Molecular identification of symbiotic dinoflagellates (Symbiodinium spp.) from Palythoa spp. (Anthozoa: Hexacorallia) in Japan

In Japan, zooxanthellate Palythoa tuberculosa Klunzinger and Palythoa mutuki Verrill (Anthozoa: Hexacorallia: Zoantharia) are found over a 1,000 + km latitudinal range, often in environments where most other zooxanthellate anthozoans are not found (i.e. tidal lagoon pools, around shallow water hydrothermal vents, subtropical rocky shorelines). Sequences of internal transcribed spacer of ribosomal DNA (ITS-rDNA) of the symbiotic dinoflagellate genus Symbiodinium (zooxanthellae) Freudenthal (Order Suessiales) from P. tuberculosa and P. mutuki from several locations in Japan (34°11′N–24°16′N) were analysed. Unexpectedly, despite the ability of the genus Palythoa to be flexible in association with different Symbiodinium subclades, most (35 of 36) Palythoa investigated here specifically associate with subclade C1 and closely related types. Symbiodinium subclade C1 has been characterized as a “generalist” in terms of the ability to associate with a range of hosts, but present results suggest that subclade C1 may also be a “generalist” in terms of being able to live in a variety of environments over a latitudinal range. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Molecular evidence suggesting species in the zoanthid genera Palythoa and Protopalythoa (Anthozoa: Hexacorallia) are congeneric

Taxonomic status of the zoanthid genera Palythoa and Protopalythoa has been in question for almost a century. Separation of the two genera has been based on traditional morphological methods (colony and polyp form, nematocyst size and form, and number of septa), with Palythoa polyps embedded in a well developed coenenchyme and Protopalythoa polyps standing free and clear of the coenenchyme. Here we sequenced two mitochondrial regions, the cytochrome oxidase I (COI) gene and 16S ribosomal DNA (16S rDNA) genes, from Palythoa and Protopalythoa samples from various parts of the world and performed phylogenetic analyses of the sequence data. The phylogenetic trees for both COI and 16S rDNA from Palythoa and Protopalythoa show four monophyletic groups (designated Palythoa tuberculosa, Palythoa heliodiscus, Palythoa mutuki 1, and Palythoa mutuki 2), with levels of sequence divergence (COI and 16S rDNA divergence approximately 0.0 approximately 1.1%) similar to or lower than that previously found among congeneric species within the closely related genus Zoanthus. Surprisingly, sequence differences among Palythoa tuberculosa, Palythoa mutuki 1, and Palythoa mutuki 2 were negligible (0.0 approximately 0.2% for both COI and 16S rDNA), potentially indicating relationships below the species level. Our sequences align well with the few Palythoa and Protopalythoa sequences reported to date. These findings strongly indicate that our samples represent a minimum of two and possibly up to four species (the Palythoa tuberculosa - P. mutuki 1 - P. mutuki 2 group, and P. heliodiscus) within the genus Palythoa, and that the genus Protopalythoa is erroneous nomenclature.     

Molecular evidence suggesting interspecific hybridization in Zoanthus spp. (Anthozoa: Hexacorallia)

Interspecific hybridization has been proposed as a possible explanation for the incredible diversity seen in reef-dwelling corals, but until now little proof of such hybridization in other reef-dwelling anthozoans has been reported. Without further observation of hybridization, the question of such a phenomenon being widespread in Anthozoa remains. Here we have examined the mitochondrial cytochrome oxidase I gene (COI) and the nuclear internal transcribed spacer of ribosomal DNA (ITS-rDNA) from three species of the mass-spawning, encrusting anemone genus Zoanthus (Z. sansibaricus, Z. kuroshio, Z. gigantus) to investigate possible hybridization. The three species coexist at two of three sampling locations in southern Japan. Zoanthus spp. ITS-rDNA region spacers (ITS-1 and ITS-2) were shown to have very high rates of divergence. At locations where all three species co-existed, several of our sampled Z. sansibaricus individuals (with identical "sansi" COI sequences) possessed two very divergent (i.e., species-level difference) ITS-rDNA alleles, the expected "sansi" allele and the divergent "B" allele. Additionally, two Z. sansibaricus individuals possessed only "B" alleles despite having "sansi" COI sequences. These results indicate that Z. sansibaricus has possibly experienced interspecific hybridization at least once with a Zoanthus partner possessing the "B" allele, and that these resulting hybrids may also sexually reproduce, demonstrating potential hybridization occurring in the order Zoantharia (Hexacorallia).     

