Sibling species of mutualistic Symbiodinium clade G from bioeroding sponges in the western Pacific and western Atlantic oceans.

Dinoflagellates in the genus Symbiodinium associate with a broad array of metazoan and protistian hosts. Symbiodinium-based symbioses involving bioeroding sponge hosts have received less attention than those involving popular scleractinian hosts. Certain species of common Cliona harbor high densities of an ecologically restricted group of Symbiodinium, referred to as Clade G. Clade G Symbiodinium are also known to form stable and functionally important associations with Foraminifera and black corals (Antipatharia) Analyses of genetic evidence indicate that Clade G likely comprises several distinct species. Here, we use nucleotide sequence data in combination with ecological and geographic attributes to formally describe Symbiodinium endoclionum sp. nov. obtained from the Pacific boring sponge Cliona orientalis and Symbiodinium spongiolum sp. nov. from the congeneric western Atlantic sponge Cliona varians. These species appear to be part of an adaptive radiation comprising lineages of Clade G specialized to the metazoan phyla Porifera and Cnidaria, which began prior to the separation of the Pacific and Atlantic Oceans.

Evolution of Sulfur Binding by Hemoglobin in Siboglinidae (Annelida) with Special Reference to Bone-Eating Worms, Osedax.

Most members of Siboglinidae (Annelida) harbor endosymbiotic bacteria that allow them to thrive in extreme environments such as hydrothermal vents, methane seeps, and whale bones. These symbioses are enabled by specialized hemoglobins (Hbs) that are able to bind hydrogen sulfide for transportation to their chemosynthetic endosymbionts. Sulfur-binding capabilities are hypothesized to be due to cysteine residues at key positions in both vascular and coelomic Hbs, especially in the A2 and B2 chains. Members of the genus Osedax, which live on whale bones, do not have chemosynthetic endosymbionts, but instead harbor heterotrophic bacteria capable of breaking down complex organic compounds. Although sulfur-binding capabilities are important in other siboglinids, we questioned whether Osedax retained these cysteine residues and the potential ability to bind hydrogen sulfide. To answer these questions, we used high-throughput DNA sequencing to isolate and analyze Hb sequences from 8 siboglinid lineages. For Osedax mucofloris, we recovered three (A1, A2, and B1) Hb chains, but the B2 chain was not identified. Hb sequences from gene subfamilies A2 and B2 were translated and aligned to determine conservation of cysteine residues at previously identified key positions. Hb linker sequences were also compared to determine similarity between Osedax and siboglinids/sulfur-tolerant annelids. For O. mucofloris, our results found conserved cysteines within the Hb A2 chain. This finding suggests that Hb in O. mucofloris has retained some capacity to bind hydrogen sulfide, likely due to the need to detoxify this chemical compound that is abundantly produced within whale bones.

Mitogenomics reveals phylogeny and repeated motifs in control regions of the deep-sea family Siboglinidae (Annelida).

Deep-sea tubeworms in the annelid family Siboglinidae have drawn considerable interest regarding their ecology and evolutionary biology. As adults, they lack a digestive tract and rely on endosymbionts for nutrition. Moreover, they are important members of chemosynthetic environments including hydrothermal vents, cold seeps, muddy sediments, and whale bones. Evolution and diversification of siboglinids has been associated with host-symbiont relationships and reducing habitats. Despite their importance, the taxonomy and phylogenetics of this clade are debated due to conflicting results. In this study, 10 complete and 2 partial mitochondrial genomes and one transcriptome were sequenced and analyzed to address siboglinid evolution. Notably, repeated nucleotide motifs were found in control regions of these mt genomes, which may explain previous challenges of sequencing siboglinid mt genomes. Phylogenetic analyses of amino acid and nucleotide datasets were conducted in order to infer evolutionary history. Both analyses generally had strong nodal support and suggest Osedax is most closely related to the Vestimentifera+Sclerolinum clade, rather than Frenulata, as recently reported. These results imply Osedax, the only siboglinid lineage with heterotrophic endosymbionts, evolved from a lineage utilizing chemoautotrophic symbionts.

Population genetic data of a model symbiotic cnidarian system reveal remarkable symbiotic specificity and vectored introductions across ocean basins.

