Molecular Phylogenetics and Evolution 53 (2009) 202–211

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

Interrelationships of chromalveolates within a broadly sampled treeof photosynthetic protists

Valérie C. Reeb a, Michael T. Peglar a, Hwan Su Yoon b, Jennifer Ruoyu Bai a, Min Wu a, Philip Shiu a,Jessie L. Grafenberg a, Adrian Reyes-Prieto a, Susanne E. Rümmele a, Jeferson Gross a,Debashish Bhattacharya a,*

a Department of Biology and Roy J. Carver Center for Comparative Genomics, University of Iowa, Iowa City, IA 52242, USAb Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine, USA

a r t i c l e i n f o

Article history:Received 25 January 2009Revised 14 April 2009Accepted 20 April 2009Available online 3 May 2009

Keywords:ChromalveolataMultigene phylogeneticsPlastidophilaRhizariaSupergroupTree of life

1055-7903/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ympev.2009.04.012

* Corresponding author. Fax: +1 319 335 1069.E-mail addresses: valerie-reeb@uiowa.edu (V.C.

(M.T. Peglar), hsyoon@bigelow.org (H.S. Yoon), jennifwumin816@gmail.com (M. Wu), philshiu@gmail.comg@uiowa.edu (J.L. Grafenberg), adrian-reyes@uiowa.edruemmele@uiowa.edu (S.E. Rümmele), jeferson-grosshi-bhattacharya@uiowa.edu (D. Bhattacharya).

a b s t r a c t

The Chromalveolata ‘‘supergroup” is a massive assemblage of single-celled and multicellular protists suchas ciliates and kelps that remains to be substantiated in molecular trees. Recent multigene analyses placechromalveolates into two major clades, the SAR (Stramenopiles, Alveolata, and Rhizaria) and the Crypto-phyta + Haptophyta. Here we determined 69 new sequences from different chromalveolates to study theinterrelationships of its constituent phyla. We included in our trees, the novel groups Telonemia and Kat-ablepharidophyta that have previously been described as chromalvoleate allies. The best phylogeneticresolution resulted from a 6-protein (actin, a-tubulin, b-tubulin, cytosolic HSP70, BIP HSP70, HSP90)and a 5-protein (lacking HSP90) alignment that validated the SAR and cryptophyte + haptophyte cladeswith the inclusion of telonemids in the former and katablepharids in the latter. We assessed the Plasti-dophila hypothesis that is based on EF2 data and suggest this grouping may be explained by horizontalgene transfers involving the EF2 gene rather than indicating host relationships.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

In recent years, significant advances have been made in molec-ular phylogenetics driven by next-generation sequencing technol-ogy, more powerful analytical methods, and the availability of agreater diversity of organisms through culture collections andenvironmental sampling. These inroads have significantly im-proved our understanding of deep-level evolutionary relationshipsamong the major eukaryotic groups and led to the discovery ofnew phyla (e.g., Not et al., 2007a; Shalchian-Tabrizi et al., 2006).Currently, eukaryote diversity is classified into six putative ‘‘super-groups”: Opisthokonta, Amoebozoa, Rhizaria, Excavata, Chromal-veolata, and Plantae (Adl et al., 2005; Cavalier-Smith, 2004).However phylogenetic support for these supergroups is highly var-iable (see Parfrey et al., 2006), depending greatly on taxon sam-pling, genetic markers considered, and phylogeneticreconstruction methods. What has however become abundantlyclear is that a single-gene approach is insufficient to robustly re-

ll rights reserved.

Reeb), mike@it-happens.neter15bai@gmail.com (J.R. Bai),

(P. Shiu), jessie-grafenber-u (A. Reyes-Prieto), susanne-

@uiowa.edu (J. Gross), debas-

solve basal splits in the eukaryote tree of life (ToL). Single gene ap-proaches are also prone to gross misinterpretation if the chosensequence has undergone highly variable rates of change in differ-ent lineages or more seriously, horizontal gene transfer (HGT) orendosymbiotic gene transfer (EGT). The latter two processes canresult in well-supported, conflicting relationships among taxawhen different genes are used to infer trees.

Two types of approaches currently exist with regard to infer-ence of the ToL. The first is to use short sequences (e.g., DNA bar-codes) to ‘place’ extant diversity within a tree and studypopulation or species-level phenomena. This approach can also in-clude the rDNA genes that generally are excellent phylogeneticmarkers and available from a broad diversity of taxa due to theirlong use in systematics and application to metagenomic and otherexploratory studies (e.g., Edgcomb et al., 2002; Huse et al., 2008).Barcodes and rDNA trees cannot however resolve the eukaryotictree in its entirety due to their limited phylogenetic resolution(e.g., Parfrey et al., 2006). The second approach has been to relyon multi-gene frameworks that incorporate from several to overa 100 genes to study supergroup relationships (e.g., Burki et al.,2008; Hackett et al., 2007; Hampl et al., 2009; Rodriguez-Ezpeletaet al., 2007). This latter approach has played a central role in cur-rent views of eukaryote evolution but until now has the major dis-advantage of limited taxon sampling. This is because completegenome data, that drives phylogenomic methods, are only now

V.C. Reeb et al. / Molecular Phylogenetics and Evolution 53 (2009) 202–211 203

slowly appearing for under-studied eukaryote groups that occupykey nodes in the ToL. Once many more of these genomes have beensequenced, then the gulf between taxon diversity and depth ofgene sampling will be adequately bridged and a robust well-sam-pled ToL will likely emerge. However genome data will offer itsown set of concerns such as identifying the ‘right’ markers to inferthe ToL that have not undergone HGT or EGT during their historyand to have available the computational power to analyze rapidlyhundreds of genes from potentially hundreds of species.

Here we take another step towards resolving the ToL, with a fo-cus on photosynthetic eukaryotes, by combining the multigene (3-gene, 5-gene, 6-gene) approach with a broad diversity of taxa. Ourfocus is on understanding the relationship between the Chromal-veolata and other eukaryotes. Plantae, the other major photosyn-thetic supergroup, and chromalveolates are defined by primaryand secondary endosymbiosis, respectively, that gave rise to theirplastids (e.g., Lane and Archibald, 2008; Reyes-Prieto et al.,2007). Understanding the phylogeny of photosynthetic groupswith respect to their non-photosynthetic and plastid-lacking coun-terparts is essential to elucidate the frequency of plastid origins.These data can be used to critically assess whether plastid endo-symbiosis comprises a useful basis for defining supergroups orother taxonomic entities.

