They can be found on forest floors, in swamps and in houses, ranging in size from smaller than the period on your smartphone’s keyboard to stretching over several city blocks.

More than a million species of fungi are estimated to live on this planet, but most of that diversity remains unknown because the fungi have avoided detection and have not been cultured for study in laboratories.

Now a team led by researchers at the U.S. Department of Energy’s Joint Genome Institute and including University of Michigan mycologist Tim James reports the development of a pipeline to generate genomes from single cells of uncultivated fungi.

The approach was tested on several uncultivated fungal species representing early diverging fungi, the earliest evolutionary branches in the fungal genealogy that provide a repertoire of important and valuable gene products. The team’s findings were published Oct. 8, 2018 in Nature Microbiology.

“Most of the phylogenetic diversity represents early diverging fungi. We know from environmental DNA surveys that they’re common in many habitats, but they’re presumably microscopic so you really have to look for them,” said James, co-senior author of the study and an associate professor in the U-M Department of Ecology and Evolutionary Biology.

“We don’t know what they look like and we know we can’t culture them, since what you can culture is not representative of what you see in environmental DNA. We would love to be able to look at a given sample and identify what the cells might look like, but we also want to look at the genomes of the organisms and infer what they’re like. That’s where single-cell genomics comes in.”

Through projects such as the Joint Genome Institute’s 1,000 Fungal Genomes, researchers aim to expand the known fraction of fungal diversity with representative genome sequences for various lineages. Even with such efforts though, the majority of available genomes belong to just two major lineages, Ascomycota and Basidiomycota. The early-diverging lineages that are closer to the base of the Fungal Tree of Life have few representative genomes.

What the study really highlights, James added, is that the single-cell approach is feasible for what he calls “fungal dark matter.” The fungal single cells yielded anywhere from 6 percent of the genome to 88 percent, but combining the single cells yielded genome co-assemblies ranging from 73 percent complete up to 99 percent complete.

There are around 2,000 described species of early diverging fungi, and about 120,000 described species of the Ascomycota and Basidiomycota, James said. “We’ve described maybe 5 percent of the fungal diversity, and we’re in an era where we can start to get at that missing piece of the diversity,” he said.

The single-cell genomics approach will be applied to a JGI Community Science Program proposal that James is leading and that involves 50 unknown early-diverging fungi from aquatic environments.

“What I’d really like to see is people take up this approach and tweak the pipeline to fit different organismal groups,” he said. “This pilot just started the exploration by looking at unicellular aquatic organisms, and yet we have organisms in soil, in plants, and so on.”

His colleagues include former U-M EEB postdoc Alisha Quandt, now at University of Colorado Boulder.

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Tim James spoke about “Leveraging genetic variation among single cells or individuals to understand the cryptic biology of Fungi” at the JGI 2018 Genomics of Energy & Environment Meeting. Watch his talk above on the JGI YouTube channel.