Phylogenetics of Trachylina (Cnidaria: Hydrozoa) with new insights ...

Journal of the Marine Biological Association of the United Kingdom, 2008, 88(8), 1673–1685.doi:10.1017/S0025315408001732 Printed in the United Kingdom#2008 Marine Biological Association of the United KingdomPhylogenetics of Trachylina (Cnidaria:Hydrozoa) with new insights on theevolution of some problematical taxaallen g. collins 1 , bastian bentlage 2 , alberto lindner 3 , dhugal lindsay 4 ,steven h.d. haddock 5 , gerhard jarms 6 , jon l. norenburg 7 , thomas jankowski 8and paulyn cartwright 21 NMFS, National Systematics Laboratory, National Museum of Natural History, MRC-153, Smithsonian Institution, PO Box 37012,Washington, DC 20013-7012, USA, 2 Department of Ecology and Evolutionary Biology, University of Kansas, 1200 SunnysideAvenue, Lawrence, KS 66045, USA, 3 Centro de Biologia Marinha—USP–Rodovia Manoel Hipólito do Rego, Km 131, 5—SãoSebastião, SP, Brazil, 4 Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan, 5 Monterey BayAquarium Research Institute, 7700 Sandholdt Road, Moss Landing, CA 95039, USA, 6 Biozentrum Grindel und ZoologischesMuseum, Universität Hamburg, Martin-Luther-King Platz 3, 20146 Hamburg, Germany, 7 Smithsonian Institution, PO Box 37012,Invertebrate Zoology, NMNH, W-216, MRC163, Washington, DC 20013-7012, USA, 8 Federal Institute of Aquatic Science andTechnology, Dübendorf 8600, SwitzerlandSome of the most interesting and enigmatic cnidarians are classified within the hydrozoan subclass Trachylina. Despite beingrelatively depauperate in species richness, the clade contains four taxa typically accorded ordinal status: Actinulida,Limnomedusae, Narcomedusae and Trachymedusae. We bring molecular data (mitochondrial 16S and nuclear smalland large subunit ribosomal genes) to bear on the question of phylogenetic relationships within Trachylina. Surprisingly,we find that a diminutive polyp form, Microhydrula limopsicola (classified within Limnomedusae) is actually a previouslyunknown life stage of a species of Stauromedusae. Our data confirm that the interstitial form Halammohydra sp. (Actinulida)is derived from holopelagic direct developing ancestors, likely within the trachymedusan family Rhopalonematidae.Trachymedusae is shown to be diphyletic, suggesting that the polyp stage has been lost independently at least two timeswithin trachyline evolution. Narcomedusae is supported as a monophyletic group likely also arising from trachymedusanancestors. Finally, some data, albeit limited, suggest that some trachyline species names refer to cryptic species that haveyet to be sorted taxonomically.Keywords: phylogenetics, Trachylina, problematic taxaSubmitted 29 November 2007; accepted 8 February 2008; first published online 8 September 2008I N T R O D U C T I O NMolecular data continue to enhance our understanding of theevolutionary history of life at all hierarchical levels.Nevertheless, many questions about cnidarian phylogeneticrelationships remain unanswered (Daly et al., 2007). Toaddress these questions, and thereby provide a robust phylogeneticframework for addressing biological questions involvingcnidarians, is the primary aim of the Cnidarian Tree of Lifeproject (http://cnidarian.info). To achieve this goal, this large,collaborative effort is compiling molecular data from bothnuclear and mitochondrial markers from as many cnidarianspecies as possible. This paper summarizes progress in understandingthe phylogeny of Trachylina and provides greaterinsight into the evolution of several problematical cnidarians.Within Hydrozoa (Cnidaria), an ancient phylogeneticdivergence appears to have given rise to two primary cladeswith extant representatives, Trachylina and HydroidolinaCorresponding author:A.G. CollinsEmail: CollinsA@SI.edu(Collins, 2002; Collins et al., 2006a). In terms of species richness,the vast majority of hydrozoan diversity is containedwithin Hydroidolina (¼Leptolina sensu Schuchert, 2007).Trachylina encompasses roughly 150 valid species, whereasHydroidolina contains more than 3000 (Schuchert, 1998).Nevertheless, because of lack of study and generally simpleand plastic morphology, the true species richness ofTrachylina remains obscure, and significant levels of crypsisare possible. Interestingly, the numbers of higher taxa classifiedwithin Trachylina and Hydroidolina are comparable.Limnomedusae, Narcomedusae, Trachymedusae andActinulida (though it has not been sampled for moleculardata until this study) are the main taxa, usually ranked asorders, within Trachylina (Figures 1–3). Hydroidolina (seeCartwright et al., this volume) contains three relativelydiverse clades, Anthoathecata (athecate hydroids and anthomedusae),Leptothecata (thecate hydroids and leptomedusae)and Siphonophora.The diversity of life history and morphological featuresacross Trachylina makes the group particularly interestingfrom an evolutionary perspective. For instance, the trachylinespecies that perhaps first come to mind are the oceanic,1673

