Selection against somatic parasitism can maintain allorecognition in fungi.

Fusion between multicellular individuals is possible in many organisms with modular, indeterminate growth, such as marine invertebrates and fungi. Although fusion may provide various benefits, fusion usually is restricted to close relatives by allorecognition, also called heterokaryon or somatic incompatibility in fungi. A possible selective explanation for allorecognition is protection against somatic parasites. Such mutants contribute less to colony functions but more to reproduction. However, previous models testing this idea have failed to explain the high diversity of allorecognition alleles in nature. These models did not, however, consider the possible role of spatial structure. We model the joint evolution of allorecognition and somatic parasitism in a multicellular organism resembling an asexual ascomycete fungus in a spatially explicit simulation. In a 1000-by-1000 grid, neighbouring individuals can fuse, but only if they have the same allotype. Fusion with a parasitic individual decreases the total reproductive output of the fused individuals, but the parasite compensates for this individual-level fitness reduction by a disproportional share of the offspring. Allorecognition prevents the invasion of somatic parasites, and vice versa, mutation towards somatic parasitism provides the selective conditions for extensive allorecognition diversity. On the one hand, if allorecognition diversity did not build up fast enough, somatic parasites went to fixation; conversely, once parasites had gone to fixation no allorecognition diversity built up. On the other hand, the mere threat of parasitism could select for high allorecognition diversity, preventing invasion of somatic parasites. Moderate population viscosity combined with weak global dispersal was optimal for the joint evolution of allorecognition and protection against parasitism. Our results are consistent with the widespread occurrence of allorecognition in fungi and the low degree of somatic parasitism. We discuss the implications of our results for allorecognition in other organism groups.

Does autophagy mediate age-dependent effect of dietary restriction responses in the filamentous fungus Podospora anserina?

Autophagy is a well-conserved catabolic process, involving the degradation of a cell's own components through the lysosomal/vacuolar machinery. Autophagy is typically induced by nutrient starvation and has a role in nutrient recycling, cellular differentiation, degradation and programmed cell death. Another common response in eukaryotes is the extension of lifespan through dietary restriction (DR). We studied a link between DR and autophagy in the filamentous fungus Podospora anserina, a multicellular model organism for ageing studies and mitochondrial deterioration. While both carbon and nitrogen restriction extends lifespan in P. anserina, the size of the effect varied with the amount and type of restricted nutrient. Natural genetic variation for the DR response exists. Whereas a switch to carbon restriction up to halfway through the lifetime resulted in extreme lifespan extension for wild-type P. anserina, all autophagy-deficient strains had a shorter time window in which ageing could be delayed by DR. Under nitrogen limitation, only PaAtg1 and PaAtg8 mediate the effect of lifespan extension; the other autophagy-deficient mutants PaPspA and PaUth1 had a similar response as wild-type. Our results thus show that the ageing process impinges on the DR response and that this at least in part involves the genetic regulation of autophagy.

High natural prevalence of a fungal prion.

Prions are infectious proteins that cause fatal diseases in mammals. Prions have also been found in fungi, but studies on their role in nature are scarce. The proposed biological function of fungal prions is debated and varies from detrimental to benign or even beneficial. [Het-s] is a prion of the fungus Podospora anserina. The het-s locus exists as two antagonistic alleles that constitute an allorecognition system: the het-s allele encoding the protein variant capable of prion formation and the het-S allele encoding a protein variant that cannot form a prion. We document here that het-s alleles, capable of prion formation, are nearly twice as frequent as het-S alleles in a natural population of 112 individuals. Then, we report a 92% prevalence of [Het-s] prion infection among the het-s isolates and find evidence of the role of the [Het-s]/het-S allorecognition system on the incidence of infection by a deleterious senescence plasmid. We explain the het-s/het-S allele ratios by the existence of two selective forces operating at different levels. We propose that during the somatic stage, the role of [Het-s]/HET-S in allorecognition leads to frequency-dependent selection for which an equilibrated frequency would be optimal. However, in the sexual cycle, the [Het-s] prion causes meiotic drive favoring the het-s allele. Our findings indicate that [Het-s] is a selected and, therefore, widespread prion whose activity as selfish genetic element is counteracted by balancing selection for allorecognition polymorphism.

Initial mutations direct alternative pathways of protein evolution.

Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 ß-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations.

Genetic analysis of the interaction between Allium species and arbuscular mycorrhizal fungi.

