Nitrogen fixation and other biogeochemically important features of Atacama Desert giant horsetail plant microbiomes inferred from metagenomic contig analysis.

BACKGROUND AND AIMS: Canyon stream beds in the hyperarid Atacama Desert surprisingly harbour magnificent groves of endemic giant horsetail wetland plants, Equisetum xylochaetum. Our previous metagenomic study of eukaryotes closely associated with this plant indicated that the microbiome included prokaryotes that might likewise influence host success and environment. We explored this possibility by using the metagenomic sequence to characterize prokaryote taxa and functional genes present in the microbiome of E. xylochaetum sampled from remote sites differing in the degree of anthropogenic disturbance. We focused on biogeochemical functions known to be important in wetland ecosystems. METHODS: To ensure that analyses were conducted on microbes most closely associated with plants, we extracted DNA from well-washed plant organs whose microbial biofilms were revealed with scanning electron microscopy. To assess the benefits of longer sequences for taxonomic and gene classifications, results of analyses performed using contigs were compared with those obtained with unassembled reads. We employed methods widely used to estimate genomic coverage of single taxa for genomic analysis to infer relative abundances of taxa and functional genes. KEY RESULTS: Key functional bacterial genera (e.g. Hydrogenophaga, Sulfuritalea and Rhodoferax) inferred from taxonomic and functional gene analysis of contigs - but not unassembled reads - to occur on surfaces of (or within) plants at relatively high abundance (>50× genomic coverage) indicated roles in nitrogen, sulfur and other mineral cycling processes. Comparison between sites revealed impacts on biogeochemical functions, e.g. reduced levels of the nifH gene marker under disturbance. Vanadium nitrogenases were more important than molybdenum nitrogenases, indicated by both functional genes and taxa such as Rhodomicrobium and Phaeospirillum inferred from contigs but not unassembled reads. CONCLUSIONS: Our contig-based metagenomic analyses revealed that microbes performing key wetland biogeochemical functions occur as tightly adherent biofilms on the plant body, not just in water or sediments, and that disturbance reduces such functions, providing arguments for conservation efforts.

Plastid Genome of Equisetum xylochaetum from the Atacama Desert, Chile and the Relationships of Equisetum Based on Frequently Used Plastid Genes and Network Analysis.

The modern pteridophyte genus Equisetum is the only survivor of Sphenopsida, an ancient clade known from the Devonian. This genus, of nearly worldwide distribution, comprises approximately 15 extant species. However, genomic information is limited. In this study, we assembled the complete chloroplast genome of the giant species Equisetum xylochaetum from a metagenomic sequence and compared the plastid genome structure and protein-coding regions with information available for two other Equisetum species using network analysis. Equisetum chloroplast genomes showed conserved traits of quadripartite structure, gene content, and gene order. Phylogenetic analysis based on plastome protein-coding regions corroborated previous reports that Equisetum is monophyletic, and that E. xylochaetum is more closely related to E. hyemale than to E. arvense. Single-gene phylogenetic estimation and haplotype analysis showed that E. xylochaetum belonged to the subgenus Hippochaete. Single-gene haplotype analysis revealed that E. arvense, E. hyemale, E. myriochaetum, and E. variegatum resolved more than one haplotype per species, suggesting the presence of a high diversity or a high mutation rate of the corresponding nucleotide sequence. Sequences from E. bogotense appeared as a distinct group of haplotypes representing the subgenus Paramochaete that diverged from Hippochaete and Equisetum. In addition, the taxa that were frequently located at the joint region of the map were E. scirpoides and E. pratense, suggesting the presence of some plastome characters among the Equiseum subgenera.

Microbiome and related structural features of Earth's most archaic plant indicate early plant symbiosis attributes.

Origin of earliest land plants from ancestral algae dramatically accelerated the evolution of Earth's terrestrial ecosystems, in which microbial symbioses have played key roles. Recent molecular diversification analyses identify the rare, geographically-limited moss Takakia as Earth's most archaic modern land plant. Despite occupying a phylogenetic position pivotal for understanding earliest plants, Takakia microbial associations are poorly known. Here, we describe symbiosis-related structural features and contig-based metagenomic data that illuminate the evolutionary transition from streptophyte algae to early embryophytes. We observed that T. lepidozioides shares with streptophyte algae secretion of microbe-harboring mucilage and bacterial taxa such as Rhizobium and genes indicating nitrogen fixation. We find that Takakia root-analogs produce lateral mucilage organs that are more complex than generally understood, having structural analogies to angiosperm lateral roots adapted for N-fixation symbioses, including presence of intracellular microbes. We also find structural and metagenomic evidence for mycorrhiza-like species of glomalean fungi (including Rhizophagus irregularis) not previously known for mosses, as well as ascomycete fungi (e.g. Rhizoscyphus ericae) that associate with other early-diverging plants. Because Takakia is the oldest known modern plant genus, this study of plants of a remote locale not strongly influenced by human activities may indicate microbiome features of early land plants.

