An introgressed gene causes meiotic drive in Neurospora sitophila.

Meiotic drive elements cause their own preferential transmission following meiosis. In fungi, this phenomenon takes the shape of spore killing, and in the filamentous ascomycete Neurospora sitophila, the Sk-1 spore killer element is found in many natural populations. In this study, we identify the gene responsible for spore killing in Sk-1 by generating both long- and short-read genomic data and by using these data to perform a genome-wide association test. We name this gene Spk-1 Through molecular dissection, we show that a single 405-nt-long open reading frame generates a product that both acts as a poison capable of killing sibling spores and as an antidote that rescues spores that produce it. By phylogenetic analysis, we demonstrate that the gene has likely been introgressed from the closely related species Neurospora hispaniola, and we identify three subclades of N. sitophila, one where Sk-1 is fixed, another where Sk-1 is absent, and a third where both killer and sensitive strain are found. Finally, we show that spore killing can be suppressed through an RNA interference-based genome defense pathway known as meiotic silencing by unpaired DNA. Spk-1 is not related to other known meiotic drive genes, and similar sequences are only found within Neurospora These results shed light on the diversity of genes capable of causing meiotic drive, their origin and evolution, and their interaction with the host genome.

Introgression maintains the genetic integrity of the mating-type determining chromosome of the fungus Neurospora tetrasperma.

Genome evolution is driven by a complex interplay of factors, including selection, recombination, and introgression. The regions determining sexual identity are particularly dynamic parts of eukaryotic genomes that are prone to molecular degeneration associated with suppressed recombination. In the fungus Neurospora tetrasperma, it has been proposed that this molecular degeneration is counteracted by the introgression of nondegenerated DNA from closely related species. In this study, we used comparative and population genomic analyses of 92 genomes from eight phylogenetically and reproductively isolated lineages of N. tetrasperma, and its three closest relatives, to investigate the factors shaping the evolutionary history of the genomes.We found that suppressed recombination extends across at least 6 Mbp (∼ 63%) of the mating-type (mat) chromosome in N. tetrasperma and is associated with decreased genetic diversity, which is likely the result primarily of selection at linked sites. Furthermore, analyses of molecular evolution revealed an increased mutational load in this region, relative to recombining regions. However, comparative genomic and phylogenetic analyses indicate that the mat chromosomes are temporarily regenerated via introgression from sister species; six of eight lineages show introgression into one of their mat chromosomes, with multiple Neurospora species acting as donors. The introgressed tracts have been fixed within lineages, suggesting that they confer an adaptive advantage in natural populations, and our analyses support the presence of selective sweeps in at least one lineage. Thus, these data strongly support the previously hypothesized role of introgression as a mechanism for the maintenance of mating-type determining chromosomal regions.

Genomic sequencing reveals historical, demographic and selective factors associated with the diversification of the fire-associated fungus Neurospora discreta.

Delineating microbial populations, discovering ecologically relevant phenotypes and identifying migrants, hybrids or admixed individuals have long proved notoriously difficult, thereby limiting our understanding of the evolutionary forces at play during the diversification of microbial species. However, recent advances in sequencing and computational methods have enabled an unbiased approach whereby incipient species and the genetic correlates of speciation can be identified by examining patterns of genomic variation within and between lineages. We present here a population genomic study of a phylogenetic species in the Neurospora discreta species complex, based on the resequencing of full genomes (~37 Mb) for 52 fungal isolates from nine sites in three continents. Population structure analyses revealed two distinct lineages in South-East Asia, and three lineages in North America/Europe with a broad longitudinal and latitudinal range and limited admixture between lineages. Genome scans for selective sweeps and comparisons of the genomic landscapes of diversity and recombination provided no support for a role of selection at linked sites on genomic heterogeneity in levels of divergence between lineages. However, demographic inference indicated that the observed genomic heterogeneity in divergence was generated by varying rates of gene flow between lineages following a period of isolation. Many putative cases of exchange of genetic material between phylogenetically divergent fungal lineages have been discovered, and our work highlights the quantitative importance of genetic exchanges between more closely related taxa to the evolution of fungal genomes. Our study also supports the role of allopatric isolation as a driver of diversification in saprobic microbes.

