A PHYLOGENOMIC FALSIFICATION OF THE CHROMALVEOLATE … · A PHYLOGENOMIC FALSIFICATION OF THE...
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A PHYLOGENOMIC FALSIFICATIONOF THE CHROMALVEOLATE HYPOTHESIS
Denis BAURAIN†, Henner BRINKMANN, Jörn PETERSEN, Naiara RODRÍGUEZ-EZPELETA, Alexandra STECHMANN,Vincent DEMOULIN, Andrew J. ROGER, Gertraud BURGER, B. Franz LANG, and Hervé PHILIPPE
Département de Biochimie, Centre Robert-Cedergren, Université de Montréal, Montréal, Québec, CanadaDépartement des Sciences de la Vie, Université de Liège, Liège, Belgium
†Present address: Unit of Animal Genomics, GIGA-R and Faculty of Veterinary Medicine, Université de Liège, Liège, Belgium
What are ’Chromalveolates’?
ANRV329-GE41-08 ARI 27 September 2007 14:38
Protist: microbialeukaryote notincluding plants andfungi
Algae:photosyntheticeukaryotes (protists)not including plants
‘Chromalveolata’:putativemonophyletic groupdescended from aprotist commonancestor thatcaptured a red algaand maintained it asa secondaryendosymbiont
INTRODUCTION
The Eukaryotic Tree of Life asBackdrop for Plastid Origin
Multigene phylogenetics and genome datafrom microbial eukaryote (protist) lineageshave provided a renewed impetus to resolv-ing the eukaryotic tree of life (e.g., 11, 71,90), culminating recently in a formal classi-fication of eukaryotes into 6 “supergroups”(3, 44). These supergroups (see Figure 1)contain the protistan roots of all multi-cellular eukaryotes and are currently de-fined as ‘Opisthokonta’ (e.g., animals, fungi,choanoflagellates), ‘Amoebozoa’ (e.g., loboseamoebae, slime molds), ‘Archaeplastida’ or‘Plantae’ [red, green (including land plants),and glaucophyte algae], ‘Chromalveolata’(e.g., diatoms, ciliates, giant kelps), ‘Rhizaria’
Excavata
Rhizaria
ChromalveolataPlantae
Amoebozoa
EuglenidsParabasalidsDiplomonads
Jakobids
RadiolariaCercozoa
AlveolatesStramenopilesHaptophytesCryptophytes
Red, GlaucophyteGreen algae
EntamoebaeAmoebae
Slime molds
Opisthokonta
AnimalsChoanozoa
FungiMicrosporidia
Figure 1Schematic view ofthe eukaryotic treeof life showing theputative sixsupergroups. Thebroken linesdenote uncertaintyof branch positionsin the tree. Forexample, the‘Rhizaria’ are likelymonophyletic butmay branch withinchromalveolatesand the ‘Excavata’may comprise atleast two distinctlineages. Thepresence ofplastid-containingtaxa in thesupergroups isshown with thecartoon of an alga.
(e.g., cercomonads, foraminifera), and ‘Ex-cavata’ (e.g., diplomonads, parabasalids). Al-though the supergroups broadly capture thediversity of eukaryotes, there are in factonly two that currently have robust sup-port from molecular phylogenetic analyses,the ‘Opisthokonta’ and the ‘Amoebozoa’ (71).Therefore in this review all supergroups aremarked with ‘ ’ to denote their provisional na-ture. Of the remaining lineages, the ‘Plantae’is gaining the most support from multigenetrees (83) and features associated with thephotosynthetic organelle (plastid) in thesetaxa (e.g., 63, 78, 99). This group is verylikely to be monophyletic, a key feature thatplays an important role in understanding plas-tid evolution. The ‘Rhizaria’ includes pho-tosynthetic amoebae (chlorarachniophytesand Paulinella chromatophora) and receives
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CHROMALVEOLATES are a large and diverse putative super-group of eukaryotes that include a mix of photosynthetic
and heterotrophic lineages. According to the Chromalveolatehypothesis (Cavalier-Smith 1999), which regards plastid lossas much more common than plastid gain, these organismsvertically descent from a single chl. c-containing ancestor thatacquired its plastid early via a secondary endosymbiosis witha red alga (Reyes-Prieto et al. 2007).
