DISTATIS: A Promising Framework to Integrate Distance Matrices in Molecular Phylogenetics.

BACKGROUND: Molecular phylogenetic algorithms frequently disagree with the approaches considering reproductive compatibility and morphological criteria for species delimitation. The question stems if the resulting species boundaries from molecular, reproductive and/or morphological data are definitively not reconcilable; or if the existing phylogenetic methods are not sensitive enough to agree morphological and genetic variation in species delimitation. OBJECTIVE: We propose DISTATIS as an integrative framework to combine alignment-based (AB) and alignment-free (AF) distance matrices from ITS2 sequences/structures to shed light whether Gelasinospora and Neurospora are sister but independent genera. METHODS: We aimed at addressing this standing issue by harmonizing genus-specific classification based on their ascospore morphology and ITS2 molecular data. To validate our proposal, three phylogenetic approaches: i) traditional alignment-based, ii) alignment-free and iii) novel distance integrative (DI)-based were comparatively evaluated on a set of Gelasinospora and Neurospora species. All considered species have been extensively characterized at both the morphological and reproductive levels and there are known incongruences between their ascospore morphology and molecular data that hampers genus-specific delimitation. RESULTS: Traditional AB phylogenetic analyses fail at resolving the Gelasinospora and Neurospora genera into independent monophyletic clades following ascospore morphology criteria. In contrast, AF and DI approaches produced phylogenetic trees that could properly delimit the expected monophyletic clades. CONCLUSION: The DI approach outperformed the AF one in the sense that it could also divide the Neurospora species according to their reproduction mode.

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia. Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance.

Large-scale suppression of recombination predates genomic rearrangements in Neurospora tetrasperma.

A common feature of eukaryote genomes is large chromosomal regions where recombination is absent or strongly reduced, but the factors that cause this reduction are not well understood. Genomic rearrangements have often been implicated, but they may also be a consequence of recombination suppression rather than a cause. In this study, we generate eight high-quality genomic data sets of the filamentous ascomycete Neurospora tetrasperma, a fungus that lacks recombination over most of its largest chromosome. The genomes surprisingly reveal collinearity of the non-recombining regions and although large inversions are enriched in these regions, we conclude these inversions to be derived and not the cause of the suppression. To our knowledge, this is the first time that non-recombining, genic regions as large as 86% of a full chromosome (or 8 Mbp), are shown to be collinear. These findings are of significant interest for our understanding of the evolution of sex chromosomes and other supergene complexes.

Mechanism study of silver nanoparticle production using Neurospora intermedia.

Elucidation of the molecular mechanism of silver nanoparticle (AgNP) synthesis is necessary to control nanoparticle size, shape, and monodispersity. In this study, the mechanism of AgNP formation by Neurospora intermedia was investigated. The higher production rate of AgNP formation using a culture supernatant heat-treated at 100° and 121°C relative to that with an un-treated culture supernatant indicated that the native form of the molecular species is not essential. The effect of the protein molecular weight (MW) on the nanoparticle size distribution and average size was studied by means of ultraviolet-visible spectroscopy and dynamic light scattering. Using un-treated and concentrated cell-free filtrate passed through 10 and 20 kDa cut-off filters led to the production of AgNPs with average sizes of 25, 30, and 34 nm, respectively. Also, using the permeate fraction of cell-free filtrate passed through a 100 kDa cut-off filter led to the formation of the smallest nanoparticles with the narrowest size distribution (average size of 16 nm and polydispersity index of 0.18). Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of the fungal extracellular proteins showed two notable bands with the MWs of 15 and 23 kDa that are involved in the reduction and stabilisation of the nanoparticles, respectively.

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.

Neurospora sp. SR8, a novel phosphate solubiliser from rhizosphere soil of Sorghum in Kachchh, Gujarat, India.

