Characterization of the lucinid bivalve-bacteria symbiotic system: the significance of the geochemical habitat on bacterial symbiont diversity and phylogeny

Extensive characterization of a single lucinid bivalve habitat was conducted to characterize the relationship between host bivalve and thiotrophic bacterial endosymbionts. For lucinids, the ecological and evolutionary relationships between hosts and endosymbionts are poorly understood. Reconstructing the evolutionary history of lucinid endosymbiosis, and the geologic significance of the association, has been hampered by insufficient knowledge of endosymbiont ecology and taxonomic diversity. Host organisms (Lucinisca nassula and Phacoides pectinatus) were collected from Cedar Keys, Florida, within the top 15-20 cm of the sediment in sea grass beds. PCR amplification and sequencing of bacterial 16S rRNA genes from lucinid gills and sediment cores retrieved ~900 sequences. Based on comparative phylogenetic methods, gill endosymbiont sequences were most closely related to uncultured Gammaproteobacteria associated with symbiosis, and specifically to lucinid endosymbionts (97-99% sequence similarity) and not to free-living organisms. Not all gill sequences were genetically identical, with intra- and inter-gill sequence diversity. Sediment diversity was high, represented by 13 major taxonomic groups, including equally dominant Chloroflexi, and Delta- and Gammaproteobacteria. Other organisms included the Bacteroidetes, Acidobacteria, Spirochetes, and Firmicutes. Rare (\u3c0.3%) sequences from the sediment were related to lucinid gill endosymbionts. Results support the hypothesis that recruitment of free-living organisms is likely. Based on habitat geochemistry, however, the bacteria are constrained to reducing conditions, and this may be reflected in the habitat types colonized by the host. Habitat-host-symbiont diversity was evaluated from other locations from Florida and The Bahamas. 16S rRNA gene sequences retrieved from those hosts revealed that not all lucinid endosymbionts belong to the Gammaproteobacteria, because some sequences were most closely related to Alphaproteobacteria. One sequence was most closely related to Methylobacterium spp., which may indicate that dual symbiosis (thiotrophy and methanotrophy) in lucinid bivalves may be possible. Together, these results are significant to paleoecological and evolutionary studies using lucinids in the fossil record (e.g. isotope studies)

Taxonomy of some Galeommatoidea (Mollusca, Bivalvia) associated with deep-sea echinoids: A reassessment of the bivalve genera Axinodon Verrill & Bush, 1898 and Kelliola Dall, 1899 with descriptions of new genera Syssitomya gen. nov. and Ptilomyax gen. nov.

The type species of Axinodon ellipticus Verrill & Bush, 1898 and Kellia symmetros Jeffreys, 1876 are re-described. It is concluded that the two species are not conspecific and that K. symmetros cannot be placed in the genus Axinodon. The family affinity of Axinodon is not resolved, although it is probable that this genus belongs to the Thyasiridae. Kellia symmetros is the type species of Kelliola and is placed in the Montacutidae. Kelliola symmetros is most probably associated with the echinoid Aeropsis rostrata and is not the species previously recorded from North Atlantic Pourtalesia echinoids under the name of Axinodon symmetros. This commensal associated with the North Atlantic Pourtalesia is here described as new and placed in the new genus as Syssitomya pourtalesiana gen. nov. sp. nov., Syssitomya gen. nov. differs from all other genera in the Montacutidae by having laminar gill filaments modified for harbouring symbiotic bacteria and it is thus assumed to be chemosymbiotic. A montacutid associated with the hadal Pourtalesia heptneri is described as Ptilomyax hadalis gen. nov. sp. nov

Taxonomic, Genetic and Functional Diversity of Symbionts Associated with the Coastal Bivalve Family Lucinidae