The Aiptasia-Symbiodinium symbiosis is a promising model for experimental studies of cnidarian-dinoflagellate associations, yet relatively little is known regarding the genetic diversity of either symbiotic partner. To address this, we collected Aiptasia from 16 localities throughout the world and examined the genetic diversity of both anemones and their endosymbionts. Based on newly developed SCAR markers, Aiptasia consisted of two genetically distinct populations: one Aiptasia lineage from Florida and a second network of Aiptasia genotypes found at other localities. These populations did not conform to the distributions of described Aiptasia species, suggesting that taxonomic re-evaluation is needed in the light of molecular genetics. Associations with Symbiodinium further demonstrated the distinctions among Aiptasia populations. According to 18S RFLP, ITS2-DGGE and microsatellite flanker region sequencing, Florida anemones engaged in diverse symbioses predominantly with members of Symbiodinium Clades A and B, but also C, whereas anemones from elsewhere harboured only S. minutum within Clade B. Symbiodinium minutum apparently does not form a stable symbiosis with other hosts, which implies a highly specific symbiosis. Fine-scale differences among S. minutum populations were quantified using six microsatellite loci. Populations of S. minutum had low genotypic diversity and high clonality (R = 0.14). Furthermore, minimal population structure was observed among regions and ocean basins, due to allele and genotype sharing. The lack of genetic structure and low genotypic diversity suggest recent vectoring of Aiptasia and S. minutum across localities. This first ever molecular-genetic study of a globally distributed cnidarian and its Symbiodinium assemblages reveals host-symbiont specificity and widely distributed populations in an important model system.

Open-ocean barriers to dispersal: a test case with the Antarctic Polar Front and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae).

Open-ocean environments provide few obvious barriers to the dispersal of marine organisms. Major currents and/or environmental gradients potentially impede gene flow. One system hypothesized to form an open-ocean dispersal barrier is the Antarctic Polar Front, an area characterized by marked temperature change, deep water, and the high-flow Antarctic Circumpolar current. Despite these potential isolating factors, several invertebrate species occur in both regions, including the broadcast-spawning nemertean worm Parborlasia corrugatus. To empirically test for the presence of an open-ocean dispersal barrier, we sampled P. corrugatus and other nemerteans from southern South America, Antarctica, and the sub-Antarctic islands. Diversity was assessed by analyzing mitochondrial 16S rRNA and cytochrome c oxidase subunit I sequence data with Bayesian inference and tcs haplotype network analysis. Appropriate neutrality tests were also employed. Although our results indicate a single well-mixed lineage in Antarctica and the sub-Antarctic, no evidence for recent gene flow was detected between this population and South American P. corrugatus. Thus, even though P. corrugatus can disperse over large geographical distances, physical oceanographic barriers (i.e. Antarctic Polar Front and Antarctic Circumpolar Current) between continents have likely restricted dispersal over evolutionary time. Genetic distances and haplotype network analysis between South American and Antarctic/sub-Antarctic P. corrugatus suggest that these two populations are possibly two cryptic species.

Endosymbionts of Siboglinum fiordicum and the phylogeny of bacterial endosymbionts in Siboglinidae (Annelida).

Siboglinid worms are a group of gutless marine annelids that are nutritionally dependent upon endosymbiotic bacteria. Four major groups of siboglinids are known-vestimentiferans, moniliferans, Osedax spp. and frenulates. Although endosymbionts of vestimentiferans and Osedax spp. have been previously characterized, little is currently known about endosymbiotic bacteria associated with frenulate and moniliferan siboglinids. This is particularly surprising given that frenulates are the most diverse and widely distributed group of siboglinids. Here, we molecularly characterize endosymbiotic bacteria associated with the frenulate siboglinid Siboglinum fiordicum by using 16S rDNA ribotyping in concert with laser-capture microdissection (LCM). Phylogenetic analysis indicates that at least three major clades of endosymbiotic gamma-proteobacteria associate with siboglinid annelids, with each clade corresponding to a major siboglinid group. S. fiordicum endosymbionts are a group of gamma-proteobacteria that are divergent from bacteria associated with vestimentiferan or Osedax hosts. Interestingly, symbionts of S. fiordicum, from Norway, are most closely related to symbionts of the frenulate Oligobrachia mashikoi from Japan, suggesting that symbionts of frenulates may share common evolutionary history or metabolic features.

Patterns of inorganic phosphate uptake in Cassiopea xamachana: a bioindicator species.

