Evolutionary Genomics of Niche-Specific Adaptation to the Cystic Fibrosis Lung in Pseudomonas aeruginosa.

The comparative genomics of the transition of the opportunistic pathogen Pseudomonas aeruginosa from a free-living environmental strain to one that causes chronic infection in the airways of cystic fibrosis (CF) patients remain poorly studied. Chronic infections are thought to originate from colonization by a single strain sampled from a diverse, globally distributed population, followed by adaptive evolution to the novel, stressful conditions of the CF lung. However, we do not know whether certain clades are more likely to form chronic infections than others and we lack a comprehensive view of the suite of genes under positive selection in the CF lung. We analyzed whole-genome sequence data from 1,000 P. aeruginosa strains with diverse ecological provenances including the CF lung. CF isolates were distributed across the phylogeny, indicating little genetic predisposition for any one clade to cause chronic infection. Isolates from the CF niche experienced stronger positive selection on core genes than those derived from environmental or acute infection sources, consistent with recent adaptation to the lung environment. Genes with the greatest differential positive selection in the CF niche include those involved in core cellular processes such as metabolism, energy production, and stress response as well as those linked to patho-adaptive processes such as antibiotic resistance, cell wall and membrane modification, quorum sensing, biofilms, mucoidy, motility, and iron homeostasis. Many genes under CF-specific differential positive selection had regulatory functions, consistent with the idea that regulatory mutations play an important role in rapid adaptation to novel environments.

Phylogenomics of braconid wasps (Hymenoptera, Braconidae) sheds light on classification and the evolution of parasitoid life history traits.

The parasitoid lifestyle is largely regarded as a key innovation that contributed to the evolutionary success and extreme species richness of the order Hymenoptera. Understanding the phylogenetic history of hyperdiverse parasitoid groups is a fundamental step in elucidating the evolution of biological traits linked to parasitoidism. We used a genomic-scale dataset based on ultra-conserved elements and the most comprehensive taxon sampling to date to estimate the evolutionary relationships of Braconidae, the second largest family of Hymenoptera. Based on our results, we propose Braconidae to comprise 41 extant subfamilies, confirmed a number of subfamilial placements and proposed subfamily-level taxonomic changes, notably the restoration of Trachypetinae stat. rev. and Masoninae stat. rev. as subfamilies of Braconidae, confirmation that Apozyx penyai Mason belongs in Braconidae placed in the subfamily Apozyginae and the recognition of Ichneutinae sensu stricto and Proteropinae as non-cyclostome subfamilies robustly supported in a phylogenetic context. The correlation between koinobiosis with endoparasitoidism and idiobiosis with ectoparasitoidism, long thought to be an important aspect in parasitoid life history, was formally tested and confirmed in a phylogenetic framework. Using ancestral reconstruction methods based on both parsimony and maximum likelihood, we suggest that the ancestor of the braconoid complex was a koinobiont endoparasitoid, as was that of the cyclostome sensu lato clade. Our results also provide strong evidence for one transition from endo- to ectoparasitoidism and three reversals back to endoparasitoidism within the cyclostome sensu stricto lineage. Transitions of koino- and idiobiosis were identical to those inferred for endo- versus ectoparasitoidism, except with one additional reversal back to koinobiosis in the small subfamily Rhysipolinae.

Apicidin biosynthesis is linked to accessory chromosomes in Fusarium poae isolates.

BACKGROUND: Fusarium head blight is a disease of global concern that reduces crop yields and renders grains unfit for consumption due to mycotoxin contamination. Fusarium poae is frequently associated with cereal crops showing symptoms of Fusarium head blight. While previous studies have shown F. poae isolates produce a range of known mycotoxins, including type A and B trichothecenes, fusarins and beauvericin, genomic analysis suggests that this species may have lineage-specific accessory chromosomes with secondary metabolite biosynthetic gene clusters awaiting description. METHODS: We examined the biosynthetic potential of 38 F. poae isolates from Eastern Canada using a combination of long-read and short-read genome sequencing and untargeted, high resolution mass spectrometry metabolome analysis of extracts from isolates cultured in multiple media conditions. RESULTS: A high-quality assembly of isolate DAOMC 252244 (Fp157) contained four core chromosomes as well as seven additional contigs with traits associated with accessory chromosomes. One of the predicted accessory contigs harbours a functional biosynthetic gene cluster containing homologs of all genes associated with the production of apicidins. Metabolomic and genomic analyses confirm apicidins are produced in 4 of the 38 isolates investigated and genomic PCR screening detected the apicidin synthetase gene APS1 in approximately 7% of Eastern Canadian isolates surveyed. CONCLUSIONS: Apicidin biosynthesis is linked to isolate-specific putative accessory chromosomes in F. poae. The data produced here are an important resource for furthering our understanding of accessory chromosome evolution and the biosynthetic potential of F. poae.