The response of Allium cepa, A. roylei, A. fistulosum, and the hybrid A. fistulosum × A. roylei to the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was studied. The genetic basis for response to AMF was analyzed in a tri-hybrid A. cepa × (A. roylei × A. fistulosum) population. Plant response to mycorrhizal symbiosis was expressed as relative mycorrhizal responsiveness (R') and absolute responsiveness (R). In addition, the average performance (AP) of genotypes under mycorrhizal and non-mycorrhizal conditions was determined. Experiments were executed in 2 years, and comprised clonally propagated plants of each genotype grown in sterile soil, inoculated with G. intraradices or non-inoculated. Results were significantly correlated between both years. Biomass of non-mycorrhizal and mycorrhizal plants was significantly positively correlated. R' was negatively correlated with biomass of non-mycorrhizal plants and hence unsuitable as a breeding criterion. R and AP were positively correlated with biomass of mycorrhizal and non-mycorrhizal plants. QTLs contributing to mycorrhizal response were located on a linkage map of the A. roylei × A. fistulosum parental genotype. Two QTLs from A. roylei were detected on chromosomes 2 and 3 for R, AP, and biomass of mycorrhizal plants. A QTL from A. fistulosum was detected on linkage group 9 for AP (but not R), biomass of mycorrhizal and non-mycorrhizal plants, and the number of stem-borne roots. Co-segregating QTLs for plant biomass, R and AP indicate that selection for plant biomass also selects for enhanced R and AP. Moreover, our findings suggest that modern onion breeding did not select against the response to AMF, as was suggested before for other cultivated species. Positive correlation between high number of roots, biomass and large response to AMF in close relatives of onion opens prospects to combine these traits for the development of more robust onion cultivars.

Mitochondrial recombination increases with age in Podospora anserina.

With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer of organelles takes place, making it possible to study mitochondrial recombination when introduced mitochondria contain different markers. A survey of wild-type isolates from a local population of the filamentous fungus Podospora anserina for the presence of seven optional mitochondrial introns indicated that mitochondrial recombination does take place in nature. Moreover the recombination frequency appeared to be correlated with age: the more rapidly ageing fraction of the population had a significantly lower linkage disequilibrium indicating more recombination. Direct confrontation experiments with heterokaryon incompatible strains with different mitochondrial markers at different (relative) age confirmed that mitochondrial recombination increases with age. We propose that with increasing mitochondrial damage over time, mitochondrial recombination - even within a homoplasmic population of mitochondria - is a mechanism that may restore mitochondrial function.

Calorie restriction in the filamentous fungus Podospora anserina.

Calorie restriction (CR) is a regimen of reduced food intake that, although the underlying mechanism is unknown, in many organisms leads to life span extension. Podospora anserina is one of the few known ageing filamentous fungi and the ageing process and concomitant degeneration of mitochondria have been well-studied. CR in P. anserina increases not only life span but also forestalls the ageing-related decline in fertility. Here we review what is known about CR in P. anserina and about possibly involved mechanisms like enhanced mitochondrial stability, reduced production of reactive oxygen species and changes in the OXPHOS machinery. Additionally, we present new microscopic data on mitochondrial dynamics under rich nutritional and CR conditions at different points in life. Lines that have grown under severe CR for more than 50x the normal life span, show no accumulation of age-related damage, though fecundity is reduced in some of these lines. Finally, we discuss the possible role of CR in P. anserina in nature and the effect of CR at different points in life.

Calorie restriction causes healthy life span extension in the filamentous fungus Podospora anserina.

Although most fungi appear to be immortal, some show systemic senescence within a distinct time frame. Podospora anserina for example shows an irreversible growth arrest within weeks of culturing associated with a destabilization of the mitochondrial genome. Here, we show that calorie restriction (CR), a regimen of under-nutrition without malnutrition, increases not only life span but also forestalls the aging-related decline in fertility. Similar to respiratory chain deficiencies the life span extension is associated with lower levels of intracellular H(2)O(2) measurements and a stabilization of the mitochondrial genome. Unlike respiratory chain deficiencies, CR cultures have a wild-type-like OXPHOS machinery similar to that of well-fed cultures as shown by native electrophoresis of mitochondrial protein complexes. Together, these data indicate that life span extension via CR is fundamentally different from that via respiratory chain mutations: Whereas the latter can be seen as a pathology, the former promotes healthy life span extension and may be an adaptive response.

High symbiont relatedness stabilizes mutualistic cooperation in fungus-growing termites.