Until recently, the chromalveolates included six phyla, dividedinto 2 subgroups: Alveolata and Chromista. The alveolates includesdinoflagellates, apicomplexans, and ciliates and most single- andmulti-gene phylogenetic analyses support their monophyly andsister group relationship to Stramenopiles (e.g., Baldauf et al.,2000; Gajadhar et al., 1991; Yoon et al., 2008). The kingdom Chro-mista (haptophytes, cryptophytes, and Stramenopiles) was pro-posed by Cavalier-Smith based on the presence of chlorophyll cand the position of the plastid in the rough endoplasmic reticulumin these taxa (Cavalier-Smith, 1981). The Chromista hypothesiswas initially supported by trees inferred from plastid and nuclearencoded plastid targeted proteins (Fast et al., 2001; Yoon et al.,2002a, 2004). However, nuclear non-plastid targeted proteinsshow it to be paraphyletic, whereby Stramenopiles groups withAlveolata and the cryptophytes and haptophytes form a monophy-letic group that is evolutionarily distantly related to the strameno-pile + alveolate clade (e.g., Hackett et al., 2007; Patron et al., 2007).Furthermore, monophyly of the Chromalveolata in toto, that is usu-ally well supported by plastid data (Bachvaroff et al., 2005; Harperand Keeling, 2003; Inagaki et al., 2004; Patron et al., 2004; Yoonet al., 2002b, 2005), is not found using alignments of non-plastidlocalized proteins (Cavalier-Smith, 2002, 2004; Harper and Keel-ing, 2003; Harper et al., 2005; Nishi et al., 2005; Stechmann andCavalier-Smith, 2003). Recently, multigene phylogenies haveshown surprisingly that Rhizaria taxa are nested within theChromalveolata (Burki et al., 2007; Hackett et al. 2007). This mas-sive assemblage of protists that is based solely on molecular datawith no unifying morphological synapomorphies has been referredto as the SAR clade (i.e., Stramenopiles + Aveolata + Rhizaria; Burkiet al., 2007). Until now, only Hackett et al., (2007), using a 16-pro-tein alignment, have shown moderate bootstrap support for theunion of the SAR clade with cryptophytes + haptophytes.

Several new phyla have been provisionally placed as sister toexisting chromalveolate members. The Katablepharidophyta(Kathablepharida) are freshwater and marine heterotrophic flagel-lates with ca. 6 described species (Clay and Kugrens, 1999). Theirmorphological characters ally them with either cryptophytes oralveolates, and small subunit (SSU) rDNA data support a sistergroup relationship to Cryptophyta (Okamoto and Inouye, 2005).The genus Telonema (Griessmann, 1913) represents a group of mar-ine heterotrophic protists that are likely related to the paraphyletic‘‘chromist” lineages (Shalchian-Tabrizi et al., 2006). Phylogeneticdata demonstrate the uniqueness of the group which led to its

transfer to the new phylum Telonemia with an apparent relation-ship to the cryptophytes + haptophytes (Shalchian-Tabrizi et al.,2006) based on HSP90 and SSU rDNA data. The phylum Chromeri-da (Moore et al., 2008), including the monotypic genus and speciesChromera velia, was recently uncovered in Australia in associationwith the coral Plesiastrea versipora (Moore et al., 2008). Both mor-phological and molecular characters suggest a sister relationshipwith apicomplexans. If confirmed, the position of the Chromeridais of high interest because it may represent a photosynthetic sisterto the Apicomplexa, thereby supporting an algal origin of theseimportant pathogens. And finally, a novel photosynthetic group,the picobiliphytes (Not et al., 2007b), apparently possesses a nucle-omorph-like structure (as in cryptophytes; Not et al., 2007b; butsee Cuvelier et al., 2008) and is related to cryptophytes and kat-ablepharids in 18S rDNA trees (Cuvelier et al., 2008). Here, we in-clude these new lineages (except picobiliphytes) in our trees to testtheir positions relative to chromalveolates.

2. Materials and methods

2.1. Taxon sampling

A total of 129 taxa representing all six eukaryotic supergroupshave been used in this study in order to better assess the mono-phyly of the Chromalveolata, Plantae, and Rhizaria and to deter-mine the phylogenetic positions of the newly described phylaChromerida, Telonemia, and Katablepharidophyta. Our sequencingeffort however focused on members of the Chromaveolata, Plantae,and Rhizaria for which 31 strains were gathered from different cul-ture collections (Supplementary Table S1): American Type CultureCollection (ATCC), The Provasoli-Guillard National Center for Cul-ture of Marine Phytoplankton (CCMP), Scottish Association forMarine Science Culture Collection of Algae and Protozoa (CCAP),Roscoff Culture Collection (RCC), Microbial Culture Collection atthe National Institute for Environmental Studies, Japan (NIES), Cul-ture Collection of Algae at the University of Göttingen (SAG), Cul-ture Collection of Algae at the University of Texas at Austin(UTEX). The Paulinella chromatophora FK01 and MO88/a isolateswere obtained from private collections.

2.2. DNA preparation and sequencing

Cells were frozen in liquid nitrogen and thawed prior to totalgenomic DNA extraction using the DNeasy kit (Qiagen, Santa Clari-ta, CA, USA). Some of the DNA samples were obtained directly fromthe ATCC. Seven nuclear genes were targeted for this study (Sup-plementary Table S1). Both the small and large ribosomal subunitgenes (SSU and LSU rDNA) and five protein coding genes were tar-geted for analysis: actin, a-tubulin (aTub), b-tubulin (bTub), andtwo Hsp70 paralogs, the luminal binding protein (Hsp70BIP) andcytosolic Hsp70 (Hsp70CYT). The sequences of heat shock protein90 (Hsp90) and translation elongation factor 2 (EF2) were obtainedfrom NCBI. Primers used for polymerase chain reaction (PCR)amplification of SSU rDNA, actin, aTub and bTub are as in Yoonet al. (2008). Primers for PCR amplification of the LSU are fromMoreira et al. (2007). Degenerate PCR primers were designed toamplify conserved regions of Hsp70BIP and Hsp70CYT from cryp-tophytes and haptophytes. The sequences of these oligos are asfollows: hsp70F2 50-ggcatcgatctnggnacnac-30 and hsp70R2 50-cgtcatggcackytcnccyt-30; CrypBIPF 50-acctactcItgcgtiggtgtgtacaa-30

and CrypBIPR 50-gtcatggctgiytciccytcraaiacctg-30; CrypCYT201F50-gagcgtctcatcggigatgc-30 and CryptCYT1581R 50-grttgtcggcgt-argtggagaag-30. Herculase� II Fusion DNA polymerase from Strata-gene (Cat. #600677) was used to amplify the different genes usingPCR. PCR fragments for the protein coding genes were cloned using

