1471-2148-8-139 1471-2148 Research article <p>Phylogenetic placement of the enigmatic parasite, <it>Polypodium hydriforme</it>, within the Phylum Cnidaria</p> Evans M Nathaniel evansnat@ku.edu Lindner Alberto alindner@usp.br Raikova V Ekaterina raikova@swipnet.se Collins G Allen COLLINSA@si.edu Cartwright Paulyn pcart@ku.edu

Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, Kansas 66045, USA

CEBIMar, University of São Paulo, São Sebastião, Brazil

Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russia

National Systematics Laboratory of NOAA Fisheries Service, National Museum of Natural History, Smithsonian Institution, Washington, DC 20013-7012, USA

 BMC Evolutionary Biology 1471-2148 2008 8 1 139 http://www.biomedcentral.com/1471-2148/8/139 18471296 10.1186/1471-2148-8-139 04 12 2007 09 5 2008 09 5 2008 2008 Evans et al; licensee 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.

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 12345. 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 678. 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 67. 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 6789.

<p>Figure 1</p>

Polypodium hydriforme

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 10111213 or the cnidarian class Scyphozoa 14; while others have suggested it belongs to a separate cnidarian class, Polypodiozoa 11516. The assignment of Polypodium to Cnidaria is based primarily on morphological evidence, most notably the fact that Polypodium possesses nematocysts 1718, 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 1920, 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 234. This hypothesis is derived from cladistic analyses utilizing 18S rDNA sequences 234. 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) 52122. Despite some attempts to overcome the effects of LBA through the use of a maximum likelihood (ML) approach 212223 and pruning long branches 522, 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).

<p>Table 1</p>

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.

<p>Additional file 1</p>

ML topology of relationships based on combined data. ML topology identical to Figure 1A but as a cladogram showing bootstrap values.

Click here for file

 <p>Figure 2</p>

Phylogenetic hypotheses of relationships among 126 metazoan taxa, based on a combined analysis of nearly complete 18S and partial 28S rDNA sequences

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 234, but raises similar concerns of long-branch attraction 5.

<p>Additional file 2</p>

ML topology of relationships based on partial 28S rDNA sequences. Relationships of 128 metazoan taxa were analyzed with partial 28S rDNA sequences.

Click here for file

 <p>Additional file 3</p>

ML topologies of relationships based on 18S data with and without myxozoans. ML topologies identical to Figure 3 but as cladograms showing bootstrap values.

Click here for file

 <p>Additional file 4</p>

Parsimony topology of relationships based on 18S rDNA sequences. This parsimony analysis of 18S rDNA sequences included 132 taxa with gaps coded as a fifth state.

Click here for file

 <p>Additional file 5</p>

Parsimony topology of relationships based on 18S rDNA sequences. This parsimony analysis of 18S rDNA sequences included 132 taxa with gaps coded as missing.

Click here for file

 <p>Figure 3</p>

ML topologies of metazoan relationships of nearly complete 18S rDNA sequences

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).

<p>Additional file 6</p>

ML topology of relationships excluding myxozoans, based on partial 28S rDNA data. This ML analysis of partial 28S rDNA sequences excluded myxozoan taxa.

Click here for file

 <p>Additional file 7</p>

ML topology of relationships excluding myxozoans, based on combined data. This ML analysis of partial 28S rDNA and 18S sequences excluded myxozoan taxa.

Click here for file

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 1718 and a cnidarian-like body plan 78912. 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 1126272829. For example, parasites belonging to the Narcomedusae (Hydrozoa) have been reported to live in the stomach cavities of other narcomedusae 1127 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 3132, increased taxonomic sampling to effectively break up long branches 3334 and utilization of best-fit models that incorporate rate variation 212223. Previous conflicting reports that show Polypodium and myxozoans form a sister taxon to Bilateria 234 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 234, 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.

<p>Additional file 8</p>

ML topology of relationships based on combined data. This analysis of 126 metazoan taxa was based on combined 18S and partial 28S rDNA with length variable sequences removed.

Click here for file

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.

Authors' contributions

This study was inspired by the work of EVR and originally conceived by EVR, AL and AGC. NME performed most of the data collection and submission of the new sequences. NME and PC performed the analyses. PC took the lead in organizing the study and drafting the manuscript with contributions from NME. All other authors provided comments and suggestions and approved the final manuscript.

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).

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