Yongcui Deng

Microbial Diversity in Hummock and Hollow Soils of Three Wetlands on the Qinghai-Tibetan Plateau Revealed by 16S rRNA Pyrosequencing

The wetlands of the Qinghai-Tibetan Plateau are believed to play an important role in global nutrient cycling, but the composition and diversity of microorganisms in this ecosystem are poorly characterized. An understanding of the effects of geography and microtopography on microbial populations will provide clues to the underlying mechanisms that structure microbial communities. In this study, we used pyrosequencing-based analysis of 16S rRNA gene sequences to assess and compare the composition of soil microbial communities present in hummock and hollow soils from three wetlands (Dangxiong, Hongyuan and Maduo) on the Qinghai-Tibetan Plateau, the world’s highest plateau. A total of 36 bacterial phyla were detected. Proteobacteria (34.5% average relative abundance), Actinobacteria (17.3%) and Bacteroidetes (11%) had the highest relative abundances across all sites. Chloroflexi, Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were also relatively abundant (1–10%). In addition, archaeal sequences belonging to Euryarchaea, Crenarchaea and Thaumarchaea were detected. Alphaproteobacteria sequences, especially of the order Rhodospirillales, were significantly more abundant in Maduo than Hongyuan and Dangxiong wetlands. Compared with Hongyuan soils, Dangxiong and Maduo had significantly higher relative abundances of Gammaproteobacteria sequences (mainly order Xanthomonadales). Hongyuan wetland had a relatively high abundance of methanogens (mainly genera Methanobacterium, Methanosarcina and Methanosaeta) and methanotrophs (mainly Methylocystis) compared with the other two wetlands. Principal coordinate analysis (PCoA) indicated that the microbial community structure differed between locations and microtopographies and canonical correspondence analysis indicated an association between microbial community structure and soil properties or geography. These insights into the microbial community structure and the main controlling factors in wetlands of the Qinghai-Tibetan Plateau provide a valuable background for further studies on biogeochemical processes in this distinct ecosystem.

Classification of pmoA amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN

The classification of high-throughput sequencing data of protein-encoding genes is not as well established as for 16S rRNA. The objective of this work was to develop a simple and accurate method of classifying large datasets of pmoA sequences, a common marker for methanotrophic bacteria. A taxonomic system for pmoA was developed based on a phylogenetic analysis of available sequences. The taxonomy incorporates the known diversity of pmoA present in public databases, including both sequences from cultivated and uncultivated organisms. Representative sequences from closely related genes, such as those encoding the bacterial ammonia monooxygenase, were also included in the pmoA taxonomy. In total, 53 low-level taxa (genus-level) are included. Using previously published datasets of high-throughput pmoA amplicon sequence data, we tested two approaches for classifying pmoA: a naïve Bayesian classifier and BLAST. Classification of pmoA sequences based on BLAST analyses was performed using the lowest common ancestor (LCA) algorithm in MEGAN, a software program commonly used for the analysis of metagenomic data. Both the naïve Bayesian and BLAST methods were able to classify pmoA sequences and provided similar classifications; however, the naïve Bayesian classifier was prone to misclassifying contaminant sequences present in the datasets. Another advantage of the BLAST/LCA method was that it provided a user-interpretable output and enabled novelty detection at various levels, from highly divergent pmoA sequences to genus-level novelty.

Aerobic methanotroph diversity in Riganqiao peatlands on the Qinghai–Tibetan Plateau

The Zoige Plateau is characterized by its high altitude, low latitude and low annual mean temperature of approximately 1°C and is a major source of atmospheric methane in the Qinghai-Tibetan Plateau. Methanotrophs play an important role in the global cycling of CH4, but the diversity, identity and activity of methanotrophs in this region are poorly characterized. Soils were collected from hummocks and hollows in the Riganqiao peatland and the methanotroph community was analysed by qPCR and sequencing methane monooxygenase (pmoA and mmoX) genes. The pmoA genes ranged between 10(7) and 10(8) copies g(-1) fresh soil, with a somewhat greater abundance in hummocks than hollows. The pmoA genes were analysed by amplicon pyrosequencing and the mmoX genes by cloning and sequencing. Methylocystis species were found to be the most abundant methanotrophs, but numerous clades were present including three novel pmoA and three novel mmoX clusters. There were differences between the methanotroph communities in the hummocks and hollows, with the most significant being an increased abundance of uncultivated type Ib methanotrophs in the hollows. The results indicate that aerobic methanotrophs are abundant in Riganqiao peatland and include previously undetected clades in this geographically isolated and distinctive environment.