1674 allen g. collins et al.Fig. 1. Images of representative trachyline species. Note that the specimens depicted are not the ones DNA samples were obtained from. (A–C) Limnomedusae;(D–I) Trachymedusae; (J –L) Narcomedusae. (A) Craspedacusta sp.; (B) Olindias sambaquiensis; (C) Gonionemus vertens; (D) Tetrorchis erythrogaster; (E) Liriopetetraphylla; (F) Halicreas minimum; (G) Botrynema brucei; (H) Pantachogon ‘white-red’; (I) Pantachogon ‘orange’; (J) Solmissus incisa; (K) Aegina rosea;(L) Solmundella bitentaculata. A, B and E from A.E. Migotto (Centro de Biologia Marinha, Universidade de São Paulo); C from P. Schuchert (Muséumd’Histoire Naturelle de la Ville de Genève); D and L from S.H.D. Haddock (images captured in California); F–K from D. Lindsay (images captured in Japan).holopelagic members of Trachymedusae and Narcomedusae,which usually develop directly from planula to adult. Evenwithin these groups there are exceptions, e.g. the bentho-pelagictrachymedusans of the family Ptychogastriidae, narcomedusanswith secondarily derived polypoid, parasitic life stages(Bouillon, 1987; Collins, 2002), and the highly unusual wormshapednarcomedusan Tetraplatia (Hand, 1955; Collins et al.,2006b) that bears four sets of swimming flaps. The only

1676 allen g. collins et al.Table 1. Samples with GenBank accession and voucher numbers when available; , derived for this study.Taxonomic hierarchy and species 16S 18S 28S VoucherStauromedusaeCleistocarpida Craterolophus convolvulus AY845344 AY920781 USNM 1073330Eleutherocarpida Haliclystus octoradiatus AY845346 AH014894ScyphozoaCoronatae Atolla vanhoeffeni AF100942 AY026368Sematostomeae Chrysaora melanaster AF358099 AY920780CubozoaCarydeida Carybdea rastonii AF358108 AY920787Chirodrpida Chironex fleckeri AF358104 AY920785HydrozoaHydroidolinaCandelabridae Candelabrum cocksii AY512520 AY920758 AY920796Proboscidactylidae Fabienna sphaerica AM183133.1 AY920767 AY920797Moerisiidae Moerisia sp. AY512534 AF358083 AY920801Porpitidae Porpita sp. AY512529 AF358086 AY920803Polyorchidae Scrippsia pacifica AY512551 AF358091 AY920804Laodiceidae Melicertissa sp. AY512515 AF358075 AY920798Prayidae Nectadamas diomedeae AY512512 AF358068 AY026377TrachylinaActinulidaHalammohydridae Halammohydra sp. EU293991 EU301622 EU301623 USNM 1109974LimnomedusaeOlindiasidae Aglauropsis aeora EU293973 AY920754 AY920793 USNM 1073327Olindiasidae Astrohydra japonica EU293975 AY920794 G. Jarms cultureOlindiasidae Craspedacusta sowerbii EU293971 AF358057 USNM 1105483Olindiasidae Craspedacusta sinensis AY512507 EU247815 Olindiasidae Craspedacusta ziguensis EU293974 USNM 1102057Olindiasidae Gonionemus vertens EU293976 Olindiasidae Limnocnida tanganyicae EU293972 AY920755 AY920795 USNM 1075114Olindiasidae Maeotias marginata AY512508 AF358056 EU247810 Olindiasidae Olindias phosphorica EU293978 AY920753 EU247808 MHNG 29811Olindiasidae Olindias sambaquiensis EU293977 EU247814 EU247809 Microhydrulidae Microhydrula limopsicola EU294003 EU247811 G. Jarms cultureMonobrachiidae Monobrachium parasiticum EU292970 AY920752 USNM 1074993NarcomedusaeAeginidae Aegina citrea EU293997 AF358058 AY920789Aeginidae Aegina rosea EU247813 Aeginidae Solmundella bitentaculata EU293998 USNM 1107456Aeginidae Solmundella bitentaculata EU247812 EU247795 MHNG 31746Cuninidae Cunina frugifera AF358059Cuninidae Sigiweddellia sp. EU293996 EU247796 Cuninidae Solmissus incisa EU294002 USNM 1111080Cuninidae Solmissus marshalli EU294001 AF358060 AY920790Tetraplatiidae Tetraplatia volitans EU293999 DQ002501 DQ002502 KUMIP 314322Tetraplatiidae Tetraplatia sp. EU294000 TrachymedusaeGeryoniidae Geryonia proboscidalis EU293979 EU247816 EU247807 Geryoniidae Liriope tetraphylla AY512510 AF358061Geryoniidae Liriope tetraphylla EU293980 AY920756 USNM 1107457Halicreatidae Botrynema brucei EU293982 EU247822 EU247798 Halicreatidae Haliscera conica EU293981 EU247825 EU247797 Halicreatidae Halicreas minimum EU293983 EU247826 Rhopalonematidae Aglantha digitale EU293985 EU247821 AY920791 USNM 1073329Rhopalonematidae Aglaura hemistoma EU247818 EU247803 MHNG 31745Rhopalonematidae Aglaura hemistoma EU293984 EU247820 EU247802 KUMIP 314323Rhopalonematidae Amphogona apicata EU293994 EU247801 Rhopalonematidae Crossota rufobrunnea EU293986 EU247824 EU247800 Rhopalonematidae Crossota rufobrunnea EU293987 EU247823 EU247799 Rhopalonematidae Pantachogon haeckeli AF358062 AY920792Rhopalonematidae Pantachogon haeckeli EU293988 USNM 1111078Rhopalonematidae Pantachogon ‘white-red’ EU293989 EU247817 EU247805 Rhopalonematidae Pantachogon ‘orange’ EU293990 EU247806 Rhopalonematidae Rhopalonema velatum EU293992 EU247819 EU247804 Rhopalonematidae Rhopalonema cf. velatum EU293993 USNM 1107461Rhopalonematidae Tetrorchis erythrogaster EU293995 USNM 1111077USNM, National Museum for Natural History, Smithsonian Institution; KUMIP, University of Kansas Natural History Museum, Division of InvertebratePaleontology; MHNG INVE, Muséum d’Histoire Naturelle de la Ville de Genève.

