Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga, a cyanobacterium, or both. About one-fifth of all known fungi lichenize, and for these fungi the symbiosis is, with few exceptions, obligate. Unlike mycorrhizal or rhizobial symbioses, lichen symbioses are not well understood. This is mainly because lichens grow very slowly (≤1 cm a year), it is very difficult to grow the fungus and the alga separately and, moreover, it is not yet possible to resynthesize the mature symbiosis in the laboratory from the fungal and photobiont partners isolated in axenic culture [1–4]. Also, it is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of any lichenizing fungus or alga. However, the lack of genetic tools for these intractable organisms has been partially compensated for by the advent of high-throughput genome sequencing.
Previously, the genomes of the model lichen Cladonia grayi, made up of the lichenizing fungus Cladonia grayi for which the association is named, and its green algal partner Asterochloris sp., were sequenced at Duke University [5, 6]. We searched the genome sequences for evidence of horizontal gene transfer [7–9] between the lichen symbionts; that is, whether there were genes of algal origin in the fungal genome or genes of fungal origin in the algal genome. A thorough homology search of all the genes in each genome revealed that two genes in the fungal genome appeared to have been horizontally transferred, although not from green algae. Both genes encoded ammonium transporters .
Ammonium transporters/ammonia permeases (AMTPs) are highly conserved proteins found in most organisms, including prokaryotes and eukaryotes. These proteins are composed of 11 transmembrane helices that fold to form a pore through which ammonia or ammonium moves [11, 12]. In their native conformation they trimerize, forming a tripartite pore . While some AMTPs have been shown to transport ammonium (NH4
+), notably those proteins in the AMT2 family of land plants [14, 15], most AMTPs have been shown to transport ammonia (NH3) [16–22]. Proteins in the related Rh family  have 12 transmembrane domains and have been shown to conduct ammonia and in some cases CO2[24–26].
The evolutionary history of this family of genes is complex, involving lineage-specific gene family expansions, contractions, and losses as well as ancient and recent horizontal gene transfer events. Fungal AMTPs are in a phylogenetic clade by themselves that includes both low-affinity [27–30] and high-affinity [31–35] AMTPs (MEP γ clade ). The history of these genes in the fungi is particularly complicated, appearing to commence with an ancient horizontal gene transfer event of high-affinity AMTPs of prokaryotic origin during the early evolution of the fungi, followed by several lineage-specific gene-family expansions, as well as a duplication and neofunctionalization event in the early evolution of the Dikarya that lead to the evolution of low-affinity AMTPs .
In addition to these events, a second horizontal gene transfer event of high-affinity AMTPs occurred in the early evolution of filamentous ascomycetes (associated with a putative adaptive radiation of the leotiomyceta [10, 36, 37]). These horizontally transferred AMTPs are distinct from the fungal high- and low-affinity AMTPs of the MEP γ clade, and in fact are most closely related to AMTPs from land plants in the AMT2 family e.g. [12, 15, 35, 38–41] and to transporters from mostly hyperacidophilic chemoautolithotrophic prokaryotes inhabiting deep sea thermal vents , volcanic hotsprings and thermal vents [43–48], acid mine drainages [49–55], and similar extreme environments (MEP α clade) .
Interestingly, only a subset of the leotiomyceta, most of which are symbiotic with green algae in lichen symbioses, have representatives of this new clade of AMTPs in their genomes. In fact, lichenizing fungi in three different taxonomic classes of fungi, including the Lecanoromycetes, the Eurotiomycetes and the Dothideomycetes [36, 56, 57], have actually duplicated these genes. By contrast, as of 2012 only four non-lichenizing fungi in two genera (Penicillium with Talaromyces teleomorphs, Fusarium with Gibberella teleomorphs) out of more than 200 publicly available sequenced fungal genomes have representatives of this new clade of AMTPs in their genomes, and these transporters are not duplicated. This result suggests that lichenized fungi have preferentially retained the MEP α gene after the initial horizontal gene transfer event during the early evolution of the filamentous ascomycetes, while non-lichenized fungi have lost this gene .
The MEP α gene was not found in all lichens surveyed. In particular, the MEP α gene was never recovered from the two orders of lichens most closely related to the order in which the original discovery was made. In one of these two orders, the Peltigerales , the lichens are symbiotic with nitrogen-fixing cyanobacteria. In the other order, the Teloschistales , many lichens inhabit high-nitrogen niches, like bird perching sites. The availability of nitrogen sources from the environment or from a symbiont, coupled with the failure to identify the AMTP gene by PCR suggests that lichens in these two orders may no longer need the MEP α AMTP and may have shed it from their genomes.
Here, we further characterize this new clade of fungal AMTPs. We sequence the genomes of eight lichenizing fungi in key lineages that may have shed the AMTPs and search the genomes for the horizontally-transferred AMTPs. We correlate the presence of the AMTPs of the new clade with nitrogen lifestyle by surveying lichen fungi that are closely related to the main lineages previously examined but that tolerate high-nitrogen habitats, or that employ nitrogen-fixing cyanobacteria rather than green algae as the primary symbionts. We also characterize the function of the AMTPs from one lichen, Cladonia grayi, by assaying for growth on ammonium as a sole nitrogen source. We present a phylogeny of fungal AMTPs to contextualize this clade.