Horizontal transfer of a nitrate assimilation gene cluster and ecological transitions in fungi: a phylogenetic study

Abstract

High affinity nitrate assimilation genes in fungi occur in a cluster (fHANT-AC) that can be coordinately regulated. The clustered genes include nrt2, which codes for a high affinity nitrate transporter; euknr, which codes for nitrate reductase; and NAD(P)H-nir, which codes for nitrite reductase. Homologs of genes in the fHANT-AC occur in other eukaryotes and prokaryotes, but they have only been found clustered in the oomycete Phytophthora (heterokonts). We performed independent and concatenated phylogenetic analyses of homologs of all three genes in the fHANT-AC. Phylogenetic analyses limited to fungal sequences suggest that the fHANT-AC has been transferred horizontally from a basidiomycete (mushrooms and smuts) to an ancestor of the ascomycetous mold Trichoderma reesei. Phylogenetic analyses of sequences from diverse eukaryotes and eubacteria, and cluster structure, are consistent with a hypothesis that the fHANT-AC was assembled in a lineage leading to the oomycetes and was subsequently transferred to the Dikarya (Ascomycota+Basidiomycota), which is a derived fungal clade that includes the vast majority of terrestrial fungi. We propose that the acquisition of high affinity nitrate assimilation contributed to the success of Dikarya on land by allowing exploitation of nitrate in aerobic soils, and the subsequent transfer of a complete assimilation cluster improved the fitness of T. reesei in a new niche. Horizontal transmission of this cluster of functionally integrated genes supports the "selfish operon" hypothesis for maintenance of gene clusters.

Figure 1. Phylogeny and clustering of the fHANT-AC.

(a) Organismal phylogeny based on rRNA and RPB2 genes (right), and gene phylogeny of the fHANT-AC cluster (left), along with data on internal gene order and extent of clustering (center). Phylograms are from 50% majority rule Bayesian consensus. Shading of genes indicates confirmed expression of sequences in GenBank (dbEST:CF878787.1, CB906451.1, CF872359, CF865713, CB895628). Thick black branches denote strong support for the complete combined sequence (Bayesian posterior probabilities [BPP]>0.95, maximum parsimony bootstrap percentages [MPB]>85), and thick gray branches denote strong support from two of three individual genes (BPP 0.99 and/or MPB 70%). Strong support from maximum likelihood bootstraps ([MLB]>80%) is indicated by an *. Actual support values for critical nodes are in Table 1. Clades labeled A (Ascomycota), B (Basidiomycota) and S (Sordariomycetes) follow James et al. (2006). Contributing phylogenetic analyses are in SI Fig. S1a,b. (b) Description of open reading frames flanking the fHANT-AC in T. reesei and U. maydis. Complete descriptions are available at genome project websites (see SI Table S1).

Figure 2. Distribution and phylogeny of HANT-AC gene homologs.

(a) Unrooted phylograms of maximum likelihood analyses performed in RaxML-VI-HPC ver. 2.2.3. Support values for critical nodes can be found in Table 2. Contributing phylogenetic analyses are in SI Fig. 2a,b,c. (b) Distribution and clustering of HANT-AC genes in eukaryotes. Clustering indicated if most or all genomes in clade show clustering. Phylogeny of eukaryotes is adapted from Baldauf . ¹A homolog similar to bacterial sequences is present in Nematostella vectensis. ² N. vectensis may also possesses bacteria-like nitrite reductase homologs.