Periodontal disease is the most widespread oral disease in dogs which if left untreated results in significant pain to the pet and loss of dentition. The objective of this study was to identify bacterial species in canine plaque that are significantly associated with health, gingivitis and mild periodontitis (<25% attachment loss). In this survey subgingival plaque samples were collected from 223 dogs with healthy gingiva, gingivitis and mild periodontitis with 72 to 77 samples per health status. DNA was extracted from the plaque samples and subjected to PCR amplification of the V1-V3 region of the 16S rDNA. Pyrosequencing of the PCR amplicons identified a total of 274 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all disease stages, particularly in health along with Moraxella and Bergeyella. Peptostreptococcus, Actinomyces, and Peptostreptococcaceae were the most abundant genera in mild periodontitis. Logistic regression analysis identified species from each of these genera that were significantly associated with health, gingivitis or mild periodontitis. Principal component analysis showed distinct community profiles in health and disease. The species identified show some similarities with health and periodontal disease in humans but also major differences. In contrast to human, healthy canine plaque was found to be dominated by Gram negative bacterial species whereas Gram positive anaerobic species predominate in disease. The scale of this study surpasses previously published research and enhances our understanding of the bacterial species present in canine subgingival plaque and their associations with health and early periodontal disease.

Plastids are descended from a cyanobacterial symbiosis which occurred over 1.2 billion years ago. During the course of endosymbiosis, most genes were lost from the cyanobacterium's genome and many were relocated to the host nucleus through endosymbiotic gene transfer (EGT). The issue of how many genes were acquired through EGT in different plant lineages is unresolved. Here, we report the genome-wide frequency of gene acquisitions from cyanobacteria in 4 photosynthetic eukaryotes--Arabidopsis, rice, Chlamydomonas, and the red alga Cyanidioschyzon--by comparison of the 83,138 proteins encoded in their genomes with 851,607 proteins encoded in 9 sequenced cyanobacterial genomes, 215 other reference prokaryotic genomes, and 13 reference eukaryotic genomes. The analyses entail 11,569 phylogenies inferred with both maximum likelihood and Neighbor-Joining approaches. Because each phylogenetic result is dependent not only upon the reconstruction method but also upon the site patterns in the underlying alignment, we investigated how the reliability of site pattern generation via alignment affects our results: if the site patterns in an alignment differ depending upon the order in which amino acids are introduced into multiple sequence alignment--N- to C-terminal versus C- to N-terminal--then the phylogenetic result is likely to be artifactual. Excluding unreliable alignments by this means, we obtain a conservative estimate, wherein about 14% of the proteins examined in each plant genome indicate a cyanobacterial origin for the corresponding nuclear gene, with higher proportions (17-25%) observed among the more reliable alignments. The identification of cyanobacterial genes in plant genomes affords access to an important question: from which type of cyanobacterium did the ancestor of plastids arise? Among the 9 cyanobacterial genomes sampled, Nostoc sp. PCC7120 and Anabaena variabilis ATCC29143 were found to harbor collections of genes which are-in terms of presence/absence and sequence similarity-more like those possessed by the plastid ancestor than those of the other 7 cyanobacterial genomes sampled here. This suggests that the ancestor of plastids might have been an organism more similar to filamentous, heterocyst-forming (nitrogen-fixing) representatives of section IV recognized in Stanier's cyanobacterial classification. Members of section IV are very common partners in contemporary symbiotic associations involving endosymbiotic cyanobacteria, which generally provide nitrogen to their host, consistent with suggestions that fixed nitrogen supplied by the endosymbiont might have played an important role during the origin of plastids.

