David J. Jacobson

A MULTILOCUS GENEALOGICAL APPROACH TO PHYLOGENETIC SPECIES RECOGNITION IN THE MODEL EUKARYOTE NEUROSPORA

Abstract 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.

Eukaryotic microbes, species recognition and the geographic limits of species: examples from the kingdom Fungi

The claim that eukaryotic micro-organisms have global geographic ranges, constituting a significant departure from the situation with macro-organisms, has been supported by studies of morphological species from protistan kingdoms. Here, we examine this claim by reviewing examples from another kingdom of eukaryotic microbes, the Fungi. We show that inferred geographic range of a fungal species depends upon the method of species recognition. While some fungal species defined by morphology show global geographic ranges, when fungal species are defined by phylogenetic species recognition they are typically shown to harbour several to many endemic species. We advance two non-exclusive reasons to explain the perceived difference between the size of geographic ranges of microscopic and macroscopic eukaryotic species when morphological methods of species recognition are used. These reasons are that microbial organisms generally have fewer morphological characters, and that the rate of morphological change will be slower for organisms with less elaborate development and fewer cells. Both of these reasons result in fewer discriminatory morphological differences between recently diverged lineages. The rate of genetic change, moreover, is similar for both large and small organisms, which helps to explain why phylogenetic species of large and small organisms show a more similar distribution of geographic ranges. As a consequence of the different rates in fungi of genetic and morphological changes, genetic isolation precedes a recognizable morphological change. The final step in speciation, reproductive isolation, also follows genetic isolation and may precede morphological change.

REPRODUCTIVE ISOLATION AND PHYLOGENETIC DIVERGENCE IN NEUROSPORA: COMPARING METHODS OF SPECIES RECOGNITION IN A MODEL EUKARYOTE

Abstract 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. crassaXN. 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.

Live-cell imaging of vegetative hyphal fusion in Neurospora crassa.

The process of hyphal fusion (anastomosis) in growing colonies of Neurospora crassa, stained with the membrane-selective dyes FM1-43 and FM4-64, was visualized by confocal microscopy. Time-lapse, live-cell imaging illustrated the dynamics of hyphal growth and anastomosis during its pre-contact, contact and post-contact, and post-fusion stages. Fusion-competent hyphae were morphologically distinct and exhibited remote sensing, resulting in branch initiation and/or re-direction of growth to facilitate contact between participating hyphae. A stained Spitzenkörper was often observed where fusion-competent hyphae met. It is suggested that this structure contains secretory vesicles responsible for the delivery of cell adhesion molecules at the point of contact, cell wall synthesizing enzymes for the swelling growth of fused hyphal tips, and digestive enzymes required for fusion pore formation. Dramatic changes in cytoplasmic flow frequently occurred between the participating hyphae following fusion. After anastomosis has taken place, septa commonly formed close to the fusion site. The live-cell imaging reported here has clearly shown the complexity of the hyphal homing and fusion process. The control and consequences of repeated anastomoses within a mycelium must be as complex as the process itself.

Variability of mitochondrial DNA as an indicator of relationships between populations of Fusarium oxysporum f. sp. melonis

Mitochondrial DNA from a reference strain of Fusarium oxysporum f. sp. melonis was cloned and filter hybridized to total DNA of 78 strains. A distinct mtDNA restriction fragment pattern corresponded to each of six vegetative compatibility groups (VCGs); two additional VCGs shared the seventh pattern. Vegetatively self-incompatible strains were associated with a VCG by restriction pattern. Length and restriction site variations were catalogued for all restriction patterns providing a quantitative measure of genetic distance among VCGs. Six VCGs congregated into three close clusters. The two remaining VCGs were separated from these clusters by a greater distance. The distant VCGs included a strain representing an isolated population and an avirulent strain which is a possible outgroup. Different VCGs containing the same race were not necessarily closely related. It could not be concluded that F. oxysporum f. sp. melonis , a group unified by pathogenicity, is monophyletic. MtDNA can be used to identify isolated populations within F. oxysporum defined by VCG.

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.

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.

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.

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.

Allelic Diversity at the het-c Locus in Neurospora tetrasperma Confirms Outcrossing in Nature and Reveals an Evolutionary Dilemma for Pseudohomothallic Ascomycetes

Abstract Vegetative cells of the filamentous ascomycete Neurospora tetrasperma are typically heterokaryotic, possessing haploid nuclei of both A and a mating types. As a consequence, N. tetrasperma is self-fertile. This life cycle, referred to as pseudohomothallism, clearly derives from true heterothallism of the type exhibited by related species such as N. crassa. Occasional homokaryotic, single-mating-type (heterothallic) isolates occur; in the laboratory, such strains can be outcrossed. The potential for outcrossing in N. tetrasperma raises the question of how this organism avoids heterokaryon incompatibility. Heterokaryon incompatability in vegetatively growing fungi is controlled by multiple loci. Two strains must be identical at each het locus (11 in N. crassa) to form a stable heterokaryon. Prior to the present survey, it seemed plausible that N. tetrasperma avoids heterokaryon incompatibility by maintaining compatible allele combinations through continual selfing. A survey of het-c variation among wild-type isolates in this study demonstrated that N. tetrasperma outcrosses in nature and that such matings can result in incompatible combinations of het-c alleles. Whereas individual wild-type isolates are invariably homoallelic for het-c, closely related strains may possess functionally different het-c alleles, which predate the origin of N. tetrasperma. Therefore, pseudohomothallic ascomycetes such as N. tetrasperma face an apparent evolutionary dilemma: the benefits of outcrossing must be balanced against the fact that matings can produce unstable heterokaryons and disrupt the pseudohomothallic life cycle.