Phylogenetic relationships within the tropical soft coral genera Sarcophyton and Lobophytum (Anthozoa, Octocorallia)

Abstract. The alcyonacean soft coral genera Sarcophyton and Lobophytum are conspicuous, ecologically important members of shallow reef communities throughout the Indo-West Pacific. Study of their ecology is, however, hindered by incomplete knowledge of their taxonomy: most species cannot be identified in the field and the two genera cannot always be distinguished reliably. We used a 735-bp fragment of the octocoral-specific mitochondrial protein-coding gene msh1 to construct a phylogeny for 92 specimens identified to 19 species of Lobophytum and 16 species of Sarcophyton . All phylogenetic methods used recovered a tree with three strongly supported clades. One clade included only morphologically typical Sarcophyton species with a stalk distinct from the polypary, poorly formed club-shaped sclerites in the colony surface, and large spindles in the interior of the stalk. A second clade included only morphologically typical Lobophytum colonies with lobes and ridges on the colony surface, poorly formed clubs in the colony surface, and interior sclerites consisting of oval forms with regular girdles of ornamental warts. The third distinct clade included a mix of Sarcophyton and Lobophytum nominal species with intermediate morphologies. Most of the species in this mixed clade had a polypary that was not distinct from the stalk, and the sclerites in the colony surface were clubs with well-defined heads. Within the Sarcophyton clade, specimens identified as Sarcophyton glaucum belonged to six very distinct genetic sub-clades, suggesting that this morphologically heterogeneous species is actually a cryptic species complex. Our results highlight the need for a complete taxonomic revision of these genera, using molecular data to help confirm species boundaries as well as to guide higher taxonomic decisions.     

Symbiodinium clade C dominates zooxanthellate corals (Scleractinia) in the temperate region of Japan

Endosymbiotic algae of the genus Symbiodinium have been divided into nine clades (A-I) following genetic classification; some clades are known to have physiological properties that enable the coral hosts to adapt to different environmental conditions. To understand the relationships of coral-alga symbioses, we focused on Symbiodinium diversity in zooxanthellate corals living under the severe environmental conditions of the temperate region (30°-35°N) of Japan. We investigated Symbiodinium clades in 346 colonies belonging to 58 coral species from six locations. We then selected three coral species-Acropora hyacinthus, Acropora japonica, and Cyphastrea chalcidicum-to investigate whether Symbiodinium clades changed during winter or summer over the course of year (May 2009-Apr 2010) in Tanabe Bay, Japan. Three Symbiodinium clades (C, D, and F) were detected in corals in the temperate region. Notably, 56 coral species contained Symbiodinium clade C. Oulastrea crispata predominantly contained clade D, but traces of clade C were also detected in all samples. The temperate-specific species Alveopora japonica contained clades C and F simultaneously. Seasonal change of symbiont clades did not occur in the three coral species during the investigation period where SSTs range on 12.5-29.2°C. However, we found Acropora (2 spp.) and Cyphastrea (1 sp.) contained different subcladal types of clade C. These results reveal that most coral species harbored Symbiodinium clade C stably throughout the year, suggesting that Symbiodinium clade C shows low-temperature tolerance, and that two hypothetical possibilities; genetic differences of subcladal types generating physiological differences or wide physiological flexibility in the clade C.     

Specificity is rarely absolute in coral-algal symbiosis: implications for coral response to climate change

Some reef-building corals have been shown to respond to environmental change by shifting the composition of their algal symbiont (genus Symbiodinium) communities. These shifts have been proposed as a potential mechanism by which corals might survive climate stressors, such as increased temperatures. Conventional molecular methods suggest this adaptive capacity may not be widespread because few (～25%) coral species have been found to associate with multiple Symbiodinium clades. However, these methods can fail to detect low abundance symbionts (typically less than 10-20% of the total algal symbiont community). To determine whether additional Symbiodinium clades are present, but are not detected using conventional techniques, we applied a high-resolution, real-time PCR assay to survey Symbiodinium (in clades A-D) from 39 species of phylogenetically and geographically diverse scleractinian corals. This survey included 26 coral species thought to be restricted to hosting a single Symbiodinium clade ('symbiotic specialists'). We detected at least two Symbiodinium clades (C and D) in at least one sample of all 39 coral species tested; all four Symbiodinium clades were detected in over half (54%) of the 26 symbiotic specialist coral species. Furthermore, on average, 68 per cent of all sampled colonies within a given coral species hosted two or more symbiont clades. We conclude that the ability to associate with multiple symbiont clades is common in scleractinian (stony) corals, and that, in coral-algal symbiosis, 'specificity' and 'flexibility' are relative terms: specificity is rarely absolute. The potential for reef corals to adapt or acclimatize to environmental change via symbiont community shifts may therefore be more phylogenetically widespread than has previously been assumed.     