It is unclear how mutualistic relationships can be stable when partners disperse freely and have the possibility of forming associations with many alternative genotypes. Theory predicts that high symbiont relatedness should resolve this problem, but the mechanisms to enforce this have rarely been studied. We show that African fungus-growing termites propagate single variants of their Termitomyces symbiont, despite initiating cultures from genetically variable spores from the habitat. High inoculation density in the substrate followed by fusion among clonally related mycelia enhances the efficiency of spore production in proportion to strain frequency. This positive reinforcement results in an exclusive lifetime association of each host colony with a single fungal symbiont and hinders the evolution of cheating. Our findings explain why vertical symbiont transmission in fungus-growing termites is rare and evolutionarily derived.

Microbial communication, cooperation and cheating: quorum sensing drives the evolution of cooperation in bacteria.

An increasing body of empirical evidence suggests that cooperation among clone-mates is common in bacteria. Bacterial cooperation may take the form of the excretion of "public goods": exoproducts such as virulence factors, exoenzymes or components of the matrix in biofilms, to yield significant benefit for individuals joining in the common effort of producing them. Supposedly in order to spare unnecessary costs when the population is too sparse to supply the sufficient exoproduct level, many bacteria have evolved a simple chemical communication system called quorum sensing (QS), to "measure" the population density of clone-mates in their close neighborhood. Cooperation genes are expressed only above a threshold rate of QS signal molecule re-capture, i.e., above the local quorum of cooperators. The cooperative population is exposed to exploitation by cheaters, i.e., mutants who contribute less or nil to the effort but fully enjoy the benefits of cooperation. The communication system is also vulnerable to a different type of cheaters ("Liars") who may produce the QS signal but not the exoproduct, thus ruining the reliability of the signal. Since there is no reason to assume that such cheaters cannot evolve and invade the populations of honestly signaling cooperators, the empirical fact of the existence of both bacterial cooperation and the associated QS communication system seems puzzling. Using a stochastic cellular automaton approach and allowing mutations in an initially non-cooperating, non-communicating strain we show that both cooperation and the associated communication system can evolve, spread and remain persistent. The QS genes help cooperative behavior to invade the population, and vice versa; cooperation and communication might have evolved synergistically in bacteria. Moreover, in good agreement with the empirical data recently available, this synergism opens up a remarkably rich repertoire of social interactions in which cheating and exploitation are commonplace.

An experimental test of the independent action hypothesis in virus-insect pathosystems.

The 'independent action hypothesis' (IAH) states that each pathogen individual has a non-zero probability of causing host death and that pathogen individuals act independently. IAH has not been rigorously tested. In this paper, we (i) develop a probabilistic framework for testing IAH and (ii) demonstrate that, in two out of the six virus-insect pathosystems tested, IAH is supported by the data. We first show that IAH inextricably links host survivorship to the number of infecting pathogen individuals, and develop a model to predict the frequency of single- and dual-genotype infections when a host is challenged with a mixture of two genotypes. Model predictions were tested using genetically marked, near-identical baculovirus genotypes, and insect larvae from three host species differing in susceptibility. Observations in early-instar larvae of two susceptible host species support IAH, but observations in late-instar larvae of susceptible host species and larvae of a less susceptible host species were not in agreement with IAH. Hence the model is experimentally supported only in pathosystems in which the host is highly susceptible. We provide, to our knowledge, the first qualitative experimental evidence that, in such pathosystems, the action of a single virion is sufficient to cause disease.

The het-c heterokaryon incompatibility gene in Aspergillus niger.

Heterokaryon incompatibility among Aspergillus niger strains is a widespread phenomenon that is observed as the inability to form stable heterokaryons. The genetic basis of heterokaryon incompatibility reactions is well established in some sexual filamentous fungi but largely unknown in presumed asexual species, such as A. niger. To test whether the genes that determine heterokaryon incompatibility in Neurospora crassa, such as het-c, vib-1 and pin-c, have a similar function in A. niger, we performed a short in silico search for homologues of these genes in the A. niger and several related genomes. For het-c, pin-c and vib-1 we did indeed identify putative orthologues. We then screened a genetically diverse worldwide collection of incompatible black Aspergilli for polymorphisms in the het-c orthologue. No size variation was observed in the variable het-c indel region that determines the specificity in N. crassa. Sequence comparison showed only minor variation in the number of glutamine coding triplets. However, introduction of one of the three N. crassa alleles (het-c2) in A. niger by transformation resulted in an abortive phenotype, reminiscent of the heterokaryon incompatibility in N. crassa. We conclude that although the genes required are present and the het-c homologue could potentially function as a heterokaryon incompatibility gene, het-c has no direct function in heterokaryon incompatibility in A. niger because the necessary allelic variation is absent.

