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     

Characterization of chemoautotrophic bacterial symbionts in a gutless marine worm Oligochaeta, Annelida) by phylogenetic 16S rRNA sequence analysis and in situ hybridization.

The phylogenetic relationships of chemoautotrophic endosymbionts in the gutless marine oligochaete Inanidrilus leukodermatus to chemoautotrophic ecto- and endosymbionts from other host phyla and to free-living bacteria were determined by comparative 16S rRNA sequence analysis. Fluorescent in situ hybridization confirmed that the 16S rRNA sequence obtained from these worms originated from the symbionts. The symbiont sequence is unique to I. leukodermatus. In phylogenetic trees inferred by both distance and parsimony methods, the oligochaete symbiont is peripherally associated with one of two clusters of chemoautotrophic symbionts that belong to the gamma subdivision of the Proteobacteria. The endosymbionts of this oligochaete form a monophyletic group with chemoautotrophic ectosymbionts of a marine nematode. The oligochaete and nematode symbionts are very closely related, although their hosts belong to separate, unrelated animal phyla. Thus, cospeciation between the nematode and oligochaete hosts and their symbionts could not have occurred. Instead, the similar geographic locations and habitats of the hosts may have influenced the establishment of these symbioses.     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Independent terrestrial origins of the Halosphaeriales (marine Ascomycota)

A phylogenetic study of marine ascomycetes was initiated to test and refine evolutionary hypotheses of marine-terrestrial transitions among ascomycetes. Taxon sampling focused on the Halosphaeriales, the largest order of marine ascomycetes. Approximately 1050 base pairs (bp) of the gene that codes for the nuclear small subunit (SSU) and 600 bp of the gene that codes for the nuclear large subunit (LSU) ribosomal RNAs (rDNA) were sequenced for 15 halosphaerialean taxa and integrated into a data set of homologous sequences from terrestrial ascomycetes. An initial set of phylogenetic analyses of the SSU rDNA from 38 taxa representing 15 major orders of the phylum Ascomycota confirmed a close phylogenetic relationship of the halosphaerialean species with several other orders of perithecial ascomycetes. A second set of analyses, which involved more intensive taxon sampling of perithecial ascomycetes, was performed using the SSU and LSU rDNA data in combined analyses. These second analyses included 15 halosphaerialean taxa, 26 terrestrial perithecial fungi from eight orders, and five outgroup taxa from the Pezizales. In these analyses the Halosphaeriales were polyphyletic and comprised two distinct lineages. One clade of Halosphaeriales comprised 12 taxa from 11 genera and was most closely related to terrestrial fungi of the Microascales. The second clade of halosphaerialean fungi comprised taxa from the genera Lulworthia and Lindra and was an isolated lineage among the perithecial fungi. Both the main clade of Halosphaeriales and the Lulworthia/Lindra clade are supported by the data as being independently derived from terrestrial ancestors.     

From volcanic origins of chemoautotrophic life to Bacteria, Archaea and Eukarya

The theory of a chemoautotrophic origin of life in a volcanic iron-sulphur world postulates a pioneer organism at sites of reducing volcanic exhalations. The pioneer organism is characterized by a composite structure with an inorganic substructure and an organic superstructure. Within the surfaces of the inorganic substructure iron, cobalt, nickel and other transition metal centres with sulphido, carbonyl and other ligands were catalytically active and promoted the growth of the organic superstructure through carbon fixation, driven by the reducing potential of the volcanic exhalations. This pioneer metabolism was reproductive by an autocatalytic feedback mechanism. Some organic products served as ligands for activating catalytic metal centres whence they arose. The unitary structure-function relationship of the pioneer organism later gave rise to two major strands of evolution: cellularization and emergence of the genetic machinery. This early phase of evolution ended with segregation of the domains Bacteria, Archaea and Eukarya from a rapidly evolving population of pre-cells. Thus, life started with an initial, direct, deterministic chemical mechanism of evolution giving rise to a later, indirect, stochastic, genetic mechanism of evolution and the upward evolution of life by increase of complexity is grounded ultimately in the synthetic redox chemistry of the pioneer organism.     

