Four new species and one new genus of zoanthids (Cnidaria, Hexacorallia) from the Galápagos Islands

Recent research has confi rmed the presence of several species of undescribed macrocnemic zoanthids (Cnidaria: Hexacorallia: Zoantharia: Macrocnemina) in the Galápagos. In this study four new species, including two belonging to a new genus, are described. Two species, Terrazoanthus onoi sp. n. and Terrazoanthus sinnigeri sp. n., both belong within the recently erected family Hydrozoanthidae to the new genus Terrazoanthus, which can be distinguished from the type genus Hydrozoanthus by being attached to abiotic substrate as opposed to hydrozoans for Hydrozoanthus. Each new species of zoanthid can be clearly distinguished by a number of characters. Antipathozoanthus hickmani sp. n. is distinguished by its exclusive association with the antipatharian Antipathes galapagensis, and has approximately 40 tentacles. Parazoanthus darwini sp. n. is distinguished by its frequent association with sponges, with approximately 24–30 tentacles and polyps embedded in a well-developed coenenchyme. T. onoi sp. n. is distinguished by its bright red oral disk color, 32–40 tentacles, and has only basitrichs and mastigophores present in the pharynx. T. sinnigeri sp. n. is distinguished by usually occurring on the underside of rubble and rocks on sandy bottoms, showing 30–36 tentacles, and numerous nematocyst types in the pharynx. Th e two Terrazoanthus species, although divergent in both morphology and ecology, are apparently very closely related, ZooKeys 42: 1–36 (2010) doi: 10.3897/zookeys.42.378 www.pensoftonline.net/zookeys Copyright J.D. Reimer, T. Fujii. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Launched to accelerate biodiversity research A peer-reviewed open-access journal James Davis Reimer & Takuma Fujii / ZooKeys 42: 1–36 (2010) 2 with identical mitochondrial 16S ribosomal DNA and cytochrome oxidase subunit I sequences. Th ese two species can be molecularly distinguished by their subtly diff erent yet distinct sequences of internal transcribed spacer of ribosomal DNA (ITS-rDNA).


Introduction
Th e Galápagos Archipelago is a group of oceanic islands in the south-east Pacifi c. Th e islands are surrounded by warm and cold ocean currents that include upwelling currents.As a result of these currents, the marine ecosystems of the Galápagos islands are isolated from other regions, and there are high levels of diversity and unique fauna (Bustamante et al. 2000(Bustamante et al. , 2002)).Th e marine area of the Galapagos islands was inscribed as a UNSECO natural World Heritage Site in 1978.However, as the Galápagos islands are becoming increasingly famous and numbers of tourist visits rise, environmental pollution and other associated problems have arisen.Since 2007 the Galápagos have been designated as a World Heritage Site in danger.
Recently, the marine fauna of the Galápagos has begun to become more intensively investigated.Some results have been published as the Galápagos Marine Life Series fi eld guides.One of these guides is focused on corals, zoanthids, octocorals and other benthic cnidarians of the Galápagos (Hickman 2008).Th e results of these investigations have demonstrated that there are many unidentifi ed species in the order Zoantharia in the Galápagos (Reimer et al. 2008a).
However, zoanthids (Cnidaria, Anthozoa, Hexacorallia, Zoantharia) form a taxonomical group for which studies are lacking in general.Zoanthids are an order of benthic cnidarians that are found in most marine ecosystems (Sinniger et al. 2005;Reimer et al. 2007).Distinguishable by their two rows of tentacles, incorporation of sand into their mesoglea, and colonial nature (for most described species) (Sinniger et al. 2005), zoanthids are increasingly becoming a subject of research as they possess unique bioactive compounds and chemicals (Behenna et al. 2008).Despite this increase in research, the classifi cation and identifi cation of many zoanthids remains diffi cult due to a myriad of problems, including but not limited to morphological variation within species, a lack of research into their species diversity, and diffi culty in internal morphological examinations due to the presence of sand and detritus (Reimer et al. 2010).Consequently, the true number of zoanthid species is currently unknown (Reimer et al. 2004).
However, studies using molecular techniques have begun to bring some standardization and reassessment to zoanthid taxonomy.Allozymes (Burnett et al. 1997), mitochondrial DNA (mt DNA) (Reimer et al. 2004;Sinniger et al. 2005) and nuclear DNA phylogenies (Reimer et al. 2007b;Swain 2009) combined with ecological data have resulted in the creation of new taxa (Sinniger and Häussermann 2008;Reimer et al. 2008a;Sinniger et al. 2009) and the merging of other taxa (e.g.Reimer et al. 2006).
With a molecular phylogenetic framework in place, recent research into zoanthids has focused on diversity in insular and previously under-investigated locations, such as New Caledonia (Sinniger 2008), the deep-sea (Reimer et al. 2007a), and the Galápagos (Reimer et al. 2008b(Reimer et al. , 2009)).
Recent investigations into the diversity of zoanthids in the Galápagos revealed the presence of several new zoanthid species groups (Reimer et al. 2008b).In this study, four species and one new genus of zoanthids from the Galápagos are formally described, and the proposed future of zoanthid research in the southeastern Pacifi c is briefl y discussed.

