Population genomics reveals historical and ongoing recombination in the Fusarium oxysporum species complex.

The Fusarium oxysporum species complex (FOSC) is a group of closely related plant pathogens long-considered strictly clonal, as sexual stages have never been recorded. Several studies have questioned whether recombination occurs in FOSC, and if it occurs its nature and frequency are unknown. We analysed 410 assembled genomes to answer whether FOSC diversified by occasional sexual reproduction interspersed with numerous cycles of asexual reproduction akin to a model of predominant clonal evolution (PCE). We tested the hypothesis that sexual reproduction occurred in the evolutionary history of FOSC by examining the distribution of idiomorphs at the mating locus, phylogenetic conflict and independent measures of recombination from genome-wide SNPs and genes. A phylogenomic dataset of 40 single copy orthologs was used to define structure a priori within FOSC based on genealogical concordance. Recombination within FOSC was tested using the pairwise homoplasy index and divergence ages were estimated by molecular dating. We called SNPs from assembled genomes using a k-mer approach and tested for significant linkage disequilibrium as an indication of PCE. We clone-corrected and tested whether SNPs were randomly associated as an indication of recombination. Our analyses provide evidence for sexual or parasexual reproduction within, but not between, clades of FOSC that diversified from a most recent common ancestor about 500 000 years ago. There was no evidence of substructure based on geography or host that might indicate how clades diversified. Competing evolutionary hypotheses for FOSC are discussed in the context of our results.

Survey of Early-Diverging Lineages of Fungi Reveals Abundant and Diverse Mycoviruses.

Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions.IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

Gene expression varies within and between enzootic and epizootic lineages of Batrachochytrium dendrobatidis (Bd) in the Americas.

While much research focus is paid to hypervirulent fungal lineages during emerging infectious disease outbreaks, examining enzootic pathogen isolates can be equally fruitful in delineating infection dynamics and determining pathogenesis. The fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd), exhibits markedly different patterns of disease in natural populations, where it has caused massive amphibian declines in some regions, yet persists enzootically in others. Here we compare in vitro gene expression profiles of a panel of Bd isolates representing both the enzootic Bd-Brazil lineage, and the more recently diverged, panzootic lineage, Bd-GPL. We document significantly different lineage-specific and intralineage gene expression patterns, with Bd-Brazil upregulating genes with aspartic-type peptidase activity, and Bd-GPL upregulating CBM18 chitin-binding genes, among others. We also find pronounced intralineage variation in membrane integrity and transmembrane transport ability within our Bd-GPL isolates. Finally, we highlight unexpectedly divergent expression profiles in sympatric panzootic isolates, underscoring microgeographic functional variation in a largely clonal lineage. This variation in gene expression likely plays an important role in the relative pathogenesis and host range of Bd-Brazil and Bd-GPL isolates. Together, our results demonstrate that functional genomics approaches can provide information relevant to studies of virulence evolution within the Bd clade.

Hybrids of amphibian chytrid show high virulence in native hosts.

Hybridization of parasites can generate new genotypes with high virulence. The fungal amphibian parasite Batrachochytrium dendrobatidis (Bd) hybridizes in Brazil's Atlantic Forest, a biodiversity hotspot where amphibian declines have been linked to Bd, but the virulence of hybrid genotypes in native hosts has never been tested. We compared the virulence (measured as host mortality and infection burden) of hybrid Bd genotypes to the parental lineages, the putatively hypovirulent lineage Bd-Brazil and the hypervirulent Global Pandemic Lineage (Bd-GPL), in a panel of native Brazilian hosts. In Brachycephalus ephippium, the hybrid exceeded the virulence (host mortality) of both parents, suggesting that novelty arising from hybridization of Bd is a conservation concern. In Ischnocnema parva, host mortality in the hybrid treatment was intermediate between the parent treatments, suggesting that this species is more vulnerable to the aggressive phenotypes associated with Bd-GPL. Dendropsophus minutus showed low overall mortality, but infection burdens were higher in frogs treated with hybrid and Bd-GPL genotypes than with Bd-Brazil genotypes. Our experiment suggests that Bd hybrids have the potential to increase disease risk in native hosts. Continued surveillance is needed to track potential spread of hybrid genotypes and detect future genomic shifts in this dynamic disease system.

