Development of clade-specific Symbiodinium primers for quantitative PCR (qPCR) and their application to detecting clade D symbionts in Caribbean corals

We developed quantitative PCR (qPCR) assays to distinguish each of the four clades (A–D) of dinoflagellate endosymbionts (genus Symbiodinium) commonly found in Caribbean corals. We applied these primer sets, which target portions of the multi-copy ribosomal DNA (rDNA) gene family, to assess the presence/absence of symbionts in clade D (as indicated by the detection of clade D DNA). We detected these symbionts in five of six Caribbean host species/genera (21% of samples analyzed, N = 10 of 47 colonies), from which clade D had rarely or never been observed. This suggests that Symbiodinium in clade D are present in a higher diversity of coral species than previously thought. This qPCR-based approach can improve our understanding of the total microbial diversity associated with corals, particularly in hosts thought to be relatively specific, and has many other potential applications for studies of coral reef ecology and conservation.     

Latitudinal symbiont zonation in <Emphasis Type="Italic">Stylophora pistillata</Emphasis> from southeast Africa

Studies on latitudinal gradients in Symbiodinium diversity on scleractinian corals are largely restricted to warm-water low latitude locations, and it appears that there is a shift in symbiont distributions with increasing latitude. The Symbiodinium assemblages of high latitude coral communities have largely been undocumented despite occupying an important transitional zone between tropical and temperate regions. Using a combination of the internal transcribed spacer region 2 (ITS2) and denaturing gradient gel electrophoresis (DGGE), we assessed the cladal and subcladal variability of Symbiodinium in the widely distributed species Stylophora pistillata along a latitudinal transect in southeast African waters which extended into high latitude locations. All colonies examined belonged to clade C. Six unique ITS2-DGGE banding profiles (designated C_spa to C_spf) were observed, which showed a latitudinal distribution from north to south, most likely a result of a gradient in water temperature and irradiance driven by riverine input in the southern regions. Sequence analysis revealed that all sequences except one did not match previously identified clade C sub-types, probably due to the lack of regional information in the Western Indian Ocean when compared to the Caribbean and Pacific. This study further supports the applicability of ITS2-DGGE in studies on Symbiodinium diversity, and highlights that potentially ecologically informative biogeographic patterns may be overlooked when only cladal designations are employed.     

Diversity and community structure of symbiotic dinoflagellates from Caribbean coral reefs

A community ecology approach to the study of the most common group of zooxanthellae, dinoflagellates in the genus Symbiodinium, was applied to symbiotic invertebrate assemblages on coral reefs in the western Caribbean, off the Yucatan peninsula (Puerto Morelos, Mexico) and over 1000 km away in the northeastern Caribbean, at Lee Stocking Island, Bahamas. Sequence differences and intragenomic variation, as determined by denaturing gradient gel electrophoresis and sequencing of the internal transcribed spacer 2 (ITS 2) region, were used to classify these symbionts. Twenty-eight genetically distinct Symbiodinium types were identified, eleven of which were found in hosts from both Caribbean locations. A single symbiont population was detected in 72% of hosts from the Yucatan and 92% of hosts from the Bahamas. The reef-wide community distribution of these symbionts is dominated by a few types found in many different host taxa, while numerous rare types appear to have high specificity for a particular host species or genus. Clade or lineage A Symbiodinium spp. was restricted to compatible hosts located within 3-4 m of the surface, while Symbiodinium spp. types from other lineages displayed differences in vertical zonation correlated with ITS type but were independent of clade designation. A comparison of the symbiont types found in field-collected hosts with types previously cultured from these hosts indicates the existence of low density or "background"-symbiont populations and cryptic, potentially non-mutualistic types in some hosts.     