Materials and methods
Sample collection: Specimens were collected by hand intertidally, or by SCUBA or snorkeling from numerous sites at the Galápagos between June 2001 and March 2007.As specimens were collected, in situ digital images were taken to assist in identifi cation and morphological analyses (oral disk/polyp diameter, color, polyp form, etc.).After collection specimens were further examined, photographed, and preserved in 75% ethanol.Specimens in this study consist of specimens initially described as Parazoanthus sp.G1, Parazoanthus sp.G2, and Parazoanthus sp.G3 in Reimer et al. (2008bReimer et al. ( , 2010)).
DNA extraction and PCR amplifi cation: DNA was extracted as described in Reimer et al. (2008a), using a spin-column Dneasy Blood and Tissue Extraction protocol (Qiagen, Santa Clarita, CA, USA).Mitochondiral 16S ribosomal DNA (mt 16S rDNA) was amplifi ed using primers and protocol described in Sinniger et al. (2005), the cytochrome oxidase subunit I (COI) gene was amplifi ed following Reimer et al. (2004Reimer et al. ( , 2007a)), and the internal transcribed spacer region of ribosomal DNA (ITS-rDNA) following Reimer et al. (2007b).PCR amplifi cation procedures for each of the molecular markers were as given in the original references above.Amplifi ed products were visualized by 1.5% agarose gel electrophoresis.
Phylogenetic analyses: New sequences obtained in the present study (Table 1) were deposited in DDBJ and GenBank (accession numbers GU357551-GU357567).By using CLUSTAL X version 1.8 (Th ompson et al. 1997), the nucleotide sequences of mt 16S rDNA, COI, and ITS-rDNA from samples were aligned with previously published sequences (see Reimer et al. 2008b) from various zoanthid species representing the genera Zoanthus, Savalia, Corallizoanthus, Mesozoanthus, Hydrozoanthus, and Parazoanthus.
Th e outgroup sequences for both mt 16S rDNA and COI trees were from the genera Zoanthus and/or Palythoa.Th e only zoanthid genera not included in both analyses were Epizoanthus, previously shown to be basal in the order Zoantharia phylogeny for both mt 16S rDNA and COI (Sinniger et al. 2005;Reimer et al. 2007a); Abyssoanthus, demonstrated to be divergent from other zoanthids for both mt 16S rDNA and COI (Reimer et al. 2007a), and Sphenopus, which is in the same family as one of the out-groups of this study, Palythoa (family Sphenopidae).Th ese represent the current full range of described zoanthid genera.New mt 16S rDNA sequences from specimens in this study were clearly divergent when compared with Mesozoanthus sequences, but Mesozoanthus sequences from Sinniger and Häussermann (2009) were not included in trees presented in this study due to their relatively short length (469 base pairs).
Hydrozoanthidae ITS-rDNA sequences (particularly ITS-1 and ITS-2 spacers) were highly divergent from other obtained ITS-rDNA sequences, and thus initially an ITS-rDNA alignment consisting only of Hydrozoanthidae sequences with sequence AB214161 from Hydrozoanthus gracilis Carlgren (Reimer et al. 2007c) plus two new H. gracilis specimen sequences as the outgroup was generated.Th e new Galapagos specimens were shown to form a very well supported monophyletic group, and therefore to improve resolution, subsequently an alignment was created with only these sequences.An ITS-rDNA phylogeny from a previous study shows the monophylies and positions of non-Hydrozoanthidae species discussed in this study (Parazoanthus sp.G1, Parazoanthus sp.G2; both sensu Reimer et al. 2008b; see Figure 7 in Reimer et al. 2008b).
