Population Biology Abstracts

430. Environmental and Clinical Populations of Cryptococcus neoformans. Anastasia P. Litvintseva1, Rytas Vilgalys2, and Thomas G. Mitchell1.1Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, 2 Department of Biology, Duke University, Durham, NC 27708

Cryptococcus neoformans is a basidiomycetous yeast and opportunistic pathogen of humans. Little is known about the genetic structure of environmental populations of this important pathogen. We collected over 600 environmental isolates of C. neoformans from North Carolina. To genotype these strains, we generated amplified fragment length polymorphism (AFLP) markers. We compared the data obtained for the environmental isolates with clinical isolates of C. neoformans from the same areas. We also compared them with environmental and clinical isolates from India and clinical isolates from Botswana. Among the North Carolina populations, AFLP analyses revealed that 81% of the isolates were serotype A and represented by a total of seven distinguishable AFLP genotypes, 8% were serotype D and represented by four distinct patterns, and 11% were serotype AD hybrids represented by five different genotypes. Analyses of the AFLP patterns by the UPGMA using arithmetic means revealed that the serotype A strains from North Carolina grouped into two well-separated clusters. Most of the clinical isolates of serotype A from North America, as well as environmental and clinical isolates from India were associated with either of these clusters. The Botswanian isolates were significantly different from both the North American and Indian populations of serotype A.

431. Sex and recombination in an imperfect world : evidence of both in Fusarium oxysporum. Keith Klein*, Veronique Edel-Hermann, Nadine Gautheron, and Christian Steinberg, , INRA UMR BBCE/IPM Dijon, France. *Dept. Of Biological Sciences, Minnesota State University, Mankato, Minnesota USA

A population sample of nonpathogenic Fusarium oxysporum from soil and plant roots was collected in 1991-92. This sample was characterized for intragenic spacer region (IGS) of rDNA by PCR-RFLP. The most numerous IGS type (106 isolates) was characterized for vegetative compatibility groups (VCG). The 106 strains analysed fall into 50 VCG types, of which 16 are multi-membered. These strains were characterized for mating type by PCR with primers specific for each of the two idiomorphs. 101 type 2 and 5 type 1 strains were found. 3 of the type 1 strains shared VCG with one or more type 2 strains. RFLP analysis of 45 of these strains was also performed. All possible recombinant types were found in the sample. These observations support the hypothesis of genetic recombination in this species. The presence of two mating types suggests that sexual recombination may be present, but parasexual recombination cannot be ruled out. The highly skewed sex ratio implies that sex is infrequent. 10 strains in the sample with the same VCG, mating-type and RFLP patttern were isolated from roots of wheat plants in a single year. This VCG was not found elsewhere, although other VCGs with wider distributions were found wheat. The RFLP pattern was shared,with minor variations, by all but three of the strains isolated from wheat. This suggests that selection at the gene level may be important in the association of non-pathogenic F. oxysporum with specific hosts.

432. Population studies of Pythium species causing greenhouse diseases. Carla D. Garzon, David M. Geiser and Gary W. Moorman Department of Plant Pathology, The Pennsylvania State University

The population genetics of Pythium species is a little explored area of research. Most studies have focused on P. ultimum, but there is no reason to consider this species as a representative model for the genus. Other important plant pathogenic Pythium species, including P. irregulare and P. aphanidermatum, have attracted little attention from geneticists despite the fact that they can be aggressive and devastating pathogens on greenhouse crops. We performed preliminary studies to evaluate the genetic diversity of P. aphanidermatum and P. irregulare using AFLP genetic markers produced by a single selective primer combination. The results of these studies indicated that these two species show patterns of variation very different from that observed in P. ultimum and from each other. Nineteen P. irregulare isolates from the US showed species structure, being divided into two groups of overlapping geographical distribution. On the other hand, P. aphanidermatum showed very little genetic variation worldwide, based on an analysis of 23 isolates from Asia, Africa, North and South America. To examine the population genetics of P. irregulare and P. aphanidermatum and to identify AFLP genetic markers associated to fungicide resistance in isolates from the United States, ninety P. irregulareand 70 P. aphanidermatum isolates were analyzed using seven additional selective AFLP primer combinations. We report our current findings and conclusions.

433. Evolutionary aspects of gibberellin biosynthesis in the Gibberella fujikuroi species complex. Stefan Malonek and Bettina Tudzynski. Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schloßgarten 3, D-48149 Münster, Germany

G. fujikuroi is a species complex of at least 36 distinct Fusarium species (section Liseola) with monophyletic origin. On the basis of sexual compatibility the species complex was divided into 8 mating populations (MP-A to MP-H). In contrast to the other MPs, only members of MP-C (F. fujikuroi), pathogenic on rice, produce high amounts of gibberellins (GAs), resulting in abnormous growth of rice seedlings. The seven genes involved in the biosynthetic pathway were found to be organised in a gene cluster in MP-C. Except for MP-A (F.moniliforme), which have only two genes left, strains of the other MPs consist of all seven genes of the cluster which are highly homologous (90-98%) to the corresponding sequences of MP-C. However, only the two genes of MP-A were shown to be highly expressed under GA producing conditions so far. In order to find the reason(s) for the lost ability to produce GAs, we started a detailed molecular characterisation of the GA biosynthetic genes from two MPs, MP-D (F.proliferatum) and MP-A, including sequencing, expression studies, cluster organisation and functional analysis. For this task, the genes were transformed into a mutant strain of MP-C which has lost the entire gene cluster due to a big deletion, but still has all the regulatory genes for GA biosynthesis. First results revealed full functionality of the so far analyzed genes of MP-D in the MP-C background. However, feeding of radiolabelled intermediates gave evidence for a genetic block at the step of ent-kaurene oxidation catalyzed by the gene P450-4. This gene of MP-D is now under detailed investigation to find the reason for non-functioning. Thus we want to investigate the evolutionary developement of this big species complex of G. fujikuroi using the dispensable gene cluster for the GA biosynthetic pathway as a molecular marker.