Mitochondria play a key role in the life and death of eukaryotic cells, yet the full spectrum of mitochondrial functions is far from being fully understood, especially in photosynthetic organisms. To advance our understanding of mitochondrial functions in a photosynthetic cell, an extensive proteomic survey of Percoll-purified mitochondria from the metabolically versatile, hydrogen-producing green alga Chlamydomonas reinhardtii was performed. Different fractions of purified mitochondria from Chlamydomonas cells grown under aerobic conditions were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry after protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis or on blue-native polyacrylamide gel electrophoresis. Of the 496 nonredundant proteins identified, 149 are known or predicted to reside in other cellular compartments and were thus excluded from the molecular and evolutionary analyses of the Chlamydomonas proteome. The mitochondrial proteome of the photosynthetic alga reveals important lineage-specific differences with other mitochondrial proteomes, reflecting the high metabolic diversity of the organelle. Some mitochondrial metabolic pathways in Chlamydomonas appear to combine typical mitochondrial enzymes and bacterial-type ones, whereas others are unknown among mitochondriate eukaryotes. The comparison of the Chlamydomonas proteins to their identifiable homologs predicted from 354 sequenced genomes indicated that Arabidopsis is the most closely related nonalgal eukaryote. Furthermore, this phylogenomic analysis shows that free-living alpha-proteobacteria from the metabolically versatile orders Rhizobiales and Rhodobacterales better reflect the gene content of the ancestor of the chlorophyte mitochondria than parasitic alpha-proteobacteria with reduced and specialized genomes.

The origin of modern wheats involved alloploidization among related genomes. To determine if Aegilops speltoides was the donor of the B and G genomes in AABB and AAGG tetraploids, we used a 3-tiered approach. Using 70 amplified fragment length polymorphism (AFLP) loci, we sampled molecular diversity among 480 wheat lines from their natural habitats encompassing all S genome Aegilops, the putative progenitors of wheat B and G genomes. Fifty-nine Aegilops representatives for S genome diversity were compared at 375 AFLP loci with diploid, tetraploid, and 11 nulli-tetrasomic Triticum aestivum wheat lines. B genome-specific markers allowed pinning the origin of the B genome to S chromosomes of A. speltoides, while excluding other lineages. The outbreeding nature of A. speltoides influences its molecular diversity and bears upon inferences of B and G genome origins. Haplotypes at nuclear and chloroplast loci ACC1, G6PDH, GPT, PGK1, Q, VRN1, and ndhF for approximately 70 Aegilops and Triticum lines (0.73 Mb sequenced) reveal both B and G genomes of polyploid wheats as unique samples of A. speltoides haplotype diversity. These have been sequestered by the AABB Triticum dicoccoides and AAGG Triticum araraticum lineages during their independent origins.

Sacoglossan sea slugs are unique in the animal kingdom in that they sequester and maintain active plastids that they acquire from the siphonaceous algae upon which they feed, making the animals photosynthetic. Although most sacoglossan species digest their freshly ingested plastids within hours, four species from the family Plakobranchidae retain their stolen plastids (kleptoplasts) in a photosynthetically active state on timescales of weeks to months. The molecular basis of plastid maintenance within the cytosol of digestive gland cells in these photosynthetic metazoans is yet unknown but is widely thought to involve gene transfer from the algal food source to the slugs based upon previous investigations of single genes. Indeed, normal plastid development requires hundreds of nuclear-encoded proteins, with protein turnover in photosystem II in particular known to be rapid under various conditions. Moreover, only algal plastids, not the algal nuclei, are sequestered by the animals during feeding. If algal nuclear genes are transferred to the animal either during feeding or in the germ line, and if they are expressed, then they should be readily detectable with deep-sequencing methods. We have sequenced expressed mRNAs from actively photosynthesizing, starved individuals of two photosynthetic sea slug species, Plakobranchus ocellatus Van Hasselt, 1824 and Elysia timida Risso, 1818. We find that nuclear-encoded, algal-derived genes specific to photosynthetic function are expressed neither in P. ocellatus nor in E. timida. Despite their dramatic plastid longevity, these photosynthetic sacoglossan slugs do not express genes acquired from algal nuclei in order to maintain plastid function.