All alignments were inspected by eye and manually edited by removing all ambiguous sites (if present) of the alignments (e.g.sites present in either only forward or reverse directions, not seen in any other sequence) from the dataset for phylogenetic analyses, and aligning mt 16S rDNA and ITS-rDNA indels as in previous studies (Reimer et al. 2008b;Sinniger et al. 2009).Consequently, three alignment datasets were generated: 1) 651 sites of 39 sequences (mt 16S rDNA); 2) 280 sites of 45 sequences (COI); and 3) 595 sites of 14 sequences (ITS-rDNA).Th e alignment data are available on request from the corresponding author.
Th e alignments of mt 16S rDNA, COI, and ITS-rDNA were tested for optimal fi t of various nucleotide substitution models using jModelTest version 0.1.1 (Posoda 2008).For the mt 16S rDNA dataset, the general time reversible (GTR) model (Rodriguez et al. 1990) incorporating variable sites and a discrete gamma distribution (GTR + I + G) was suggested by jModelTest under Akaike Information Criterion (AIC), while the Hasegawa, Kishino and Yano model (Hasegawa et al. 1985) incorporating variable sites (HKY + I) was suggested for the COI dataset, and the K80 model (Kimura 1980) was suggested for the ITS-rDNA dataset.Th e maximum-likelihood (ML) analyses with PhyML (Guindon and Gascuel 2003) of these datasets were independently performed using an input tree generated by BIONJ (Gascuel 1997) with the models selected by jModelTest.PhyML bootstrap trees (1000 replicates) were constructed using the same parameters as the individual ML tree.
Bayesian trees were reconstructed by using the program MrBayes 3.1.2(Ronquist and Huelsenbeck 2003) under the K80 model incorporating variable sites of nucleotide substitution (K80 + I) for the mt DNA 16S rDNA and COI datasets, and under the Jukes and Cantor model (JC69; Jukes and Cantor 1969) for the ITS-rDNA dataset [all models selected by jModelTest under Bayes Information Criterion].One cold and three heated Markov chain Monte Carlo (MCMC) chains with default-chain temperatures were run for 20 million generations, sampling log-likelihoods (InLs), and trees at 1000-generation intervals (20,000 InLs and trees were saved during MCMC).Th e fi rst 15% of all runs were discarded as "burn-in" for all datasets.Th e likelihood plots for all three datasets also showed that MCMC reached the stationary phase by this time.Th us, the remaining 17,000 trees (17 million generations) of mt 16S rDNA, COI and ITS-rD-NA were used to obtain posterior probabilities and branch-length estimates, respectively.
Morphological analyses: Th e external morphology of specimens was examined using both preserved specimens and in situ images.Polyp dimensions (oral disk diameter, polyp height) for both in situ and preserved specimens were obtained, as were the following data: tentacle number, color of polyp, color(s) of oral disk, relative amount of sand encrustation, associated/substrate species.Additionally, the relative development of the coenenchyme was examined.
For internal morphological examinations, some specimens underwent initial decalcifi cation followed by desilifi cation as outlined in Reimer et al. (2010).After these treatments, specimens were dehydrated through an ethanol-xylene series.Some specimens in 100% ethanol were placed in vacuo for approximately 30 minutes to remove bubbles in the coelenteron.Th en, they were embedded in paraffi n.Serial sections of 5-10 μm thick were prepared with a rotary microtome and stained with Delafi eld's hematoxylin and eosin.