Quantifying functional heterothallism in the pseudohomothallic ascomycete Neurospora tetrasperma.

Neurospora tetrasperma is a pseudohomothallic filamentous ascomycete that has evolved from heterothallic ancestors. Throughout its life cycle, it is predominantly heterokaryotic for mating type, and thereby self-fertile. However, studies of N. tetrasperma have revealed the occasional production of self-sterile asexual and sexual spores of a single-mating type, indicating that it can be functionally heterothallic. Here, we report the extensive sampling and isolation of natural, heterokaryotic, strains of N. tetrasperma from the United Kingdom (UK): 99 strains were collected from Surrey, England, and four from Edinburgh, Scotland. We verified by phylogenetic analyses that these strains belong to N. tetrasperma. We isolated cultures from single germinated asexual spores (conidia) from 17 of these newly sampled UK strains from Surrey, and 16 previously sampled strains of N. tetrasperma from New Zealand (NZ). Our results show that the N. tetrasperma strains from the UK population produced a significantly greater proportion of self-sterile, homokaryotic conidia than the NZ population: the proportion of homokaryotic conidia was 42.6 % (133/312 spores) and 15.3 % (59/386) from the UK and the NZ populations, respectively. Although homokaryons recovered from several strains show a bias for one of the mating types, the total ratio of mat A to mat a mating type in homokaryons (UK: 72/61, NZ 28/31) did not deviate significantly from the expected 1:1 ratio for either of these populations. These results indicate that different populations exhibit differences in their life cycle characteristics, and that a higher degree of outcrossing might be expected from the UK population. This study points to the importance of studying multiple strains and populations when investigating life history traits of an organism with a complex life cycle, as previously undetected differences between populations may be revealed.

Genetic architecture of a reinforced, postmating, reproductive isolation barrier between Neurospora species indicates evolution via natural selection.

A role for natural selection in reinforcing premating barriers is recognized, but selection for reinforcement of postmating barriers remains controversial. Organisms lacking evolvable premating barriers can theoretically reinforce postmating isolation, but only under restrictive conditions: parental investment in hybrid progeny must inhibit subsequent reproduction, and selected postmating barriers must restore parents' capacity to reproduce successfully. We show that reinforced postmating isolation markedly increases maternal fitness in the fungus Neurospora crassa, and we detect the evolutionary genetic signature of natural selection by quantitative trait locus (QTL) analysis of the reinforced barrier. Hybrid progeny of N. crassa and N. intermedia are highly inviable. Fertilization by local N. intermedia results in early abortion of hybrid fruitbodies, and we show that abortion is adaptive because only aborted maternal colonies remain fully receptive to future reproduction. In the first QTL analysis of postmating reinforcement in microbial eukaryotes, we identify 11 loci for abortive hybrid fruitbody development, including three major QTLs that together explain 30% of trait variance. One of the major QTLs and six QTLs of lesser effect are found on the mating-type determining chromosome of Neurospora. Several reinforcement QTLs are flanked by genetic markers showing either segregation distortion or non-random associations with alleles at other loci in a cross between N. crassa of different clades, suggesting that the loci also are associated with local effects on same-species reproduction. Statistical analysis of the allelic effects distribution for abortive hybrid fruitbody development indicates its evolution occurred under positive selection. Our results strongly support a role for natural selection in the evolution of reinforced postmating isolation in N. crassa.

Massive changes in genome architecture accompany the transition to self-fertility in the filamentous fungus Neurospora tetrasperma.

A large region of suppressed recombination surrounds the sex-determining locus of the self-fertile fungus Neurospora tetrasperma. This region encompasses nearly one-fifth of the N. tetrasperma genome and suppression of recombination is necessary for self-fertility. The similarity of the N. tetrasperma mating chromosome to plant and animal sex chromosomes and its recent origin (<5 MYA), combined with a long history of genetic and cytological research, make this fungus an ideal model for studying the evolutionary consequences of suppressed recombination. Here we compare genome sequences from two N. tetrasperma strains of opposite mating type to determine whether structural rearrangements are associated with the nonrecombining region and to examine the effect of suppressed recombination for the evolution of the genes within it. We find a series of three inversions encompassing the majority of the region of suppressed recombination and provide evidence for two different types of rearrangement mechanisms: the recently proposed mechanism of inversion via staggered single-strand breaks as well as ectopic recombination between transposable elements. In addition, we show that the N. tetrasperma mat a mating-type region appears to be accumulating deleterious substitutions at a faster rate than the other mating type (mat A) and thus may be in the early stages of degeneration.