Extant bivalve members from the family Lucinidae harbor chemosynthetic gammaproteobacterial gill endosymbionts capable of thioautotrophy. These endosymbionts are environmentally acquired and belong to a paraphyletic group distantly related to other marine chemosymbionts. In coastal habitats, lucinid chemosymbionts participate in facilitative interactions with their hosts and surrounding seagrass habitat that results in symbiotic sulfide detoxification, oxygen release from seagrass roots, carbon fixation, and/or symbiotic nitrogen fixation. Currently, the structural and functional complexity of whole lucinid gill microbiomes, as well as their interactions with lucinid bivalves and their surrounding environment, have not been comprehensively characterized. This dissertation focuses on the taxonomic, genetic, and functional diversity in the gill microbiomes of three Floridian coastal lucinid bivalve species, Phacoides pectinatus, Ctena orbiculata, and Stewartia floridana, in the context of environmental data where appropriate. Analyses of these lucinid gill microbiomes showed taxonomic diversity that was unaffected by spatial distribution patterns. Phacoides pectinatus gill microbiomes sampled from a coastal mangrove habitat contained, in order of relative abundances, a chemosynthetic symbiont species that was taxonomically and functionally distinct from seagrass-associated chemosynthetic lucinid symbionts, a heterotrophic Kistimonas-like species, and a heterotrophic Spirochaeta-like species. In comparison, gill microbiomes of a seagrass-dwelling C. orbiculata population comprised four strains of chemosymbionts that belonged to two separate species and low abundances of an uncharacterized Endozoicomonas-like operational taxonomy unit (OTU). Gill microbiomes of a separate seagrass-dwelling S. floridana population consisted of another chemosynthetic symbiont species and low abundances of a heterotrophic Spirochaeta-like species that was distantly related to the Spirochaeta-like species in P. pectinatus. Functional characterization of host- and microbiome-related genes/transcripts in these bivalve species revealed previously unreported C1-compound oxidation functions in some chemosymbionts and other functions relevant to microbe-microbe competition, symbiont selection, metabolism support, and symbiont-to-host nutrient transfer. Preliminary differential expression analyses on host- and microbiome genes across micro-habitats with different vegetation coverages showed potential upregulation of C. orbiculata functions involved in aerobic respiration, aerobic stress, electron transport, and mitochondrial sulfide detoxification, as well as downregulation of a sulfurtransferase gene encoded by its chemosynthetic symbionts, in a seagrass-covered quadrat compared to an algae-covered quadrat. In comparison, very few genes mappable to S. floridana and its chemosymbiont were differentially expressed between predominantly sand-covered and seagrass-covered quadrats, but the Spirochaeta-like species over-expressed carbon, nitrogen, phosphate, transport, synthesis, transcriptional regulation, and protein degradation functions in predominantly sand-covered quadrats. These findings reaffirm the overlooked notion of heterogeneous lucinid gill microbiomes that can vary within and between host species and populations. At the same time, this project advances understanding of the functional diversity across chemosynthetic lucinid symbionts and offers insights on lucinid-microbiome-environment interactions

Environmental Controls on the Diversity and Distribution of Endosymbionts Associated with \u3ci\u3ePhacoides pectinatus\u3c/i\u3e (Bivalvia: Lucinidae) from Shallow Mangrove and Seagrass Sediments, St. Lucie County, Florida

Lucinid bivalves are capable of colonizing traditionally inhospitable shallow marine sediments due to metabolic functions of bacterial endosymbionts located within their gills. Because lucinids can often be the dominant sediment infauna, defining their roles in sediment and pore fluid geochemical cycling is necessary to address concerns related to changes in coastal biological diversity and to understanding the sensitivity of threatened coastal ecosystems over time. However, there has been limited research done to understand the diversity and distribution of many lucinid chemosymbiotic systems. Therefore, the goals of this thesis were to evaluate the distribution of Phacoides pectinatus and its endosymbiont communities from Ft. Pierce, St. Lucie County, Florida, and to define the environmental controls on potential free-living P. pectinatus endosymbionts to understand symbiont distribution patterns and host acquisition. Nearly all of the encountered P. pectinatus hosts were confined to within one meter of mangrove canopy. The distribution of P. pectinatus correlated to concentrations of organic carbon in the sediments, but not to total dissolved sulfide or sea grass vegetation densities. Sequencing of bacterial 16S rRNA genes from sediment and pore fluids from the P. pectinatus habitat revealed that Proteobacteria dominated the communities, including Alpha-, Delta-, and Gammaproteobacteria classes. Other major phyla included the Bacteroidetes, Chloroflexi, Planctomycetes, and Spirochaetes. 16S rRNA genes for both P. pectinatus gills and feet were closely related to novel bacterial communities comprised of Sedimenticola, Kistimonas, Methylomarinum, and Spirochaeta spp., as well as unclassified Rickettsiales (Alphaproteobacteria) and unclassified Lentisphaerae. This level of potential endosymbiont diversity has not been previously reported for lucinids. Moreover, potential endosymbiont populations differed by lucinid location, as gills containing higher Rickettsiales occurred in areas with the lowest clam density and gills in deeper sediments contained a higher proportion of Methylomarinum. Despite broad sediment and pore water bacterial diversity, no environmental sequences genetically matched those from P. pectinatus at the genus level. These results, while strengthening our understanding of a lucinid-symbiont system, still highlight how limited our knowledge is about these systems. These results provide new directions for future research and can be used to help understand how sensitive these systems are to environmental change

