Cultivation and cloning allow phylogenetic placement of a prominent fungal lineage.

We combined gene divergence data, classical genetics, and phylogenetics to study the evolution of the mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma. In this species, a large non-recombining region of the mating-type chromosome is associated with a unique fungal life cycle where self-fertility is enforced by maintenance of a constant state of heterokaryosis. Sequence divergence between alleles of 35 genes from the two single mating-type component strains (i.e. the homokaryotic mat A or mat a-strains), derived from one N. tetrasperma heterokaryon (mat A+mat a), was analyzed. By this approach we were able to identify the boundaries and size of the non-recombining region, and reveal insight into the history of recombination cessation. The non-recombining region covers almost 7 Mbp, over 75% of the chromosome, and we hypothesize that the evolution of the mating-type chromosome in this lineage involved two successive events. The first event was contemporaneous with the split of N. tetrasperma from a common ancestor with its outcrossing relative N. crassa and suppressed recombination over at least 6.6 Mbp, and the second was confined to a smaller region in which recombination ceased more recently. In spite of the early origin of the first “evolutionary stratum”, genealogies of five genes from strains belonging to an additional N. tetrasperma lineage indicate independent initiations of suppressed recombination in different phylogenetic lineages. This study highlights the shared features between the sex chromosomes found in the animal and plant kingdoms and the fungal mating-type chromosome, despite fungi having no separate sexes. As is often found in sex chromosomes of plants and animals, recombination suppression of the mating-type chromosome of N. tetrasperma involved more than one evolutionary event, covers the majority of the mating-type chromosome and is flanked by distal regions with obligate crossovers. In fungi, mating occurs between individuals of alternative mating-types and there is no dichotomy of individuals into two morphologically different sexes. Nevertheless, in this paper we show that chromosomal regions controlling mating-type identity in fungi share features with the more complex sex chromosomes found in the other eukaryote kingdoms. We have specifically studied the mating-type chromosome in an emerging model-species of filamentous ascomycetes, Neurospora tetrasperma, and show that it resembles the sex chromosomes of animals and plants both in failing to recombine with its homologous chromosome over the majority of its length, and having obligate crossovers at the flanking “pseudoautosomal” regions. Furthermore, our data indicate that the evolution of the mating-type chromosome in this species involved more than one successive evolutionary event, each defining an “evolutionary stratum”, a term initially introduced by to represent different sequential steps whereby recombination became arrested between the proto-sex chromosomes in humans. We argue that insight into the evolution of chromosomal sex determination can be gained through the study of alternative, simple, systems, such as N. tetrasperma, in which the genomic consequences of reduced recombination per se can be disentangled from sex-biased evolutionary forces such as male-biased mutation and dispersal.