Three new phylogenetic and biological Neurospora species: N. hispaniola, N. metzenbergii and N. perkinsii.

The recent recognition of provisional Neurospora phylogenetic species (PS) 1-3 gave us the opportunity to compare genetic isolation, which underlies phylogenetic species recognition (PSR), with reproductive isolation as criteria for recognizing new species. This investigation involved first finding new individuals of PS 1-3 from a search of the Perkins culture collection, then assessing genetic isolation by PSR for old and new members of PS 1-3 and finally comparing species recognition by genetic isolation as determined by PSR to species recognition by reproductive isolation as determined by biological species recognition (BSR) and geographic distribution. To aid the search for additional members of the PS we used the genetic variation originally used to discover Neurospora PS 1-3 to easily distinguish members of Neurospora PS 1-3 from the closely related species N. crassa and N. intermedia. To increase our chance of success the analysis was performed on N. crassa and N. intermedia isolates that were either not clearly assignable to species by BSR using tester strains or were from the same geographic locations as the known members of PS1-3. Eleven new members of Neurospora PS 1-3 were identified: one new PS1, nine new PS2 and one new PS3. To complement PSR we investigated reproductive isolation with BSR in PS1-3 and the two other most closely related species, N. intermedia and N. crassa, with intraspecific and interspecific crosses. PS1 and PS2 appear reproductively isolated because they successfully mated intraspecifically and not interspecifically. PS3 isolates successfully crossed with other PS3 isolates, however they also successfully crossed with N. crassa, as previously reported, indicating that genetic isolation can precede reproductive isolation. We compared phylogenetic, mating and geographical data to challenge the use of PSR as the main criterion in the formal description of species and, having failed to discredit the approach, describe the new species, N. hispaniola (PS1), N. metzenbergii (PS2) and N. perkinsii (PS3).

Vaccine production in Neurospora crassa.

We have chosen to use the filamentous fungus Neurospora crassa to produce subunit vaccines. Here we describe the production and purification of Influenza hemagglutinin and neuraminidase antigens in N. crassa. The N. crassa system used by Neugenesis offers many advantages over other systems for production of recombinant protein. In contrast to mammalian cell culture, N. crassa can be grown in a rapid and economic manner, generating large amounts of recombinant protein in simple, defined medium. Vaccines, therefore, can be produced more rapidly and at lower cost than conventional cell culture or egg-based systems. This has important applications to tailoring the seasonal vaccine supply and responding to new pandemics.

The mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma represents a model for early evolution of sex chromosomes.

We combined gene divergence data, classical genetics, and phylogenetics to study the evolution of the mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma. In this species, a large non-recombining region of the mating-type chromosome is associated with a unique fungal life cycle where self-fertility is enforced by maintenance of a constant state of heterokaryosis. Sequence divergence between alleles of 35 genes from the two single mating-type component strains (i.e. the homokaryotic mat A or mat a-strains), derived from one N. tetrasperma heterokaryon (mat A+mat a), was analyzed. By this approach we were able to identify the boundaries and size of the non-recombining region, and reveal insight into the history of recombination cessation. The non-recombining region covers almost 7 Mbp, over 75% of the chromosome, and we hypothesize that the evolution of the mating-type chromosome in this lineage involved two successive events. The first event was contemporaneous with the split of N. tetrasperma from a common ancestor with its outcrossing relative N. crassa and suppressed recombination over at least 6.6 Mbp, and the second was confined to a smaller region in which recombination ceased more recently. In spite of the early origin of the first "evolutionary stratum", genealogies of five genes from strains belonging to an additional N. tetrasperma lineage indicate independent initiations of suppressed recombination in different phylogenetic lineages. This study highlights the shared features between the sex chromosomes found in the animal and plant kingdoms and the fungal mating-type chromosome, despite fungi having no separate sexes. As is often found in sex chromosomes of plants and animals, recombination suppression of the mating-type chromosome of N. tetrasperma involved more than one evolutionary event, covers the majority of the mating-type chromosome and is flanked by distal regions with obligate crossovers.