phylogenetics of trachylina 1677SSU (approximately 1800 bp) was amplified and sequencedaccording to Medlin et al. (1988). The original PCR profilewas modified to 948C/4 min; (948C/20 s, 578C/20 s, 728C/1 min 45 s) 35; 728C/7 min. Nearly complete LSU (approximately3200 bp) was amplified in two parts using the primersF63mod (Medina et al., 2001) and R2084 (5 0 -AGA GCC AATCCT TTT CC-3 0 ) in one PCR reaction and the primers F1383(5 0 -GGA CGG TGG CCA TGG AAG T-3 0 ) and R3238(5 0 -SWA CAG ATG GTA GCT TCG-3 0 ) in a second reaction.The temperature profile of each PCR was the following: 948C/5 min; (948C/30 s, 458C/60 s, 728C/3 min) 30; 728C/10 min(modified from Medina et al., 2001). The LSU sequences wereobtained with the previously published primers F63sq, F635,R635, R1411sq, R1630, F2076sq, F2766 (all from Medinaet al., 2001), R2800 (Voigt et al., 2004) and the newly developedprimers F1383, F1689 (5 0 -CTA AGM SRY AGG GAAAYT C-3 0 ), R2084 and R3238. Purification of PCR productsand sequencing were carried out at the laboratories ofCogenics (Houston, Texas). Sequences and collection datafor the source specimens have been deposited to GenBank(see Table 1).DatasetsIn order to investigate different phylogenetic questions, assesslevels of correspondence among the different phylogeneticmarkers, and derive the most robust phylogenetic hypothesisof Trachylina possible, we created five datasets. First, partialmitochondrial 16S, nuclear SSU, and nuclear LSU data forall taxa (Table 1) were aligned using MUSCLE (multiplesequence alignment with high accuracy and high throughput)(Edgar, 2004). No adjustments were made by eye. ‘Conserved’positions for each of the alignments were identified usingGblocks v0.91 (Castresana, 2000) with default parameters,except that half the taxa were allowed to be gaps for anygiven position. Positions deemed non-conserved by Gblockswere excluded from phylogenetic analysis.The first dataset analysed was constructed in order toconduct initial investigations of the phylogenetic placementof the actinulidan Halammohydra sp., which had beenhypothesized by some authors to represent an anciently diverginglineage of Hydrozoa (Swedmark & Tessier, 1966).Therefore, for this dataset, we combined both SSU and LSUdata (for the regions over which we were able to derive datafrom Halammohydra, 1298 and 1032 nucleotides, respectively)from a diverse set of trachylines, seven representativesof Hydroidolina, and six non-hydrozoan representatives ofMedusozoa, used as outgroups (see Figure 4). Non-hydrozoanswere not included in any of the other datasets, whichwere constructed to conduct more detailed analyses ofTrachylina phylogeny and to investigate the degree ofcorrespondence between the different markers. The secondthrough fourth datasets consisted of all available trachylinesequences for mitochondrial 16S, SSU, and LSU markers(Table 1), along with seven species representing the breadthof hydroidolinan (non-trachyline hydrozoan) diversity foroutgroups (Figures 5–7).The fifth and final dataset was used to derive the most robustphylogenetic hypothesis possible for Trachylina. This datasetcombined data from all three markers for which at least SSUFig. 4. Phylogenetic hypothesis with non-hydrozoan medusozoan outgroups to investigate the position of Halammohydra sp. within Hydrozoa. Note also theposition of Microhydrula limopsicola. Phylogram of ML topology based on combined SSU and LSU data (dataset 1) with bootstrap indices under both MLand MP at each node. ‘ , ’ indicates a bootstrap index of less than 61; a single 100 or , was placed at nodes if indices under both criteria were 100 or lessthan 61, respectively. For ML analyses, the assumed model of nucleotide evolution was GTR þ I þ G. Scale bar is equivalent to 0.1 substitutions per site.