The social evolution of somatic fusion.

The widespread potential for somatic fusion among different conspecific multicellular individuals suggests that such fusion is adaptive. However, because recognition of non-kin (allorecognition) usually leads to a rejection response, successful somatic fusion is limited to close kin. This is consistent with kin-selection theory, which predicts that the potential cost of fusion and the potential for somatic parasitism decrease with increasing relatedness. Paradoxically, however, Crozier found that, in the short term, positive-frequency-dependent selection eliminates the required genetic polymorphism at allorecognition loci. The 'Crozier paradox' may be solved if allorecognition is based on extrinsically balanced polymorphisms, for example at immune loci. Alternatively, the assumption of most models that self fusion is mutually beneficial is wrong. If fusion is on average harmful, selection will promote unconditional rejection. However, we propose that fusion within individuals is beneficial, selecting for the ability to fuse, but fusion between individuals on average costly, selecting for non-self recognition (rather than non-kin recognition). We discuss experimental data on fungi that are consistent with this hypothesis.

Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence.

Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with terminal inverted repeats and encodes a DNA and a RNA polymerase. Here we test whether pAL2-1 homologs cause rapid aging independent of intrinsic and external conditions. We first analyzed a natural population of P. anserina and in 40% of the 112 isolates we detected pAL2-1 homologous plasmids. Though the lifespan varied considerably among the strains, plasmid-infected wild-type strains are on average shorter lived than plasmid-free strains and typically show a reduced lifespan extending effect of calorie restriction (CR). However, interesting exceptions were found, inviting further study. To further investigate the effect of pAL2-1 homologs under various conditions, we constructed and analyzed isogenic lines with and without the plasmid. We found that the presence of pAL2-1 homologs did not significantly affect growth rate as suggested by the population analysis, but reduced lifespan under all conditions. This effect was particularly clear for the lifespan extending conditions tested (CR, low temperature, antibiotics) supporting the idea that pAL2-1 homologs are additional senescence factors independent of the intrinsic senescence determinants.

Adaptation to the cost of resistance in a haploid clonally reproducing organism: the role of mutation, migration and selection.

A model of compensatory evolution with respect to fungicide resistance in a haploid clonally reproducing fungus is developed in which compensatory mutations mitigate fitness costs associated with resistance. The role of mutation, migration and selection in invasion of rare genotypes when the environment changes from unsprayed to sprayed and from sprayed to unsprayed is analysed in detail. In some circumstances (ignoring back mutations) stable internal steady-state values for multiple genotypes can be obtained. In these cases a threshold value (f*) for the fraction of the population exposed to the fungicide can be derived for the transition between different steady-state conditions. Conditions are derived for invasion-when-rare of resistant genotypes at boundary equilibria established sometime after the onset of spraying and conversely of sensitive genotypes sometime after the cessation of spraying are derived. In these cases conditions are presented for (a) the invasion of a resistant genotype with a compensatory mutation (resistant-compensated) into a sensitive-uncompensated population that has re-equilibrated following the onset of spraying and (b) the invasion of a susceptible-uncompensated genotype into a resistant-compensated population that has re-equilibrated following the cessation of spraying, provided certain conditions are met. A resistant-compensated genotype may be fixed (or at near-fixation) in the population following a period of spraying, provided the mean intrinsic growth rate of the resistant-compensated genotype in a sprayed environment (over exposed and non-exposed parts of the population) is greater than that of the susceptible-uncompensated genotype. The fraction of the population exposed (the efficiency of spraying) is critical in this respect. However, it is possible for a sensitive-uncompensated genotype to invade provided there is no fitness gain associated with the resistant-compensated genotype, introduction by migration occurs following equilibration of the population to the new environment, and competitive effects are re-imposed when spraying ceases. We further derive a threshold level for the resident resistant-compensated population to reduce to following the cessation of spraying, such that the introduced susceptible-uncompensated genotype will invade. These results will be of use in determining the long-term persistence of resistance in a pathogen population once a fungicide is no longer effective and removed from use.

Effect of dispersal and nutrient availability on the competitive ability of toxin-producing yeast.

