Phylogeny of Capitata and Corynidae (Cnidaria, Hydrozoa) in light of mitochondria! 16S rDNA data ALLEN G. COLLINS*, SILKE WINKELMANN, HEIKE HADRYS & BERND SCHIERWATER Accepted: 14 April 2004 Collins, A. G., Winkelmann, S., Hadiys, H. & Schiei-water, B. (2004). Phylogeny of Capitata (Cnidaria, Hydrozoa) and Coiynidae (Capitata) in light of mitochondrial 16S rDNA data. ? Zoologica Scripts, 34, 91-99. New sequences of the partial rDNA gene coding for the mitochondrial large ribosomal sub- unit, 16S, are derived ffom 47 diverse hydrozoan species and used to investigate phylogenetic relationships among families of the group Capitata and among species of the capitate family Corynidae. Our analyses identiiy a well-supported clade, herein named Aplanulata, of capitate hydrozoans that are united by the synapomorphy of undergoing direct development without the ciliated planula stage that is typical of cnidarians. Aplanulata includes the important model organisms of the group Hydridae, as well as species of Candelabiidae, Corymorphidae, and Tubulariidae. The hypothesis that Hydridae is closely related to brackish water species of Moerisiidae is strongly controverted by 16S rDNA data, as has been shown for nuclear 18S rDNA data. The consistent phylogenetic signal derived from 16S and 18S data suggest that both markers would be useful for broad-scale multimarker analyses of hydrozoan relation- ships. Coiynidae is revealed as paraphyletic with respect to Polyorchidae, a group for which information about the hydroid stage is lacking. Bicorona, which has been classified both within and outside of Corynidae, is shown to have a close relationship with all but one sampled species of Coryne. The coiynid genera Coiyne, Dipurena, and Sarsia are not revealed as mono- phyletic, further calling into question the morphological criteria used to classiiy them. The attached gonophores of the coiynid species Sarsia lovenii are confirmed as being derived fi-om an ancestral state of liberated medusae. Our results indicate that the 16S rDNAmarker could be useful for a DNA-based identification system for Cnidaria, for which it has been shown that the commonly used cytochrome c oxidase subunit 1 gene does not work. Allen G. Collins, Silke Winkelmann, Heike Hadijs if Bernd Schieiivater, ITZ, Ecology ix Evolution, Biinteweg lid, 30SS9 Hannover, Germany. E-?nail: allen.collins@ecolevol.de Silke Winkelmann, German Research Center for Biotechnology/Epigenetic Regulation, Mascheroder Weg 1, 38124 Braunschweig, Germany * Present address: NMFS, National Museum of Natural Histoty, MRC-1 S3 Smithsonian Institution, PO Box 37012, Washington DC 20013-7012, USA. E-mail: CollinsA@SI.edu Introduction In order to understand the evolutionaiy causes and conse- quences of the great diversity of cnidarian hfe cycles, robust phylogenetic hypotheses at all hierarchical levels within Cnidaria are needed. At the broadest scale, progress is being made as analyses of both morphology (Bouillon & Boero 2000; Marques & Collins 2004) and 18S rDNA (Collins 2000, 2002) converge in indicating that the two cnidarian groups having undergone the most frequent and dramatic life history evolution, Anthoathecata and Leptothecata, form a clade (along with Siphonophorae) within Hydrozoa known as Hydroidolina. Hydroidolinan hydrozoans provide excellent opportunities for generating hypotheses explaining evolu- tionary shifts in life cycle (Bouillon et al. 1991; Boero et al. 1992, 1997). However, these ideas have not yet been tested because they require sound phylogenetic hypotheses for multiple groups within Hydroidolina in order to establish whether or not broad-scale associations exist among hydro- zoan life-history characteristics, such as geographical range, speciation rates, ecological circumstances, population genetic structures, etc. Two groups that could eventually be key to understanding life-cycle evolution in Hydroidolina are Capitata and the capitate