Amphibian-killing chytrid in Brazil comprises both locally endemic and globally expanding populations.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is the emerging infectious disease implicated in recent population declines and extinctions of amphibian species worldwide. Bd strains from regions of disease-associated amphibian decline to date have all belonged to a single, hypervirulent clonal genotype (Bd-GPL). However, earlier studies in the Atlantic Forest of southeastern Brazil detected a novel, putatively enzootic lineage (Bd-Brazil), and indicated hybridization between Bd-GPL and Bd-Brazil. Here, we characterize the spatial distribution and population history of these sympatric lineages in the Brazilian Atlantic Forest. To investigate the genetic structure of Bd in this region, we collected and genotyped Bd strains along a 2400-km transect of the Atlantic Forest. Bd-Brazil genotypes were restricted to a narrow geographic range in the southern Atlantic Forest, while Bd-GPL strains were widespread and largely geographically unstructured. Bd population genetics in this region support the hypothesis that the recently discovered Brazilian lineage is enzootic in the Atlantic Forest of Brazil and that Bd-GPL is a more recently expanded invasive. We collected additional hybrid isolates that demonstrate the recurrence of hybridization between panzootic and enzootic lineages, thereby confirming the existence of a hybrid zone in the Serra da Graciosa mountain range of Paraná State. Our field observations suggest that Bd-GPL may be more infective towards native Brazilian amphibians, and potentially more effective at dispersing across a fragmented landscape. We also provide further evidence of pathogen translocations mediated by the Brazilian ranaculture industry with implications for regulations and policies on global amphibian trade.

Inbreeding depression in urban environments of the bird's nest fungus Cyathus stercoreus (Nidulariaceae: Basidiomycota).

Many organisms display codispersal of offspring, but fewer display codispersal of compatible gametes. This mechanism enhances the ability of a species to colonize after long distance dispersal as a mechanism of reproductive assurance, but it also fosters inbreeding and potential reduction in fitness. Here we investigated both long distance dispersal and inbreeding in the bird's nest fungus Cyathus stercoreus, a dung and mulch-associated fungus with a splash cup fruiting body appearing like a miniature bird's nest of 'eggs' or peridioles that contain thousands of mating compatible meiotic spores. To investigate the genetic structure in the species, six North American urban populations were hierarchically sampled and genotyped using 10 microsatellite markers. We detected significant levels of inbreeding through heterozygote deficiencies at four loci, with global FIS=0.061. Dispersal limitation was suggested by both spatial autocorrelation and the detection of population structure between Louisiana and Michigan using clustering and F-statistics. Although inbreeding may facilitate colonization by the fungus, it has a negative effect on the fitness of populations as estimated from a 15% reduction in growth rates of inbred strains relative to outcrossed. Mating tests revealed that C. stercoreus has a higher estimated number of mating-type alleles (MAT-A= 39, MAT-B= 24) than other species of bird's nest fungi, which would increase its outcrossing efficiency. We speculate that the increased number of mating-type alleles is the result of a recent range and population size expansion into urban environments.

First Report of False Rust Caused by Synchytrium minutum on Kudzu in Korea.

Kudzu (Pueraria montana var. lobata (Willd.) Maesen & S. Almeida) is a weedy, fabaceous vine that is native to and widely distributed in Asia where it is used for various medicinal purposes such as treating convulsions and fever (2). In the United States, especially the southeastern states, kudzu has become a problematic invasive species that overgrows nearly every substrate on which it occurs. Thus, biological control strategies for controlling this vine are of great interest (4). From October to November 2004, a disease of kudzu was observed in Gwangju and Pyeongtaek in Gyeonggi Province, Korea. The disease appeared on leaves and stems as numerous, discrete, small galls, which enlarged, becoming yellowish orange and eventually erupting into orange, pulverulent sori. Galls were scattered or gregarious, amphigenous, predominately hypophyllous, and sometimes formed along veins as well as on petioles and stems. Sori that formed from galls were solitary but sometimes became confluent, 0.1 to 1 mm in diameter, globose to subglobose, and orange to dark orange; walls were hyaline and thin. Sporangia were copious in sori, typically polyhedral due to compression or globose, 16 to 32 µm in diameter, with smooth, hyaline walls and orange contents. Zoospores were not observed during several failed attempts to germinate sporangia. On the basis of morphological descriptions and keys (3), the fungus was identified as Synchytrium minutum (Pat.) Gäum. (Chytridiomycota), the only species of Synchytrium known to occur on Pueraria (1,3). Comparison with specimens from China and New Guinea (BPI 794733 and BPI 1109528) confirmed this identification. Portions of the nLSU and nSSU rDNA from one of the two Korean specimens deposited as voucher material in the U.S. National Fungus Collections (BPI 880898 and BPI 880899) were sequenced (GenBank Accession Nos. HQ324138 and HQ324139), and a subsequent BLAST search against GenBank confirmed placement in the genus Synchytrium with 95% similarity to S. decipiens. S. minutum is widespread in Asia and Oceania and also has been reported from California (1,3). To our knowledge, this is the first report of S. minutum in Korea (1) and is noteworthy to those interested in biological control of kudzu because S. minutum may have potential in this regard. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , September, 2010. (2) H. S. Jung. M.S. thesis. Seoul National University, Seoul, Korea, 1997. (3) J. S. Karling. Synchytrium. Academic Press Inc., New York, NY, 1964. (4) M. A. Weaver et al. Biol. Control 50:150, 2009.