Symbiodiniaceae diversity and characterization of palytoxin in various zoantharians (Anthozoa,Hexacorallia)

Organisms Diversity & Evolution - Anemone-like animals belonging to the order Zoantharia are common anthozoans widely distributed from shallow to deep tropical and subtropical waters. Some...     

Genomic diversity of vibrios associated with the Brazilian coral Mussismilia hispida and its sympatric zoanthids (Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi)

Aims: A taxonomic survey of the vibrios associated with the Brazilian endemic coral Mussismilia hispida and the sympatric zoanthids (i.e. Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi). Methods and Results: Mucus of 54 cnidarian specimens collected in three different places at São Sebastião in two consecutive years (i.e. 2005 and 2006) was used for taxonomic characterization of the cnidarian microbiota. Ninety‐eight of the 151 vibrio isolates fell within the vibrio core group according to partial 16S rDNA sequences. We performed the sequencing of recA and pyrH genes of all vibrio isolates. The most abundant taxa belonged to the vibrio core group (Vibrio harveyi, Vibrio rotiferianus, Vibrio campbellii and Vibrio alginolyticus), Vibrio mediterranei (=Vibrio shillonii) and Vibrio chagasii. With the exception of V. chagasii which was found only in the mucus of M. hispida, the other species appeared in different hosts with no evidence for the presence of host‐specific clones or species. Using rep‐PCR analysis, we observed a high genomic heterogeneity within the vibrios. Each vibrio isolate generated a different rep‐PCR fingerprint pattern. There was a complete agreement between the grouping based on rep‐PCR and concatenated sequences of pyrH, recA and 16S rDNA, but the pyrH gene has the highest discriminatory power for vibrio species identification. Conclusion: The vibrio core group is dominant in the mucus of these cnidarians. There is a tremendous diversity of vibrio lineages within the coral mucus. pyrH gene sequences permit a clear‐cut identification of vibrios. Significance and Impact of the Study: The taxonomic resolution provided by pyrH (but not recA) appears to be enough for identifying species of vibrios and for disclosing putative new taxa. The vibrio core group appears to be dominant in the mucus of the Brazilian cnidarians. The overrepresentation of these vibrios may reflect as yet unknown ecological functions in the coral holobiont.     

Phylogeny of the highly divergent zoanthid family Microzoanthidae (Anthozoa,Hexacorallia) from the Pacific

Fujii, T. & Reimer, J. D. (2011). Phylogeny of the highly divergent zoanthid family Microzoanthidae (Anthozoa, Hexacorallia) from the Pacific. —Zoologica Scripta, 40, 418–431. In this study, one new family, one new genus and two new species of zoanthids from rubble zones spanning the temperate, subtropical and tropical Pacific Ocean are described. Two new species are described, Microzoanthus occultus sp. n. and Microzoanthus kagerou sp. n., both belonging to the new genus Microzoanthus and new family Microzoanthidae, and they can be clearly distinguished both morphologically and genetically from each other and other zoanthids by their very small size, reduced or absent stolon, habitat usually on the bottom side of rubble zone rocks, and divergent and distinct DNA (cytochrome oxidase subunit I, mitochondrial 16S ribosomal DNA, internal transcribed spacer region of ribosomal DNA) sequences. The phylogenetic analyses clearly show Microzoanthidae fam. n. to be genetically far different from all other hexacorallians at the order level, but the macrocnemic arrangement of mesenteries and other morphological characters (colonial specimens with narrow stolons, two rows of tentacles sand encrustation) clearly place these specimens within the order Zoantharia. This study demonstrates how it is highly likely the existence of many marine invertebrate taxa remains overlooked, and that widely distributed groups such as Microzoanthidae fam. n. remain to be discovered.     

