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Phylogenetic relationships among sea anemones (Cnidaria: Anthozoa: Actiniaria)

M. Daly *, A. Chaudhuri, L. Gusmão, E. RodríguezThe Ohio State University, Department of Evolution, Ecology, and Organismal Biology, 1315 Kinnear Road, Columbus, OH 43212, USA

a r t i c l e i n f o

Article history:Received 27 November 2007Revised 17 February 2008Accepted 28 February 2008Available online 10 March 2008

Keywords:HexacoralliaNynantheaeEvolutionMolecular systematics

a b s t r a c t

Sea anemones (order Actiniaria) are among the most diverse and successful members of the anthozoansubclass Hexacorallia, being found at all depths and latitudes and in all marine habitats. Members of thisgroup exhibit the greatest variation in anatomy, biology, and life history in Hexacorallia, and lack anymorphological synapomorphy. Nonetheless, previous molecular phylogenetic studies have found thatActiniaria is monophyletic with respect to other extant hexacorallians. However, relationships withinActiniaria have remained unresolved, as none of these earlier works have included sufficient taxon sam-pling to estimate relationships within Actiniaria. We have analyzed sequences from two mitochondrialand two nuclear markers for representatives of approximately half of the family-level diversity withinthe order, and present the first phylogenetic tree for Actiniaria. We concur with previous studies thathave suggested that molecular evolution is unusually slow in this group. We determine that taxonomicgroups based on the absence of features tend not to be recovered as monophyletic, but that at least someclassical anatomical features define monophyletic groups.

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1. Introduction

Sea anemones (order Actiniaria) are among the most diverseand successful members of the anthozoan subclass Hexacorallia,being found in all marine habitats and at all depths and latitudes.They play an important role in benthic–pelagic coupling as part ofthe benthic suspension feeding community (Sebens and Paine,1978), transferring energy to the benthos from the water columnand releasing metabolites, gametes, and offspring back into thewater column. An individual polyp may live a relatively long time,with estimations of lifespan exceeding 50 years in nature and 150years in captivity (Shick, 1991). Their ecological success isundoubtedly facilitated by their propensity for engaging in symbi-otic relationships with other animals, including hermit crabs, mol-luscs, and clown fish.

The evolutionary component of this ecological success is poorlyunderstood. In some instances, ecologically important variableslike ability to engage in aggressive intraspecific interactions corre-spond to historical hypotheses of relatedness (e.g., Francis, 1988),but other features, including reproductive mode or symbiotic state,seem not to follow a consistent pattern (e.g., Geller and Walton,2001; Geller et al., 2005). This lack of correspondence between tax-onomy and ecological, morphological, or biological variables is notsurprising, given that relationships among the Actiniaria are some-times based on the absence of features rather than synapomor-

phies, and that phylogenetic relationships have never beenexplicitly or rigorously explored through phylogenetic analysis.

2. Classification and previous hypotheses of relationship

Most sea anemones belong to the suborder Nynantheae Carl-gren, 1899. The other suborders, Endocoelantheae Carlgren, 1925,Protantheae Carlgren, 1891, and Ptychodacteae Stephenson,1921, together comprise fewer than 20 of the approximately1100 described species of Actiniaria. Members of these subordersare not commonly encountered in shallow temperate or tropicalwaters at SCUBA diving depths, and are generally unfamiliar exceptto specialists. Nynantheae is the group to which nearly all seaanemones used in comparative and ecological studies belong,and its members are abundant and diverse at all depths andlatitudes.

All Nynantheae have ciliated tracts on at least some mesenterialfilaments, and have mesenteries that develop as pairs in the spacebetween the paired mesenteries of previous cycles (exocoels).These attributes are also seen in members of Ptychodacteae, whichis a clade within Nynantheae (e.g., Berntson et al., 1999; Cappolaand Fautin, 2000; Daly et al., 2003), despite its taxonomic rank.Multiple molecular phylogenetic analyses have demonstrated themonophyly of Nynantheae (France et al., 1996; Song and Won,1997; Berntson et al., 1999; Won et al., 2001; Daly et al., 2002,2003) with respect to other hexacorallian orders.

The current classification of Actiniaria (e.g., Fautin, 2007) wascodified by Carlgren (1949) in his influential monograph on

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* Corresponding author. Fax: +1 614 292 7774.E-mail address: daly.66@osu.edu (M. Daly).

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actiniarians, corallimorpharians, and ptychodactiarians. WithinNynantheae, Carlgren recognized three ‘‘tribes” (actually infra-or-ders), based on the presence or absence of basilar muscles. AllNynantheae with a defined and adherent base were placed inThenaria (also called Basilaria); all those lacking an adherent basewere placed in Athenaria (=Abasilaria) or Boloceroidaria. Bolocero-idaria and Athenaria differ in the morphology of the aboral end,which is always disc-like in boloceroidarians and typically roundedin athenarians, and in the musculature of the column, with boloce-roidarians having ectodermal longitudinal muscles and athenari-ans (and thenarians) lacking them. Although Carlgren (1949)considered it of lesser importance than the morphology of the base,the distinction between those taxa with ectodermal longitudinalmuscles and those without was deemed of great importance byother authors (e.g., Stephenson, 1922).

Thenaria is the most diverse and speciose group within Nynan-theae. It was divided by Stephenson (1921) into two subgroups,Endomyaria and Mesomyaria, based on the morphology of themarginal sphincter muscle. Thenarians with a mesogleal marginalsphincter were placed in Mesomyaria; those with an endodermalmarginal sphincter were placed in Endomyaria. The marginalsphincter muscle encircles the distal column and allows a polypto constrict its diameter; ‘‘endodermal” and ‘‘mesogleal” refer towhether the sphincter muscle forms a cord that extends into thecoelenteron from the distal column endoderm or comprises fibersand cells embedded in the distal column mesoglea. Stephenson(1935) erected Acontiaria for thenarians with acontia, nemato-cyst-dense, threadlike extensions of the mesenterial filaments.Although Stephenson initially considered Acontiaria to be part ofMesomyaria (e.g., Stephenson, 1921, 1922; see Fig. 1B), when herecognized it formally (Stephenson, 1935), he accorded it equalrank to Mesomyaria, referring to Acontiaria, Endomyaria, andMesomyaria as ‘‘subtribes” (actually superfamilies). This classifica-tion was implemented by Carlgren (1949), although Carlgren didnot accept Stephenson’s (1935) treatment of Athenaria as equalin rank to Acontiaria, Endomyaria, or Mesomyaria, recognizing in-stead the Thenaria–Athenaria split as primary and according thosegroups higher taxonomic rank.

