The Mating-Type Chromosome in the Filamentous Ascomycete Neurospora tetrasperma Represents a Model for Early Evolution of Sex Chromosomes Audrius Menkis 1,2 , David J. Jacobson 3¤ , Tim Gustafsson 2 , Hanna Johannesson 2 * 1 Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2 Department of Evolutionary Biology, Uppsala University, Uppsala, Sweden, 3 Department of Biological Sciences, Stanford University, Stanford, United States of America Abstract 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. Citation: Menkis A, Jacobson DJ, Gustafsson T, Johannesson H (2008) The Mating-Type Chromosome in the Filamentous Ascomycete Neurospora tetrasperma Represents a Model for Early Evolution of Sex Chromosomes. PLoS Genet 4(3): e1000030. doi:10.1371/journal.pgen.1000030 Editor: Gil McVean, University of Oxford, United Kingdom Received September 21, 2007; Accepted February 8, 2008; Published March 14, 2008 Copyright: ß 2008 Menkis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support to AM from Sven and Lilly Lawski Foundation, to HJ from Carl Trygger Foundation and Nilsson-Ehle donations is greatly acknowledged. DJJ was supported by US National Science Foundation grant MCB-0417282, awarded to David D. Perkins (deceased), Stanford University. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Hanna.Johannesson@ebc.uu.se ¤ Current address: Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, United States of America Introduction Many diverse systems for sex determination have evolved in plants and animals [1–3]. One involves physically distinct sex chromosomes, a system thought to have evolved independently many times by suppression of recombination around the sex determination genes, followed by differentiation and degeneration of the non-recombining chromosome [4]. In the fungal kingdom, there is no dichotomy of individuals into sexes bearing different gametes, but instead mating-type identity is determined by inheritance of alleles at mating-type loci. Nevertheless, chromo- somal regions controlling mating-type identity in fungi share features with the more complex sex chromosomes of algae, plants and animals [5]. Although mating-type loci consist of one to a few linked genes, and are thus limited to a small genomic region, alleles at the mating-type loci of fungi often differ to the extent that there is no sequence similarity between them [e.g. 6,7]. Furthermore, complete recombination cessation in the region around the mating-type loci have been reported from several fungal taxa [7–9]. However, fungi generally have much smaller regions of suppressed recombination than animal dimorphic chromosomal regions. For example, in Cryptococcus neoformans recombination is suppressed on only 6% of a 1.8 Mb chromo- some, or ca. 100 kb [8]. The filamentous ascomycete Neurospora tetrasperma constitutes an exception in which recombination is blocked over the majority of the chromosome containing the mating-type loci, referred to as the mating-type (mat) chromosome. Moreover, the non-recombining region is flanked by distal regions where obligate crossovers are observed [10,11]. In this species, the large non-recombining region is associated with a uniquely fungal life cycle, called pseudoho- mothallism, where self-fertility is enforced by maintenance of a constant state of heterokaryosis, normally only observed post- fertilization in this group of fungi. Modified programs of meiosis and sexual spore development lead to the packaging of two haploid nuclei of opposite mating-type (mat A and mat a) into each N. tetrasperma ascospore progeny [12,13]. The species maintains its ability to outcross by the occasional production of homokaryotic, self-sterile (mat A or mat a) propagules, both asexual and sexual, which may be isolated to obtain single mating-type component PLoS Genetics | www.plosgenetics.org 1 2008 | Volume 4 | Issue 3 | e1000030