434. Exploring the fitness of filamentous fungi using a lichen, Xanthoparmelia cumberlandia, and Neurospora crassa as model systems. A. Pringle and J.W. Taylor. UC Berkeley, Plant & Microbial Biology, Berkeley, CA

There is no standard measure of fungal fitness. Mycologists have choosen to understand fitness as either a combination of survival and reproduction, or as a comparative variable which contrasts the reproductive success of two individuals. We argue that fitness should be understood as the combination of survival and reproduction, equivalent to: R = *lxmx where R is fitness, lx is the probability of survival to age x, and mx is the number of progeny produced by an individual at age x. Measuring R is laborious; it may be useful to choose a single aspect of fitness as a surrogate of R. Choosing which aspect of fitness to measure can be facilitated by an understanding of how fitness measures are correlated, unfortunately, few data record the correlations between different aspects of fitness. In two separate studies we demonstrate significant correlations between different aspects of fitness. First, Xanthoparmelia cumberlandia is used to demonstrate a significant correlation between thallus size and reproductive effort, showing that size is an easily measured surrogate of fitness. Published data of other species show a similar correlation, indicating that it may be a general feature of sexual lichens. Second, Neurospora crassa is used to explore correlations between spore germination, mycelial growth, biomass, and sexual and asexual sporulation.

435. The human pathogen Aspergillus fumigatus is a single globally distributed fungal species. A. Pringle, D. M. Baker, J. L. Platt, J. P. Wares, J.P. Latge and J.W. Taylor. UC Berkeley, Plant & Microbial Biology, Berkeley, CA

Aspergillosis is a typically fatal fungal infection of immunocompromised humans caused by species in the genus Aspergillus, including A. fumigatus. The fungus is assumed to be asexual as neither mating, sexual structures, nor meiospores, have been observed. Investigations of the species status and reproductive habit of other fungi have shown that morphological species may harbor cryptic genetic species, and that apparently asexual fungi can be sexual. Understanding whether or not A. fumigatus comprises multiple species, and is cryptically sexual, is a matter of biological interest; the data will also provide critical information to the medical community, which must manage the evolution of resistance to widely prescribed drugs and design effective anti-fungal therapies. Sixty-one cultures of A. fumigatus were collected from Africa, Asia, Europe, and North and South America. Using a phylogenetic species concept, we analyzed sequence data of five microsatellite loci and conclude that A. fumigatus is a single globally distributed fungal species. We are currently using these same data to investigate the sexuality of the fungus.

436. Creation of Congenic Serotype A Isolates of Cryptococcus neoformans. S.M. Keller1, M.A. Viviani2 and B.L. Wickes1. 1 Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 2Laboratorio di Micologia Medica, Istituto di Igiene e Medicina Preventiva, Universita degli Studi, IRCCS Ospedale Maggiore, Milano, Italy

Cryptococcus neoformans, an opportunistic human fungal pathogen, is comprised of two varieties (neoformans and gattii) and 4 serotypes (A, B, C, D). Recently, the first fertile serotype A MATa isolate (IUM 96-2828) was described in detail, making classical genetic analysis using serotype A strains, the most frequent clinical isolate, possible. Congenic strains are beneficial for mating studies and to clean up the backgrounds of gene disruptant strains for in vivo testing. At present, only congenic serotype D strains are available for use in classical genetic analysis, which was the major reason why this serotype was chosen for the genome sequencing project. Using H99 (standard serotype A MATalpha laboratory isolate) and IUM 96-2828 (serotype A MATa environmental isolate) as parents, pairs of serotype A congenic strains were prepared in each background by successive backcrossing through 10 generations. Each F10 congenic strain was compared to its respective parent strain using AFLP analysis. Preliminary data suggests that these strains differ only at the mating type locus. Genetic markers were created and then moved into these congenic pairs of strains to facilitate efficient mating and to provide host strains for transformation. The serotype A congenic strains created in this study will prove a valuable tool for addressing questions about mating type and the effects of genetic background on characteristics like virulence.

437. Nuclear reassortment between vegetative mycelia of the basidiomycete Heterobasidion annosum in nature. Hanna Johannesson1 and Jan Stenlid2.1 Department of Plant and Microbial Biology, Berkeley, USA.2 Department of Forest Mycology and Pathology, Uppsala, Sweden.

The recognition of self from non-self in basidiomycetes is mediated by the somatic incompatibility (SI) system, a multigenic system that prevents free exchange of nuclei and cytoplasm between heterokaryotic individuals of a population. SI in the basidiomyceteHeterobasidion annosum is controlled at a series of three to four multiallelic discrete loci. Previous laboratory studies have indicated that SI reactions are not an absolute block to nuclear exchange between unrelated heterokaryotic strains of H. annosum, and in this study we present evidence for nuclear reassortment between vegetative mycelia in natural populations of the species. By using six highly variable microsatellite markers we genotyped single nuclei obtained from 18 somatically incompatible heterokaryotic strains of H. annosum originating from a single stump of Picea abies. We found that 28 nuclei compose the 18 heterokaryotic genotypes found in the stump; 19 of the nuclei were found in more than one heterokaryotic mycelia. Furthermore, in one of the heterokaryotic mycelia we verified the coexistence of four different nuclei. Heterokaryotic single-conidial mycelia, each containing two nuclei from that mycelium in different combinations, were somatically incompatible when paired on Petri-dishes. These results show that the SI system is not preventing nuclear migration in heterokaryotic mycelia of H. annosum.

