Universität Potsdam

Christoph Bleidorn, Lars Podsiadlowski, Min Zhong, Igor Eeckhaut, Stefanie Hartmann, Kenneth M. Halanych, Ralph Tiedemann

On the phylogenetic position of Myzostomida : can 77 genes get it wrong?

first published in: BMC Evolutionary Biology (2009), 9, Art. 150, DOI: 10.1186/1471-2148-9-150

Postprint published at the Institutional Repository of the Potsdam University: In: Postprints der Universitat¨ Potsdam Mathematisch-Naturwissenschaftliche Reihe ; 123 http://opus.kobv.de/ubp/volltexte/2010/4489/ http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-44893

Postprints der Universitat¨ Potsdam Mathematisch-Naturwissenschaftliche Reihe ; 123

() BMC Evolutionary I~iology BioMed Central

Research article Open Access On the phylogenetic position of Mlyzostomida: can 77 genes get it wrong? Christoph Bleidorn * I, Lars Podsiadlovvski2, Min Zhong3, Igor Eeckhaut4, Stefanie Hartmann5, Kenneth M Halanych3 and Ralph Tiedemann1

Address: lUnit of Evo lutionary Biology/Systematic Zoology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, Haus 26, 0-14476 Potsdam-Golm, Germany, 2lnstitute of Evo lutionary Biology and Ecology, Rheinische Friedrich-Wilhelms-Universitat Bonn, An der lmmenburg 1, 0-53121 Bonn, Germany, 30 epartment of Biological Sciences, Auburn University, 101 Life Science Building, AL 36849, USA, 4Marine Biology Laboratory, Natural Sciences Building, University of Mons-Hainaut, Av. Champs de Mars 6, B-7000 Mons, Belgium and SUnit of Bioinformatics, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, Haus 26, 0-14476 Potsdam-Golm, Germany Email: ChristophBleidorn*- [email protected];[email protected]; Min Zhong - [email protected]; 19or Eeckhaut - [email protected]; Stefanie Hartmann - [email protected]; Kenneth M Halanych - [email protected]; Ralph Tiedemann - [email protected] * Corresponding author

Published: I July 2009 Received: 28 January 2009 Accepted: I July 2009 BMC Evolutionary Biology 2009.9:150 doi:10.1186/ 1471-2148-9-150 This article is available from: http://www.biomedcentral.com/ 1471-2148/9/ 15C1 © 2009 Bleidorn et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (htt[;,-lIcreativecommons org/licenses/by/2 0). which permits unrestricted use. distribution. and reproduction in any medium. provided the original work is properly cited.

Abstract Background: Phylogenomic analyses recently b,ecame popular to address questions about deep metazoan phylogeny. Ribosomal proteins (RP) dominate many of these analyses or are. in some cases. the only genes included. Despite initial hopes. phylogenomic analyses including tens to hundreds of genes still fail to robustly place many bilaterian taxa. Results: Using the phylogenetic position of myzostomids as an example. we show that phylogenies derived from RP genes and mitochondrial genes produce incongruent results. Whereas the former support a position within a clade of platyzoan taxa. mitochondrial data recovers an annelid affinity. which is strongly supported by the gene order data and is congruent with morphology. Using hypothesis testing. our RP data significantly rejects the annelids affinity. whereas a platyzoan relationship is significantly rejected by the mitochondrial data. Conclusion: We conclude (i) that reliance of a set of markers belonging to a single class of macromolecular complexes might bias the analysis. and (ii) that concatenation of all available data might introduce conflicting signal into phylogenetic analyses. We therefore strongly recommend testing for data incongruence in phylogenomic analyses. Furthermore. judging all available data. we consider the annelid affinity hypothesis more plausible than a possible platyzoan affinity for myzostomids. and suspect long branch attraction is influencing the RP data. However. this hypothesis needs further confirmation by future analyses.