<Emphasis Type="Italic">Symbiodinium</Emphasis> sp. associations in the gorgonian<Emphasis Type="Italic"> Pseudopterogorgia elisabethae</Emphasis> in the Bahamas: high levels of genetic variability and population structure in symbiotic dinoflagellates

Little is known concerning the fine-scale diversity, population structure, and biogeography for Symbiodinium spp. populations inhabiting particular invertebrate species, including the gorgonian corals, which are prevalent members of reef communities in the Gulf of Mexico, the Caribbean, and the western Atlantic. This study examined the Symbiodinium sp. clade B symbionts hosted by the Caribbean gorgonian Pseudopterogorgia elisabethae (Bayer). A total of 575 colonies of P. elisabethae were sampled in 1995 and 1998–2000 from 12 populations lying along an ~450 km transect in the Bahamas and their Symbiodinium sp. clade B symbionts genotyped at two polymorphic dinucleotide microsatellite loci. Twenty-three unique, two-locus genotypes were identified in association with these P. elisabethae colonies. Most colonies hosted only a single Symbiodinium sp. clade B genotype; however, in some instances ( n=25), two genotypes were harbored simultaneously. For 10 of the 12 populations, 66–100% of the P. elisabethae colonies hosted the same symbiont genotype. Added to this, in 9 of the 12 populations, a Symbiodinium sp. clade B genotype was either unique to a population or found infrequently in other populations. This distribution of Symbiodinium sp. clade B genotypes resulted in statistically significant ( P<0.05 or <0.001) differentiation in 62 of 66 pairwise comparisons of P. elisabethae populations. Tests of linkage disequilibrium suggested that a combination of clonal propagation of the haploid phase and recombination is responsible for maintaining these distinct Symbiodinium sp. clade B populations.     

Molecular characterization of symbiotic algae (Symbiodinium spp.) in soritid foraminifera (Sorites orbiculus) and a scleractinian coral (Orbicella annularis) from St John,US Virgin Islands

The exchange of Symbiodinium symbionts among scleractinian and soritid hosts could facilitate acclimatization to changing conditions by establishing novel symbiotic unions better tuned to prevailing conditions. In this study, we compare the communities of Symbiodinium spp. in neighboring populations of Orbicella annularis and Sorites orbiculus from St. John, US Virgin Islands, using operational taxonomic unit (OTU) clustering of cloned internal transcribed spacer 2 (ITS-2) rDNA sequences. We tested for partitioning of Symbiodinium OTUs by host and depth within and between two sites to explore the potential for symbiont exchange between hosts and light-dependent microhabitat specialization. An apparent lack of overlap in Symbiodinium communities (13 OTUs representing 7 clades) hosted by O. annularis and S. orbiculus suggests that exchange among these hosts does not occur. A low number of novel clade G ITS-2 sequences were found in one O. annularis and one S. orbiculus. A phylogenetic analysis of these sequences revealed them to be sub-clade G2 Symbiodinium, which are most commonly hosted by excavating clionid sponges. A permutational MANOVA revealed within host differences in the partitioning of Symbiodinium OTUs by site but not depth. This finding highlights the potential roles of either dissimilar environmental conditions between sites, or at least partial separation between populations, in determining the types of Symbiodinium contained in different hosts on a spatial scale of a few kilometers.     

Temporal and spatial infection dynamics indicate recognition events in the early hours of a dinoflagellate/coral symbiosis

The obligate symbiotic relationship between dinoflagellates, Symbiodinium spp. and reef building corals is re-established each host generation. The solitary coral Fungia scutaria Lamarck 1801 harbors a single algal strain, Symbiodinium ITS2 type C1f (homologous strain) during adulthood. Previous studies have shown that distinct algal ITS2 types in clade C correlate with F. scutaria—Symbiodinium specificity during the onset of symbiosis in the larval stage. The present study examined the early specificity events in the onset of symbiosis between F. scutaria larvae and Symbiodinium spp., by looking at the temporal and spatial infection dynamics of larvae challenged with different symbiont types. The results show that specificity at the onset of symbiosis was mediated by recognition events during the initial symbiont—host physical contact before phagocytosis, and by subsequent cellular events after the symbionts were incorporated into host cells. Moreover, homologous and heterologous Symbiodinium sp. strains did not exhibit the same pattern of localization within larvae. When larvae were infected with homologous symbionts (C1f), ~70% of the total acquired algae were found in the equatorial area of the larvae, between the oral and aboral ends, 21 h after inoculation. In contrast, no spatial difference in algal localization was observed in larvae infected with heterologous symbionts. This result provides evidence of functional differences among gastrodermal cells, during development of the larvae. The cells in the larval equator function as nutritive phagocytes, and also appear to function as a region of enhanced symbiont acquisition in F. scutaria.     