The ecological role of interference competition through toxin production is not well understood. In particular, it is unclear under what conditions the benefits of toxic killing outweigh the metabolic costs involved. A killer advantage has been suggested to rely on local competitive interactions where the benefits of killing accrue to the toxin producer preferentially, but this notion has little empirical support. In addition, contrasting predictions exist about the effect of resource abundance on the benefits of toxin production; this benefit should either be highest when resources are abundant and metabolic costs are relatively low or when resources are scarce and toxic killing is a 'last resort strategy' to obtain nutrients. Here, we test these predictions for one aspect of competitive ability, that is, the ability of toxin producers to invade a population of sensitive non-producers from a low initial frequency. We use competition experiments between isogenic K1 toxin-producing and non-producing strains of Saccharomyces cerevisiae, where we manipulate dispersal under two extreme nutrient conditions: one environment with and the other without replenishment of nutrients. We find that toxin production is beneficial when dispersal is limited under both nutrient conditions, but only when resources are abundant these outweigh its cost and allow invasion of the producer.

The evolution of obligate mutualism: if you can't beat 'em, join 'em.

Wolbachia is best known as a facultative endosymbiotic parasite, manipulating host reproduction. However, it has also evolved as an obligate mutualist at least twice. In a recent paper, Pannebakker et al. identify a possible mechanism for such a transition from facultative parasitism to obligate mutualism in a parasitic wasp in which Wolbachia are required for producing eggs (oogenesis). Their proposed mechanism suggests that compensatory evolution in the host to counter the harmful effects of Wolbachia is the basis of this evolutionary transition.

Spatial structure inhibits the rate of invasion of beneficial mutations in asexual populations.

Populations in spatially structured environments may be divided into a number of (semi-) isolated subpopulations due to limited offspring dispersal. Limited dispersal and, as a consequence, local competition could slow down the invasion of fitter mutants, allowing the short-term coexistence of ancestral genotypes and mutants. We determined the rate of invasion of beneficial mutants of Escherichia coli, dispersed to different degrees in a spatially structured environment during 40 generations, experimentally and theoretically. Simulations as well as experimental data show a decrease in the rate of invasion with increasingly constrained dispersal. When a beneficial mutant invades from a single spot, competition with the ancestral genotype takes place only along the edges of the growing colony patch. As the colony grows, the fitness of the mutant will decrease due to a decrease in the mutant's fraction that effectively competes with the surrounding ancestor. Despite its inherently higher competitive ability, increased intragenotype competition prevents the beneficial mutant from rapidly taking over, causing short-term coexistence of superior and inferior genotypes.

Mitotic recombination accelerates adaptation in the fungus Aspergillus nidulans.

Understanding the prevalence of sexual reproduction in eukaryotes is a hard problem. At least two aspects still defy a fully satisfactory explanation, the functional significance of genetic recombination and the great variation among taxa in the relative lengths of the haploid and diploid phases in the sexual cycle. We have performed an experimental study to explore the specific advantages of haploidy or diploidy in the fungus Aspergillus nidulans. Comparing the rate of adaptation to a novel environment between haploid and isogenic diploid strains over 3,000 mitotic generations, we demonstrate that diploid strains, which during the experiment have reverted to haploidy following parasexual recombination, reach the highest fitness. This is due to the accumulation of recessive deleterious mutations in diploid nuclei, some of which show their combined beneficial effect in haploid recombinants. Our findings show the adaptive significance of mitotic recombination combined with flexibility in the timing of ploidy level transition if sign epistasis is an important determinant of fitness.

A mitochondrial mutator plasmid that causes senescence under dietary restricted conditions.

BACKGROUND: Calorie or dietary restriction extends life span in a wide range of organisms including the filamentous fungus Podospora anserina. Under dietary restricted conditions, P. anserina isolates are several-fold longer lived. This is however not the case in isolates that carry one of the pAL2-1 homologous mitochondrial plasmids. RESULTS: We show that the pAL2-1 homologues act as 'insertional mutators' of the mitochondrial genome, which may explain their negative effect on life span extension. Sequencing revealed at least fourteen unique plasmid integration sites, of which twelve were located within the mitochondrial genome and two within copies of the plasmid itself. The plasmids were able to integrate in their entirety, via a non-homologous mode of recombination. Some of the integrated plasmid copies were truncated, which probably resulted from secondary, post-integrative, recombination processes. Integration sites were predominantly located within and surrounding the region containing the mitochondrial rDNA loci. CONCLUSION: We propose a model for the mechanism of integration, based on innate modes of mtDNA recombination, and discuss its possible link with the plasmid's negative effect on dietary restriction mediated life span extension.