Background netic signal, and stochastic error related to gene length Molecular phylogenies based on a single or a few genes might be problematic (1) . Use ofphylogenomics (molec­ often lead to apparently conflicting signals. Violation of ular phylogenetic studies using a genome-scale approach) orthology assumption, biases leading to non-phyloge- has been thought to overcome these problems, and "end-

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ing incongruence" was in sight (2). However, poor taxon yses of all these 200 newly generated datasets were con­ sampling (3) and systematic error that is positively mis­ ducted. We found by calculating the branch attachment leading (4) can cause phylogenomic analyses to yield frequency (BAF) for Myzostomida using Phyutilitly (17), incorrect trees with high support. that myzostomids group with Turbanella in 33% of the 35- gene datasets, and in 41 % of the 50 gene dataset (see Use of phylogenomic analyses to address deep metazoan Additional File 1). Alternatively, myzostomids grouped as relationships has recently increased. Many of these analy­ sister to Bilateria (24%/ 13%), with gnathostumulids ses consist of con catenated sets of ribosomal proteins (24%/22%), or with chaetognaths (S%/ 17%). Interest­ (RP) [5-S) or of data sets dominated by RP data (3). RP ingly, these taxa are suspected of having high rates of genes are highly expressed and therefore often outnumber nucleotide substitution. In none of these analyses did other genes in EST-data sets. They are assumed to be myzostomids group with annelids. These analyses also largely free of paralogy across metazoans [9,10) and as shows that the high amount of missing data (as typical for such seem to represent good candidates for phylogenetic EST-based datasets), seems to have no influence regarding analyses. the phylogenetic position of the myzostomids.

The phylogenetic posItion of myzostomids, parasitic These results were additionally supported by a Bayesian organisms typically found on echinoderms, has been analysis under a site-heterogeneous model (see Addi­ highly disputed over centuries, and possible relationships tional File 1). Congruent to the ML-analysis, myzosto­ with flatworms (11) or syndermatans (12) have been sug­ mids grouped with Turbanella and cluster between long­ gested by single gene analyses. However, analyses of mito­ branched platyzoan taxa. Additionally, we performed chondrial gene order and sequence data show strong hypothesis-testing to evaluate if single gene topologies are evidence that myzostomids are part of the annelid radia­ congruent with the best ML tree of the initial concatenated tion [13), a result that is congruent with morphological 77 -RP analysis. For these analyses, we pruned taxa missing investigations (14). These results are contrasted by phyl­ in single gene datasets from the best tree and used these ogenomic analyses based on an EST-borne 150 gene data­ trees as a constraint for ML-analyses. Using AU-tests as set (15) that group myzostomids within a clade of implemented in CONS EL (1S), we found that all 77 single platyzoan taxa including flatworms, rotifers, gnathostom­ gene analyses are congruent with the best tree. Moreover, ulids, and gastrotrichs. Nevertheless, the position of the AU-test significantly rejects monophyly of a clade con­ Myzostomida, and some other taxa, has been regarded as sisting of Myzostomida and Annelida sensu lato (s.l.) unstable, and Dunn et al. (15) excluded these taxa from when analysing the complete dataset. Summarising these further analyses with these EST data. Taxa that defy robust analyses, the RP dataset weakly supports a platyzoan/ phylogenetic placement are called "problematic taxa" myzostomid association, without any support for an (16). annelid origin. This relationship was also suggested by earlier molecular analyses based on a few genes [11,12). Here we compare analyses of two independent datasets to elucidate the phylogenetic position of Myzostomida: RP For the second data set, we sequenced another nearly genes and mitochondrial genomes. We show that markers complete mitochondrial genome. Within myzostomids, belonging to a single class of macromolecular complexes two major clades can be identified (19), and both are rep­ might bias the analysis and discuss implications for phyl­ resented by the available myzostomids mitochondrial ogenomic analyses in general. genomes (Endomyzostoma sp. reported here and Myzostoma seymourcollegiorum from Bleidorn et al. (13)). The gene Results and discussion order (Figure 2) of the endoparasitic Endomyzostoma spe­ Analysing an alignment consisting of 77 RP genes, the cies is similar to that of the ectocommensal Myzostoma sey­ best tree of the ML-analysis (Figure 1) supports mono­ mourcollegiorum and as such reveals an order of protein phyly of Myzostomida (ML-bootstrap-support (MLB) coding and rRNA genes which is identical to the conserved 100%). They are recovered as sister group of the gas trot- pattern of (most) annelids, while no other animal taxon rich Turbanella (support <50%), and together placed in a shares this pattern with myzostomids and annelids clade containing platyzoan taxa with long branches, [13,20,21). including Syndermata (Acanthocephala + Rotifera) and Platyhelminthes (support <50%). Annelids (including ML-analysis of the 7S-taxa mitochondrial genome dataset echiurids and sipunculids) are recovered as monophyletic (Figure 3), including data for three myzostomids (the two (MLB 7S%). To test if this result is driven by only few mentioned above, plus mitochondrial genes found in the genes, we performed two partition jackknifing analyses EST-library of Myzostoma cirriferum) , recovers mono­ where we generated 100 concatenated datasets containing phyletic Myzostomida (MLB 100%) as sister group to all either 35 or 50 randomly drawn gene partitions. ML anal- other annelids (MLB <50%). Included platyzoan taxa

