Evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous fungi

Abstract

In filamentous fungi, het loci (for heterokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. As filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. Hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. However, heterokaryotic cells are viable only if the individuals involved have identical alleles at all het loci. One het locus, het-c, has been characterized at the molecular level in Neurospora crassa and encodes a glycine-rich protein. In an effort to understand the role of this locus in filamentous fungi, we chose to study its evolution by analyzing het-c sequence variability in species within Neurospora and related genera. We determined that the het-c locus was polymorphic in a field population of N. crassa with close to equal frequency of each of the three allelic types. Different species and even genera within the Sordariaceae shared het-c polymorphisms, indicating that these polymorphisms originated in an ancestral species. Finally, an analysis of the het-c specificity region shows a high occurrence of nonsynonymous substitution. The persistence of allelic lineages, the nearly equal allelic distribution within populations, and the high frequency of nonsynonymous substitutions in the het-c specificity region suggest that balancing selection has operated to maintain allelic diversity at het-c. Het-c shares this particular evolutionary characteristic of departing from neutrality with other self/nonself-recognition systems such as major histocompatibility complex loci in mammals and the S (self-incompatibility) locus in angiosperms.

Figure 1

Inferred amino acid sequences of PCR amplified het-c allelic specificity motif from 40 isolates. The alignment was obtained by clustalw 6.1 and modified manually. Nc, Neurospora crassa; Ni, N. intermedia; Ndi, N. discreta; Ns, N. sitophila; Nt, N. tetrasperma; Np, N. pannonica; Ndo, N. dodgei; Ss, Sordaria sclerogenia; Sh, S. heterothallis; Sb, S. brevicollis; Gsp, Gelasinospora sp. Numbers following indicate the Fungal Genetics Stock Center (FGSC) number or P number (4481, 4486, and 4499 strains). PA (Panama), GR (Groveland), and OR (Oakridge) refer to the three molecularly characterized alleles that confer different het-c specificities (20). The het-c specificity of alleles designated in the figure was determined by partial diploid analysis or heterokaryon tests (refs. and and unpublished results). Asterisk and dashes indicate identical and deletion residue sites, respectively. Underlined section is het-c specificity variable region. Bold region is polymorphic block.

Figure 2

Neighbor-joining tree using 390-bp nucleotide sequences spanning the het-c specificity motif from 39 isolates (N. crassa FGSC 1130 (Panama) and 430 (Ivory Coast) have identical nucleotide sequences in this region). The het-c sequence from the homothallic species, N. dodgei (Ndo1692), was used as an outgroup. Distance was calculated by Kimura’s two parameter method in the phylogenetic analysis package phylip 3.5 (24). Numbers are bootstrap percentages (out of 1,000 resamplings) in support of each node. Bootstrap percentages below 70% were not included. Each insertion and deletion was treated as a single event in the analysis. Species abbreviations are indicated as in Fig. 1. The designation OR, het-c^OR allelic-type; PA, het-c^PA allelic-type; and GR, het-c^GR allelic-type is based on alignment in Fig. 1.

Figure 3

Histograms of d_N and d_S substitutions per site mapped onto the codons for both the polymorphic and conserved regions in het-c from the 40 isolates. Sites 40–129 correspond to the sequences aligned in Fig. 1. Filled bar sites (codons 65–119) are polymorphic and correspond to the underlined region in Fig. 1. (a) The average number of nonsynonymous substitutions per nonsynonymous site (d_N) grouped by codons, and (b) the average number of synonymous substitutions per synonymous site (d_S) grouped by codons. Calculations were done by mega according to the Cantor–Jukes correction (25).