Phylogenetic placement of "zoochlorellae" (Chlorophyta), algal symbiont of the temperate sea anemone Anthopleura elegantissima

At northern latitudes the sea anemones Anthopleura elegantissima and its congener A. xanthogrammica contain unidentified green chlorophytes (zoochlorellae) in addition to dinophytes belonging to the genus Symbiodinium. This dual algal symbiosis, involving members of distinct algal phyla in one host, has been extensively studied from the perspective of the ecological and energetic consequences of hosting one symbiotic type over the other. However, the identity of the green algal symbiont has remained elusive. We determined the phylogenetic position of the marine zoochlorellae inhabiting A. elegantissima by comparing sequence data from two cellular compartments, the nuclear 18S ribosomal RNA gene region and the plastid-encoded rbcL gene. The results support the inclusion of these zoochlorellae in a clade of green algae that form symbioses with animal (Anthopleura elegantissima), fungal (the lichen genus Nephroma), and seed plant (Ginkgo) partners. This clade is distinct from the Chlorella symbionts of Hydra. The phylogenetic diversity of algal hosts observed in this clade indicates a predisposition for this group of algae to participate in symbioses. An integrative approach to the study of these algae, both within the host and in culture, should yield important clues about how algae become symbionts in other organisms.     

Symbiotic green algae in eggs of Hynobius nigrescens,an amphibian endemic to Japan

The egg capsules of some amphibians' eggs are known to become green colored before hatching. This is due to the increase of green symbionts in the egg capsule surrounding the embryo. The green symbionts in North American amphibian eggs were reported to be unicellular green algae in the Oophila‐clade of Volvocales, Chlorophyceae. However, it remains unclear whether this is also the case in other parts of the world. In this study, we analyzed the green symbionts in green‐colored eggs of Hynobius nigrescens, an amphibian endemic to Japan, obtained from five distinct locations. Microscopic observations revealed that the green symbionts were similar in appearance to Oophila amblystomatis, which was reported in some amphibian eggs in North America, in which non‐motile cells of the algae had thick cell walls with reticulate protuberances. PCR‐DGGE followed by phylogenetic analyses of partial 18S rRNA sequences revealed that the symbionts from the five locations were identical and most likely unialgal in each egg capsule. They formed an independent subclade within the Oophila‐clade, indicating that H. nigrescens has a unique symbiont. Our data are consistent with the previous report on North American amphibian eggs and support the specific symbiotic relationships between Oophila‐clade symbionts and the eggs of amphibians. This is the first report on the specific symbiont‐and‐host association between an Oophila‐clade symbiont and an amphibian outside of North America. We also discuss several possibilities regarding the origin of green symbionts (vertical transmission or invasion) on the basis of the discovery and detailed observation of H. nigrescens eggs without any green symbionts.     

SYMBIODINIUM (DINOPHYTA) DIVERSITY AND STABILITY IN AQUARIUM CORALS1

Indo‐Pacific reef corals growing for years in closed‐system aquaria provide an alternate means to investigate host–symbiont specificity and stability. The diversity of dinoflagellate endosymbionts (Symbiodinium spp.) from coral communities in private and public aquaria was investigated using molecular‐genetic analyses. Of the 29 symbiont types (i.e., species) identified, 90% belonged to the most prevalent group of Symbiodinium harbored by Indo‐Pacific reef corals, Clade C, while the rest belonged to Clade D. Sixty‐five percent of all types were known from field surveys conducted throughout the Pacific and Indian oceans. Because specific coral–dinoflagellate partnerships appear to have defined geographic distributions, correspondence of the same symbionts in aquarium and field‐collected specimens identifies regions where particular colonies must have been collected in the wild. Symbiodinium spp. in clade D, believed to be “stress‐tolerant” and/or “opportunistic,” occurred in a limited number of individual colonies. The absence of a prevalent, or “weedy,” symbiont suggests that conditions under which aquarium corals are grown do not favor competitive replacements of their native symbiont populations. The finding of typical and diverse assemblages of Symbiodinium spp. among aquarium corals living many years under variable chemical/physical conditions, artificial and natural light, while undergoing fragmentation periodically, indicates that individual colonies maintain stable, long‐term symbiotic associations.     