Spatial Variation of Cladophora Epiphytes in the Nan River, Thailand.

Cladophora is an algal genus known to be ecologically important. It provides habitats for microorganisms known to provide ecological services such as biosynthesis of cobalamin (vitamin B12) and nutrient cycling. Most knowledge of microbiomes was obtained from studies of lacustrine Cladophora species. However, whether lotic freshwater Cladophora microbiomes are as complex as the lentic ones or provide similar ecological services is not known. To illuminate these issues, we used amplicons of 16S rDNA, 18S rDNA, and ITS to investigate the taxonomy and diversity of the microorganisms associated with replicate Cladophora samples from three sites along the Nan River, Thailand. Results showed that the diversity of prokaryotic and eukaryotic members of Cladophora microbiomes collected from different sampling sites was statistically different. Fifty percent of the identifiable taxa were shared across sampling sites: these included organisms belonging to different trophic levels, decomposers, and heterotrophic bacteria. These heterogeneous assemblages of bacteria, by functional inference, have the potential to perform various ecological functions, i.e., cellulose degradation, cobalamin biosynthesis, fermentative hydrogen production, ammonium oxidation, amino acid fermentation, dissimilatory reduction of nitrate to ammonium, nitrite reduction, nitrate reduction, sulfur reduction, polyphosphate accumulation, denitrifying phosphorus-accumulation, and degradation of aromatic compounds. Results suggested that river populations of Cladophora provide ecologically important habitat for microorganisms that are key to nutrient cycling in lotic ecosystems.

Shotgun metagenomics and microscopy indicate diverse cyanophytes, other bacteria, and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria).

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free-living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.

Bacteria Associated with Echinodorus cordifolius and Lepironia articulata Enhance Nitrogen and Phosphorus Removal from Wastewater.

Phytoremediation and bioremediation are eco-friendly methods of wastewater treatment that are widely used throughout the world to reduce anthropogenic water contamination. This study was conducted to assess the effectiveness of symbiotic bacteria in phytoremediation using two aquatic plants, Echinodorus cordifolius and Lepironia articulata, that were tested in sterilized and unsterilized groups. The results showed that unsterilized plants removed more phosphate, ammonium, nitrate and nitrite than the sterilized plants. In untreated and unsterilized E. cordifolius groups, the dominant bacterium was Calothrix (46.90 and 49.69%, respectively), which was higher than in the sterilized E. cordifolius group (38.88%). In untreated and unsterilized groups of L. articulata, Clostridium was a dominant bacterium. The proportion of Clostridium was much lower in the sterilized L. articulata group (1.31%) than in the untreated (13.71%) and unsterilized (49.02%) groups. Our results suggested that root-associated bacteria in E. cordifolius and L. articulata were effective in the removal of phosphorus and nitrogen from domestic wastewater.

Evolution of organellar genes of chlorophyte algae: Relevance to phylogenetic inference.

Protein-coding genes in organellar genomes have been widely used to resolve relationships of chlorophyte algae. The mode of evolution of these protein-coding genes affects relationship estimations, yet selection effects on genes commonly used as markers in phylogenetic analyses are insufficiently well understood. To gain more understanding about the effects of green algal organelle protein-coding genes on phylogenies, more information is needed about the mode of gene evolution. We used phylogenetic frameworks to examine evolutionary relationships of 58 protein-coding genes present in the organellar genomes of chlorophyte and streptophyte algae at multiple levels: organelle, biological function, and individual gene, and calculated pairwise dN/dS ratios of algal organellar protein-coding genes to investigate mode of evolution. Results indicate that mitochondrial genes have evolved at a higher rate than have chloroplast genes. Low dN/dS ratios indicating relatively high level of conservation indicate that nad2, nad5, atpA, atpE, psbC, and psbD might be particularly good candidates for use as markers in chlorophyte phylogenies. Chlorophycean atp6, nad2, atpF, clpP, rps2, rps3, rps4, and rps7 protein-coding sequences exhibited selective mutations, suggesting that changes in proteins encoded by these genes might have increased fitness in Chlorophyceae.

Genome-wide analysis of carbohydrate-active enzymes in Pyramimonas parkeae (Prasinophyceae).