Spinaxinus (Bivalvia: Thyasiroidea) from sulfide biogenerators in the Gulf of Mexico and hydrothermal vents in the Fiji Back Arc: Chemosymbiosis and Taxonomy

Two new species of the thyasirid genus Spinaxinus (S. emicatus Oliver n. sp. and S. phrixicus Oliver n. sp.) are described from the Gulf of Mexico and the southwest Pacific, respectively. Both are compared with the type species of the genus, the eastern Atlantic S. sentosus Oliver and Holmes, 2006. Living specimens from the Gulf of Mexico were retrieved from artificial sulfide bio-generators on the upper Louisiana Slope. Gill morphology and molecular markers from the symbiotic bacteria confirm that Spinaxinus is chemosynthetic and that the chemoautotrophic bacteria are related to sulfide oxidizing Gammaproteobacteria. Living specimens from the southwest Pacific were retrieved from hydrothermal vent sites in the Fiji and Lau Back Arc Basins. In the Atlantic Spinaxinus is now recorded from two anthropogenic situations and appears to be generally absent from natural cold seep sites and not yet recorded at any hydrothermal sites. The primarily anthropogenic distribution of Spinaxinus in the Atlantic is discussed with reference to the natural hydrothermal vent habitat of the Pacific S. phrixicus.Spinaxinus (Bivalvia: Thyasiroidea) de bio-generadores artificiales de sulfuro situados en el Golfo de Méjico y en fuentes hidrotermales de las Islas Fiji: quimiosimbiosis y taxonomía. – En este trabajo se describen dos especies nuevas de tisárido del género Spinaxinus (S. emcatus Oliver n. sp. y S. phrixicus Oliver n. sp.) encontradas res- pectivamente en el Golfo de Méjico y en el sureste del Pacífico. Se comparan estas dos especies nuevas con la especie tipo del género, S. sentous Oliver y Holmes, 2006 descrita en el Este del Atlántico. Para describir estas dos especies, se observaron ejemplares vivos recolectados sobre bio-generadores artificiales de sulfuro situados en la parte alta de la plataforma conti- nental de Louisiana, en el Golfo de Méjico. Las observaciones realizadas de las branquias de Spinaxinus y la caracterización genética de las bacterias simbiontes en estos ejemplares confirmaron que Spinaxinus es un género quimiosintético que con- tiene bacterias quimioautótrofas cercanas a las Gammaproteobacterias responsables de la oxidación del sulfuro. También se recolectaron ejemplares vivos de fuentes hidrotermales situadas en las Islas Fiji y en ‘Lau Back Arc Basins’ ambas localiza- das en el Pacífico suroccidental. La especie atlántica de Spinaxinus se encontró en dos tipos de sustratos artificiales mientras que parece que esta especie no se encuentra en ambientes naturales equivalentes como serían las surgencias frías y las fuentes hidrotermales. La distribución aparentemente limitada de la especie atlántica se discute en relación con la distribución de S. phrixicus en las fuentes hidrotermales del Pacífico