Chemical and molecular characterization of Phomopsis and Cytospora-like endophytes from different host plants in Brazil

Phomopsis and related taxa comprise important endophytic and plant pathogenic species, and are known for the production of a diverse array of secondary metabolites. Species concepts within this group based on morphological characters and assumed host specificity do not reflect phylogenetic affinities. Additional phenotypic characters, such as profiles of secondary metabolites, are needed for practical species recognition. We investigated 36 strains of Phomopsis spp. and Cytospora-like fungi, obtained as endophytes of different host plants in Brazil, using metabolite profiling based on HPLC-UV/liquid chromatography -mass spectrometry (LC-MS) combined with cluster analysis of the results. Strains were also subjected to phylogenetic analyses based on internal transcribed spacer (ITS) rDNA. Six chemotypes were identified. Chemotypes 1-5 contained Phomopsis strains, while Cytospora-like strains formed the chemotype 6. Strains of chemotype 1 typically produced alternariols, altenusin, altenuene, cytosporones, and dothiorelones. Alternariol and seven unknown compounds were consistently produced by strains of chemotype 2. Members of chemotypes 3-5 produced poor metabolite profiles containing few chemical markers. Cytospora-like endophytes (chemotype 6) produced a characteristic set of metabolites including cytosporones and dothiorelones. Bayesian and Maximum Parsimony (MP) trees classified strains of each chemotype into single phylogenetic lineages or closely related groups. Strains of chemotypes 1 and 2 formed a monophyletic group along with Diaporthe neotheicola. The remaining Phomopsis strains formed monophyletic (chemotype 4) or polyphyletic (chemotypes 3 and 5) lineages inside a large and well supported clade. Cytospora-like strains formed a monophyletic lineage located at an intermediary position between Diaporthe/Phomopsis and Valsa/Cytospora clades. The combined results show that the production of secondary metabolites by Phomopsis and related Diaporthales may be species-specific, giving support to the use of metabolite profiling and chemical classification for phenotypic recognition and delimitation of species.     

Patterns of coral-dinoflagellate associations in Acropora: significance of local availability and physiology of Symbiodinium strains and host-symbiont selectivity

Like other reef-building corals, members of the genus Acropora form obligate endosymbioses with dinoflagellates (zooxanthellae) belonging to the genus Symbiodinium. Both Symbiodinium and its hosts are diverse assemblages, and the relationships between host and algal genotypes are unclear. In this study, we determined phylogenetic relationships between Symbiodinium isolates from a wide range of Acropora species and plotted the algal genotypes onto a molecular phylogeny of 28 Acropora species, using the same samples for the host and symbiont genotyping. In addition, we performed a preliminary survey of zooxanthella distribution in Acropora species from the central Great Barrier Reef. Three of the four known major zooxanthellae clades were represented in the 168 samples examined, and within the major clade C, three distinct subclades were identified. No evidence was found for coevolution, but several clear patterns of specificity were identified. Moreover, composition of the zooxanthella pool varied among locales and in one host species we found light-related patterns of zooxanthella distribution.     

Semisynthetic analogues of the marine cembranoid sarcophine as prostate and breast cancer migration inhibitors

Sarcophine (1) is a bioactive cembranoid diterpene isolated from the Red Sea soft coral Sarcophyton glaucum. Previous semisynthesis attempts resulted in decreased or complete loss of 1's anticancer activity. Sarcophine and analogues showed antimigratory activity against breast and prostate cancer cell lines. This encouraged further semisynthestic optimizations to improve its activity and establish a preliminary structure-activity relationship. Eight new and five known semisynthetic analogues were generated. These compounds were evaluated for their ability to inhibit growth, proliferation, and migration of the prostate and breast metastatic cancer cell lines PC-3 and MDA-MB-231, respectively. Most analogues exhibited enhanced antimigratory activity.     

