Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships

Abstract

Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes.

Keywords: N-cycle; evolution; functional genes; phylogenetics; phylogeny; phylogeography; terrestrial subsurface.

Figure 1

Principle component analysis of physical and chemical characteristics of the fracture water samples.

Figure 2

Phylum-level taxonomic distribution of eight common functional genes detected in the subsurface metagenomes. N denotes the total number of gene sequences detected in the metagenomes. The taxonomic classification of these sequences was assigned based on the lineage of their NR-best hit. The common functional genes are denoted by letters: a: NarV gene; b: NPD gene; c: PAPS reductase gene; d: NifH gene; e: NifD gene; f: NifK gene; g: NifE gene; h: NifN gene. Sequence counts were also overlain with scaled color intensity for visual effect. The last row gives the number of phyla represented by each common functional gene.

Figure 3

Multi-dimensional scaling plots of dissimilarity in microbial compositions represented by the eight common functional genes within and between metagenomes at the phylum (A) and genus (B) level. Filled circles denote the location of the centroid of each metagenomes. The closer the centroids, the more similar are the taxonomic compositions of the represented metagenomes. Each centroid is joined to the eight common functional genes (denoted by metagenome-specific symbols) by black lines that represent the distance between the centroid and each gene. The dashed lines encapsulate the “distance space” of each metagenome. The size and shape of the “distance space” indicate within-sample variance in the taxonomic profiles. The inter-sample variances were significantly different at the phylum level but not at the genus level.

Figure 4

Maximum likelihood trees of deduced amino acid sequences of NarV and NifH genes detected in assembled metagenomes. Sequences from this study are highlighted by color lines, with blue for BE2011, cyan for BE2012, orange for DR5, green for FI88, magenta for TT107, dark purple for TT109, and yellow for MM5. P-test significance values of unweighted UniFrac distances among metagenomes are given. Scale bars represent the amino acid substitution rate per site. Number of taxa and characters, and amino acid evolutionary models used are as follow: NarV gene: 273, 188, LG+G; NPD gene: 584, 248, LG+G; PAPS reductase gene: 479, 159, LG+G; NifH gene: 191, 268, LG+I+G; NifD gene: 154, 415, LG+I+G; NifK gene: 196, 419, LG+I+G; NifE gene: 137, 427, LG+I+G; and NifN gene: 84, 423, LG+I+G.

Figure 5

Phylogenetic relatedness of the studied metagnomes revealed by each of the common functional genes. Branch support of the dendrograms was derived from 1000 permutations of Jackknife resampling. Nodes supported by 50–69% (open circles), 70–89% (half-filled circles), and 90–100% (filled circles) of the permutated trees are indicated.

Figure 6

Bayesian likelihood tree of deduced amino acid sequences of the PorC-AB operon. The same color scheme as in Figure 4 is used to highlight our sequences. Our metagenomes are followed by contig IDs, whereas reference taxa are suffixed by GenBank accession numbers and, if available, the source of isolates. Phyla are given to the right of the tree. Nodes are supported by posterior probability and scale bar represents the amino acid substitution rate per site.

Figure 7

Schematic diagram of the proposed N cycle in deep terrestrial subsurface sites in South Africa. Solid lines indicate processes suggested by molecular (this study) and geochemistry analyses (Silver et al., 2012). Dashed lines indicate processes that are known in the global N cycle but their occurrence in deep terrestrial subsurface sites is yet to be proven. Asterisks denote microbially mediated processes.