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Title: Independent origins of diploidy in the entomopathogen Metarhizium

item Kepler, Ryan
item CHEN, YUAN - Duke University
item KILCREASE, JAMES - Orise Fellow
item Shao, Jonathan
item Rehner, Stephen

Submitted to: Mycologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/7/2016
Publication Date: 1/30/2017
Citation: Kepler, R.M., Chen, Y., Kilcrease, J., Shao, J.Y., Rehner, S.A. 2017. Independent origins of diploidy in the entomopathogen Metarhizium. Mycologia. 108(6):1091-1103.

Interpretive Summary: Insect pests annually cause billions of dollars of agricultural losses. The use of beneficial fungi to control pest insects is needed to reduce reliance on chemical pesticides that are expensive and damaging to the environment and human health. In this study we used molecular methods (i.e., DNA) for determining the relationships and differences within a group of 90 fungal isolates parasitic on pest beetles. Using DNA sequencing, DNA fragment analysis and microscopy, we demonstrate that what was thought to be three species is actually at least nine species. In addition we show that the genomes of two species are twice the size of others due to hybridization between closely related individuals. The results are significant because the genome doubling that has occurred in these species provide new opportunities for investigating how these fungi cause disease in insects. This information will be used by insect mycologists and plant pathologists discovering and implementing integrated pest management strategies of field crop pests.

Technical Abstract: Understanding of ploidal variation in fungi lags behind that for plants and animals because cytogenetic tools are often unable to accurately resolve and size the typically small genomes of fungi by traditional optical methods. Variation in ploidal status is frequently associated with changes in phenotype, and sudden changes in chromosome number may promote rapid speciation and alter adaptive potential. With the advent of whole genome sequences for a broad diversity of fungal species it is now possible to develop taxon-specific genotyping tools that enable comprehensive assessment of ploidy across previously uncharacterized genomes. Here we present evidence based on nuclear DNA sequence, microsatellite polymorphisms and the configuration of the mating type locus to demonstrate independent origins of diploidy in Metarhizium majus and M. guizhouense, a sister species pair of soil-borne entomopathogens. A multilocus phylogeny revealed that each species lineage is phylogenetically complex and contains two or more distinct subclades. The majority of isolates in both species possess only a single sequence type or microsatellite allele at the 16 loci queried, indicating most individuals are haploid. However, the multilocus phylogeny also revealed single subclades within each species lineage whose members display more than one allele at multiple loci. After establishing single spore isolations to rule out mixed or heterokaryotic cultures, these same individuals displayed the same pattern of polymorphism as the parental cultures from which they originated, a finding consistent with a stable diploid genome structure. A PCR assay for diagnosis of mating type showed that haploid individuals were of a single mating type whereas presumed diploid isolates contained both MAT1-1 and MAT1-2 idiomorphs. Sequences spanning the intergenic spacer between APN2, which flanks the MAT locus, and the adjacent MAT1-1-3 and MAT1-2-1 idiomorphs differed in sequence, demonstrating that the different MAT loci in diploid strains are distinct from one another. Taken together, these data suggest that both diploid lineages are autopolyploids that likely arose by crosses between two closely related individuals of opposite mating type. The effect of this unique genomic arrangement on sexual reproduction remains to be determined.