Do ’Chromalveolates’ really exist?
would have to have been acquired before the split betweenthe haptophyte + cryptophyte clade from alveolates + -stramenopiles + Rhizaria (Figure 3b). Plastids would thenpresumably have been lost, independently, in Rhizaria,some stramenopiles, ciliates, early diverging dinoflagel-lates (e.g. Oxyrrhis) and many or most apicomplexans[39]. Finally, the subsequent uptake of a green algal endo-symbiont in the ancestor of chlorarachniophytes wouldproduce the distribution of plastids observed today(Figure 3b). Like the original chromalveolate hypothesis(Box 2), this scenario would require that plastid loss be farmore common than gain. Although the prevalence of plas-tid loss (as opposed to loss of photosynthesis) amongeukaryotes is unknown, the nuclear genomes of two Phy-tophthora species [29] (stramenopiles) and the apicom-plexan Cryptosporidium [41] encode plastid-derivedgenes, despite these organisms lacking plastids, an indica-tion of at least two instances of plastid loss in the ancestorsof these different organisms. Additionally, the recentlydiscovered photosynthetic eukaryote Chomera velia [42],which is closely related to apicomplexans, stronglyindicates a shared photosynthetic ancestor of both Apicom-plexa and dinoflagellates and subsequent loss in the plas-tid-lacking members of these groups.
If the new position of Rhizaria as a part of Chromalveo-lata reflects the true evolutionary history of this lineage,one would predict that genes of red algal ancestry mightpersist in the nuclear genomes of this group as remnants ofthe red algal genomes that were present in the rhizarian
common ancestor. Interestingly, red algal-derived plastidgenes were discovered in the nuclear genome of the greenalgal plastid-containing rhizarian Bigelowiella natans[43], and were interpreted as having been acquired bylateral gene transfer rather than vertically inherited froma red algal plastid-containing ancestor. A complete genomesequence for B. natans will soon be available (http://www.jgi.doe.gov) and will make it possible to test whetheror not this red algal ‘footprint’ is (at least in part) the resultof ancient endosymbiotic gene transfer. However, mostRhizaria are recalcitrant to laboratory experimentation,and significant amounts of sequence data from diversemembers of this lineage will be slow in coming. At anyrate, if analyses eventually show that two (ormore) distinctplastids were harbored by the ancestors of extant organ-isms, as has been previously shown in some dinoflagellates(see Ref. [37]), then determining the organismal history ofsuch eukaryotes might be even more difficult than cur-rently appreciated.
Phylogenetic hope in light of EGT?Although we have focused on chromalveolates and ignoredthe potentially significant role of lateral gene transfer ineukaryotic evolution (e.g. Ref. [44]), the reality of EGT andits phylogenetic implications can be extended to many ofthe eukaryotic supergroups. The relationships within andbetween chromalveolate and rhizarian taxa are not onlyimportant for understanding amajor component of the treeof life but also for understanding organelle evolution and
Figure 3. Two hypotheses to explain the distribution of secondary plastids, based on competing scenarios of eukaryotic evolution. A green algal-derived secondary plastidhas been acquired by two separate lineages, in independent endosymbiotic events (thin dashed lines). (a) A single red algal endosymbiosis occurred in the commonancestor of Chromalveolata, necessitating multiple plastid losses at the base of the various nonphotosynthetic lineages. (b) If Rhizaria evolved fromwithin chromalveolates,it is most parsimonious to assume that the red algal secondary plastid was lost before the diversification of this lineage. A green algal secondary plastid has been acquiredby chlorarachniophytes more recently.
Opinion Trends in Ecology and Evolution Vol.23 No.5
273
Amoebozoa
Opisthokonta
Plantae
!Chromalveolata"(Hacrobia)
!Chromalveolata"(Stramenopiles+Alveolates)
Rhizaria
Excavata
SAR
RECENT phylogenomic studies have led to dramatically ex-panded ‘Chromalveolates’. To account for the growing
collection of heterotrophic lineages apparently related to chl.c-containing algae, the Chromalveolate hypothesis has to pos-tulate ever more plastid losses. Further, it suffers from a re-current lack of support in most molecular phylogenies, eventhose based on large data sets. Therefore, alternative evolu-tionary scenarios have been proposed to explain the origin of’Chromalveolate’ organisms (Lane and Archibald 2008).
HERE, we present a falsifying experiment that comparesthe Chromalveolate hypothesis to serial models invoking
higher-order eukaryote-eukaryote endosymbioses (EEEs).
How old are chl. c-containing plastids?