The properties of spontaneous mutations in the opportunistic pathogen Pseudomonas aeruginosa.

BACKGROUND: Natural genetic variation ultimately arises from the process of mutation. Knowledge of how the raw material for evolution is produced is necessary for a full understanding of several fundamental evolutionary concepts. We performed a mutation accumulation experiment with wild-type and mismatch-repair deficient, mutator lines of the pathogenic bacterium Pseudomonas aeruginosa, and used whole-genome sequencing to reveal the genome-wide rate, spectrum, distribution, leading/lagging bias, and context-dependency of spontaneous mutations. RESULTS: Wild-type base-pair mutation and indel rates were ~10(-10) and ~10(-11) per nucleotide per generation, respectively, and deficiencies in the mismatch-repair system caused rates to increase by over two orders of magnitude. A universal bias towards AT was observed in wild-type lines, but was reversed in mutator lines to a bias towards GC. Biases for which types of mutations occurred during replication of the leading versus lagging strand were detected reciprocally in both replichores. The distribution of mutations along the chromosome was non-random, with peaks near the terminus of replication and at positions intermediate to the replication origin and terminus. A similar distribution bias was observed along the chromosome in natural populations of P. aeruginosa. Site-specific mutation rates were higher when the focal nucleotide was immediately flanked by C:G pairings. CONCLUSIONS: Whole-genome sequencing of mutation accumulation lines allowed the comprehensive identification of mutations and revealed what factors of molecular and genomic architecture affect the mutational process. Our study provides a more complete view of how several mechanisms of mutation, mutation repair, and bias act simultaneously to produce the raw material for evolution.

Evolutionary genomics of epidemic and nonepidemic strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen of humans and is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Prolonged infection of the respiratory tract can lead to adaptation of the pathogen to the CF lung environment. To examine the general patterns of adaptation associated with chronic infection, we obtained genome sequences from a collection of P. aeruginosa isolated from airways of patients with CF. Our analyses support a nonclonal epidemic population structure, with a background of unique, recombining genotypes, and the rare occurrence of successful epidemic clones. We present unique genome sequence evidence for the intercontinental spread of an epidemic strain shared between CF clinics in the United Kingdom and North America. Analyses of core and accessory genomes identified candidate genes and important functional pathways associated with adaptive evolution. Many genes of interest were involved in biological functions with obvious roles in this pathosystem, such as biofilm formation, antibiotic metabolism, pathogenesis, transport, reduction/oxidation, and secretion. Key factors driving the adaptive evolution of this pathogen within the host appear to be the presence of oxidative stressors and antibiotics. Regions of the accessory genome unique to the epidemic strain were enriched for genes in transporter families that efflux heavy metals and antibiotics. The epidemic strain was significantly more resistant than nonepidemic strains to three different antibiotics. Multiple lines of evidence suggest that selection imposed by the CF lung environment has a major influence on genomic evolution and the genetic characteristics of P. aeruginosa isolates causing contemporary infection.

A global multilocus analysis of the model fungus Neurospora reveals a single recent origin of a novel genetic system.