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A crop ora I r----- Nematostella Cnidaria ,------Hydra Strongylocentrotus I .------Ciona Deuterostomia Fugu Homo .------Gnathostomula Gnathostomulida 100 I 100 Flaccisagitta IChaetognatha Spade/fa .------Priapulus .------I-fypsibius 100 .------Xiphinema L__ l1Q O ~======~A~:s~c~a~r~~~_ 91 Caenorhabditis Ecdysozoa r------Ixodes r------Hom8l rus .------Oaphnia .----- Apis L..-______Anopheles .------Turbanella '-______1.:...:0'-"-10 ,--- M. seymourcollegiorum "C '--- M. cirriferum - 88 ,------Pomphorhynchus ~ ,------Philodina '< 73'------Brachionus oN 76 .------Paraplanocera Q) .------.---- Macrostomum ::::l ---J1l.(;0~OC==~======:s Echinococcus I Schistosoma Q) oo Ougesia japonica ->< '------Ougesia ryukenis Q) ~93 Schmidtea 100 Bugula Bryozoa Flustra I Pedicellina Barentsia IKamptozoa 100 Idiosepis Euprymna 50 ,------Aplysia Mollusca .---- Crassostrea '---Argopecten ,------Terebratalia I Brachiopoda Carinoma I Lineus Nemertea Cerebratulus 99 ,---- Eurythoe '---- Chaetopterus ,----- Sipunculus » '--- Themiste ::::l .------Platynereis ::::l ,---- Urechis CD '----- Capitella _. .----- Arenicola C. ,---- Tubifex Q) ,----- Lumbricus ,----- Hirudo Helobdella Haementeria

Figure I ML analysis of the RP-dataset using RAxML with mixed models. Bootstrap support estimated from 100 replicates is given at the nodes.

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Orbinia (Anneli da)

Urechis (Echiura, Anne lida)

Plolynereis (A nne l ida)

MYZOS I0l110 seYl11ourcollegiorul11 (Myzo to mi da)

Endomyzostol11o sp. (Myzostom ida)

Figure 2 Mitochondrial gene order of Myzostoma seymourcollegiorum compared with annelids. Protein-coding genes and ribosomal RNA genes were identified by blasting on the NCBI Entrez databases. Transfer RNA genes were identified by their potential secondary structures using the tRNAscan-SE Search Server (Lowe and Eddy 1997). Identical patterns between taxa are highlighted. Abbreviations are as follow: ATP synthase subunits (atp6, atpB) cytochrome c oxidase subunits (cox l-cox3), apocytochrome b (cob), nicotinamide adenine dinucleotide ubiquinone oxireductase subunits (nad l-nad6), small and large ribosomal subunit (rmS, rmL). Transfer RNA genes are denominated by the corresponding amino acid (one letter code).

(Platyhelminthes, Acanthocephala, Rotifera) form a When accepting the results of the RP analyses, we have to monophyletic group (MLB 81 %). Very similar results are assume convergent evolution of many morphological revealed by Bayesian analysis under a site-heterogeneous characters (e.g. chaetae, parapodia, trochophore larvae) model (see Additional File 1). Here, a clade containing and an exceptional case of convergence in mitochondrial Annelida s.l. and Myzostomida is supported by a poste­ gene order between annelids and myzostomids. In the rior probability of l.0. other case, we have to assume that 77 RP genes are mis­ leading phylogenetic analysis. Reasons for incongruence Using hypothesis testing, we were able to significantly between markers might be either biological (e.g., selec­ reject monophyly of a clade containing platyzoan taxa tion, incomplete lineage sorting), or methodological (e.g., (Platyhelminthes and Syndermata) and Myzostomida. inaccurate phylogenetic reconstruction due to model mis­ specification) [26,27). In the case of lineage sorting we The conflict regarding the phylogenetic position of would expect mixed signal when comparing the 77 RP myzostomids between analyses of the RP and the mito­ genes. But this is not the case, as there is not any support chondrial dataset is obvious - but only one of these for an annelid affinity in this dataset. Due to lack of con­ hypotheses can be true. Consistent with the mitochon­ cordance in the taxon sampling we were not able to com­ drial data, an annelid affinity is also supported by the bine both sets of markers into a single supermatrix and as nuclear Myosin II gene [13), Hox genes (22), and is in line such methods estimating species trees from gene trees with morphological data [14,23-25). (e.g. BEST, (28)) were not applicable. However, Ewing et