1678 allen g. collins et al.Fig. 5. Phylogenetic hypothesis based on mitochondrial 16S data (dataset 2). Phylogram of one of the 20 most parsimonious trees, with bootstrap indices underboth ML and MP at each node. ‘ , ’ indicates a bootstrap index of less than 61; a single 100 or , was placed at nodes if indices under both criteria were 100 or lessthan 61, respectively. For ML analyses, the assumed model of nucleotide evolution was K81uf þ I þ G. Scale bar is equivalent to 20 substitutions.or LSU had successfully been derived (Table 1; Figure 8), as wellas the seven outgroup hydroidolinans for which all threemarkers were available. For this dataset, two chimericalsequences were constructed. Mitochondrial 16S fromSolmundella bitentaculata from Antarctica was combinedwith nuclear SSU and LSU of S. bitentaculata from theMediterranean Sea. Mitochondrial 16S from Pantachogonhaeckeli from the central coast of California was combinedwith un-vouchered Genbank SSU and LSU sequences derivedfrom P. haeckeli from Californian coastal waters.Phylogenetic analysesPhylogenetic hypotheses were constructed under bothmaximum likelihood (ML) and maximum parsimony (MP)optimality criteria. Modeltest (Posada & Crandall, 1998) wasused, employing the Akaike information criterion, to choosethe most appropriate model for each of the datasets.Assuming these models, PAUP (Swofford, 2002) was usedto search (with 100 random addition replicates) for the topologywith the greatest likelihood and GARLI v0.951(Zwickl, 2006) was used to calculate ML bootstrap indicesfrom searches for 500 replicate datasets. PAUP was furtheremployed to find most parsimonious topologies, with 100random replicate searches and to calculate bootstrap indices(500 replicates, with 10 searches per replicate).R E S U L T SIn general, ML and MP topologies are congruent, only differingwhere bootstrap values are low under one or both criteria.Topologies, either ML or MP, for each of the five datasets arepresented as Figures 4–8. In addition, both ML and MP bootstrapindices are reported on these figures at each of the relevantnodes. Two main results are drawn from Figure 4 showing theML topology for the combined SSU and LSU analysis includingnon-hydrozoan taxa as outgroups. First, the representative ofActinulida, Halammohydra sp., does not appear to be an earlydiverging lineage of Hydrozoa, but is instead revealed as amemberofTrachylina.Second,Microhydrulalimopsicola(classifiedin the family Microhydrulidae within Limnomedusae) isshown to not be a member of Hydrozoa at all, but instead tohave a very close relationship to the stauromedusanHaliclystus octoradiatus.Figure 5 presents one of 20 MP trees based on our 16S data.Generally, nodes in the topology do not receive high support.There is a well-supported split between two clades, one containingspecies of Limnomedusae plus species of the trachymedusanfamily Geryoniidae and the other comprising allthe remaining trachymedusans, narcomedusans and the actinulidanHalammohydra sp. Other phylogenetic associationsthat receive method-independent (i.e. both MP and ML)support include: the limnomedusan genera Limnocnida and

phylogenetics of trachylina 1679Fig. 6. Phylogenetic hypothesis based on SSU data (dataset 3). Strict consensus of the 21 most parsimonious trees, with bootstrap indices under both ML and MPat each node. ‘ , ’ indicates a bootstrap index of less than 61; a single 100 or , was placed at nodes if indices under both criteria were 100 or less than 61,respectively. For ML analyses, the assumed model of nucleotide evolution was GTR þ I þ G.Fig. 7. Phylogenetic hypotheses based on LSU data (dataset 4). Left: strict consensus of the 12 most parsimonious trees with bootstrap indices under both ML andMP at each node. ‘ , ’ indicates a bootstrap index of less than 61; a single 100 or , was placed at nodes if indices under both criteria were 100 or less than 61,respectively. For ML analyses, the assumed model of nucleotide evolution was GTR þ I þ G. Right: ML topology with ML bootstrap indices for three nodesreceiving support under ML but not under MP; terminal taxa in the same order as shown on the left.

