Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria

  • Nathaniel M Evans1,

    Affiliated with

    • Alberto Lindner2,

      Affiliated with

      • Ekaterina V Raikova3,

        Affiliated with

        • Allen G Collins4 and

          Affiliated with

          • Paulyn Cartwright1Email author

            Affiliated with

            BMC Evolutionary Biology20088:139

            DOI: 10.1186/1471-2148-8-139

            Received: 04 December 2007

            Accepted: 09 May 2008

            Published: 09 May 2008

            Abstract

            Background

            Polypodium hydriforme is a parasite with an unusual life cycle and peculiar morphology, both of which have made its systematic position uncertain. Polypodium has traditionally been considered a cnidarian because it possesses nematocysts, the stinging structures characteristic of this phylum. However, recent molecular phylogenetic studies using 18S rDNA sequence data have challenged this interpretation, and have shown that Polypodium is a close relative to myxozoans and together they share a closer affinity to bilaterians than cnidarians. Due to the variable rates of 18S rDNA sequences, these results have been suggested to be an artifact of long-branch attraction (LBA). A recent study, using multiple protein coding markers, shows that the myxozoan Buddenbrockia, is nested within cnidarians. Polypodium was not included in this study. To further investigate the phylogenetic placement of Polypodium, we have performed phylogenetic analyses of metazoans with 18S and partial 28S rDNA sequences in a large dataset that includes Polypodium and a comprehensive sampling of cnidarian taxa.

            Results

            Analyses of a combined dataset of 18S and partial 28S sequences, and partial 28S alone, support the placement of Polypodium within Cnidaria. Removal of the long-branched myxozoans from the 18S dataset also results in Polypodium being nested within Cnidaria. These results suggest that previous reports showing that Polypodium and Myxozoa form a sister group to Bilateria were an artifact of long-branch attraction.

            Conclusion

            By including 28S rDNA sequences and a comprehensive sampling of cnidarian taxa, we demonstrate that previously conflicting hypotheses concerning the phylogenetic placement of Polypodium can be reconciled. Specifically, the data presented provide evidence that Polypodium is indeed a cnidarian and is either the sister taxon to Hydrozoa, or part of the hydrozoan clade, Leptothecata. The former hypothesis is consistent with the traditional view that Polypodium should be placed in its own cnidarian class, Polypodiozoa.

            Background

            Polypodium hydriforme is an endocellular parasite whose unusual life cycle, peculiar morphology, and high rates of DNA evolution, have led to much controversy regarding its phylogenetic position within metazoans [15]. Polypodium spends most of its life inside the oocytes of acipenseriform fishes (sturgeons and paddlefish). During this time, Polypodium develops from a binucleate cell into an inside-out planuliform larva and then into an elongate inside-out stolon; the epidermal cell layer is located internal to the body and the gastrodermis is located externally [68]. The embryo, larva and stolon are surrounded by a protective polyploid cell, which also functions in digestion [7]. Just prior to host spawning, Polypodium everts to the normal position of cell layers, revealing tentacles scattered along the stolon. During eversion, the yolk of the host oocyte fills the gastral cavities of the parasite, supplying the future free-living stage with nutrients [6, 7]. Finally, upon emerging from the host egg in fresh water, the free-living stolon (Figure 1A) fragments into individual medusoid-like forms (Figure 1B) that go on to multiply by means of longitudinal fission, form sexual organs, and ultimately infect host fish with their gametophores [69].
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-8-139/MediaObjects/12862_2007_Article_708_Fig1_HTML.jpg
            Figure 1

            Polypodium hydriforme. A) Stolon stage just after emerging from the host oocyte. B) Four specimens of free-living Polypodium with 12 tentacles. Photos by E. Raikova.

            Two conflicting hypotheses have been proposed regarding the phylogenetic placement of Polypodium. The first, more traditional hypothesis is that Polypodium is a cnidarian. Some have suggested it is nested within a derived group of hydrozoans, the Narcomedusae [1013] or the cnidarian class Scyphozoa [14]; while others have suggested it belongs to a separate cnidarian class, Polypodiozoa [1, 15, 16]. The assignment of Polypodium to Cnidaria is based primarily on morphological evidence, most notably the fact that Polypodium possesses nematocysts [17, 18], the stinging structures characteristic of all cnidarians. In addition, the presence of tentacles and overall body-plan organization of Polypodium are reminiscent of cnidarians, although it is unclear if the adult free-living stage is homologous to a polyp or medusa stage. This hypothesis is supported by a cladistic analysis of small subunit nuclear ribosomal DNA (18S rDNA) sequences in conjunction with morphological characters (including nematocysts) [2]. In this study, Polypodium falls within the medusozoan clade of cnidarians, although the non-cnidarian placozoan, Trichoplax [19, 20], also fell within this clade, rendering Cnidaria paraphyletic.

