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The use of compounds produced by hosts or symbionts for defence against antagonists has been identified in many organisms, including in fungus-farming termites (Macrotermitinae). The obligate mutualistic fungus Termitomyces plays a pivotal role in plant biomass decomposition and as the primary food source for these termites. Despite the isolation of various specialized metabolites from different Termitomyces species, our grasp of their natural product repertoire remains incomplete. To address this knowledge gap, we conducted a comprehensive analysis of 39 Termitomyces genomes, representing 21 species associated with members of five termite host genera. We identified 754 biosynthetic gene clusters (BGCs) coding for specialized metabolites and categorized 660 BGCs into 61 biosynthetic gene cluster families (GCFs) spanning five compound classes. Seven GCFs were shared by all 21 Termitomyces species and 21 GCFs were present in all genomes of subsets of species. Evolutionary constraint analyses on the 25 most abundant GCFs revealed distinctive evolutionary histories, signifying that millions of years of termite-fungus symbiosis have influenced diverse biosynthetic pathways. This study unveils a wealth of non-random and largely undiscovered chemical potential within Termitomyces and contributes to our understanding of the intricate evolutionary trajectories of biosynthetic gene clusters in the context of long-standing symbiosis., (© 2024. The Author(s).)

The higher classification of termites requires substantial revision as the Neoisoptera, the most diverse termite lineage, comprise many paraphyletic and polyphyletic higher taxa. Here, we produce an updated termite classification using genomic-scale analyses. We reconstruct phylogenies under diverse substitution models with ultraconserved elements analyzed as concatenated matrices or within the multi-species coalescence framework. Our classification is further supported by analyses controlling for rogue loci and taxa, and topological tests. We show that the Neoisoptera are composed of seven family-level monophyletic lineages, including the Heterotermitidae Froggatt, Psammotermitidae Holmgren, and Termitogetonidae Holmgren, raised from subfamilial rank. The species-rich Termitidae are composed of 18 subfamily-level monophyletic lineages, including the new subfamilies Crepititermitinae, Cylindrotermitinae, Forficulitermitinae, Neocapritermitinae, Protohamitermitinae, and Promirotermitinae; and the revived Amitermitinae Kemner, Microcerotermitinae Holmgren, and Mirocapritermitinae Kemner. Building an updated taxonomic classification on the foundation of unambiguously supported monophyletic lineages makes it highly resilient to potential destabilization caused by the future availability of novel phylogenetic markers and methods. The taxonomic stability is further guaranteed by the modularity of the new termite classification, designed to accommodate as-yet undescribed species with uncertain affinities to the herein delimited monophyletic lineages in the form of new families or subfamilies., (© 2024. The Author(s).)

In fungi, fusion between individuals leads to localized cell death, a phenomenon termed heterokaryon incompatibility. Generally, the genes responsible for this incompatibility are observed to be under balancing selection resulting from negative frequency-dependent selection. Here, we assess this phenomenon in Aspergillus fumigatus, a human pathogenic fungus with a very low level of linkage disequilibrium as well as an extremely high crossover rate. Using complementation of auxotrophic mutations as an assay for hyphal compatibility, we screened sexual progeny for compatibility to identify genes involved in this process, called het genes. In total, 5/148 (3.4%) offspring were compatible with a parent and 166/2,142 (7.7%) sibling pairs were compatible, consistent with several segregating incompatibility loci. Genetic mapping identified five loci, four of which could be fine mapped to individual genes, of which we tested three through heterologous expression, confirming their causal relationship. Consistent with long-term balancing selection, trans-species polymorphisms were apparent across several sister species, as well as equal allele frequencies within A. fumigatus. Surprisingly, a sliding window genome-wide population-level analysis of an independent dataset did not show increased Tajima's D near these loci, in contrast to what is often found surrounding loci under balancing selection. Using available de novo assemblies, we show that these balanced polymorphisms are restricted to several hundred base pairs flanking the coding sequence. In addition to identifying the first het genes in an Aspergillus species, this work highlights the interaction of long-term balancing selection with rapid linkage disequilibrium decay., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

This paper addresses the stability of mycelial growth in fungi and differences between ascomycetes and basidiomycetes. Starting with general evolutionary theories of multicellularity and the role of sex, we then discuss individuality in fungi. Recent research has demonstrated the deleterious consequences of nucleus-level selection in fungal mycelia, favoring cheaters with a nucleus-level benefit during spore formation but a negative effect on mycelium-level fitness. Cheaters appear to generally be loss-of-fusion (LOF) mutants, with a higher propensity to form aerial hyphae developing into asexual spores. Since LOF mutants rely on heterokaryosis with wild-type nuclei, we argue that regular single-spore bottlenecks can efficiently select against such cheater mutants. We then zoom in on ecological differences between ascomycetes being typically fast-growing but short-lived with frequent asexual-spore bottlenecks and basidiomycetes being generally slow-growing but long-lived and usually without asexual-spore bottlenecks. We argue that these life history differences have coevolved with stricter nuclear quality checks in basidiomycetes. Specifically, we propose a new function for clamp connections, structures formed during the sexual stage in ascomycetes and basidiomycetes but during somatic growth only in basidiomycete dikaryons. During dikaryon cell division, the two haploid nuclei temporarily enter a monokaryotic phase, by alternatingly entering a retrograde-growing clamp cell, which subsequently fuses with the subapical cell to recover the dikaryotic cell. We hypothesize that clamp connections act as screening devices for nuclear quality, with both nuclei continuously testing each other for fusion ability, a test that LOF mutants will fail. By linking differences in longevity of the mycelial phase to ecology and stringency of nuclear quality checks, we propose that mycelia have a constant and low lifetime cheating risk, irrespective of their size and longevity., Competing Interests: The authors declare no conflict of interest.