Although Carlgren (1949) described his classification as taxo-nomic rather than phylogenetic, he seems to have accorded histaxonomic groups some phylogenetic status (e.g., Carlgren, 1942).In addition, Carlgren’s (1949) classification has been used to informstudies of phylogeny: Schmidt (1974) used some of Carlgren’s tax-onomic categories as terminal taxa in his phylogenetic analysis ofnematocyst ultrastructure in Hexacorallia. However, Schmidt(1974) recognized only three groups within Nynantheae: Bolocero-idaria, Mesomyaria, and Endomyaria. He treated Acontiaria as aparaphyletic grade within Mesomyaria and considered Athenariapolyphyletic, assigning some members to Mesomyaria and othersto Endomyaria (Schmidt, 1974).

The relationships among Acontiaria, Athenaria, Boloceroidaria,Endomyaria, and Mesomyaria are not clear. Members of eachgroup are recognized by a partially overlapping mosaic of features.For example, in acontiarians, the marginal sphincter muscle is typ-ically mesogleal, being absent in a few groups whose membershave very small polyps with generally reduced musculature. Thus,the morphology of the sphincter seems to unite Mesomyaria andAcontiaria, with the latter characterized by acontia. However, de-spite the name of the group, acontia cannot be construed as a syn-apomorphy for Acontiaria, as these structures are found in a fewAthenaria (e.g., Andvakiidae, Haliactiidae). Similarly, Endomyariaincludes a handful of species without distinct marginal sphincters(e.g., Actinodendronidae), and at least one whose sphincter is par-tially mesogleal (e.g., Isosicyonis, see Riemann-Zürneck, 1980; Fau-tin, 1984; Rodríguez and López-González, 2008). This mosaic

distribution of features among currently recognized groups makesunderstanding the evolution of nynanthean sea anemones difficult.

Molecular phylogenetic studies have demonstrated that Carl-gren’s classification is flawed as a phylogenetic hypothesis, butthe precise nature of the conflict between phylogeny and classifica-tion is not clear. For example, several molecular phylogenetic stud-ies (Berntson et al., 1999; Won et al., 2001; Daly et al., 2002, 2003)have shown that Athenaria is not monophyletic with respect toThenaria: some Athenaria are more closely related to thenarianendomyarians or to acontiarians than they are to one another.However, because these previous studies were concerned withthe relationships among anthozoan orders, and did not samplebroadly within Actiniaria, they fail to provide a robust test of themonophyly of the major subordinal groups within Actiniaria.

The absence of a working phylogenetic framework for Actinia-ria, and for Nynantheae in particular, inhibits study of the evolu-tion and diversification of anemones. Identifying monophyletic,paraphyletic, and polyphyletic groups among the currently recog-nized subordinal taxa is a necessary first step in revising actiniar-ian taxonomy, and will facilitate subsequent, more detailedphylogenetic study of less-inclusive groups. To these ends, we se-quenced more than 3.5 kb from 48 species of Nynantheae. Ouranalyses include two mitochondrial markers (partial 16S and 12SrDNA) and two nuclear markers (18S and partial 28S rDNA) from45 genera in 23 families, including many of the most commonlyencountered and well-studied taxa. We find that both Athenariaand Mesomyaria are polyphyletic. There is some support for themonophyly of Acontiaria and Endomyaria, but the polyphyly ofAthenaria and Mesomyaria make interpreting these groupsdifficult.

3. Materials and methods

3.1. Taxonomic sampling and data collection

We provide new sequences for multiple representatives of eachproposed group within Nynantheae, representing approximatelyhalf of the family-level diversity within Actiniaria (Table 1). Multi-ple species were sampled for large or potentially heterogeneoustaxa such as Actiniidae, Actinostolidae, Edwardsiidae, Hormathii-dae, and Sagartiidae. Specimens were collected by hand intertid-ally, through SCUBA diving, or via trawls. All specimens wereidentified using polyp anatomy and the distribution and size of cni-dae in various regions of the polyp. Voucher specimens in formalinhave been deposited at the American Museum of Natural History(AMNH), the Bavarian State Collection of Zoology (ZSM), the collec-tion of Biodiversidad y Ecología de Invertebrados Marinos (BEIM)at the University of Seville, California Academy of Sciences (CAS),University of Kansas Natural History Museum (KUNHM), and USNational Museum of Natural History (USNM).

We have included only those sequences for which we were ableto amplify at least three of the four markers, and thus have ana-lyzed a total of 184 sequences for 49 taxa. Comparative sequencesfrom GenBank were also included as appropriate (Table 1). No pre-vious analysis has examined all four of these markers for Actiniaria,so there were few comparative sequences available in GenBankthat met our criterion of having three or more markers per species.

Relationships among orders of Hexacorallia are unclear (Dalyet al., 2003; Medina et al., 2006; Brugler and France, 2007), witheither Zoanthidea or a clade comprised of Zoanthidea, Antipatha-ria, Corallimorpharia, and Scleractinia forming the sister group ofActiniaria. Of these orders, Scleractinia and Corallimorpharia arebest represented in GenBank, but they are also the most divergentin terms of sequence similarity to Actiniaria, especially for themitochondrial markers. No member of Zoanthidea is represented

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in GenBank by more than one of our four markers. Thus, we choserepresentatives of order Antipatharia to root our analyses.