Bleaching response of corals and their <Emphasis Type="Italic">Symbiodinium</Emphasis> communities in southern Africa

The high-latitude coral communities of southern Africa suffered minimal impacts during past mass bleaching events. Recent reports indicate an increase in bleaching frequency during the last decade, yet the actual levels of thermal stress and contributing factors in these bleaching events, and the degree of acclimatisation or adaptation on these reefs are poorly understood. During the 2005 warm-water anomaly in the southern Indian Ocean we conducted bleaching surveys and collected samples for genotyping of the algal symbiont communities at 21 sites in southern Mozambique and South Africa. Coral bleaching reached unprecedented levels and was negatively correlated with both latitude and water depths. Stylophora pistillata and Montipora were the most susceptible taxa, whereas three common branching corals had significantly different bleaching responses (Stylophora > Acropora > Pocillopora). Temperature records indicated that localised strong upwelling events coupled with persistent above-average seawater temperatures may result in accumulated thermal stress leading to bleaching. Symbiodinium in 139 scleractinian corals belonged almost exclusively to clade C, with clade D symbionts present in only 3% of the colonies. Two atypical C subclades were present in Stylophora and Pocillopora colonies and these were more abundant in shallow than deeper sites. Taxon-specific differences in bleaching responses were unrelated to different clades of algal symbionts and suggest that Symbiodinium C subtypes with diverse thermal tolerance, coupled with acclimatisation and morphology of the host colony influence the bleaching response. Additionally, the predominance of putatively thermal-sensitive Symbiodinium in southern African corals may reflect a limited experience of bleaching and emphasises the vulnerability of these reefs to moderate levels of thermal stress.     

Phylogenetic analyses of potentially free-living <Emphasis Type="Italic">Symbiodinium</Emphasis> spp. isolated from coral reef sand in Okinawa,Japan

The existence of “free-living” Symbiodinium that can form symbioses with hosts is implied by the presence of hosts that produce Symbiodinium-free gametes and expulsion and/or expelled symbiotic algae from host. However, it is still unclear if potentially symbiotic Symbiodinium are found “free-living” in the coral reef environment. Sixteen Symbiodinium strains were established from samples taken from three sampling locations of coral reef sand in Okinawa, Japan. Phylogenetic analyses of the partial large subunit ribosomal DNA (28S-rDNA) and the internal transcribed spacer of ribosomal DNA (ITS-rDNA) conclusively showed that all 16 isolates belonged to Symbiodinium clade A sensu Rowan and Powers (1991). The lack of other Symbiodinium clades besides clade A in this study may be due to other clades not being readily culturable under culture conditions used here. The new isolates could be phylogenetically divided into four groups, though no sequences were identical to previously reported Symbiodinium. Two of the four groups were closely related to symbiotic Symbiodinium clade A isolated from a variety of host species. One isolate group formed a highly supported monophyly with Symbiodinium types that have previously been characterized as “free-living”. The remaining isolate group, although within clade A, was quite divergent from other clade A Symbiodinium. These results indicate that novel diversity of free-living Symbiodinium exists in coral sand.     