Phosphorus (P) is abundant in soils in both inorganic and organic forms; nevertheless, it is unavailable to plants due to its fixation. Phosphate solubilising microorganisms including fungi play a pivotal role in making P available for plants by the process of solubilisation and mineralisation. Among the fungi that solubilize phosphate, the genera Aspergillus and Penicillium are the most representative although strains of Trichoderma and Rhizoctonia solani have also been reported as P solubilizers. Here, we report Neurospora discreta strain SR8 (NCCS Pune accession No. MCC1096 and NCBI accession No. KJ676544) as a P solubiliser as the first report. The strain was isolated from rhizospheric soil of Sorghum bicolor (L.) Moench. grown in semi-arid climate of a unique ecological zone of Kachchh, western India. The organism was identified on the basis of morphological characterization and by sequencing of ITS region. The strain SR8 survived the stressed environment in terms of high salinity and low precipitation rate in this area and could be a potent P solubiliser in stressed environments.

Culture and molecular identification of fungal contaminants in edible bird nests.

Widespread food poisoning due to microbial contamination has been a major concern for the food industry, consumers and governing authorities. This study is designed to determine the levels of fungal contamination in edible bird nests (EBNs) using culture and molecular techniques. Raw EBNs were collected from five house farms, and commercial EBNs were purchased from five Chinese traditional medicine shops (companies A-E) in Peninsular Malaysia. The fungal contents in the raw and commercial EBNs, and boiled and unboiled EBNs were determined. Culturable fungi were isolated and identified. In this study, the use of these methods revealed that all EBNs had fungal colony-forming units (CFUs) that exceeded the limit set by Standards and Industrial Research Institute of Malaysia (SIRIM) for yeast and moulds in EBNs. There was a significant difference (p < 0.05) in the number of types of fungi isolated from raw and commercial EBNs, but no significant difference in the reduction of the number of types of fungi after boiling the EBNs (p > 0.05). The types of fungi isolated from the unboiled raw EBNs were mainly soil, plant and environmental fungi, while the types of fungi isolated from the boiled raw EBNs, unboiled and boiled commercial EBNs were mainly environmental fungi. Aspergillus sp., Candida sp., Cladosporium sp., Neurospora sp. and Penicillum sp. were the most common fungi isolated from the unboiled and boiled raw and commercial EBNs. Some of these fungi are mycotoxin producers and cause opportunistic infections in humans. Further studies to determine the mycotoxin levels and methods to prevent or remove these contaminations from EBNs for safe consumption are necessary. The establishment and implementation of stringent regulations for the standards of EBNs should be regularly updated and monitored to improve the quality of the EBNs and consumer safety.

Intron evolution in Neurospora: the role of mutational bias and selection.

We used comparative and population genomics to study intron evolutionary dynamics in the fungal model genus Neurospora. For our investigation, we used well-annotated genomes of N. crassa, N. discreta, and N. tetrasperma, and 92 resequenced genomes of N. tetrasperma from natural populations. By analyzing the four well-annotated genomes, we identified 9495 intron sites in 7619 orthologous genes. Our data supports nonhomologous end joining (NHEJ) and tandem duplication as mechanisms for intron gains in the genus and the RT-mRNA process as a mechanism for intron loss. We found a moderate intron gain rate (5.78-6.89 × 10(-13) intron gains per nucleotide site per year) and a high intron loss rate (7.53-13.76 × 10(-10) intron losses per intron sites per year) as compared to other eukaryotes. The derived intron gains and losses are skewed to high frequencies, relative to neutral SNPs, in natural populations of N. tetrasperma, suggesting that selection is involved in maintaining a high intron turnover. Furthermore, our analyses of the association between intron population-level frequency and genomic features suggest that selection is involved in shaping a 5' intron position bias and a low intron GC content. However, intron sequence analyses suggest that the gained introns were not exposed to recent selective sweeps. Taken together, this work contributes to our understanding of the importance of mutational bias and selection in shaping the intron distribution in eukaryotic genomes.

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.

Analyses of expressed sequence tags in Neurospora reveal rapid evolution of genes associated with the early stages of sexual reproduction in fungi.