Genomic DNA was isolated from tentacle or column tissue usingthe Quiagen DNAeasy� kit, or by standard CTAB extraction. Tem-plate DNA was amplified from genomic samples using publishedprimers (Table 2) and standard techniques (e.g., Daly et al.,2003). Samples which could not be readily amplified using stan-dard protocols were amplified with the high-fidelity enzyme Her-culase� (Stratagene, La Jolla, CA), using manufacturer suppliedprotocols. All PCR products were cleaned using AmPure� magneticbead solution (AgenCourt, Beverly, MA) and rehydrated withdeionized, double-distilled water. Sequencing reactions used a to-tal of 10 lL of cleaned PCR product, at a concentration of 25 ngproduct for every 200 bp of marker length. Cleaned PCR productswere sequenced using amplification primers on an ABI 3730xl bystaff at the sequencing facilities of Genaissance (New Haven, CT

and Cogenics Houston, TX). Forward and reverse sequences wereassembled in Sequencher v4.7 (Gene Codes Corporation, Ann Ar-bor, MI) and blasted against the nucleotide database of GenBankto determine whether the target locus and organism were se-quenced rather than a symbiont or other contaminant. All se-quences have been deposited in GenBank (Table 1).

3.2. Data analysis

Sequences for each marker were aligned in MUSCLE (Edgar,2004) using the default parameters. The Incongruence Length Dif-ference test (ILD: Farris et al., 1994) was used to identify instancesof incongruence within and between the nuclear and mitochon-drial markers. The combined data set is available as a Nexus filethrough Treebase (http://www.treebase.org/treebase/index.html).

Fig. 1. Classification schemes for nynanthean sea anemones. Each of these has been re-interpreted as a phylogenetic tree based on its hierarchical structure. Only Nynantheaeand its subclades have been shown; Protantheae is included because it has been construed by some authors to include Boloceroidaria. (A) Classification of Carlgren (1949). (B)Classification of Stephenson (1920, 1921, 1922). Stephenson’s Stichodactylinae includes members of family Stichodactylidae, considered by Carlgren (1949) and Schmidt(1974) to belong within Endomyaria, and also members of order Corallimorpharia. (C) Classification of Schmidt (1974).

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The resulting alignments were analyzed separately and in com-bination using parsimony as implemented in TNT v1.1 (Goloboffet al., 2000), using random and consensus sectorial searches, treedrifting, and 10 rounds of tree fusing. In all analyses, gaps weretreated as ambiguous (?) rather than as a fifth state. Trees of min-imum length were found at least three times. The combined data

were subjected to 1000 rounds of jackknife resampling (36% prob-ability of removal, collapse clades with <50% support) to assessclade support.

ModelTest 3.7 was used to identify an appropriate model forboth the nuclear and mitochondrial subsets (Posada and Crandall,1998; Posada and Buckley, 2004); the model was chosen basedon the Akaike information criterion. Because the same model(GTR + gamma) was deemed appropriate for both data sets and be-cause the ILD tests detected no significant incongruence betweenmarkers, a combined matrix of all four markers was analyzed inRAxML-vi-HPC 2.2.3 (Stamatakis, 2006) using 10 replicate searchesof 100 runs, implementing GTR with the gamma model of rate het-erogeneity as the model of nucleotide substitution (per Model-Test); model parameters were estimated by RAxML. Thecombined data were subjected to 1000 rounds of bootstrap resam-pling in RAxML to assess support for clades.

Table 1Taxa included in this study, with voucher location and accession numbers

Higher taxon Family Genus Species Voucher 12S 16S 18S 28S

Athenaria Edwardsiidae Edwardsia elegans AMNH EU190726 EU190770 AF254376 AY34570Edwardsianthus gilbertensis AMNH EU190728 EU190772 EU190859 EU190817Nematostella vectensis AMNH EU190750 AY169370 AF254382 EU190838

Halcampidae Halcampa duodecimcirrata AMNH –– EU190776 AF254375 EU190820Halcampoididae Halcampoides purpurea AMNH EU190735 EU190780 AF254380 EU190824Haloclavidae Haloclava producta AMNH EU190734 EU190779 AF254370 EU190823

Peachia cylindrica ZSM EU190743 EU190789 –– EU190832Andvakiidae Andvakia boninensis KUNHM EU190717 EU190759 EU190848 EU190805

Acontiaria Aiptasiidae Aiptasia pulchella KUNHM EU190715 EU190757 EU190846 EU190803Paraiptasia Sp. KUNHM EU190742 EU190788 EU190869 EU190831Bartholomea annulata KUNHM EU190721 EU190763 EU190851 EU190809

Diadumenidae Diadumene cincta KUNHM EU190725 EU190769 EU190856 EU190814Haliplanellidae Haliplanella lineata KUNHM EU190730 EU190774 EU190860 EU190819Hormathiidae Actinauge richardi KUNHM EU190719 EU190761 EU190850 EU190807

Calliactis parasitica KUNHM EU190711 EU190752 EU190842 EU190799Hormathia armata BEIM EU190731 EU190775 EU190861 ––

Kadosactidae Kadosactis antarctica BEIM –– EU190782 EU190865 EU190825Metridiidae Metridium senile KUNHM EU190740 EU190786 AF052889 EU190829Nemathidae Nemanthus nitidus KUNHM EU190741 EU190787 EU190868 EU190830Sagartiidae Cereus pedunculatus KUNHM EU190724 EU190767 EU190855 EU190813

Phellia gausapata ZSM EU190744 EU190790 EU190870 EU190833Sagartia troglodytes KUNHM EU190746 EU190792 EU190872 EU190834Sagartiogeton laceratus KUNHM EU190748 EU190794 EU190874 EU190836

Boloceroidaria Boloceroididae Boloceroides mcmurrichi KUNHM –– EU190764 EU190852 EU190810Endomyaria Actiniidae Actinia fragacea KUNHM EU190714 EU190756 EU190845 EU190802