Nutritional role of two algal symbionts in the temperate sea anemone Anthopleura elegantissima brandt

The intertidal sea anemone Anthopleura elegantissima in the Pacific Northwest may host a single type of algal symbiont or two different algal symbionts simultaneously: zooxanthellae (Symbiodinium muscatinei) and zoochlorellae (green algae; Trebouxiophyceae, Chlorophyta). A seasonal comparison of zooxanthellate and zoochlorellate anemones showed stable symbiont population densities in summer and winter, with densities of zoochlorellae about 4 times those of zooxanthellae. Photosynthesis-irradiance curves of freshly isolated symbionts show that the productivity (P(max) cell) of freshly isolated zooxanthellae was about 2.5 times that of zoochlorellae during July; comparable rates were obtained in other months. Models of algal carbon flux show that zoochlorellae may supply the host with more photosynthetic carbon per unit anemone biomass than zooxanthellae supply. Zooxanthellate anemone tissue was 2 per thousand ((13)C) and 5 per thousand ((15)N) enriched and zoochlorellate anemone tissue was 6 per thousand ((13)C) and 8 per thousand ((15)N) enriched over their respective symbionts, suggesting that zoochlorellate anemones receive less nutrition from their symbionts than do zooxanthellate individuals. The disparity between predicted contributions from the algal carbon budgets and the stable isotopic composition suggests that short-term measures of algal contributions may not reflect actual nutritional inputs to the host. Isotopic data support the hypothesis of substantial reliance on external food sources. This additional nutrition may allow both algae to persist in this temperate intertidal anemone in spite of differences in seasonal photosynthetic carbon contributions.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

Gut symbiotic bacteria in the cabbage bugs <Emphasis Type="Italic">Eurydema rugosa</Emphasis> and <Emphasis Type="Italic">Eurydema dominulus</Emphasis> (Heteroptera: Pentatomidae)

The cabbage bugs Eurydema rugosa Motschulsky and Eurydema dominulus (Scopoli) (Heteroptera: Pentatomidae: Strachiini) possess a number of crypts in a posterior region of the midgut, which are filled with bacterial symbiont cells. Here we characterized the gut symbionts of Eurydema stinkbugs using molecular phylogenetic and histological techniques. Specific gammaproteobacteria were consistently identified from the posterior midgut of E. rugosa representing nine populations and E. dominulus representing six populations, respectively. The bacterial 16S rRNA gene sequences were identical within the species but slightly different (98.2% sequence identity) between the species. Molecular phylogenetic analysis revealed that the Eurydema symbionts formed a well-defined monophyletic group in the Gammaproteobacteria. The symbionts were phylogenetically distinct from the gut symbionts of the stinkbug families Acanthosomatidae, Plataspidae, Parastrachiidae, Scutelleridae, and other pentatomid species, suggesting multiple evolutionary origins of the gut symbiotic bacteria among diverse stinkbugs. In situ hybridization confirmed that the symbiont is located in the cavity of the midgut crypts. Aposymbiotic insects of E. rugosa, which were produced by egg surface sterilization, were viable but suffered retarded growth, reduced body weight, and abnormal body color, suggesting the biological importance of the symbiont for the host.     

Flexibility of nutritional strategies within a mutualism: food availability affects algal symbiont productivity in two congeneric sea anemone species

Mutualistic symbioses are common, especially in nutrient-poor environments where an association between hosts and symbionts can allow the symbiotic partners to persist and collectively out-compete non-symbiotic species. Usually these mutualisms are built on an intimate transfer of energy and nutrients (e.g. carbon and nitrogen) between host and symbiont. However, resource availability is not consistent, and the benefit of the symbiotic association can depend on the availability of resources to mutualists. We manipulated the diets of two temperate sea anemone species in the genus Anthopleura in the field and recorded the responses of sea anemones and algal symbionts in the family Symbiodiniaceae to our treatments. Algal symbiont density, symbiont volume and photosynthetic efficiency of symbionts responded to changes in sea anemone diet, but the responses depended on the species of sea anemone. We suggest that temperate sea anemones and their symbionts can respond to changes in anemone diet, modifying the balance between heterotrophy and autotrophy in the symbiosis. Our data support the hypothesis that symbionts are upregulated or downregulated based on food availability, allowing for a flexible nutritional strategy based on external resources.     