204 V.C. Reeb et al. / Molecular Phylogenetics and Evolution 53 (2009) 202–211

the pCR-blunt II Topo vector (Invitrogen catalog No. 45-0245). Upto 8 clones per gene and per taxon were sequenced in order to de-tect potential paralogs. Sequencing of cloned plasmid DNA wasdone using vector or gene-specific primers. PCR products of rRNAcoding regions were sequenced directly using gene-specific prim-ers. Sequencing reactions were done using the BigDyeTM terminatorkit (PE-Applied Biosystems, Norwalk, CT, USA) and run on an ABI3730 automated DNA sequencer at the Roy J. Carver Center forComparative Genomics at the University of Iowa. Because katable-pharids and telonemids are predators that feed on haptophyte al-gae, we also sequenced the respective algal prey provided by theculture collections to insure that we obtained sequences of the tar-get taxa.

2.3. Phylogenetic analyses

Phylogenetic analyses were carried out on several datasetsincluding sequences generated from this study (Supplementary Ta-ble S1) and sequences obtained from different public databases(e.g., NCBI, Joint Genome Institute). The datasets are as follows:(1) 108 taxa for 3 protein coding genes, actin, aTub, and bTub(108taxa_3protComb); (2) 112 taxa for 5 genes, SSU, LSU, actin,aTub, and bTub (112taxa_5genesComb); (3) 75 taxa for 6 proteincoding genes, actin, aTub, bTub, Hsp70BIP, Hsp70CYT, and Hsp90(75taxa_6protComb); (4) 64 taxa for EF2 (64taxa_EF2); (5) 75 taxafor 7 proteins coding genes actin, aTub, bTub, Hsp70BIP, Hsp70CYT,Hsp90, and EF2 (75taxa_7protComb); (6) 66 taxa for Hsp90 (66tax-a_Hsp90); and (7) 75 taxa for 5 protein coding genes, actin, aTub,bTub, Hsp70BIP and Hsp70CYT. Before combining each dataset,individual genes were analyzed and checked for incongruencyusing a RAxML bootstrap proportion >70% for conflicting groupingsas the criterion.

Ribosomal RNA sequences were aligned as nucleotides and pro-tein coding genes were aligned as amino acids in MacClade v4.07(Maddison and Maddison, 2005). Ambiguously aligned regions thatviolated positional homology were assessed manually and ex-cluded from further analysis. Genealogies were inferred using theparallel version of MrBayes 3.1.1 (Ronquist and Huelsenbeck,2003) and RAxML v 7.0.4 (Stamatakis et al., 2008) on the Cyberin-frastructure for Phylogenetic Research (CIPRES) portal v 1.13 athttp://www.phylo.org/. Models of sequence evolution for the dif-ferent datasets were determined using Modeltest 3.7 (Posada andCrandall, 1998) for nucleotides sequences and ProtTest 1.4 (Aba-scal et al., 2005) for amino acid sequences. For Bayesian analyses,the WAG + I + C model was applied to all partitions that were com-bined in the 75taxa_6protComb, 64taxa_EF2, 75taxa_7protComb,66taxa_Hsp90, and 75taxa_5protComb datasets. One model wasapplied per partition for the 108taxa_3protComb (WAG + I + Cfor actin and bTub, RtREV + C for aTub) and 112taxa_5genesComb(GTR + I + C for SSU and LSU, WAG + I + C for actin and bTub,RtREV + C for aTub) datasets, using the unlinked option. Two timesfour MCMCMC chains were run simultaneously for 2 million gen-erations sampling trees every 100 generations for each of the data-sets under investigation. Stationarity in likelihood scores wasdetermined by plotting the tree �lnL values against generationsunder Tracer v1.3 (Rambaut and Drummond, 2005). All trees belowthe stationarity level were discarded (i.e., as ‘burnin’). A majorityrule consensus tree was generated from the post-burnin trees.RAxML bootstrap analysis was done with each dataset (exceptthe 112taxa_5genesComb) for 100 iterations using the WAG sub-stitution matrix, empirical base frequencies, and estimated propor-tion of invariable sites. A majority rule consensus tree wascalculated to determine the support values for each node.

Because of their high rate of evolution, and resulting long-branch attraction, foraminifers were excluded from all analyses,thus only Cercozoa remain in our dataset to represent Rhizaria.

Nevertheless, foraminifers showed a sister relationship to the coreRhizaria clade in the actin gene analysis (e.g., Nikolaev et al., 2004),whereas in other single-gene analyses they grouped with the rho-dophytes which also have long branches (see also Yoon et al.,2008). For the same reasons, SSU and LSU rRNA from excavateswere excluded from the 112taxa_5genesComb analyses.