Diversity and evolution in the zoanthid genus Palythoa (Cnidaria: Hexacorallia) based on nuclear ITS-rDNA

Previous phylogenetic studies based on mitochondrial DNA markers have suggested that the zoanthid genus Palythoa may consist of both Palythoa species (Palythoa tuberculosa) and species formerly assigned to the genus Protopalythoa (Palythoa mutuki, Palythoa heliodiscus). In the present study various Palythoa spp. samples collected primarily from southern Japan with additional samples from the Indo-Pacific and Caribbean Sea were examined. The nuclear internal transcribed spacer of ribosomal DNA (ITS-rDNA) was sequenced and aligned for phylogenetic analyses to further investigate the relationship between P. tuberculosa, P. mutuki, and P. heliodiscus. ITS-rDNA analyses showed species groups forming monophylies with similar topology but with much higher resolution than seen for mitochondrial phylogenetic analyses. The results also confirmed the very close relationship of P. tuberculosa and P. mutuki. Some specimens appeared to be a potentially undescribed Palythoa species (designated Palythoa sp. sakurajimensis). Additionally, ITS-rDNA sequences of P. mutuki and P. tuberculosa showed additive polymorphic site, demonstrating for the first time a potential history of reticulate evolution in Palythoa.     

Descriptions of two azooxanthellate Palythoa species (Subclass Hexacorallia,Order Zoantharia) from the Ryukyu Archipelago,southern Japan

Two new species of zoantharians (Hexacorallia, Zoantharia, Sphenopidae), Palythoa mizigama sp. n. and Palythoa umbrosa sp. n., are described from the Ryukyu Archipelago, southern Japan. Unlike almost all other known Palythoa spp., both species are azooxanthellate and inhabit low-light environments such as floors or sides of caves, crevasses, or hollows of shallow coral reefs. The two species were initially considered to be the same species from their similar habitat environments and highly similar morphological features. However, phylogenetic analyses of nuclear internal transcribed spacer (ITS) ribosomal DNA, mitochondrial 16S ribosomal DNA, and cytochrome oxidase subunit I (COI) sequences revealed that these two species have a genetically distant relationship within the genus Palythoa. Morphological characteristics, including polyp size, tentacle number, external/internal coloration, and types and sizes of cnidae were examined in this study. As a result, only tentacle coloration was found to be useful for the morphological distinction between the two species. Palythoa mizigama possesses white tentacles with black horizontal stripes while Palythoa umbrosa possesses white tentacles without any stripe patterns. Considering their distant phylogenetic relationship, it can be assumed that their unique yet similar morphological and ecological characteristics developed independently in each species as an example of parallel evolution.     

Identification of a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea)

beta-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal beta-d-N-glucosamine and beta-d-N-galactosamine residues from oligosaccharides. In this report, we purified a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from the marine zoanthid Palythoa caribaeorum by applying ammonium sulfate fractionation, affinity chromatography on a chitin column, followed by two rounds of size exclusion chromatography. SDS-PAGE analysis indicated a single band protein of apparent homogeneity with a molecular mass of 25kDa. The purified enzyme preferentially hydrolyzed p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylgalactosamide (pNP-GalNAc). Detailed kinetic analysis using pNP-GlcNAc resulted in a specific activity of 57.9 U/mg, a K(m) value of 0.53 mM and a V(max) value of 88.1 micromol/h/mg and k(cat) value of 0.61s(-1). Furthermore, purified Pcb-NAHA1 enzyme activity was decreased by Hg Cl(2) or maltose and stimulated in the presence of Na(2)SeO(4,) BaCl(2), MgCl(2,) chondroitin 6-sulfate, and phenylmethylsulfonylfluoride. The optimum activity of Pcb-NAHA1 was observed at pH 5.0 and elevated temperatures (45-60 degrees C). Direct sequencing of proteolytic fragments generated from Pcb-NAHA1 revealed remarkable similarities to plant chitinases, which belong to family 18, although no chitinase activity was detected with Pcb-NAHA1. We conclude that beta-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor.     

Reproductive biology of Palythoa caribaeorum and Protopalythoa valilbilis (Cnidaria, Anthozoa, Zoanthidea) from the southeastern coast of Brazil.

The reproductive biology of Palythoa caribaeorum (Duchassaing & Michelotti 1860) and Protopalythoa variabilis (Duerden 1898) was studied through monthly samples from tagged colonies from June 1996 to June 1997, in Sao Sebastiao channel, S?o Paulo, Brazil (45 degrees 26'W, 23 degrees 50'S). The gametogenesis was similar to that of other zoanthids as shown by histological preparations. Oocyte diameters and maturation stages of testis vesicles were evaluated on squash preparations. Both species showed sequential protogynic hermaphroditism, with high frequency of fertile polyps (83% in P. variabilis and 72% in P. caribaeorun), high frequency of colonies in female sex condition (65.3% of P variabilis and 41.7% of P caribaeorum), and apparently continuous gametogenesis. In P. caribaribaeonrum, egg release was continuous and sperm release took place during half of the analyzed period. In P. variabilis, egg and sperm release occurred in April-May and February-March 1997, respectively.     