            The second hypothesis is that Polypodium is the sister taxon to Myxozoa, a diverse group of parasites in aquatic animals, and that Polypodium + Myxozoa is the sister group to Bilateria [24]. This hypothesis is derived from cladistic analyses utilizing 18S rDNA sequences [24]. However, because Polypodium and myxozoans have unusually high divergence rates in their 18S rDNA sequences, these cladistic analyses have been criticized by a number of authors who suggest that the data might be unduly affected by long-branch attraction (LBA) [5, 21, 22]. Despite some attempts to overcome the effects of LBA through the use of a maximum likelihood (ML) approach [2123] and pruning long branches [5, 22], these results have been largely silent on the placement of Polypodium. For instance, Kim et al. [22] applied a maximum likelihood approach to 18S rDNA sequence data and found that myxozoans and Polypodium did not group together. Instead, Polypodium was part of an unresolved polytomy that included several cnidarian lineages and Trichoplax, as well as myxozoans + Bilateria. Most recently, Jimenez-Guri et al. [24] utilized multiple protein-coding gene sequences in a ML analysis and found the myxozoan, Buddenbrockia plumatellae nested within cnidarians. Unfortunately, this study had relatively limited sampling of cnidarians and did not include Polypodium.

            In an attempt to resolve this controversy, we sequenced an additional marker in Polypodium, a partial gene sequence of the large nuclear ribosomal unit (28S rDNA), and greatly expanded the taxonomic sampling of cnidarian sequences. Using this approach, we provide evidence that Polypodium is nested within Cnidaria and does not group with myxozoans.

            Results

            Sampled taxa

            All taxa used in this study are arranged taxonomically in Table 1. 155 sequences were obtained from GenBank. 45 new cnidarian sequences for 18S and 59 for 28S (including 2 new 18S and 2 new partial 28S from Polypodium taxa) were generated for this study and deposited in GenBank (see Table 1 for accession numbers). Polypodium hydriforme sequences were obtained from both North American and Eurasian hosts. Eurasian samples were collected from two individuals of Acipenser ruthenus. North American samples were collected from Polyodon spathula and Scaphirhynchus platorynchus. This is the first reported presence of Polypodium infection in Scaphirhynchinae. While Polypodium was recovered from the oocytes of S. platorynchus, the sample from which we extracted sequence data was found externally attached to its presumed host. More specific collection data for Polypodium specimens are associated with each sequence submitted to GenBank (see Table 1 for accession numbers).
            Table 1

            Taxon and sequence list

              

            Accession numbers

             

            Higher classification

            Taxon ID

            28S

            18S

            Voucher

            Bilateria

                

            Annelida

            Proceraea cornuta

            AF212165

            AF212179

             

            Annelida

            Urechis caupo

            AF342804

            AF342805

             

            Arthropoda

            Limulus polyphemus

            AF212167

            U91490

             

            Arthropoda

            Tenebrio sp. /Tenebrio molitor

            AY210843

            X07801

             

            Brachiopoda

            Phoronis vancouverensis

            AF342797

            U12648

             

            Chordata

            Oncorhynchus sp. /O. kisutch

            U34341

            AF030250

             

            Chordata

            Petromyzon marinus

            AF061798

            M97575.1

             

            Chordata

            Raja schmidti

            AF278683

            AF278682

             

            Chordata

            Triakis semifasciata

            AF212182

            AF212180

             

            Echinodermata

            Strongylocentrotus purpuratus

            AF212171

            L28056.

             

            Hemichordata

            Cephalodiscus gracilis

            AF212172

            AF236798

             

            Hemichordata

            Harrimania sp.

            AF212173

            AF236799

             

            Hemichordata

            Ptychodera flava

            AF212176

            AF278681

             

            Hemichordata

            Ptychoderidae

            AF278684

            D14359

             

            Hemichordata

            Saccoglossus kowalevskii

            AF212175

            L28054

             

            Kinorhyncha

            Pycnophyes sp. Tjarno

            AY859597

            AY859598

             

            Mollusca

            Parvicardium minimum

            DQ279966

            DQ279942

             

            Mollusca

            Placopecten magellanicus

            AF342798

            X53899

             

            Nematoda

            Caenorhabditis elegans

            X03680

            X03680

             

            Nematomorpha

            Chordodes morgani

            AF342787

            AF036639

             

            Nemertea

            Amphiporus sp.

            AF342786

            AF119077

             

            Nemertodermatida

            Meara stichopi

            AY157605

            AF119085

             

            Onychophora

            Peripatus sp.