1680 allen g. collins et al.Fig. 8. Phylogenetic hypothesis based on combined 16S, SSU, and LSU data (dataset 5). Phylogram of ML topology, with bootstrap indices under bothML and MP at each node. ‘ , ’ indicates a bootstrap index of less than 61; a single 100 or , was placed at nodes if indices under both criteria were 100or less than 61, respectively. For ML analyses, the assumed model of nucleotide evolution was GTR þ I þ G. Scale bar is equivalent to 0.1 substitutionsper site.Craspedacusta; the trachymedusan genera Geryonia and Lirope(Geryoniidae); the trachymedusan genera Haliscera, Halicreasand Botrynema (Halicreatidae); the trachymedusan generaRhopalonema and Pantachogon; the trachymedusan generaAmphogona, Aglaura and Aglantha; and, the narcomedusangenera Aegina, Solmundella and Solmissus.Figure 6 shows the strict consensus of 21 MP trees found insearches using the SSU dataset. With three exceptions, all ofthe relationships strongly supported by the mitochondrial16S analyses (Figure 5) receive method independent supportfrom the SSU data. The first exception is that there is nosupport for the splitting of Trachylina into two main clades.The second exception is that the ML bootstrap support forthe node linking Haliscera, Halicreas and Botrynema(Halicreatidae) is only 54. The third exception is that theclade linking Aegina citrea, Solmundella and Solmissus alsoincludes Tetraplatia volitans, which had an uncertain positionbased on the 16S data. Additional clades receiving support, asmeasured by both MP and ML bootstraps are: the limnomedusantaxa Aglauropsis (marine) and Maeotias (brackish)with the freshwater Craspedacusta and Limnocnida; the limnomedusangenus Olindias with Geryoniidae; and, the twotrachmedusan clades (Rhaopalonema þ Pantachogon andAmphogona þ Aglaura þ Aglantha).The strict consensus of 12 MP trees based on the LSUdata supports many of the relationships also indicated bythe 16S and SSU analyses (Figure 7). One of the two maintrachyline clades (non-geryoniid trachymedusans, narcomedusansand the actinulidan Halammohydra sp.) suggestedby 16S is also supported by LSU data. Two additional cladesthat receive high bootstrap support from LSU data underboth MP and ML criteria are: all sampled representatives ofRhopalonematidae plus Halammohydra sp.; and, all sampledrepresentatives of Narcomedusae.Finally, Figure 8 shows the ML topology using the combineddataset. Given the great amount of correspondencebetween the different single-marker analyses, it is not surprisingthat this combined analysis contains nearly all ofthe associations enumerated above. In total comparingacross all the single-marker analyses, there is only oneinstance of relationships receiving strong contradictorysupport. The SSU, LSU and combined data support an allianceof Aegina citrea, Solmundella, Solmissus andTetraplatia volitans, whereas the 16S analyses suggestedthat the first three taxa form a well-supported clade exclusiveof Tetraplatia.D I S C U S S I O NScope of LimnomedusaeLimnomedusae has a somewhat complicated taxonomic historythat reflects uncertainty about the phylogenetic affinities of thegroups that constitute it, as well as confusion arising from thehistorical use of separate taxonomic systems by experts onhydromedusae and hydroids. The taxon was created in thelate 1930s (Kramp, 1938; Browne & Kramp, 1939) for species

phylogenetics of trachylina 1681of the families Moerisiidae, Olindiasidae (¼Olindiidae) andProboscidactylidae. The underlying concept of Limnomedusaewas that it should receive species with a biphasic lifecycle, whose medusae did not fit the Anthoathecata(¼Anthomedusae or Athecata) because they had eitherecto-endodermal statocysts or gonads along their radialcanals, and whose polyps did not fit the Leptothecata(¼Leptomedusae or Thecata) because they lacked a theca.Subsequently, Monobrachiidae, which also meets the criteriaabove, has been added to Limnomedusae (Naumov, 1960),as have the small and simple species of Armorhydridae andMicrohydrulidae, for lack of better alternatives (Bouillon,1985). More detailed analysis that included information onthe cnidome indicated that Moerisiidae is more likely toshare a recent common ancestry with members of theanthoathecate group Capitata (Rees, 1958; Petersen, 1990).Likewise, the absence of statocysts and the presence of desmonemesstrongly favour the hypothesis that Proboscidactylidaeis closely related to anthoathecate species classified as Filifera(Edwards, 1973; Schuchert, 1996). Recently, molecular datahave confirmed that Moerisidae and Proboscidactylidae donot belong in Limnomedusae (Collins et al., 2006a), therebylimiting its scope to Armorhydridae, Microhydrulidae,Monobrachidae and Olindiasidae. To a certain extent, ourresults (Figures 4–8) challenge this view of Limnomedusae,as further discussed below.Microhydrula limopsicola is a species ofStauromedusaeMicrohydrula limopsicola (originally classified withinLimnomedusae) is