2.4. Testing tree topologies

We implemented the approximately unbiased (AU-) test (Shi-modaira, 2002) to assess the likelihoods of tree topologies thatsupport alternative positions of Telonemia or those that enforcethe monophyly of the supergroup Plastidophila. For testing theplacement of Telonemia, we generated different backbone phylog-enies that were identical to the best RAxML topologies using the108taxa_3protComb, 75taxa_5protComb (excluding Hsp90),75taxa_6protComb, and 66taxa_Hsp90 datasets, but we re-posi-tioned Telonemia at the base and at the first split within every phy-lum, as well as at every branch defining higher taxonomic clades.To test Plastidophila monophyly on phylogenies that did not re-cover this group, we forced monophyly of Viridiplantae, Rhodo-phyta, Cryptophyta, Haptophyta and Katablepharidophyta on theRAxML best trees for the 75taxa_6protComb, 70taxa_actin, 72tax-a_aTub, 72taxa_bTub, 45taxa_Hsp70CYT, and 66taxa_Hsp90 data-sets. RAxML analysis of the Hsp70 (BIP) dataset recoveredmonophyly of Plastidophila (without bootstrap support) thus wasnot tested further. We repeated the same set of tests with theinclusion of Telonemia in the Plastidophila. In addition, we as-sessed alternative positions for each of the Plastidophila phyla(i.e., Viridiplantae, Rhodophyta + cryptophytes nucleomorphs,Cryptophyta, and Katablepharidophyta + Haptophyta) on the EF2best RAxML topology. For this purpose, we placed each of thesephyla at the base and at the first split within other phyla, as wellas at every branch defining higher taxonomic clades. The site-by-site likelihoods for trees in the different analyses were calculatedusing the respective dataset and TREEPUZZLE v5.2 (Schmidtet al., 2002) with the WAG + C + F evolutionary model (the alphavalue for the gamma distribution was obtained from RAxML).The AU-test was implemented using CONSEL v 0.1i (Shimodairaand Hasegawa, 2001) to identify the pool of probable trees in eachtest and to assign their probabilities.

3. Results and discussion

3.1. Results of multi-gene phylogenetic analyses

The major goal of our multi-gene analysis was to determine theinterrelationships of known and putative chromalveolate phylawithin a broadly sampled eukaryotic ToL. To this end, we gener-ated 69 sequences from 31 taxa. Our novel data comprised 16 ac-tin, 16 aTub, 10 bTub, 3 Hsp70BIP, 2 Hsp70CYT, 5 SSU rDNA, and 17LSU rDNA coding regions (Supplementary Table S1). The 3-protein(108taxa_3protComb; 1088 aa) RAxML tree inferred from thesedata is shown in Fig. 1. This phylogeny was rooted on the branchleading to the Opisthokonta. The only supergroups that receive sig-nificant bootstrap and Bayesian support in this tree are the unikontlineages (i.e., Opisthokonta and Amoebozoa; Stechmann and Cava-lier-Smith, 2003; see also Yoon et al., 2008) with strong support forthe separation of unikont and bikont eukaryotes (RAxML bootstrapproportions [RBP] = 94%; Bayesian posterior probability [BPP] =1.0). In general, among bikonts the ‘deepest’ nodes that are consis-tently supported are at the level of phylum (e.g., Alveolata[RBP = 77%; BPP = 1.0]; Viridiplantae [RBP = 97%; BPP = 1.0]; Cryp-tophyta [RBP = 100%; BPP = 1.0]). Two interesting results of notewith respective to chromalveolates are the association in the

Alexandrium tamarense Lingulodinium polyedrum Heterocapsa triquetra

Amphidinium carterae Karlodinium veneficum

Karenia brevis Oxyrrhis marina

Perkinsus marinus Plasmodium berghei Plasmodium yoelii

Plasmodium falciparum Toxoplasma gondii

Theileria parva Cryptosporidium hominis Cryptosporidium parvum

Tetrahymena pyriformis Tetrahymena thermophila

Paramecium tetraurelia Thalassiosira pseudonana

Phaeodactylum tricornutum Mallomonas rasilis

Heterosigma akashiwo Ochromonas danica

Ankylochrysis lutea Aureococcus anophagefferens

Pinguiococcus pyrenoidosus Bolidomonas pacifica

Cafeteria roenbergensis Plectospira myriandra

Thraustotheca clavata Phytophthora palmivora

Apodachlya brachynema Phytophthora ramorum Phytophthora sojae

Telonema subtile Telonema antarcticum

Trypanosoma brucei Neobodo designis

Euglena gracilis Jakoba libera

Naegleria gruberi Acrasis rosea

Streblomastix strix Trichomonas vaginalis

Bodomorpha minima Heteromita globosa

Cercomonas sp. Paulinella chromatophora FK01

Paulinella chromatophora MO88 Thaumatomonas sp.

Cholamonas cyrtodiopsidis Bigelowiella natans

Bigelowiella sp. Gymnophrys sp.

Dunaliella sp. Chlamydomonas reinhardtii Volvox carteri Scenedesmus obliquus

Ostreococcus tauri Ostreococcus ‘lucimarinus’

Micromonas pusilla Micromonas pusilla

Arabidopsis thaliana Populus trichocarpa

Glycine max Zea mays

Oryza sativa Physcomitrella patens

Mesostigma viride Chlorella sp.

Helicosporidium sp. Glaucocystis nostochinearum

Cyanophora paradoxa Chrysochromulina polylepis Prymnesium parvum Imantonia rotunda

Pleurochrysis carterae Chrysochromulina sp.

Isochrysis galbana Emiliania huxleyi

Diacronema sp. Pavlova gyrans

Pavlova lutheri Katablepharis japonica Leucocryptos marina

Chroomonas mesostigmatica Storeatula sp. Guillardia theta Cryptomonas paramaecium Rhodomonas sp.

Rhodomonas salina Goniomonas truncata

Flintiella sanguinaria Porphyra yezoensis

Chondrus crispus Cyanidioschyzon merolae

Rhodochaete parvula Galdieria sulphuraria

Dictyostelium purpureum Dictyostelium discoideum

Physarum polycephalum Danio rerio

Mus musculus Drosophila melanogaster

Strongylocentrotus purpuratus Sclerotinia sclerotiorum

Neurospora crassa Ustilago maydis

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Rhizaria

Telonemia

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Haptophyta

Katablepharidophyta

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0.1 substitutions/site

Fig. 1. Phylogeny of the major eukaryotic groups inferred from maximum likelihood (RAxML) analysis of the combined amino acid sequences of actin, a-tubulin, and b-tubulin from 108 taxa (108taxa_3protComb data set). The phylogram shows the RAxML tree with the highest likelihood. Numbers above the branches are RAxML bootstrapproportions when P50%. Thick branches indicate Bayesian posterior probabilities P95%. Branch lengths are proportional to the number of substitutions per site (see scalebar).

V.C. Reeb et al. / Molecular Phylogenetics and Evolution 53 (2009) 202–211 205

Bayesian analysis of katablepharids with haptophytes (BPP = 1.0)and the positioning of telonemids as sister to Stramenopiles(BPP = 1.0) that are monophyletic with alveolates (BPP = 1.0).