            AY210836

            AY210837

             

            Platyhelminthes

            Diclidophora denticulata

            AY157169

            AJ228779

             

            Platyhelminthes

            Stenostomum leucops

            AY157151

            D85095

             

            Platyhelminthes

            Stylochus zebra

            AF342800

            AF342801

             

            Priapulida

            Priapulus caudatus

            AY210840

            Z38009

             

            Sipuncula

            Phascolopsis gouldii

            AF342795

            AF342796

             

            Tardigrada

            Milnesium.sp.\M. tardigradum

            AY210826

            U49909

             

            Urochordata

            Styela plicata

            AF158724

            L12444

             

            Urochordata

            Thalia democratica

            AF158725

            D14366

             

            Cnidaria

                

            Polypodiozoa

            Polypodium (Host: Acipenser ruthenus)

            EU272585

            EU272630

             

            Polypodiozoa

            Polypodium (Host: Polyodon spathula)

             

            EU272629

             

            Polypodiozoa

            Polypodium (Host:Scaphirhynchus platorynchus)

            EU272586

              

            Anthozoa, Antipatharia

            Antipathes galapagensis

            AY026365

            AF100943

             

            Anthozoa, Scleractinia

            Montastraea franksi

            AY026375

            AY026382

             

            Cubozoa, Carybdeidae

            Carybdea rastonii

            AY920787

            AF358108

             

            Cubozoa, Carybdeidae

            Darwin carybdeid sp.

            AY920788

            AF358105

             

            Cubozoa, Carybdeidae

            Tripedalia cystophora

            EU272595

            EU272637

             

            Cubozoa, Chirodropidae

            Chironex fleckeri

            AY920785

            AF358104

             

            Cubozoa, Chirodropidae

            Chiropsalmus sp.

            AY920786

            AF358103

             

            Hydrozoa, Capitata

            Dipurena ophiogaster

            EU272560

            EU272615

            KUNHM 2803

            Hydrozoa, Capitata

            Ectopleura dumortieri

            EU272561

            EU272616

             

            Hydrozoa, Capitata

            Euphysora bigelowi

            EU272563

            EU272618

            KUNHM 2829

            Hydrozoa, Capitata

            Moerisia sp.

            AY920801

            AF358083

             

            Hydrozoa, Capitata

            Pennaria disticha

            EU272581

            AY920762

             

            Hydrozoa, Capitata

            Polyorchis penicillatus

             

            AF358090

             

            Hydrozoa, Capitata

            Porpita sp.

            AY920803

            AF358086

             

            Hydrozoa, Capitata

            Ralpharia gorgoniae

            EU272590

            EU272633

            KUNHM 2778

            Hydrozoa, Capitata

            Scrippsia pacifica

            AY920804

            AF358091

             

            Hydrozoa, Capitata

            Solanderia ericopsis

            EU272593

            EU272636

            MHNG INVE29593

            Hydrozoa, Capitata

            Velella sp.

            EU272597

            AF358087

             

            Hydrozoa, Capitata

            Zanclea prolifera

            EU272598

            EU272639

            KUNHM 2793

            Hydrozoa, Capitata

            Zyzzyzus warreni

            EU272599

            EU272640

            KUNHM 2777

            Hydrozoa, Capitata

            Candelabrum cocksii

            AY920796

            AY920758

            MHNG INVE29531

            Hydrozoa, Capitata

            Cladocoryne floccosa

            EU272551

            EU272608

             

            Hydrozoa, Filifera

            Bimeria vestita

            EU272548

            EU272605

             

            Hydrozoa, Filifera

            Bougainvillia carolinensis

            EU272549

            EU272606

             

            Hydrozoa, Filifera

            Brinckmannia hexactinellidophila

            EU272550

            EU272607

            MHNG INVE38148

            Hydrozoa, Filifera

            Clava multicornis

            EU272552

            EU272609

             

            Hydrozoa, Filifera

            Clavactinia gallensis

            EU272553

            EU272610

            MHNG INVE33470

            Hydrozoa, Filifera

            Cordylophora caspia

            EU272556

            EU272612

             

            Hydrozoa, Filifera

            Corydendrium sp.

            EU272557

            EU272613

            KUNHM 2764

            Hydrozoa, Filifera

            Dicoryne conybearei

            EU272559

            EU272614

            MHNG INVE32949

            Hydrozoa, Filifera

            Eudendrium.racemosum

            EU272562

            EU272617

             

            Hydrozoa, Filifera

            Fabienna sphaerica

            AY920797

            AY920767

             

            Hydrozoa, Filifera

            Garveia annulata/Garveia sp.