Obtained slides of HF-treated zoanthid specimens were examined with a light microscope (Nikon Express E50i).Th e following morphological characters and conditions were examined; mesentery condition, number, and form (in particular fi fth mesentery from dorsal directive complete or incomplete); presence or absence of drag marks from debris; presence or absence of sand and debris in mesoglea; overall condition of tissue and cells; and in particular ectoderm and endoderm; any other morphological characters of note (e.g.presence of gametes, etc.) (described in Reimer et al. 2010).
Nematocyst observation: Undischarged nematocysts were measured from tentacles, column, actinopharynx, and mesenterial fi laments of polyps (specimens examined n=2-4; polyp n=4-8) for all new species.400x images of the nematocysts were obtained by optical microscope, and measured using the software ImageJ (National Institutes of Health, USA).Nematocyst nomenclature generally followed England (1991), however both Schmidt (1974) and Hidaka and co-workers (1987;1992) have previously suggested basitrichs and mastigophores are same type of the nematocyst, and thus in this study, these two types were dealt with as the same type, unless they could be clearly distinguished from one another, in which case they were analyzed separately.
Other morphologically similar and undescribed zoanthids (epizoic on antipatharians, similar sizes, yellowish in color) have been recorded from Madagascar and Japan (specimens in JDR's collection), although these other specimens were found on different antipatharian species than Antipathozoanthus hickmani, and were never red or cream in color.
Antipathozoanthus hickmani is the only zoanthid in the Galápagos found on living Antipathes galapagensis (Table 3).
Habitat and distribution.All collected samples from Galapagos were on the black coral Antipathes galapagensis, at depths of 12 m to 35 m.Although A. galapagensis is found throughout the archipelago, Antipathozoanthus hickmani colonies were observed only at Santiago, Floreana, Isabela and Pinzon Islands, and it may be that this genus has a patchy distribution in the Galápagos.A. hickmani is potentially also found at Isla del Coco (Costa Rica) on the same antipatharian species, based on Museo de Zoologia, University of Costa Rica specimen UCR 827, although this has yet to be confi rmed with detailed examinations.
Biology and associated species.Antipathozoanthus hickmani may cover only a portion of a living Antipathes galapagensis black coral colony, or cover the entire colony, suggesting this species may be parasitic.Some A. hickmani specimens were found on completely dead A. galapagensis colonies or branches.
Notes.Previously mentioned in Reimer et al. (2008bReimer et al. ( , 2010) ) and Hickman (2008) as Parazoanthus sp.G1.Habitat and distribution.Similar to Terrazoanthus onoi sp.n. (below), specimens of Parazoanthus darwini are found on rock walls, in crevices, or at the base of rocks, and were found from depths of 2 m to ~30 m, and may extend deeper.Colonies of P. darwini were seen at Wolf, Marchena, Isabela, Fernandina, Santa Cruz, San Cristobal, Española and Floreana Islands, and its range is likely throughout the archipelago.