Three party symbiosis: acoelomorph worms,corals and unicellular algal symbionts in Eilat (Red Sea)

Epizoic worms were found to occur on certain coral colonies from reefs off the coast of Eilat (Red Sea). We identified 14 coral species infested by acoelomorph worms at a depth range of 2–50 m. The host corals were all zooxanthellate and included both massive and branching stony corals and a soft coral. Worms from all hosts were identified as belonging to the genus Waminoa and contained two distinct algal symbionts differing in size. The smaller one was identified as Symbiodinium sp. and the larger one is presumed to belong to the genus Amphidinium. Worm-infested colonies of the soft coral, Stereonephthya cundabiluensis, lacked a mucus layer and exhibited distinct cell microvilli, a phenotype not present in colonies lacking Waminoa sp. In most cases, both cnidarian and Acoelomorph hosts displayed high specificity for genetically distinctive Symbiodinium spp. These observations show that the epizoic worms do not acquire their symbionts from the “host” coral.     

Mode of zooxanthella transmission does not affect zooxanthella diversity in acroporid corals

Two distinct modes of algal endosymbiont acquisition exist in corals, a direct transmission from the parental colony to the eggs and a larval or post-larval uptake from the environment. The former, maternal-transmission mode is expected to be a more closed system, while the latter is believed to be an open system. Here we test the hypothesis that the diversity of symbionts in closed systems is lower than that in open systems. We examine the identity and diversity of the algal endosymbionts (zooxanthellae) in 25 Montipora species sampled from Irian Jaya (Indonesia) and Magnetic Island (central Great Barrier Reef) and compare the results with those previously obtained from Acropora species, which belong to the same family. All Montipora colonies examined harbour clade C zooxanthellae, with two colonies harbouring both clade C and D zooxanthellae simultaneously. Two algal strains (named C· and D· in this study) present in Montipora have not been observed in Acropora, and may have co-evolved with Montipora. Symbiodinium C· shows approximately 5% sequence divergence from C strains observed in Acropora spp. and occurs in 76% of the colonies examined. Nevertheless, several other C strains commonly found in other corals occur in some of the Montipora colonies. Montipora species transmit their algal endosymbionts directly to the eggs, while Acropora species have to acquire zooxanthellae from the environment every generation. Contrary to our expectations, the diversity of zooxanthellae is similar for the two genera, indicating that the mode of symbiont transmission (i.e. maternal versus horizontal) does not affect symbiont diversity in acroporid corals.Communicated by M.S. Johnson, Crawley     

Symbiont specificity and bleaching susceptibility among soft corals in the 1998 Great Barrier Reef mass coral bleaching event

Considerable variability in bleaching was observed within and among soft coral taxa in the order Alcyonacea (Octocorallia: Cnidaria) on the central Great Barrier Reef (GBR, latitude 18.2°–19.0°S, longitude 146.4°–147.3°E) during the 1998 mass coral bleaching event. In April 1998, during a period of high sea surface temperatures, tissue samples were taken from bleached and unbleached colonies representative of 17 soft coral genera. The genetic identities of intracellular dinoflagellates (Symbiodinium spp.) in these samples were analyzed using PCR-denaturing gradient gel electrophoresis fingerprinting analysis of the internal transcribed spacer regions 1 and 2. Alcyonaceans from the GBR exhibited a high level of symbiont specificity for Symbiodinium types mostly in clade C. A rare clade D type (D3) was associated only with Clavularia koellikeri, while Nephthea sp. hosted symbionts in clade B (B1n and B36). Homogenous Symbiodinium clade populations were detected in all but one colony. Colonies that appeared bleached possessed symbiont types that were genetically indistinguishable from those in nonbleached conspecifics. These data suggest that parameters other than the resident endosymbionts such as host identity and colony acclimatization are important in determining bleaching susceptibility among soft corals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Specificity of two temperate dinoflagellate–anthozoan associations from the north-western Pacific Ocean