            EU272564

            AY920766

            KUNHM 2860

            Hydrozoa, Filifera

            Hydra circumcincta

            AY026371

            AF358080

             

            Hydrozoa, Filifera

            Hydractinia symbiolongicarpus

            EU272568

            EU272621

             

            Hydrozoa, Filifera

            Hydrichthella epigorgia

            EU272569

            EU272622

            KUNHM 2665

            Hydrozoa, Filifera

            Hydrichthys boycei

            EU272570

             

            MHNG INVE37417

            Hydrozoa, Filifera

            Koellikerina fasciculata

            EU272571

            EU272623

             

            Hydrozoa, Filifera

            Leuckartiara octona

            EU272573

            EU272624

             

            Hydrozoa, Filifera

            Lizzia blondina

            EU272574

            EU272625

             

            Hydrozoa, Filifera

            Pachycordyle pusilla

            EU272579

            EU272627

            MHNG INVE32953

            Hydrozoa, Filifera

            Pandea sp.

            EU272580

            AY920765

             

            Hydrozoa, Filifera

            Podocoryne carnea

            AY920802

            AF358092

             

            Hydrozoa, Filifera

            Proboscidactyla ornata

            EU272587

            EU272631

            KUNHM 2767

            Hydrozoa, Filifera

            Pruvotella grisea

            EU272588

            EU272632

            MHNG INVE34436

            Hydrozoa, Filifera

            Rathkea octopunctata

            EU272591

            EU272634

            KUMIP 314321

            Hydrozoa, Filifera

            Rhizogeton nudus

            EU272592

            EU272635

            MHNG INVE35757

            Hydrozoa, Filifera

            Turritopsis dohrnii

            EU272596

            EU272638

            MHNG INVE29753

            Hydrozoa, Leptothecata

            Abietinaria filicula

            EU272540

            EU272600

            MHNG INVE29947

            Hydrozoa, Leptothecata

            Aglaophenia tubiformis

            EU272543

            EU272601

            MHNG INVE29967

            Hydrozoa, Leptothecata

            Amphisbetia minima

            EU272544

            EU272602

            MHNG INVE25071

            Hydrozoa, Leptothecata

            Anthohebella parasitica

            EU272545

            EU272603

            MHNG INVE29762

            Hydrozoa, Leptothecata

            Clytia noliformis

            EU272554

            EU272611

             

            Hydrozoa, Leptothecata

            Halecium muricatum

            EU272565

            EU272619

            MHNG INVE29028

            Hydrozoa, Leptothecata

            Halopteris minuta

            EU272567

            EU272620

            MHNG INVE25073

            Hydrozoa, Leptothecata

            Melicertum octocostatum

            EU272575

            AY920757

            USNM 1073342

            Hydrozoa, Leptothecata

            Octophialucium indicum

            EU272577

            EU272626

            MHNG INVE29970

            Hydrozoa, Leptothecata

            Plumularia setacea

            EU272583

            EU272628

            MHNG INVE36298

            Hydrozoa, Siphonophorae

            Agalma elegans

            EU272542

            AY937313

            YPM 35029

            Hydrozoa, Siphonophorae

            Apolemia sp.

            EU272546

            AY937331

            YPM 35090

            Hydrozoa, Siphonophorae

            Cordagalma cordiforme

            EU272555

            AY937317

            YPM 35032

            Hydrozoa, Siphonophorae

            Halistemma rubrum

            EU272566

            AY937358

            YPM 35359

            Hydrozoa, Siphonophorae

            Nanomia bijuga

            EU272576

            AY937338

            YPM 35043

            Hydrozoa, Siphonophorae

            Nectopyramis sp.

            AY026377

            AF358068

             

            Hydrozoa, Siphonophorae

            Physophora hydrostatica

            EU272582

            AY937342

            YPM 35046

            Hydrozoa, Siphonophorae

            Sulculeolaria quadrivalvis

            EU272594

            AY937353

            YPM 35357

            Hydrozoa, Stylasteridae

            Crypthelia cryptotrema

            EU272558

            EU272641

            USNM1027758

            Hydrozoa, Stylasteridae

            Lepidopora microstylus

            EU272572

            EU272644

            USNM1027724

            Hydrozoa, Stylasteridae

            Pseudocrypthelia pachypoma

            EU272589

            EU272643

            USNM1027728

            Hydrozoa, Stylasteridae

            Adelopora crassilabrum

            EU272541

            EU272642

            USNM1027760

            Hydrozoa, Trachylina

            Limnocnida tanganyicae

            AY920795

            AY920755

             

            Hydrozoa, Trachylina

            Maeotias marginata

            EU247810

              

            Hydrozoa, Trachylina

            Olindias phosphorica

            EU247808

            AY920753

            MHNG INVE29811

            Scyphozoa, Coronatae

            Atolla vanhoeffeni

            AY026368

            AF100942

             

            Scyphozoa, Coronatae

            Nausithoe rubra

            AY920776

            AF358095

             

            Scyphozoa, Rhizostomea

            Catostylus sp.