Resolving the closest relatives of Gnetales has been an enigmatic problem in seed plant phylogeny. The problem is known to be difficult because of the extent of divergence between this diverse group of gymnosperms and their closest phylogenetic relatives. Here, we investigate the evolutionary properties of conifer chloroplast DNA sequences. To improve taxon sampling of Cupressophyta (non-Pinaceae conifers), we report sequences from three new chloroplast (cp) genomes of Southern Hemisphere conifers. We have applied a site pattern sorting criterion to study compositional heterogeneity, heterotachy, and the fit of conifer chloroplast genome sequences to a general time reversible + G substitution model. We show that non-time reversible properties of aligned sequence positions in the chloroplast genomes of Gnetales mislead phylogenetic reconstruction of these seed plants. When 2,250 of the most varied sites in our concatenated alignment are excluded, phylogenetic analyses favor a close evolutionary relationship between the Gnetales and Pinaceae—the Gnepine hypothesis. Our analytical protocol provides a useful approach for evaluating the robustness of phylogenomic inferences. Our findings highlight the importance of goodness of fit between substitution model and data for understanding seed plant phylogeny.

Microorganisms from all domains of life establish associations with plants. Although some harm the plant, others antagonize pathogens or prime the plant immune system, support the acquisition of nutrients, tune plant hormone levels, or perform additional services. Most culture-independent plant microbiome research has focused on amplicon sequencing of the 16S rRNA gene and/or the internal transcribed spacer (ITS) of rRNA genomic loci, which show the relative abundance of the microbes to each other. Here, we describe shotgun sequencing of 275 wild Arabidopsis thaliana leaf microbiomes from southwest Germany, with additional bacterial 16S and eukaryotic ITS1 rRNA amplicon data from 176 of these samples. Shotgun data, which unlike the amplicon data capture the ratio of microbe to plant DNA, enable scaling of microbial read abundances to reflect the microbial load on the host. In a more cost-effective hybrid strategy, we show they also allow a similar scaling of amplicon data to overcome compositionality problems. Our wild plants were dominated by bacterial sequences, with eukaryotes contributing only a minority of reads. Microbial membership showed weak associations with both site of origin and plant genotype, both of which were highly confounded in this dataset. There was large variation among microbiomes, with one extreme comprising samples of low complexity and a high load of microorganisms typical of infected plants, and the other extreme being samples of high complexity and a low microbial load. Critically, considering absolute microbial load led to fundamentally different conclusions about microbiome assembly and the interaction networks among major taxa.

Archaeological remains indicate that the origin of western agriculture occurred in a brief period about 10,500 years ago in a region of the Middle East known as the Fertile Crescent, where the wild progenitors of several key agricultural cereal species are endemic. Domestication entailed the appearance of agronomic traits such as seed size and threshability. For a representative sample of 20 domesticated barley (Hordeum vulgare) lines, including 13 two-rowed and 7 six-rowed varieties, we determined the haplotypes at seven loci-Adh2, Adh3, Amy1, Dhn9, GAPDH, PEPC and WAXY encompassing 5,616 bases per line-and compared them to the haplotypes at the same loci for 25 wild forms (Hordeum spontaneum) collected within and outside the Fertile Crescent. In comparisons of wild versus domesticated barley, the number of haplotypes (70 vs. 17), average nucleotide diversity, , (0.0077 vs. 0.0028), and Watterson's theta at silent sites (0.0104 vs. 0.0028) was reduced in domesticated lines. Two loci, Amy1 and PEPC, were monomorphic in domesticated lines; Amy1 and GAPDH produced signiWcant values of Tajima's D. At GAPDH, was slightly higher in domesticated than wild forms, due to divergent high-frequency haplotypes; for the remaining six loci, 87% of nucleotide diversity has been lost in the domesticated forms. Bottlenecks acting on neutrally evolving loci either during the domestication process, during subsequent breeding, or both, are suYcient to account for reduced diversity and the results of Tajima's test, without the need to evoke selection at these loci. Phylogenetic networks data uncover distinct wild and domesticated barley genotypes and suggest that barley may have been domesticated in the Jordan valley. Because, based on AFLP data, the domesticated Turkish cultivars had a genetic basis as large as that present in large germplasm collections, all comparisons provided in this paper are of general value more than being restricted to the Turkish barley germplasm.