The evolution of non-reciprocal nuclear exchange in mushrooms as a consequence of genomic conflict.

Heterothallic mushrooms accomplish sex by exchanging nuclei without cytoplasm. Hyphal fusions occur between haploid mycelia resulting from germinated spores and subsequent reciprocal nuclear exchange without cytoplasmic mixing. The resulting dikaryon is therefore a cytoplasmic mosaic with uniformly distributed nuclei (two in each cell). Cytoplasmic inheritance is doubly uniparental: both mated monokaryons can potentially transmit their cytoplasm to the sexual spores, but normally only a single type per spore is found.Intracellular competition between mitochondria is thus limited, but at the dikaryon level, the two types of mitochondria compete over transmission. This creates the conditions for genomic conflict: within the dikaryon, a selfish mitochondrial mutant with increased relative transmission can be favoured, but selection between dikaryons will act against such a mitochondrial mutant. Moreover, because nuclear fitness is directly dependent on dikaryon fitness, a reduction in dikaryon fitness directly conflicts with nuclear interests. We propose that genomic conflict explains the frequent occurrence of non-reciprocal nuclear exchange in mushrooms. With non-reciprocal exchange, one monokaryon donates a nucleus and the other accepts it, but not vice versa as in the typical life cycle. We propose a model where non-reciprocal nuclear exchange is primarily driven by mitochondria inducing male sterility and the evolution of nuclear suppressors