Patterns of distribution of calcite crystals in soft corals sclerites

The gross morphology of soft coral surface sclerites has been studied for taxonomic purposes for over a century. In contrast, sclerites located deep in the core of colonies have not received attention. Some soft coral groups develop massive colonies, in these organisms tissue depth can limit light penetration and circulation of internal fluids affecting the physiology of coral tissues and their symbiotic algae; such conditions have the potential to create contrasting calcifying conditions. To test this idea, we analyzed the crystal structure of sclerites extracted from different colony regions in selected specimens of zooxanthellate and azooxanthellate soft corals with different colony morphologies, these were: Sarcophyton mililatensis, Sinularia capillosa, Sinularia flexibilis, Dendronephthya sp. and Ceeceenus levis. We found that the crystals that constitute polyp sclerites differ from those forming stalk sclerites. We also observed different crystals in sclerites located at various depths in the stalk including signs of sclerite breakdown in the stalk core region. These results indicate different modes of calcification within each colonial organism analyzed and illustrate the complexity of organisms usually regarded as repetitive morphological and functional units. Our study indicates that soft corals are ideal material to study natural gradients of calcification conditions. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Low genetic diversity of symbiotic dinoflagellates (Symbiodinium) in scleractinian corals from tropical reefs in southern Hainan Island, China

Endosymbiotic dinoflagellates in the genus Symbiodinium are among the most abundant and important group of photosynthetic protists found in coral reef ecosystems.In order to further characterize this diversity and compare with other regions of the Pacific,samples from 44 species of scleractinian corals representing 20 genera and 9 families,were collected from tropical reefs in southern Hainan Island,China.Denaturing gradient gel electrophoresis fingerprinting of the ribosomal internal transcribed spacer 2 identified 11 genetically distinct Symbiodinium types that have been reported previously.The majority of reef-building coral species (88.6％) harbored only one subcladal type of symbiont,dominated by host-generalist C1 and C3,and was influenced little by the host’s apparent mode of symbiont acquisition.Some species harbored more than one clade of Symbiodinium (clades C,D) concurrently.Although geographically isolated from the rest of the Pacific,the symbiont diversity in southern Hainan Island was relatively low and similar to both the Great Barrier Reef and Hawaii symbiont assemblages (dominated by clade C Symbiodinium).These results indicate that a specialist symbiont is not a prerequisite for existence in remote and isolated areas,but additional work in other geographic regions is necessary to test this idea.     

Diversity and distribution of symbiodinium associated with seven common coral species in the Chagos Archipelago, central Indian Ocean

The Chagos Archipelago designated as a no-take marine protected area in 2010, lying about 500 km south of the Maldives in the Indian Ocean, has a high conservation priority, particularly because of its fast recovery from the ocean-wide massive coral mortality following the 1998 coral bleaching event. The aims of this study were to examine Symbiodinium diversity and distribution associated with scleractinian corals in five atolls of the Chagos Archipelago, spread over 10,000 km(2). Symbiodinium clade diversity in 262 samples of seven common coral species, Acropora muricata, Isopora palifera, Pocillopora damicornis, P. verrucosa, P. eydouxi, Seriatopora hystrix, and Stylophora pistillata were determined using PCR-SSCP of the ribosomal internal transcribed spacer 1 (ITS1), PCR-DDGE of ITS2, and phylogenetic analyses. The results indicated that Symbiodinium in clade C were the dominant symbiont group in the seven coral species. Our analysis revealed types of Symbiodinium clade C specific to coral species. Types C1 and C3 (with C3z and C3i variants) were dominant in Acroporidae and C1 and C1c were the dominant types in Pocilloporidae. We also found 2 novel ITS2 types in S. hystrix and 1 novel ITS2 type of Symbiodinium in A. muricata. Some colonies of A. muricata and I. palifera were also associated with Symbiodinium A1. These results suggest that corals in the Chagos Archipelago host different assemblages of Symbiodinium types then their conspecifics from other locations in the Indian Ocean; and that future research will show whether these patterns in Symbiodinium genotypes may be due to local adaptation to specific conditions in the Chagos.     