Anemonia viridis KUNHM EU190718 EU190760 EU190849 EU190806Anthopleura elegantissima KUNHM EU190713 EU190755 EU190844 EU190801Anthopleura krebsi KUNHM EU190716 EU190758 EU190847 EU190804Anthopleura kurogane KUNHM EU190737 EU190783 Z21671 EU190826Bunodactis verrucosa KUNHM EU190723 EU190766 EU190854 EU190812Bunodosoma grandis KUNHM EU190722 EU190765 EU190853 EU190811Epiactis lisbethae KUNHM EU190727 EU190771 EU190858 EU190816Macrodactyla doreenensis KUNHM EU190739 EU190785 EU190867 EU190828Isosicyonis striata BEIM EU190736 EU190781 EU190864 ––Urticina coriacea KUNHM –– EU190797 EU190877 EU190840

Actinodendridae Actinostephanus haeckeli KUNHM EU190720 EU190762 –– EU190808Aliciidae Triactis producta KUNHM EU490525 EU190796 EU190876 EU190839Liponematidae Lipomena brevicornis USNM EU190738 EU190784 EU190866 EU190827Phymanthidae Phymanthus loligo KUNHM EU190745 EU190791 EU190871 ––Stichodactylidae Heteractis aurora KUNHM EU190729 EU190773 –– EU190818

Heteractis magnifica KUNHM EU190732 EU190777 EU190862 EU190821Stichodactyla gigantea KUNHM EU190747 EU190793 EU190873 EU190835

Mesomyaria Actinoscyphiidae Actinoscyphia plebeia BEIM EU190712 EU190754 –– EU190800Actinostolidae Actinostola crassicornis USNM –– EU190753 EU190843 EU272904

Anthosactis pearseae CAS EU190751 EU190798 EU190878 EU190841Hormosoma scotti BEIM EU190733 EU190778 EU190863 EU190822Stomphia diademon KUNHM EU190749 EU190795 EU190875 EU190837

Ptychodacteae Preactiidae Dactylanthus antarcticus KUNHM –– AY345877 AF052896 AY345873Antipatharia Antipathidae Antipathes galapagensis N/A –– –– AF100943 AY026365

Cladopathidae Chrysopathes formosa N/A DQ304771 DQ304771 –– ––

Generic names in bold indicate type genera; binomens in bold indicate type species of type genera. Markers not sequenced for a particular taxon are indicated by a dashedline. New sequences generated for this study indicated in bold. See ‘Section 3’ for full voucher location information. Note: antipatharians are used as the outgroup.

Table 2Genetic markers used in this study, with sources for primer sequences, fragmentlength (as a range), and aligned length of the sequences

Marker Primer source Unaligned length (bp) Aligned length (bp)

12S Chen et al. (2002) 764–821 127516S Geller and Walton (2001) 416–473 92518S Apakupakul et al. (1999) 1712–1778 242528S Chen and Yu (2000) 930–960 1332

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4. Results

4.1. Markers and congruence

The markers ranged in length from 416 to 1778 bases, andcontained 13–29% parsimony informative sites after alignment(Table 3). The longest marker (18S) was the least variable and re-quired relatively fewer gaps to align. In general, the nuclear mark-ers were less variable than the mitochondrial markers. Themitochondrial markers were more variable both in observed se-quence (base differences) and in length, requiring more gaps toalign sequences across Nynantheae. As might be expected giventheir co-linearity, there is no significant (p = 0.05) incongruencebetween 18S and 28S, nor is there between 12S and 16S. Further-more, the ILD test detected no significant incongruence betweenthe nuclear and mitochondrial markers, nor between any pair wisecombination of markers.

4.2. Parsimony analyses

All five primary trees derived from parsimony analysis of thecombined data set agreed on the same basic topology: a polyphy-letic Athenaria, with the three members of the athenarian familyEdwardsiidae basal to a clade (hereafter the ‘‘primary clade”) con-sisting of Boloceroidaria, Thenaria, and the remaining Athenaria(Fig. 2). The primary clade has two subclades: one correspondsroughly to Endomyaria; the other includes all members of Acontia-ria, Boloceroidaria, and Mesomyaria. Athenaria is polyphyletic: theEdwardsiidae are at the base of the tree; Peachia and Haloclava(family Haloclavidae) nest within Endomyaria; Halcampa, Hal-campoides, and Andvakia (families Halcampidae, Halcampoididae,and Andvakiidae, respectively) cluster among Acontiaria.Mesomyaria is polyphyletic, with at least three lineages: mostmembers of family Actinostolidae are at the base of the acontiateclade; the actinostolid Anthosactis is basal to one of the two acon-tiate clades; and the actinoscyphiid Actinoscyphia is nested withinthe acontiate clade (Fig. 2).

Basilar muscles are inferred to have evolved at the base of theprimary clade and to have been lost in several lineages (Fig. 2).All members of the Endomyaria clade have endodermal marginalsphincters, except Actinostephanus, Dactylanthus, Haloclava, andPeachia, which lack marginal musculature. No species with endo-dermal marginal sphincters belong to any of the other clades. Sim-ilarly, the taxa with mesogleal marginal sphincters all belong to theAcontiaria–Boloceroidaria–Mesomyaria clade, although manymembers of this clade (e.g., Boloceroides, Halcampa, Halcampoides,Triactis) lack marginal musculature. Acontia are inferred to havearisen once, and to have been lost.