Endosymbionts of two species of mediterranean lucinid clams

Symbiosen stellen, wie anhand einiger Invertebraten belegt, eine potentiell wichtige Überlebensstrategie vieler Tiere in unwirtlichen Lebensräumen dar. Solche Lebensräume können etwa schwefelhaltige Sedimentschichten sein. Um mit den lebensfeindlichen Bedingungen hier zu Recht zu kommen, besitzen Muscheln der Familie Lucinidae (dt. Mondmuscheln) Sulfid-oxidierende Endosymbionten. Außerdem schließen die Symbionten durch den chemischen Prozess die energiereichen reduzierten Schwefelverbindungen als zusätzliche Nahrungsquelle auf. Da bislang vorwiegend tropische Vertreter der Familie untersucht wurden, beschäftigt sich meine Diplomarbeit mit den kleineren Mittelmeerarten Loripes lacteus (Linneaus, 1758) und Anodontia (Loripinus) fragilis (Philippi, 1836). Ziel der Arbeit war eine Beschreibung der Endosymbionten beider Muschelarten. Die analysierten Tiere stammten aus Sulfid-reichen Sedimentschichten unterhalb einer Cymodocea nodosa-Seegraswiese. Sequenzanalysen hatten das 16S rRNA Gen des bakteriellen Symibonten zum Ziel. Niedrige Sequenzähnlichkeiten, basierend auf der Berechnung von paarweisen Distanzen (P-distances), gaben einen ersten Hinweis, dass die Endosymbionten von L. lacteus und A. fragilis zwei unterschiedlichen Bakterienarten angehören. Basierend auf der Sequenz wurden klonspezifische Sonden für die Muschelsymbionten entwickelt und mittels Fluoreszenz in situ Hybridisierung (FISH) die Spezifität geprüft. Außerdem konnte mittels confocaler Laser-Scannung Mikroskopie eine gleichmäßige Verteilung des fluoreszierenden Symbiontensignals in der gesamten Lateralzone der Kiemenfilamente beobachtet werden. Die Untersuchung ergab, dass L. lacteus und A. fragilis eine einzige, spezifische Endosymbiontenpopulation aus der Gruppe der γ-Proteobacteria beherbergen, die über drei Monate unveränderlich blieb. Transmissionselektronenmikroskopische Aufnahmen zeigen einzelne Endosymbionten eingeschlossen in Vakuolen, die innerhalb großer Bacteriocyten über die gesamte Lateralzone verteilt sind. Außerdem variieren die zusätzlichen Zelltypen in den untersuchten Bivalven, was sich vermutlich auf unterschiedliche physiologische Anpassungen an die Symbiose zurückführen lässt. Im Bakterioplasma der Endosymbionten von L. lacteus befinden sich zahlreiche Vakuolen, die möglicherweise der Sulfidspeicherung dienen. Phylogenetische Analysen unterstützen eine Monophylie von Endosymbionten der Familie Lucinidae nicht. Stattdessen zeigen symbiontische Sequenzen basierend auf Kiemen-Extrakten von L. lacteus eine nahe Verwandtschaft mit Symbionten von Lucina floridana und Codakia costata. 16S rRNA Sequenzen aus A. fragilis bilden dagegen eine Schwesterngruppe mit A. phillipiana und Solemya terraeregina. Diese Gruppe clustert innerhalb eines allgemeinen Symbiontenclades, das neben der Endosymbiontensequenz der Lucinidae Phacoides (Lucina) pectinata auch die Symbionten der Bivalvenfamilien Solemyidae und Thyasiridae, sowie solche von vestimentiferen Röhrenwürmern enthält. Damit ist durch die phylogenetische Analyse die Existenz zweier unterschiedlicher endosymbiontischer Arten in L. lacteus und A. fragilis untermauert.Symbioses are a potent survival solution for organisms in hostile environments like sulphide rich sediments, as was proven in several invertebrates. Among these, the bivalve family Lucinidae was reported to harbour sulphide oxidising endosymbionts to cope with the harsh conditions of their habitats and exploit the energy rich resources. Since mainly large tropical representatives of this family were examined in the past, my diploma thesis focused on the small Mediterranean species Loripes lacteus (Linnaeus, 1758) and Anodontia (Loripinus) fragilis (Philippi, 1836). The study aimed to provide a description of endosymbionts of both clam species. Therefore, in each of these bivalves collected from sulphide rich sediment layers below a Cymodocea nodosa sea-grass bed, sequences of the bacterial 16S rRNA gene were identified. Low sequence similarity based on the calculation of pairwise distances (P-distances) gave a first indication that endosymbionts of L. lacteus and A. fragilis belong to different bacterial strains. Using clone specific probes, fluorescence in situ hybridisation (FISH) proved the specificity of potential endosymbiotic sequences. Further, an even distribution of the symbionts throughout the lateral zone of gill filaments was observed using the confocal laser scanning microscope. L. lacteus and A. fragilis were shown to harbour a single, specific endosymbiont population of γ-Proteobacteria, which was stable over three months. On transmission electron micrographs, single endosymbionts enclosed by vacuoles located in large bacteriocytes along the lateral zone were detected. Additional cell-types varied slightly between the examined bivalves, probably due to different physiological adaptations of symbiont and host. Endosymbionts of L. lacteus further possess several vacuoles within the bacterial cytoplasm, most likely for sulphide storage. Phylogenetic analyses do not supported monophyly of lucinid endosymbionts. Symbiotic sequences originating from L. lacteus are related to endosymbionts of Lucina floridana and Codakia costata. 16S rRNA sequences obtained from A. fragilis form a sistergroup with A. phillipiana and Solemya terraeregina clustering within a general symbiotic clade of symbiont sequences from the lucinid Phacoides (Lucina) pectinata, Solemyidae, Thyasiridae and Vestimentifera. Thus, the phylogenetic analysis supported the existence of two different endosymbiotic species in L. lacteus and A. fragilis