Body size and invasion success in marine bivalves

The role of body size in marine bivalve invasions has been the subject of debate. Roy et al . found that large-bodied species of marine bivalves were more likely to be successful invaders, consistent with patterns seen during Pleistocene climatic change, but Miller et al . argued that such selectivity was largely driven by the inclusion of mariculture species in the analysis and that size-selectivity was absent outside of mariculture introductions. Here we use data on non-mariculture species from the north-eastern Pacific coast and from a global species pool to test the original hypothesis of Roy et al . that range limits of larger bivalves are more fluid than those of smaller species. First, we test the hypothesis that larger bivalve species are more successful than small species in expanding their geographical ranges following introduction into new regions. Second, we compare body sizes of indigenous and non-indigenous species for 299 of the 303 known intertidal and shelf species within the marine bivalve clade that contains the greater number of non-mariculture invaders, the Mytilidae. The results from both tests provide additional support for the view that body size plays an important role in mediating invasion success in marine bivalves, in contrast to Miller et al . Thus range expansions in Recent bivalves are consistent with patterns seen in Pleistocene faunas despite the many differences in the mechanisms.     

Ecosystem productivity is associated with bacterial phylogenetic distance in surface marine waters

Understanding the link between community diversity and ecosystem function is a fundamental aspect of ecology. Systematic losses in biodiversity are widely acknowledged but the impact this may exert on ecosystem functioning remains ambiguous. There is growing evidence of a positive relationship between species richness and ecosystem productivity for terrestrial macro‐organisms, but similar links for marine micro‐organisms, which help drive global climate, are unclear. Community manipulation experiments show both positive and negative relationships for microbes. These previous studies rely, however, on artificial communities and any links between the full diversity of active bacterial communities in the environment, their phylogenetic relatedness and ecosystem function remain hitherto unexplored. Here, we test the hypothesis that productivity is associated with diversity in the metabolically active fraction of microbial communities. We show in natural assemblages of active bacteria that communities containing more distantly related members were associated with higher bacterial production. The positive phylogenetic diversity–productivity relationship was independent of community diversity calculated as the Shannon index. From our long‐term (7‐year) survey of surface marine bacterial communities, we also found that similarly, productive communities had greater phylogenetic similarity to each other, further suggesting that the traits of active bacteria are an important predictor of ecosystem productivity. Our findings demonstrate that the evolutionary history of the active fraction of a microbial community is critical for understanding their role in ecosystem functioning.     

Characterization of methanotrophic bacterial populations in soils showing atmospheric methane uptake.

The global methane cycle includes both terrestrial and atmospheric processes and may contribute to feedback regulation of the climate. Most oxic soils are a net sink for methane, and these soils consume approximately 20 to 60 Tg of methane per year. The soil sink for atmospheric methane is microbially mediated and sensitive to disturbance. A decrease in the capacity of this sink may have contributed to the approximately 1%. year(-1) increase in the atmospheric methane level in this century. The organisms responsible for methane uptake by soils (the atmospheric methane sink) are not known, and factors that influence the activity of these organisms are poorly understood. In this study the soil methane-oxidizing population was characterized by both labelling soil microbiota with (14)CH(4) and analyzing a total soil monooxygenase gene library. Comparative analyses of [(14)C]phospholipid ester-linked fatty acid profiles performed with representative methane-oxidizing bacteria revealed that the soil sink for atmospheric methane consists of an unknown group of methanotrophic bacteria that exhibit some similarity to type II methanotrophs. An analysis of monooxygenase gene libraries from the same soil samples indicated that an unknown group of bacteria belonging to the alpha subclass of the class Proteobacteria was present; these organisms were only distantly related to extant methane-oxidizing strains. Studies on factors that affect the activity, population dynamics, and contribution to global methane flux of "atmospheric methane oxidizers" should be greatly facilitated by use of biomarkers identified in this study.     

Polyphyletic origins of bacterial Nramp transporters.

The redox-active metals iron and manganese are required for energy metabolism, protection against oxidative stress and defense against infections. In eukaryotes, both divalent metals are transported by Nramp transporters. The sequence of these transporters was remarkably conserved during evolution. Several bacterial Nramp homologs (MntH) are also proton-dependent manganese transporters. Here, we present phylogenetic evidence for the polyphyletic origins of three groups of MntH proteins and for possible Nramp horizontal gene transfer with eukaryotes. We propose that the evolution of the MntH/Nramp family is related to adaptation to oxidative environments, including those arising during infection of animals and plants.     