Cyanobacteria
Viridiplantae
Glaucophyta
Rhodophyta
Cryptophyta
Haptophyta
Stramenopiles
Euglenophyta
Chlorarachniophyta
0.0 1.00.2 0.4 0.6 0.8Time
Phaeodactylum tricornutumOdontella sinensisThalassiosira pseudonanaEctocarpus siliculosusPavlova lutheriEmiliania huxleyiGuillardia thetaGracilaria tenuistipitataPorphyra yezoensisPorphyra purpureaGaldieria sulphurariaCyanidioschyzon merolaeCyanidium caldariumCyanophora paradoxaChlorokybus atmophyticusMesostigma virideNephroselmis olivaceaOstreococcus tauriEuglena gracilisChlorella vulgarisStigeoclonium helveticumScenedesmus obliquusChlamydomonas reinhardtiiOltmannsiellopsis viridisPseudendoclonium akinetumBigelowiella natansChara vulgarisStaurastrum punctulatumZygnema circumcarinatumChaetosphaeridium globosumAnthoceros formosaeMarchantia polymorphaPhyscomitrella patensNostoc sp. pcc7120Nostoc punctiforme pcc73102Synechococcus elongatus pcc6301Trichodesmium erythraeum ims101Synechococcus sp. pcc7002Synechocystis sp. pcc6803Crocosphaera watsonii wh8501Thermosynechococcus elongatus bp1Synechococcus sp. ja33abSynechococcus sp. ja23baGloeobacter violaceus pcc7421
Supplementary Figure 2.PHYLOGENETIC inference under the CAT model on 55plastid-encoded proteins (44 OTUs x 10,805 AA) resulted
in a tree that was relatively dated using a log-normal auto-correlated model. In this analysis, chl. c-containing plastidsappear to have diversified at 38.8% of the time elapsed sincethe last common ancestor of Plantae and ’Chromalveolates’.
How to test ’Chromalveolates’?
ChromalveolateHypothesis
Alternative (Serial)Hypothesis
Time
Alveolata
Haptophyta
Stramenopiles
Cryptophyta
other Rhodophyta
Cyanidiales
Streptophyta
Glaucophyta
Opisthokonta
Cyanobacteria
Chlorophyta
0.0 0.2 1.00.4 0.6 0.8
Chlorophyta
Cyanobacteria
Opisthokonta
Glaucophyta
Streptophyta
Cyanidiales
other Rhodophyta
Cryptophyta
Stramenopiles
Haptophyta
Alveolata
plastid / mitochondrion / nucleus histories
Alveolata
Haptophyta
Stramenopiles
Cryptophyta
Streptophyta
Glaucophyta
Opisthokonta
Cyanobacteria
Chlorophyta Chlorophyta
Cyanobacteria
Opisthokonta
Glaucophyta
Streptophyta
Cryptophyta
Stramenopiles
Haptophyta
Alveolata
1
3
21
23
ChromalveolateHypothesis
AlternativeHypothesis
ChromalveolateHypothesis
AlternativeHypothesis
Streptophyta
Glaucophyta
Opisthokonta
Cyanobacteria
Chlorophyta
Haptophyta
Stramenopiles
Cryptophyta
Streptophyta
Glaucophyta
Opisthokonta
Cyanobacteria
Chlorophyta
Cryptophyta
Stramenopiles
Haptophyta1 + 2 + 3
1 + 2 + 3
BOTH Chromalveolate and serial hypotheses postulate aninitial, single secondary endosymbiosis of a red alga
within a eukaryotic host (dotted arrows), leading to the emer-gence of a chl. c–containing founder. The Chromalveolate hy-pothesis then assumes that this alga gives rise to ’Chromalve-olates’ by vertical descent (top left tree). In contrast, serial hy-potheses posit multiple subsequent EEEs (plain arrows), whichhorizontally spread plastids among otherwise unrelated eu-karyotes (top right tree). As chl. c plastids emerge from withinred algae, overall histories of plastid (orange), mitochondrial(blue), and nuclear (black) genomes cannot be superimposed.However, removing red algae ‘regularizes’ plastid history bycreating a single branch out of three smaller ones (middletrees). Now, the Chromalveolate hypothesis predicts that thesignal for the monophyly of ’Chromists’ (discarding alveolates)should be similarly strong across all genomic compartments(orange arrowhead in bottom left tree), while it should bestrong only with plastid genomes in serial hypotheses (orangearrowhead in bottom right tree). To validate our approach,green plants were used as a test case, since the signal for theirmonophyly is expected to be similarly strong, regardless of thecompartment or the hypothesis (green arrowheads).
Greenalgae
Greenalgae
Greenalgae
Greenalgae
Greenalgae
Greenalgae
6 ‘Chromists’
6 green plants
1 glaucophyte 55 plastid 13 mitochondrion encoded proteins108 nucleus
ingroup
outgroup
11 cyanobacteria 11 opisthokonts
Preydigestion
Preydigestion
Preydigestion
Preydigestion
Preydigestion
Preydigestion
Preydigestion
Preydigestion
Preydigestion
Preydigestion
Preydigestion
USING the same sampling of ingroup species, we assem-bled one concatenated protein data set per compart-
ment. The strength of the phylogenetic signal was then es-timated through a Variable Length Bootstrap (VLB) strategy.