The genus Podaxis was first described from India by Linnaeus in 1771, but several revisions of the genus have left the taxonomy unclear. Forty-four Podaxis species names and nine intraspecific varieties are currently accepted, but most fungarium specimens are labelled Podaxis pistillaris . Recent molecular analyses based on barcoding genes suggest that the genus comprises several species, but their status is largely unresolved. Here we obtained basidiospores and photographs from 166 fungarium specimens from around the world and generated a phylogeny based on rDNA internal transcribed spacer ITS1,5.8S and ITS2 (ITS), and a phylogenomic analysis of 3 839 BUSCO genes from low-coverage genomes for a subset of the specimens. Combining phylogenetics, phylogenomics, morphology, ecology, and geographical distribution, spanning 250 years of collections, we propose that the genus includes at least 16 unambiguous species. Based on 10 type specimens (holotype, paratype, and syntype), four recorded species were confirmed, P. carcinomalis , P. deflersii , P. emerici , and P. farlowii . Comparing phylogenetic analysis with described species, including morphology, ecology, and distribution, we resurrected P. termitophilus and designated neotypes, epitypes, or lectotypes for five previously described species, P. aegyptiacus , P. africana , P. beringamensis , P. calyptratus , and P. perraldieri . Lastly, based on phylogenies and morphology of type material, we synonymized three reported species, P. algericus , P. arabicus , and P. rugospora with P. pistillaris , and described five new species that we named P. desolatus , P. inyoensis , P. mareebaensis , P. namaquensis , and P. namibensis . Citation : Li GS, Leal-Dutra CA, Cuesta-Maté A, et al. 2023. Resolution of eleven reported and five novel Podaxis species based on ITS phylogeny, phylogenomics, morphology, ecology, and geographic distribution. Persoonia 51: 257-279. doi: 10.3767/persoonia.2023.51.07., Competing Interests: The authors declare that there is no conflict of interest., (© 2023 Naturalis Biodiversity Center & Westerdijk Fungal Biodiversity Institute.)

Background: The termite-fungus symbiosis is an ancient stable mutualism of two partners that reproduce and disperse independently. With the founding of each termite colony the symbiotic association must be re-established with a new fungus partner. Complementarity in the ability to break down plant substrate may help to stabilize this symbiosis despite horizontal symbiont transmission. An alternative, non-exclusive, hypothesis is that a reduced rate of evolution may contribute to stabilize the symbiosis, the so-called Red King Effect., Methods: To explore this concept, we produced the first linkage map of a species of Termitomyces, using genotyping by sequencing (GBS) of 88 homokaryotic offspring. We constructed a highly contiguous genome assembly using PacBio data and a de-novo evidence-based annotation. This improved genome assembly and linkage map allowed for examination of the recombination landscape and its potential effect on the mutualistic lifestyle., Results: Our linkage map resulted in a genome-wide recombination rate of 22 cM/Mb, lower than that of other related fungi. However, the total map length of 1370 cM was similar to that of other related fungi., Conclusions: The apparently decreased rate of recombination is primarily due to genome expansion of islands of gene-poor repetitive sequences. This study highlights the importance of inclusion of genomic context in cross-species comparisons of recombination rate., (© 2023. The Author(s).)

Fungal and plant mitochondria are known to exchange DNA with retroviral plasmids. Transfer of plasmid DNA to the organellar genome is best known and occurs through wholesale insertion of the plasmid. Less well known is the transfer of organellar DNA to plasmids, in particular tRNA genes. Presently, it is unknown whether fungal plasmids can adopt mitochondrial functions such as tRNA production through horizontal gene transfer. In this paper, we studied the exchange of DNA between fungal linear plasmids and fungal mtDNA, mainly focusing on the basidiomycete family Lyophyllaceae. We report at least six independent transfers of complete tRNA genes to fungal plasmids. Furthermore, we discovered two independent cases of loss of a tRNA gene from a fungal mitochondrial genome following transfer of such a gene to a linear mitochondrial plasmid. We propose that loss of a tRNA gene from mtDNA following its transfer to a plasmid creates a mutualistic dependency of the host mtDNA on the plasmid. We also find that tRNA genes transferred to plasmids encode codons that occur at the lowest frequency in the host mitochondrial genomes, possibly due to a higher number of unused transcripts. We discuss the potential consequences of mtDNA transfer to plasmids for both the host mtDNA and the plasmid., (© 2022. The Author(s).)

Blastosporella zonata is one of the few basidiomycete fungi that produce asexual spores (conidia) on the mushroom. The role of these conidia in the fungal lifecycle is not known. We tested whether conidia are being utilized in local dispersal by looking for signatures of clonality in 21 samples from three localities separated by about three kilometres in Murillo, Colombia. To identify clonally related individuals, we sequenced three polymorphic markers at two unlinked loci (nuclear rRNA: ITS and LSU, and TEFI α) for all collections plus three herbarium samples. We identified two sets of clonally related individuals growing closely together in one of the three localities, and only one pair shared between localities. In all three localities we observed multiple non-clonally related dikaryons showing that sexual reproduction is also important. Our results indicate that the conidia on the mushroom are primarily important for local dispersal. Unexpectedly, our results also indicate two reproductively isolated populations, possibly representing cryptic biological species. Citation : Van de Peppel LJJ, Baroni TJ, Franco-Molano AE, et al. 2022. Genetic population structure of the agaric Blastosporella zonata (Lyophyllacea) reveals cryptic species and different roles for sexual and asexual spores in dispersal. Persoonia 49: 195-200. https://doi.org/10.3767/persoonia.2022.49.06., Competing Interests: The authors declare that there is no conflict of interest., (© 2022 Naturalis Biodiversity Center & Westerdijk Fungal Biodiversity Institute.)