The large diversity of mating systems observed in the fungal kingdom underlines the importance of mating system change in fungal evolution. The selfing species Neurospora tetrasperma has evolved a novel method of achieving self-fertility by a mating system referred to as pseudohomothallism. However, little is known about the origin of N. tetrasperma and its relationship to the self-sterile, heterothallic, Neurospora species. In this study, we used a combination of phylogenetic and population genetic analyses to reconstruct the evolutionary history of N. tetrasperma and its heterothallic relatives. We sequenced 9 unlinked nuclear loci from 106 strains of N. tetrasperma sampled from across the globe, and a sample of 28 heterothallic strains of Neurospora. Our analyses provide strong support for monophyly of N. tetrasperma, but reject the monophyly of N. crassa. We estimate that N. tetrasperma is of a recent origin and that it diverged from the heterothallic species ∼1 million years ago. We also extend previous findings on the diversification within the N. tetrasperma clade, with 10 lineages identified. Taken together, these findings indicate that N. tetrasperma is younger than has been previously reported and that a rapid diversification of lineages has occurred within the N. tetrasperma clade.

Alternaria sections Infectoriae and Pseudoalternaria: New genomic resources, phylogenomic analyses, and biodiversity.

Species in Alternaria sections Infectoriae and Pseudoalternaria are commonly isolated from agricultural crops and a variety of other plant hosts. With the increasing appreciation that species from these two sections are often the dominant taxa recovered from important cereal crops, the need for improved understanding of their biodiversity and taxonomy has grown. Given that morphological characteristics and existing molecular markers are not sufficient for distinguishing among species, we expanded the genomic resources for these sections to support research in biosystematics and species diagnostics. Whole genome assemblies for 22 strains were generated, including the first genomes from section Infectoriae or Pseudoalternaria strains sampled from Canada, which significantly increases the number of publicly released genomes, particularly for section Pseudoalternaria. We performed comprehensive phylogenomic analyses of all available genomes (n = 39) and present the first robust phylogeny for these taxa. The segregation of the two sections was strongly supported by genomewide data, and multiple lineages were detected within each section. We then provide an overview of the biosystematics of these groups by analyzing two standard molecular markers from the largest sample of section Infectoriae and Pseudoalternaria strains studied to date. The patterns of relative diversity suggest that, in many cases, multiple species described based on minor morphological differences may actually represent different strains of the same species. A list of candidate loci for development into new informative molecular markers, which are diagnostic for sections and lineages, was created from analyses of phylogenetic signals from individual genes across the entire genome.

Competition both drives and impedes diversification in a model adaptive radiation.

Competitors are known to be important in governing the outcome of evolutionary diversification during an adaptive radiation, but the precise mechanisms by which they exert their effects remain elusive. Using the model adaptive radiation of Pseudomonas fluorescens, we show experimentally that the effect of competition on diversification of a focal lineage depends on both the strength of competition and the ability of the competitors to diversify. We provide evidence that the extent of diversification in the absence of interspecific competitors depends on the strength of resource competition. We also show that the presence of competitors can actually increase diversity by increasing interspecific resource competition. Competitors that themselves are able to diversify prevent diversification of the focal lineage by removing otherwise available ecological opportunities. These results suggest that the progress of an adaptive radiation depends ultimately on the strength of resource competition, an effect that can be exaggerated or impeded by the presence of competitors.

Incipient speciation by divergent adaptation and antagonistic epistasis in yeast.

Establishing the conditions that promote the evolution of reproductive isolation and speciation has long been a goal in evolutionary biology. In ecological speciation, reproductive isolation between populations evolves as a by-product of divergent selection and the resulting environment-specific adaptations. The leading genetic model of reproductive isolation predicts that hybrid inferiority is caused by antagonistic epistasis between incompatible alleles at interacting loci. The fundamental link between divergent adaptation and reproductive isolation through genetic incompatibilities has been predicted, but has not been directly demonstrated experimentally. Here we empirically tested key predictions of speciation theory by evolving the initial stages of speciation in experimental populations of the yeast Saccharomyces cerevisiae. After replicate populations adapted to two divergent environments, we consistently observed the evolution of two forms of postzygotic isolation in hybrids: reduced rate of mitotic reproduction and reduced efficiency of meiotic reproduction. This divergent selection resulted in greater reproductive isolation than parallel selection, as predicted by the ecological speciation theory. Our experimental system allowed controlled comparison of the relative importance of ecological and genetic isolation, and we demonstrated that hybrid inferiority can be ecological and/or genetic in basis. Overall, our results show that adaptation to divergent environments promotes the evolution of reproductive isolation through antagonistic epistasis, providing evidence of a plausible common avenue to speciation and adaptive radiation in nature.