Whilst many studies of symbiotic dinoflagellate diversity have focused on tropical reef environments, only a few have explored the degree and pattern of divergence of these endosymbionts at high latitudes. In this study, the genetic diversity and specificity of symbiotic dinoflagellates associated with two common anthozoan hosts in the north-western Pacific Ocean was studied in four different seasons during a period of 1 year. Partial nucleotide sequences of 28S and complete ITS1 ribosomal DNA regions were used to identify, genetically, the endosymbionts extracted from the scleractinian Alveopora japonica and the actinarian Heteractis sp. A. japonica harbours symbionts belonging to Symbiodinium of clade F, while Heteractis sp. associates with Symbiodinium of clade C. Moreover, no seasonal changes in the endosymbiont community were detected in these two associations during this study. This is the first evidence that these two temperate cnidarian–microalgae symbioses are stable. Furthermore, we tested the apparent specificity of the Heteractis sp.– Symbiodinium sp. clade C association, by performing alga-infection experiments with aposymbiotic hosts, and monitoring the uptake and persistence of homologous and heterologous symbionts. The findings confirm the association patterns detected in the field and show that Heteractis sp. only establishes a successful association with Symbiodinium cells of clade C, at least among the heterologous symbionts occurring in the study area. Our results are consistent with the idea that selective pressures in highly fluctuating temperate environments might have granted symbiosis-specificity an adaptive value.Communicated by T. Ikeda, Hakodate     

Molecular characterization of the zoanthid genus <Emphasis Type="Italic">Isaurus</Emphasis> (Anthozoa: Hexacorallia) and associated zooxanthellae (<Emphasis Type="Italic">Symbiodinium</Emphasis> spp.) from Japan

The zoanthid genus Isaurus (Anthozoa: Hexacorallia) is known from both the Indo-Pacific and Atlantic Oceans, but phylogenetic studies examining Isaurus using molecular markers have not yet been conducted. Here, two genes of markers [mitochondrial cytochrome oxidase subunit I (COI) and mitochondrial 16S ribosomal DNA (mt 16S rDNA)] from Isaurus specimens collected from southern Japan (n = 19) and western Australia (n = 3) were sequenced in order to investigate the molecular phylogenetic position of Isaurus within the order Zoantharia and the family Zoanthidae. Additionally, obtained sequences and morphological data (polyp size, mesentery numbers, mesogleal thickness) were utilized to examine Isaurus species diversity and morphological variation. By comparing our obtained sequences with the few previously acquired sequences of genera Isaurus as well as with Zoanthus, Acrozoanthus (both family Zoanthidae), and Palythoa spp. (family Spenophidae) sequences, the phylogenetic position of Isaurus as sister to Zoanthus within the Family Zoanthidae was suggested. Based on genetic data, Isaurus is most closely related to the genus Zoanthus. Despite considerable morphological variation (in particular, polyp length, mesentery numbers, external coloration) between collected Isaurus specimens, all specimens examined are apparently conspecific or very closely related based on molecular data and observed morphological variation within colonies. Additionally, obtained internal transcribed spacer of ribosomal DNA (ITS-rDNA) sequences from symbiotic zooxanthellae (Symbiodinium spp.) from all Isaurus specimens were shown to be subclade C1-related Symbiodinium. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nutrient enrichment and the ultrastructure of zooxanthellae from the giant clam Tridacna maxima

The separate and combined effects of ammonium (10M) and phosphate (2M) on the ultrastructure of zooxanthellae (Symbiodinium sp.) from giant clams, Tridacna maxima, were examined in the field. Nitrogen addition significantly changed the ultrastructure of the zooxanthellae inhabiting the clams. After 9 mo exposure, the cross-sectional area of zooxanthellae from N-treated clams was significantly lower than that from other treatments [N=39.3 m²; C=47.9 m²; P=43.2m²; N+P=44.5 m²; (P=0.001)]. There was also a significant decrease in the size of starch bodies, especially around the pyrenoid of the zooxanthellae from N and N+P treatments [N=1.2 m²; C=2.0 m²; P=1.8 m²; N+P=1.2 m²; (P=2.08E-11)]. This presumably occurs as a result of the mobilization of organic carbon stores in response to stimulated amino acid synthesis under enriched nutrient conditions. These data strongly suggest that the symbiotic zooxanthellae of clams are limited to some extent by the availability of inorganic nitrogen, and that relatively minor changes to the nutrient loading of the water column can have substantial effects on the biochemistry of symbioses such as that which exists between clams and zooxanthellae.     