Cryptic diversity hides host and habitat specialization in a gorgonian‐algal symbiosis

Shallow water anthozoans, the major builders of modern coral reefs, enhance their metabolic and calcification rates with algal symbionts. Controversy exists over whether these anthozoan–algae associations are flexible over the lifetimes of individual hosts, promoting acclimative plasticity, or are closely linked, such that hosts and symbionts co‐evolve across generations. Given the diversity of algal symbionts and the morphological plasticity of many host species, cryptic variation within either partner could potentially confound studies of anthozoan‐algal associations. Here, we used ribosomal, organelle and nuclear sequences, along with microsatellite variation, to study the relationship between lineages of a common Caribbean gorgonian and its algal symbionts. The gorgonian Eunicea flexuosa is a broadcast spawner, composed of two recently diverged, genetically distinct lineages largely segregated by depth. We sampled colonies of the two lineages across depth gradients at three Caribbean locations. We find that each host lineage is associated with a unique Symbiodinium B1/184 phylotype. This relationship between host and symbiont is maintained when host colonies are reciprocally transplanted, although cases of within phylotype switching were also observed. Even when the phylotypes of both partners are present at intermediate depths, the specificity between host and symbiont lineages remained absolute. Unrecognized cryptic diversity may mask host‐symbiont specificity and change the inference of evolutionary processes in mutualistic associations. Symbiotic specificity thus likely contributes to the ecological divergence of the two partners, generating species diversity within coral reefs.     

Cytochrome oxidase I sequences reveal possible cryptic diversity in the cosmopolitan symbiotic copepod Nesippus orientalis Heller, 1868 (Pandaridae: Siphonostomatoida) on elasmobranch hosts from the KwaZulu-Natal coast of South Africa

Over the past decade, numerous molecular phylogenetic studies uncovered cryptic diversity within the Copepoda, yet very few investigations focused on symbiotic copepods. Here we report mitochondrial DNA cytochrome oxidase I diversity in the cosmopolitan elasmobranch symbiont Nesippus orientalis off the KwaZulu-Natal coast of South Africa. Analysis of partial COI sequences of copepods sampled from a diversity of shark hosts, revealed the presence of two divergent clades. Diversity within the clades does not appear to be structured based on host species, host individual, geographic locality or time of sampling. However, divergence between the two clades seems to be related to host species. Phylogenetic analyses of representatives from the two clades, along with Nesippus spp., Caligus spp. and Lepeophtheirus spp. outgroups, further supports the distinction between the two clades. Future molecular phylogenetic investigations of widespread copepod symbionts most likely will reveal far greater levels of biodiversity than currently recognized.     

Phylogeny and biogeography of Anthopleura in the North Atlantic Ocean

Two species of Anthopleura, A. ballii and A. thallia, co-occur in Northern Europe with Bunodosoma biscayensis, a species closely allied to Anthopleura. The historical factors underlying their distribution are here investigated as part of a preliminary phylogenetic analysis of morphological data. Based on this analysis, Bunodosoma is nested within Anthopleura, and B. biscayensis is more closely related to members of Anthopleura than to other members of Bunodosoma. Because B. biscayensis also has adhesive verrucae, rather than non-adhesive, rounded vesicles, it is transferred to Anthopleura.The tree consists of two major clades, each of which includes species from the Atlantic and the Pacific. The tree shows geographic structure: one clade includes species found only in the Northern Hemisphere, and the other includes widespread species and species found only in the Southern Hemisphere. Although all of the European species of Anthopleura are part of the Northern Hemisphere clade, none are one another’s closest relative.     