438. Genetic diversity of Fusarium graminearum from maize in Korea. Jae-Jin Jeon1, Hun Kim1, Hye-Sun Kim1, Kurt A. Zeller3, Theresa Lee1, Sung-Hwan Yun2, Robert L. Bowden3, John F. Leslie3, and Yin-Won Lee1.1School of Agricultural biotechnology, Seoul National University, Suwon 441-744, Korea, 2Division of Life Sciences, Soonchunhyang University, Asan 336-745, Korea, 3Department of Plant Pathology, 4017D Throckmorton Plant Sciences Center, Kansas State University, Manhattan KS 66506-5502.

A total of 584 isolates of Fusarium graminearum (Shwein.) Petch (teleomorph:Gibberella zeae) were obtained from maize in Gangwon province of Korea during 1999-2000. Of these isolates, 500 were self-fertile and 84 were female-sterile. A phylogenetic tree of the isolates was constructed by using amplified fragment length polymorphism (AFLP). AFLP showed polymorphic bands and these bands, haplotypic loci, were used to analyze population genetic diversity. Population structure of the isolates consists of four lineages (7, 6, 3 and 2). Lineage 7 was the major group (74%) and followed by lineage 3 (13%), lineage 6 (12%) and lineage 2 (1%). Estimate of Nei¡̅s Gst and Nm values showed a significant difference in allelic frequencies among lineages. Maximum parsimony trees based on selected sequencing data from Tri101, Tri7 and MAT-1-1-1 genes was found to be concordant with AFLP data. Each lineage showed a significant difference in fertility. Fertility of lineage 7 isolates was 100% followed by lineage 6 and lineage 3 isolates that showed 69% and 25%, respectively. When the representative isolates of each lineage were inoculated to barley, they were virulent regardless of lineages. Trichothecene production of F. graminearum isolates was variable; lineage 7 and lineage 3 isolates produced deoxynivalenol (DON), whereas lineage 6 and lineage 2 isolates produced nivalenol (NIV). The results of this study provide the difference of genetic variation among lineages in F. graminearum population from maize in Korea.

439. Molecular Phylogenetic Analysis Indicates Cephalosporium maydis a Distinct Taxon in the Gaeumannomyces-Phialophora Complex. A.A. Saleh1 and J.F. Leslie2. 1Agricultural Genetic Engineering Research Institute, ARC, Giza, Egypt. 2Dept. of Plant Pathology, Kansas St. University, Manhattan, KS 66506.

A recent hypothesis is that the maize pathogen Cephalosporium maydis is closely related to, if not a part of, the Gaeumannomyces-Phialophora (GP) complex. We evaluated strains of C. maydis, other Cephalosporium spp., and the GP complex using several DNA-based techniques. Based on AFLP data, C. maydis is a unique species that is distinct from other plant pathogenic species of Cephalosporium andGaeumannomyces. We obtained DNA sequences of four nuclear genes from four strains representing C. maydis and 21 additional, potentially related, species. These data are consistent with the hypothesis that C. maydis is a distinct taxon that is closely related to the GP complex. Although no sexual stage is known for C. maydis, the MAT-2allele is > 77% similar to MAT-2 sequences from other Gaeumannomyces strains. The clade formed by strains from the GP species complex was monophyletic in our analysis, but the clade formed by strains from the Acremonium-Cephalosporium (AC) complex was not. We also found that C. gramineum, the causal agent of Cephalosporium stripe disease of wheat, is distantly related to both the AC and the GP species complexes. Thus the taxonomic treatment of both C. maydis and C. gramineum needs revisions, with C. maydis assigned to the GP complex and C. gramineum assigned to another genus.

440. Biological Species in the Gibberella fujikuroi species complex (Fusarium section Liseola) recovered from Maize and Sorghum in Egypt. A.A. Saleh and J.F. Leslie. Department of Plant Pathology, Kansas State University, Manhattan, KS 66506-5502.

Fusarium species in section Liseola, with teleomorphs in the Gibberella fujikuroi species complex, cause stalk, ear and kernel rots of maize and sorghum and produce mycotoxins such as fumonisins and moniliformin. We have examined 353 isolates within section Liseola, isolated from both maize and sorghum. Species among these isolates were identified with AFLP markers and sexual fertility testing. We recovered representatives of G. fujikuroi mating populations (MPs), MP-A (F. verticillioides, teleomorph G. moniliformis), MP-D (F. proliferatum, teleomorph G. intermedia), MP-F (F. thapsinum, teleomorph G. thapsina), and MP-G (F. nygamai, teleomorph G. nygamai), along with members of an undescribed biological species closely related to F. andiyazi. MP-A was the most frequently recovered MP from maize (73% of recovered isolates), and MP-D was the most frequently recovered MP from sorghum (51% of recovered strains from sorghum). Female fertile strains were most common within MP-A (68%). The inbreeding effective population sizes (Ne), based on mating type frequencies, for MP-A, MP-D, and MP-F were 85%, 100%, and 88%, respectively. Based on the observed frequency of female fertility, estimated Ne of the MP-A, MP-D, and MP-F isolates were 98%, 70%, and 31%, respectively. Our results suggest that sexual reproduction occurs more frequently within MP-A than within MP-D or MP-F. The relatively low female fertility within MP-D and MP-F may limit genetic exchange among individuals within these species relative to that possible in MP-A.