The influence of symbiont type on photosynthetic carbon flux in a model cnidarian–dinoflagellate symbiosis

We measured the relationship between symbiont diversity, nutritional potential, and symbiotic success in the cnidarian–dinoflagellate symbiosis, by infecting aposymbiotic (i.e. symbiont-free) specimens of the model sea anemone Aiptasia sp. with a range of Symbiodinium types. Four cultured heterologous Symbiodinium types (i.e. originally isolated from other host species) were used, plus both cultured and freshly isolated homologous zooxanthellae (i.e. from Aiptasia sp.). Rates of photosynthesis, respiration, and symbiont growth were measured during symbiosis establishment and used to estimate the contribution of the zooxanthellae to the animal’s respiratory carbon demands (CZAR). Anemones containing Symbiodinium B1 (both homologous and heterologous) tended to attain higher CZAR values and hence benefit most from their symbiotic partners. This was despite Symbiodinium B1 not achieving the highest cell densities, though it did grow more quickly during the earliest stages of the infection process. Rather, the heterologous Symbiodinium types A1.4, E2, and F5.1 attained the highest densities, with populations of E2 and F5.1 also exhibiting the highest photosynthetic rates. This apparent success was countered, however, by very high rates of symbiosis respiration that ultimately resulted in lower CZAR values. This study highlights the impact of symbiont type on the functionality and autotrophic potential of the symbiosis. Most interestingly, it suggests that certain heterologous symbionts may behave opportunistically, proliferating rapidly but in a manner that is energetically costly to the host. Such negative host–symbiont interactions may contribute to the host–symbiont specificity seen in cnidarian–dinoflagellate symbioses and potentially limit the potential for partner switching as an adaptive mechanism.     

Inter-polyp genetic and physiological characterisation of <Emphasis Type="Italic">Symbiodinium</Emphasis> in an <Emphasis Type="Italic">Acropora valida</Emphasis> colony

Corals harbouring genetically mixed communities of endosymbiotic algae (Symbiodinium) often show distribution patterns in accordance with differences in light climate across an individual colony. However, the physiology of these genetically characterised communities is not well understood. Single stranded conformation polymorphism (SSCP) and real time quantitative polymerase chain reaction (qPCR) analyses were used to examine the genetic diversity of the Symbiodinium community in hospite across an individual colony of Acropora valida at the spatial scale of single polyps. The physiological characteristics of the polyps were examined prior to sampling with a combined O₂ microelectrode with a fibre-optic microprobe (combined sensor diameter 50–100 μm) enabling simultaneous measurements of O₂ concentration, gross photosynthesis rate and photosystem II (PSII) quantum yield at the coral surface as a function of increasing irradiances. Both sun- and shade-adapted polyps were found to harbour either Symbiodinium clade C types alone or clades A and C simultaneously. Polyps were grouped in two categories according to (1) their orientation towardps light, or (2) their symbiont community composition. Physiological differences were not detected between sun- and shade-adapted polyps, but O₂ concentration at 1,100 μmol photons m⁻² s⁻¹ was higher in polyps that harboured both clades A and C symbionts than in polyps that harboured clade C only. These results suggest that the acclimatisation of zooxanthellae of individual polyps of an A. valida colony to ambient light levels may not be the only determinant of the photosynthetic capacity of zooxanthellae. Here, we found that photosynthetic capacity is also likely to have a strong genetic basis and differs between genetically distinct Symbiodinium types.     