The Biogeography and Phylogeny of Unicellular Cyanobacterial Symbionts in Sponges from Australia and the Mediterranean

The distribution, host associations, and phylogenetic relationships of the unicellular cyanobacterial symbionts of selected marine sponges were investigated with direct 16s rDNA sequencing. The results indicate that the symbionts of the marine sponges Aplysina aerophoba, Ircinia variabilis, and Petrosia ficiformis from the Mediterranean, four Chondrilla species from Australia and the Mediterranean, and Haliclona sp. from Australia support a diversity of symbionts comprising at least four closely related species of Synechococcus. These include the symbionts presently described as Aphanocapsa feldmannii from P. ficiformis and Chondrilla nucula. A fifth symbiont from Cymbastela marshae in Australia is an undescribed symbiont of sponges, related to Oscillatoria rosea. One symbiont, Candidatus Synechococcus spongiarum, was found in diverse sponge genera in the Mediterranean Sea and the Indian, Pacific, and Southern oceans, whereas others were apparently more restricted in host association and distribution. These results are discussed in terms of the biodiversity and biogeographic distributions of cyanobacterial symbionts.This revised version was published online in November 2004 with corrections to Volume 48.     

Fungal specificity and selectivity for algae play a major role in determining lichen partnerships across diverse ecogeographic regions in the lichen‐forming family Parmeliaceae (Ascomycota)

Microbial symbionts are instrumental to the ecological and long‐term evolutionary success of their hosts, and the central role of symbiotic interactions is increasingly recognized across the vast majority of life. Lichens provide an iconic group for investigating patterns in species interactions; however, relationships among lichen symbionts are often masked by uncertain species boundaries or an inability to reliably identify symbionts. The species‐rich lichen‐forming fungal family Parmeliaceae provides a diverse group for assessing patterns of interactions of algal symbionts, and our study addresses patterns of lichen symbiont interactions at the largest geographic and taxonomic scales attempted to date. We analysed a total of 2356 algal internal transcribed spacer (ITS) region sequences collected from lichens representing ten mycobiont genera in Parmeliaceae, two genera in Lecanoraceae and 26 cultured Trebouxia strains. Algal ITS sequences were grouped into operational taxonomic units (OTUs); we attempted to validate the evolutionary independence of a subset of the inferred OTUs using chloroplast and mitochondrial loci. We explored the patterns of symbiont interactions in these lichens based on ecogeographic distributions and mycobiont taxonomy. We found high levels of undescribed diversity in Trebouxia, broad distributions across distinct ecoregions for many photobiont OTUs and varying levels of mycobiont selectivity and specificity towards the photobiont. Based on these results, we conclude that fungal specificity and selectivity for algal partners play a major role in determining lichen partnerships, potentially superseding ecology, at least at the ecogeographic scale investigated here. To facilitate effective communication and consistency across future studies, we propose a provisional naming system for Trebouxia photobionts and provide representative sequences for each OTU circumscribed in this study.     

srDna-based taxonomic affinities of algal symbionts from a planktonic foraminifer and a solitary radiolarian

Planktonic sarcodines (acantharia, radiolaria, and planktonic foraminifera) are oceanic amoeboid protozoa that often harbor a variety of microalgae as intracellular symbionts. The identity and function of these endosymbiotic algae have intrigued and perplexed biologists for more than a century. The most conspicuous and well‐studied symbiotic algae of planktonic foraminifera and radiolaria are dinoflagellates, but a variety of nondinoflagellate taxa have also been reported. Ultrastructural features have been used to characterize some of these nondinoflagellate algae, but rarely has this led to clear taxonomic affiliations. We analyzed the nuclear small subunit ribosomal DNA (srDNA) isolated from the symbionts of the spinose planktonic foraminiferan Globigerinella siphonifera d'Orbigny (=Globigerinella aequilateralis Brady) and a solitary radiolarian (Spongodrymus sp. Haeckel) in order to determine the identity of these symbionts. The small coccoid algae isolated from G. siphonifera correspond to the Type I symbionts described by Faber et al. (1988) . Phylogenetic analysis of the srDNA sequences places these symbionts within the prymnesiophyte (haptophyte) lineage, closer to Prymnesium Conrad than to Phaeocystis Lagerheim. To our knowledge, this is the first confirmed case of a symbiotic prymnesiophyte. In addition, we were able to examine the level of sequence heterogeneity between symbionts isolated from different individuals of a single host species. The three isolates in this study had srDNA sequences that were almost identical, indicating that the three were all of the same species. Very green symbiotic algae were isolated from three solitary radiolaria identified as species of Spongodrymus. The symbiont srDNA sequences from the three individual hosts were identical to each other, again implicating a single species of algae in that symbiotic association. These symbionts are prasinophytes most closely related to the clade containing Tetaselmis convolutae Norris, Hori et Chihara. Tetraselmis convolutae is the algal symbiont of the marine flatworm, Convolutae roscoffensis Graff.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