441. Might the B-alpha and B-beta mating-type gene products of Schizophyllum commune really cross-talk? Thomas J. Fowler, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT USA 05405

Two of the four loci that determine mating type in the heterothallic mushroom-bearing fungus Schizophyllum commune are the linked and redundantly functioning multigenic loci called B-alpha and B-beta. Within the species there are multiple versions of both B-alpha and B-beta. Until now, only portions of a few of the versions of these loci had been cloned, sequenced, and characterized in biological assays. DNA containing the linked B-alpha3-B-beta2 versions has been sequenced. Eleven genes encoding lipopeptide pheromones and two genes encoding seven-transmembrane domain pheromone receptors are located in this B-mating-type complex. All of these genes have been confirmed to confer mating activity. It had always been thought that pheromone and receptor components encoded by B-alpha do not activate or respond to the pheromone and receptor components encoded by B-beta, and vice versa. However, some pheromones encoded by B-beta2 do activate receptors associated with the B-alpha versions B-alpha8 and B-alpha9 in addition to activating B-beta receptors. The B-alpha8 receptor gene was subsequently isolated and shown to predict a receptor very similar to the B-beta1 receptor. These findings indicate that some cross-talk between the signaling components encoded by the two loci probably does occur, but may also indicate that the B-complexes containing B-alpha8 and B-alpha9 do not consist of a simple alpha-beta pair. Previously published studies of recombination between B-alpha and B-beta support this latter idea, but more molecular information will be required for confirmation. And where do these cross-talking versions fit into the evolutionary history of the S. commune B-loci?

442. Molecular diversity between maize and banana populations of F. verticillioides. Mulè G. , Mirete, S., M.T. González-Jaén, A. Moretti, C. Vázquez, B. Patiño, A. Logrieco. Is. Scienze Produzioni Alimentari, Bari, Italy, and Univ. Complutense Madrid, Madrid, Spain

 Fusarium verticillioides is one of the most prevalent Fusarium species on maize, causing stalk and ear rot worldwide. The organism, associated with various animal diseases, is able to produce potent mycotoxins, the fumonisins. A population morphologically identified as F. verticillioides has been also reported from banana fruits. This latter population showed different toxigenic profile, being able to produce in vitro moniliformin and not fumonisins, and although produced fertile perithecia of mating population A, time for obtaining perithecia and size of them differed significantly from usual fertile crosses among strains from maize. Two different molecular approaches, amplified fragment length polymorphism (AFLP) and the TEF-a (translational elongation factor alfa) were used to strengthen these differences. All molecular methods showed two different clusters, supporting the fact that they represent two distinct populations.

443. Phylogeny and Biogeography of Morchella. Kerry O'Donnell1, Nancy S. Weber2, Steve Rehner3 and Gary Mills4. 1USDA/ARS/NCAUR, Peoria, IL,2Oregon State University, Corvallis, OR,3USDA/ARS/BARC, Beltsville, MD and 4Michigan State University, East Lansing, MI.

Species of the filamentous ascomycete genus Morchella, better known as the true morels or morel ‘mushrooms', are among the most highly-prized macrofungi collected by mycophiles during Spring in the northern hemisphere. Field guides to morels typically recognize 6 or fewer morphospecies and these are generally assumed to be cosmopolitan in their distribution. To test these hypotheses, multigene genealogies were constructed from a global collection of approximately 600 individuals to investigate the phylogeny and biogeography ofMorchella, using a genealogical concordance version of phylogenetic species recognition. Early in the study, allelic variation within the nuclear rDNA internal transcribed spacer region was used as a genetic screen for the detection of putative phylogenetically distinct species. Parsimony analysis of the combined multilocus DNA sequence data provided a nearly fully resolved phylogeny in which a monophyletic Morchella comprised two sister clades: the ‘yellow-tan-grey' morels (Esculenta clade) and the ‘black' morels (Elata clade) with 13 and 15 species, respectively. Biogeographical interpretation of the phylogenetic data suggests that the ancestral area for Morchella is North America, the most phylogenetically diverse area studied with 13 endemics [4 Esculenta clade, 9Elata clade]. The most surprising result of this study is that at least 24 of the 28 species exhibit continental endemism. Results of this study have practical implication for morel breeding and conservation biology and provide a robust phylogenetic framework for studying the evolution of mating systems.

444. Hop,an active Mutator-like element isolated in Fusarium oxysporum transposes in the heterologous species Fusarium culmorum. Fabienne Chalvet, Christelle Vasnier and Marie-Josée Daboussi Institut de Génétique et Microbiologie, Université Paris-Sud, 91405 Orsay Cedex, France

A new type of active DNA transposon has been identified in the genome of Fusarium oxysporumthrough its transposition into the niaD target gene (Chalvet et al.). Its structural features (length, ITRs size, 9 bp duplication) as well as the presence of conserved domains in the transposase are those characterizing Mutator-like elements (MULEs). Therefore, Hop is the first active member of this family found outside the plants. The high rate of excision observed in spite of its location in an exon indicates that Hop is very active and thus represents a promising efficient tagging system for fungal gene isolation. TheHop copy inserted in the niaD gene has been introduced in different genetic contexts free of endogenous elements in order to check for its autonomy. Evidence for excision was found in Fusarium culmorum. The properties of transposition of such element (reinsertion frequency, target site duplication, propensity to insert into genes) are under investigation. The fact that Hop-related elements were found in different distant related species suggest that Hop would be able to function in a wide range of ascomycetes.

445. Tracking DNA polymorphisms in field populations of Aphanomyces cochlioides. John J. Weiland, USDA-Agricultural Research Service, Red River Valley Agricultural Research Center, Fargo, N.D 58105

Root and seedling diseases caused by Aphanomyces cochlioides are serious impediments to sugarbeet production in wet growing regions, yet information on the genetics and the inheritance of virulence in this organism is lacking. No race structure for A. cochlioides has been reported and preliminary studies have revealed limited genetic diversity in this oomycete using DNA-based technologies. In the present study, application of random amplified polymorphic DNA (RAPD) analysis to single zoospore isolates obtained from sugarbeet fields in the U.S. identified 2 polymorphisms that assorted randomly within local populations; some field populations harbored only one polymorphic type. The data indicate that these polymorphisms are found in A. cochlioides isolates ranging from the northern Red River Valley of the U.S. to the historic regions of sugarbeet production in Texas. Implications of this result in the development of novel virulence and fungicide resistance in A. cochlioides are discussed.