Identification of active aerobic methanotrophs in plateau wetlands using DNA stable isotope probing

Sedge-dominated wetlands on the Qinghai-Tibetan Plateau are methane emission centers. Methanotrophs at these sites play a role in reducing methane emissions, but relatively little is known about the composition of active methanotrophs in these wetlands. Here, we used DNA stable isotope probing to identify the key active aerobic methanotrophs in three sedge-dominated wetlands on the plateau. We found that Methylocystis species were active in two peatlands, Hongyuan and Dangxiong. Methylobacter species were found to be active only in Dangxiong peat. Hongyuan peat had the highest methane oxidation rate, and cross-feeding of carbon from methanotrophs to methylotrophic Hyphomicrobium species was observed. Owing to a low methane oxidation rate during the incubation, the labeling of methanotrophs in Maduo wetland samples was not detected. Our results indicate that there are large differences in the activity of methanotrophs in the wetlands of this region.

Anthropogenic protection alters the microbiome in intertidal mangrove wetlands in Hainan Island

Intertidal mangrove wetlands are of great economic and ecological importance. The regular influence of tides has led to the microbial communities in these wetlands differing significantly from those in other habitats. In this study, we investigated the microbiomes of the two largest mangrove wetlands in Hainan Island, China, which have different levels of anthropogenic protection. Soil samples were collected from the root zone of 13 mangrove species. The microbial composition, including key functional groups, was assessed using Illumina sequencing. Bioinformatics analysis showed that there was a significant difference in the microbiomes between the protected Bamen Bay and the unprotected Dongzhai Bay. The overall microbiome was assigned into 78 phyla and Proteobacteria was the most abundant phylum at both sites. In the protected wetland, there were fewer marine-related microbial communities, such as sulfate-reducing bacteria, and more terrestrial-related communities, such as Verrucomicrobia methanotrophs. We also observed distinct microbial compositions among the different mangrove species at the protected site. Our data suggest that the different microbiomes of the two mangrove wetlands are the result of a complex interaction of the different environmental variables at the two sites.

Autotrophic and symbiotic diazotrophs dominate nitrogen-fixing communities in Tibetan grassland soils

Biological nitrogen fixation, conducted by soil diazotrophs, is the primary nitrogen source for natural grasslands. However, the diazotrophs in grassland soils are still far from fully investigated. Particularly, their regional-scale distribution patterns have never been systematically examined. Here, soils (0-5 cm) were sampled from 54 grasslands on the Tibetan Plateau to examine the diazotroph abundance, diversity, and community composition, as well as their distribution patterns and driving factors. The diazotroph abundance was expressed as nifH gene copies, measured using real-time PCR. The diversity and community composition of diazotrophs were analyzed through MiSeq sequencing of nifH genes. The results showed that Cyanobacteria (47.94%) and Proteobacteria (45.20%) dominated the soil diazotroph communities. Most Cyanobacteria were classified as Nostocales which are main components of biological crusts. Rhizobiales, most of which were identified as potential symbiotic diazotrophs, were also abundant in approximately half of the soil samples. The soil diazotroph abundance, diversity, and community composition followed the distribution patterns in line with mean annual precipitation. Moreover, they also showed significant correlations with prokaryotic abundance, plant biomass, vegetation cover, soil pH values, and soil nutrient contents. Among these environmental factors, the soil moisture, organic carbon, available phosphorus, and inorganic nitrogen contents could be the main drivers of diazotroph distribution due to their strong correlations with diazotroph indices. These findings suggest that autotrophic and symbiotic diazotrophs are the predominant nitrogen fixers in Tibetan grassland soils, and highlight the key roles of water and nutrient availability in determining the soil diazotroph distribution on the Tibetan Plateau.