Evidence for the absence of meiotic silencing by unpaired DNA in Neurospora tetrasperma.

Meiotic silencing by unpaired DNA is a posttranscriptional gene silencing process in Neurospora crassa. Any gene without a homolog in the same chromosomal position during meiotic prophase generates a sequence-specific signal that prevents expression of all copies of that gene, but only during meiosis. Meiotic silencing is epigenetic and involves components of a meiosis-specific RNA silencing machinery. Although N. tetrasperma is closely related to N. crassa, its sexual biology is significantly different. N. tetrasperma was used here to evaluate both the generality of meiotic silencing within the genus and its possible evolutionary significance. A reporter gene for meiotic silencing, a histone H1-GFP fusion construct, was introgressed from N. crassa into various chromosome locations in N. tetrasperma. Whereas we did not observe meiotic silencing in four out of five introgression series, we obtained inconclusive results in the fifth series. Thus, we propose that meiotic silencing in N. tetrasperma is either absent or is substantially reduced when compared to N. crassa, possibly because the sad-1 gene (RNA-directed RNA polymerase, RdRP) is naturally unsynapsed (although "paired") and self-silenced during meiosis by structural differences between N. tetrasperma mating-type chromosomes. In N. crassa, wild-type sad-1 function is essential for meiotic silencing. Many point mutations in or deletion of sad-1 result in self-silencing of RdRP, and consequently suppression of meiotic silencing in heterozygous asci. The apparent absence or reduced meiotic silencing in N. tetrasperma raises the possibility that this form of silencing is not necessarily a major genome defense mechanism or responsible for reproductive isolation among the species of the genus Neurospora.

Ancestral polymorphism and linkage disequilibrium at the het-6 region in pseudohomothallic Neurospora tetrasperma.

In species of Neurospora, non-self recognition is mediated by at least 11 heterokaryon (het) incompatibility loci. Previously, we identified ancient allelic variation at het-c in pseudohomothallic N. tetrasperma, which confirmed outcrossing in this species. Here, we report distinct ancestral alleles at het-6 and un-24, two closely linked genes with het incompatibility function in N. crassa. The pattern of variation at het-6 and un-24 in N. tetrasperma is similar to that observed for N. crassa, where two ancestral allele specificities exist for each locus, Oak Ridge (het-6(OR), un-24(OR)) and Panama (het-6(PA), un-24(PA)). Only het-6(OR)/un-24(OR) and het-6(PA)/un-24(PA) allele combinations have been observed. The absence of recombinant haplotypes (e.g., het-6(OR)/un-24(PA)) appears to derive from an ancestral chromosomal rearrangement that limits recombination. Allelic variation at het-6 and un-24 in N. tetrasperma provides further evidence of outcrossing in this predominantly selfing species and indicates that selection maintains ancient allelic diversity at het loci.

New findings of Neurospora in Europe and comparisons of diversity in temperate climates on continental scales.

The life cycles of the conidiating species of Neurospora are adapted to respond to fire, which is reflected in their natural history. Neurospora is found commonly on burned vegetation from the tropic and subtropical regions around the world and through the temperate regions of western North America. In temperate Europe it was unknown whether Neurospora would be as common as it is in North America because it has been reported only occasionally. In 2003 and 2004 a multinational effort surveyed wildfire sites in southern Europe. Neurospora was found commonly from southern Portugal and Spain (37 degrees N) to Switzerland (46 degrees N). Species collected included N. crassa, N. discreta, N. sitophila and N. tetrasperma. The species distribution and spatial dynamics of Neurospora populations showed both similarities and differences when compared between temperate Europe and western North America, both regions of similar latitude, climate and vegetation. For example the predominant species in western North America, N. discreta phylogenetic species 4B, is common but not predominant in Europe, whereas species rare in western North America, N. crassa NcB and N. sitophila, are much more common in Europe. The meiotic drive element Spore killer was also common in European populations of N. sitophila and at a higher proportion than anywhere else in the world. The methods by which organisms spread and adapt to new environments are fundamental ecosystem properties, yet they are little understood. The differences in regional diversity, reported here, can form the basis of testable hypotheses. Questions of phylogeography and adaptations can be addressed specifically by studying Neurospora in nature.