446. Population Genetic Differentiation and Lineage Composition Among Gibberella zeaein North and South America. K.A. Zeller1, J.I. Vargas1, G. Valdovinos-Ponce1, J.F. Leslie1, & R.L. Bowden2. 1Dept. of Plant Pathology, Kansas State Univ., Manhattan, KS;2USDA-ARS Plant Sci & Entomology Res. Unit, Manhattan, KS.

G. zeae (Fusarium graminearum) causes Fusarium Head Blight (FHB) of wheat and barley, and has been responsible for severe economic losses worldwide. Sequence analyses of G. zeae have been interpreted to mean that populations of G. zeae are composed of eight phylogenetic lineages, with a phylogeographic structure among these lineages. We compared AFLP polymorphisms in populations of G. zeae from the United States, Mexico, Brazil, and Uruguay. Populations of G. zeae causing FHB in the United States include only a single phylogenetic lineage (lineage 7). Subpopulations across the United States have high genotypic diversity, do not deviate from expectations of random mating, and are interconnected by extensive gene-flow. South American populations of G. zeae from both wheat and from sorghum include a minority component of isolates that cluster with other phylogenetic lineages (lineages 1, 2, and 6), but are dominated by genotypically diverse populations of isolates from lineage 7. Populations of G. zeae causing FHB on wheat from two locations in Mexico are dominated by isolates from lineage 3. Intercontinental gene flow probably occurs from North to South America, but the amount of gene flow between the continents is much less than that occuring within each continent.

447. Interfertility and Marker Segregation in Hybrid Crosses of Gibberella fujikuroi and Gibberella intermedia. K.A. Zeller1, M.A Wohler1, L.V. Gunn2, S. Bullock2, B.A. Summerell2 and J.F. Leslie1. 1 Department of Plant Pathology, Kansas State University, Manhattan, KS;2 Royal Botanic Gardens, Sydney, NSW, Australia.

Gibberella fujikuroi and Gibberella intermedia [mating populations (MP) C and D of the Gibberella fujikuroi species complex] generally can be distinguished by differences in the spectrum of mycotoxins produced, the lack of sexual cross fertility, and diagnostic differences in DNA sequences. Some isolates from these two biological species, however, can interbreed and complete meiosis to produce viable progeny. Analysis of marker segregation amongst such hybrid progeny can be used to estimate the degree of genomic rearrangement and genetic incompatibility that has accumulated since these sibling species diverged. We isolated recombinant progeny from crosses of MP-C × MP-C, MP-D × MP-D, and MP-C × MP-D. These progeny segregate for the MAT locus, a homolog of theFum1 locus, and numerous loci defined by amplified fragment length polymorphisms (AFLPs). In intra-specific crosses, most of the AFLP loci segregate in the expected 1:1 ratios. In MP-C × MP-D crosses more of the AFLP loci appear to deviate from the expected 1:1 segregation ratios. We are evaluating the fertility of the hybrid progeny in backcrosses with their parents to identify genetic loci that are critical to the evolutionary differentiation of these two sibling species.

448. Genetic consequences of habitat fragmentation in rare wood decay fungi. Jan Stenlid, Mårten Gustafsson, and Nils Högberg Department of Forest Mycology and Pathology, SwedishUniversity of Agricultural Sciences, Box 7026, S-75007 Uppsala, Sweden.

Fragmentation of habitat is a major threat to species diversity in forest ecosystems. In boreal forests, many wood-inhabiting organisms, including fungi, are threatened due to scarcity of localities containing sufficient amount of substrates. We here present studies in two species with a fragmented habitat in parts of their natural distribution range. Based on variation in variable regions of coding genes and in RAPD profiles, studies in the rare polypore Fomitopsis rosea suggested a reduced heterozygosity in small populations in Southern Sweden and Russia, but heterozygosity was close to expectations in larger populations in Northern Sweden, Norway and Finland. Small populations may experience reduced fitness. In F. rosea and Phlebia centrifuga, spore germination was reduced in small populations outside the core range of distribution in Sweden. Dispersal in Basidiomycete fungi can be followed using a species specific spore trapping approach with homokaryotic mycelia. The dispersal range was correlated with the strength of spore source, and in a recent study, the spore dispersal outside the regional range in Sweden of fruiting bodies in F. rosea and P. centrifuga was undetectable. Future work will involve studies on the effect of inbreeding on several fitness parameters e.g. wood decay ability and interspecific competition.

449. Microsatellite evolution in Neurospora. Jeremy Dettman and John Taylor Plant and Microbial Biology, UC Berkeley

Microsatellite loci have become one of the most popular classes of markers for population genetic analyses. Despite this fact, the evolutionary dynamics and mutational processes of microsatellites are still not fully understood. To address this issue, we sequenced 4 unlinked microsatellites and their flanking regions from 147 strains of Neurospora. Representatives from eight phylogenetic species were included, with most emphasis on N. crassa and N. intermedia. To elucidate the genealogical relationships among alleles, repeat number was mapped onto trees constructed from sequence data. This allowed us to place the microsatellite mutations in the evolutionary context of the less rapidly evolving flanking regions. Within populations and species, distributions of allele repeat number were generally consistent with the stepwise mutational model proposed for microsatellites. Indels in the flanking regions were observed, but they caused only small amounts of intraspecific allele length homoplasy, confirming the usefulness of microsatellites for population level analyses. While flanking sequences were quite divergent among species, allele repeat number distributions commonly overlapped, i.e., alleles that were identical in state, but not identical by descent, were shared among species. As well, several mutations in the tandem repeats themselves were observed within certain lineages. These high levels of interspecific homoplasy indicated that several more microsatellite loci must be characterized before we can test their efficacy in phylogenetic reconstruction among Neurospora species.