BACKGROUND: The broadly accepted pattern of rapid evolution of reproductive genes is primarily based on studies of animal systems, although several examples of rapidly evolving genes involved in reproduction are found in diverse additional taxa. In fungi, genes involved in mate recognition have been found to evolve rapidly. However, the examples are too few to draw conclusions on a genome scale. RESULTS: In this study, we performed microarray hybridizations between RNA from sexual and vegetative tissues of two strains of the heterothallic (self-sterile) filamentous ascomycete Neurospora intermedia, to identify a set of sex-associated genes in this species. We aligned Expressed Sequence Tags (ESTs) from sexual and vegetative tissue of N. intermedia to orthologs from three closely related species: N. crassa, N. discreta and N. tetrasperma. The resulting four-species alignments provided a dataset for molecular evolutionary analyses. Our results confirm a general pattern of rapid evolution of fungal sex-associated genes, compared to control genes with constitutive expression or a high relative expression during vegetative growth. Among the rapidly evolving sex-associated genes, we identified candidates that could be of importance for mating or fruiting-body development. Analyses of five of these candidate genes from additional species of heterothallic Neurospora revealed that three of them evolve under positive selection. CONCLUSIONS: Taken together, our study represents a novel finding of a genome-wide pattern of rapid evolution of sex-associated genes in the fungal kingdom, and provides a list of candidate genes important for reproductive isolation in Neurospora.

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.

A relationship between carotenoid accumulation and the distribution of species of the fungus Neurospora in Spain.

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.

Array CGH phylogeny: how accurate are comparative genomic hybridization-based trees?

BACKGROUND: Array-based Comparative Genomic Hybridization (CGH) data have been used to infer phylogenetic relationships. However, the reliability of array CGH analysis to determine evolutionary relationships has not been well established. In most CGH work, all species and strains are compared to a single reference species, whose genome was used to design the array. In the accompanying work, we critically evaluated CGH-based phylogeny using simulated competitive hybridization data. This work showed that a limited number of conditions, principally the tree topology and placement of the reference taxon in the tree, had a strong effect on the ability to recover the correct tree topology. Here, we add to our simulation study by testing the use of CGH as a phylogenetic tool with experimental CGH data from competitive hybridizations between N. crassa and other Neurospora species. In the discussion, we add to our empirical study of Neurospora by reanalyzing of data from a previous CGH phylogenetic analysis of the yeast sensu stricto complex. RESULTS: Array ratio data for Neurospora and related species were normalized with loess, robust spline, and linear ratio based methods, and then used to construct Neighbor-Joining and parsimony trees. These trees were compared to published phylogenetic analyses for Neurospora based on multilocus sequence analysis (MLSA). For the Neurospora dataset, the best combination of methods resulted in recovery of the MLSA tree topology less than half the time. Our reanalysis of a yeast dataset found that trees identical to established phylogeny were recovered only by pruning taxa - including the reference taxon - from the analysis. CONCLUSION: Our results indicate that CGH data can be problematic for phylogenetic analysis. Success fluctuates based on the methods utilized to construct the tree and the taxa included. Selective pruning of the taxa improves the results - an impractical approach for normal phylogenetic analysis. From the more successful methods we make suggestions on the normalization and post-normalization methods that work best in estimating genetic distance between taxa.

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.

Evolution of synonymous codon usage in Neurospora tetrasperma and Neurospora discreta.

Neurospora comprises a primary model system for the study of fungal genetics and biology. In spite of this, little is known about genome evolution in Neurospora. For example, the evolution of synonymous codon usage is largely unknown in this genus. In the present investigation, we conducted a comprehensive analysis of synonymous codon usage and its relationship to gene expression and gene length (GL) in Neurospora tetrasperma and Neurospora discreta. For our analysis, we examined codon usage among 2,079 genes per organism and assessed gene expression using large-scale expressed sequenced tag (EST) data sets (279,323 and 453,559 ESTs for N. tetrasperma and N. discreta, respectively). Data on relative synonymous codon usage revealed 24 codons (and two putative codons) that are more frequently used in genes with high than with low expression and thus were defined as optimal codons. Although codon-usage bias was highly correlated with gene expression, it was independent of selectively neutral base composition (introns); thus demonstrating that translational selection drives synonymous codon usage in these genomes. We also report that GL (coding sequences [CDS]) was inversely associated with optimal codon usage at each gene expression level, with highly expressed short genes having the greatest frequency of optimal codons. Optimal codon frequency was moderately higher in N. tetrasperma than in N. discreta, which might be due to variation in selective pressures and/or mating systems.

A comprehensive phylogeny of Neurospora reveals a link between reproductive mode and molecular evolution in fungi.