            AY920777

            AF358100

             

            Scyphozoa, Semaeostomeae

            Chrysaora melanaster

            AY920780

            AF358099

             

            Scyphozoa, Semaeostomeae

            Aurelia sp.

            EU272547

            EU272604

             

            Scyphozoa, Semaeostomeae

            Phacellophora camtschatica

            AY920778

            AF358096

             

            Staurozoa, Stauromedusae

            Craterolophus convolvulus

            AY920781

            AY845344

             

            Staurozoa, Stauromedusae

            Haliclystus octoradiatus

            AH014894

            AY845346

             

            Staurozoa, Stauromedusae

            Haliclystus sanjuanensis

            AY920782

            AF358102

             

            Myxozoa

                

            Malacosporea

            Buddenbrockia plumatellae

             

            AJ937883

             

            Myxosporea

            Henneguya salminicola

            AY302726

              

            Myxosporea

            Kudoa trifolia

            AM490336

            AM183300

             

            Myxosporea

            Kudoa unicapsula

            AM490335

            AM490334

             

            Myxosporea

            Myxobolus cerebralis

             

            EF370481

             

            Myxosporea

            Myxobolus dogieli

             

            EU003978

             

            Myxosporea

            Parvicapsula limandae

             

            EF429096

             

            Outgroups

                

            Choanoflagellida

                

            Codonosigidae

            Monosiga brevicollis

            AY026374

            AF084618

             

            Salpingoecidae

            Salpingoeca infusionum

            AY026380

            AF100941

             

            Ctenophora,

                

            Cyclocoela

            Beroe ovata

            AY026369

            AF293694

             

            Cyclocoela

            Mnemiopsis leidyi

            AY026373

            AF293700

             

            Typhlocoela

            Pleurobrachia bachei

            AY026378

            AF293677

             

            Fungi

                

            Ascomycota

            Candida albicans

            X70659

            X53497

             

            Ascomycota

            Saccharomyces cerevisiae

            J01355

            M27607

             

            Basidiomycota

            Tricholoma matsutake

            U62964

            U62538.1

             

            Mucoromycotina

            Mucor racemosus

            AJ271061

            AJ271061

             

            Porifera,

                

            Calcarea

            Leucosolenia sp.

            AY026372

            AF100945

             

            Demospongia

            Mycale fibrexilis

            AY026376

            AF100946

             

            Demospongia

            Suberites ficus

            AY026381

            AF100947

             

            A complete list of sequences used in the analyses with GenBank accession numbers and museum voucher numbers. Bold numbers indicate new sequences generated for this study. KUMIP = University of Kansas Museum of Invertebrate Paleontology, KUNHM = University of Kansas Natural History Museum, MHNG = Muséum d'histoire naturelle de Genève, YPM = Yale Peabody Museum, USNM = US National Museum of Natural History.

            All Polypodium sequences were newly generated for this study. We did not include the previously published 18S Polypodium sequence (GenBank accession number U37526) because of concern over the quality of the sequence which included a number of ambiguities. Furthermore, while the two new Polypodium 18S sequences (from hosts Acipenser ruthensus and Polyodon spathula) differed from each other by a total of 8 sites they differed from #U37526 by 77 and 83 sites respectively. These differences included a large number of insertions and deletions. The two new 28S sequences (from hosts Acipenser ruthensus and Scaphirhynchus platorynchus) only differed from each other by 2 sites.

            Position of Polypodium

            The complete combined dataset of 18S rDNA and partial 28S rDNA contains 4842 characters, 2901 of which are variable and 2124 parsimony informative. Both the ML and parsimony topologies reconstructed from the combined dataset suggest that Polypodium is nested within a monophyletic Cnidaria, and myxozoans are the sister taxon to bilaterians (Figure 2). The ML bootstrap values supporting a monophyletic Cnidaria (including Polypodium), a monophyletic Medusozoa (including Polypodium) and the Polypodium + hydrozoan clade are 73, 67 and 73 respectively (Figure 2A, and Additional file 1). Parsimony analysis of the combined dataset differs from that of ML in that Polypodium is nested within a group of hydrozoans, the leptothecates (Figure 2B). The parsimony bootstrap values supporting a monophyletic Cnidaria and Hydrozoa, with Polypodium nested within these clades are 50 and 51 respectively (Figure 2B). The clade nested within hydrozoans, that includes Polypodium + leptothecates is weakly supported in the sub-sampling tests with a bootstrap value of less than 50.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-8-139/MediaObjects/12862_2007_Article_708_Fig2_HTML.jpg
            Figure 2

            Phylogenetic hypotheses of relationships among 126 metazoan taxa, based on a combined analysis of nearly complete 18S and partial 28S rDNA sequences. Arrow indicates Polypodium taxa. A) Maximum likelihood topology. The assumed model (GTR+I + G) has six substitutions rates estimated from the data (A-C, 1.1786; A-G, 3.3654; A-T, 1.7283; C-G, 0.7403; C-T, 4.7803; G-T, 1.0000), an assumed proportion of invariant sites (0.1692) and a gamma shaped parameter or (0.5584). The length of the bar indicates 0.1 substitutions per site. Bootstrap values for this topology are indicated on the cladogram in Additional file 1. B) Strict consensus of 32 trees of length 25141 from a parsimony analyses. Bootstrap values of 50 or greater are indicated.