The data sets differ in the clades for which they provide strongsupport. The 12S sequences consistently support (jackknife val-ues > 50%) several key nodes, including nynanthean monophyly(100%), the basal position of Edwardsiidae relative to othernynantheans (100%), the primary clade (80%), the Endomyaria

clade (69%), and the acontiate clade (80%), in addition to severalinternal relationships in each of the larger clades. In comparison,the 16S data provide strong support for relatively few groups,recovering only nynanthean monophyly (80%) and a sister grouprelationship between several pairs of taxa with jackknife frequencyexceeding 50%. The nodes receiving high support in analysis of only18S or 28S sequences are the same ones well supported by 16S se-quences, although support tends to be higher in the nuclear datasets: for example, nynanthean monophyly has jackknife supportin excess of 95% in both the 18S and 28S analyses but support of80% in the 16S analysis. Neither the 18S nor 28S data sets aloneprovide substantial support for relationships between families.

4.3. Model-based analyses

In the tree of highest likelihood, Nynantheae comprises twoclades (Fig. 3). One of these is identical in composition to the end-omyarian clade of the parsimony analyses. The other clade con-tains all members of Acontiaria, Boloceroidaria, and Mesomyaria,but also includes the Edwardsiidae. Although most of its memberslie within the Acontiaria–Boloceroidaria–Mesomyaria clade, Athe-naria is polyphyletic, comprising at least three distantly relatedclades: Haloclava and Peachia nest within Endomyaria; Edwardsii-dae is sister to one of the mesomyarian lineages; and Halcampa andHalcampoides are basal members of a larger clade that also includesAndvakia. Similarly, Mesomyaria is polyphyletic. All Actinostolidaeexcept Anthosactis group with Edwardsiidae, as the sister to theclade containing the acontiate species, some athenarians, and theremaining mesomyarians. Anthosactis is the most basal memberof this latter clade, and the mesomyarian Actinoscyphia nests with-in it. Boloceroides nests within the acontiate clade, near the base ofone of the two main lineages of acontiate taxa.

Branch lengths are not distributed evenly across the tree, orwithin clades. The internal branches tend to be shorter in the Acon-tiaria–Boloceroidaria–Mesomyaria clade than in the Endomyariaclade. However, branch lengths overall show more heterogeneityin the Acontiaria–Boloceroidaria–Mesomyaria clade: this clade in-cludes most of the very long branches and very short branches. Thelongest-branched ingroup taxa (Edwardsianthus and Hormosoma,see Fig. 3) are each the sister of a much shorter-branched taxon.

5. Discussion

5.1. Signal, agreement, and incongruence

The basic topologies of the combined parsimony and likelihoodanalyses are similar. Nynantheae comprises two major clades. Oneof these corresponds to Endomyaria, with the addition of the athe-narians Peachia and Haloclava and the ptychodactean Dactylanthus.The other clade includes members of Acontiaria, Boloceroidariaand Mesomyaria, plus several lineages of Athenaria. The resultsof the parsimony and likelihood analyses disagree about whethermembers of the athenarian family Edwardsiidae belong withinthe Acontiaria–Boloceroidaria–Mesomyaria clade or as the sisterto it plus the Endomyaria clade. Because they differ in the place-ment of Edwardsiidae, the trees resulting from parsimony and like-lihood analyses differ in their interpretation of the evolution ofbasilar muscles: in the parsimony analysis, these muscles evolveat the base of the primary clade (Fig. 2), in the likelihood analysis,they evolve at the base of Nynantheae (Fig. 3).

The 12S fragment was most effective at recovering groups, sup-porting more groups at all levels of the tree than any of the othermarkers. Although this may not be surprising given that it is themost variable marker (Table 3), the 12S fragment is only slightlymore variable than the 28S fragment, and, despite this slightly

Table 3Results of parsimony analysis of each data set

Marker ordata set

# Parsimony informativecharacters (% informative)

# Equallyparsimonious trees

Length

12 372 (29%) 17 117416 195 (21%) 9 92118 305 (13%) 2 210728 373 (28%) 4 3315Mitochondrial 567 (26%) 9 2177Nuclear 678 (18%) 5 5547Combined 1245 (21%) 5 7968

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higher percent variation, it actually contains fewer variable posi-tions because its raw sequence is approximately 100 bases shorterthan the 28S fragment. Despite this promising degree of variation,the 12S fragment is problematic: the relationships seen in the anal-ysis of this fragment alone were most divergent from those seen incombined analysis, and there was significant incongruence(p = 0.6) between 12S and a combined data set containing the otherthree markers, although no significant incongruence was detectedbetween 12S and any individual marker. Based on comparison oftopologies, most of this incongruence is in relatively terminal rela-tionships. Thus, despite greater variability and more support forcritical nodes, 12S also shows more conflict with the patterns ofrelationship recovered by combined analysis. This incongruencebetween 12S and other markers is not seen in scleractinian corals(e.g., Chen et al., 2002), for which the patterns of relationshiprecovered by 12S closely mirror those obtained though analysisof 16S, 28S, and 18S, both alone and in combination (e.g., Romanoand Palumbi, 1996; Romano and Cairns, 2000; Daly et al., 2003).

Although many important intra-ordinal relationships were notwell-supported by any single analysis, these nodes are not dis-puted, and are weakly supported by each separate analysis. Allmarkers contribute character support to the nodes of primaryinterest. The one major node not universally supported is theplacement of Edwardsia, Edwardsianthus, and Nematostella at thebase of the combined parsimony tree (Fig. 2). The separate parsi-mony analyses of 16S, 18S, and 28S each reconstruct Edwardsiidaeas a clade rather than a grade, and place it either within the Acon-tiaria–Boloceroidaria–Mesomyaria clade or unresolved at the splitbetween this clade and Endomyaria, placements consistent withthe results of the likelihood analysis (Fig. 3). This conflict is likelyan artifact related to the extremely divergent 12S sequence ofEdwardsianthus, and the resulting ‘‘long branch” of this taxon, evi-dent in the scaled branch lengths of Fig. 3. Because Edwardsiidaecontains an extremely long branch, the placement of its taxa maybe suspect (Anderson and Swofford, 2004; Bergsten, 2005): theparsimony analysis may be grouping Edwardsianthus with the long

Fig. 2. Strict consensus tree of five primary trees resulting from combined parsimony analysis of 12S, 16S, 18S, and 28S data sets. Species epithets are given only for generarepresented by more than one species; for complete list of taxa, see Table 1. Numbers above the branches are jackknife resampling values expressed as a percent;numbers < 50 are not indicated. Taxa in bold lack basilar muscles and thus are those traditionally considered among Athenaria. The acontiarian clade is indicated by thedashed box; lineages marked with a solid circle are inferred to have lost acontia. The most parsimonious interpretation of the evolution of basilar muscles is indicated; thesemuscles are inferred to have been lost in Boloceroidarians and Athenarians.