The filamentous ascomycete genus Neurospora encompasses taxa with a wide range of reproductive modes. Sexual reproduction in this genus can be divided into three major modes; heterothallism (self-incompatibility), homothallism (self-compatibility) and pseudohomothallism (partial self-compatibility). In addition to the sexual pathway, most of the heterothallic taxa propagate with morphologically distinct, vegetative dissemination propagules (macroconidia), while this feature is undetected in the majority of the homothallic taxa. In this study, we used sequence information of seven nuclear gene loci from 43 taxa (295 of the possible 301 locus-by-taxon combinations) to create a phylogeny of Neurospora. The results suggest that transitions in reproductive mode have occurred at multiple times within this group of fungi. Although a homothallic ancestor would imply fewer switches in reproductive mode, we argue that the ancestor of Neurospora was likely heterothallic and that homothallism has evolved independently at least six times in the evolutionary history of the genus. Furthermore, the two pseudohomothallic taxa of Neurospora (N. tetrasperma and N. tetraspora) represent two independent origins of pseudohomothallism. Likelihood ratio tests of substitution rates among branches in the phylogeny indicate that reproductive mode is an important factor driving genome evolution in Neurospora. First, an increased level of non-synonymous/synonymous substitutions in branches delineating homothallic taxa was found, suggesting a reduced efficiency of purifying selection in these taxa. Furthermore, elevated nucleotide substitution rates were found in heterothallic, conidia-producing, lineages as compared to the homothallic non-conidiating lineages. The latter finding is likely due to the presence of conidia, i.e., a higher rate of mitotic divisions inducing mutations, and/or that the homothallic taxa have evolved a lower mutation rate to avoid genomic degeneration.

Conflict between reproductive gene trees and species phylogeny among heterothallic and pseudohomothallic members of the filamentous ascomycete genus Neurospora.

In this study, we investigated the genealogies of genes important for sexual identity, i.e. mating-type (mat) and pheromone-receptor (pre) genes, among heterothallic and peudohomothallic taxa of Neurospora. The resulting genealogies were compared with the species phylogeny derived from non-coding sequences. We found conflicting topologies between the reproductive genealogies and the species phylogeny, and these conflicts were supported by both node support analyses and likelihood tests on the relative fit of datasets to alternative phylogenetic hypotheses. We argue that reproductive genes are more permeable to gene flow, i.e. are more often introgressed between species of Neurospora, than other parts of the genome. Certain conflicts between the species phylogeny and both mat genealogies were observed, suggesting that the two mating-type idiomorphs were selectively introgressed into a species from a single ancestral source. Taken together, the results presented here highlight complex evolutionary trajectories of reproductive genes in the fungal kingdom, which may be of importance for reproductive behavior in natural populations.

Genome-wide investigation of reproductive isolation in experimental lineages and natural species of Neurospora: identifying candidate regions by microarray-based genotyping and mapping.

Inherent incompatibilities between genetic components from genomes of different species may cause intrinsic reproductive isolation. In evolution experiments designed to instigate speciation in laboratory populations of the filamentous fungus Neurospora, we previously discovered a pair of incompatibility loci (dfe and dma) that interact negatively to cause severe defects in sexual reproduction. Here we show that the dfe-dma incompatibility also is a significant cause of genetic isolation between two naturally occurring species of Neurospora (N. crassa and N. intermedia). The strong incompatibility interaction has a simple genetic basis (two biallelic loci) and antagonistic epistasis occurs between heterospecific alleles only, consistent with the Dobzhansky-Muller model of genic incompatibility. We developed microarray-based, restriction-site associated DNA (RAD) markers that identified approximately 1500 polymorphisms between the genomes of the two species, and constructed the first interspecific physical map of Neurospora. With this new mapping resource, the approximate genomic locations of the incompatibility loci were determined using three different approaches: genome scanning, bulk-segregant analyses, and introgression. These population, quantitative, and classical genetics methods concordantly identified two candidate regions, narrowing the search for each incompatibility locus to only approximately 2% of the nuclear genome. This study demonstrates how advances in high-throughput, genome-wide genotyping can be applied to mapping reproductive isolation genes and speciation research.

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).