A preliminary report of phylogenetic diversity of bacterial strains isolated from marine creatures

Bacterial diversity among marine creatures, especially molluscs, as a source for searching out novel lineages of bacteria, was studied. Marine creatures were collected at the coasts of the Kanto area in Japan. A total of 116 strains of bacteria were isolated from the intestines of 19 species of marine creatures includings molluscs, pisces and protochordata. Partial sequencing of 16S rDNA revealed that most of the isolates belonged to the gamma subclass of the Proteobacteria and Cytophaga-Flavobacterium-Bacteroides group. The BLAST searches revealed that the complete 16S rDNA sequence of 17 strains out of 116 isolates showed less than 94% similarity with 16S rDNA sequences deposited in the database. Four strains out of the 17 isolates belonged to the Rhodobacter group, 8 strains to the Alteromonas group, and the remaining 5 strains to the Cytophaga-Flavobacterium-Bacteroides group. Phylogenetic positions of 6 strains belonging to the Alteromonas group, which were isolated from different marine creatures, were close to each other, and represented a novel 16S rDNA lineage within the gamma subclass of Proteobacteria. Therefore, it may be inferred that these 6 strains belong to a new genus of Proteobacteria. Phylogenetic positions of the other strains are also independent from neighboring taxa, and they were suggested to respectively form a novel lineage. From these results, it is clear that the biodiversity of bacteria in marine creatures is much wider than was previously thought, and unknown microbiological resources are buried in these organisms.     

Independent origins of self-compatibility in Arabidopsis thaliana

The evolution from outcrossing based on self-incompatibility (SI) to a selfing system is one of the most prevalent transitions in flowering plants. It has been suggested that the loss of SI in Arabidopsis thaliana is associated with pseudogene formation at the SCR male component of the S locus. Recent work, however, suggests that alternative alleles with large deletions at the S locus are also present and may be responsible for the evolution of self-compatibility in this species. We demonstrate that most of these deletion alleles are evolutionarily derived from an S haplotype (haplogroups A) that already possessed the SCR pseudogene. This haplotype and its deletion variants are nearly fixed in Europe. Together with previous transgenic data, these results suggest that the pseudogenization of PsiSCR1 gene changed the SI phenotype in the majority of A. thaliana accessions, and was a critical step in the evolution of selfing in this species. Two other haplogroups (B and C) were also identified, the former of which contains a novel and possibly functional SCR allele. In contrast to haplogroups A, these two haplogroups are found primarily in Africa and Asia. These results suggest that self-compatibility, which appears to be fixed in this species, arose multiple times with different genetic bases, and indicates that a species-specific trait is associated with parallel evolution at the molecular level.     

Characterization of the methanotrophic bacterial community present in a trichloroethylene-contaminated subsurface groundwater site.

Groundwater, contaminated with trichloroethylene (TCE) and tetrachloroethylene (PCE), was collected from 13 monitoring wells at Area M on the U.S. Department of Energy Savannah River Site near Aiken, S.C. Filtered groundwater samples were enriched with methane, leading to the isolation of 25 methanotrophic isolates. The phospholipid fatty acid profiles of all the isolates were dominated by 18:1 omega 8c (60 to 80%), a signature lipid for group II methanotrophs. Subsequent phenotypic testing showed that most of the strains were members of the genus Methylosinus and one isolate was a member of the genus Methylocystis. Most of the methanotroph isolates exhibited soluble methane monooxygenase (sMMO) activity. This was presumptively indicated by the naphthalene oxidation assay and confirmed by hybridization with a gene probe encoding the mmoB gene and by cell extract assays. TCE was degraded at various rates by most of the sMMO-producing isolates, whereas PCE was not degraded. Savannah River Area M and other groundwaters, pristine and polluted, were found to support sMMO activity when supplemented with nutrients and then inoculated with Methylosinus trichosporium OB3b. The maximal sMMO-specific activity obtained in the various groundwaters ranged from 41 to 67% compared with maximal rates obtained in copper-free nitrate mineral salts media. This study partially supports the hypothesis that stimulation of indigenous methanotrophic communities can be efficacious for removal of chlorinated aliphatic hydrocarbons from subsurface sites and that the removal can be mediated by sMMO.     