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outgroup branch and splitting it from its presumed close relativesEdwardsia and Nematostella (e.g., Daly, 2002).

The tree resulting from likelihood analysis of combined data hasmore nodes with support values greater than 50% than does theparsimony analysis of the combined data set. Nodes well sup-ported in the likelihood analysis but not in the parsimony analysisinclude Endomyaria, several basal nodes within the Acontiaria–

Boloceroidaria–Mesomyaria clade, the Edwardsiidae clade, Hormo-soma plus the Stomphia–Actinostola clade, in addition to severalterminal relationships within Endomyaria and Acontiaria–Boloce-roidaria–Mesomyaria. Many of these relationships are recoveredin the parsimony analysis, but are only weakly supported. Thesesupport values are calculated using different procedures: the jack-knife resampling procedure used for the parsimony results may be

Fig. 3. Maximum likelihood tree resulting from combined analysis of 12S, 16S, 18S, and 28S data sets under the model GTR + gamma. Relative branch lengths are indicated.Species epithets are given only for genera represented by more than one species; for complete list of taxa, see Table 1. Numbers above the branches are bootstrap resamplingvalues expressed as a percent; numbers < 50 are not indicated. Taxa in bold lack basilar muscles and thus are those traditionally considered among Athenaria. The acontiarianclade is indicated by the dashed box; lineages marked with a solid circle are inferred to have lost acontia. The most parsimonious interpretation of the evolution of basilarmuscles is indicated; these muscles are inferred to have been lost in Boloceroidarians and Athenarians.

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expected to have slightly lower support values than the bootstrapresampling of the likelihood results because it samples withoutreplacement. Nonetheless, although a bootstrap analysis of theparsimony results provides slightly higher values at each of thewell-supported nodes (data not shown), this alone cannot accountfor the differences in support for many of the nodes.

5.2. Relationships among Nynantheae

Because there is general agreement between the individual datasets and between the parsimony and likelihood analyses of thecombined data sets, these results provide a broad framework foraddressing relationships between the major groups of Nynantheae.No analysis supports monophyly of any of the proposed groupswithin Nynantheae; the monophyly of Boloceroidaria, which wasrepresented by a single taxon, remains untested. In all treatments,Athenaria is polyphyletic, with some members at the base of thetree, and others nested within each of the two subsets of the mainnynanthean clade. This interpretation has been suggested by manyauthors when they hypothesized that the absence of basilar mus-cles of athenarians must be an adaptation to a burrowing habit(Hand, 1966; Schmidt, 1974; Daly et al., 2002). The relationshipsindicated for the various athenarian groups are largely compatiblewith morphology. For example, haloclavids have either no mar-ginal sphincter or an endodermal one, and Haloclava has columnarvesicles similar to those seen in Bunodosoma and other endomyar-ian taxa. Moreover, Schmidt (1974) proposed that haloclavids wereclosely related to endomyarians based on their cnidom. Andvakiagroups with the acontiate thenarians, a placement that is plausiblegiven that it also bears acontia. The relationship between Halcam-pa, Halcampoides, and the Acontiaria–Boloceroidaria–Mesomyariaclade is less readily interpretable. The sphincter is absent in Hal-campoides and mesogleal in Halcampa, supporting their inclusionin this larger clade. However, neither has any trace of acontia,and thus their placement within an acontiate branch of this cladeis surprising.

The phylogenetic position of the athenarian family Edwardsii-dae has long been the subject of debate (see Daly et al., 2002),and it remains unclear. These taxa are placed inconsistently, withparsimony analysis recovering a paraphyletic Edwardsiidae asthe sister to other nynantheans, and likelihood analysis recoveringa monophyletic Edwardsiidae as the sister to one of the mesomy-arian lineages of the Acontiaria–Boloceroidaria–Mesomyaria clade.Previous morphological and molecular analyses using parsimonyhave supported monophyly of Edwardsiidae (Daly, 2002; Dalyet al., 2002), but these have included relatively more members ofthe family than the current study. Denser taxon sampling may helpclarify the affinities of this group. The polyphyly of the family andits placement at the base of the tree may be an artifact of the rel-atively divergent sequence for 12S in Edwardsianthus.

In all analyses, Boloceroidaria groups with Acontiaria. In thecombined parsimony analysis, Boloceroides is the sister to Triactis,and these together are the sister group of the Acontiaria; in thelikelihood analysis, the Boloceroides–Triactis clade nests withinAcontiaria. Aliciidae, the family to which Triactis belongs, has beentraditionally considered among the Endomyaria (Carlgren, 1949;England, 1987; Fautin, 2007), although its members are generallyconsidered to lack marginal sphincter muscles (e.g., Carlgren,1949). England (1987) identified a weak sphincter in one specimenof Triactis, but failed to illustrate the structure or indicate whetherit is mesogleal or endodermal. Schmidt (1972, 1974) proposed aclose relationship between Boloceroidaria and Aliciidae, notingsimilarities in the cnidom and in the ectodermal longitudinal mus-cles (restricted to the distal column in Aliciidae). Our results sup-port his hypothesis: the aliciid Triactis does not group with theother endomyarians, clustering instead with Boloceroides.