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Oscarella Porifera Geodla I °9J:grgfuC;:: I Cnidaria ,--- Xenoturbella 81 Branchiostoma Lampetra 89 la("\... Saccoglossus Deuterostomia 1:jalanog7ossus 97 Florometra 95 100 Paracentrotus Asterias Epiperipatus IOnychophora Priapulus I Priapulida Llthobius Narceus 100 Limulus 9 99 Hepthatela """--- Ixodes 96 . Artemia Tnops EuarthroplOda Squilla l°'Penaeus 75 Drosophila 100 84 Locus!a TnbollUm 100 100 Endomyzostoma M. seymourcollegiorum M. cirriferum 100 Riftia Galathealinum Helobdella Perionyx Lumbncus Platynereis Nepf1tys Clymenella . Eclysippe Plsta Terebellides 94 Urechis Phascolosoma Orbinia 1 Scoloplos Phoronis I Phoronida Katharina 94 Octopus 100 I 89 Nautilus Mollusca 100 HallOtls Conus 100 Terebratulina I 100 Terebratalia BrachiolPoda Laqueus 100 Loxocorone I Loxosomella Kamptozoa 100 Flustrellidra B Bugula I ryozoa Spadella I Paraspadella Chae ~ tognatha 100 Trichinella 51 --- Agamermis '------Xiphinema Nematoda ___--.Jl.Q.Qj~~ =~=:~~~ Onchocerca L Caenorhabditis Anisakis r------Brachionus ,------Leptorhynchoides ,------Microstomum r------Microcotyle 100 Gyrodactylus '------.---'-=--=-i 89 100 Trichobilharzia Schistosoma Paragonimus FascIOla Diphyllobotrium Hymenolepis Echinococcus Taenia ....P.1 Figure 3 ML analysis of the mitochondrial gene dataset. Analysis was conducted with RAxML using mixed models. Bootstrap val ­ ues from 100 replicates are given at the nodes.