Generally, sexual organisms contain two haploid genomes, one from each parent, united in a single diploid nucleus of the zygote which links their fate during growth. A fascinating exception to this is Basidiomycete fungi, where the two haploid genomes remain separate in a dikaryon, retaining the option to fertilize subsequent monokaryons encountered. How the ensuing nuclear competition influences the balance of selection within and between individuals is largely unexplored. We test the consequences of the dikaryotic life cycle for mating success and mycelium-level fitness components. We assume a trade-off between mating fitness at the level of the haploid nucleus and fitness of the fungal mycelium. We show that the maintenance of fertilization potential by dikaryons leads to a higher proportion of fertilized monokaryons, but that the ensuing intradikaryon selection for increased nuclear mating fitness leads to reduced mycelium fitness relative to a diploid life cycle. However, this fitness reduction is lower compared to a hypothetical life cycle where dikaryons can also exchange nuclei. Prohibition of fusion between dikaryons therefore reduces the level of nuclear parasitism. The number of loci influencing fitness is an important determinant of the degree to which average mycelium-level fitness is reduced. The results of this study crucially hinge upon a trade-off between nucleus and mycelium-level fitness. We discuss the evidence for this assumption and the implications of an alternative that there is a positive relationship between nucleus and mycelium-level fitness., Competing Interests: BA, TC, DA No competing interests declared, (© 2022, Auxier et al.)

A new study shows that trail pheromones produced by an invasive ant species suppress the dispersal and stimulate the reproduction of cotton aphids that the ants can 'milk' for honeydew. Aphids use these pheromones as a signal of ant presence and respond adaptively, analogous to early stages of animal husbandry where animals were attracted to human settlements., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Ophiostomatales (Ascomycota) accommodates more than 300 species characterized by similar morphological adaptations to arthropod dispersal. Most species in this order are wood-inhabiting fungi associated with bark or ambrosia beetles. However, a smaller group of species occur in other niches such as in soil and Protea infructescences. Recent surveys of Termitomyces fungus gardens (fungus combs) of fungus-growing termites led to the discovery of characteristic ophiostomatalean-like fruiting structures. In this study, these ophiostomatalean-like fungi were identified using morphological characteristics, conventional molecular markers, and whole genome sequencing. In addition, the influence of the extracts derived from various parts of Termitomyces combs on the growth of these fungi in culture was considered. Based on phylogenomic analyses, two new genera ( Intubia and Chrysosphaeria ) were introduced to accommodate these ophiostomatalean species. Phylogenetic analyses revealed that the isolates resided in three well-supported lineages, and these were described as three new species ( Intubia macrotermitinarum, I. oerlemansii , and Chrysosphaeria jan-nelii ). Culture-based studies showed that these species do not depend on the Termitomyces comb material for growth.

The ancestor of termites relied on gut symbionts for degradation of plant material, an association that persists in all termite families. 1 , 2 However, the single-lineage Macrotermitinae has additionally acquired a fungal symbiont that complements digestion of food outside the termite gut. 3 Phylogenetic analysis has shown that fungi grown by these termites form a clade-the genus Termitomyces-but the events leading toward domestication remain unclear. 4 To address this, we reconstructed the lifestyle of the common ancestor of Termitomyces using a combination of ecological data with a phylogenomic analysis of 21 related non-domesticated species and 25 species of Termitomyces. We show that the closely related genera Blastosporella and Arthromyces also contain insect-associated species. Furthermore, the genus Arthromyces produces asexual spores on the mycelium, which may facilitate insect dispersal when growing on aggregated subterranean fecal pellets of a plant-feeding insect. The sister-group relationship between Arthromyces and Termitomyces implies that insect association and asexual sporulation, present in both genera, preceded the domestication of Termitomyces and did not follow domestication as has been proposed previously. Specialization of the common ancestor of these two genera on an insect-fecal substrate is further supported by similar carbohydrate-degrading profiles between Arthromyces and Termitomyces. We describe a set of traits that may have predisposed the ancestor of Termitomyces toward domestication, with each trait found scattered in related taxa outside of the termite-domesticated clade. This pattern indicates that the origin of the termite-fungus symbiosis may not have required large-scale changes of the fungal partner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Nonself recognition leading to somatic incompatibility (SI) is commonly used by mycologists to distinguish fungal individuals. Despite this, the process remains poorly understood in basidiomycetes as all current models of SI are based on genetic and molecular research in ascomycete fungi. Ascomycete fungi are mainly found in a monokaryotic stage, with a single type of haploid nuclei, and only briefly during mating do two genomes coexist in heterokaryotic cells. The sister phylum, Basidiomycota, differs in several relevant aspects. Basidiomycete fungi have an extended heterokaryotic stage, and SI is generally observed between heterokaryons instead of between homokaryons. Additionally, considerable nuclear migration occurs during a basidiomycete mating reaction, introducing a nucleus into a resident homokaryon with cytoplasmic mixing limited to the fused or neighboring cells. To accommodate these differences, we describe a basidiomycete model for nonself recognition using post-translational modification, based on a reader-writer system as found in other organisms. This post-translational modification combined with nuclear migration allows for the coexistence of two genomes in one individual while maintaining nonself recognition during all life stages. Somewhat surprisingly, this model predicts localized cell death during mating, which is consistent with previous observations but differs from the general assumptions of basidiomycete mating. This model will help guide future research into the mechanisms behind basidiomycete nonself recognition.

Insights into the genomic consequences of symbiosis for basidiomycete fungi associated with social insects remain sparse. Capitalizing on viability of spores from centuries-old herbarium specimens of free-living, facultative, and specialist termite-associated Podaxis fungi, we obtained genomes of 10 specimens, including two type species described by Linnaeus >240 years ago. We document that the transition to termite association was accompanied by significant reductions in genome size and gene content, accelerated evolution in protein-coding genes, and reduced functional capacities for oxidative stress responses and lignin degradation. Functional testing confirmed that termite specialists perform worse under oxidative stress, while all lineages retained some capacity to cleave lignin. Mitochondrial genomes of termite associates were significantly larger; possibly driven by smaller population sizes or reduced competition, supported by apparent loss of certain biosynthetic gene clusters. Our findings point to relaxed selection that mirrors genome traits observed among obligate endosymbiotic bacteria of many insects., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Some multicellular organisms can fuse because mergers potentially provide mutual benefits. However, experimental evolution in the fungus Neurospora crassa has demonstrated that free fusion of mycelia favours cheater lineages, but the mechanism and evolutionary dynamics of this exploitation are unknown. Here we show, paradoxically, that all convergently evolved cheater lineages have similar fusion deficiencies. These mutants are unable to initiate fusion but retain access to wild-type mycelia that fuse with them. This asymmetry reduces cheater-mutant contributions to somatic substrate-bound hyphal networks, but increases representation of their nuclei in the aerial reproductive hyphae. Cheaters only benefit when relatively rare and likely impose genetic load reminiscent of germline senescence. We show that the consequences of somatic fusion can be unequally distributed among fusion partners, with the passive non-fusing partner profiting more. We discuss how our findings may relate to the extensive variation in fusion frequency of fungi found in nature.