Multilocus sequence data reveal extensive phylogenetic species diversity within the Neurospora discreta complex.

Previous observations of morphological, reproductive and genetic variation have suggested that Neurospora discreta, as presently circumscribed, might represent a diverse complex of multiple species. To investigate this hypothesis we examined the phylogenetic relationships among 73 fungal strains traditionally identified as N. discreta. Strains were chosen from across the morphological, ecological and geographical ranges of the species. Sequence data were obtained from three unlinked nuclear loci, and phylogenetic species recognition was applied to the dataset using protocols that have been shown to be reliable for identifying independent lineages and delineating species of Neurospora. The results demonstrate that the present circumscription of N. discreta includes at least eight separate phylogenetic species. This research also reveals an abundance of previously unrecognized genetic diversity within the genus, characterizes the interspecific evolutionary relationships and contributes to a fuller understanding of species diversity in Neurospora.

Blocked recombination along the mating-type chromosomes of Neurospora tetrasperma involves both structural heterozygosity and autosomal genes.

The Neurospora tetrasperma mating-type chromosomes have been shown to be structurally heterozygous by reciprocal introgression of these chromosomes between N. tetrasperma and N. crassa. This structural heterozygosity correlates with both a previously described recombination block and cytologically visible unpaired chromosomes at pachytene. Genes on the autosomes are also implicated in blocking recombination.

The so locus is required for vegetative cell fusion and postfertilization events in Neurospora crassa.

The process of cell fusion is a basic developmental feature found in most eukaryotic organisms. In filamentous fungi, cell fusion events play an important role during both vegetative growth and sexual reproduction. We employ the model organism Neurospora crassa to dissect the mechanisms of cell fusion and cell-cell communication involved in fusion processes. In this study, we characterized a mutant with a mutation in the gene so, which exhibits defects in cell fusion. The so mutant has a pleiotropic phenotype, including shortened aerial hyphae, an altered conidiation pattern, and female sterility. Using light microscopy and heterokaryon tests, the so mutant was shown to possess defects in germling and hyphal fusion. Although so produces conidial anastomosis tubes, so germlings did not home toward wild-type germlings nor were wild-type germlings attracted to so germlings. We employed a trichogyne attraction and fusion assay to determine whether the female sterility of the so mutant is caused by impaired communication or fusion failure between mating partners. so showed no defects in attraction or fusion between mating partners, indicating that so is specific for vegetative hyphal fusion and/or associated communication events. The so gene encodes a protein of unknown function, but which contains a WW domain; WW domains are predicted to be involved in protein-protein interactions. Database searches showed that so was conserved in the genomes of filamentous ascomycete fungi but was absent in ascomycete yeast and basidiomycete species.

Neurospora in temperate forests of western North America.

The fungal genus Neurospora has a distinguished history as a laboratory model in genetics and biochemistry. The most recent milestone in this history has been the sequencing of the genome of the best known species, N. crassa. The hope and promise of a complete genome sequence is a full understanding of the biology of the organism. Full understanding cannot be achieved, however, in the absence of fundamental knowledge of natural history. We report that species of Neurospora, heretofore thought to occur mainly in moist tropical and subtropical regions, are common primary colonizers of trees and shrubs killed by forest fires in western North America, in regions that are often cold and dry. Surveys in 36 forest-fire sites from New Mexico to Alaska yielded more than 500 cultures, 95% of which were the rarely collected N. discreta. Initial characterization of genotypes both within a site and on a single tree showed diversity consistent with sexual reproduction of N. discreta. These discoveries fill important gaps in knowledge of the distribution of members of the genus on both large and small spatial scales and provide the framework for future studies in new regions and microhabitats. The overall result is that population biology and genetics now can be combined, placing the genus Neurospora in a unique position to expand its role in experimental biology as a useful model organism for ecology, population genetics and evolution.

Variation among natural isolates of Neurospora on small spatial scales.