These results support expansion of the Chromalveolata to includethese taxa although this awaits definitive proof of overall chromal-veolate monophyly (see below) that is not supported here because

206 V.C. Reeb et al. / Molecular Phylogenetics and Evolution 53 (2009) 202–211

neither the SAR nor the cryptophyte + haptophyte clades are recov-ered in the tree. Given these results, it is clear that even at this rel-atively broad sampling of taxon diversity, these 3 conservedeukaryotic proteins do not encode adequate phylogenetic signalto resolve basal chromalveolate relationships because most ofthese nodes receive little or no bootstrap or Bayesian support. Thissuggests that future analyses must either use other, more powerful(e.g., longer) phylogenetic markers for this purpose or includeadditional gene data to infer the ToL in its entirety. Given ouruse of a relatively broad sample of taxa, it is unclear whether addedtaxon diversity will alleviate this paucity in deep phylogeneticsignal.

3.2. Does the addition of rDNA data help the situation?

To test the effect of adding two conserved gene sequences toour 3-protein data, we generated a 5-gene, 112 taxon alignmentthat included SSU and LSU rDNA (112taxa_5genesComb; 3109nt + 1088 aa). These data were analyzed using a mixed-model(DNA, amino acids; see Methods) approach under Bayesian infer-ence. The topology of this tree (Supplementary Fig. S1) is similarto the 3-protein phylogeny with some important differences. Thekatablepharids are associated with cryptophytes (BPP = 1.0) inthe 5-gene tree rather than with Haptophyta as in the 3-proteinphylogeny. Association of Katablepharidophyta with the crypto-phytes is generally recovered from rDNA data (Cuvelier et al.,2008; Not et al., 2007a; Okamoto and Inouye, 2005; Slapetaet al., 2006), but not with protein data (this study, Kim and Gra-ham, 2008; Kim et al., 2006). In addition, it should be noted thatunlike cryptophytes that have flat mitochondrial cristae, hapto-phytes and katablepharids both contain tubular cristae.

We included Chromera velia rDNA sequences in this tree andshow it to be affiliated with dinoflagellates (BPP = 0.99) rather thanwith apicomplexans as previously reported (Moore et al., 2008),and there is Bayesian support (BPP = 0.98) for the union of two ofthe three Plantae phyla (Viridiplantae and Rhodophyta). RegardingC. velia, according to (Moore et al., 2008) this species is clearly analveolate because it contains three of the diagnostic features:micropore, subsurface alveoli supported by microtubules, andmitochondria with ampulliform and tubular cristae. The Chromeri-da possess a single large cone-shaped plastid bound by four mem-branes. This group also has unique and novel features: e.g., itcontains a photosynthetic (likely red algal-derived as in most otherchromalveolates) plastid containing chlorophyll a, but not chloro-phyll c (Moore et al., 2008); the taxon uses UGA codons to encodetryptophan in the psbA plastid gene (also found in apicoplasts ofcoccidians and in various mitochondria), whereas other algae useUGG. Phylogenetic analyses show a close relationship of C. veliato apicomplexans (LSU rDNA), or to colpodellids (SSU rDNA) withrejection using various topology tests of the alternative placement(found here) as sister to dinoflagellates (Moore et al., 2008). Inter-estingly, the psbA phylogeny (i.e., in absence of apicomplexans thatlack photosynthesis) places C. velia close to the peridinin dinoflag-ellates. A close relationship of the C. velia plastid to that of dino-flagellates is also supported by the arrangement of thylakoidlamellae in stacks of three, but this may be the ancestral conditionfor the putative photosynthetic ancestor of apicomplexans anddinoflagellates. Given these considerations, it is likely that the affil-iation of C. velia with dinoflagellates found here reflects the ab-sence of protein data in our alignment from Chromerida.

With regard to the Plantae, this putative supergroup has untilnow been recovered only in some multigene analyses (Burkiet al., 2008; Hackett et al., 2007; Rodriguez-Ezpeleta et al., 2005).However in the 5-gene tree, the third Plantae phylum, Glauco-phyta, is associated with cryptophytes and katablepharids(BPP = 1.0). The monophyly of glaucophytes and cryptophytes has

long been noted in rDNA trees (e.g., Bhattacharya et al., 1995; Shal-chian-Tabrizi et al., 2007; Van de Peer and De Wachter, 1997) andis generally not supported (but see Rensing et al., 1997) when pro-tein data are used to infer phylogeny. The rDNA sequences are pre-sumably responsible for the 5-gene result in this case, although thebasis for the divergent phylogenetic signal is currently unknown.Taken together, the rDNA data, although, adding significant lengthto the alignment fail to improve appreciably our ability to resolvebasal splits in the eukaryotic tree.

3.3. A 6-protein data set provides a reasonably well-resolved ToL

Given the results described above, we added 3 conserved pro-teins (Hsp70CYT, Hsp70BIP, and Hsp90]) to the existing 3-proteinalignment (75taxa_6protComb; 2302 aa) with the goal of gainingadded phylogenetic resolution. The extent of missing data for thisalignment is shown in Supplementary Table S2. This tree (Fig. 2),despite extensive missing data for some protists (e.g., ca. 50% inthe dinoflagellate Alexandrium tamarense), provides robust supportfor two important existing hypotheses based on larger, relativelytaxon-depauperate data sets (e.g., Burki et al., 2008; Hackettet al., 2007). First, the SAR clade with Rhizaria as sister to Stra-menopiles + Alveolata (RBP = 84%; BPP = 1.0 [as in Hackett et al.(2007)] is well supported (RBP = 88%; BPP = 1.0) and second, thecryptophyte + haptophyte clade, now with the addition of the kat-ablapharids (RBP = 65%; BPP = 1.0) is also resolved in our tree. As inthe 3-protein tree, katablepharids are monophyletic with hapto-phytes, but without strong support (RBP = 64%). In contrast how-ever to the 3-protein and 5-gene trees, the telonemids (ca. 45%missing data) form an independent split at the base of the tree withno obvious affiliation to Stramenopiles or alveolates. The Glauco-phyta and Rhodophyta are sisters in the RAxML tree but with nobootstrap or Bayesian support and the Viridiplantae do not receivesupport for their phylogenetic position within the eukaryoticradiation.