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al. (29) found no evidence that lineage sorting is mislead­ mitochondrial sequences support an annelid affinity for ing phylogenetic reconstruction by analysing a 216 gene myzostomids. This result is in line with some nuclear deep metazoan phylogeny dataset. genes (Myosin II, Hox genes) and morphology, and is strongly supported by mitochondrial gene order and as But it might not be far fetched that analyses of RP genes such we consider this hypothesis more plausible than a are misleading. It has been shown that phylogenetic anal­ possible platyzoan affinity. yses of rRNA genes are affected by long-branch attraction regarding the position of myzostomids [13), and co-evo­ Irrespective of which hypothesis will confirmed by future lution between ribosomal proteins and its rRNA binding analyses, we conclude (i) that reliance of a set of markers sites have been already demonstrated [30). Moreover, in a belonging to a single class of macromolecular complexes phylogenomic analysis regarding Ecdysozoa, analysing might bias the analysis, and (ii) that concatenation of all different macro molecular complexes individually recover data might introduce conflicting signal into the analyses. different hypotheses (e.g., RP genes supported a different We therefore strongly recommend testing for data incon­ hypothesis than Chaperonins) (31). Another study on the gruence in phylogenomic analyses, as this might be the same topic found that ribosomal proteins might be mis­ key for robust phylogenetic placement of problematic leading due to evolutionary biases (10). The existence of taxa. systematic functional or structural signal that competes with ancestral signal has been recently demonstrated for Methods phylogenetic datasets (32) . Individuals of Myzostoma cirriferum were collected from its host, the crinoid Antedon bifida, sampled in Morgat Analyses by Rokas et al. (2) suggested that combining (France). Total RNA of .vl00 frozen individuals was many genes in large molecular datasets will overcome extracted using the Qiagen RNeasy Plant Mini Kit (Qia­ problems of single gene analyses and end incongruence gen, Hilden, Germany). An amplified cDNA library was (33). Despite these hopes, subsequent analysis using phy­ constructed at the Max Planck Institute for Molecular logenomic datasets [3, 15) largely supported the backbone Genetics in Berlin using CloneMiner (Invitrogen). cDNA of the "New animal phylogeny" (34), but failed to resolve was size fractioned and directional cloned using the vector the phylogenetic position of many so-called problematic pDNR-LIB. Clones containing cDNA inserts were taxa (15,35,36). Moreover, such analyses disagree in sequenced from the 5' end on the automated capillary resolving relationships at the base of the metazoan tree sequencer systems ABI 3730 XL (Applied Biosystems, (15,37). Darmstadt, Germany) and MegaBace 4500 (GE Health­ care, Miinchen, Germany) using BigDye chemistry. EST In the case of myzostomids, our analyses show that differ­ processing was done at the Center for Integrative Bioinfor­ ent marker sets can resolve different topologies and usage matics in Vienna. Sequencing chromatograms were evalu­ of complete macromolecular complexes might bring con­ ated using Phred [40,41). Vector-, adapter-, poly-A-, and flicting signal into supermatrices and as such mislead bacterial sequences were removed using Lucy (42), Seq­ analyses. Interestingly, we do not find any conflict within Clean (43), and CrossMatch (44). Sequences were then our RP dataset, but all incongruence is between both sets clustered and assembled using the TIGCL package (43) by of markers. As such, reliance on a set of sequences belong­ performing pairwise comparisons (MGIBlast) and a sub­ ing to a single macro molecular complex might give a sequent clustering using CAP3 (45) . All M. cirriferum EST's biased picture, as these genes might share a common evo­ have been deposited in the EMBL sequence database (46) . lutionary bias. This holds true for either mitochondrial or ribosomal proteins. For future work, we strongly recom­ We generated an additional nearly complete mitochon­ mend careful inspection of phylogenomic datasets for drial genome for the endoparasitic myzostomid incongruent signals [38,39) in order to refine phyloge­ Endomyzostoma sp. Individuals were collected in Antarctic nomic analyses, as this might be the key for the placement peninsula region area by dredge from the R/V Laurence M. of so-called problematic taxa. Could and frozen at -80 0 C after collection. Total genomic DNA extractions employed the DNeasy Tissue Kit (Qia­ Conclusion gen) according to the manufacture's instructions. The Analysing a 77 gene RP-dataset, we found that a grouping genome of Endomyzostoma sp. was amplified in four over­ of myzostomids within platyzoan taxa is favoured. Statis­ lapping fragments. First, we used taxonomically inclusive tical tests have shown that this is congruent with every sin­ primers (47) to amplify the conserved regions of mLSU, gle gene partition of this dataset and jackknifing analysis cox], cob and nad5 genes. PCR products were purified with subsequent investigation of the branch attachment using QIAquick PCR purification kit (Qiagen) and frequency of myzostomids revealed no sign of support for sequenced using a CEQ8000 (Beckmann). Three pairs of an annelid affinity. Contrasting these results, analyses of specific long-PCR primers (Table 1) were designed to

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Table I: Long peR Primers for amplifications of Endomyzostoma mt:DNA:

Fragments Primer name Sequence Annealing Temp.

coxl-cob CO I-Myz-longF 5'---ATT TIT TCC TTA CAT TTA GCT GGG GCT AGG-3' 53 Cytb-Myz-longR 5'---TGT TTA ACT CCT AAA GGG TIT GAT GAC CCG C---3' 53 cob-nodS Cytb-Myz-longF 5'---TCC TCA TTA ATA AAA ATC CCG TTC CAC CCG---3' 54 Nad5-Myz-618R 5'---TAC TAG TGC AGA AAC GGG TGT AGG TGC TGC---3' 54 nadS-mLSU Nad5-Myz-615F 5'---GTA CAC TCA TCA ACA TTA GTA ACA GCA GGC---3' 54 16S-Myz-longR 5'---CTT TAG AAA AAT AAA CCT GTT ATC CCT GTG G---3' 54