Sexual selection in Fungi

Sexual selection is an important factor that drives evolution, in which fitness is increased, not by increasing survival or viability, but by acquiring more or better mates. Sexual selection favours traits that increase the ability of an individual to obtain more matings than other individuals that it is in competition with. For many sexually reproducing organisms, obtaining mates is an essential part of the lifecycle, sexual selection can therefore be very strong. A trait that leads to more matings can be selected, even if it strongly reduces other components of fitness, for instance predator escape. Often sexual selection leads to sex specific traits, which can become very extravagant. In animals and plants, it has been well established that this form of selection is an important evolutionary force, but it has not been considered for fungi. This thesis revolves around the idea that in this aspect, fungi are not fundamentally different from animals and plants and that also for species from this kingdom sexual selection influences evolution. Many fungi reproduce sexually and need to find a partner before reproduction can proceed. Furthermore, it is likely that not all individuals that benefit from mating can perform mating, hence a struggle for mate acquisition will occur. In my research I have investigated how likely it is that in fungi such struggles occur and which mechanisms might act during competitions. For these studies I used the mushroom forming basidiomycete fungus Schizophyllum commune as a model organism. I studied the potential for mate competition in natural populations, performed laboratory mating essays to test competition and preference, and experimentally tested if sexual selection can increase competitive ability. Sexual reproduction in fungi is highly regulated. Many molecular mechanisms are known that modulate each step, from meiosis to gamete production and from mate finding to gamete fusion. In most fungi these characteristics are regulated by genes located on the mating type locus or loci. These genes do not only regulate mating, but also define compatibility between the gametes: gametes with the same alleles at a mating type locus cannot fuse. Because of this double function, fungal mating types are potentially very important for sexual selection. Besides that mating types are a target for sexual selection because they affect traits that might increase competitive ability, since the mating types determine compatibility, they also define who competes with whom. Sexual selection describes how within one sex mating occurs for individuals of the other sex and is therefore always intra-sexual. Fungi do not have different sexes, but do have sex roles. Sexual selection will therefore act if there is competition for mating in the male or female role. Compatibility between sex roles is different from compatibility between mating types. The first is defined by the size of the mycelium, and the second by a genetic recognition mechanism. This difference is of importance to understand how sexual selection can act in fungi and is explained in Chapter 2. Mating in mushroom fungi occurs by reciprocal exchange of nuclei. In the female role nuclei from a compatible mate are incorporated into the haploid mycelium. These nuclei migrate though the mycelium until in each cell of the mycelium two haploid nuclei are present; it becomes a so called dikaryon. Only a dikaryon can produce mushrooms that produce spores. In the male role, a mycelium can donate nuclei to a haploid mycelium. A mycelium can thus be considered hermaphroditic. After fertilize, the dikaryon can still act as a nucleus donor, but not incorporate more nuclei – this type of mating is known as the Buller phenomenon. Also spores can act as male, but as they have no mycelium, not as a female. Due to the presence of more individuals that can mate in the male role than there are female mycelia (monokaryons), competition over fertilizations is expected. Competition can only occur when there are multiple individuals. Fungi are sessile organisms that can only meet other individuals when they are in the same locality. To test whether there is potential for sexual selection in nature, the number of individuals that meet each other needs to be defined. Not much knowledge on numbers of individuals is known, because mushroom fungi generally grow by mycelium expansion inside a substratum and each part of the mycelium can produce mushrooms. Therefore, all mushrooms on a tree can be one genetic individual, but it is also possible that each mushroom is a separate individual. We sampled 24, 12 and 24 mushrooms from the same substrate of three natural populations to analyse how mating occurred (Chapter 3). We determined the identity of the two different nuclei in each mushroom, as well as the mitochondria. Because mitochondria do not migrate during mating, they are specific for each female mycelium. We found that multiple genetic individuals (3, 3 and 8) are present in a small area, and that many matings must have occurred. Even though it is generally assumed that matings occur between two monokaryons, none such matings were found. The data suggest that mating in nature occurs between a monokaryon and a spore, or a monokaryon and a dikaryon. During a dikaryon-monokaryon (di-mon) mating only one of the two nucleus types from the dikaryon is successful in fertilizing the monokaryon. The nucleus type that is successful will likely increase its fitness considerably, as the entire female mycelium becomes colonized. Sexual selection is expected to select for nuclei that are better in performing this fertilization. Furthermore, during mating the receiving monokaryon meets two different nuclei, and might be able to choose between them. We performed crosses between 15 dikaryons and six different monokaryons to test if selection occurs, and whether selection occurs by male-male competition, or by female choice (Chapter 4). When confronting the same dikaryon with different monokaryon, in some of the cases the female mycelium decided which of the two nuclei won. In most cases however, the same nucleus always fertilized the monokaryon, irrespective of which monokaryon. This suggests that nuclei are able to either manipulate the monokaryon in incorporating them into the mycelium and not the other type, or that the nuclei of one type can directly suppress mating by the other nuclei in the dikaryon. Nuclei in a dikaryon have a strict way of cell division in which the different types divide in synchrony. Probably the two nuclei keep each other in check to assure this synchrony. Experiments in which the two nuclei in a dikaryon are separated into monokaryons suggest that the two nuclei suppress each other’s mitotic division, and that one of the two nuclei is better in suppression than the other. Consequently, after de-dikaryotisation more monokaryons of one type are recovered than of the other. We tested if this mechanism of suppression might be responsible for the dominant nuclei in the di-mon matings (Chapter 5). Separating the two nuclei confirmed earlier findings that always one of the two nuclei is dominant and that a hierarchy in dominance exists. This pecking order did not correspond with the results from the winner in the di-mon matings, which suggests that the mechanism of suppressed mitotic division is not responsible for dominance in di-mon matings. Nevertheless, we argue that the hypothesis that a link between the two mechanisms exists should not be completely written off. Because the interactions that take place during di-mon matings are very complex, the functioning of this mechanism might be obscured during mating. The observed variance in mating success described above might lead to sexual selection, however, it does not show that sexual selection actually led to traits that improve increased mate acquisition. To show that traits can evolve that increase fitness by higher mating success, we performed an evolution experiment (Chapter 6). An evolving population of nuclei was continuously mated with a non-evolving monokaryon. This setup selected for novel traits that increase competitive ability over matings. After 20 transfers, four out of twelve evolved lines had increased in competitive fitness and one line had decreased. Different fitness components were measured to investigate which traits had resulted in changed fitness. Fertilization success was mainly determined at the moment of fusion with and in initial migration into the receiving monokaryon. Two strains showed increased spores production, but this did not add to the increased fitness caused by fusion and initial migration. Little fitness change occurred during migration or in the dikaryon phase. We observed no clear trade-offs between the competitive ability of fertilizing in the male role, and female characteristics. This experiment showed that sexual selection can act in mushroom fungi. Sexual selection can also play a role in other groups of fungi than the mushroom forming fungi. So far this has not been considered, and little research has been done to show how mate competition might influence evolution. We reinterpreted the current knowledge on mating in fungi and assessed whether and when sexual selection might play a role (Chapter 7). Sexual selection is most likely to occur when sex roles can be observed during mating, as this can lead to skewed sex ratios. Also when there is large difference in quality between potential mates sexual selection might lead to evolution of choice. Directions are given where sexual selection is expected to function in fungal mating. Examples are given of how sexual selection might have led to for instance the evolution of micro-conidia in ascomycetes and pheromone redundancy in basidiomycetes. Furthermore, the existence of different sex roles in fungi, can lead to sexual conflict between the genomes derived from the paternal and the maternal gametes of which examples are given. The realization that sexual selection can also act in fungi gives great opportunity to test how universal general theories of sexual selection are in another important group of organisms. Additionally, because fungi are easy to manipulate, predictions on sexual selection can be tested experimentally using fungi. Mushroom forming fungi have a life history which differs from animals and plants. Sexual selection will therefore affect mushroom fungi in a different manner than it would animals and plants. In the general discussion of this thesis (Chapter 8) I will assess how mating influences fungal fitness, teasing apart the benefits and costs of mating in the male and female roles. I give directions for future research and discuss a setup to directly measure the effect of pheromones on female choice in mushroom fungi. There are still many unanswered fundamental questions about sexual selection. Adding knowledge from a third important kingdom can help increase the understanding of the principles that drive evolution by sexual selection. Furthermore, applying sexual selection theory to fungi might elucidate the functioning of the sometimes very complex mechanisms that have evolved for fungal mating. </p

Tree diversity and species identity effects on soil fungi, protists and animals are context dependent

Plant species richness and the presence of certain influential species (sampling effect) drive the stability and functionality of ecosystems as well as primary production and biomass of consumers. However, little is known about these floristic effects on richness and community composition of soil biota in forest habitats owing to methodological constraints. We developed a DNA metabarcoding approach to identify the major eukaryote groups directly from soil with roughly species-level resolution. Using this method, we examined the effects of tree diversity and individual tree species on soil microbial biomass and taxonomic richness of soil biota in two experimental study systems in Finland and Estonia and accounted for edaphic variables and spatial autocorrelation. Our analyses revealed that the effects of tree diversity and individual species on soil biota are largely context dependent. Multiple regression and structural equation modelling suggested that biomass, soil pH, nutrients and tree species directly affect richness of different taxonomic groups. The community composition of most soil organisms was strongly correlated due to similar response to environmental predictors rather than causal relationships. On a local scale, soil resources and tree species have stronger effect on diversity of soil biota than tree species richness per se