Population genetic structure of escolar (<Emphasis Type="Italic">Lepidocybium flavobrunneum</Emphasis>)

Escolar (Lepidocybium flavobrunneum) is a large, mesopelagic fish that inhabits tropical and temperate seas throughout the world, and is a common bycatch in pelagic longline fisheries that target tuna and swordfish. Few studies have explored the biology and natural history of escolar, and little is known regarding its population structure. To evaluate the genetic basis of population structure of escolar throughout their range, we surveyed genetic variation over an 806 base pair fragment of the mitochondrial control region. In total, 225 individuals from six geographically distant locations throughout the Atlantic (Gulf of Mexico, Brazil, South Africa) and Pacific (Ecuador, Hawaii, Australia) were analyzed. A neighbor-joining tree of haplotypes based on maximum likelihood distances revealed two highly divergent clades (δ = 4.85%) that were predominantly restricted to the Atlantic and Indo-Pacific ocean basins. All Atlantic clade individuals occurred in the Atlantic Ocean and all but four Pacific clade individuals were found in the Pacific Ocean. The four Atlantic escolar with Pacific clade haplotypes were found in the South Africa collection. The nuclear ITS-1 gene region of these four individuals was subsequently analyzed and compared to the ITS-1 gene region of four individuals from the South Africa collection with Atlantic clade haplotypes as well as four representative individuals each from the Atlantic and Pacific collections. The four South Africa escolar with Pacific mitochondrial control region haplotypes all had ITS-1 gene region sequences that clustered with the Pacific escolar, suggesting that they were recent migrants from the Indo-Pacific. Due to the high divergence and geographic separation of the Atlantic and Pacific clades, as well as reported morphological differences between Atlantic and Indo-Pacific specimens, consideration of the Atlantic and Indo-Pacific populations as separate species or subspecies may be warranted, though further study is necessary.     

Molecular evidence for multiple lineages in the gorgonian family Plexauridae (Anthozoa: Octocorallia)

Octocorals are diverse and abundant on many marine hard substrates, and, within this group, members of the family Plexauridae are an important component of tropical reef assemblages, especially in the Caribbean. To understand historical relationships within this large and diverse assemblage, and to test the monophyly of the family and some of its genera, DNA sequences of two mitochondrial loci (msh1 and ND2, ~1,185 bp) were analyzed from 46 species in 21 genera from deep and shallow waters in the tropical western Atlantic and in the tropical western and eastern Pacific (plus 9 taxa in the closely related Gorgoniidae and 1 species of the more distantly related Alcyoniidae). Five strongly supported clades were recovered. Three large clades correspond roughly to the Plexauridae, Paramuriceidae, and Gorgoniidae, and two smaller clades were comprised of taxa previously assigned to several families. Astrogorgia sp. did not group with any of the clades. The mutual relationships among the five clades remain unclear. Several genera previously regarded as unrelated appear to be grouped among the three families; e.g. Hypnogorgia sp. (Paramuriceidae) falls within a clade consisting of both Pacific and Atlantic Muricea spp. (Plexauridae), while Swiftia sp., Scleracis sp., and an Atlantic Thesea sp. (all Paramuriceidae) group with the gorgoniids. In several instances, genera containing Atlantic and Pacific species were recovered as monophyletic (Muricea spp., Bebryce spp.). However, in at least three cases (Echinomuricea spp., Thesea spp., Villogorgia spp.), placement of Atlantic and Pacific species in the same genus may reflect convergence of sclerite morphology. The results indicate a strong need for reexamination of octocoral taxonomy using a combination of molecular, morphological, and chemical evidence.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00227-005-1592-y.Communicated by J.P. Grassle, New Brunswick     

Egg laying and embryonic-larval development in the snail<Emphasis Type="Italic"> Thais (Stramonita) chocolata</Emphasis> (Duclos, 1832) with observations on its evolutionary relationships within the Muricidae

