Ancient trans-specific polymorphism at pheromone receptor genes in basidiomycetes

Abstract

In the majority of sexual organisms, reproduction occurs almost exclusively through the combination of distinct and alternate forms, called sexes or mating types. In some fungi, there can be dozens to hundreds of alternate alleles that determine compatible mating types. Such extensive polymorphism is expected to be maintained by balancing selection, and in extreme cases may give rise to trans-specific polymorphism. Here, we analyzed sequences of two pheromone receptors in the Microbotryum fungal species complex (Basidiomycota), which has only two alternate mating types. Several lines of evidence strongly suggest that the pheromone receptors are two allelic sequences acting to determine the alternate A1 and A2 mating types required for mating in Microbotryum. Phylogenetic trees of pheromone receptors in the Microbotryum species complex indicated a trans-specific polymorphism: the Microbotryum sequences from a given mating type were all more similar to the pheromone receptors of distantly related classes of fungi than to the alternate pheromone receptor in the Microbotryum species. A phylogenetic tree built using other known pheromone receptors from basidiomycetes showed that trans-specific polymorphism is widespread. The pheromone receptor alleles from Microbotryum appeared as the oldest, being at least 370 million years old. This represents the oldest known trans-specific polymorphism known in any organism so far, which may be due to the existence of sex chromosomes, obligate sexuality, mitochondrial inheritance linked to the mating type, and a highly selfing mating system in Microbotryum.

Figure 1.—

Organization of the locus encompassing the pheromone (narrow vertical rectangles) and pheromone receptor (wide horizontal rectangles) genes in Ustilago maydis (A) and Coprinopsis cinerea (B). Arrows indicate receptor and pheromone combinations that are compatible. Different degrees of shading (solid, shaded, open) indicate paralogs and different filled motifs indicate alleles. The a locus in U. maydis has one a-receptor gene (pra) and one a-pheromone gene (mfra). The products of Iga2 and Rga2 (squares) have no obvious role in sexual development but are involved in mitochondrial function (Bortfeld et al. 2004). The B locus of C. cinerea is composed of three subloci, each containing a receptor gene and two pheromone genes. For two haploid cells to be compatible, it is required and sufficient that they have different alleles for at least one of the sublocus. Pheromones can only activate receptors within the same sublocus class. This figure has been adapted from Casselton and Kues (2007) and Stanton and Hull (2007).

Figure 2.—

Illustration of the fragments of the pheromone receptors pr-MatA1 and pr-MatA2 that were sequenced in multiple Microbotryum species. Two overlapping fragments were amplified per receptor using the primers indicated in Table 2.

Figure 3.—

Phylogeny based on the amino acid sequences of parts of the pheromone receptors pr-MatA1 and pr-MatA2 of Microbotryum and other fungi. Nodes not strongly supported are represented as unresolved. Statistical supports indicate NJ bootstraps. Taxa labels for Microbotryum sequences correspond to the mating type (A1 or A2) and to the host plant on which fungal strains were collected.

Figure 4.—

Phylogeny of all known complete STE3-like pheromone receptors from basidiomycete fungi, obtained from the PHYML analysis. Statistical supports indicate ML bootstraps. The calibration point of 450 MY is indicated (node A), as well as the dates for nodes B and C inferred from the molecular clock analysis, whose resulting phylogeny is shown in supplemental Figure S3. The phylogeny of the same genera based on nuclear gene sequences obtained in previous works (Roux et al. 1998; Lutzoni et al. 2004; Blackwell et al. 2006; James et al. 2006; Garnica et al. 2007; James 2007; Matheny et al. 2007) is given as an inset. The calibration point of 450 MY, which represents the divergence between ascomycetes and basidiomycetes, is also indicated, as well as the duplication of pheromone receptor genes at the mating-type locus. The time calibration was determined using a molecular clock analysis performed with PAML (supplemental Figure S3) and was placed on the PHYML tree for convenience.