            The analyses using partial 28S rDNA sequences alone (129 sampled taxa) contains 1756 characters, 1196 of which are parsimony informative. The ML topology using this dataset reveals Polypodium nested within Cnidaria, specifically within leptothecate hydrozoans, (Additional file 2). This analysis however fails to recover a monophyletic Cnidaria, as the anthozoans are placed outside the Cnidaria + Bilateria clade. Analysis of the 18S rDNA dataset alone (132 taxa, 3038 characters, 1469 parsimony informative) under both optimality criteria conflicts with the combined and partial 28S topologies. The 18S rDNA topology for both criteria place Polypodium at the base of Bilateria (Figure 3A, Additional files 3, 4 and 5). However, the ML topology also reflects a sister relationship between Polypodium and myxozoans (Figure 3A and Additional file 3A) while the parsimony topology does not (Additional files 4 and 5). Moreover, under parsimony criteria the position of myxozoans is dependent upon how gaps are coded: if gaps are coded as a fifth character state, myxozoans are placed as a highly derived clade of bilaterians (Additional file 4); if gaps are coded as missing, myxozoans are placed as sister to all metazoans (Additional file 5). The 18S analysis showing placement of Polypodium with Bilateria, and more specifically as sister to myxozoans, is consistent with previously reported studies using the same marker [24], but raises similar concerns of long-branch attraction [5].
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-8-139/MediaObjects/12862_2007_Article_708_Fig3_HTML.jpg
            Figure 3

            ML topologies of metazoan relationships of nearly complete 18S rDNA sequences. Arrow indicates Polypodium taxa. Bootstrap values for both topologies are indicated on the cladograms in Additional file 3. A) 132 taxa including 6 myxozoan taxa and two Polypodium taxa. The assumed model (GTR+I + G) has six substitutions rates estimated from the data (A-C, 1.4071; A-G, 3.3470; A-T, 1.6901; C-G, 0.84888; C-T, 4.7638; G-T, 1.0000), an assumed proportion of invariant sites (0.1757) and a gamma shaped parameter or (0.5837). B) Same dataset as (A) but with the 6 myxozoan taxa removed. The assumed model (GTR+I + G) has six substitutions rates estimated from the data (A-C, 1.4115; A-G, 3.3559; A-T, 1.7502; C-G, 0.8342; C-T, 4.8554; G-T, 1.0000), an assumed proportion of invariant sites (0.2464) and a gamma shaped parameter or (0.6326). The length of the bar indicates 0.1 substitutions per site.

            Test of long-branch attraction

            Myxozoans and Polypodium have unusually high rates of evolution in their 18S and 28S rDNA sequences relative to the other sampled taxa. To investigate the influence of myxozoans on the placement of Polypodium, we removed the myxozoans from our three datasets and re-ran each analysis. Under the ML analysis of 18S rDNA, the removal of myxozoans results in the placement of Polypodium nested within Cnidaria (Figure 3B and Additional file 3B). This result suggests that the placement of Polypodium at the base of bilaterians in the 18S analysis (Figure 3A) was indeed an artifact of LBA. The placement of Polypodium within Cnidaria was not effected by the removal of myxozoans in the 28S (Additional file 6) and combined datasets (Additional file 7).

            To investigate the possible role of LBA on myxozoan placement, we removed Polypodium from the combined ML analyses and found that it did not affect the position of Myxozoa at the base of the Bilateria (not shown). Given that bilaterians also form long branches, we tried removing all bilaterian sequences in the combined ML analysis. This resulted in a Myxozoa + Polypodium clade nested within Cnidaria (not shown). However, when Polypodium and bilaterians were removed, myxozoans fell outside the cnidarians (not shown). Similar effects of myxozoan placement to long-branches were also found in parsimony analyses of the combined dataset (not shown).