Genus
Biology and associated species.Collected Parazoanthus darwini specimens from Galapagos are often (but not always) associated with diff erent species of bright yellow-orange or red sponges, possibly in the groups Poecilosclerida and/or Hadromerida (T.Swain, personal communication).P. darwini colonies often grow in patches over the sponge, or may even cover it entirely, and often extend to surrounding rock substrate.Despite being covered by P. darwini, the sponge is always alive, suggesting this association may be symbiotic.
Notes.Despite COI and mt 16S rDNA sequences of this species being identical to sequences from Parazoanthus swiftii from the Caribbean (Figures 5a, 5b), slightly longer mt 16S rDNA sequences (Figure 2 in Reimer et al. 2007) were not identical.Additionally, due to the morphology of P. swiftii (rarely not on sponges, relatively shorter tentacles, large [6 mm] diameter polyps that often extend well out from coenenchyme) and large geographic distances between P. swiftii and P. darwini localities, it is clear that these are two diff erent species.
Previously mentioned in Reimer et al. (2008b) and Hickman (2008) as Parazoanthus sp.G2.Diagnosis.Sub-tropical to tropical Hydrozoanthidae that are found on rocky substrates, (e.g., not obligate symbionts with hydrozoans).Some species in this genus are brightly colored.

Family
Etymology.Named for the latin "terra" meaning "rock", the substrate on which species of this genus are commonly found on, with ending in concordance to other zoanthid genera.Gender neuter, as with other zoanthid genera ending in "-zoanthus".
Material examined.In addition, T. onoi is bigger (oral disk diameter and polyp height) than T. sinnigeri, and forms much larger colonies (Table 3).T. onoi commonly has only basitrichs and microbasic p-mastigophores in its pharynx, and no large or small holotrichs at all, unlike T. sinnigeri (Table 2).
Phylogenetically, Terrazoanthus onoi is very closely related to T. sinnigeri, with identical COI and mt 16S rDNA sequences, but consistently diff ers by four base pairs in ITS-rDNA, and forms a clade separate from T. sinnigeri.
An extensive literature search revealed no other described Parazoanthidae species from the Pacifi c that are non-epizoic and bright red in color.An undescribed zoanthid species inhabiting rock and coral reef substrata from Indonesia often referred to as "yellow polyps" (sensu Sinniger et al. 2005) is likely also a Terrazoanthus sp., but is distinct from T. onoi in terms of color and distribution, and is phylogenetically diff erent.
Habitat and distribution.Specimens of Terrazoanthus onoi were found on rock substrate in areas of high current (i.e., the base of large rocks, rock walls, etc.).Colonies were found at Darwin, Marchena, Genovesa, Isabela, Pinzon, Española, and Floreana Islands, and it is likely T. onoi is found throughout the archipelago.Th is species has been found from the low infra-littoral to depths of over 35 m, and is likely to be at even deeper depths.Biology and associated species.Found on the top surfaces of rocks and biogenic non-living substrate, Terrazoanthus onoi is often found close to sponges, seaweed, and other benthos, but is not epizoic and does not have an association with any particular species.
It should be noted that specimen MISE 02-27 was found to have an ITS-rDNA sequence inconsistent with other Terrazoanthus onoi specimens (Figure 6), although other data (morphology, mt 16S rDNA and COI data) fi t well with T. onoi.For these reasons, this specimen has not been conclusively assigned to T. onoi or to the other new Terrazoanthus species below.Th ese results indicate there may be other Terrazoanthus species in the Galápagos that await discovery and description.
Holotype: MHNG-INVE-67498.Colony divided into three pieces, on rocks of approximately 2.5 × 2.5 cm, 2.5 × 1.0 cm, and 2.0 × 1.5 cm, with heights of approximately 1.0 cm.Total of approximately 40 polyps connected by stolons.Polyps approximately 1.5-2.0mm in diameter, and approximately 1.0-2.0mm in height from coenenchyme.Polyps and coenenchyme encrusted with relatively large pieces of sand clearly visible to the naked eye, tissue of polyps and coenenchyme light brown/ grey in color.In situ, colony was on bottom of rock.Collected from Roca Espejo, Marchena I., Galapagos, Ecuador, at 9.1 m, collected by JDR, FL, and BR, March 3, 2007.Preserved in 99.5% ethanol.