Acute impact of agriculture on high-affinity methanotrophic bacterial populations

Exposure of mineral soils to atmospherically relevant concentrations of ¹³CH₄ (2 ppmv) followed by ¹³C-phospholipid fatty acid stable isotope probing allows assessment of the high-affinity methanotrophic bacterial sink in hitherto unattainable detail. Utilizing this approach, inorganic fertilizer-treated soils from a long-term agricultural experiment were shown to display dramatic reduction, by > 70%, of the methanotrophic bacterial cell numbers. Reduction in the methane sink capacity of the soils was slightly lower than the directly observed reduction in methanotrophic bacterial counts, indicating that the inhibitory effects on high-affinity methanotrophic bacteria are not fully expressed through CH₄ oxidation rates. The results emphasize the need to rigorously assess commonly applied agricultural practices with respect to their unseen negative impacts on soil microbial diversity in relation to terrestrial sinks for atmospheric trace gases.     

Physiological, ecological, and phylogenetic characterization of Stappia, a marine CO-oxidizing bacterial genus

Bacteria play a major role in marine CO cycling, yet very little is known about the microbes involved. Thirteen CO-oxidizing Stappia isolates obtained from existing cultures, macroalgae, or surf samples representing geographically and ecologically diverse habitats were characterized using biochemical, physiological, and phylogenetic approaches. All isolates were aerobic chemoorganotrophs that oxidized CO at elevated (1,000 ppm) and ambient-to-subambient concentrations (<0.3 ppm). All contained the form I (OMP) coxL gene for aerobic CO dehydrogenase and also the form II (BMS) putative coxL gene. In addition, some strains possessed cbbL, the large subunit gene for ribulose-1,5-bisphosphate carboxylase/oxygenase, suggesting the possibility of lithotrophic or mixotrophic metabolism. All isolates used a wide range of sugars, organic acids, amino acids, and aromatics for growth and grew at salinities from 5 to 45 ppt. All but one isolate denitrified or respired nitrate. Phylogenetic analyses based on 16S rRNA gene sequences indicated that several isolates could not be distinguished from Stappia aggregata and contributed to a widely distributed species complex. Four isolates (of strains GA15, HI, MIO, and M4) were phylogenetically distinct from validly described Stappia species and closely related genera (e.g., Ahrensia, Pannonibacter, Pseudovibrio, and Roseibium). Substrate utilization profiles, enzymatic activity, and membrane lipid composition further distinguished these isolates and supported their designations as new Stappia species. The observed metabolic versatility of Stappia likely accounts for its cosmopolitan distribution and its ability to contribute to CO cycling as well as other important biogeochemical cycles.     

Diversification of chemosymbiotic bivalves: origins and relationships of deeper water Lucinidae

Although species of the chemosymbiotic bivalve family Lucinidae are often diverse and abundant in shallow water habitats such as seagrass beds, new discoveries show that the family is equally speciose at slope and bathyal depths, particularly in the tropics, with records down to 2500 m. New molecular analyses including species from habitats down to 2000 m indicate that these cluster in four of seven recognized subfamilies: Leucosphaerinae, Myrteinae, Codakiinae, and Lucininae, with none of these comprising exclusively deep‐water species. Amongst the Leucosphaerinae, Alucinoma, Epidulcina, Dulcina, and Myrtina live mainly at depths greater than 200 m. Most Myrteinae inhabit water depths below 100 m, including Myrtea, Notomyrtea, Gloverina, and Elliptiolucina species. In the Codakinae, only the Lucinoma clade live in deep water; Codakia and Ctena clades are largely restricted to shallow water. Lucininae are the most speciose of the subfamilies but only four species analyzed, Troendleina sp., ‘Epicodakia’ falkandica, Bathyaustriella thionipta, and Cardiolucina quadrata, occur at depths greater than 200 m. Our results indicate that slope and bathyal lucinids have several and independent originations from different clades with a notable increased diversity in Leucosphaerinae and Myrteinae. Some of the deep‐water lucinids (e.g. Elliptiolucina, Dulcina, and Gloverina) have morphologies not seen in shallow water species, strongly suggesting speciation and radiation in these environments. By contrast, C. quadrata clusters with a group of shallow water congenors. Although not well investigated, offshore lucinids are usually found at sites of organic enrichment, including sunken vegetation, oxygen minimum zones, hydrocarbon seeps, and sedimented hydrothermal vents. The association of lucinids with hydrocarbon seeps is better understood and has been traced in the fossil record to the late Jurassic with successions of genera recognized; Lucinoma species are particularly prominent from the Oligocene to present day. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 401–420.     