            Discussion

            Polypodium is a cnidarian

            Our metazoan dataset of 18S and partial 28S rDNA sequences, with a large taxonomic sample of cnidarians, places Polypodium within a monophyletic Cnidaria. This accords with the fact that Polypodium possesses nematocysts [17, 18] and a cnidarian-like body plan [79, 12]. The precise placement of Polypodium within Cnidaria is less certain. The ML combined analysis places Polypodium as sister to Hydrozoa (Figure 2A), a hypothesis consistent with the suggestion that Polypodium be considered a separate class of cnidarians, Polypodiozoa [1]. By contrast, the combined parsimony analysis (Figure 2B) and the ML analyses of 28S alone (Additional file 2 and 6) place Polypodium within the hydrozoan clade Leptothecata. Given that leptothecates have relatively high rates of evolution within hydrozoans, one possible explanation for the conflicting hypotheses is that the placement of Polypodium within leptothecates is an artifact of LBA and that the combined data, in conjunction with the ML approach (Figure 2A), overcame this localized LBA artifact.

            Evolution of Polypodium life-history characters

            Although the fresh water habitat of Polypodium is unusual for cnidarians, it is not unheard of, especially within hydrozoans. For instance, the model organism Hydra and the jellyfish Craspedacusta are both exclusively fresh-water hydrozoans. Hydra and Craspedacusta are distantly related [25] and our analyses do not indicate a close phylogenetic affinity of Polypodium to either of the clades containing these taxa. Thus, it appears that in the evolution of cnidarians, invasion to fresh-water habitats has happened at least three separate times.

            Although Polypodium is the only known intracellular cnidarian parasite, other cnidarians have adopted parasitic life-styles [11, 2629]. For example, parasites belonging to the Narcomedusae (Hydrozoa) have been reported to live in the stomach cavities of other narcomedusae [11, 27] and anthomedusae [27]. In addition, the sea anemone Edwardsiella lineata parasitizes the stomach cavity of the ctenophore Mnemiopsis leidyi [28] and the anemone Peachia quinquecapitata is reported to parasitize the stomachs of hydromedusa [29].

            Effects of long-branch attraction

            The well-documented effects of long-branch attraction artifacts (reviewed in Bergsten [30]) are particularly concerning when investigating relationships amongst early-diverging metazoans, where rates between lineages vary greatly [22]. Suggestions for avoiding LBA artifacts include choice of appropriate markers [31, 32], increased taxonomic sampling to effectively break up long branches [33, 34] and utilization of best-fit models that incorporate rate variation [2123]. Previous conflicting reports that show Polypodium and myxozoans form a sister taxon to Bilateria [24] can be explained by limited taxon sampling and an inadequate number of informative characters in their analyses, both of which confound long-branch problems. In this study, the increased taxonomic sampling of cnidarians and the addition of 28S rDNA sequence data proved critical to placing the highly divergent Polypodium taxon within Cnidaria. The choice of optimality criteria (ML vs. parsimony) both supported Polypodium as a cnidarian but did affect the placement within Cnidaria.

            Polypodium and Myxozoa

            Our analyses are inconclusive in the placement of Myxozoa within metazoans. We found that myxozoans consistently grouped with long-branched taxa and that removal of long-branches resulted in myxozoans being placed to the next longest branch. For example myxozoans group with Polypodium in the absence of Bilateria and group with Bilateria in the absence of Polypodium (not shown).

            Jimenez-Guri et al. [24] sampled the myxozoan, Buddenbrockia, and found it to fall within Cnidaria, as the sister group to two hydrozoan representatives and a single scyphozoan. Previous studies have suggested a sister group relationship between cnidarians and myxozoans [24], and some morphological evidence has been used to support this view [35]. Although our present study does not support this relationship, further investigation is merited. Myxozoans are a highly diverse group (reviewed in Kent et al. [36]) that comprise two clades, the Myxosporea and the Malacosporea [37]. We were only able to include 28S rDNA sequences from myxosporeans, although the malacosporean Buddenbrockia was included in our 18S analysis and found to group with other myxozoans and outside of Cnidaria. Future studies with a comprehensive sampling of myxozoans together with Polypodium, in a dataset that includes a large taxonomic sampling of cnidarians, should shed further light on the relationships between myxozoans and Polypodium.

            Conclusion

            Although previous molecular phylogenetic hypotheses conflicted with the traditional interpretation of cnidarian affinity for Polypodium, the molecular evidence we present, using an augmented dataset, ultimately confirms and reconciles this traditional hypothesis and suggests that Polypodium is indeed a cnidarian. This study also reaffirms the importance to large taxonomic sampling and inclusion of additional informative characters for avoiding long-branch attraction artifacts.

            Methods

            DNA isolation, amplification and sequencing

            Genomic DNA was extracted using Qiagen DNeasy kits according to manufacturer's protocol (QIAGEN Inc., Mississauga, ON) or a standard phenol/chloroform protocol. The latter method involved tissue digestion with proteinase K (20 mg/ml) in a lysis buffer (20 mM Tris-CL pH 8.0, 5 mM EDTA pH 8.0, 400 mM NaCl, 2%SDS), extraction with phenol/chloroform (1:1), precipitation with 2.5 vol. 95% EtOH, and elution in TE or H2O.