The wall-less green flagellate Pyramimonas parkeae is classified in clade I of the prasinophytes, a paraphyletic assemblage representing the last common ancestor of Viridiplantae, a monophyletic group composed of the green algae and land plants. Consequently, P. parkeae and other prasinophytes illuminate early-evolved Viridiplantae traits likely fundamental in the systems biology of green algae and land plants. Cellular structure and organellar genomes of P. parkeae are now well understood, and transcriptomic sequence data are also publically available for one strain of this species, but corresponding nuclear genomic sequence data are lacking. For this reason, we obtained shotgun genomic sequence and assembled a draft nuclear genome for P. parkeaeNIES254 to use along with existing transcriptomic sequence to focus on carbohydrate-active enzymes. We found that the P. parkeae nuclear genome encodes carbohydrate-active protein families similar to those previously observed for other prasinophytes, green algae, and early-diverging embryophytes for which full nuclear genomic sequence is publically available. Sequences homologous to genes related to biosynthesis of starch and cell wall carbohydrates were identified in the P. parkeae genome, indicating molecular traits common to Viridiplantae. For example, the P. parkeae genome includes sequences clustering with bacterial genes that encode cellulose synthases (Bcs), including regions coding for domains common to bacterial and plant cellulose synthases; these new sequences were incorporated into phylogenies aimed at illuminating the evolutionary history of cellulose production by Viridiplantae. Genomic sequences related to biosynthesis of xyloglucans, pectin, and starch likewise shed light on the origin of key Viridiplantae traits.

Complete mitochondrial genomes of prasinophyte algae Pyramimonas parkeae and Cymbomonas tetramitiformis.

Mitochondria are archetypal eukaryotic organelles that were acquired by endosymbiosis of an ancient species of alpha-proteobacteria by the last eukaryotic common ancestor. The genetic information contained within the mitochondrial genome has been an important source of information for resolving relationships among eukaryotic taxa. In this study, we utilized mitochondrial and chloroplast genomes to explore relationships among prasinophytes. Prasinophytes are represented by diverse early-diverging green algae whose physical structures and genomes have the potential to elucidate the traits of the last common ancestor of the Viridiplantae (or Chloroplastida). We constructed de novo mitochondrial genomes for two prasinophyte algal species, Pyramimonas parkeae and Cymbomonas tetramitiformis, representing the prasinophyte clade. Comparisons of genome structure and gene order between these species and to those of other prasinophytes revealed that the mitochondrial genomes of P. parkeae and C. tetramitiformis are more similar to each other than to other prasinophytes, consistent with other molecular inferences of the close relationship between these two species. Phylogenetic analyses using the inferred amino acid sequences of mitochondrial and chloroplast protein-coding genes resolved a clade consisting of P. parkeae and C. tetramitiformis; and this group (representing the prasinophyte clade I) branched with the clade II, consistent with previous studies based on the use of nuclear gene markers.

Comparative DNA sequence analyses of Pyramimonas parkeae (Prasinophyceae) chloroplast genomes.

Prasinophytes form a paraphyletic assemblage of early diverging green algae, which have the potential to reveal the traits of the last common ancestor of the main two green lineages: (i) chlorophyte algae and (ii) streptophyte algae. Understanding the genetic composition of prasinophyte algae is fundamental to understanding the diversification and evolutionary processes that may have occurred in both green lineages. In this study, we sequenced the chloroplast genome of Pyramimonas parkeae NIES254 and compared it with that of P. parkeae CCMP726, the only other fully sequenced P. parkeae chloroplast genome. The results revealed that P. parkeae chloroplast genomes are surprisingly variable. The chloroplast genome of NIES254 was larger than that of CCMP726 by 3,204 bp, the NIES254 large single copy was 288 bp longer, the small single copy was 5,088 bp longer, and the IR was 1,086 bp shorter than that of CCMP726. Similarity values of the two strains were almost zero in four large hot spot regions. Finally, the strains differed in copy number for three protein-coding genes: ycf20, psaC, and ndhE. Phylogenetic analyses using 16S and 18S rDNA and rbcL sequences resolved a clade consisting of these two P. parkeae strains and a clade consisting of these plus other Pyramimonas isolates. These results are consistent with past studies indicating that prasinophyte chloroplast genomes display a higher level of variation than is commonly found among land plants. Consequently, prasinophyte chloroplast genomes may be less useful for inferring the early history of Viridiplantae than has been the case for land plant diversification.

Complete Chloroplast Genome Sequence of Phagomixotrophic Green Alga Cymbomonas tetramitiformis.

We report here the complete chloroplast genome sequence of Cymbomonas tetramitiformis strain PLY262, which is a prasinophycean green alga that retains a phagomixotrophic mode of nutrition. The genome is 84,524 bp in length, with a G+C content of 37%, and contains 3 rRNAs, 26 tRNAs, and 76 protein-coding genes.