Species diversity and molecular characterization of Alternaria section Alternaria isolates collected mainly from cereal crops in Canada.

Alternaria is often one on the most abundant fungal genera recovered from a wide array of plant hosts and environmental substrates. Many species within the sub-generic Alternaria section Alternaria are common plant pathogens that cause pre-harvest losses due to reduced productivity and post-harvest losses due to spoilage and contamination with mycotoxins. As certain species of Alternaria may have distinct mycotoxin profiles, and very broad host ranges, understanding the distribution of species by geography and host is critical for disease prediction, toxicological risk assessment, and guiding regulatory decisions. In two previous reports, we performed phylogenomic analyses to identify highly informative molecular markers for Alternaria section Alternaria, and validated their diagnostic ability. Here, we perform molecular characterization of 558 section Alternaria strains, collected from 64 host genera in 12 countries, using two of these section-specific loci (ASA-10 and ASA-19) along with the RNA polymerase II second largest subunit (rpb2) gene. The majority of strains (57.4%) originated from various cereal crops in Canada, which formed the main focus of our study. Phylogenetic analyses were used to classify strains into section Alternaria species/lineages, demonstrating that the most common species on Canadian cereal crops are Alternaria alternata and A. arborescens. Further population genetic analyses were consistent with A. alternata being a widely distributed species with relatively low levels of geographic isolation (i.e., Canadian isolates did not form distinct clades when compared to other regions). Our expanded sampling of A. arborescens has greatly increased the known diversity of this group, with A. arborescens isolates forming at least three distinct phylogenetic lineages. Proportionally, A. arborescens is more prevalent in Eastern Canada than in Western Canada. Sequence analyses, putative hybrids, and mating-type distributions provided some evidence for recombination events, both within and between species. There was little evidence for associations between hosts and genetic haplotypes of A. alternata or A. arborescens.

Evolutionary insight from whole-genome sequencing of experimentally evolved microbes.

Experimental evolution (EE) combined with whole-genome sequencing (WGS) has become a compelling approach to study the fundamental mechanisms and processes that drive evolution. Most EE-WGS studies published to date have used microbes, owing to their ease of propagation and manipulation in the laboratory and relatively small genome sizes. These experiments are particularly suited to answer long-standing questions such as: How many mutations underlie adaptive evolution, and how are they distributed across the genome and through time? Are there general rules or principles governing which genes contribute to adaptation, and are certain kinds of genes more likely to be targets than others? How common is epistasis among adaptive mutations, and what does this reveal about the variety of genetic routes to adaptation? How common is parallel evolution, where the same mutations evolve repeatedly and independently in response to similar selective pressures? Here, we summarize the significant findings of this body of work, identify important emerging trends and propose promising directions for future research. We also outline an example of a computational pipeline for use in EE-WGS studies, based on freely available bioinformatics tools.

Geographic isolation drives speciation in Nearctic aphids.

Across herbivorous insect clades, species richness and host-use diversity tend to positively covary. This could be because host-use divergence drives speciation, or because it raises the ecological limits on species richness. To evaluate these hypotheses, we performed phylogenetic path model analyses of the species diversity of Nearctic aphids. Here, we show that variation in the species richness of aphid clades is caused mainly by host-use divergence, whereas variation in speciation rates is caused more by divergence in non-host-related niche variables. Aphid speciation is affected by both the evolution of host and non-host-related niche components, but the former is largely caused by the latter. Thus, our analyses suggest that host-use divergence can both raise the ecological limits on species richness and drive speciation, although in the latter case, host-use divergence tends to be a step along the causal path leading from non-host-related niche evolution to speciation.

Untargeted metabolomics screening reveals unique secondary metabolite production from Alternaria section Alternaria.

Alternaria section Alternaria is comprised of many species that infect a broad diversity of important crop plants and cause post-harvest spoilage. Alternaria section Alternaria species, such as A. alternata and A. arborescens, are prolific producers of secondary metabolites that act as virulence factors of disease and are mycotoxins that accumulate in infected tissues-metabolites that can vary in their spectrum of production between individuals from the same fungal species. Untargeted metabolomics profiling of secondary metabolite production using mass spectrometry is an effective means to detect phenotypic anomalies in secondary metabolism within a species. Secondary metabolite phenotypes from 36 Alternaria section Alternaria isolates were constructed to observe frequency of production patterns. A clear and unique mass feature pattern was observed for three of the strains that were linked with the production of the dehydrocurvularin family of toxins and associated detoxification products. Examination of corresponding genomes revealed the presence of the dehydrocurvularin biosynthesis gene cluster associated with a sub-telomeric accessory region. A comparison of sequence similarity and occurrences of the dehydrocurvularin biosynthetic gene cluster within Pleosporalean fungi is presented and discussed.

