Distribution and Environmental Drivers of Fungal Denitrifiers in Global Soils

Abstract

The microbial process of denitrification is the primary source of the greenhouse gas nitrous oxide (N₂O) from terrestrial ecosystems. Fungal denitrifiers, unlike many bacteria, lack the N₂O reductase, and thereby are sources of N₂O. Still, their diversity, global distribution, and environmental determinants, as well as their relative importance, compared to bacterial and archaeal denitrifiers, remain unresolved. Employing a phylogenetically informed approach to analyze 1,980 global soil and rhizosphere metagenomes for the denitrification marker gene nirK, which codes for the copper dependent nitrite reductase in denitrification, we show that fungal denitrifiers are sparse, yet cosmopolitan and that they are dominated by saprotrophs and pathogens. Few showed biome-specific distribution patterns, although members of the Fusarium oxysporum species complex, which are known to produce substantial amounts of N₂O, were proportionally more abundant and diverse in the rhizosphere than in other biomes. Fungal denitrifiers were most frequently detected in croplands, but they were most abundant in forest soils when normalized to metagenome size. Nevertheless, the overwhelming dominance of bacterial and archaeal denitrifiers suggests a much lower fungal contribution to N₂O emissions than was previously estimated. In relative terms, they could play a role in soils that are characterized by a high carbon to nitrogen ratio and a low pH, especially in the tundra as well as in boreal and temperate coniferous forests. Because global warming predicts the proliferation of fungal pathogens, the prevalence of potential plant pathogens among fungal denitrifiers and the cosmopolitan distribution of these organisms suggest that fungal denitrifier abundance may increase in terrestrial ecosystems. IMPORTANCE Fungal denitrifiers, in contrast to their bacterial counterparts, are a poorly studied functional group within the nitrogen cycle, even though they produce the greenhouse gas N₂O. To curb soil N₂O emissions, a better understanding of their ecology and distribution in soils from different ecosystems is needed. Here, we probed a massive amount of DNA sequences and corresponding soil data from a large number of samples that represented the major soil environments for a broad understanding of fungal denitrifier diversity at the global scale. We show that fungal denitrifiers are predominantly cosmopolitan saprotrophs and opportunistic pathogens. Fungal denitrifiers constituted, on average, 1% of the total denitrifier community. This suggests that earlier estimations of fungal denitrifier abundance, and, thereby, it is also likely that the contributions of fungal denitrifiers to N₂O emissions have been overestimated. Nevertheless, with many fungal denitrifiers being plant pathogens, they could become increasingly relevant, as soilborne pathogenic fungi are predicted to increase with ongoing climate change.

Keywords: biogeography; denitrification; nirK; nitrous oxide; pathogenic fungi; phyloecology; terrestrial fungi.

FIG 1

Origins of metagenomes and abundance of fungal nirK in terrestrial biomes. (A) 1,980 metagenomes representing 608 sampling locations across the globe. The sampling locations of 41 rhizosphere samples are not indicated due to the absence of associated geographic coordinates. Made with Natural Earth. (B) Fungal nirK (fnirK) gene fragment counts, normalized to the total number of reads per metagenome. (C) Abundance of fnirK, relative to fungal 18S rRNA gene (18S) fragment counts. (D) Abundance of fungal, relative to prokaryotic, nirK (pnirK) gene fragment counts. Different letters indicate significant differences between biomes (ANOVA, Šidák-corrected pairwise comparisons, P < 0.05). The numbers above the boxplots in panels B to D indicate the number of metagenomes within each biome with at least one fungal nirK hit. The box limits represent the interquartile range (IQR), with the median values being represented by the centerline. Whiskers represent values that are ≤1.5 times the upper and lower quartiles, whereas points indicate values outside this range. The shaded areas show kernel density estimations, indicating the distribution of the data.

FIG 2

Reference phylogeny of prokaryotic and fungal nirK, created from 6,732 full-length genomic nirK gene sequences. The phylogeny was determined based on a maximum likelihood analysis of the amino acid sequences of nirK, using the LG+G substitution model. The phylogeny was subsequently used as the reference tree for the phylogenetic placement of nirK gene fragments that were retrieved from 1,980 metagenomes. Clades were collapsed, and the number of species per collapsed clade is indicated. The outgroups consist of multicopper oxidase (MCO) sequences originating from cyanobacterial and thaumarcheotal genomes.

FIG 3

Phylogenetic placements of fungal nirK gene fragments detected in soil and rhizosphere biomes within the nirK reference cladogram tree. The leaf color indicates the fungal class, and the outgroup sequences are collapsed. The most likely phylogenetic placement for each read is represented by a circle and is colored according to the biome classification at Level 2. The circle size indicates the number of placements on a given tree edge. Stars mark the branches that are enriched in fungal nirK placements within a biome, compared to other biomes. Biome-specific placements at Level 2 are shown in Fig. S2.

FIG 4

Spearman ranked correlations of fungal nirK abundance as well as the ratio to 18S rRNA gene abundance (fnirK:18S) and prokaryotic nirK (fnirK: pnirK) with edaphic variables. Correlations are shown for all terrestrial biomes and for five terrestrial biomes at Level 2. Rhizosphere metagenomes were not included due to the absence of associated metadata. The correlations are colored if significant (P < 0.05), according to their strength, with red for negative correlations and blue for positive correlations. Nonsignificant correlations are colored in gray. Those not determined due to an insufficient sample number (n < 25) are marked with an asterisk. Sample sizes differed among metagenomes, as indicated in Table S2. SOC, soil organic carbon; C:N, total carbon to total nitrogen ratio; N, total nitrogen; NH₄⁺, ammonium; NO₃⁻, nitrate; moisture, soil moisture; clay, soil clay content; Cu, soil copper content; pH, soil pH measured in CaCl₂. NO₃⁻ in tundra soils was reported in μM.