450. Sympatric biological and phylogenetic species among pseudohomothallic isolates identified as Neurospora tetrasperma. G. S. Saenz1, D. J. Jacobson2, W. H. Dvorachek1, and D. O. Natvig1.1University of New Mexico, Albuquerque, 2Stanford University

Isolates assigned to Neurospora tetrasperma are distinct within the genus in possessing four-spored asci. Each large ascospore gives rise to a heterokaryotic (mat A +mat a), self-fertile thallus, which is pseudohomothallic. Our previous work showed that: 1) pseudohomothallism is monophyletic in Neurospora, and 2) outbreeding is common within populations of N. tetrasperma, despite predominant selfing and frequent sexual dysfunction during outbreeding. Phylogenetic and reproductive relationships were assessed among 14 strains of N. tetrasperma from one population in a single 5 ha field. Upstream regions of four genes, het-c, frq, sod-2, and pdx-2 were sequenced. Reproductive isolation was assessed by outcrossing. Tree-building analyses revealed three separate, well-supported sympatric phylogenetic species within this local population. However, only one of the three lineages was reproductively isolated in laboratory crosses. The results suggest that phylogenetic differentiation can precede the development of genetic barriers to sexual reproduction, as also seen in N. crassa and N. intermedia (Dettman et al. FGN 49S:19). This implies a common speciation trend within Neurospora, but is particularly enigmatic for N. tetrasperma given the sympatric nature of the closely related putative species.

451. Phylogenetic, biogeographic and population genetic approaches to the analysis of cryptic speciation in the entomopathogen.

Stephen A. Rehner and Ellen P. Buckley. USDA/ARS, Insect Biocontrol Laboratory, Beltsville, MD.

Beauveria is a genus of globally distributed entomopathogenic hyphomycetes of practical interest as biological control agents of pest insects. Species recognition in Beauveria is difficult due to a lack of informative morphological structures. A gene-genealogical concordance approach to species recognition was used to investigate phylogenetic diversity of Beauveria, particularly B. bassiana, a cosmopolitan species widely used in biological control of agricultural pest insects. Phylogenetic analyses of 7 loci revealed five major clades, each of which was possessed fairly uniform spore characteristics, however the clades do not correspond well to prevailing species concepts for this genus. Most notably, the morphological species B. bassiana is biphyletic with morphologically similar strains divided among two unrelated clades. All clades consisted of multiple endemic lineages indicative of an underlying pattern of cryptic diversification. Biogeographic structuring in the B. bassiana complex is consistent with a mode of divergence in allopatry. Several Cordyceps species are derived from within Beauveria phylogeny, thus Beauveria species are either inherently sexual or, if strictly asexual, are likely to be recently derived from Cordyceps. Polymorphic microsatellite markers have been developed for the type B. bassiana clade for inferring population genetic structure and investigating speciation and species boundaries.

452. Identification of a mating-type gene in the homothallic fungus Aspergillus nidulans. Paul S Dyer, Mathieu Paoletti, Marcos J Alcocer and David B Archer. School of Life and Environmental Sciences, University of Nottingham, Nottingham NG7 2RD, UK.

Mating-type (MAT) genes have been identified from the pyrenomycete, loculoascomycete and discomycete classes of ascomycete fungi. By using hot-start PCR with degenerate primers, together with thermal asymmetric interlaced (TAIL)-PCR, it has been possible to identify a characteristic MAT-2 gene from the plectomycete fungus Aspergillus nidulans. It includes a conserved high mobility group (HMG)-domain. RACE-PCR analysis has been used to investigate transcription of the gene, confirming the presence of an intron in a conserved position within MAT-2 genes. Further analysis of the flanking regions of the MAT-2 gene has revealed three further genes, including a putative gene with homology to an element of the anaphase-promoting complex (APC) of Schizosaccharomyces pombe. The presence of an APC homologue provides evidence of microsyteny around the MAT locus. However, no MAT-1alpha-domain gene could be detected. This suggests either that a MAT-1 homologue may be present elsewhere in the genome, not directly adjacent to the MAT-2 locus, or thatA. nidulans may contain only a MAT-2 mating-type gene. This would be a unique situation among homothallic euascomycetes so far analysed, which contain either aMAT-1 gene or both MAT-1 /MAT-2 genes.

453. Evaluating the likelihood of genetic recombination between introduced and indigenous strains of the entomopathogenic fungus Beauveria bassiana in agricultural fields. Louela Castrillo1, John Vandenberg2, and Michael Griggs2. 1Department of Entomology, Cornell University, Ithaca, NY 14853 and 2 USDA-ARS, US Plant, Soil and Nutrition Lab., Ithaca, NY 14853.