an Antarctic species that possessesminute polyps found living on bivalve shells (Jarms &Tiemann, 1996; Figure 2C). The polyps are less than 0.5 mmin length and 0.35 mm in diameter and solitary, though theydo form aggregates. The morphology of the polyps is quitesimple as they lack tentacles and possess just a single typeof nematocyst, microbasic euryteles. These polyps wereobserved to rest on a thin periderm and produce creeping,asexual frustules (Jarms & Tiemann, 1996). The polypsof Limnomedusae (where known) are tiny, often withouttentacles (e.g. Figure 2A), capable of producing frustules,and typically armoured with microbasic euryteles. This suiteof features therefore suggests that M. limopsicola should bepart of Limnomedusae. Thus, it is quite surprising thatgenetic data taken from samples of M. limopsicola stronglyindicate that it is actually part of the non-hydrozoanStauromedusae (Figure 4).Concern that this result was a product of contaminationprompted a re-sampling of a culture of Microhydrula limopsicolamaintained in the Jarms laboratory. This yielded the samedata, indicating that M. limopsicola is a member ofStauromedusae. In fact, in an ongoing study ofStauromedusae, mt16S data (Collins et al., unpublisheddata) identify M. limopsicola as a member of the genusHaliclystus, with a very close relationship to the speciesof Haliclystus from southern Chile reported on by Zagal(2004).The most straightforward way to reconcile the interpretationof M. limopsicola as described by Jarms & Tiemann(1996) is that it likely represents a microscopic life cyclestage not previously known in Stauromedusae. This wouldexplain why no gonads were observed by Jarms & Tiemann(1996). Life cycles of stauromedusans (and indeed mostcnidarians) are not well known or documented. In particular,the juvenile stages of very few stauromedusan species areknown. The only case for which the actual habitat of juvenilestauropolyps is known is for two species of Stylocoronella,whose polyps live interstitially (Salvini-Plawen, 1987;Kikinger & Salvini-Plawen, 1995). Euryteles are widelydistributed across Medusozoa, including Stauromedusae,so this character is not difficult to reconcile. However,this would appear to be the first time that a periderm andfrustules have been recognized in any species ofStauromedusae. Both features are known from specieswithin Discomedusae (Scyphozoa) and Limnomedusae(Hydrozoa) suggesting that they have evolved independentlyat least two times.It is unclear if this result could be extended to other speciesof Microhydrulidae, the limnomedusan family to whichM. limopsicola was assigned. The family contains just threespecies in two genera, and no adults or sexual reproductionhave been reported from any of them. Microhydrula ponticais very similar in morphology to M. limopsicola and it maybe that this species too, known from the northern Atlantic,represents another species of Stauromedusae. Rhaptapagiscantacuzeni, the third representative of the family, is differentiatedfrom the other species by the possession of an unusualform of microbasic euryteles known as semiophores (Bouillon &Deroux, 1967). DNA sampling of these taxa should help toresolve phylogenetic affinities of these species.Freshwater limnomedusansWe present some new mt16S data for freshwater limnomedusans.These data further reinforce (albeit with low support) thehypothesis that freshwater limnomedusans had a single evolutionaryorigin (Figure 5, but note the robust support inFigure 8). Among our 16S samples is a representative ofCraspedacusta ziguensis. As reviewed by Jankowski (2001),the validity of this species has been in doubt. However, ourmt16S data, which include representatives of two otherreadily accepted species of Craspedacusta, C. sowerbii andC. sinensis, indicate that C. ziguensis has comparable divergencesfrom these two species (P distances of 7.4% and6.1%, respectively) as they do from each other (8.8%). TheSSU and 16S data show Craspedacusta to have a close relationshipto the freshwater genus Limnocnida. The polyps of thetwo genera are nearly indistinguishable: they are diminutive(1 mm or less in length), form small colonies up to seven individuals,lack tentacles, possess papillae with eurytele nematocystsat their oral ends, and produce creeping frustules thatdevelop into additional polyps (Bouillon, 1957). The otherfreshwater species sampled here, Astohydra japonica, also producesfrustules, but the polyps are smaller (up to 0.3 mm inlength), solitary, and have 10 to 20 nematocyst-rich tentaclescomposed of single cells that may be homologous with thepapillae of Craspedacusta and Limnocnida polyps(Hashimoto, 1981). The polyps of Astrohydra japonica andthose of another freshwater genus, Calpasoma (not yetsampled for molecular data), are indistinguishable, and if itwere not for the presence of a poorly known medusa stagein the former (Hashimoto, 1985), the two would have to beconsidered conspecific.