Timing of spawning and early development of Palythoa tuberculosa (Anthozoa, Zoantharia, Sphenopidae) in Okinawa, Japan

The spawning behavior and early embryogenesis of Palythoa tuberculosa (Anthozoa, Zoantharia) were observed in August 2009 off Okinawa Island, Japan. P. tuberculosa released zygotes just after high tide around new moon nights. The mean diameter of zygotes was 365.6 ± s.d.14.8 μm, and zygotes did not contain any symbiotic algae (zooxanthellae). About 2 h after spawning, the first cleavage furrow appeared on one side of the zygotes, although it was uncertain when eggs were fertilized. After second cleavage, the arrangement of blastomeres was pseudospherical. At 9 h after spawning, the embryo became a concave-convex dish shape, then gastrulation occurred and the blastopore was formed. Seven-day old larvae were ellipsoid and about 700 μm long, with an open mouth at one end. Two weeks after spawning, the larvae developed a longitudinal band of long cilia (= ventral ciliate band) that is characteristic of zoanthella larvae. In P. tuberculosa, larvae show a non-radial body plan and then metamorphose to almost-radial (in outward appearance) polyps after settlement. These results may support a hypothesis that a common ancestor of Cnidaria had a bilateral body plan that has been secondarily lost in some extant cnidarians.     

Karyotypic mosaicism and molecular cytogenetic markers in the scleractinian coral Acropora pruinosa Brook, 1982 (Hexacorallia,Anthozoa, Cnidaria)

Coral Reefs - Molecular cytogenetic investigation was carried out on the scleractinian coral, Acropora pruinosa (A. pruinosa). Conventional Giemsa staining techniques for karyotyping, such as G-...     

Unexpected diversity and a new species of Epizoanthus (Anthozoa,Hexacorallia) attached to eunicid worm tubes from the Pacific Ocean

Epizoanthus species are generally found in association with other marine invertebrates such as hermit crabs and gastropods. Although Epizoanthus spp. are relatively common, there is limited information about their diversity and ecology due to their habitats or hosts, often being below the depths of SCUBA diving (>~50 m). In particular, the Epizoanthus fauna of the Indo-Pacific Ocean remains poorly understood. In this study, the diversity of Epizoanthus species associated with eunicid worm tubes from shallow waters in the Pacific Ocean we investigated using molecular analyses (mitochondrial cytochrome oxidase subunit 1 = COI, mitochondrial 16S ribosomal DNA = mt 16S-rDNA, nuclear internal transcribed spacer region of ribosomal DNA = ITS-rDNA) combined with morphological and ecological data. The combined data set leads us to describe two new species; Epizoanthus inazuma sp. n. and Epizoanthus beriber sp. n. Both new species are found in low-light environments: Epizoanthus inazuma sp. n. on mesophotic coral reef slopes and reef floors, or on the sides of overhangs; Epizoanthus beriber sp. n. has only been found in caves. Morphological characteristics of these two new species are very similar to Epizoanthus illoricatus Tischbierek, 1930 but the two new species are genetically distinct. Mesentery numbers and coloration of polyps may be useful diagnostic characteristics among eunicid-associated Epizoanthus species. These results demonstrate that there is high potential for other potentially undescribed zoantharian species, particularly in underwater cave habitats.     

Zoanthids (Anthozoa, Hexacorallia) from the Great Barrier Reef and Torres Strait, Australia: systematics, evolution and a key to species