The fungal pathogen Beauveria bassiana is widely used as a mycoinsecticide for control of several insect pests, providing a biological alternative to synthetic chemical insecticides. A key advantage for microbial control agents is their potential to replicate and persist in the environment, offering continued suppression of insect pest populations. However, exploiting this advantage is commensurate with the need to determine the impact of mass releases of this fungus on non-target organisms and to assure safety and long-term efficacy. To date, no information is available on the potential for genetic recombination between introduced and indigenous strains of B. bassiana in agricultural fields and whether this can result in recombinants with altered virulence and host range. In this study we evaluated the likelihood of genetic recombination by determining 1) vegetative compatibility groups (VCG) among B. bassiana strains indigenous to the US and strains that have been mass released for insect control and 2) the frequency of recombination between co-infecting strains of B. bassianain an insect host, where recombination is likely to occur in nature. Our data revealed a group of genetically similar strains isolated from Colorado potato beetles belonging to the same VCG. These strains originated from the northeastern part of the US and from Quebec and Ontario, Canada. Co-inoculations of beetle larvae with complementary nit mutants resulted in heterokaryon formation only between strains of the same VCG, suggesting that the self/non-self recognition system of the parasexual process is an effective barrier preventing genetic exchange between dissimilar strains in the field. Further studies are being conducted to determine stability and virulence of recombinants.

454. Population subdivision in Fusarium graminearum lineage 7 in the U.S. is correlated with toxin chemotype. Liane Rosewich Gale1, Todd Ward2, Virgilio Balmas3, and H. Corby Kistler1.1USDA-ARS, University of Minnesota, MN 55108, 2USDA-ARS Microbial Genomics Research Unit, Peoria, IL 61604, 3Università degli Studi di Sassari, I-07100 Sassari.

Isolates of Fusarium graminearum, causing head blight of wheat, from nine U.S. states, representing 86 fields in 53 counties, were characterized using ten single-copy RFLP probes, a telomeric probe and RFLP probes diagnostic for species and lineage. In addition, isolates were assigned to one of three profiles of trichothecene metabolites (chemotypes) using a PCR-based approach. Most of the 712 isolates examined were confirmed as lineage 7, though four isolates from ND had slightly different RFLP patterns and were excluded from further analysis. The telomeric probe was used for clone determination, leaving 587 isolates for subsequent data analyses. Most lineage 7 isolates (94.6%) from the U.S. were of 15acetyl deoxynivalenol (15ADON) chemotype. The 3acetyl deoxynivalenol (3ADON) chemotype was found at 5% and was only identified in samples from ND and MN. The nivalenol chemotype was infrequent at 0.4%. Gene flow analysis demonstrates that the 15ADON population in the U.S. is genetically isolated from the 3ADON population (Nm = 0.5). In comparison, a representative collection consisting of 19 isolates of lineage 7 from Italy was genetically similar to the 3ADON population from the U.S. (Nm > 2). Regarding diversity, no major differences could be discerned between the 3ADON and 15ADON populations from the U.S., even though the 3ADON population was nearly fixed at some RFLP.

455. Multi-gene phylogenies reveal taxonomic confusion in the genus Magnaporthe. Stephen B. Goodwin1, Morris Levy2, Jessica R. Cavaletto1 and Yang Tian3. 1 USDA-ARS,2 Department of Biological Sciences, 3 Department of Botany and Plant Pathology, 915 West State Street, Purdue University, West Lafayette, IN 47907-2054, USA

The genus Magnaporthe contains a number of species infecting monocot hosts. One of these, which causes blast of rice, formerly was called M. grisea. However, rice-infecting isolates of M. grisea were separated recently into the new species, M. oryzae. This analysis cleared up some taxonomic confusion, but did not address the relationships of the M. grisea complex and other species of Magnaporthe to related genera, such as Gaeumannomyces. To determine in more detail the phylogenetic relationships of M. grisea, the internal transcribed spacer region of isolates from diverse hosts was sequenced. Sequences from related species, primarily Magnaporthe and Gaeumannomyces, were downloaded from GenBank, along with sequences representing the Diaporthales as an outgroup. Neighbor-joining analysis revealed that the genusMagnaporthe was polyphyletic; M. poae, M. rhizophila and the type species for the genus, M. salvinii, all clustered within Gaeumannomyces rather than with M. grisea and M. oryzae. The species M. grisea comprised a monophyletic group of several genetically distinct taxa that are mostly host delimited. In addition to M. oryzae, clusters with high bootstrap support that may represent separate species include: most isolates from Cenchrus/Pennisetum; isolates fromPennisetum typhoideum; isolates from Digitaria spp. (including the type forM. grisea); and some isolates from Eleusine hosts. These conclusions were confirmed by analysis of beta-tubulin and calmodulin sequences which gave the same results but with varying levels of resolution. The only exception was translation elongation factor alpha which did not resolve the Gaeumannomyces group from M. oryzae. The evolutionary diversification of M. grisea appears to be recent and rapid, and the multi-continental distribution of several forms likely reflects human-aided dispersal of infested seed stocks. Clustering of the type species for the genus, M. salvinii, with Gaeumannomyces rather than with M. oryzae and M. grisea increases the taxonomic confusion surrounding this genus.

456. A population genetic study of the Ustilago maydis virus, a dsRNA killer virus associated with Ustilago maydis. Voth, Peter and May, Georgiana. Dept. of Plant Biological Sciences, University of Minnesota, St. Paul, MN USA

Ustilago maydis (corn smut) infects vegetative and reproductive tissues of maize and the teosintes. Associated with U. maydis is the Ustilago maydis virus (UMV), a double-stranded RNA virus that causes the fungus to produce a "killer" toxin. The proteinaceous UMV toxin is lethal to susceptible U. maydis individuals and can inhibit mating between compatible U. maydis individuals. UMV is only transmitted through cytoplasmic fusion during mating of compatible U. maydis individuals. U. maydis individuals are protected from the virus through nuclear resistance or cytoplasmic immunity. The interactions between UMV and U. maydis may affect the reproductive biology of U. maydis, by enforcing assortative mating in this obligately sexual organism, and subsequently the population genetic structure of UMV. The aim of the current study is to characterize the population genetic structure of UMV throughout the Western Hemisphere through sequence analysis of the viral genome. Using immuno-capture RT-PCR, I have amplified a 500 bp region of the viral capsid protein in isolates from the United States, Mexico, and South America. Sequence analysis of this region revealed nucleotide sequence polymorphisms associated with large scale geographic distance.