Other material (all from Galapagos, Ecuador): MISE 464, Gardner, Floreana I., 27 m, collected by JDR and AC, March 13, 2007;MISE 471, Devil's Crown, Floreana I., 7 m, collected by JDR and AC, March 13, 2007; MISE 418, Punta Espejo, Marchena I., 7 m, collected by JDR, FL, CH, Sequences for new species in this study in larger font; sequences newly obtained in this study and new taxa described in this study in bold.Sequences/species names from previous studies in regular font.For specimen information see Morphology: Terrazoanthus sinnigeri has dull brown, white, or clear oral disks and the outer surface of polyps is heavily encrusted with large particles, with polyps clear of the stolon.Stolons are also heavily encrusted, and approximately the width of polyp diameters.T. sinnigeri has 30 to 36 tentacles that are almost as long or sometimes longer as the diameter of the expanded oral disk (Figure 4).Tentacles often much more transparent than oral disks (when colored).
Diff erential diagnosis.In the Galápagos, Terrazoanthus sinnigeri diff ers from Parazoanthus darwini and Antipathozoanthus hickmani by substrate preference (rock as opposed to sponges and anthipatharians, respectively), as well as from Terrazoanthus onoi sp.n. (above) by both color (brown, white or transparent as opposed to bright red) and microhabitat (under rocks and rubble as opposed to exposed rock surfaces).In addition, T. sinnigeri is smaller (oral disk diameter and polyp height) than congener T. onoi.T. sinnigeri colonies are stoloniferous and generally much smaller than colonies of T. onoi (Table 3).Terrazoanthus sinnigeri can be further distinguished from T. onoi by the presence of many types of nematocysts in the pharynx, unlike T. onoi, which only commonly possesses basitrichs and microbasic p-mastigophores with rare mediumsized holotrichs in the pharynx (Table 2).Terrazoanthus sinnigeri also has small holot-   2).Encrustations on the scapus of T. sinngeri are generally much larger than on T. onoi (compare Figures 3 and 4).
Terrazoanthus sinnigeri is phylogenetically very closely related to T. onoi, but has diff erent and unique ITS-rDNA (see T. onoi description; Figure 6).
Similar to Terrazoanthus sinnigeri, there have been reports of other small zoanthids inhabiting cryptic habitats under coral rubble and rock from the Galápagos, Singapore and Japan (J.D. Reimer, T. Fujii, personal observation), but these zoanthids are clearly diff erent in DNA sequence from all known Hydrozoanthidae and Parazoanthidae, and will be described elsewhere.Morphologically, these undescribed zoanthids look very similar to T. sinnigeri, but are often unitary (not colonial), are encrusted with very large pieces of sand, have very little coloring (usually lacking any color asides from around the oral opening) and have fewer tentacles (<26, usually 20-22; data not shown) than T. sinnigeri.
Habitat and distribution.Specimens located at depths of 7 to over 27 meters at Floreana, Marchena, Darwin, North Seymour Islands, and Bainbridge Rocks, with other potential specimens observed at other islands.It is likely that this species is widely distributed throughout the Galápagos, and its distribution may extend into deeper waters as it was often found at the lowest depth searched during collection dives.Generally found on the underside of rocks, rubble, or dead shells, often in small cracks or crevices.
Biology and associated species.Found under rocks and rubble, Terrazoanthus sinnigeri is often found nearby bryozoans and coralline algae, but appears to not be epizoic on any particular organism.
Notes.In Reimer et al. (2008b) it was originally thought that Terrazoanthus sinnigeri (specimens 02-09, 03-560) was a diff erent, white morphotype of T. onoi (mentioned in the paper and Hickman (2008) as Parazoanthus sp.G3) based on COI and mt 16S rDNA sequence data, but given the species' divergent morphologies, cnidae, and ecologies, as well as diff erent ITS-rDNA sequences, we describe them as closely related but distinct species.It is likely that these two sibling species have recently diverged from one another.
Although speculative, it may be that Terrazoanthus sinnigeri's preferred habitat under rocks has resulted in its lack of bright pigmentation or occasional total lack of pigments compared to bright red T. onoi, which is found in areas more exposed to light, similar as to seen in subterranean invertebrates (e.g.Leys et al. 2003), and this should be investigated in the future.