family Corynidae. The former comprises roughly 25 families, many of which exhibit marked life-cycle variation, and is classified along with its putative sister group, Filifera, in the hydrozoan order Anthoathecata. Capitata has been the subject of just a single character-based phylogenetic analysis 5 The Norwegian Academy of Science and Letters ? Zoologica Scripta, 34,1, January 2005, pp91-99 91 Phylogeny of Capitata and Corynidae ? A. G. Collins et al. (Petersen 1990). Corynidae is a capitate family of approxim- ately 90 species (following Schuchert 2001), whose colonial hydroids are common, though often not appreciated, compon- ents of shallow water marine communities around the world. Subsequent to the hydroid stage, corynid life cycles vary considerably. Whereas some species liberate free-swimming medusae, others have fixed gonophores, and still others lack any semblance of medusae. Classical taxonomy of Corynidae separated Coryne spp. from other corynid species based on absence and presence, respectively, of the medusa stage (Rees 1957; Brinckmann-Voss 1970; Bouillon 1985). However, this criterion for classification, while practical, appears to be artificial in the sense that it is unlikely to be reflective of evolutionary history (Petersen 1990; Schuchert 2001), a con- clusion already confirmed for another hydroidolinan group, the filiferan family Hydractiniidae, with a similar taxonomic history (Cunningham & Buss 1993). Despite recognizing that past taxonomy most likely fails to reflect the phylogeny of the group, Schuchert (2001), in his comprehensive review of Corynidae, concluded that evolu- tionary relationships, and even species identifications, are dif- ficult if not impossible to determine based on morphological characters because they are limited in number and likely to be highly labile. Therefore, molecular data are necessary in order to accomplish the basic goals of systematics for Corynidae. In order to address this need, we have derived sequences (420-520 bp) from the mitochondrial 16S rRNA gene (16S) for 11 corynid species, 24 species representing 12 capitate families that may potentially be closely allied to Corynidae, 9 species of noncapitate hydroidolinans, and 4 trachyline hydrozoans as definitive outgroups. We evaluate whether the mitochondrial 16S gene iragment contains suitable information to assess the phylogenetic status of Corynidae, identify species groups that are closely related to cotynids, and generate reliable hypotheses of phylogenetic relationships among capitate families and corynid species. Materials and methods DNA was obtained from a wide diversity of hydrozoan species, arranged taxonomicaUy in Table 1. For higher-level hydrozoan taxonomy, we followed Marques & Collins (2004), whereas for Capitata we followed Petersen (1990) with changes effected by Schuchert (1996). DNA was extracted by using either the DNA extraction kits, DNAzol (Alolecular Research Center Inc., Cincinnati) or Invisorb (Invitek GmbH, Berlin), or through a basic protocol involving tissue homogenization, proteinase K digestion, extraction with phenol/chloroform/isoamylal- cohol (25 : 24 : 1), centrifugation at 8000 g for 15 min, and precipitation with 0.1 vol. 5 M NH^Ac and 2.5 vol. ice-cold 98% EtOH. Tvo pairs of primer sets were used to amplify nearly identical regions of mitochondrial 16S rDNA. For some samples, hydrozoan specific primers (fwd: GGTGWHRCC ^ ^ 4 FILIFERA Polyorchidae Moerisiidae Hydridae Hydrocorynidae Paragotoeidae Sphaerocorynidae Zancleopsidae Porpitidae (=Velellidae) Cladocorynidae Zancleidae Milleporidae Teissieridae Pennariidae (=Halocordylidae) Dicyclocorynidae Solanderiidae Corynidae Cladonematidae Tricyclusidae Acaulidae Corymorphidae Candelabridae (=Myriothelidae) Margelopsidae Paracorynidae Tubulariidae Fig. 1 Phylogenetic hypothesis of relationships among capitate hydrozoans from Petersen (1990). Taxon names in bold are sampled in the present analysis