All acontiate taxa included in this analysis group in the samelarger clade, but are not members of an exclusive clade withinthe Acontiaria–Boloceroidaria–Mesomyaria clade. Instead, theacontiate taxa form two clades, one of which includes severalnon-acontiate taxa (see lineages marked by solid circles in Figs. 2and 3). Although the precise relationships between the acontiateand non-acontiate taxa differ slightly between the parsimonyand likelihood analyses, the basic pattern is robust: the clade thatincludes the acontiarians Actinauge, Andvakia, Calliactis, Hormathia,Kadosactis, Nemanthus, and Phellia also includes the non-acontiategenera Actinoscyphia, Halcampa, and Halcampoides. Families inAcontiaria have been differentiated based on the types of nemat-ocysts in their acontia (Carlgren 1949). We find that the taxa withboth basitrichs and microbasic p-mastigophores (sometimes calledmicrobasic amastigophores; see Östman, 2000) in their acontiagenerally cluster. For example, one of the two acontiate clades in-cludes members of Aiptasiidae, Diadumenidae, Haliplanellidae,Metridiidae, and most Sagartiidae; these families are characterizedas having both basitrichs and microbasic p-mastigophores in theacontia. However, Phellia, Andvakia, and Kadosactis, all of whichhave basitrichs and microbasic p-mastigophores, are consistentlyplaced with Nemanthus, Actinauge, Calliactis, and Hormathia, generain which the acontia contain only basitrichs. We find no support formonophyly of Sagartiidae: Phellia clusters with Andvakia and Nem-anthus in the larger clade that contains members of Hormathiidaeand the non-acontiate taxa. All interpretations and analyses sup-port monophyly of Aiptasiidae and Hormathiidae, two of the threeacontiate families represented by multiple species.

A close relationship between some members of Acontiaria andthe mesomyarians Actinoscyphia and Anthosactis is seen in the like-lihood tree and in the results from all parsimony analyses. Thisrelationship, in addition to the inclusion of acontiate and non-acontiate Athenaria, renders Acontiaria polyphyletic. The logicalinterpretation of this result, that acontia have been lost (or haveevolved) multiple times, is not a novel proposal. Schmidt (1972,1974) did not recognize acontiarian anemones as a group, insteadgrouping them with non-acontiate species in either ‘‘earlyMesomyaria” or ‘‘late Mesomyaria.” According to Schmidt (1972,1974), ‘‘early” and ‘‘late” mesomyarians differ in that ‘‘late”mesomyarians have a kind of nematocyst (which he termed p-rhabdoids A) absent in the ‘‘early” lineage. Despite our finding ofpolyphyly of Acontiaria, our results fail to support the distinctionbetween ‘‘early” and ‘‘late” mesomyarians. Riemann-Zürneck(1978) proposed that Actinoscyphiidae was more closely relatedto members of Acontiaria than to other Mesomyaria, and inferredthat members of this group had lost acontia. Our results bolsterher interpretation of the phylogenetic affinities of Actinoscyphii-dae, as we consistently recover Actinoscyphia among the Acontia-ria. Other possible instances of loss of acontia include the lineageleading to Halcampa and Halcampoides, and in the lineage ofAnthosactis.

Mesomyaria can be divided into at least two lineages: a cladethat is either basal to the endomyarian and acontiate clades or ba-sal to only the acontiarians and a clade nested within the acontia-rians. The former clade, which includes Actinostola, Hormosoma,and Stomphia, had been recognized as a group to the exclusion ofAnthosactis by earlier workers (e.g., Carlgren, 1899; Stephenson,1921; Fautin and Hessler, 1989). Morphological evidence also sup-ports a clade comprised of Actinostola, Hormosoma, and Stomphia(Rodríguez et al., in review). Like most mesomyarian taxa, thesegenera belong to Actinostolidae, a family defined by absence ofcharacters rather than by synapomorphies (Carlgren, 1949) andwhich is likely to comprise a paraphyletic grade or a polyphyleticassemblage rather than a monophyletic group. Sanamyan and San-amyan (2007) proposed that some actinostolids from chemosyn-thetic habitats were closely related to acontiarians; this proposal

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might explain the placement of the actinostolid Anthosactis pear-seae (endemic to whale falls; see Daly and Gusmão, 2007) withacontiarians. However, further studies are necessary to test theutility and generality of the characters used by Sanamyan and San-amyan (2007).

As is the case with Acontiaria, most of the taxa historically con-sidered part of Endomyaria group together, but this clade also in-cludes members of Athenaria (Haloclava, Peachia) andPtychodacteae (Dactylanthus). The monophyly of this larger End-omyaria clade is found in all analyses, even though it is notstrongly supported by either jackknife or bootstrap resampling.Schmidt (1974) proposed that Haloclavidae belonged within End-omyaria because of its relatively sparse complement of nemat-ocysts; we find that Haloclava and Peachia nest withinEndomyaria. Haloclavidae is not monophyletic. Haloclava groupswith Bunodosoma to the exclusion of Peachia in all analyses; thisrelationship has bootstrap or jackknife support of 100%. Cappolaand Fautin (2000) indicated that the ptyochodactiarian Dactylan-thus was closely related to members of the endomyarian familyActiniidae, but maintained that the ptyochodactiarians belongedin their own suborder, for which they resurrected the name Pty-chodacteae (Stephenson, 1922). We concur with their interpreta-tion of the phylogenetic affinity of Dactylanthus: it is consistentlyrecovered as part of the clade that includes Actinia, the type genusof Actiniidae. Because of the derived position of Dactylanthus with-in a clade of nynanthean anemones, we interpret its family, Preac-tiidae, as a family of Nynantheae rather than part of a separatesuborder.