The telonemid result is worthy of further consideration. The ini-tial description of this protist phylum used Hsp90 alone and incombination with SSU rDNA to suggest monophyly with crypto-phytes with this clade being sister to haptophytes (Shalchian-Tab-rizi et al., 2006). The sequences of aTub and bTub were generatedby this study but the addition of these genes to the Hsp90 datadid not provide added resolution of the placement of Telonemiaamong eukaryotes. Subsequent analyses using rDNA either pro-vided no resolution (Shalchian-Tabrizi et al., 2007) or suggestedan affiliation with the SAR clade (Lefevre et al., 2008; BPP = 1.0).In our analyses, although the individual actin, aTub, and bTub RAx-ML trees do not place Telonemia in the tree with support (resultsnot shown; see also Shalchian-Tabrizi et al., 2006), the concate-nated data tree supports the affiliation of telonemids with Stra-menopiles (Fig. 1), and the 5-gene tree that includes rDNA placesthem within the Stramenopiles + Alveolata clade (Fig. S1). It istherefore very surprising that the 6-protein alignment providesno resolution at all with respect to this clade (Fig. 2) given the in-creased phylogenetic support that is found for other groups in thetree. The 6-protein phylogeny shows telonemids to comprise anindependent split in the ToL. The reason for this odd result be-comes apparent when one inspects the Hsp90 tree that (as in Shal-chian-Tabrizi et al., 2006) places telonemids sister to cryptophytes(RBP = 50%, Supplementary Fig. S2). The effect of the removal ofHsp90 from the 6-protein alignment is predictable, with telone-mids again being monophyletic with Stramenopiles (RBP = 51%;BPP = 1.0) and the remainder of the tree remaining relatively un-changed (Supplementary Fig. S3). What this suggests to us is thatthe telonemid genes that have been studied thus far provide (atleast) two types of phylogenetic signal with Hsp90 as the outlier,suggesting a specific relationship with cryptophytes that is not

Dinoflagellata

Apicomplexa

Ciliophora

Stramenopiles

Rhizaria

Telonemia

Excavata

Viridiplantae

Glaucophyta

Rhodophyta

Cryptophyta

Haptophyta

Katablepharidophyta

Amoebozoa

Ophistokonta

100

100

88

84

100

100

100

100 91

100 78

86 72 Heterocapsa triquetra

Crypthecodinium cohnii Alexandrium tamarense Amphidinium carterae 100

Karlodinium veneficum Karenia brevis

Oxyrrhis marina Perkinsus marinus

100 73

100 100 Plasmodium berghei Plasmodium falciparum

100 Babesia sp. Theileria parva

Toxoplasma gondii Tetrahymena thermophila Paramecium tetraurelia

100 51

97 100 Thalassiosira pseudonana

Phaeodactylum tricornutum 52 Aureococcus anophagefferens

Heterosigma akashiwo 100 Phytophthora ramorum

Phytophthora sojae Spumella uniguttata

100 98 Thaumatomonas sp.

Cercomonas sp. 100 Bigelowiella sp.

Bigelowiella natans

95 87

100 100

100 Trypanosoma cruzi Trypanosoma brucei

Leishmania major Euglena gracilis

Jakoba libera Naegleria gruberi

100

100 76

93 70

50 Glycine max Arabidopsis thaliana

Nicotiana tabacum Zea mays

Physcomitrella patens Selaginella moellendorfii

65 100

100 Ostreococcus ‘lucimarinus’ Ostreococcus tauris

Micromonas pusilla 100 Chlamydomonas reinhardtii

Volvox carteri

99 94

82 Porphyra yezoensis Chondrus crispus

Flintiella sanguinaria 78 Galdieria sulphuraria

Cyanidioschyzon merolae 100 Glaucocystis nostochinearum

Cyanophora paradoxa

65

64 100

100 100 Emiliania huxleyi Isochrysis galbana

Prymnesium parvum Pavlova lutheri

100 Katablepharis japonica Leucocryptos marina

100 100

75 65 Chroomonas mesostigmatica

Storeatula sp. Guillardia theta

Cryptomonas paramaecium Rhodomonas salina

Goniomonas truncata 100 Telonema subtile

Telonema antarcticum 100 Dictyostelium purpureum

Physarum polycephalum

100

100 100

100 Xenopus laevis Danio rerio

100 Drosophila melanogaster Apis mellifera

Monosiga brevicollis

100 100 Neurospora crassa

Magnaporthe grisea Debaryomyces hansenii

Plasmodium yoelii

0.1 substitutions/site

Fig. 2. Phylogeny of the major eukaryotic groups inferred from maximum likelihood (RAxML) analysis of the combined amino acid sequences of 6 nuclear proteins (actin,aTub, bTub, Hsp70BIP, Hsp70CYT, and Hsp90) from 75 taxa (75taxa_6protComb data set). The phylogram shows the RAxML tree with the highest likelihood. Numbers abovethe branches correspond to RAxML bootstrap proportions when P50%. Thick branches indicate Bayesian posterior probabilities P95%. Branch lengths are proportional to thenumber of substitutions per site (see scale bar).

V.C. Reeb et al. / Molecular Phylogenetics and Evolution 53 (2009) 202–211 207

readily apparent with the other 5 genes. Genome data from telone-mids is required to determine whether this result is explained byHGT events in these taxa (e.g., hsp90 and other genes come from

a cryptophyte source) as an explanation for the topological conflict.With respect to cell ultrastructure, some telonemid characters (i.e.,cortical alveoli-like peripheral vacuoles and tubular mitochondrial

208 V.C. Reeb et al. / Molecular Phylogenetics and Evolution 53 (2009) 202–211

cristae) indicate a relationship to alveolates and some chromists(Shalchian-Tabrizi et al., 2006), whereas the presence in Telonemaantarcticum of tripartite tubular flagellar hairs on the immature fla-gellum (Klaveness et al., 2005) provides a character that is shared