 Zoanthid taxonomy is currently in a state of chaos, with many described species very few of which can be reliably identified. As part of a genetically based, objective reappraisal of the number of northern Australian species, a total of 355 zoanthid specimens were collected from 19 localities in the Great Barrier Reef and Torres Strait during 1992–1994. Specimens, initially assigned to one of ten morphological or ecological forms, were subjected to allozyme electrophoretic analysis. Analysis of genetic data revealed only seven discrete (i.e., non-interbreeding) groups in the family Zoanthidae. These groups, which are delimited by fixed gene differences, are considered species under a biological species concept. Some species show considerable morphological variation and have broad environmental tolerances. We provide the first key to Great Barrier Reef zoanthid species based on our results and observations. Species can be differentiated in the field on the basis of gross colony morphology, sand encrusting habit, polyp form and habitat. Genetic data are also used to generate a phylogenetic hypothesis of relationships among the Zoanthidae, which is compared to previous morphologically based systems. Division of the group on the basis of mesenterial arrangement appears justified, but nematocyst data appear less valuable in phylogenetic studies of the group than has been suggested previously. Accepted: 29 January 1996     

Morphological and molecular revision of Zoanthus (Anthozoa: Hexacorallia) from southwestern Japan, with descriptions of two new species

No clear method of identifying species in the zoanthid genus Zoanthus has been established, due in part to the morphological plasticity of this genus (e.g., in polyp and colony form, oral disk color, tentacle number). Previous research utilizing the mitochondrial cytochrome oxidase I gene (COI) as a phylogenetic marker indicated that Zoanthus spp. in Japan may consist of only one or two species, despite a bewildering variety of observed morphotypes. Here we have utilized not only COI but also mitochondrial 16S ribosomal DNA (mt 16S rDNA) in order to clarify the extent of Zoanthus species diversity in southern Japan. Our molecular genetic results clearly show the presence of three monophyletic Zoanthus species groups with varying levels of morphological plasticity, including the new species Z. gigantus n. sp. and Z. kuroshio n. sp. We describe all three species found in this study, and identify potential morphological characters (coenenchyme and polyp structure as well as polyp external surface pigmentation patterns) useful in Zoanthus species identification. A morphological dichotomous key is provided to assist in field species identification.     

High levels of morphological variation despite close genetic relatedness between Zoanthus aff. vietnamensis and Zoanthus kuroshio (Anthozoa: Hexacorallia)

Recent investigations into the encrusting anemone genus Zoanthus using molecular and morphological techniques have begun to bring order to this taxonomically neglected group. Previous studies have confirmed the existence of three distinct species present in southern Japan: Z. sansibaricus, Z. kuroshio, and Z. gigantus. Results from such studies show species of Zoanthus to be highly morphologically plastic, often incorporating morphotypes with varying oral disk color and oral disk diameter. Literature lists the species Z. aff. vietnamensis as occurring in southern Japan and throughout the western Pacific Ocean, but due to the morphological plasticity of Zoanthus species, a re-examination of Z. aff. vietnamensis using molecular techniques was needed. Here, using mitochondrial 16S rDNA and the nuclear internal transcribed spacer of ribosomal DNA (ITS-rDNA) sequences, as well as morphological data, we have examined several nominal Z. aff. vietnamensis samples collected from Kagoshima Bay and Yakushima Island, Japan. Based on polyp length and diameter, oral disk diameter, mesentery and tentacle numbers, and colony form, Z. aff. vietnamensis is easily distinguishable from Z. sansibaricus, Z. kuroshio, and Z. gigantus. However, despite these clear morphological differences, our mitochondrial and nuclear sequence-based phylogenies indicate that Z. aff. vietnamensis and Z. kuroshio are very closely related (perhaps conspecific), highlighting the morphological plasticity of this genus and the difficulty of species identification based on morphological data alone.     

Shallow-water zoantharians (Cnidaria,Hexacorallia) from the Central Indo-Pacific

Despite the Central Indo-Pacific (CIP) and the Indonesian Archipelago being a well-known region of coral reef biodiversity, particularly in the ‘Coral Triangle’, little published information is available on its zoantharians (Cnidaria: Hexacorallia: Zoantharia). In order to provide a basis for future research on the Indo-Pacific zoantharian fauna and facilitate comparisons between more well-studied regions such as Japan and the Great Barrier Reef, this report deals with CIP zoantharian specimens in the Naturalis collection in Leiden, the Netherlands; 106 specimens were placed into 24 morpho-species and were supplemented with 88 in situ photographic records from Indonesia, the Philippines, and Papua New Guinea. At least nine morpho-species are likely to be undescribed species, indicating that the region needs more research in order to properly understand zoantharian diversity within the CIP. The Naturalis’ zoantharian specimens are listed by species, as well as all relevant collection information, and in situ images are provided to aid in future studies on zoantharians in the CIP.