1682 allen g. collins et al.Geryoniidae (Trachymedusae) is derived fromwithin LimnomedusaeThe medusae of Trachymedusae and Limnomedusae arequite similar, with gonads typically on the radial canalsand statocysts of ecto-endodermal origin. The two differencesbetween the groups are the presence or absence of apolyp stage and hollow or solid marginal tentacles inLimnomedusae and Trachymedusae, respectively (Schuchert,2007). As a practical matter, the two groups can often be separatedbased on the number of radial canals, with most speciesof Limnomedusae possessing four (rarely six) and mostspecies of Trachymedusae having eight (but some with fouror six). We have found strong evidence that Geryoniidae isthe sister group to species of the limnomedusan genusOlindias (Figures 4–8), whereas our analyses weakly favourthe hypothesis that Limnomedusae plus Geryoniidae formsa clade with Monobrachium as its earliest diverging lineage(Figures 4 & 8).Interestingly, Geryoniidae is one of the two families ofTrachymedusae with species not characterized by havingeight radial canals, but rather four (Liriope) or six (Geryonia).Moreover, species of Geryoniidae have both solid and hollowmarginal tentacles, as well as centripetal canals. Therefore, inmost respects Geryoniidae can readily be incorporated withinLimnomedusae. It has long been known that members ofGeryoniidae lack a polyp stage (e.g. Brooks, 1886) and thisappears to be the major difference between the group andmembers of Limnomedusae. Given our phylogenetic results,it would appear that a polyp stage was likely lost independentlyin Geryoniidae and the lineage leading to the othertrachymedusans.Scope of TrachymedusaeAs presently constituted, Trachymedusae comprises five families,Geryoniidae, Halicreatidae, Petasidae, Ptychogastriidea andRhopalonematidae. Our analyses contain members of threeof these families; we have yet to sample Petasidae andPtychogastridae. Besides Geryoniidae, the other group of trachymedusanswithout eight radial canals is Petasidae. Petasidshave four radial canals, raising the possibility that they couldpotentially fall with Geryoniidae within Limnomedusae.However, they lack other features (hollow tentacles, centripetalcanals) that could give credence to such ahypothesis. Ptychogastriids are somewhat more typical ofTrachymedusae, possessing eight radial canals. But thesespecies are bentho-pelagic and have features (such as tentacleswith adhesive discs) that appear to be adapted for such anexistence. Several members of Limnomedusae (Olindias,Gonionemus and Vallentinia, etc.) also spend considerabletime resting on substrates, prompting one to question if ptychogastriidstoo might be closely related to or derived fromwithin Limnomedusae.Our analyses find strong support for the hypothesis thatHalicreatidae and Rhopalonematidae form a clade withNarcomedusae and the interstitial Halammohydra sp. (Figures7 & 8). The best sampled family in our analyses isRhopalonematidae. A close alliance appears to exist betweenAglantha, Aglaura and Amphogona, all of which have agastric peduncle and pendant gonads. Our data also suggest arelatively close relationship between Pantachogon andRhopalonema, and that these two genera form a clade withAglantha plus Aglaura plus Amphogona. Among our samplesof Rhopalonematidae, Crossota appears to be the earliest diverginglineage. Interestingly, in many of our analyses, the unusualinterstitial Halammohydra (Actinulida) is shown as having hadan origin within Rhopalonematidae. In all of our analyses,representatives of Halicreatidae form a well supported clade.Our combined data analyses suggest that this family is thesister group of a clade containing Narcomedusae,Halammohydra and the rhopalonematids (Figure 8).Halammohydra may be derived from withinRhopalonematidae (Trachymedusae)Species of Halammohydra were among the first cnidariansdiscovered to inhabit the interstices of sand grains (Remane,1927). Subsequent studies have led to the discovery ofadditional species of interstitial hydrozoans (e.g. Swedmark,1964; Clausen, 1971; Norenburg & Morse, 1983). Otohydra,discovered off Roscoff, was grouped with Halammohydra inthe order Actinulida by Swedmark & Tessier (1958). In theirview (Swedmark & Tessier, 1966), these small and simpleanimals were representatives of the earliest diverging hydrozoanlineages, which themselves were inhabitants of the interstitialenvironment. In contrast, the presence of featuresincluding solid tentacles, ecto-endodermally derived statocysts,and lack of asexual reproduction, have been regardedas evidence that actinulids may be related to free-swimmingNarco- and Trachymedusae (e.g. Remane, 1927; Werner,1965; Salvini-Plawen, 1987; Bouillon & Boero, 2000;Marques & Collins, 2004). In particular, it has been suggestedthat the small and simple bodies of adult actinulids resembleearly developmental stages of trachylines (e.g. Swedmark,1964; Bouillon & Boero, 2000). Gould (1977), for example,holds that actinulids and other interstitial organisms mayhave attained small body sizes by accelerating sexual maturation(paedomorphosis through progenesis), which heregarded as an important adaptive response for inhabitingminute interstitial spaces.Paedomorphic species have been notoriously difficult forphylogenetic analyses based on morphology (Wiens et al.,2005) and molecular data can be particularly important forsuch cases. Our analyses consistently place Halammohydrawithin Trachylina (Figures 4–8) and confirm earlier suggestionsthat actinulids are related to Narco- and Trachymedusae.Specifically, ML analyses of LSU (Figure 7, right) and combineddata (Figure 8) indicate that Halammohydra falls within thefamily Rhopalonematidae (Trachymedusae). Although adultrhopalonematids are typical planktonic trachyline medusae,some early developmental stages are similar to the adultHalammohydra. For example, developing medusae of the rhopalonematidAglaura hemistoma resemble adult actinulids inhaving an elongated and flagellated actinuloid body shapebearing long tentacles (Metschnikoff, 1886). Although trachylinemedusae are usually planktonic, the evolution of the interstitialHalammohydra from a trachyline ancestor may havefollowed a shift from a holopelagic to a pelago-benthic or holobenthiclife cycle. As mentioned above, several trachyline specieslive pelago-benthic lifestyles. It is conceivable that the evolutionof a similar epibenthic habitat in the ancestor of actinulids mayhave preceded the invasion of the interstitial environment.It remains to be seen how our understanding of actinulidanevolution will be refined when samples of Otohydra become