Phylogenomic analyses of Alternaria section Alternaria: A high-resolution, genome-wide study of lineage sorting and gene tree discordance.

The genus Alternaria contains a diversity of saprobic and pathogenic species that can be found in a wide range of environments. Alternaria is currently divided into 26 subgeneric sections, and the "small-spored" Alternaria section Alternaria includes many species that are economically important agricultural pathogens. Recognizing that a stable framework for systematics and species identification is essential for management and regulation purposes, this section has experienced much taxonomic debate and systematic revision in recent years. Molecular phylogenetic studies have challenged the reliability of using morphological characteristics to differentiate Alternaria species but have also suggested that commonly used molecular markers for fungal phylogenetics may not be sufficiently informative at this taxonomic level. To allow the assessment of molecular variation and evolutionary history at a genome-wide scale, we present an overview and analysis of phylogenomic resources for Alternaria section Alternaria. We review the currently available genomic resources and report five newly sequenced genomes. We then perform multiple comparative genomic analyses, including macrosynteny assessment and inference of phylogenetic relationships using a variety of data sets and analysis methods. Fine-scale, genome-wide phylogenetic reconstruction revealed incomplete lineage sorting and the genomic distribution of gene/species tree discordance. Based on these patterns, we propose a list of candidate genes that may be developed into informative markers that are diagnostic for the main lineages. This overview identifies gaps in knowledge and can guide future genome sequencing efforts for this important group of plant pathogenic fungi.

Development and evaluation of a target enrichment bait set for phylogenetic analysis of oomycetes.

Target enrichment is a term that encompasses multiple related approaches where desired genomic regions are captured by molecular baits, leaving behind redundant or non-target regions in the genome, followed by amplification and next-generation sequencing of those captured regions. A molecular bait set was developed based on 426 single-copy, oomycete-specific orthologs and 3 barcoding genes. The bait set was tested on 27 oomycete samples (belonging to the Saprolegniales, Albuginales, and Peronosporales) derived from live and herbarium specimens, as well as control samples of true fungi and plants. Results show that (i) our method greatly enriches for the targeted orthologs on oomycete samples, but insignificantly on fungal and plant samples; (ii) an average of 263 out of 429 orthologs (61%) were recovered from oomycete live and herbarium specimens; (iii) sequencing roughly 100 000 read pairs per sample is sufficient for optimal ortholog recovery while maintaining low sequencing costs; and (iv) the expected relationships were recovered by phylogenetic analysis from the data generated. This is the first report of an oomycete-specific target enrichment method with broad potential applications for evolutionary and taxonomic studies. A key benefit of our target enrichment method is that it allows researchers to easily unlock the vast and unexplored oomycete genomic diversity stored in natural history collections.

Anchored Phylogenomics, Evolution and Systematics of Elateridae: Are All Bioluminescent Elateroidea Derived Click Beetles?

Click-beetles (Coleoptera: Elateridae) are an abundant, diverse, and economically important beetle family that includes bioluminescent species. To date, molecular phylogenies have sampled relatively few taxa and genes, incompletely resolving subfamily level relationships. We present a novel probe set for anchored hybrid enrichment of 2260 single-copy orthologous genes in Elateroidea. Using these probes, we undertook the largest phylogenomic study of Elateroidea to date (99 Elateroidea, including 86 Elateridae, plus 5 non-elateroid outgroups). We sequenced specimens from 88 taxa to test the monophyly of families, subfamilies and tribes. Maximum likelihood and coalescent phylogenetic analyses produced well-resolved topologies. Notably, the included non-elaterid bioluminescent families (Lampyridae + Phengodidae + Rhagophthalmidae) form a clade within the otherwise monophyletic Elateridae, and Sinopyrophoridae may not warrant recognition as a family. All analyses recovered the elaterid subfamilies Elaterinae, Agrypninae, Cardiophorinae, Negastriinae, Pityobiinae, and Tetralobinae as monophyletic. Our results were conflicting on whether the hypnoidines are sister to Dendrometrinae or Cardiophorinae + Negastriinae. Moreover, we show that fossils with the eucnemid-type frons and elongate cylindrical shape may belong to Eucnemidae, Elateridae: Thylacosterninae, ancestral hard-bodied cantharoids or related extinct groups. Proposed taxonomic changes include recognition of Plastocerini as a tribe in Dendrometrinae and Hypnoidinae stat. nov. as a subfamily within Elateridae.