Nutrient levels in the nearshore waters of the Florida Keys have increased over the past few decades concomitant with a decline in the health of Florida's reef system. Phosphorus is a particular concern in the Florida Keys as it may be the limiting nutrient in nearshore waters. We demonstrate that the upside-down jellyfish, Cassiopea xamachana, decreases its rate of phosphate uptake following exposure to elevated levels of dissolved inorganic phosphate. We also show that this subsequent suppression of uptake rates persists for some time following exposure to elevated phosphates. Using these attributes, we experimentally investigated the use of C. xamachana as a bioindicator for dissolved inorganic phosphates in seawater. Our results show that these animals reveal comparative differences in environmental phosphates despite traditional testing methods yielding no detectable phosphates. We propose that C. xamachana is a bioindicator useful for integrating relevant information about phosphate availability in low nutrient environments.

Host-specialist lineages dominate the adaptive radiation of reef coral endosymbionts.

Bursts in species diversification are well documented among animals and plants, yet few studies have assessed recent adaptive radiations of eukaryotic microbes. Consequently, we examined the radiation of the most ecologically dominant group of endosymbiotic dinoflagellates found in reef-building corals, Symbiodinium Clade C, using nuclear ribosomal (ITS2), chloroplast (psbA(ncr)), and multilocus microsatellite genotyping. Through a hierarchical analysis of high-resolution genetic data, we assessed whether ecologically distinct Symbiodinium, differentiated by seemingly equivocal rDNA sequence differences, are independent species lineages. We also considered the role of host specificity in Symbiodinium speciation and the correspondence between endosymbiont diversification and Caribbean paleo-history. According to phylogenetic, biological, and ecological species concepts, Symbiodinium Clade C comprises many distinct species. Although regional factors contributed to population-genetic structuring of these lineages, Symbiodinium diversification was mainly driven by host specialization. By combining patterns of the endosymbiont's host specificity, water depth distribution, and phylogeography with paleo-historical signals of climate change, we inferred that present-day species diversity on Atlantic coral reefs stemmed mostly from a post-Miocene adaptive radiation. Host-generalist progenitors spread, specialized, and diversified during the ensuing epochs of prolonged global cooling and change in reef-faunal assemblages. Our evolutionary reconstruction thus suggests that Symbiodinium undergoes "boom and bust" phases in diversification and extinction during major climate shifts.

Adaptive radiation in extremophilic Dorvilleidae (Annelida): diversification of a single colonizer or multiple independent lineages?

Metazoan inhabitants of extreme environments typically evolved from forms found in less extreme habitats. Understanding the prevalence with which animals move into and ultimately thrive in extreme environments is critical to elucidating how complex life adapts to extreme conditions. Methane seep sediments along the Oregon and California margins have low oxygen and very high hydrogen sulfide levels, rendering them inhospitable to many life forms. Nonetheless, several closely related lineages of dorvilleid annelids, including members of Ophryotrocha, Parougia, and Exallopus, thrive at these sites in association with bacterial mats and vesicomyid clam beds. These organisms are ideal for examining adaptive radiations in extreme environments. Did dorvilleid annelids invade these extreme environments once and then diversify? Alternatively, did multiple independent lineages adapt to seep conditions? To address these questions, we examined the evolutionary history of methane-seep dorvilleids using 16S and Cyt b genes in an ecological context. Our results indicate that dorvilleids invaded these extreme habitats at least four times, implying preadaptation to life at seeps. Additionally, we recovered considerably more dorvilleid diversity than is currently recognized. A total of 3 major clades (designated "Ophryotrocha," "Mixed Genera" and "Parougia") and 12 terminal lineages or species were encountered. Two of these lineages represented a known species, Parougia oregonensis, whereas the remaining 10 lineages were newly discovered species. Certain lineages exhibited affinity to geography, habitat, sediment depth, and/or diet, suggesting that dorvilleids at methane seeps radiated via specialization and resource partitioning.

Improved resolution of reef-coral endosymbiont (Symbiodinium) species diversity, ecology, and evolution through psbA non-coding region genotyping.