            An approximately 1.8 kb portion of the gene coding for 18S was amplified and sequenced with universal eukaryotic primers as described by Medlin et al. [38], with the annealing temperature modified to 57°C. With the exception of Polypodium samples, a nearly complete, roughly 3 kb portion of the gene coding for 28S was amplified and sequenced with an approach modified from that reported in Collins et al. [25]. 28S was directly amplified in two fragments with combinations of primers F63mod+R2077sq and F1379+R3264 from Medina et al. [39] or newly developed medusozoan specific primers F97+R2084 and F1383+R3238 (F97: CCYYAGTAACGGCGAGT, R2084: AGAGCCAATCCTTTTCC, F1383: GGACGGTGGCCATGGAAGT, and R3238: SWACAGATGGTAGCTTCG). Amplifications of 28S were conducted with the following thermal profile: 4 minutes at 94°C; 30 cycles of 30 seconds at 94°C, 1 minute at 45°C, and 3 minutes at 72°C; and 10 minutes at 72°C. For Polypodium, a portion of the 5' end of 28S (approx. 0.8–1.0 kbps) was amplified using two universal metazoan primers (fw1and rev2) as reported by Sonnenberg et al. [40]. Sequencing was carried out using amplification primers and F635sq and R635sq from Medina et al. [39].

            All gene fragments were purified and sequenced by Cogenics, Inc. (Houston, TX) and assembled and edited using Sequencher v4.5 (Gene Code Co., 2005). Sequences for each marker were aligned using the program MUSCLE [41]. The 28S sequence alignment was then trimmed to reflect only that region which included sequence data for Polypodium. This trimmed 28S dataset was analyzed separately and used in conjunction with the complete 18S sequences to create the combined dataset.

            Phylogenetic analyses

            Phylogenetic analyses were performed using both maximum likelihood (ML) and parsimony criteria. ML searches were performed using GARLI v0.951.OsX-GUI [42] under an assumed GTR model with rates estimated from the data. The assumed model of nucleotide substitution was selected by using the Akaike Information Criterion (AIC) as implemented in ModelTest [43]. Each run was repeated 10 times from random starting trees using default termination conditions. Each run gave identical topologies and similar likelihood scores. 100 bootstrap replications were run in GARLI v0.951.0sX-GUI [42] under the same parameters.

            To assess the effect that omitting length-variable regions has on topology, we removed these regions from the combined dataset, using the less stringent settings of Gblocks [44]. This dataset contained 126 metazoan taxa, 2415 characters, 1391 of which are parsimony informative. We found that removal of length-variable regions had no effect on the placement of Polypodium and minimal effect on overall topology in our combined ML analyses (Additional file 8). Therefore we performed all other analyses with the complete datasets, including the more variable regions.

            Parsimony analyses were performed using TNTv.1.1 [45]. Separate tree searches were performed with gaps coded as missing and gaps coded as a fifth state. However, with one exception (see results for myxozoan placement with 18S data) there was no significant difference in topology. Numerous search methods available in TNT were utilized to search the tree space but the following approach was found to consistently recover trees with minimum lengths from our datasets. The implemented search was a driven new technology search with a random seed of 0 (where 0 = time). Default settings for sectorial searches (RSS and CSS) and tree fusing were used [46], with 5 replicates per repetition, and a requirement that the global optimum be found 20 times. TBR branch swapping was performed on the resulting trees and a strict consensus was calculated. TNT was used to calculate standard bootstrap values (1000 replicates). Alignments and trees for 18S, 28S and combined datasets have been submitted to TreeBASE http://​www.​treebase.​org/​treebase/​index.​html.

            Declarations

            Acknowledgements

            We thank S. Ash Bullard, Jan Dean, Bobby Reed and Ron Bruch for contributions of Polypodium specimens. We also thank Peter Schuchert, Casey Dunn and Michael Dawson for contributions of other cnidarian specimens. NME acknowledges the instructors at the 2007 Ohio State Cladistics workshop, especially P. Goloboff, for help with TNT. We thank M. Holder for discussions, B. Bentlage, B. Lieberman, A. Nawrocki, and five anonymous reviewers for helpful comments on earlier versions of this manuscript. This work was supported by grants from NSF AToL EF-0531779 (to PC and AGC) and support for AL from NSF (PEET DEB-9978086) and FAPESP (06/02960-8/05821-9/60327-0).

            Authors’ Affiliations

            (1)
            Department of Ecology and Evolutionary Biology, University of Kansas
            (2)
            CEBIMar, University of São Paulo
            (3)
            Institute of Cytology of the Russian Academy of Sciences
            (4)
            National Systematics Laboratory of NOAA Fisheries Service, National Museum of Natural History, Smithsonian Institution

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            Copyright

            © Evans et al. 2008

            This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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