Pseudo-homothallism is a reproductive strategy elected by some fungi producing heterokaryotic sexual spores containing genetically different but sexually compatible nuclei. This lifestyle appears as a compromise between true homothallism (self-fertility with predominant inbreeding) and complete heterothallism (with exclusive outcrossing). However, pseudohomothallic species face the problem of maintaining heterokaryotic mycelia to fully benefit from this lifestyle, as homokaryons are self-sterile. Here, we report on the structure of chromosome 1 in mat+ and mat2 isolates of strain S of the pseudohomothallic fungus Podospora anserina. Chromosome 1 contains either one of the mat+ and mat2 mating types of P. anserina, which is mostly found in nature as a mat+/mat2 heterokaryotic mycelium harboring sexually compatible nuclei. We identified a "mat" region 0.8 Mb long, devoid of meiotic recombination and containing the mating-type idiomorphs, which is a candidate to be involved in the maintenance of the heterokaryotic state, since the S mat+ and S mat2 strains have different physiology that may enable hybrid-vigor-like phenomena in the heterokaryons. The mat region contains 229 coding sequences. A total of 687 polymorphisms were detected between the S mat+ and S mat2 chromosomes. Importantly, the mat region is colinear between both chromosomes, which calls for an original mechanism of recombination inhibition. Microarray analyses revealed that 10% of the P. anserina genes have different transcriptional profiles in S mat+ and S mat2, in line with their different phenotypes. Finally, we show that the heterokaryotic state is faithfully maintained during mycelium growth of P. anserina, yet mat+/mat+ and mat2/mat2 heterokaryons are as stable as mat+/mat2 ones, evidencing a maintenance of heterokaryosis that does not rely on fitness-enhancing complementation between the S mat+ and S mat2 strains.A dikaryotic stage during a significant portion of the lifecycle is the hallmark of the higher fungi (Ascomycota and Basidiomycota), called for this reason the Dikarya. The dikaryotic part of the life cycle is different in the two groups. In Basidiomycota, mating-competent mycelia fuse and yield the secondary dikaryotic mycelium, upon which basidiosporebearing dikaryotic fruiting bodies are differentiated. In Ascomycota, fruiting bodies are differentiated around a monokaryotic female gametangium (the ascogonium), which is fertilized by a male gamete (antheridium or spermatium) to yield the dikaryon, which undergoes further development and produces numerous ascospore-containing asci. In Ascomycota, the dikaryotic stage is thus restricted to the sexual lineage inside the fruiting body. There is one exception to this in the Taphrinomycetes, where a dikaryotic mycelium is formed as part of the life cycle (Martin 1940). Ascomycota are nonetheless able to exhibit heterokaryotic mycelia following somatic fusion between genetically different individuals (Buller 1933). In Basidiomycota, a special structure (the clamp) enables the mai...