76

60

79

98

58

100

64 62

98 94

90

68 GlycA

NicoZea m

Physco

Mes100 Volvo

Chlam100

100 OstreococOstreococ

Micromon

99

57

97

100 86 Ch

BrotryocladiHildenbrandia

100 PorphyPorphyr

84 Galdieria sulphCyanidio

100 GHemise

58 100

100 Emi

PavlovaLeucocryptos m

Guillardia tGon

80 100

100 100

56 TrypanosLeish

TrypanosomKaren

Euglena gracilis 62 Nae

Jakoba libera

74

100

82

96 69

100 Theileria parva Babesia bovis

Toxoplas100 Plasmodium b

Plasmodium f

99 99

73

AlexandrPerkinsus

55

99 86

100 PhaeoThal

Aureococcus ano100 Phytophthora sojae

Phytophthora ramorum71

100 Dictyostelium purpureuPhysarum polycephalum

Cyanophora paradoxa

91

100 87

100 Xenopus laevis Danio rerio

100 Drosophila melanogaster Apis mellifera Monosiga brevicollis

100 100 Neurospora crassa

Magnaporthe grisea Debaryomyces hansenii

91

0.1 substitutions/site

Fig. 3. Phylogeny of the major eukaryotic groups inferred from maximum likelihood (RARAxML tree with the highest likelihood. Numbers above the branches correspond to RAxprobabilities P95%. Branch lengths are proportional to the number of substitutions per

with Stramenopiles (Andersen, 2004). Both of these observationsare in accord with our results and may indicate that Telonemia isan independent lineage within the SAR clade. Given these data, ifone were to discount the Hsp90 data for telonemids, then all of

Ophistokonta

Glaucophyta Amoebozoa

Stramenopiles

Rhizaria

Ciliophora

ine max rabidopsis thaliana

tiana tabacum ays mitrella patens

Selaginella moellendorfii ostigma viride x carteri ydomonas reinhardtii cus ‘lucimarinus’ cus tauris

as pusilla ondrus crispus

Gelidium canariensis a uvarioides rubra ra yezoensis a miniata uraria schyzon merolae uillardia theta

lmis andersenii Isochrysis galbana

liania huxleyi lutheri arina heta iomonas truncata oma cruzi mania major a brucei ia brevis ( Dinoflagellata)

gleria gruberi

ma gondii erghei alciparum

Karlodinium micrum Amphidinium carterae ium tamarense marinus

Paramecium tetraurelia Tetrahymena thermophila

Thaumatomonas sp. Bigelowiella natans

dactylum tricornutum assiosira pseudonana phagefferens

m

Viridiplantae

Rhodophyta

Haptophyta

Katablepharidophyta Cryptophyta

Excavata

Apicomplexa

Dinoflagellata

Cryptophyta nucleomorph

xML) analysis of EF2 from 64 taxa (64taxa_EF2 data set). The phylogram shows theML bootstrap proportions when P50%. Thick branches indicate Bayesian posteriorsite (see scale bar).

V.C. Reeb et al. / Molecular Phylogenetics and Evolution 53 (2009) 202–211 209

the chromalveolates in our trees can be grouped into two majorclades, the SAR clade that putatively includes telonemids and thecryptophyte + haptophyte clade that includes katablepharids. Onlythe picobiliphytes remain to be placed in such a multigene frame-work. Therefore, our results that are based on relatively sparse se-quence data but reasonable taxon sampling significantly simplifiesviews of chromalveolate evolution. The potential union of the twomajor clades, that comprises a vastly different view than formu-lated in the original chromalveolate hypothesis (Cavalier-Smith,1999), remains as a possible future development that may arisefrom the use of genome-level data to address this challengingissue.

To test the phylogenetic position of Telonemia we used the AU-test to assess the likelihoods of tree topologies that placed Telon-emia in alternate positions in the 108taxa_3protComb,75taxa_5protComb, 75taxa_6protComb, and 66taxa_Hsp90 trees.This analysis shows that many alternate positions for telonemidsare not rejected at a significance value of P < 0.05: 13 for108taxa_3protComb, 6 for 75taxa_5protComb, 3 for 75taxa_6prot-Comb, and 21 for 66taxa_Hsp90 trees. It is therefore clear thatthese datasets are unable to robustly resolve the phylogenetic po-sition of Telonemia in the ToL. Nevertheless, the AU-test using thetwo datasets that excluded Hsp90 and showed a Telonemia–Stra-menopiles relationship (i.e., 108taxa_3protComb and75taxa_5protComb) do reject sisterhood of telonemids with cryp-tophytes (P = 0.026 and P = 0.021), haptophytes (P = 0.037 and

Table 1Bayesian posterior probabilities and RAxML bootstrap proportions for major clades in the

Groups 108taxa_3protComb 112taxa_5genesComb 75taxa_6pr

BPP RBP BPP RBP BPP

Dinoflagellata 100 100 100 NA 100Dinoflagellata +

ChromeridaNA NA 99 NA NA

Apicomplexa 100 90 100 NA 100Dinoflagellata +

Apicomplexa100 80 100a NA 100

Ciliophora 100 100 100 NA 100Alveolata 100 77 100a NA 100Stramenopiles / / 100 NA 100Telonemia / 100 100 NA 100Stramenopiles +

Telonemia100 / / NA /

Stramenopiles +Alveolata

100b /b 97b NA 100

Rhizaria 100 74 100 NA 100SAR (Stramenopiles +

Alveolata + Rhizaria)/ / / NA 100

Viridiplantae 100 97 100 NA 100Rhodophyta 100 88 100 NA 100Viridiplantae +

Rhodophyta/ / 95 NA /

Glaucophyta 100 75 100 NA 100Plantae / / / NA /Cryptophyta 100 100 100 NA 100Katablepharidophyta 100 100 100 NA 100Cryptophyta +

Katablepharidophyta/ / 100 NA /

Haptophyta 100 100 100 NA 100Haptophyta +

Katablepharidophyta97 / / NA /

Cryptophyta +Katablepharidophyta +Haptophyta

/ / / NA 100

Chromalveolata / / / / /Plastidophila / / / / /

/ = RPB < 60%; BPP < 95%.NA, not applicable

a Including Chromerida.b Including Telonemia.c Including Cryptophyta nucleomorph.

P = 0.005), and marginally fails to reject their alliance with Kat-ablepharidophyta in the 108taxa_3protComb tree (P = 0.051). Theplacement of Telonemia at the base of the Cryptophyta was not re-jected (P = 0.099) using the 75taxa_6protComb (including Hsp90)but its placement with Stramenopiles was rejected (P = 0.01). A sis-ter group relationship between Telonemia and Stramenopiles isalso rejected in the 66taxa_Hsp90 tree (P = 0.011). Taken together,the results of the AU-tests do not support a specific Telonemia–Stramenopiles sister group relationship because several alternateplacements of telonemids are not significantly rejected. Howeverdatasets containing Hsp90 always reject Telonemia + Strameno-piles suggesting this gene may provide conflicting phylogeneticsignal when compared to the other genes used in our study.