The agricultural mutualistic symbiosis between macrotermitine termites and Termitomyces fungi is obligate for both partners. The termites provide a protective growth environment for the fungus by cultivating it inside their colony and providing it with foraged plant material. The termites use the fungus for plant substrate degradation, and the production of asexual fruiting bodies for nourishment and re-inoculation of the fungus garden. The termite colony can reach an age of up to several decades, during which time it is believed that a single fungal monoculture is asexually propagated by the offspring of a single founding royal pair. The termite-fungus mutualism has a long evolutionary history dating back more than 30 million years. Both on the time-scale of a termite colony lifespan and that of the mutualistic symbiosis, questions arise about stability. We address the physical stability of the mound, the termite colony and the monoculture fungal garden during a colony's lifetime. On the long-term evolutionary scale, we address the stability of the symbiosis, where horizontal transmission of the symbiotic fungus raises the question of how the mutualistic interaction between host and symbiont persists over generations.

Although mutualistic symbioses per definition are beneficial for interacting species, conflict may arise if partners reproduce independently. We address how this reproductive conflict is regulated in the obligate mutualistic symbiosis between fungus-growing termites and Termitomyces fungi. Even though the termites and their fungal symbiont disperse independently to establish new colonies, dispersal is correlated in time. The fungal symbiont typically forms mushrooms a few weeks after the colony has produced dispersing alates. It is thought that this timing is due to a trade-off between alate and worker production; alate production reduces resources available for worker production. As workers consume the fungus, reduced numbers of workers will allow mushrooms to 'escape' from the host colony. Here, we test a specific version of this hypothesis: the typical asexual structures found in all species of Termitomyces -nodules-are immature stages of mushrooms that are normally harvested by the termites at a primordial stage. We refute this hypothesis by showing that nodules and mushroom primordia are macro- and microscopically different structures and by showing that in the absence of workers, primordia do, and nodules do not grow out into mushrooms. It remains to be tested whether termite control of primordia formation or of primordia outgrowth mitigates the reproductive conflict.

Mutation accumulation in long-lived fairy-ring mushrooms is orders of magnitude lower than predicted based on per-cell division mutation rates in other organisms. A possible explanation is the maintenance of 'immortal' template-DNA in the active periphery of the fairy ring., (Copyright © 2019. Published by Elsevier Ltd.)

Mitochondria retain their own genome, a hallmark of their bacterial ancestry. Mitochondrial genomes (mtDNA) are highly diverse in size, shape, and structure, despite their conserved function across most eukaryotes. Exploring extreme cases of mtDNA architecture can yield important information on fundamental aspects of genome biology. We discovered that the mitochondrial genomes of a basidiomycete fungus (Termitomyces spp.) contain an inverted repeat (IR), a duplicated region half the size of the complete genome. In addition, we found an abundance of sequences capable of forming G-quadruplexes (G4DNA); structures that can disrupt the double helical formation of DNA. G4DNA is implicated in replication fork stalling, double-stranded breaks, altered gene expression, recombination, and other effects. To determine whether this occurrence of IR and G4DNA was correlated within the genus Termitomyces, we reconstructed the mitochondrial genomes of 11 additional species including representatives of several closely related genera. We show that the mtDNA of all sampled species of Termitomyces and its sister group, represented by the species Tephrocybe rancida and Blastosporella zonata, are characterized by a large IR and enrichment of G4DNA. To determine whether high mitochondrial G4DNA content is common in fungi, we conducted the first broad survey of G4DNA content in fungal mtDNA, revealing it to be a highly variable trait. The results of this study provide important direction for future research on the function and evolution of G4DNA and organellar IRs., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Fungus-growing termites engage in an obligate mutualistic relationship with Termitomyces fungi, which they maintain in monocultures on specialised fungus comb structures, without apparent problems with infectious diseases. While other fungi have been reported in the symbiosis, detailed comb fungal community analyses have been lacking. Here we use culture-dependent and -independent methods to characterise fungus comb mycobiotas from three fungus-growing termite species (two genera). Internal Transcribed Spacer (ITS) gene analyses using 454 pyrosequencing and Illumina MiSeq showed that non-Termitomyces fungi were essentially absent in fungus combs, and that Termitomyces fungal crops are maintained in monocultures as heterokaryons with two or three abundant ITS variants in a single fungal strain. To explore whether the essential absence of other fungi within fungus combs is potentially due to the production of antifungal metabolites by Termitomyces or comb bacteria, we performed in vitro assays and found that both Termitomyces and chemical extracts of fungus comb material can inhibit potential fungal antagonists. Chemical analyses of fungus comb material point to a highly complex metabolome, including compounds with the potential to play roles in mediating these contaminant-free farming conditions in the termite symbiosis.

The mutation rate is a fundamental factor in evolutionary genetics. Recently, mutation rates were found to be strongly reduced at high density in a wide range of unicellular organisms, prokaryotic and eukaryotic. Independently, cell division was found to become more asymmetrical at increasing density in diverse organisms; some 'mother' cells continue dividing, while their 'offspring' cells do not divide further. Here, we investigate how this increased asymmetry in cell division at high density can be reconciled with reduced mutation-rate estimates. We calculated the expected number of mutant cells due to replication errors under various modes of segregation of template-DNA strands and copy-DNA strands, both under symmetrical (exponential) and asymmetrical (linear) growth. We show that the observed reduction in the mutation rate at high density can be explained if mother cells preferentially retain the template-DNA strands, since new mutations are then confined to non-dividing daughter cells, thus reducing the spread of mutant cells. Any other inheritance mode results in an increase in the number of mutant cells at higher density. The proposed hypothesis that patterns of DNA-strand segregation are density-dependent fundamentally challenges our current understanding of mutation-rate estimates and extends the distinction between germline and soma to unicellular organisms.