Evolutionary origins of novel conchologic growth patterns in tropical American corbulid bivalves

We conducted a combined sclerochronologic and phylogenetic analysis to document patterns and rates of shell accretion in several subclades of related corbulids, and to explore the evolutionary origins of novel conchologic developmental patterns. We found three disparate patterns of valve development in Neogene tropical American corbulid genera. These patterns include growth through primarily radial accretion along the sagittal plane, and two derivative patterns: one characterized by initial deposition of a thin shell followed by valve thickening with little increase in valve height, and another producing a well-defined nepioconch through a marked change in the primary growth direction. We conducted a species-level phylogenetic analysis of the taxa surveyed for growth patterns, focusing on the ([Bothrocorbula+Hexacorbula]+Caryocorbula) clade. The phylogenetic distribution of shell growth patterns suggests that this clade is characterized by derivative patterns of growth. Oxygen-isotope calibrated ontogenetic age estimates of species in the derived Bothrocorbula subclade further suggest that transitions from the ancestral radial (sagittal) growth pattern to a derived pattern of growth are a function of heterochrony (peramorphosis by acceleration). These findings are significant because they link previously observed patterns of morphological constraint with a specific evolutionary process, demonstrate how morphologic constraint and innovation can be interrelated, and serve as a model for understanding the evolution of morphologic diversity in the clade as a whole. Furthermore, this study highlights the utility of sclerochronologic records as an important component of evolutionary developmental research on organisms with accretionary skeletal growth.     

Apparent overdominance of enzyme specific activity in two marine bivalves

Electrophoretic examination of a natural population sample of 332 mussels (Mytilus trossulus) revealed ten active allozyme alleles for the octopine dehydrogenase (Odh) locus and a statistically significant (P<0.005) departure from expected genotypic proportions caused by a deficiency of heterozygous genotypes. In vitro specific activity for octopine dehydrogenase (E.C. 1.5.1.11) was determined for 207 mussels representing 17 different Odh genotypes. Odh heterozygotes had an average specific activity that was 19% greater than that of apparently homozygous genotypes, a significant (P<0.05) difference. Electrophoretic examination of a natural population sample of 209 oysters (Crassostrea virginica) revealed 23 active allozyme alleles for the leucine aminopeptidase-2 (Lap-2) locus and a non-significant (P>0.05) deficiency of heterozygous genotypes. In vitro specific activity for leucine aminopeptidase (E.C. 3.4.-.-) was determined for 89 oysters representing 19 different Lap-2 genotypes. Lap-2 heterozygotes had an average specific activity that was 56% greater than that of homozygous genotypes, a significant (P<0.0001) difference. Possible explanations for the apparent overdominance in enzyme specific activity and the deficiency of heterozygotes include null alleles, molecular imprinting and aneuploidy.     

Molecular evidence and marine snake origins

A molecular phylogeny was used to refute the marine scenario for snake origins. Nuclear gene sequences suggested that snakes are not closely related to living varanid lizards, thus also apparently contradicting proposed relationships between snakes and marine mosasaurs (usually considered to be varanoids). However, mosasaurs share derived similarities with both snakes and living varanids. A reanalysis of the morphological data suggests that, if the relationships between living taxa are constrained to the proposed molecular tree, with fossil forms allowed to insert in their optimal positions within this framework, mosasaurs cluster with snakes rather than with varanids. Combined morphological and molecular analyses also still unite marine lizards with snakes. Thus, the molecular data do not refute the phylogenetic evidence for a marine origin of snakes.