Divergent adaptation promotes reproductive isolation among experimental populations of the filamentous fungus Neurospora.

BACKGROUND: An open, focal issue in evolutionary biology is how reproductive isolation and speciation are initiated; elucidation of mechanisms with empirical evidence has lagged behind theory. Under ecological speciation, reproductive isolation between populations is predicted to evolve incidentally as a by-product of adaptation to divergent environments. The increased genetic diversity associated with interspecific hybridization has also been theorized to promote the development of reproductive isolation among independent populations. Using the fungal model Neurospora, we founded experimental lineages from both intra- and interspecific crosses, and evolved them in one of two sub-optimal, selective environments. We then measured the influence that initial genetic diversity and the direction of selection (parallel versus divergent) had on the evolution of reproductive isolation. RESULTS: When assayed in the selective environment in which they were evolved, lineages typically had greater asexual fitness than the progenitors and the lineages that were evolved in the alternate, selective environment. Assays for reproductive isolation showed that matings between lineages that were adapted to the same environment had greater sexual reproductive success than matings between lineages that were adapted to different environments. Evidence of this differential reproductive success was observed at two stages of the sexual cycle. For one of the two observed incompatibility phenotypes, results from genetic analyses were consistent with a two-locus, two-allele model with asymmetric (gender-specific), antagonistic epistasis. The effects of divergent adaptation on reproductive isolation were more pronounced for populations with greater initial genetic variation. CONCLUSION: Divergent selection resulted in divergent adaptation and environmental specialization, consistent with fixation of different alleles in different environments. When brought together by mating, these alleles interacted negatively and had detrimental effects on sexual reproductive success, in agreement with the Dobzhansky-Muller model of genetic incompatibilities. As predicted by ecological speciation, greater reproductive isolation was observed among divergent-adapted lineages than among parallel-adapted lineages. These results support that, given adequate standing genetic variation, divergent adaptation can indirectly cause the evolution of reproductive isolation, and eventually lead to speciation.

Genomics of Compensatory Adaptation in Experimental Populations of Aspergillus nidulans.

Knowledge of the number and nature of genetic changes responsible for adaptation is essential for understanding and predicting evolutionary trajectories. Here, we study the genomic basis of compensatory adaptation to the fitness cost of fungicide resistance in experimentally evolved strains of the filamentous fungus Aspergillus nidulans The original selection experiment tracked the fitness recovery of lines founded by an ancestral strain that was resistant to fludioxonil, but paid a fitness cost in the absence of the fungicide. We obtained whole-genome sequence data for the ancestral A. nidulans strain and eight experimentally evolved strains. We find that fludioxonil resistance in the ancestor was likely conferred by a mutation in histidine kinase nikA, part of the two-component signal transduction system of the high-osmolarity glycerol (HOG) stress response pathway. To compensate for the pleiotropic negative effects of the resistance mutation, the subsequent fitness gains observed in the evolved lines were likely caused by secondary modification of HOG pathway activity. Candidate genes for the compensatory fitness increases were significantly overrepresented by stress response functions, and some were specifically associated with the HOG pathway itself. Parallel evolution at the gene level was rare among evolved lines. There was a positive relationship between the predicted number of adaptive steps, estimated from fitness data, and the number of genomic mutations, determined by whole-genome sequencing. However, the number of genomic mutations was, on average, 8.45 times greater than the number of adaptive steps inferred from fitness data. This research expands our understanding of the genetic basis of adaptation in multicellular eukaryotes and lays out a framework for future work on the genomics of compensatory adaptation in A. nidulans.

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.