Ribosomal DNA sequence data abounds from numerous studies on the dinoflagellate endosymbionts of corals, and yet the multi-copy nature and intragenomic variability of rRNA genes and spacers confound interpretations of symbiont diversity and ecology. Making consistent sense of extensive sequence variation in a meaningful ecological and evolutionary context would benefit from the application of additional genetic markers. Sequences of the non-coding region of the plastid psbA minicircle (psbA(ncr)) were used to independently examine symbiont genotypic and species diversity found within and between colonies of Hawaiian reef corals in the genus Montipora. A single psbA(ncr) haplotype was recovered in most samples through direct sequencing (~80-90%) and members of the same internal transcribed spacer region 2 (ITS2) type were phylogenetically differentiated from other ITS2 types by substantial psbA(ncr) sequence divergence. The repeated sequencing of bacterially-cloned fragments of psbA(ncr) from samples and clonal cultures often recovered a single numerically common haplotype accompanied by rare, highly-similar, sequence variants. When sequence artifacts of cloning and intragenomic variation are factored out, these data indicate that most colonies harbored one dominant Symbiodinium genotype. The cloning and sequencing of ITS2 DNA amplified from these same samples recovered numerically abundant variants (that are diagnostic of distinct Symbiodinium lineages), but also generated a large amount of sequences comprising PCR/cloning artifacts combined with ancestral and/or rare variants that, if incorporated into phylogenetic reconstructions, confound how small sequence differences are interpreted. Finally, psbA(ncr) sequence data from a broad sampling of Symbiodinium diversity obtained from various corals throughout the Indo-Pacific were concordant with ITS lineage membership (defined by denaturing gradient gel electrophoresis screening), yet exhibited substantially greater sequence divergence and revealed strong phylogeographic structure corresponding to major biogeographic provinces. The detailed genetic resolution provided by psbA(ncr) data brings further clarity to the ecology, evolution, and systematics of symbiotic dinoflagellates.

A connection between colony biomass and death in Caribbean reef-building corals.

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994-2007), eleven years in the Exuma Cays, Bahamas (1995-2006), and four years in Puerto Morelos, Mexico (2003-2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1-4 m) compared to deeper-dwelling conspecifics (12-15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels.

Secondary structure models for the internal transcribed spacer (ITS) region 1 from symbiotic dinoflagellates.

Ribosomal genes and their spacers have been extensively utilized to examine the biodiversity and phylogenetics of protists. Among these, the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) are known to form secondary structures that are critically important for proper processing of the pre-rRNA into mature ribosomes. Although the secondary structure of ITS2 has been widely investigated, considerably less is known about ITS1 and its secondary structure. Here, secondary structures of the ITS1 were modeled for 46 ITS "types" from Symbiodinium, a diverse dinoflagellate genus that forms symbioses with many protists and metazoans, using comparative phylogenetic and minimum free energy approaches. The predicted ITS1 secondary structures for each Symbiodinium "type" were highly stable (DeltaG=-46.40 to -85.30 kcal mol(-1) at 37 degrees C) and consisted of an open loop with five helices separated by single-stranded regions. Several structural characteristics were conserved within monophyletic sub-groups, providing additional support for the predicted structures and the relationships within this genus. Finally, the structures were applied to identify potential pseudogenes from five Symbiodinium ITS1 datasets. Consequently, ITS1 secondary structures are useful in understanding the biology and phylogenetics, as well as recognizing and excluding questionable sequences from datasets, of protists such as Symbiodinium.

Reef endemism, host specificity and temporal stability in populations of symbiotic dinoflagellates from two ecologically dominant Caribbean corals.

BACKGROUND: The dinoflagellate genus Symbiodinium forms symbioses with numerous protistan and invertebrate metazoan hosts. However, few data on symbiont genetic structure are available, hindering predictions of how these populations and their host associations will fair in the face of global climate change. METHODOLOGY/PRINCIPAL FINDINGS: Here, Symbiodinium population structure from two of the Caribbean's ecologically dominant scleractinian corals, Montastraea faveolata and M. annularis, was examined. Tagged colonies on Florida Keys and Bahamian (i.e., Exuma Cays) reefs were sampled from 2003-2005 and their Symbiodinium diversity assessed via internal transcribed spacer 2 (ITS2) rDNA and three Symbiodinium Clade B-specific microsatellite loci. Generally, the majority of host individuals at a site harbored an identical Symbiodinium ITS2 "type" B1 microsatellite genotype. Notably, symbiont genotypes were largely reef endemic, suggesting a near absence of dispersal between populations. Relative to the Bahamas, sympatric M. faveolata and M. annularis in the Florida Keys harbored unique Symbiodinium populations, implying regional host specificity in these relationships. Furthermore, within-colony Symbiodinium population structure remained stable through time and environmental perturbation, including a prolonged bleaching event in 2005. CONCLUSIONS/SIGNIFICANCE: Taken together, the population-level endemism, specificity and stability exhibited by Symbiodinium raises concerns about the long-term adaptive capacity and persistence of these symbioses in an uncertain future of climate change.

CORRESPONDENCE BETWEEN COLD TOLERANCE AND TEMPERATE BIOGEOGRAPHY IN A WESTERN ATLANTIC SYMBIODINIUM (DINOPHYTA) LINEAGE(1).

