Mating between sympatric species is a regular occurrence in natural populations, and increasing anthropogenic re-distributions of organisms is driving ever greater frequencies of species contact and hybridization [1, 2]. Whether giving rise to new, independent hybrid lineages or to the movement of alleles between species (i.e. introgression), recent population genetics and experimental studies show that inter-specific mating can be a major force behind adaptation and speciation [3–5]. The potential for hybridization to contribute to biodiversity, however, involves the interactions of multiple processes that remain incompletely understood . In particular, hybridization is limited by a complex interplay of pre- and post-mating reproductive barriers that can decrease mating compatibility between species or the fitness of hybrid individuals .
Especially in sympatric species, isolating mechanisms that depend on mating behaviors (i.e. compatibilities of different sexes or mating types) play a central role in reproductive isolation. For animals and plants, it has been shown that mating (mate interaction) and gametic (gamete interaction) behavior are effective mechanisms to keep species separated . In fungi the influence of mating patterns is less transparent, partly because of the complexity and variety of fungal mating systems . Many fungi persist as haploid individuals, where compatibility is determined by molecular signals, rather than a reliance on genotypically determined anisogamy. In most fungi, mating depends on the compatibility of pheromones and pheromone receptors, and thus, those proteins and their genes might play a central role for reproductive isolation. Although studies show the importance of assortative mating, and even reinforcement for reproductive isolation in fungi, evidence that these are due to the pheromone and pheromone receptor specifically is indirect [9–11].
Genetics of the mating compatibility system might also be effective in the post-mating stage and thus influence hybrid’s fitness potential. For instance, in mating systems, where homogametic and heterogametic sexes occur (e.g. XY or ZW systems in mammals or birds, respectively), hybrid inviability occurs more often in the heterogametic sex than in the homogametic sex , which often attributed to the hemizygosity nature of the structurally divergent sex chromosomes [13–15]. In fungi, where structural heterozygosity of sex chromosomes also occurs, asymmetrical effects upon hybrid fitness depending on the particular combination of sex chromosomes has also been observed  - in analogy to Darwin's corollary to hybrid viability .
In the present study we utilize members of the basidiomycete fungi in the genus Microbotryum to analyze the effect of the mating type on reproductive isolation. Microbotryum comprises many fungal species that typically specialize to a given host plant species [18–22]. The sibling species M. lychnidis-dioicae and M. silenes-dioicae (hereafter referred to as M-Sl and M-Sd) infecting Silene latifolia and Silene dioica, respectively, can hybridize in natural overlapping habitats, but frequency of hybrids is low [23, 24]. As typical for fungi, mating in Microbotryum occurs during the haploid stage and is controlled by a special region in the genome (MAT), which is responsible for the production of pheromones and pheromone receptors. In Microbotryum, the MAT region is located on a pair of non-recombining and size-dimorphic mating type chromosomes . The different mating types are referred to as a1 and a2 and haploid conjugation occurs exclusively between cells of opposite form .
The role of the mating system and mating type during reproductive isolation, and especially effects that are linked to the MAT region, remain unclear in Microbotryum. Generally, most Microbotryum species have high selfing rates , thus limiting the probability of interspecific gene flow [26–28]. Mechanisms of pre-mating barriers in the form of assortative mating have been investigated but not found [29, 30]. When hybridization is achieved experimentally, it often leads to the production of unbalanced meiotic products with limited growth and reduced infection ability . This loss of F1-hybrid’s fitness in Microbotryum seems to increase with the genetic distance among crossed species [31, 32]. In addition, maladaptation to the extrinsic host environment seems to be important in Microbotryum, where hybrids are less successful in producing complete infection symptoms than non-hybrids on the parental host environment .
Here, we aim to analyze determinants of mating type effects on reproductive isolation between the recently-derived Microbotryum species M. lychnidis-dioicae and Microbotryum silenes-dioicae. This is achieved by backcrossing experiments that can manipulate the identity between paired mating partners at the MAT regions and the rest of the genome. First, we test whether assortative mating occurs with regard to the mating type locus in the hybrid-produced gametes backcrossing combinations with gametes from parental species. Secondly, we quantify the fitness of backcrosses of F1-hybrids on different host environments to assess the contribution of mating type effects and extrinsic factors.