mt 16S rDNA
Th e phylogenetic tree from analyses of mt 16S rDNA showed two large clades; one consisting of Parazoanthidae specimens, and another of Hydrozoanthidae specimens  .00)that also included "yellow polyps" sensu Sinniger et al. (2005) and "Parazoanthus" sp.302, which both had slightly divergent sequences.

COI
COI phylogenetic results are shown in Figure 5b.Th e phylogenetic tree showed two large clades: one consisting of Parazoanthidae specimens, and another of Hydrozoanthidae specimens.Antipatharian-associated specimens previously informally described as Parazoanthus sp.G1 (04-140, 04-184) formed a well-supported (ML=98%, Bayes=1.00)clade together with an Antipathozoanthus macaronesicus specimen from Cape Verde.Th is clade was sister to Parazoanthus puertoricense West 1979 from Honduras.

"Parazoanthid" diversity
Traditionally, the higher-level taxonomy of zoanthids has relied on a wide variety of diagnostic characteristics, including mesenterial arrangement (Haddon and Shackleton 1891) and nematocysts (Schmidt 1974).Although suborders are organized based on the position of the sphincter muscle (mesodermal or endodermal), genera have been historically designated based on not only morphology, but also ecology and species associations.Th us, the recent reexamination and reclassifi cation of zoanthid taxa utilizing DNA sequences along with their ecology as diagnostic characters is not without historical precedent.From the results of this study along with data in Sinniger et al. (2009), Sinniger and Häussermann (2009) and Reimer et al. (2008a) the "parazoanthids" (the family Parazoanthidae as it formerly existed) are now divided into two families and eight genera (Parazoanthidae, including Parazoanthus Haddon & Shackleton, 1891, Savalia Nardo, 1814, Isozoanthus Chun, 1903, Corallizoanthus Reimer et al., 2008, Mesozoanthus Sinniger & Häussermann, 2009, Antipathozoanthus Sinniger et al., 2009;and Hydrozoanthidae, including Hydrozoanthus Sinniger et al., 2009 and Terrazoanthus gen. n.), refl ecting the formerly unknown levels of generic and family diversity that are present in this zoanthid group.It is apparent that Parazoanthidae as it formally existed was clearly a "catch-all" for many diff erent zoanthid species, as previously hypothesized (Reimer et al. 2008a).
It is also becoming increasingly apparent that zoanthid diversity is higher than previously thought in "ignored" or understudied regions, ecosystems (Reimer et al. 2007a) or even microhabitats (Reimer et al. 2008b, this study).Th is is clearly demonstrated by Terrazoanthus sinnigeri, which inhabits the underside of rocks and dead coral, a very common yet relatively understudied microhabitat.In subtropical seas, biological studies of zoanthids have focused mainly on coral reef areas, and in contrast studies of boulder/rubble areas have been neglected.Th is microhabitat has also recently been shown to host other previously undescribed invertebrate species, such as the comatulid Dorametra sesokonis Obuchi, Kogo & Fujita, 2009 in southern Japan (Obuchi et al. 2009).Additional specimens collected from the Galápagos also found from the undersides of rocks (specimen 03-103 in Reimer et al. 2008b;specimens 427, 455, 460 in JDR's collection from the 2007 expedition) apparently belong to one or more other undescribed zoanthid taxa, and will be described elsewhere.Until the discovery of such zoanthids (Reimer et al. 2008b), no information on zoanthids found in such a cryptic microenvironment had been reported, and it may be that this and other understudied microhabitats also harbor zoanthid species new to science.

Proposed future zoanthid research in the southeastern Pacifi c
Despite a few reports, zoanthid diversity over the entire southeastern Pacifi c remains understudied.Th is study and recent investigations from Chile (Sinniger and Häussermann 2009) demonstrate that undescribed zoanthid diversity exists in this region.In particular, very little data exist for the Pacifi c coast of South and Central America, and eff orts should be made to promote investigations in this area.With more knowledge of zoanthid diversity and distribution in the southeastern Pacifi c, more accurate biogeographical discussions of the evolution of parazoanthids and hydrozoanthids will become possible.