There are few environments more hostile and species-poor than deserts and the mounds of Nasutitermitinae termites. However, despite the very different adaptations required to survive in such extreme and different environments, the fungal genus Podaxis is capable of surviving in both: where few other fungi are reported to grow. Despite their prominence in the landscape and their frequent documentation by early explorers, there has been relatively little research into the genus. Originally described by Linnaeus in 1771, in the early 20th Century, the then ∼25 species of Podaxis were almost entirely reduced into one species: Podaxis pistillaris. Since this reduction, several new species of Podaxis have been described but without consideration of older descriptions. This has resulted in 44 recognised species names in Index Fungorum but the vast majority of studies and fungarium specimens still refer to P. pistillaris. Studies of Podaxis' extremely different lifestyles is hampered by its effective reduction to a single-species genus. Here we examine the history of the taxonomy of Podaxis before focusing on its extreme lifestyles. From this, we consider how the muddled taxonomy of Podaxis may be resolved; opening up further avenues for future research into this enigmatic fungal genus., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

When many gametes compete to fertilize a limited number of compatible gametes, sexual selection will favour traits that increase competitive success during mating. In animals and plants, sperm and pollen competition have yielded many interesting adaptations for improved mating success. In fungi, similar processes have not been shown directly yet. We test the hypothesis that sexual selection can increase competitive fitness during mating, using experimental evolution in the mushroom-forming fungus Schizophyllum commune (Basidiomycota). Mating in mushroom fungi occurs by donation of nuclei to a mycelium. These fertilizing 'male' nuclei migrate through the receiving 'female' mycelium. In our setup, an evolving population of nuclei was serially mated with a non-evolving female mycelium for 20 sexual generations. From the twelve tested evolved lines, four had increased and one had decreased fitness relative to an unevolved competitor. Even though only two of those five remained significant after correcting for multiple comparisons, for all five lines we found a correlation between the efficiency with which the female mycelium is accessed and fitness, providing additional circumstantial evidence for fitness change in those five lines. In two lines, fitness change was also accompanied by increased spore production. The one line with net reduced competitive fitness had increased spore production, but reduced fertilisation efficiency. We did not find trade-offs between male reproductive success and other fitness components. We compare these findings with examples of sperm and pollen competition and show that many similarities between these systems and nuclear competition in mushrooms exist., Competing Interests: The authors have declared that no competing interests exist.

Sodiomyces alkalinus is one of the very few alkalophilic fungi, adapted to grow optimally at high pH. It is widely distributed at the plant-deprived edges of extremely alkaline lakes and locally abundant. We sequenced the genome of S. alkalinus and reconstructed evolution of catabolic enzymes, using a phylogenomic comparison. We found that the genome of S. alkalinus is larger, but its predicted proteome is smaller and heavily depleted of both plant-degrading enzymes and proteinases, when compared to its closest plant-pathogenic relatives. Interestingly, despite overall losses, S. alkalinus has retained many proteinases families and acquired bacterial cell wall-degrading enzymes, some of them via horizontal gene transfer from bacteria. This fungus has very potent proteolytic activity at high pH values, but slowly induced low activity of cellulases and hemicellulases. Our experimental and in silico data suggest that plant biomass, a common food source for most fungi, is not a preferred substrate for S. alkalinus in its natural environment. We conclude that the fungus has abandoned the ancestral plant-based diet and has become specialized in a more protein-rich food, abundantly available in soda lakes in the form of prokaryotes and small crustaceans., (2018 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

Sexual reproduction is costly compared to asexual reproduction, in particular because males generally contribute little to offspring. Research published today in BMC Biology shows that some populations of a termite species have disposed of males altogether. However, this need not necessarily be seen as a victory for the females, since males in most termite societies are active colony members that contribute their fair share to colony tasks.

One of the most important fungal agents of pistachio dieback disease belongs to the ascomycete genus Paecilomyces that has been identified as P. variotii. In 2012-2014, 700 plant samples from pistachio trees and 27 other plant species with dieback symptoms were collected from 10 provinces of Iran. Of the 567 pistachio samples, 277 Paecilomyces strains were obtained and from the 133 samples of other plants (except pistachio and including Pistacia mutica, Punica granatum, Prunus amygdalus, Caesalpinia gilliesii, Nerium oleander, Tamarix aphylla, Tamarix hispida and Haloxylon sp.), 23 fungal isolates were recovered and five isolates were obtained from the air of infected pistachio orchards. Based on morphology, all 305 isolates were identified as P. variotii. Physiological studies revealed that 299 isolates belong to P. formosus. Three isolates were assigned to P. variotii, while three isolates could not be assigned to any of the known species. Of the 305 isolates, 62 were selected for phylogenetic analysis based on DNA variation (ITS, β-tubulin and calmodulin). This analysis showed that all of our isolates form a clade with P. formosus. P. formosus consists of the three former species P. formosa, P. lecythidis and P. maximus. This study shows that our isolates form a strongly supported clade with strains of P. lecythidis. So, the causal agent of dieback disease of pistachio and other examined trees is P. formosus which is closely related to the former species P. lecythidis and has some differences with the former species P. formosa and P. maximus. Based on phylogenetic studies P. formosus thus seems to be a species complex that could be divided into three separate species., Competing Interests: The authors have declared that no competing interests exist.

Workers in an ant colony can kill fungus-infected brood, thereby protecting the rest of the colony from fungal infection. This form of social immunity is analogous to the immune system of multicellular organisms where immune cells kill infected cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites. IMPORTANCE Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished., (Copyright © 2018 da Costa et al.)