amplify these long fragments: coxl-cob, cob-nad5 and quently used to mask all alignments prior to computing nad5-mLSU. Long PCRs were employed on Eppendorf phylogenies: columns with many gaps or highly diverse Mastercycler (Eppendorf) PCR machines using Takara 1A­ amino acids were removed from the peptide alignments. Taq PCR System. 50 III long PCR reactions were set up A concatenated alignment of all 77 single gene alignments

including 5 III 10xbuffer, 8 III dNTP (2 mM), 5 III MgCl 2 was constructed. The alignment has been deposited at (25 mM), 2 III of each long PCR specific primers (10 IlM treebase [55). each), 0.5 III Takara IA-Taq (5 il/ lll), 2 III DNA template and 25.5 III sterilized distilled water. The long PCR proto­ We used the AIC as implemented in ProtTest l.3 [56) for col was 94 ° C for 3 min, followed by 35 cycles with 94 ° C model selection of the con catenated dataset. For Maxi­ for 30 sec, 53 or 54°C for 30 s, and 70 °C for 12 min; final mum Likelihood (ML) analysis, we used RAxML [57) with extension at 72 ° C for 10 min and hold at 4 ° C. The coxl­ the PROTGAMMARTREV model to analyse single gene cob fragment was around 8 kb; the cob-nad5 was 2 kb, partitions, as well as the concatenated dataset. The con­ while nad5-mLSU was about 4.5 kb in size. These three catenated dataset was analysed using mixed models for 77 fragments were purified using QiaQuick Gel Extraction single gene partitions. Clade stability was estimated by Kit (Qiagen) and then cloned into the pGEM-T Easy vector 100 replicates of non-parametric bootstrapping. (Promega). Positive clones were screened by PCRs and plasmids were isolated by QIAprep Spin Miniprep Kit In a second step, we performed partition jackknifing anal­ (Qiagen). Then EcoR! was used to digest the isolated plas­ yses where we generated 100 con catenated datasets each mids to check the insert size. Primer walking was containing either 35 or 50 randomly drawn gene parti­ employed to sequence this plasmid with large inserts. tions. ML analyses of all these 200 newly generated data­ sets were analysed under mixed models with the settings Sequences were joined together and edited using DNAS­ as described above. We calculated the Branch Attachment TAR'" Lasergene programs SeqMan and MegAlign (48). Frequency (BAF) for Myzostomida using Phyutilitly (17) Blast searches were used to identify protein-coding genes for the 100 35-gene datasets, as well as for the 100 50- and ribosomal RNA genes; tRNA genes were identified gene datasets. BAF visualizes alternative positions of par­ using tRNAscan-SE web server (49) under default settings ticular taxa across a set of trees. and source = "mito/chloroplast", or drawn by hand based on their potential secondary structures and anticodon We conducted Bayesian inference based on the site-heter­ sequences. The GenBank accession number for the partial ogeneous CAT model using PhyloBayes v2.1c [58) . Two mitochondrial genome is FI975144. independent chains were run were run for 17814 and 14209 points. To check for convergence, the program Phylogenetic analyses of the ribosomal protein dataset bpcomp [58) was used to compare the bipartitions We used the published alignments [5,7) as backbone for between the two runs. With a burn-in of 1000 and taking our analysis. Human ribosomal protein genes retrieved every two trees, the largest discrepancy observed between from the Ribosomal Protein Gene Database [50) as search bipartitions was 0.129. After discarding the burn-in, a template for local tblastN searches using an e value

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Table 2: List of taxa included in the ribosomal protein dataset.

