst,(Coll,Amoe)),Mala),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.003 2310204
9 ((((Opst,Coll),Amoe),Mala),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.002 0.001
10 (((Opst,Amoe),Mala),((Exca,Coll),(Plan,(SAR,((Cryp,Hapt),TelRap))))) 3310204 7310205
11 (((Opst,Amoe),Mala),(Exca,((Plan,Coll),(SAR,((Cryp,Hapt),TelRap))))) 2310204 8310205
12 (((Opst,Amoe),Mala),(Exca,(Plan,(SAR,((Cryp,Hapt),(Coll,TelRap)))))) 1310204 0.001
13 (((Opst,Amoe),Mala),(Exca,(Plan,(SAR,(((Cryp,Coll),Hapt),TelRap))))) 8310205 8310206
14 (((Opst,Amoe),Mala),(Exca,(Plan,(SAR,((Cryp,(Coll,Hapt)),TelRap))))) 2310205 6310207
15 ((((Opst,Amoe),Coll),Mala),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 1310205 2310207
16 ((Opst,Amoe),((Exca,(Mala,Coll)),(Plan,(SAR,((Cryp,Hapt),TelRap))))) 6310215 5310214
17 ((Opst,Amoe),((Exca,Mala),Coll)),(Plan,(SAR,((Cryp,Hapt),TelRap))))) 4310212 3310211
PValueb
Rank TreeTopologyBasedonaSampleof77Taxaa(i.e.,twoMalawimonasexcluded) Allc 20%e
1 (((Opst,Amoe),Coll),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.532 0.614
2 (((Opst,Amoe),Coll),(Exca,(SAR,(Plan,((Cryp,Hapt),TelRap))))) 0.630 0.529
3 (((Opst,Coll),Amoe),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.071 0.046
4 (((Opst,Coll),Amoe),(Exca,(SAR,(Plan,((Cryp,Hapt),TelRap))))) 0.076 0.045
5 ((Opst,(Amoe,Coll)),(Exca,(Plan,(SAR,((Cryp,Hapt),TelRap)))) 0.284 0.040
6 ((Opst,(Amoe,Coll)),(Exca,(SAR,(Plan,((Cryp,Hapt),TelRap)))) 0.372 0.037
7 ((Opst,Amoe),((Exca,Coll),(Plan,(SAR,((Cryp,Hapt),TelRap))))) 0.001 0.001
8 ((Opst,Amoe),((Exca,Coll),(SAR,(Plan,((Cryp,Hapt),TelRap))))) 0.003 2310204
9 ((Opst,Amoe),(Exca,((Plan,Coll),(SAR,((Cryp,Hapt),TelRap))))) 2310206 4310207
10 ((Opst,Amoe),(Exca,((SAR,Coll),(Plan,((Cryp,Hapt),TelRap))))) 7310206 3310208
11 ((Opst,Amoe),(Exca,(Plan,(SAR,(((Cryp,Coll),Hapt),TelRap))))) 6310207 1310204
12 ((Opst,Amoe),(Exca,(SAR,(Plan,(((Cryp,Coll),Hapt),TelRap))))) 2310205 6310205
13 ((Opst,Amoe),(Exca,(Plan,(SAR,((Cryp,(Coll,Hapt)),TelRap))))) 6310209 7310239
14 ((Opst,Amoe),(Exca,(SAR,(Plan,((Cryp,(Coll,Hapt)),TelRap))))) 8310205 2310247
15 ((Opst,Amoe),(Exca,(Plan,(SAR,((Cryp,Hapt),(Coll,TelRap)))))) 1310274 5310251
16 ((Opst,Amoe),(Exca,(SAR,(Plan,((Cryp,Hapt),(Coll,TelRap)))))) 1310269 8310254
17 ((Opst,Amoe),(Exca,(Plan,((SAR,Coll),((Cryp,Hapt),TelRap))))) 2310263 7310240
18 ((Opst,Amoe),(Exca,(SAR,((Plan,Coll),((Cryp,Hapt),TelRap))))) 2310251 2310243
a The abbreviation of major groups: Opst, Opisthokonta; Amoe, Amoebozoa; Exca, Excavata; Plan, Archaeplastida; SAR, Stramenopila þ Alveolata þ Rhizaria; Cryp,
GuillardiaþPlagioselmis;Hapt,Haptophyta;TelRap,TelonemiaþRaphidiophrys;Mala,Malawimonas;andColl,Collodictyon.
bPvaluesinwhichthetopologiescannotberejectedatthe5%levelofsignificancewereunderlined.
cPvalueswerecalculatedfromtheoriginalalignment(i.e.,nositesremoved).
dPvalueswerecalculatedfromthetrimmedalignmentwithremovalofthe20%fastestevolvingsites(markedbygrayrectanglesinfig.4A).
ePvalueswerecalculatedfromthetrimmedalignment(i.e.,twoMalawimonasexcluded)withremovalofthe20%fastestevolvingsites(markedbygrayrectanglesinfig.4B).
examination of the Excavata phylogeny (Rodriguez-Ezpeleta therootoftheeukaryotetreeiscontroversialandnoclear
etal.2007;Hampletal.2009).Inaddition,thealternativeposi- evidenceexistsforitsposition,alineagethatisnotincluded
tionofMalawimonaswithinExcavatawasnotrejectedbythe withineitherunikontsorbikontsislikelyofearlyorigin.The
AUtest(P50.064;table2),altogethersuggestingthatthepo- poor diversity of known Diphyllatia (Collodictyon and Di-
sitionofMalawimonaswasnotstableandhighlysensitiveto phylleia) is striking in this respect as one would expect
taxonomic sampling.Hence,althoughthe groupingofCollo- to find more related lineages along its branch, but it re-
dictyonandMalawimonasremainsunclearafterouranalyses, mainstoseeifDiphyllatiainfactrepresentalargergroup:
the unstable position of Malawimonas and low support in theycouldbecloselyrelatedtoothergroupsthatareyetto
Bayesian analyses applying the CAT model indicates be sequenced or discovered. Regardless of these possible
that these two lineages may belong to different groups of sister groups, interpretations of the evolutionary origin
eukaryotes. of Collodictyon are largely dependent on the position of
the root of the eukaryote tree.
Collodictyon Is Placed Near the ‘‘Unikont–Bikont’’ Two rare genomic changes have suggested an ancient
Bifurcation split between the unikonts and bikonts; the bikonts have
Our phylogenetic inferences suggest that Collodictyon beenshowntoshareafusionofthedihydrofolatereductase
diverged near the unikont—bikont bifurcation. Although (DHFR) and thymidylate synthase (TS) genes, whereas all
1566