0
20
40
60
80
100
0 2,000 4,000 6,000 8,000 10,000
Boo
tstr
ap s
uppo
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%)
plastid
0 3,000
# positions (AA)
mitochondrion
0 3,000 6,000 9,000 12,000 15,000
nucleus
WHATEVER the compartment considered, the monophylyof green plants is easily recovered, whereas the mono-
phyly of ‘Chromists’ is only recovered with plastid genomes.This observation falsifies the Chromalveolate hypothesis.
How to check our assumptions?
RNA Polymerase1,911 AA
Photosynthesis5,717 AA
Ribosome2,074 AA
Others1,103 AA
Ribosome8,697 AA
Proteasome3,102 AA
Varia3,980 AA
Plastid (10,805 AA)
Nucleus (15,392 AA)
SINCE heterogeneous rates may affect inference, we decidedto subdivide each of our plastid and nuclear data sets into
smaller data sets according to functional class.
THEN, to allow easy com-parison, we defined n70
as the number of positions re-quired to reach a VLB support≥70% for the monophyly ofthe group put to test (by fittinga monomolecular model). 0
20
40
60
80
100
0 400 800 1,200 1,600 2,000
Boo
tstr
ap s
uppo
rt (
%)
# positions (AA)
n70 = 172
VLBs n 70 Values# Pos f(Sub) Trees
Greens ‘Chromists’ Gre ‘Chr’
PlastidPolymerase 1,911 4.79
BS (%
)
365 1,802
Photosynthesis 5,717 1.63
BS (%
)
101 95
Ribosome 2,074 3.56
BS (%
)
208 172
Mitochondrion 3,106 3.51
BS (%
)
176 n.c.
NucleusProteasome 3,102 2.34
BS (%
)
501 n.c.
Ribosome 8,697 2.72
BS (%
)
130 n.c.
Varia 3,980 1.99
BS (%
)
305 n.c.
ALTHOUGH plastid genomes display extreme rate varia-tions, the monophyly of ’Chromists’ is recovered with n70
values that remain small relative to the number of positionsavailable. In contrast, VLB support is so low in mitochondrialand nuclear compartments that it is not even possible to fit themodel. Thus, rate heterogeneity does not impair our test.
0.20Theileria annulataBabesia bovisPlasmodium falciparum
Toxoplasma gondiiEimeria tenella
Cryptosporidium parvumPerkinsus marinusDinophyceae
Sterkiella histriomuscorumTetrahymena thermophilaParamecium tetraurelia
Blastocystis hominisPhytophthora sojaeSaprolegnia parasitica
Nannochloropsis oculataEctocarpus siliculosus
Phaeodactylum tricornutumThalassiosira pseudonana
Pavlova lutheriPrymnesium parvumIsochrysis galbanaEmiliania huxleyi
Chondrus crispusPorphyra yezoensis
G.t. nmGaldieria sulphuraria
Cyanidioschyzon merolaeGuillardia theta
Scenedesmus obliquusChlamydomonas reinhardtiiDunaliella salina
Ostreococcus tauriMicromonas sp.
Mesostigma virideClosterium peracerosum-strigosum-littorale complex
Physcomitrella patensPinus taeda
Cyanophora paradoxaGlaucocystis nostochinearum
Puccinia graminisUstilago maydis
Schizosaccharomyces pombeRhizopus oryzae
Blastocladiella emersoniiSpizellomyces punctatus
Sphaeroforma arcticaCapsaspora owczarzaki
Monosiga brevicollisMonosiga ovataReniera sp.Hydra magnipapillata
Nematostella vectensisAcanthamoeba castellanii
Hartmannella vermiformisMastigamoeba balamuthiDictyostelium discoideum
Physarum polycephalum
71
83
98 40
84
87
99
85
67
33
79
96
82
98
88
76
Figure 2.
Amoebozoa
Opisthokonta
Glaucophyta
Viridiplantae
Cryptophyta
Rhodophyta
Haptophyta
Stramenopiles
Alveolata
FINALLY, using an extended nuclear data set (57 OTUs x15,392 AA), we tested the affinity of the fast-evolving and
compositionally biased nucleomorph of Guillardia theta. In-ference under the CAT model yielded 100% bootstrap sup-port for the grouping of the nucleomorph with red algae,while providing no support for the monophyly of ’Chroma-lveolates’. This helps us to exclude phylogenetic artifacts andto conclude that the Chromalveolate hypothesis is falsified.
Want to know more?BAURAIN D. et al. (2010) Phylogenomic evidence for sepa-rate acquisition of plastids in cryptophytes, haptophytes, andstramenopiles. Mol. Biol. Evol. 27:1698-1709.
SMBE2010, Annual Meeting of the Society for Molecular Biology and Evolution, Lyon, France, July 4–8, 2010