Conclusions
As shown by this research and other recent investigations (Sinniger et al. 2009), the levels of higher level (e.g.>genus) diversity of zoanthids are much higher than has previously been thought.
Insular and relatively unexplored marine regions of the world such as the Galapagos likely harbor many undiscovered and undescribed zoanthid species.
Phylogenetic analyses as performed here provide a powerful identifi cation tool that can determine relative levels of relationships between zoanthids that would not be possible with only morphological and ecological data.Th is is most strongly demonstrated by the very close evolutionary relationship between the two new Terrazoanthus species, which are genetically very close and morphologically and ecologically quite distinct.constructive comments throughout manuscript preparation.Drs.Euichi Hirose and Mamiko Hirose (University of the Ryukyus) are acknowledged for slide preparations.Timothy Swain (Florida State University) performed preliminary sponge spicule examinations.Th e fi rst author was supported in part by the Biological Institute on Kuroshio (Otsuki, Japan), the Rising Star Program at the University of the Ryukyus, and a grantin-aid ("Wakate B") from the Japan Society for the Promotion of Science.Th e second author was supported in part by the Coral Reef and Island Educational Research Fund from the University of the Ryukyus.

Figure 1 .
Figure 1.Antipathozoanthus hickmani sp.n. in situ in the Galapagos.a holotype MHNG-INVE-67495 showing the entire colony covering an Antipathes galapagensis, with living antipatharians visible in the background.Image by Angel Chiriboga (AC) b specimen MISE 441 at Don Ferdi, Bainbridge Rocks, Santiago I., at 23 m by JDR, March 9, 2007 c and d specimen MISE 474, Roca Onan.Pinzon I., at 35 m by AC.All scale bars: 1 cm except in a (10 cm).
available or data not acquired.a Specimens with the designations such as 03-560 are from 2001-2004 surveys (see Reimer et al. 2008b).Other specimens are from 2007 and have either specimen numbers (e.g.471) in JDR's collection, or museum type specimen numbers as given.Abbreviations: USNM: National Museum of Natural History, Smithsonian Institution, Washington, D.C., USA, CMNH: Chiba Prefectural Natural History Museum, Japan, MHNG: Natural History Museum of Geneva, Switzerland, MISE: Molecular Invertebrate Systematics and Ecology Laboratory, University of the Ryukyus, Nishihara, Okinawa, Japan.b Latitude and longitude values that are negative represent South and West values respectively, while positive values (latitude only) represent North values.c Collector abbreviations: CH = C. Hickman, Jr., LV = L. Vinueza, AC = A. Chiriboga, GE = G.Edgar, JDR = JD Reimer, RP = R. Pepolas, FL = F. Liss, BR = B. Riegl, DR = D. Ruiz, FR = F. Riveiria, OB = O.Breedy, MV = M. Vera.

Figure 5 .
Figure 5. Maximum likelihood (ML) trees of a mitochondrial 16S ribosomal DNA, and b cytochrome oxidase subunit I (COI) sequences for zoanthid specimens.Values at branches represent ML probabilities (>50%).Monophylies with more than 95% Bayesian posterior probabilities are shown by thick branches.Sequences for new species in this study in larger font; sequences newly obtained in this study and new taxa described in this study in bold.Sequences/species names from previous studies in regular font.For specimen information see Table1.
T. onoi does not (Table

Systematics Family Parazoanthidae Delage & Hérouard, 1901 Diagnosis:
Macrocnemic zoanthids that have an endodermal sphincter muscle.Many species in this family associated with other organisms as substrate.

Table 1 .
Examined zoanthid specimens for new species from the Galapagos Islands, and GenBank Accession Numbers.

Table 3 .
Sinniger et al. 2005 four new species of zoanthids from the Galapagos.specificinsertions and deletions in mt 16S rDNA, especially in the V5 region (as defi ned inSinniger et al. 2005) of this gene.Phylogenetically species are more closely related to brachycnemic zoanthids (especially from the genus Palythoa Lamouroux 1816) than to other parazoanthids.

Table 1 .
Description.Size: Polyps are approximately 2-8 mm in diameter when open, and rarely more than 10 mm in height.Colonies small, consisting of one polyp (unitary) to less than 50 polyps.