Biogeochemical evidence for chemosymbiosis in the fossil record

Chemosymbiotic invertebrates obtain nutrition from harbouring bacteria that oxidize reduced chemicals to produce energy for carbon fixation. This allows the animals to thrive in the extreme conditions of the deep sea, because the high concentrations of sulphide (thiotrophy) and methane (methanotrophy) at cold seeps and hydrothermal vents can be utilized by the symbiotic bacteria. This research investigates whether the key role of chemosymbiosis in shaping modern deep sea ecosystems can be traced through geological time, by using the stable isotope composition (δ13C, δ15N, δ34S) of organic matter in invertebrate shells. Shell-bound organic matter (SBOM) was isolated using various shell removal techniques, and method comparison suggests that the original isotopic signal is least affect by using EDTA or acetic acid. Multi-isotope analysis of SBOM obtained from (deep sea) molluscs and brachiopods confirms that the main types of chemosymbiosis can be differentiated from non-symbiotic heterotrophic nutritional strategies. In particular chemosymbiotic SBOM δ13C is characteristically depleted, with defined ranges for the presence of either methanotrophic or thiotrophic symbionts across environmental settings. In suspected thiotrophic taxa from ancient cold seeps, the preservation of this modern range (SBOM δ13C -35‰ to -29‰) is limited to young subfossil specimens, but the upper threshold is only exceeded in pre-Pliocene samples. Moreover, the protected intra-crystalline SBOM pool retains a distinct δ13C signal up to the Miocene, and available δ34S and δ15N data of intra-crystalline SBOM do not overlap between heterotrophy and thiotrophy. For methanotrophy (δ13C -65‰ to -36‰ at modern cold seeps) a residual δ13C biosignature does appear to be present in total SBOM from Miocene samples. This encouraging finding, together with the discovery of intra-crystalline original proteins in a fossil of Cretaceous age, suggests that future work on other well-preserved specimens could trace the evolution of chemosymbiosis deep into geological time

Resilience of infaunal ecosystems during the Early Triassic greenhouse Earth

The Permian-Triassic mass extinction severely depleted biodiversity, primarily observed in the body fossil of well-skeletonized animals. Understanding how whole ecosystems were affected and rebuilt following the crisis requires evidence from both skeletonized and soft-bodied animals; the best comprehensive information on soft-bodied animals comes from ichnofossils. We analyzed abundant trace fossils from 26 sections across the Permian-Triassic boundary in China and report key metrics of ichnodiversity, ichnodisparity, ecospace utilization, and ecosystem engineering. We find that infaunal ecologic structure was well established in the early Smithian. Decoupling of diversity between deposit feeders and suspension feeders in carbonate ramp-platform settings implies that an effect of trophic group amensalism could have delayed the recovery of nonmotile, suspension-feeding epifauna in the Early Triassic. This differential reaction of infaunal ecosystems to variable environmental controls thus played a substantial but heretofore little appreciated evolutionary and ecologic role in the overall recovery in the hot Early Triassic ocean