3.4. EF2 and birth of the Plastidophila

An intriguing result that was recently published postulates thepossible existence of a novel supergroup, the ‘‘Plastidophila” (Kimand Graham, 2008) that includes cryptophytes, katablepharids,haptophytes, rhodophytes, and Viridiplantae. This supergroup isbased primarily on analysis of a single gene, the conserved trans-lation elongation factor EF2. The affiliation of these Plantae andchromalveolate lineages is also supported by a unique two aminoacid signature sequence shared by these EF2 proteins. Thereforethe gene tree result appears to be robust. We had previously sug-gested (Hackett et al., 2007; Nosenko and Bhattacharya, 2007)

different phylogenetic analyses.

otComb 64taxa_EF2 75taxa_7protComb 66taxa_Hsp90 75taxa_5protComb

RBP BPP RBP BPP RBP BPP RBP BPP RBP

100 100 99 100 100 100 99 100 100NA NA NA NA NA NA NA NA NA

100 100 96 100 100 99 65 100 100100 100 82 100 100 100 88 100 97

100 100 100 100 100 100 100 100 100100 100 74 100 100 100 100 100 97100 100 99 100 100 100 100 100 99100 NA NA 100 100 100 100 100 100/ NA NA / / / / 100 /

84 / / 100 97 100 82 100b /

100 100 / 100 100 100 97 100 9788 100 / 100 99 99 / 100 /

100 100 100 100 100 100 95 100 9799 100c 99c 100 100 100 99 100 99/ 100 / / / / / / /

100 NA NA 100 100 100 99 100 75/ / / / / / / / /100 100 / 100 100 100 93 100 100100 NA / 100 100 NA NA 100 100/ / / / / / / / /

100 100 100 100 100 100 100 100 10064 100 / 100 87 / / 96 /

65 / / 100 86 / / 100 /

/ / / / / / / / // 100 98 / / / / / /

210 V.C. Reeb et al. / Molecular Phylogenetics and Evolution 53 (2009) 202–211

that the EF2 topology is best explained by HGT from a cell that isancestral to red and green algae into the haptophyte ancestor(cryptophyte and katablepharid data were not available at thattime), rather than HGT or alternatively EGT from the red algalendosymbiont to the nucleus of chromalveolates. This latter pointis consistent with the positioning of red algal nucleomorph EF2sequences with red algae rather than at the base of these chrom-alveolates. In addition, the putative HGT origin of EF2 is not un-ique in our analyses because the dinoflagellate Karenia brevisalso appears to have gained its EF2 gene from an excavate source(Nosenko and Bhattacharya, 2007). Therefore how do we inter-pret the Plastidophila hypothesis? In our opinion, this idea ismost likely be explained by HGT because no other single genetree in our analyses (results not shown) reproduces robustly thisresult. This is a common feature of HGT (i.e., strong support for aconflicting topology).

To study this issue further, we generated a well-sampled EF2tree to validate the results of Kim and Graham (2008) and to assessthe effects of EF2 inclusion on our 6-protein alignment. Our predic-tion for the latter analysis was that it would disrupt existing cladesin the tree (i.e., Fig. 2) and skew the tree towards Plastidophilamonophyly, but much more weakly than when EF2 was analyzedalone. This is another potential indication of HGT origin; i.e., a sin-gle gene more strongly supports a result than when it is combinedwith other conserved phylogenetic markers. The EF2 tree is shownin Fig. 3. Here, we also recover robustly the Plastidophila(RBP = 98%; BPP = 1.0; see Table 1) using our taxonomically morebroadly sampled data set than that used in Kim and Graham(2008). Many other groupings agree with accepted ideas abouteukaryote phylogeny (e.g., opisthokont monophyly [RBP = 91%;BPP = 1.0], alveolate monophyly [RBP = 74%; BPP = 1.0]) and areconsistent with our 3-protein, 5-gene, and 6-protein analyses.Essentially, only the Plastidophila conflicts strongly with these re-sults. When Kim and Graham (2008) combined EF2 with 5 otherproteins (actin, Hsp70CYT, Hsp90, aTub, bTub), they again recov-ered the Plastidophila but with lower bootstrap support(RBP = 88% versus 98%) for nodes and no longer found monophylyof Viridiplantae and Rhodophyta. When we add EF2 to our 6-pro-tein alignment, maximum likelihood support for the SAR cladeand Excavata rise significantly (RBP = 99%, 100%, respectively),but support for Plastidophila falls to 57% and now includes Telon-emia (Supplementary Fig. S4). These results are consistent with ourprediction described above for an HGT origin of EF2 in Crypto-phyta/Haptophyta/Katablepharidophyta and similar to Hsp90,seems to suggest that caution needs to be used when interpretingsingle gene trees that argue strongly for or against a particularview of ancient (e.g., supergroup) eukaryotic relationships.

To test these results, we again used the AU-test to assess Plast-idophila monophyly in two ways. First, we forced the monophylyof the 5 Plastidophila lineages and also the monophyly of the 5phyla + Telonemia on the 75taxa_6protComb, 70taxa_actin, 72tax-a_aTub, 72taxa_bTub, and 66taxa_Hsp90 trees and assessed thelikelihoods of these competing trees. All of these topologies wererejected at P < 0.05, except for Plastidophila monophyly includingTelonemia using the 66taxa_Hsp90 data set. For the second ap-proach that tested alternative positions of each of the Plastidophilaphyla on the 64taxa_EF2 topology, only two positions were not re-jected in each of the 4 analyses, but these represent rearrange-ments within the Plastidophila. In summary, our resultsdemonstrate that Plastidophila monophyly arises from the EF2dataset, and that alternative topologies that force Plastidophilapolyphyly with this data set are significantly rejected. Other genes,whether single or combined significantly reject the Plastidophilahypothesis, thereby supporting the idea that monophyly of this no-vel supergroup when inferred using EF2 is most likely explained bya gene-specific phenomenon such as HGT.

Acknowledgments

This research was funded by a Grant from the National ScienceFoundation Assembling the Tree of Life program (EF 04-31117). Weare grateful for the constructive comments of three anonymousreviewers.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2009.04.012.

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