The polyphyly of Actiniidae, one of the largest families in Actin-iaria, is not unexpected based on previous molecular phylogenies(e.g., Won et al., 2001; Daly et al., 2003). This group has been prob-lematic for morphologists, and, like the mesomyarian family Actin-ostolidae, is defined by the absence of features rather thansynapomorphies. We find Actiniidae to be broadly paraphyletic,with members distributed throughout the endomyarian clade. Nodata set or analysis recovers monophyly of Actiniidae. Further-more, we concur with previous findings based on molecules (Gellerand Walton, 2001) and morphology (Daly, 2004) that the actiniidgenus Anthopleura is polyphyletic, with some members closely al-lied to members of other actiniid genera, and others associatedwith representatives of other families (e.g., Isosicyonis, Dactylan-thus, Heteractis, Phymanthus). The definition and composition ofactiniid genera such as Anthopleura, Bunodosoma, and Bunodactishas changed over time as more anatomical knowledge has accu-mulated (e.g., Daly and den Hartog, 2004) and the definitions ofcharacters used to differentiate actiniid genera has been very con-fused (e.g., England, 1987; Daly, 2003). The higher taxonomic posi-tion of the actiniid genus Isosicyonis has been controversial becausethe marginal sphincter has both mesogleal and endodermal com-ponents (Riemann-Zürneck, 1980; Fautin, 1984). However, mor-phological investigation of a recently described species suggeststhat Isosicyonis has a primarily endodermal sphincter and thus be-longs among endomyarians (Rodríguez and López-González, 2008),a placement consistent with our phylogenetic results.

5.3. Summary and conclusions

The relationships we recover among the main lineages ofNynantheae are not compatible with the taxonomic hierarchy orwith some previous concepts of relationship. Carlgren’s (1949) dis-tinction between Athenaria, Boloceroidaria, and Thenaria (e.g.,Fig. 1A) is not supported: Boloceroidaria and Athenaria are subsetsof Thenaria. The groups within Thenaria fare somewhat better:most taxa with a mesogleal sphincter cluster together, as do mostforms with an endodermal sphincter. The clade of forms with amesogleal sphincter also includes the acontiate taxa. However,

each of these clades includes some members lacking marginalsphincter muscles and some members of Athenaria, and the cladeof acontiate taxa includes a few non-acontiate forms. Based onthese results, we recommend that ranks and taxonomic categoriesabove family be abandoned for Nynantheae pending further phylo-genetic investigations. These categories are difficult to implementbecause of the conflicting distribution of characters, and have nophylogenetic significance. Furthermore, for many of the larger fam-ilies (e.g., Actiniidae, Actinostolidae, Edwardsiidae), neither mono-phyly nor phylogenetic position are well resolved.

These results bear on previous interpretations of character evo-lution in Actiniaria. Carlgren (1942) hypothesized that the ancestorof Actiniaria had no marginal sphincter, and postulated that a mes-ogleal sphincter had arisen several times directly from undifferen-tiated endodermal circular muscles or differentiated endodermalsphincter. He also interpreted endodermal sphincters as conver-gent among Actiniaria, in part because they occur in some otherhexacoral lineages (Carlgren, 1942). Thus, he allowed for the possi-bility that actiniarians with mesogleal marginal sphincter musclesand those with endodermal sphincter muscles might be close rel-atives, despite their anatomical dissimilarity. However, Carlgren’s(1942) explanation was strongly influenced by his interpretationof basilar muscles as of primary importance; he considered thepresence of basilar muscles more important than the morphology(or presence) of a marginal sphincter. In contrast, Schmidt (1974)considered basilar muscles relatively unimportant, and interpretedthe endodermal sphincter muscle as being derived from an ances-tral mesogleal sphincter muscle (e.g., Fig. 1C). Based on our phylo-genetic analyses, we interpret basilar muscles as a relatively labilefeature among Actiniaria. Basilar muscles appear to have been lostmultiple times within both the endomyarian and Acontiaria–Bolo-ceroidaria–Mesomyaria clades. The position of Edwardsiidae bearsdirectly on the question of basilar muscles; if the parsimony resultis correct and Edwardsiidae is basal to other Nynantheae, then bas-ilar muscles are a synapomorphy for the crown clade of Nynan-theae; if the likelihood tree is correct and Edwardsiidae nestswithin the Acontiaria–Boloceroidaria–Mesomyaria clade, then bas-ilar muscles are primitive for Nynantheae.

Our results consistently depict a dichotomy between taxa withan endodermal (or no) marginal sphincter and those with a mesog-leal (or no) marginal sphincter. Thus, we have no basis for inferringthe ancestral type of marginal sphincter, and cannot determinehomology of the endodermal and mesogleal sphincter muscles inNynantheae. Therefore, we cannot refute Carlgren’s (1942) inter-pretation of the origin of the sphincter. However, our results areincompatible with his hypothesis of repeated switches betweenendodermal and mesogleal sphincters: we find two clades, eachof which is characterized by a different kind of sphincter muscle.These results are also incompatible with Schmidt (1974) hypothe-sis that forms with an endodermal sphincter are most derived. Fullresolution of these issues can only be achieved upon inclusion ofmembers of the actiniarian suborders Endocoelantheae and Pro-tantheae, both of which are characterized by the absence of bothmarginal sphincter and basilar musculature.

Acknowledgments

This study would not have been possible without specimens do-nated by or collected with the assistance of Adorian Ardelean, HaRim Cha, Michel Claereboudt, Allen Collins, Daphne Fautin, RogerGoodwill, Ken Halanych, Verena Häussermann, Stephane Hourdez,Annie Lindgren, Pablo López-González, Alvaro Migotto, AntonioMarques, Bernard Picton, Janet Voight, and Kensuke Yanagi. Muchof the material was collected during field work in association withSeto Marine Lab of Kyoto University, Japan; the Chiba Museum ofNatural History, Japan; and Sultan Qaboos University, Oman; the

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support and hospitality of these institutions and their staff aregratefully acknowledged. Conversations with Paulyn Cartwrightand Daphne Fautin helped focus our attention on issues of taxonsampling, gene choice, and analysis. Annie Lindgren and EspritHeestand provided help in the lab; Dan Janies and John Freuden-stein provided help with analysis. This work was supported byNSF DEB 9978106 (to D. G. Fautin), NSF DEB 0415277, EF-0531763, and an AAAS WISC grant to M. Daly.

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