457. Population biology of mutations conferring resistance to QoI fungicides in Pyricularia grisea. Yun-Sik Kim, Paul Vincelli and Mark Farman, Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA.

Strobilurin-based (QoI) fungicides are used to control gray leaf spot disease of perennial ryegrass caused by Pyricularia grisea. QoI fungicides bind to cytochrome b, thereby poisoning the cytochrome bc1 respiratory complex. In 2000, QoI-resistant P. griseaisolates were identified in Kentucky and Illinois. Characterization of the mitochondrial cytochrome b gene in these cultures revealed two nucleotide substitutions associated with resistance. These cause F129L and G143A substitutions in the cytochrome b protein. Here, we describe experiments to investigate the population biology of QoI resistance in P. grisea. The cytochrome b gene was amplified from more than 200 isolates collected in2001and 2002 from perennial ryegrass throughout the eastern and central US. Cleavable Amplified Polymorphic Sequence (CAPS) analysis revealed cytochrome b mutations in isolates from Illinois, Indiana, Kentucky, Maryland and Virginia. Mutant isolates possessed the F129L or G143A mutation but never both. Comparison of sporulation and disease severity revealed no significant differences between mutants and wild-types. A field experiment was conducted to determine if a QoI-resistant population can be superceded by sensitive one(s) after fungicide-mediated selection is lifted. Two years after the last application of a QoI fungicide, we were able to recover only resistant isolates, with a vast predominance of the G143ACYTB allele. However, the following year, we recovered significant numbers of F129L mutants and wild-type isolates. We interpret this population shift to mean that than the latter isolates are more fit than G143A mutants, at least under the conditions present in our field experiment.

458. Organization of genetic variation and relaxed concerted evolution in individuals of arbuscular mycorrhizal fungi. Teresa E. Pawlowska and John W. Taylor. University of California, Plant & Microbial Biology, Berkeley, CA 94720, USA.

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) form symbioses with 85% of terrestrial plants, including many crop species. These obligate biotrophs facilitate plant mineral nutrition and impart to their host tolerance to pathogens and abiotic stress. Coenocytic mycelium and laden with hundreds of nuclei spores of AM fungi yield no morphological evidence of sexual reproduction. Polymorphism of rDNA arrays observed within individual spores inspired a speculation that diverse genomes distributed among different nuclei are maintained and propagated throughout the AM fungal life history. Using a population genetics approach to study transmission of a protein-coding polymorphic genetic marker in single-spore cultures of Glomus etunicatum, and PCR-amplification of rDNA variants from individually microdissected nuclei, we demonstrated that nuclei populating hyphae and spores of AM fungi are genetically uniform. Intrasporal rDNA polymorphism is contained within individual nuclei and evinces a relaxation of concerted evolution, a recombination-driven process responsible for homogenization of rDNA arrays dispersed within a genome. Polyploid organization of AM fungal genomes, indicated by our data, could ameliorate deleterious mutations that cannot be purged by recombination in these presumably asexual fungi. Our insights into the organization of genetic variation within glomeromycotan individuals provide foundation for the developmental and population genetics of AM fungi.

459. Heterozygosity in Chytrids. Yajuan Liu, Matt Hodson and Benjamin Hall Biology Department, University of Washington, Seattle, Washington

Chytrids (Chytridiomycota) are the only fungi that , at one stage in the life cycle, produce motile reproductive cells with a single and posteriorly directed flagellum. Molecular phylogenetic studies based on rDNA, RPB1 and RPB2 sequences show that chytrids are the basal lineage of fungi. In our studies of the RPB1 and RPB2 genes from representatives of four fungal phyla, extremely divergent haplotypes of RPB1 and RPB2 are found only in chytrids. Exceptions to this are Coelomomyces stegomyiae and Monoblepharis macraudra where in each case only one RPB1 and one RPB2 sequence has been found. Each of these organisms is known to have a sexual stage and meiosis in its life cycle. In contrast, two highly divergent haplotypes of RPB1 and RPB2 have been found in Neocallimastix frontallis and Chytridium confervae, organisms with neither a sexual stage nor meiosis and in Chytriomyces sp. 345D, for which zygotes have been observed but meiosis is not confirmed. For example, the RPB2 haplotypes have 0.2% amino acid and 2.5% nucleotide divergence in Chytridium and are 3.8% amino acid and 5.5% nucleotide divergence in Neocallimastix. This findings suggest that, free from the homogenizing influence of meiotic recombination and allelic gene conversion because they have existed for millions of years without sexual reproduction, in these latter chytrids formerly allelic pairs of genes have diverged extensively, as a result of random independent mutations.

460. Genetic differentiation and phylogeographic structure of Fusarium graminearum in Canada. P.K. Mishra1, J.P. Tewari1, R.M. Clear2, and T.K. Turkington3. 1Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5; 2Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, MB, R3C 3G8; and 3Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB, T4L 1W1, Canada.

The fungus Fusarium graminearum is a serious pathogen of a range of crop plants in many parts of the world inflicting significant losses. It is also known to produce estrogenic and carcinogenic mycotoxins, which pose an acute risk to human and animal health. We have analyzed 341 isolates of F. graminearum collected from different provinces of Canada over many years using restriction digestion of polymerase chain reaction amplified nuclear ribosomal DNA intergenic spacer region (IGS) and inter simple sequence repeats (ISSR) analysis. A substantial genetic diversity was found within the populations. This study will allow deducing various dispersal mechanisms of F. graminearum in Canada.