Many corals form obligate symbioses with photosynthetic dinoflagellates of the genus Symbiodinium Freudenthal (1962). These symbionts vary genotypically, with their geographical distribution and abundance dependent upon host specificity and tolerance to temperature and light variation. Despite the importance of these mutualistic relationships, the physiology and ecology of Symbiodinium spp. remain poorly characterized. Here, we report that rDNA internal transcribed spacer region 2 (ITS2) defined Symbiodinium type B2 associates with the cnidarian hosts Astrangia poculata and Oculina arbuscula from northerly habitats of the western Atlantic. Using pulse-amplitude-modulated (PAM) fluorometry, we compared maximum photochemical efficiency of PSII of type B2 to that of common tropical Symbiodinium lineages (types A3, B1, and C2) under cold-stress conditions. Symbiont cultures were gradually cooled from 26°C to 10°C to simulate seasonal temperature declines. Cold stress decreased the maximum photochemical efficiency of PSII and likely the photosynthetic potential for all Symbiodinium clades tested. Cultures were then maintained at 10°C for a 2-week period and gradually returned to initial conditions. Subsequent to low temperature stress, only type B2 displayed rapid and full recovery of PSII photochemical efficiency, whereas other symbiont phylotypes remained nonfunctional. These findings indicate that the distribution and abundance of Symbiodinium spp., and by extension their cnidarian hosts, in temperate climates correspond significantly with the photosynthetic cold tolerance of these symbiotic algae.

Siboglinid-bacteria endosymbiosis: A model system for studying symbiotic mechanisms.

Siboglinid worms are a group of gutless marine annelids which are nutritionally dependent upon endosymbiotic bacteria.1,2 Four major groups of siboglinids are known including vestimentiferans, Osedax spp., frenulates and moniliferans.3-5 Very little is known about the diversity of bacterial endosymbionts associated with frenulate or monoliferan siboglinids. This lack of knowledge is surprising considering the global distribution of siboglinids; this system is likely among the most common symbioses in the deep sea. At least three distinct clades of endosymbiotic gamma-proteobacteria associate with siboglinid annelids.6 Frenulates harbor a clade of gamma-proteobacteria that are divergent from both the thiotrophic bacteria of vestimentiferans and monoliferans as well as the heterotrophic bacteria of Osedax spp.6,7 We also discuss priorities for future siboglinid research and the need to move beyond descriptive studies. A promising new method, laser-capture microdissection (LCM), allows for the precise excision of tissue regions of interest.8 This method, when used in concert with molecular and genomic techniques, such as Expressed Sequence Tag (EST) surveys using pyrosequencing technology, will likely enable investigations into physiological processes and mechanisms in these symbioses. Furthermore, adopting a comparative approach using different siboglinid groups, such as worms harboring thiotrophic versus methanotrophic endosymbionts, may yield considerable insight into the ecology and evolution of the Siboglinidae.

Measuring rDNA diversity in eukaryotic microbial systems: how intragenomic variation, pseudogenes, and PCR artifacts confound biodiversity estimates.

Molecular approaches have revolutionized our ability to study the ecology and evolution of micro-organisms. Among the most widely used genetic markers for these studies are genes and spacers of the rDNA operon. However, the presence of intragenomic rDNA variation, especially among eukaryotes, can potentially confound estimates of microbial diversity. To test this hypothesis, bacterially cloned PCR products of the internal transcribed spacer (ITS) region from clonal isolates of Symbiodinium, a large genus of dinoflagellates that live in symbiosis with many marine protists and invertebrate metazoa, were sequenced and analysed. We found widely differing levels of intragenomic sequence variation and divergence in representatives of Symbiodinium clades A to E, with only a small number of variants attributed to Taq polymerase/bacterial cloning error or PCR chimeras. Analyses of 5.8S-rDNA and ITS2 secondary structure revealed that some variants possessed base substitutions and/or indels that destabilized the folded form of these molecules; given the vital nature of secondary structure to the function of these molecules, these likely represent pseudogenes. When similar controls were applied to bacterially cloned ITS sequences from a recent survey of Symbiodinium diversity in Hawaiian Porites spp., most variants (approximately 87.5%) possessed unstable secondary structures, had unprecedented mutations, and/or were PCR chimeras. Thus, data obtained from sequencing of bacterially cloned rDNA genes can substantially exaggerate the level of eukaryotic microbial diversity inferred from natural samples if appropriate controls are not applied. These considerations must be taken into account when interpreting sequence data generated by bacterial cloning of multicopy genes such as rDNA.