Symbiogenesis, literally 'becoming by living together', refers to the crucial role of symbiosis in major evolutionary innovations. The term usually is reserved for the major transition to eukaryotes and to photosynthesising eukaryotic algae and plants by endosymbiosis. However, in some eukaryote lineages endosymbionts have been lost secondarily, showing that symbiosis can trigger a major evolutionary innovation, even if symbionts were lost secondarily. This leads to the intriguing possibility that symbiosis has played a role in other major evolutionary innovations as well, even if not all extant representatives of such groups still have the symbiotic association. We evaluate this hypothesis for two innovations in termites (Termitoidae, also known informally as "Isoptera"): i) the role of flagellate gut protist symbionts in the transition to eusociality from cockroach-like ancestors, and ii) the role of non-gut associated symbionts in the transition to 'higher' termites, characterized by the absence of flagellate gut protists. In both cases we identify a crucial role for symbionts, even though in both cases, subsequently, symbionts were lost again in some lineages. We also briefly discuss additional possible examples of symbiogenesis. We conclude that symbiogenesis is more broadly applicable than just for the endosymbiotic origin of eukaryotes and photosynthetic eukaryotes, and may be a useful concept to acknowledge the important role of symbiosis for evolutionary innovation. However, we do not accept Lynn Margulis's view that symbiogenesis will lead to a paradigm shift from neoDarwinism, as the role of symbiosis in evolutionary change can be integrated with existing theory perfectly., (Copyright © 2017. Published by Elsevier Ltd.)

In the vast majority of sexual life cycles, fusion between single-celled gametes is directly followed by nuclear fusion, leading to a diploid zygote and a lifelong commitment between two haploid genomes. Mushroom-forming basidiomycetes differ in two key respects. First, the multicellular haploid mating partners are fertilized in their entirety, each cell being a gamete that simultaneously can behave as a female, i.e. contributing the cytoplasm to a zygote by accepting nuclei, and a male gamete, i.e. only donating nuclei to the zygote. Second, after gamete union, the two haploid genomes remain separate so that the main vegetative stage, the dikaryon, has two haploid nuclei per cell. Only when the dikaryon produces mushrooms, do the nuclei fuse to enter a short diploid stage, immediately followed by meiosis and haploid spore formation. So in basidiomycetes, gamete fusion and genome mixing (sex) are separated in time. The 'living apart together' of nuclei in the dikaryon maintains some autonomy for nuclei to engage in a relationship with a different nucleus. We show that competition among the two nuclei of the dikaryon for such 'extramarital affairs' may lead to genomic conflict by favouring genes beneficial at the level of the nucleus, but deleterious at that of the dikaryon.This article is part of the themed issue 'Weird sex: the underappreciated diversity of sexual reproduction'., (© 2016 The Author(s).)

Although frequently found on mounds of the grass-cutting termite genus Trinervitermes, virtually nothing is known about the natural history of the fungal genus Podaxis (Agaricaceae) nor why it associates with termite mounds. More than 40 species of this secotioid genus have been described since Linnaeus characterised the first species in 1771. However, taxonomic confusion arose when most of these species were reduced to synonymy with Podaxis pistillaris in 1933. Although a few more species have since been described, the vast majority of specimens worldwide are still treated as P. pistillaris. Using 45 fresh and herbarium specimens from Southern Africa, four from North America and one each from Ethiopia, and Kenya, we constructed the first comprehensive phylogeny of the genus. Four of the genotyped specimens were more than 100 y old. With the exception of the type specimen of Podaxis rugospora, all herbarium specimens were labelled as P. pistillaris or Podaxis sp. However, our data shows that the genus contains at least five well-supported clades with significant inter-clade differences in spore length, width and wall thickness, and fruiting body length, supporting that clades likely represent distinct Podaxis species. Certain clades consistently associate with termites while others appear entirely free-living., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Based on fungus-fungus pairing assays and HRMS-based dereplication strategy, six new cyclic tetrapeptides, pseudoxylallemycins A-F (1-6), were isolated from the termite-associated fungus Pseudoxylaria sp. X802. Structures were characterized using NMR spectroscopy, HRMS, and Marfey's reaction. Pseudoxylallemycins B-D (2-4) possess a rare and chemically accessible allene moiety amenable for synthetic modifications, and derivatives A-D showed antimicrobial activity against Gram-negative human-pathogenic Pseudomonas aeruginosa and antiproliferative activity against human umbilical vein endothelial cells and K-562 cell lines.

Based on molecular dating, the origin of insect agriculture is hypothesized to have taken place independently in three clades of fungus-farming insects: the termites, ants or ambrosia beetles during the Paleogene (66-24 Ma). Yet, definitive fossil evidence of fungus-growing behavior has been elusive, with no unequivocal records prior to the late Miocene (7-10 Ma). Here we report fossil evidence of insect agriculture in the form of fossil fungus gardens, preserved within 25 Ma termite nests from southwestern Tanzania. Using these well-dated fossil fungus gardens, we have recalibrated molecular divergence estimates for the origins of termite agriculture to around 31 Ma, lending support to hypotheses suggesting an African Paleogene origin for termite-fungus symbiosis; perhaps coinciding with rift initiation and changes in the African landscape.

In multicellular organisms, there is a potential risk that cheating mutants gain access to the germline. Development from a single-celled zygote resets relatedness among cells to its maximum value each generation, which should accomplish segregation of cheating mutants from non-cheaters and thereby protect multicellular cooperation. Here we provide the crucial direct comparison between high- and low-relatedness conditions to test this hypothesis. We allow two variants of the fungus Neurospora crassa to evolve, one with and one without the ability to form chimeras with other individuals, thus generating two relatedness levels. While multicellular cooperation remains high in the high-relatedness lines, it significantly decreases in all replicate low-relatedness lines, resulting in an average threefold decrease in spore yield. This reduction is caused by cheating mutants with reduced investment in somatic functions, but increased competitive success when fusing with non-cheaters. Our experiments demonstrate that high genetic relatedness is crucial to sustain multicellular cooperation.