Distribution of sarcophytol A in soft coral of the Sarcophyton genus

The distribution of sarcophytol-A in the Sarcophyton genus was investigated in seven samples belonging to S. glaucum (3 samples), S. infundibulifurme (2 samples), S. crassocaule (1 sample) and S. trocheliophorum (1 sample) that were collected on Ishigaki Island in Okinawa Prefecture. Sarcophytol-A was present in one sample each of S. glaucum and S. infundibulifurme. This study indicates that the composition of cembranoids in the Sarcophyton genus is not related with the respective species, but with the individual samples collected.     

Symbiodinium diversity among host clionaid sponges from Caribbean and Pacific reefs: Evidence of heteroplasmy and putative host-specific symbiont lineages

Among the Porifera, symbiosis with Symbiodinium spp. (i.e., zooxanthellae) is largely restricted to members of the family Clionaidae. We surveyed the diversity of zooxanthellae associated with sponges from the Caribbean and greater Indo-Pacific regions using chloroplast large subunit (cp23S) domain V sequences. We provide the first report of Clade C Symbiodinium harbored by a sponge (Cliona caesia), and the first report of Clade A Symbiodinium from an Indo-Pacific sponge (C. jullieni). Clade A zooxanthellae were also identified in sponges from the Caribbean, which has been reported previously. Sponges that we examined from the Florida Keys all harbored Clade G Symbiodinium as did C. orientalis from the Indo-Pacific, which also supports earlier work with sponges. Two distinct Clade G lineages were identified in our phylogenetic analysis; Symbiodinium extracted from clionaid sponges formed a monophyletic group sister to Symbiodinium found in foraminiferans. Truncated and 'normal' length variants of 23S rDNA sequences were detected simultaneously in all three morphotypes of C. varians providing the first evidence of chloroplast-based heteroplasmy in a sponge. None of the other sponge species examined showed evidence of heteroplasmy. As in previous work, length variation in cp23S domain V sequences was found to correspond in a highly precise manner to finer resolution of phylogenetic topology among Symbiodinium clades. On a global scale, existing data indicate that members of the family Clionaidae that host zooxanthellae can form symbiotic associations with at least four Symbiodinium clades. The majority of sponge hosts appear to harbor only one cladal type of symbiont, but some species can harbor more than one clade of zooxanthellae concurrently. The observed differences in the number of partners harbored by sponges raise important questions about the degree of coevolutionary integration and specificity of these symbioses. Although our sample sizes are small, we propose that one of the Clade G lineages identified in this study is comprised of sponge-specialist zooxanthellae. These zooxanthellae are common in Caribbean sponges, but additional work in other geographic regions is necessary to test this idea. Sponges from the Indo-Pacific region harbor zooxanthellae from Clades A, C, and G, but more sponges from this region should be examined.     

Recognizing diversity in coral symbiotic dinoflagellate communities

A detailed understanding of how diversity in endosymbiotic dinoflagellate communities maps onto the physiological range of coral hosts is critical to predicting how coral reef ecosystems will respond to climate change. Species-level taxonomy of the dinoflagellate genus Symbiodinium has been predominantly examined using the internal transcribed spacer (ITS) region of the nuclear ribosomal array (rDNA ITS2) and downstream screening for dominant types using denaturing gradient gel electrophoresis (DGGE). Here, ITS2 diversity in the communities of Symbiodinium harboured by two Hawaiian coral species was explored using direct sequencing of clone libraries. We resolved sixfold to eightfold greater diversity per coral species than previously reported, the majority of which corresponds to a novel and distinct phylogenetic lineage. We evaluated how these sequences migrate in DGGE and demonstrate that this method does not effectively resolve this diversity. We conclude that the Porites spp. examined here harbour diverse assemblages of novel Symbiodinium types and that cloning and sequencing is an effective methodological approach for resolving the complexity of endosymbiotic dinoflagellate communities harboured by reef corals.     

Macroalgal-associated dinoflagellates belonging to the genus Symbiodinium in Caribbean reefs

Coral-algal symbiosis has been a subject of great attention during the last two decades in response to global coral reef decline. However, the occurrence and dispersion of free-living dinoflagellates belonging to the genus Symbiodinium are less documented. Here ecological and molecular evidence is presented demonstrating the existence of demersal free-living Symbiodinium populations in Caribbean reefs and the possible role of the stoplight parrotfish (Sparisoma viride) as Symbiodinium spp. dispersers. Communities of free-living Symbiodinium were found within macroalgal beds consisting of Halimeda spp., Lobophora variegata, Amphiroa spp., Caulerpa spp. and Dictyota spp. Viable Symbiodinium spp. cells were isolated and cultured from macroalgal beds and S. viride feces. Further identification of Symbiodinium spp. type was determined by length variation in the Internal Transcribed Spacer 2 (ITS2, nuclear rDNA) and length variation in domain V of the chloroplast large subunit ribosomal DNA (cp23S-rDNA). Determination of free-living Symbiodinium and mechanisms of dispersal is important in understanding the life cycle of Symbiodinium spp.     