of paitial mitochondrial 16S rDNA sequences. TGCCCAVTG; rev: TAAAGGTCGAACAGACCTAC; Werner Schroth, Hannover) were used to amplify approxim- ately 420-480 bp of the mitochondrial 16S rRNA gene. Alternatively, the forward primer from Cunningham & Buss (1993) was combined with the reverse primer from Schroth etal. (2002) to ampHfy roughly 450-520 bp. PCR products were subsequently purified and sequenced in both directions using an automated sequencer (either an ABI 310 or a Alegabace 500). Fdited 16S sequences were aligned by eye, along with sequences from 5 additional hydrozoans obtained from Gen- Bank (Table 1), using the software SEAVIEW. Our aligned data set (available upon request) covers 14 of 24 families treated in Petersen's (1990) phylogenetic analysis of Capitata (Fig. 1). One of Petersen's (1990) families, Halocotynidae, is assumed to fall within Zancleidae (following Schuchert 1996). We have not been able to sample Protohydridae, which may be allied to Corymorphidae (Stepanjants et al. 2000), the recendy erected Boeromedusidae (Bouillon 1995), or Halimedusidae, which was recently transferred from Filifera (Mills 2000). Both of the latter two groups may be closely related to Polyorchidae (Mills 2000). Sites of ambiguous homology within the alignment were excluded from phylogenetic analyses, which were carried out using PAUP*4.0 (Swofford 1998). Maximum parsimony (MP), minimum evolution (MF), and maximum likelihood (AIL) 92 Zoologica Scripta, 34, 1, January 2005, pp91-99 t The Norwegian Academy of Science and Letters A. G. Collins et al. ? Phylogeny of Capitata and Corynidae Table 1 Hydrozoan samples used in this analysis, with life cycle notes for capitate species, GenBank accession numbers, and locality of source material. Life cycle notes: cp ? colonial polyp stage; sp ? solitaiy polyp stage; ap ? absence of polyp stage; fm ? free swimming medusa stage; eu ? liberated eumedusoids; fg ? fixed gonophores; am ? complete absence of medusa stage and/or gonophores; nk ? not known. Higher Taxonomy Species; Life cycle notes Ace. No. Locality of material Trachylina Limnomedusae Craspedacusta sinensis AY512507 China Limnomedusae Maeotias marginata AY512508 Northern California Limnomedusae Olindias phosphorica AYS12509 Mallorca Narcomedusae Aegina citrea AY512510 Coast of California Trachymedusae Liriope tetraphylla U19377 not known Hydroidolina Leptothecata Aequorea aquorea AY512518 Woods Hole, MA Leptothecata Blackfordia virginica AY512516 Northern California Leptothecata Clytia sp. AY512519 Coast of California Leptothecata Melicertissa sp. AY512515 Guam Leptothecata Tiaropsidium kelseyi AY512517 Coast of California Siphonophorae Nectopyramis sp AY512512 Coast of California Siphonophorae Physalia utriculus AY512511 Tasmania Anthoathecata Filifera Podocoryna carnea AY512513 not known Filifera Thecocodium quadratum AY512514 not known Capitata Candelabridae Candelabrum cocksii; sp, fg AY512520 France, Atlantic Cladocorynidae Cladocoryne floccosa; cp, fg AY512535 Mallorca Cladonematidae Cladonema radiatum; cp fm AY512539 France, Atlantic Cladonematidae Eleutheria dichotoma; cp, fm AY512538 France IVIediterranean Cladonematidae Staurodadia bilateralis; nk, fm AY512537 Japan Cladonematidae S. oahuensis; nk, fm AY512536 Japan Cladonematidae S. wellingtoni; cp, fm AJ580934 New Zealand Corymorphidae Corymorpha intermedia; nk, fm AY512526 New Zealand Corymorphidae C nutans; sp, fm AY512527 France, Atlantic Corynidae Bicorona tricyda; cp, fg AJ508540 New Zealand Corynidae Coryne eximia; cp fm AY512541 France, Atlantic Corynidae C. japonica; cp, fm AY512540 New Zealand Corynidae C. muscoides; cp, fg AY512553 France, Atlantic Corynidae C. pintneri; cp fg AY512542 France, Mediterannean Sea Corynidae C. producta; cp fm AYS12543 Sandgerdi, Iceland Corynidae C. pusilla; cp, fg AYS12SS2 France, Atlantic Corynidae Dipurena reesi; cp, fm AYS12S46 Brazil Corynidae D. simulans; cp fm AYS12S47 France, Atlantic