phylogenetics of trachylina 1683available for DNA-based analyses. Species of the latter genusshare with Halammohydra the flagellated/ciliated actinuloidbody with ontogenetically similar (ecto-endodermal) statocysts.However, the two groups have some significant differences,with Otohydra lacking a nerve ring and an adhesiveorgan, being hermaphroditic, and possessing two types of tentacles(Swedmark & Teissier, 1958; Swedmark, 1964; Clausen,1971). Elucidating the phylogenetic placement of Otohydraremains an important step towards a better understandingof the patterns and processes that resulted in the invasion ofthe interstitial environment within Trachylina.Scope of NarcomedusaeThe enigmatic intracellular parasitic cnidarian Polypodiumhydriforme (see Raikova, 1994) has sometimes been referredto Narcomedusae (e.g. Hyman, 1940), but unpublished analysessuggest that it is not part of this group (Evans et al., inpreparation). Therefore, just four families make upNarcomedusae (Schuchert, 2007). Three families, Aeginidae,Cuninidae and Tetraplatiidae, are sampled here, whereasSolmarisidae is not. The name Tetraplatiidae first appearedin print in Daly et al. (2007) as a nomen nudum, but the taxonomicauthority was erroneously stated to be Schuchert, 2007in reference to Schuchert’s (2007) Hydrozoan Directorywebsite. We take the opportunity to formally establish thenew family Tetraplatiidae, with its type genus beingTetraplatia Busch, 1851. Its members are readily distinguishedby the possession of four distinctive swimming lappets arisingfrom a groove that divides their worm-like bodies into aboraland oral halves.Our analyses suggest that our sampled representatives ofNarcomedusae are monophyletic, though support for thishypothesis is only moderate (Figures 6–8). We find nostrong evidence for the monophyly of Aeginidae orCuninidae. However, support for most relevant nodes is weakand so it does not appear that our analyses contradict monophylyof these families, with one exception. In most of our analyses,the worm-shaped Tetraplatia is shown to have a closerelationship with Aegina citrea and Solmundella bitentaculata.The sister to these three species, among our samples, appearsto be Solmissus marshalli. Thus, Tetraplatia and by extensionthe family Tetraplatiidae appears to fall within Aeginidae. Aswith all the subgroups dealt with in the present paper,additional sampling should further enhance understanding ofthe phylogeny and evolution of Narcomedusae.Concluding remarksImportant questions remain about how to best classify trachylinetaxa in light of the phylogenetic analyses presented here.Because our taxon sampling is relatively sparse, formalrecommendations seem inappropriate at this time. However,our limited sampling suggests that Trachymedusae is (atleast) diphyletic, suggesting that this taxon will need toeither have its meaning modified or be discarded. BecauseLimnomedusae is paraphyletic with respect to the trachymedusanfamily Geryoniidae, Limnomedusae will also needto have its meaning altered to take these, and forthcoming,findings into account.Finally, future sampling of molecular data will no doubt beused to clarify species boundaries within trachyline cnidarians.Our data are extremely limited in this regard, butreporting of a few observations are warranted. We noteseveral substantial genetic divergences within what arecurrently thought to be single species. First, both mitochondrial16S and nuclear SSU data taken from Liriope tetraphyllacollected in California and the Caribbean (Panama) showsignificant divergences (P distances .11% for 16S and.0.3% for SSU) likely indicating at least some crypsis inthis cosmopolitan species. Similarly, Tetraplatia volitans andTetraplatia sp. from California and Japan, respectively, havemitochondrial 16S sequences that differ by nearly 6%, exceedingtypical intraspecific differences measured for other hydrozoans(Schuchert, 2005; Govindarajan et al., 2005; Migliettaet al., 2007). Visual inspection of Tetraplatia sp. specimensfrom Japan suggests that these do not fit the description ofT. chuni, the only other named species in the genus, butrather that of T. volitans, suggesting that this latter namemay be being used to refer to at least two distinct species. Incontrast, very little to no genetic divergence was revealedin samples of Crossota rufobrunnea and Pantachogon haeckeli(including some unusual colour forms; Figure 1H, I) fromCalifornia and Japan, and Aglaura hemistoma samples fromJapan and the Mediterranean Sea. The geographical distributionsof trachyline hydrozoans, as well as the factorscontrolling them, remain open and interesting questions forfurther research.A C K N O W L E D G E M E N T SWe gratefully acknowledge support from the US NationalScience Foundation (NSF) Assembling the Tree of Life Grant0531779 (to P.C., A.G.C., and D. Fautin). We also thank:three anonymous referees for reviewing an earlier version ofthe MS; P. Schuchert for providing DNA extractions; C. Mahand K. Halanych for collecting medusae in Antarcticathrough support from a NSF grant (OPP-0338218 to KH); theSmithsonian’s Marine Science Network and R. Collin for supportingthe collection of specimens; the Smithsonian’sLaboratory of Analytical Biology for the use of its computercluster to conduct phylogenetic analyses; E. Strong for the useof a computer to conduct phylogenetic analyses; N. 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