Although species of Neurospora are among the most studied model organisms in genetics and biochemistry, basic questions remain with respect to their ecology and population biology. In this study, we sought to clarify relationships among individuals over a small spatial scale, toward assessing both local variation and mode of colonization. Isolates of Neurospora were collected after fires in the Florida Everglades (May 1999), where abundant colonies appeared on diverse plants, including grasses and woody shrubs. Colonies were sampled in a linear fashion from two adjacent scorched sugarcane stems at one site and from a burned woody shrub at a distant second site. Species and mating types were assigned based on crossing behavior. Variation at two loci, het-c and frq, was determined by direct sequencing of PCR products. The results demonstrated substantial within- and among-species variation on a small scale, with up to three species and six different haplotypes occurring on a single stem. In total, four species and more than 10 genetically distinct individuals (haplotypes) were present across the three stems, often with multiple individuals occupying the same position. A permutation analysis revealed that individuals were not distributed randomly and that adjacent nodes on cane stems were more likely than chance to be colonized by the same haplotype. This suggests that visible eruptions of conidia on burned plants reflect substantial vegetative mycelial spread through subsurface tissues after primary colonization. Results also revealed that adjacent isolates from a single plant can possess different functional alleles at het-c, an observation meaningful in the context of the proposed role of het-c in self recognition.

A multilocus genealogical approach to phylogenetic species recognition in the model eukaryote Neurospora.

To critically examine the relationship between species recognized by phylogenetic and reproductive compatibility criteria, we applied phylogenetic species recognition (PSR) to the fungus in which biological species recognition (BSR) has been most comprehensively applied, the well-studied genus Neurospora. Four independent anonymous nuclear loci were characterized and sequenced from 147 individuals that were representative of all described outbreeding species of Neurospora. We developed a consensus-tree approach that identified monophyletic genealogical groups that were concordantly supported by the majority of the loci, or were well supported by at least one locus but not contradicted by any other locus. We recognized a total of eight phylogenetic species, five of which corresponded with the five traditional biological species, and three of which were newly discovered. Not only were phylogenetic criteria superior to traditional reproductive compatibility criteria in revealing the full species diversity of Neurospora, but also significant phylogenetic subdivisions were detected within some species. Despite previous suggestions of hybridization between N. crassa and N. intermedia in nature, and the fact that several putative hybrid individuals were included in this study, no molecular evidence in support of recent interspecific gene flow or the existence of true hybrids was observed. The sequence data from the four loci were combined and used to clarify how the species discovered by PSR were related. Although species-level clades were strongly supported, the phylogenetic relationships among species remained difficult to resolve, perhaps due to conflicting signals resulting from differential lineage sorting.

Reproductive isolation and phylogenetic divergence in Neurospora: comparing methods of species recognition in a model eukaryote.

We critically examined methods for recognizing species in the model filamentous fungal genus Neurospora by comparing traditional biological species recognition (BSR) with more comprehensive applications of both BSR and phylogenetic species recognition (PSR). Comprehensive BSR was applied to a set of 73 individuals by performing extensive crossing experiments and delineating biological species based on patterns of reproductive success. Within what were originally considered two species, N. crassa and N. intermedia, we recognized four reproductively isolated biological species. In a concurrent study (Dettman et al. 2003), we used genealogical concordance of four independent nuclear loci to recognize phylogenetic species in Neurospora. Overall, the groups of individuals identified as species were similar whether recognized by reproductive success or by phylogenetic criteria, and increased genetic distance between parents was associated with decreased reproductive success of crosses, suggesting that PSR using genealogical concordance can be used to reliably recognize species in organisms that are not candidates for BSR. In one case, two phylogenetic species were recognized as a single biological species, indicating that significant phylogenetic divergence preceded the development of reproductive isolation. However, multiple biological species were never recognized as a single phylogenetic species. Each of the putative N. crassa x N. intermedia hybrids included in this study was confidently assigned to a single species, using both PSR and BSR. As such, no evidence for a history of hybridization in nature among Neurospora species was observed. By performing reciprocal mating tests, we found that mating type, parental role, and species identity of parental individuals could all influence the reproductive success of matings. We also observed sympatry-associated sexual dysfunction in interspecific crosses, which was consistent with the existence of reinforcement mechanisms.