Farming is done not only by humans, but also by some ant, beetle and termite species. With the discovery of a stingless bee farming a fungus that provides benefits to its larvae, bees can be added to this list., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Innovative evolutionary developments are often related to gene or genome duplications. The crop fungi of attine fungus-growing ants are suspected to have enhanced genetic variation reminiscent of polyploidy, but this has never been quantified with cytological data and genetic markers. We estimated the number of nuclei per fungal cell for 42 symbionts reared by 14 species of Panamanian fungus-growing ants. This showed that domesticated symbionts of higher attine ants are polykaryotic with 7-17 nuclei per cell, whereas nonspecialized crops of lower attines are dikaryotic similar to most free-living basidiomycete fungi. We then investigated how putative higher genetic diversity is distributed across polykaryotic mycelia, using microsatellite loci and evaluating models assuming that all nuclei are either heterogeneously haploid or homogeneously polyploid. Genetic variation in the polykaryotic symbionts of the basal higher attine genera Trachymyrmex and Sericomyrmex was only slightly enhanced, but the evolutionarily derived crop fungi of Atta and Acromyrmex leaf-cutting ants had much higher genetic variation. Our opposite ploidy models indicated that the symbionts of Trachymyrmex and Sericomyrmex are likely to be lowly and facultatively polyploid (just over two haplotypes on average), whereas Atta and Acromyrmex symbionts are highly and obligatorily polyploid (ca. 5-7 haplotypes on average). This stepwise transition appears analogous to ploidy variation in plants and fungi domesticated by humans and in fungi domesticated by termites and plants, where gene or genome duplications were typically associated with selection for higher productivity, but allopolyploid chimerism was incompatible with sexual reproduction., (© 2015 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.)

Allorecognition, the ability to distinguish "self" from "nonself" based on allelic differences at allorecognition loci, is common in all domains of life. Allorecognition restricts the opportunities for social parasitism, and is therefore crucial for the evolution of cooperation. However, the maintenance of allorecognition diversity provides a paradox. If allorecognition is costly relative to cooperation, common alleles will be favored. Thus, the cost of allorecognition may reduce the genetic variation upon which allorecognition crucially relies, a prediction now known as "Crozier's paradox." We establish the relative costs of allorecognition, and their consequences for the short-term evolution of recognition labels theoretically predicted by Crozier. We use fusion among colonies of the fungus Neurospora crassa, regulated by highly variable allorecognition genes, as an experimental model system. We demonstrate that fusion among colonies is mutually beneficial, relative to absence of fusion upon allorecognition. This benefit is due not only to absence of mutual antagonism, which occurs upon allorecognition, but also to an increase in colony size per se. We then experimentally demonstrate that the benefit of fusion selects against allorecognition diversity, as predicted by Crozier. We discuss what maintains allorecognition diversity., (© 2015 The Author(s).)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate.

Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass-degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus-growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus-growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454-pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus-level taxa was present in all termite species and accounted for 56-68% of the species-specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus-level ecological niches. Finally, we show that gut communities of fungus-growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus-growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus-growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites., (© 2014 John Wiley & Sons Ltd.)

The replication and segregation of multi-copy mitochondrial DNA (mtDNA) are not under strict control of the nuclear DNA. Within-cell selection may thus favour variants with an intracellular selective advantage but a detrimental effect on cell fitness. High relatedness among the mtDNA variants of an individual is predicted to disfavour such deleterious selfish genetic elements, but experimental evidence for this hypothesis is scarce. We studied the effect of mtDNA relatedness on the opportunities for suppressive mtDNA variants in the fungus Neurospora carrying the mitochondrial mutator plasmid pKALILO. During growth, this plasmid integrates into the mitochondrial genome, generating suppressive mtDNA variants. These mtDNA variants gradually replace the wild-type mtDNA, ultimately culminating in growth arrest and death. We show that regular sequestration of mtDNA variation is required for effective selection against suppressive mtDNA variants. First, bottlenecks in the number of mtDNA copies from which a 'Kalilo' culture started significantly increased the maximum lifespan and variation in lifespan among cultures. Second, restrictions to somatic fusion among fungal individuals, either by using anastomosis-deficient mutants or by generating allotype diversity, prevented the accumulation of suppressive mtDNA variants. We discuss the implications of these results for the somatic accumulation of mitochondrial defects during ageing., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is relaxed. Furthermore, because mtDNA replication is not strictly regulated, within-cell selection may favour mtDNA variants with a replication advantage, but a deleterious effect on cell fitness. The opportunities for selfish mtDNA mutations to spread are restricted by various organism-level adaptations, such as uniparental transmission, germline mtDNA bottlenecks, germline selection and, during somatic growth, regular alternation between fusion and fission of mitochondria. These mechanisms are all hypothesized to maintain functional mtDNA. However, the strength of selection for maintenance of functional mtDNA progressively declines with age, resulting in age-related diseases. Furthermore, organismal adaptations that most probably evolved to restrict the opportunities for selfish mtDNA create secondary problems. Owing to predominantly maternal mtDNA transmission, recombination among mtDNA from different individuals is highly restricted or absent, reducing the scope for repair. Moreover, maternal inheritance precludes selection against mtDNA variants with male-specific effects. We finish by discussing the consequences of life-history differences among taxa with respect to mtDNA evolution and make a case for the use of microorganisms to experimentally manipulate levels of selection., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Eukaryotic cells typically contain numerous mitochondria, each with multiple copies of their own genome, the mtDNA. Uniparental transmission of mitochondria, usually via the mother, prevents the mixing of mtDNA from different individuals. While on the one hand, this should resolve the potential for selection for fast-replicating mtDNA variants that reduce organismal fitness, maternal inheritance will, in theory, come with another set of problems that are specifically relevant to males. Maternal inheritance implies that the mitochondrial genome is never transmitted through males, and thus selection can target only the mtDNA sequence when carried by females. A consequence is that mtDNA mutations that confer male-biased phenotypic expression will be prone to evade selection, and accumulate. Here, we review the evidence from the ecological, evolutionary and medical literature for male specificity of mtDNA mutations affecting fertility, health and ageing. While such effects have been discovered experimentally in the laboratory, their relevance to natural populations--including the human population--remains unclear. We suggest that the existence of male expression-biased mtDNA mutations is likely to be a broad phenomenon, but that these mutations remain cryptic owing to the presence of counter-adapted nuclear compensatory modifier mutations, which offset their deleterious effects., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Background: We investigate the scope for selection at the level of nuclei within fungal individuals (mycelia) of the mutualistic Termitomyces cultivated by fungus-growing termites. Whereas in most basidiomycete fungi the number and kind of nuclei is strictly regulated to be two per cell, in Termitomyces mycelia the number of nuclei per cell is highly variable. We hypothesised that natural selection on these fungi not only occurs between mycelia, but also at the level of nuclei within the mycelium. We test this hypothesis using in vitro tests with five nuclear haplotypes of a Termitomyces species., Results: First, we studied the transition from a mixture of five homokaryons (mycelia with identical nuclei) each with a different nuclear haplotype to heterokaryons (mycelia with genetically different nuclei). In vitro cultivation of this mixture for multiple asexual transfers led to the formation of multiple heterokaryotic mycelia, and a reduction of mycelial diversity over time. All heterokaryotic mycelia contained exactly two types of nucleus. The success of a heterokaryon during in vitro cultivation was mainly determined by spore production and to a lesser extent by mycelial growth rate. Second, heterokaryons invariably produced more spores than homokaryons implying that homokaryons will be outcompeted. Third, no homokaryotic 'escapes' from a heterokaryon via the formation of homokaryotic spores were found, despite extensive spore genotyping. Fourth, in contrast to most studied basidiomycete fungi, in Termitomyces sp. no nuclear migration occurs during mating, limiting the scope for nuclear competition within the mycelium., Conclusions: Our experiments demonstrate that in this species of Termitomyces the scope for selection at the level of the nucleus within an established mycelium is limited. Although 'mate choice' of a particular nuclear haplotype is possible during mating, we infer that selection primarily occurs between mycelia with two types of nucleus (heterokaryons).