Marine sterols. VIII. Isolation and structure of sarcosterol, a new sterol with a delta17(20)-double bond from the soft coral Sarcophyton glaucum.

The sterol mixture of the southern Japan's soft coral, Sarcophyton glaucum, was found to contain 11 sterols including a novel sterol, 23,24 xi-dimethylcholesta-5,22-dien-3 beta-ol and a new diunsaturated C29 sterol. 22,23-Dihydrobrassicasterol and gorgosterol were the major components in free- and esterified sterols respectively. Brassicasterol was found in S. glaucum, in contrast to the ubiquity of 24-epibrassicasterol in the marine invertebrates in the northern districts. The new sterol (sarcosterol) was isolated; its structure as 23 xi, 24 xi-dimethylcholesta-5, 17(20)-trans-dien-3 beta-ol was based on spectra evidence and comparison with cholesta-5, 17(20)-trans-dien-3 beta-ol.     

Phylogenetic relationships among zooxanthellae (Symbiodinium) associated to excavating sponges (Cliona spp.) reveal an unexpected lineage in the Caribbean

Phylogenetic relationships of symbiotic dinoflagellate lineages, distributed in all tropical and subtropical seas, suggest strategies for long distance dispersal but at the same time strong host specialization. Zooxanthellae (Symbiodinium: Dinophyta), which are associated to diverse shallow-water cnidarians, also engage in symbioses with some sponge species of the genus Cliona. In the Caribbean, zooxanthellae-bearing Cliona has recently become abundant due to global warming, overfishing, and algae abundance. Using molecular techniques, the symbionts from five excavating species (Clionacaribbaea, C. tenuis, C. varians, C. aprica and C. laticavicola) from the southern and southwestern Caribbean were surveyed. Several DNA sequence regions were used in order to confirm zooxanthellae identity; 18S rDNA, domain V of chloroplast large subunit (cp23S), internal transcribed spacer 2 (ITS2), and ITS2 secondary structure. Sequence analyses corroborated the presence of three zooxanthellae clades: A, B, and G. Presence of clades A and B in common boring sponges of the Caribbean fit with the general pattern of the province. The discovery of clade G for the first time in any organism of the Atlantic Ocean leads us to consider this unusual finding as a phylogenetic relict through common ancestors of sponge clades or an invasion of the sponge from the Indo-Pacific.     

Real-time PCR reveals a high incidence of <Emphasis Type="Italic">Symbiodinium </Emphasis>clade D at low levels in four scleractinian corals across the Great Barrier Reef: implications for symbiont shuffling

Reef corals form associations with an array of genetically and physiologically distinct endosymbionts from the genus Symbiodinium. Some corals harbor different clades of symbionts simultaneously, and over time the relative abundances of these clades may change through a process called symbiont shuffling. It is hypothesized that this process provides a mechanism for corals to respond to environmental threats such as global warming. However, only a minority of coral species have been found to harbor more than one symbiont clade simultaneously and the current view is that the potential for symbiont shuffling is limited. Using a newly developed real-time PCR assay, this paper demonstrates that previous studies have underestimated the presence of background symbionts because of the low sensitivity of the techniques used. The assay used here targets the multi-copy rDNA ITS1 region and is able to detect Symbiodinium clades C and D with >100-fold higher sensitivity compared to conventional techniques. Technical considerations relating to intragenomic variation, estimating copy number and non-symbiotic contamination are discussed. Eighty-two colonies from four common scleractinian species (Acropora millepora, Acropora tenuis, Stylophora pistillata and Turbinaria reniformis) and 11 locations on the Great Barrier Reef were tested for background Symbiodinium clades. Although these colonies had been previously identified as harboring only a single clade based on SSCP analyses, background clades were detected in 78% of the samples, indicating that the potential for symbiont shuffling may be much larger than currently thought.