OTU higher taxon Genes % AAs present

Acroporo mileporo Cnidaria 59 60. 15 Anopheles gombioe Arthropoda 77 99.61 Apis mellifero Arthropoda 77 99.07 Aplysio colifornico Mollusca 76 96.46 Arenicolo morino Annelida 60 66.44 Argopecten irrodions Mollusca 70 93.71 Ascoris suum Platyhelminthes 76 95.36 Borentsio elongoto Kamptozoa 46 54. 19 Brochionus plicotilis Rotifera 70 90.82 Bugu/o neritino Bryozoa 77 98.09 Coenorhobditis elegons Nematoda 77 98.99 Copitello sp. I Annelida 76 86.63 Corinomo mutobilis Nemertea 73 93.57 Cerebrotu/us locteus Nemertea 71 90.23 Choetopterus voriegotus Annelida 67 84.95 Ciono intestinolis Tunicata 77 99.49 Crossostreo gigos Mollusca 75 94.16 Dophnio mogno Arthropoda 77 97.63 Dugesio joponico Platyhelminthes 67 75.20 Dugesio ryukyuensis Platyhelminthes 62 75.76 Echinococcus gronu/otus Platyhelminthes 73 92.17 Euprymno scolopes Mollusca 58 78. 15 Eurythoe complonoto Annelida 41 39.93 Floccisogitto enfloto Chaetognatha 61 69.58 Flustro folioceo Bryozoa 76 89.93 Gnothostomu/um porodoxo Gnathostomulida 59 69.44 Hoementerio depresso Annelida 54 53 .32 Helobdello robusto Annelida 75 78.78 Hirudo medicinolis Annelida 64 85. 13 Homorus omericonus Arthropoda 57 70.38 Homo sopiens Vertebrata 77 99.70 Hydro mognipopilloto Cnidaria 77 98.79 Hypsibius dujordini Tardigrada 74 86.16 Idiosepius porodoxus Mollusca 43 57.63 Ixodes scopu/oris Arthropoda 71 87.07 Lineus viridis Nemertea 57 73.05 Lumbricus rubellus Annelida 76 98.32 Mocrostomum lignono Platyhelminthes 56 70.06 Myzostomo cirriferum Myzostomida 47 64.84 Myzostomo seymourcollegiorum Myzostomida 62 75.47 Nemotostello vectensis Cnidaria 72 85.36 Poroplonoco sp. Platyhelminthes 70 88.46 Pedicellino cernuo Kamptozoa 71 89.31 Philodino roseolo Rotifera 28 32.29 Plotynereis dumerilli Annelida 26 40.54 Pomphorhynchus loevis Acanthocephala 63 63 .04 Priopu/us coudotus Priapulida 37 36.12 Schistosomo monsoni Platyhelminthes 77 98.42 Schmidteo mediterroneo Platyhelminthes 77 97.14 Sipuncu/us nudus Annelida 49 47. 11 Spodello cepholoptero Chaetognatha 66 79.94 Strongylocentrotus purpurotus Echinodermata 76 94.80 Tokifugu rubripes Vertebrata 77 99.86 Terebrotolio tronsverso Brachiopoda 64 78.17 Themiste logeniformes Annelida 64 78.06 Tubifex tubifex Annelida 76 96.90 Turbonello ombronensis Gastrotricha 57 57.32 Urechis coupo Annelida 73 92.73 Xiphinemo index Nematoda 70 90.44

Number of ribosomal protein genes and percentage of amino acids present in the concatenated dataset are given.

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Table 3: List of species included in the mitochondrial genome Table 3: List of species included in the mitochondrial genome dataset. Incomplete mitochondrial genomes are indicated with dataset. Incomplete mitochondrial genomes are indicated with an asterik (*). an asterik (*). (Continued)