In filamentous fungi, allorecognition takes the form of heterokaryon incompatibility, a cell death reaction triggered when genetically distinct hyphae fuse. Heterokaryon incompatibility is controlled by specific loci termed het-loci. In this article, we analyzed the natural variation in one such fungal allorecognition determinant, the het-c heterokaryon incompatibility locus of the filamentous ascomycete Podospora anserina. The het-c locus determines an allogenic incompatibility reaction together with two unlinked loci termed het-d and het-e. Each het-c allele is incompatible with a specific subset of the het-d and het-e alleles. We analyzed variability at the het-c locus in a population of 110 individuals, and in additional isolates from various localities. We identified a total of 11 het-c alleles, which define 7 distinct incompatibility specificity classes in combination with the known het-d and het-e alleles. We found that the het-c allorecognition gene of P. anserina is under diversifying selection. We find a highly unequal allele distribution of het-c in the population, which contrasts with the more balanced distribution of functional groups of het-c based on their allorecognition function. One explanation for the observed het-c diversity in the population is its function in allorecognition. However, alleles that are most efficient in allorecognition are rare. An alternative and not exclusive explanation for the observed diversity is that het-c is involved in pathogen recognition. In Arabidopsis thaliana, a homolog of het-c is a pathogen effector target, supporting this hypothesis. We hypothesize that the het-c diversity in P. anserina results from both its functions in pathogen-defense, and allorecognition.

Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.

Before a mycelium of a mushroom-forming basidiomycete develops mushrooms, the monokaryotic mycelium needs to become fertilized. Although the mechanistic details of mating in mushrooms have been studied thoroughly in laboratory research, very little is known on mating patterns in nature. In this study, we performed fine-scale analyses of three populations of Schizophyllum commune from their natural substrate (i.e. dead beech branches). From the three branches, 24, 12, and 24 fruiting bodies were isolated and for each mushroom, the origins of its nuclei and cytoplasm were reconstructed using DNA markers. Nuclear genotypes were determined using sequencing data and mating types, and mitochondrial haplotypes using SNP markers. From these combined data we reconstructed colonization and mating patterns of the mycelia. On each branch, we found multiple dikaryons (3, 3, and 8, respectively); in two instances one nuclear haplotype was shared between two dikaryons and in two other cases a nuclear haplotype was shared between three dikaryons. Each dikaryon always had a single mitochondrial haplotype. These findings indicate that mating usually is not symmetrical and that a monokaryon is most likely fertilized by a small monokaryon, a spore or a dikaryon. Sharing of nuclear haplotype between different dikaryons resulted either from multiple fertilizations of a single monokaryon, if the dikaryons had identical mitochondrial haplotypes, or, if the dikaryons had different mitochondrial haplotypes, most likely from secondary matings between a monokaryon and a dikaryon (Buller phenomenon). We conclude that mating in S. commune between same-sized monokaryons with reciprocal migration, as generally described in textbooks, is rare in nature. We discuss the implications of non-symmetric mating for basidiomycete evolution., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Basidiomycete fungi perform fertilizations by incorporation of nuclei into a monokaryotic mycelium to establish a dikaryon. The dikaryon cannot incorporate another type of nucleus, but can still act as a nucleus donor in a dikaryon-monokaryon (di-mon) mating, known as the Buller phenomenon. Previously, it has been observed that: (1) in a particular di-mon mating, one of the nuclear types of the dikaryon generally performs better as a donor than the other, and (2) when nuclei from a dikaryon are separated to form monokaryons again (dedikaryotisation), recovery of monokaryons of the two nuclear types is usually unequal. In this study, we investigated if these two observations of asymmetry are functionally related. We tested this hypothesis by performing both di-mon matings and dedikaryotisation of dikaryons derived from five different monokaryons. When a single mechanism controls both processes, the nucleus better at fertilizing a monokaryon in a Buller pairing should also be recovered upon dedikaryotisation with a higher frequency. The results showed a hierarchical structure for recovery among nuclei in dedikaryotisation, but this hierarchy did not correspond to the fertilization success during di-mon mating. These findings thus show that the mechanism causing asymmetric regeneration of nuclei, is most likely not the same as the mechanism responsible for increased chance of fertilization in di-mon matings. We discuss the complexity of the interactions that occur during di-mon matings with regards to the mating type loci., (Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)