Anne Pringle

CRYPTIC SPECIATION IN THE COSMOPOLITAN AND CLONAL HUMAN PATHOGENIC FUNGUS ASPERGILLUS FUMIGATUS

Abstract Microbes and other organisms smaller than one to a few millimeters in size are hypothesized to have global populations, in contrast to the geographically restricted ranges of larger organisms. However, fungi, which routinely have reproductive propagules no larger than 10 micrometers, challenge the generality of this hypothesis because recent studies have shown that globally distributed morphological species embrace two or more geographically restricted phylogenetic species. We used the concordance of gene genealogies to recognize phylogenetic species in the globally distributed opportunistic human pathogenic fungus, Aspergillus fumigatus. Based on DNA sequence data of five loci for each of 63 individuals collected from five continents, we have delineated two phylogenetic species in this single morphological species. Unlike all other fungi examined to date, both genetically isolated groups showed a global distribution with no evidence of a correlation between genotype and geographic location. Sexual reproduction has never been observed in A. fumigatus, but when the same data were used to explore the association of alleles at the five loci for one of the phylogenetic species, evidence was found to support recombination. The discovery of a cryptic species is medically relevant because different species are likely to differ in virulence or drug resistance. The discovery of a globally distributed A. fumigatus species clade highlights the need for ecological studies of the fungus to either document global dispersal or propose alternative mechanisms by which it persists as single, global phylogenetic population.

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.

The fitness of filamentous fungi

Fitness is a common currency in comparative biology. Without data on fitness, hypotheses about the adaptive significance of phenotypes or basic mechanisms of evolution, for example natural selection, remain speculative. Experiments with fungi can address questions specific to fungi or questions with a broader significance. Fungi can challenge the generality of fundamental evolutionary principles, yet there are no standard measures of fungal fitness. We argue that focusing on a single aspect of a complex life cycle, or a single measure of fitness (e.g. the number of asexual spores) is appropriate. Choosing which aspect of fitness to measure can be facilitated by an understanding of how fitness measures are correlated. Choices can also be based on the ecology of a species, for example whether a fungus is semelparous and reproduces once, or iteroparous and reproduces multiple times.

Global patterns of ectomycorrhizal introductions

Plants have often been moved across the globe with intact root systems. These roots are likely to have housed symbiotic ectomycorrhizal (EM) fungi and the movement of plants may have facilitated the introduction of EM fungi.Here, we report data compiled from a newly created database of EM fungal introductions.We estimate the magnitude of EM fungal introductions around the world and examine patterns associated with these introductions. We also use the data to develop a framework for understanding the invasion biology of EM fungi.At least 200 species of basidiomycete and ascomycete EM fungi have been moved from native ranges to novel habitats. The majority of recorded introductions are associated with Pinus or Eucalyptus plantations in the southern hemisphere. Most introduced species appear to be constrained from spreading in novel habitats and associate only with their introduced hosts. Aspects of life history, including host range, may influence the ability of EM species to establish or invade. Human-caused introductions of EM fungi are a common and global phenomenon.The mechanisms controlling EM fungi in novel habitats and potential impacts of EM fungal introductions are almost entirely unknown.

Divergent phenologies may facilitate the coexistence of arbuscular mycorrhizal fungi in a North Carolina grassland.

Interest in the diversity of arbuscular mycorrhizal (AM) fungal communities has been stimulated by recent data that demonstrate that fungal communities influence the competitive hierarchies, productivity, diversity, and successional patterns of plant communities. Although natural communities of AM fungi are diverse, we have a poor understanding of the mechanisms that promote and maintain that diversity. Plants may coexist by inhabiting disparate temporal niches; plants of many grasslands are either warm or cool season specialists. We hypothesized that AM fungi might be similarly seasonal. To test our hypothesis, we tracked the sporulation of individual AM fungal species growing within a North Carolina grassland. Data were collected in 1996 and 1997; in 1997, sampling focused on two common species. We found that AM fungi, especially Acaulospora colossica and Gigaspora gigantea, maintained different and contrasting seasonalities. Acaulospora colossica sporulated more frequently in the warm season, but Gi. gigantea sporulated more frequently in the cool season. Moreover, AM fungal species were spatially aggregated at a fine scale. Contrasting seasonal and spatial niches may facilitate the maintenance of a diverse community of AM fungi. Furthermore, these data may illuminate our understanding of the AM fungal influence on plant communities: various fungal species may preferentially associate with different plant species and thereby promote diversity in the plant community.