OTU higher taxon Pisto cristoto Annelida Plotynereis dumerilli Annelida Acropora tenuis Cnidaria Priopu/us coudotus Priapulida Agomermis sp. Nematoda Riftio pochyptilo * Annelida Anisokis simplex Nematoda Soccoglossus kowolevskii Hemichordata Artemio franciscono Arthropoda Schistosomo monsoni Platyhelminthes Asterios omurensis Echinodermata Scoloplos ormiger * Annelida Bolonoglossus cornosus Hemichordata Spodello cepholoptera Chaetognatha Brachionus plicotilis Rotifera Squillo montis Arthropoda Branchiostomo florido Cephalochordata Toenio osiotico Platyhelminthes Bugu/o neritino Bryozoa Terebellides stroemi Annelida Coenorhobditis elegons Nematoda Terebratolio transverso Brachiopoda Clymenello torquoto Annelida Terebratulino retuso Brachiopoda Conus textile Mollusca Tribolium costoneum Arthropoda Diphyllobotrium lotum Platyhelminthes Trichinello spiralis Nematoda Drosophilo melonogoster Arthropoda Trichobilhorzio regenti Platyhelminthes Echinococcus granolosus Platyhelminthes Triops concriformis Arthropoda Ec/ysippe vonelli * Annelida Urechis coupo Annelida Endomyzostomo sp. * Myzostomida Xenoturbello bocki Xenoturbellida Epiperipotus biolleyi Onychophora Xiphinemo omericonum Nematoda Fosciolo hepotico Platyhelminthes Florometra serrotissimo Echinodermata Flustrellidra hispido Bryozoa per-site log-likelihoods with RAxML for both, the topol­ Golotheolinum brachiosum * Annelida ogy inferred by the single gene analysis and the con­ Geodio neptuni Porifera strained topology from the best tree, and used an Ail-test Gyrodoctylus soloris Platyhelminthes as implemented in CONSEL (18) to test if these hypothe­ Holiotis rubra Mollusca ses differ significantly. Moreover, we constrained the Helobdello robusto Annelida monophyly of clade consisting of Annelida sensu lato (i.e. Heptothelo hongzhouensis Arthropoda including echiurids, siboglinids, and sipunculids) and HymenolepiS diminuto Platyhelminthes Ixodes hexogonus Arthropoda myzostomids and tested with the method mentioned Kothorino tunicoto Mollusca above if this hypothesis differs significantly from the best Lompetra fluviotilis Vertebrata tree. Loqueus rubellus Brachiopoda Leptorhynchoides thecotus Acanthocephala Phylogenetic analysis of mitochondrial genome sequences Limu/us polyphemus Arthropoda Amino acid alignments of protein-coding genes from 78 Lithobius forftcotus Arthropoda complete and partial mitochondrial genomes (Table 3) Locusto migratorio Arthropoda Loxocorone ollox Kamptozoa were computed using ClustalW as implemented in Bioedit Loxosomello oloxioto Kamptozoa ver. 7.0.1 [59). Mitochondrial sequences were down­ Lumbricus terrestris Annelida loaded from OGRe database (60) . Additionally, we per­ Metridium senile Cnidaria formed BLAST searches to find mitochondrial genes Microcotyle sebostis Platyhelminthes within the newly generated EST-library of Myzostoma cir­ Microstomum lineore * Platyhelminthes riferum. Myzostomo cirriferum * Myzostomida Myzostomo seymourcollegiorum * Myzostomida Norceus onnu/orus Arthropoda Gblocks, ver. 0.91 (61) was used to identify unambigu­ Noutilus mocrompho/us Mollusca ously aligned proportions of the alignments. Parameters Nephtys sp. Annelida used were: minimum number of sequences for a con­ Octopus vulgoris Mollusca served position = 41, minimum number of sequences for Onchocerco volvulus Nematoda a flank position: 41, maximum number of contiguous Orbinio lotreillii Annelida non-conserved positions: 8, minimum length of a block: Oscorello cormelo Porifera 10, allowed gap positions: with half, use similarity matrix: Poracentrotus lividus Echinodermata Poragonimus westermoni Platyhelminthes yes. Gblocks treatment recovered 51 % of the original Porospodello gotoi Chaetognatha alignment, leading to a concatenated alignment of 2295 Penoeus monodon Arthropoda amino acids, with all genes except atp8 being partially rep­ Perionyx excovoto Annelida resented in the final alignment. The alignment has been Phoscolosomo gouldii * Annelida deposited at treebase [55). Phoronis psommophilo * Phoronida

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Maximum likelihood analysis was performed with 2. Rokas A, Williams BL, King N, Caroll SB: Genome-scale approaches to resolving incongruence in molecular phyloge­ RaxML, veL 7.03 [57). MtRev + CAT was chosen as model nies. Nature 2003, 425:798-804. for amino acid substitutions. The dataset was partitioned 3. Philippe H, Lartillot N, Brinkmann H: Multigene analyses of bilat­ according to single gene sequences, so that model param­ erian animals corroborate the monophyly of Ecdysozoa, Lophotrochozoa, and Protostomia. Mol Bioi Evol 2005, eters and amino acid frequencies were optimized for each 22: 1246-1253. single gene alignment. 100 bootstrap replicates were per­ 4. Hedtke SM, Townsend TM, Hillis DM: Resolution of phylogenetic formed to infer the support of clades from the best tree. conflict in large data sets by increased taxon sampling. Syst Bioi 2006, 55:522-529. Additionally, we constrained monophyly of a clade con­ 5. 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