Oviposition and embryonic-larval development are described for the muricacean snail Thais (Stramonita) chocolata from the Southeast Pacific coast. As with numerous other muricacean snails, this species engages in communal egg laying, with females depositing egg capsules in clusters on subtidal rocks. Each cluster of capsules contains 100–150 pedunculate, ampulliform egg capsules, with each capsule containing an average of 2,600 small (130 m) eggs. Intracapsular development was followed using light and scanning electron microscopy to describe the successive embryonic stages of the species. Free-swimming veliger larvae of about 225 m length were released from capsules after 49 days incubation at 13.6°C. The planktotrophic larvae were cultured in seawater aquaria by feeding with pure cultures of phytoplankton, recording growth and form of the larvae. Larvae reached competence after 4 months at 22°C, at 1,450–1,740 m in size, and a few larvae were observed through metamorphosis and early definitive growth. The embryonic-larval development of T. chocolata coincides with the general characteristics of the ontogeny observed in other Thais species as well as of other genera of the Rapaninae such as Concholepas. This lent support to grouping these genera into a single clade. The lack of knowledge of the development of free larvae of Thais spp. means that we do not know whether these similarities also include an extensive larval phase as generally characteristic of other members of the clade. The mode of development may be useful in characterizing some clades of this family. Thus for example, the transference of some Thais to the genus Nucella (Subfamily Ocenebrinae) is supported by differences in the mode of embryonic development, which differentiates these subfamilies. Paleobiological data reported for Neogastropoda allow postulation of primitiveness in planktotrophic larval development compared to more recent developmental strategies such as direct development of different types, which characterize various clades of this family.Communicated by O. Kinne, Oldendorf/Luhe     

The effects of feeding or addition of dissolved inorganic nutrients in maintaining the symbiosis between dinoflagellates and a tropical marine cnidarian

The availability of solid food (Artemia nauplii) and dissolved inorganic nutrients (ammonium, nitrate, phosphate) to the shallow-water marine hydroid Myrionema amboinense was manipulated for 1-8 days in order to investigate their role in the growth of intracellular symbiotic dinoflagellates (zooxanthellae) of the genus Symbiodinium. Symbionts from hydroids collected from the field or maintained under laboratory conditions (25°C, 12 h:12 h light:dark cycle, 80 µE m^-2 s^-1 fluorescent lighting) always exhibited a single peak in mitotic index (MI) at dawn. Symbionts in freshly collected field animals had an MI peak of about 15%. Symbiotic dinoflagellates in hydroids fed Artemia nauplii twice daily in the laboratory maintained this dawn peak of MI between 10% and 15%, but in the absence of feeding or added inorganic nutrients, this peak declined to less than 1% within 2-4 days. In contrast, when hydroids were placed in solutions containing ammonium (20 µM NH₄Cl), nitrate (10 µM NaNO₃), and a combination of ammonium and phosphate (2 µM Na₂HPO₄) immediately after collection, the algal MI remained between 5% and 15% for 4-7 days; the addition of 2 µM phosphate did not increase MI relative to unfed rates. When unfed animals were placed in dissolved nitrogen or fed Artemia, the symbiont MI increased from <1% to 10-17% within 2-3 days; P alone had no effect. However, the increase resulting from added inorganic nutrients was temporary, lasting only 5-7 days. These observations suggest that algal division in the host is maintained indefinitely in the field or by feeding particulate foods twice daily in the laboratory, but the addition of inorganic nutrients alone (ammonium, nitrate and ammonium/phosphate) appeared to support the completion of a maximum of one additional round of cell division. Nutrients required for continued growth and division of symbiotic dinoflagellates are linked to host feeding and host growth; without external food, neither host nor symbiont continue to grow. The same phenomenon is seen in zooxanthellate anemones, clams and corals, where total numbers of symbionts appear to be linked to changes in host-tissue biomass (protein), achieving relatively stable densities in M. amboinense, corals and other cnidarian symbioses, depending on their local environmental conditions. The results of the present study help explain the cellular responses of algal symbionts in reef-dwelling invertebrates to additions of dissolved inorganic nutrients to coral-reef ecosystems.