Phylogenetic Analysis and Fluorescence <Emphasis Type="Italic">In Situ</Emphasis> Hybridization Detection of Archaeal and Bacterial Endosymbionts in the Anaerobic Ciliate <Emphasis Type="Italic">Trimyema Compressum</Emphasis>

The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings, a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that Trimyema species do not require very specific methanogenic symbionts, and symbiont replacement could have occurred in the history of Trimyema species. On the other hand, the bacterial symbiont was located near the lineage of the family Syntrophomonadaceae in the phylum Firmicutes. The sequence similarity between the bacterial symbiont and the nearest species was 85%, indicating that bacterial symbionts may be specific to the Trimyema species. The elimination of bacterial symbionts from the ciliate cell by antibiotic treatment resulted in considerably decreased host growth. However, it was not restored by stigmasterol addition (<2 μg ml⁻¹), which was different from the previous report that showed that the symbiont-free strain required exogenous sterols for growth. In addition, the decline of host growth was not accompanied by host metabolism shift toward the formation of more reduced products, which suggested that the contribution of bacterial symbionts to the host ciliate was not a dispose of excessive reducing equivalent arising from the host’s fermentative metabolism as methanogenic symbionts do. This study showed that bacterial symbionts make a significant contribution to the host ciliate by an unknown function and suggested that interactions between bacterial symbionts and T. compressum are more complicated than hitherto proposed.     

<Emphasis Type="Italic">Elliptochloris bilobata</Emphasis> var. <Emphasis Type="Italic">corticola</Emphasis> var. nov. (Trebouxiophyceae,Chlorophyta), a novel subaerial coccal green alga

We investigated a previously unidentified subaerial corticolous strain of the genus Elliptochloris Tschermak-Woess. The alga shares the generic morphological characters with Elliptochloris bilobata, the type species of the genus, but it has a thicker cell wall of adult globular cells, different chloroplast structure and it also differs in shape of elliptical autospores. The differences of the autospore shape between both species were evaluated using landmark-based geometric morphometrics. The 18S rDNA gene sequence of the new alga forms a monophyletic clade with the authentic strain of E. bilobata within the green algal class Trebouxiophyceae close to representatives of the genus Coccomyxa. We describe the new alga as Elliptochloris bilobata var. corticola var. nov. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Many corals host thermally resistant symbionts in high-temperature habitat

Physiologically distinct lines of dinoflagellate symbionts, Symbiodinium spp., may confer distinct thermal tolerance thresholds on their host corals. Therefore, if a coral can alternately host distinct symbionts, changes in their Symbiodinium communities might allow corals to better tolerate increasing environmental temperatures. However, researchers are currently debating how commonly coral species can host different symbiont types. We sequenced chloroplast 23 s rDNA from the Symbiodinium communities of nine reef-building coral species across two thermally distinct lagoon pools separated by ~500 m. The hotter of these pools reaches 35°C in the summer months, while the other pool’s maximum temperature is 1.5°C cooler. Across 217 samples from nine species, we found a single haplotype in both Symbiodinium clades A and D, but four haplotypes in Symbiodinium clade C. Eight of nine species hosted a putatively thermally resistant member of clade D Symbiodinium at least once, one of which hosted this clade D symbiont exclusively. Of the remaining seven that hosted multiple Symbiodinium types, six species showed higher proportions of the clade D symbiont in the hotter pool. Average percentage rise in the frequency of the clade D symbiont from the hotter to cooler pool was 52% across these six species. Even though corals hosted members of both the genetically divergent clades D and C Symbiodinium, some showed patterns of host–symbiont specificity within clade C. Both Acropora species that hosted clade C exclusively hosted a member of sub-clade C2, while all three Pocillopora species hosted a member of sub-clade C1 (sensu van Oppen et al. 2001). Our results suggest that coral–algal symbioses often conform to particular temperature environments through changes in the identity of the algal symbiont.     

Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt)

We have analyzed the genetic profiles of dinoflagellate populations obtained from the Pacific coast sea anemone Anthopleura elegantissima (Brandt) at collection sites from Washington to California. Genetic differences within the symbiont populations of California anemones have been uncovered by restriction length polymorphism (RFLP) analysis of the small subunit (SSU) and large subunit (LSU) ribosomal RNA genes, and by denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer region 2 (ITS 2). The existence of two Symbiodinium species is substantiated by sequence analysis of the variable regions V1, V2, and V3 of the SSUrDNA, which also establishes their phylogenetic relatedness to other members of the genus Symbiodinium. Anemones from Washington and Oregon harbor a single dinoflagellate species, for which we propose the name S. muscatinei sp. nov. At these northern locations, S. muscatinei either exists alone or co-occurs with the Chlorella-like green algal symbiont. Our results indicate that S. muscatinei co-occurs with a second dinoflagellate, S. californium, in mixed populations in central and southern California. We suggest that the geographic distribution of these dinoflagellates is related to the temperature cline created by latitude.     

Coexistence of novel gammaproteobacterial and Arsenophonus symbionts in the scale insect Greenisca brachypodii (Hemiptera,Coccomorpha: Eriococcidae)

Scale insects are commonly associated with obligate, intracellular microorganisms which play important roles in complementing their hosts with essential nutrients. Here we characterized the symbiotic system of Greenisca brachypodii, a member of the family Eriococcidae. Histological and ultrastructural analyses have indicated that G. brachypodii is stably associated with coccoid and rod‐shaped bacteria. Phylogenetic analyses have revealed that the coccoid bacteria represent a sister group to the secondary symbiont of the mealybug Melanococcus albizziae, whereas the rod‐shaped symbionts are close relatives of Arsenophonus symbionts in insects – to our knowledge, this is the first report of the presence of Arsenophonus bacterium in scale insects. As a comparison of 16S and 23S rRNA genes sequences of the G. brachypodii coccoid symbiont with other gammaprotebacterial sequences showed only low similarity (～90%), we propose the name ‘Candidatus Kotejella greeniscae’ for its tentative classification. Both symbionts are transovarially transmitted from one generation to the next. The infection takes place in the neck region of the ovariole. The bacteria migrate between follicular cells, as well as through the cytoplasm of those cells to the perivitelline space, where they form a characteristic ‘symbiont ball’. Our findings provide evidence for a polyphyletic origin of symbionts of Eriococcidae.     

GENE SEQUENCE DIVERSITY AND THE PHYLOGENETIC POSITION OF ALGAE ASSIGNED TO THE GENERA PHAEOPHILA AND OCHLOCHAETE (ULVOPHYCEAE,CHLOROPHYTA)1

The phylogenetic position of microfilamentous marine green algae assigned to the species Phaeophila dendroides, Entocladia tenuis (Phaeophila tenuis, and Ochlochaete hystrix was examined through phylogenetic analyses of nuclear‐encoded small subunit rDNA and chloroplast‐encoded tufA gene sequences. These analyses placed the P. dendroides strains within the Ulvophyceae, at the base of a clade that contains representatives of the families Ulvaceae, Ulvellaceae, and the species Bolbocoleon piliferum, supporting an earlier hypothesis that P. dendroides constitutes a distinct lineage. Substantial divergence in both nuclear and plastid DNA sequences exists among strains of P. dendroides from different geographic localities, but these isolated strains are morphologically indistinguishable. The lineage may have an accelerated rate of gene sequence evolution relative to other microfilamentous marine green algae. Entocladia tenuis and O. hystrix are placed neither in the P. dendroides clade nor in the Ulvellaceae as previous taxonomic schemes predicted but instead form a new clade or clades at the base of the Ulvaceae. Ruthnielsenia gen. nov. is proposed to accommodate Kylin's species, which cannot be placed in Entocladia (=Acrochaete), Phaeophila, or Ochlochaete. Ruthnielsenia tenuis (Kylin) comb. nov., previously known only from Atlantic coasts, is reported for the first time from the Pacific coast of North America (San Juan Island, WA, USA). Isolates of R. tenuis from the Atlantic and Pacific coasts of North America have identical small subunit rDNA and tufA gene sequences.