The ectomycorrhizal fungus Amanita phalloides was introduced and is expanding its range on the west coast of North America

The deadly poisonous Amanita phalloides is common along the west coast of North America. Death cap mushrooms are especially abundant in habitats around the San Francisco Bay, California, but the species grows as far south as Los Angeles County and north to Vancouver Island, Canada. At different times, various authors have considered the species as either native or introduced, and the question of whether A. phalloides is an invasive species remains unanswered. We developed four novel loci and used these in combination with the EF1α and IGS loci to explore the phylogeography of the species. The data provide strong evidence for a European origin of North American populations. Genetic diversity is generally greater in European vs. North American populations, suggestive of a genetic bottleneck; polymorphic sites of at least two loci are only polymorphic within Europe although the number of individuals sampled from Europe was half the number sampled from North America. Endemic alleles are not a feature of North American populations, although alleles unique to different parts of Europe were common and were discovered in Scandinavian, mainland French, and Corsican individuals. Many of these endemic European haplotypes were found together at single sites in California. Early collections of A. phalloides dated prior to 1963 and annotated using sequences of the ITS locus proved to be different species of Amanita. The first Californian collections that we confirmed as A. phalloides were made from the Del Monte Hotel (now the Naval Postgraduate School) in Monterey, and on the campus of the University of California, Berkeley, in 1938 and in 1945. These historical data are used in combination with data on A. phalloides’ current distribution to estimate a rate of spread for A. phalloides in California. Many species of ectomycorrhizal (EM) fungi have been introduced across and among continents, but with this evidence, the death cap becomes the only known invasive EM fungus in North America.

The Irreversible Loss of a Decomposition Pathway Marks the Single Origin of an Ectomycorrhizal Symbiosis

Microbial symbioses have evolved repeatedly across the tree of life, but the genetic changes underlying transitions to symbiosis are largely unknown, especially for eukaryotic microbial symbionts. We used the genus Amanita, an iconic group of mushroom-forming fungi engaged in ectomycorrhizal symbioses with plants, to identify both the origins and potential genetic changes maintaining the stability of this mutualism. A multi-gene phylogeny reveals one origin of the symbiosis within Amanita, with a single transition from saprotrophic decomposition of dead organic matter to biotrophic dependence on host plants for carbon. Associated with this transition are the losses of two cellulase genes, each of which plays a critical role in extracellular decomposition of organic matter. However a third gene, which acts at later stages in cellulose decomposition, is retained by many, but not all, ectomycorrhizal species. Experiments confirm that symbiotic Amanita species have lost the ability to grow on complex organic matter and have therefore lost the capacity to live in forest soils without carbon supplied by a host plant. Irreversible losses of decomposition pathways are likely to play key roles in the evolutionary stability of these ubiquitous mutualisms.

High levels of variation in ribosomal DNA sequences within and among spores of a natural population of the arbuscular mycorrhizal fungus Acaulospora colossica

Sequences of the nuclear rDNA ITS re- gion were sampled from a natural population of an arbuscular mycorrhizal fungus, Acaulospora colossica. Genetic diversity was explored through three levels of sampling: within an individual (a single spore), between individuals of a single soil sample, and be- tween individuals of different soil samples across a 36 m2 area. High levels of ITS sequence variation were discovered, and sequences from different spores and different soil samples were sometimes more similar than sequences of the same spore or soil sample. These results demonstrate a complex pattern of rDNA organization within a natural population of Glomalean fungi, and challenge ideas of individuality and collectivism in fungi.

Distribution and abundance of the introduced ectomycorrhizal fungus Amanita phalloides in North America.

Despite a growing awareness of the global reach of ectomycorrhizal (EM) fungal introductions, little is known about the fate of introduced EM fungi in novel ranges. Using herbarium specimens, species distribution models, and field collections of sporocarps, root tips and extramatrical mycelia, we assessed the distribution and abundance of the European species Amanita phalloides in North America. There are two distinct ranges of the fungus, one along the West Coast (California to British Columbia) and the second on the East Coast (Maryland to Maine). As predicted by a species distribution model, the West Coast range is larger. Amanita phalloides is more frequently found in native forests on the West Coast than on the East Coast. At Point Reyes Peninsula in California, A. phalloides dominates community sporocarp biomass, and is frequent as root tips. In individual soil cores at Point Reyes, root tips of A. phalloides make up 50% of total root tip biomass. Hyphae of A. phalloides are frequent, but make up only 2% of total hyphal biomass. The contrasting patterns of the distribution and abundance of A. phalloides on the East and West Coasts of North America may influence both its future spread and its impacts.

Bacterial Diversity across Individual Lichens

ABSTRACT Symbioses are unique habitats for bacteria. We surveyed the spatial diversity of bacterial communities across multiple individuals of closely related lichens using terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing. Centers of lichens house richer, more consistent assemblages than species-poor and compositionally disparate lichen edges, suggesting that ecological succession plays a role in structuring these communities.