Corynidae Sarsia lovenii; cp, fg AJ508795 Sandgerdi, Iceland Corynidae S. marii; cp fm AYS12S44 France, Mediterannean Sea Corynidae S. mirabilis; cp fm AYS12S48 Woods Hole, MA Corynidae S. tubulosa; cp, fm AYS12S4S not known Hydridae Hydra drcumdnta; sp, am AYS12S21 Switzerland Hydridae H. vulgaris; sp., am AYS12S22 Switzerland Moerisiidae Moerisia sp; sp, fm AYS12S34 Northern California Pennariidae Pennaria disticha; cp, eu AYS12S33 Mallorca Polyorchidae Polyorchis tiaplus; nk, fm AYS12S49 Northern California Polyorchidae P. penidllatus; nk, fm AYS12SS0 Friday Harbor, WA Polyorchidae Scrippsia padfica; nk, fm AYS12SS1 San Diego, CA Porpitidae Porpita sp; cp fm AYS12S29 Guam Porpitidae Velella velella; cp fm AYS12S28 Coast of California Solanderiidae Solanderia ericopsis; cp fg AYS12S30 New Zealand Tubulariidae Ectopleura larynx; cp fg AYS12S23 France, Atlantic Tubulariidae E. wrighti; cp fm AYS12S24 Mallorca Tubulariidae Hybocodon prolifer; sp, fm AYS12S2S France, Atlantic Tubulariidae Tubularia indivisa; sp., fg U19379 not known Zancleidae Zandea costata; cp fm AYS12S31 France, Mediterannean Sea Zancleidae Z sessilis; cp fm AYS12S32 Mallorca 5 The Norwegian Academy of Science and Letters ? Zoologica Scripta, 34,1, January 2005, pp91-99 93 Phylogeny of Capitata and Corynidae ? A. G. Collins et al. criteria were used to perform replicate searches (500, 500, and 10, respectively) for optimal trees. Likelihood ratio tests employed by AIodelTest (Posada & Crandall 1998) were used to determine an appropriate model of nucleotide evolution assumed for the ME and AIL searches. Bootstrap analyses with 500 replicates under MP and ME were conducted in order to assess node support. Finally, parsimonious searches were carried out to find optimal trees that are constrained to conform to prior hypotheses of relationships among capitate hydrozoan groups. Results After excluding sites of ambiguous alignment, the data set used for phylogenetic analysis contains 415 nucleotide char- acters, of which 161 do not vary across our samples. Of the 254 variable characters, 226 are parsimony informative. This partial mitochondrial 16S fragment is AT-rich for the sam- pled hydrozoans, with mean nucleotide frequencies of 40.5, 11.6, 15.6, and 32.3% for A, C, G, andT, respectively. Hier- archical likelihood ratio tests implemented using ModelTest (Posada & Crandall 1998) indicate that the model that best fits our data (TVM + I + G) has different rates for each type of transversion, one rate for transitions, an assumed proportion of invariable sites (0.337), and a gamma shape parameter (0.656). The ML topology (Fig. 2) is broadly similar to the consensus MP (nine MP trees of length 1756) and ME topologies (not shown). Numerous nodes throughout the topologies do not receive strong support from the partial mitochondrial 16S sequences. Not surprisingly, these are the nodes that most often differ when the various criteria are used for evaluation. Many taxa are revealed as nonmonophyletic, e.g. Capitata, Cladonematidae, Corymorphidae, Corynidae, Tubulariidae, and Zancleidae. Although there is a well-supported break between Trachylina and Hydroidolina, relationships among the hydroidolinan taxa are resolved, but with little support. Anthoathecata (Capitata + Eilifera) is revealed as paraphyletic, as is Capitata with respect to Eilifera, Leptothecata and Siphonophorae. 391 Craspedacusla sinensis~\ I? Maeotias marginata I . Olmdias phosphorica {? TRACHYLINE HYDROZOANS ' Liriope tetraphylla ^^^^^^? Physalia utricutus \ ? Nectopyramis s\yj SIPHONOPHORAE Podocoryna camea ~\ p.. ipcoA Thecodium quadratum_^ Melicertissa sp. "^ Blackfordia virginica ? Aequorea aequorea ? LEPTOTHECATA ? Tiaropsidium kelseyi ? Clytia sp. _y ? Pennaria distich^y- Pennariidae jmnm ^e/e//a velella?\ porpitidae Porpita sp. J I ciadocoryne fioccosa^ Cladocorynidae JI soiandeha ericopsisjy- SolanderJidae \i-Zancleasessilis-X zancleidae I I Z costata J 64/