Hidden among Sea Anemones: The First Comprehensive Phylogenetic Reconstruction of the Order Actiniaria (Cnidaria, Anthozoa, Hexacorallia) Reveals a Novel Group of Hexacorals

<div><p>Sea anemones (order Actiniaria) are among the most diverse and successful members of the anthozoan subclass Hexacorallia, occupying benthic marine habitats across all depths and latitudes. Actiniaria comprises approximately 1,200 species of solitary and skeleton-less polyps and lacks any anatomical synapomorphy. Although monophyly is anticipated based on higher-level molecular phylogenies of Cnidaria, to date, monophyly has not been explicitly tested and at least some hypotheses on the diversification of Hexacorallia have suggested that actiniarians are para- or poly-phyletic. Published phylogenies have demonstrated the inadequacy of existing morphological-based classifications within Actiniaria. Superfamilial groups and most families and genera that have been rigorously studied are not monophyletic, indicating conflict with the current hierarchical classification. We test the monophyly of Actiniaria using two nuclear and three mitochondrial genes with multiple analytical methods. These analyses are the first to include representatives of all three currently-recognized suborders within Actiniaria. We do not recover Actiniaria as a monophyletic clade: the deep-sea anemone <i>Boloceroides daphneae,</i> previously included within the infraorder Boloceroidaria, is resolved outside of Actiniaria in several of the analyses. We erect a new genus and family for <i>B. daphneae</i>, and rank this taxon <i>incerti ordinis</i>. Based on our comprehensive phylogeny, we propose a new formal higher-level classification for Actiniaria composed of only two suborders, Anenthemonae and Enthemonae. Suborder Anenthemonae includes actiniarians with a unique arrangement of mesenteries (members of Edwardsiidae and former suborder Endocoelantheae). Suborder Enthemonae includes actiniarians with the typical arrangement of mesenteries for actiniarians (members of former suborders Protantheae, Ptychodacteae, and Nynantheae and subgroups therein). We also erect subgroups within these two newly-erected suborders. Although some relationships among these newly-defined groups are still ambiguous, morphological and molecular results are consistent enough to proceed with a new higher-level classification and to discuss the putative functional and evolutionary significance of several morphological attributes within Actiniaria.</p></div

Ancestral character state reconstruction of morphological characters within Hexacorallia.

<p>Representation of ancestral character state reconstruction for seven morphological characters within Hexacorallia (acontia, basilar muscles, longitudinal ectodermal muscles, deciduous tentacles, marginal sphincter muscle, microorganism endosymbiosis, apical flaps). Characters mapped onto the Maximum Likelihood (ML) analysis of concatenated 12S, 16S, 18S, 28S and <i>cox</i>3. Characters absent unless specified in the figure. Directionality of acontia not inferred due to lack of support of nodes. Colored boxes indicate clades defined in the text; name of each clade is next to the colored box. Species epithets are given only for genera represented by more than one species; for a complete list of taxa, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096998#pone.0096998.s001" target="_blank">Table S1</a>. Only bootstrap resampling values (parsimony [TNT] and ML, respectively) >90% and values of posterior probabilities of 0.9 are indicated (by filled-in circles) for legibility; refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096998#pone-0096998-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096998#pone-0096998-g002" target="_blank">2</a> for other support values.</p

Phylogenetic reconstruction of Actiniaria.

<p>Tree resulting from the Maximum Likelihood (ML) analysis of concatenated 12S, 16S, 18S, 28S and <i>cox</i>3. Colored boxes indicate clades defined in the text; the name of each clade is next to the colored box. Species epithets are given only for genera represented by more than one species; for a complete list of taxa, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096998#pone.0096998.s001" target="_blank">Table S1</a>. Numbers above the branches are bootstrap resampling values (parsimony [TNT] and ML, respectively) expressed as a percent followed by posterior probabilities (multiplied by100 for legibility); values <50 indicated by “–”; filled-in circles indicate nodes with support of 100% for all inferences.</p

Relationships and interpretation of higher-level taxa in Carlgren’s [12] classification.

<p>Carlgren <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096998#pone.0096998-Carlgren1" target="_blank">[12]</a> considered Ptychodacteae an order; Cappola and Fautin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096998#pone.0096998-Cappola1" target="_blank">[15]</a> re-classified this as a suborder. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096998#pone.0096998-Rodrguez2" target="_blank">[11]</a> for extensive discussion of the composition and classification of Mesomyaria and Acontiaria.</p

Cnidae capsules and apical flaps.

<p>A) SEM image of a discharged capsule of a basitrich from the tentacle of <i>Relicanthus daphneae</i> comb. nov. showing apical flaps. B) Detail of the apical flaps (arrows) of <i>R. daphneae</i> comb. nov. C) SEM image of a discharged capsule of a microbasic <i>p</i>-mastigophore from a member of Scleractinia; notice the absence of apical flaps. D) SEM image of a discharged capsule of a microbasic <i>p</i>-amastigophore from a member of Actiniaria (<i>Metridium</i>). E) SEM image of a discharged capsule of a microbasic <i>p</i>-mastigophore from a member of Actiniaria (<i>Bunodosoma</i>); notice differential disposition of larger spines between microbasic <i>p</i>-amastigophores (distal to capsule, E) and <i>p</i>-mastigophores (proximal to capsule, C, D). Abbreviations: af, Apical flaps; cp, Capsule; tb, tubule. Scale bars: A, 30 µm; B, E, 3 µm; C, 5 µm; D, 10 µm.</p

Representation of conflicts in topologies among markers and phylogenetic inferences.

<p>Comparisons made using results from ML of the concatenated dataset as reference; A, agree; U, unresolved; D, disagree in placement but agree on monophyly; X, disagree in placement and monophyly. Relationships among groups are considered at the higher-level (i.e., Ptychodacteae is considered as a group although is never recovered monophyletic, see text). Conct, concatenated; (−), data not available (SS alignment only available for 12S, 18S and 28S; some datasets did not converge in Phylobayes). Newly-erected higher-level groups in bold.</p

Appendix 11 Lilies-character-taxon-results

Online Appendix 11. Lilies character scores

Appendix 12 Lilies-user-results

Online Appendix 12. Lilies user scores

Appendix 15 all-users-results

line Appendix 15. Combined results for all users. Details for Figure 3

Appendix 20 Instructions to Crowd

Online Appendix 20. Instruction sheets for the study participants (undergraduate students at Ohio State University)