Controlling two-dimensional movement of microparticles over an electrode array surface.

This paper presents an original microchip device that manipulates planar x- and y-positions of dielectric microbeads over an electrode array with dielectrophoresis (DEP) effects. Implemented with a simple single-layer metal process, the microchip device consists of two parallel arrays of triangular-shaped electrodes. The two arrays of electrodes are arranged such that they locked into each other to form interdigitated electrodes. The unique geometry and placement of the electrodes can produce different configurations of DEP waveforms. The different DEP waveforms will, in turn, allow the strength and the locations of the electric field maxima and minima to be manipulated. Our experiments showed that with a quad-pole traveling wave, the microbeads can be moved across the electrode array surface in directions parallel to the fluid flow to establish their x-positions via traveling wave dielectrophoresis (TWD) effect. In addition, the microbeads can be moved in directions perpendicular to the fluid flow to establish their y- positions via activating one or both sets of electrode arrays with DEP waveforms. A line of microbeads can be held indefinitely at the predetermined location or be immediately moved to an arbitrary location on the electrode array. With a functional transportation of particles along two planar axes, the "teeth-like" electrode structure can be easily integrated into a microfluidic system where an accurate position control of particles is required.

The chromosomal region containing pab-1, mip, and the A mating type locus of the secondarily homothallic homobasidiomycete Coprinus bilanatus.

In this paper we describe the cloning of the DNA region containing the A1 mating type genes of the secondarily homothallic mushroom Coprinus bilanatus and compare its organization to that of heterothallic homobasidiomycetes. As in other species, the C. bilanatus A factor contains several different genes that encode two different types of homeodomain transcription factor (HD1 and HD2); and some of these genes are active in the heterologous host C. cinereus. The HD1 and HD2 genes are distributed over two closely linked subloci, Aalpha and Abeta. A gene coding for a mitochondrial intermediate peptidase (mip) directly flanks the Aalpha sublocus. The pab-1 gene, required for para-aminobenzoic acid synthesis, is found 39 kb upstream of mip. The structural arrangement of this chromosomal region closely resembles the heterothallic C. cinereus. In contrast, the Aalpha and Abeta subloci of Schizophyllum commune are further separated, with pab-1 located between the two subloci, suggesting that a translocation event may have occurred during evolution.

Abundance and diversity of Schizophyllum commune spore clouds in the Caribbean detected by selective sampling.

Selective spore trapping and molecular genotyping methods were employed to examine potential long-distance gene flow among Caribbean populations of the common mushroom Schizophyllum commune. Spore-trap samples from five locations were analysed using restriction fragment polymorphisms of five enzymatically amplified gene regions. Successful trappings suggested S. commune spores to be abundant in the air, with an estimated sedimentation rate of approximately 18 spores/m2/h. High levels of genetic diversity characterized the spore-trap samples, with as many as 12 alleles observed at a single locus (chitin synthase) over all samples. In addition, spore-trap samples showed significant among sample heterogeneity including geographical population substructure. The ribosomal DNA (rDNA) intergenic spacer displayed the greatest allele frequency differences among samples, clearly separating the samples into those possessing only a South American-type allele and those segregating for both North and South American-type alleles. The molecular variation provided no clear evidence for dispersal over large, aquatic barriers within the Caribbean region, and instead suggested that spore-trapping experiments are primarily reflective of the local, established population.

Polymorphism at the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune.

The common split-gilled mushroom Schizophyllum commune is found throughout the world on woody substrates. This study addresses the dispersal and population structure of this fungal species by studying the phylogeny and evolutionary dynamics of ribosomal DNA (rDNA) spacer regions. Extensive sampling (n = 195) of sequences of the intergenic spacer region (IGS1) revealed a large number of unique haplotypes (n = 143). The phylogeny of these IGS1 sequences revealed strong geographic patterns and supported three evolutionarily distinct lineages within the global population. The same three geographic lineages were found in phylogenetic analysis of both other rDNA spacer regions (IGS2 and ITS). However, nested clade analysis of the IGS1 phylogeny suggested the population structure of S. commune has undergone recent changes, such as a long distance colonization of western North America from Europe as well as a recent range expansion in the Caribbean. Among all spacer regions, variation in length and nucleotide sequence was observed between but not within the tandem rDNA repeats (arrays). This pattern is consistent with strong within-array and weak among-array homogenizing forces. We present evidence for the suppression of recombination between rDNA arrays on homologous chromosomes that may account for this pattern of concerted evolution.