If you want to discover a hidden world of new life-forms, you don’t have to scour dark caves or slog through remote rainforests. Just look under your feet. When then-graduate student Anna Rosling went to northern Sweden to map the distribution of a particular root-loving fungus, she found something much more intriguing: Many of her root samples contained traces of DNA from unknown species. Weirder still, she never encountered a complete organism. When the field season ended, she had only isolated bits of raw genetic material. The fragments clearly belonged to the fungal kingdom, but they revealed little else. “I got obsessed,” recalls Rosling, now a professor of evolutionary biology at Uppsala University in Sweden.
Since then mycologists have realized that such phantoms are everywhere. Point to a patch of dirt, a body of water, even the air you’re breathing, and odds are that it is teeming with mushrooms, molds and yeasts (or their spores) that no one has ever seen. In ocean trenches, Tibetan glaciers and all habitats between, researchers are routinely detecting DNA from obscure fungi. By sequencing the snippets, they can tell they’re dealing with new species, thousands of them, that are genetically distinct from any known to science. They just can’t match that DNA to tangible organisms growing out in the world.
These slippery beings are so widespread that scientists are calling them “dark fungi.” It’s a comparison to the equally elusive dark matter and dark energy that make up 95 percent of our universe and exert tremendous influence on, well, everything. Like those invisible entities, dark fungi are hidden movers and shakers. Scientists are convinced they perform the same vital functions as known fungi, directing the flow of energy through ecosystems as they break down organic matter and recycle nutrients. Dark fungi are prime examples of what biologist E. O. Wilson called “the little things that run the world.” But their cryptic lifestyle has made it a maddening challenge for scientists trying to show how exactly they run it.
Taxonomists have described just 150,000 of the millions of fungi predicted by global biodiversity estimates, and recent discoveries suggest a huge portion of what’s left may be off-limits to routine biological investigation. “We have not even started to scratch the surface,” says Henrik Nilsson, a mycologist at the University of Gothenburg in Sweden. “I’d be willing to bet that the clear majority will be dark.” Given the central place of fungi in the web of life that sustains us, experts argue we should get a better grasp on them.
Everything we know about dark fungi comes from environmental DNA, or eDNA. That term refers to strings of base pairs—the building blocks of DNA that are constantly sloughing off all living things. Researchers can analyze these free-floating bits of double helix to determine which species have been hanging around an area without seeing them. To identify fungi specifically, scientists look to a handy genetic marker called the internal transcribed spacer (ITS), which consists of several hundred base pairs that evolve quickly and thus help distinguish between species. Although the ITS is only a tiny fraction of the genome, researchers can single it out and amplify it with the same polymerase chain reaction technology used in COVID lab tests. If an ITS sequence is different enough from all others in genetic databases, it is thought to represent a new species, whether scientists lay eyes on its physical form or not.
At the turn of the millennium, eDNA sequencing burst onto the scene as a new way to discover species. Scientists suddenly found themselves awash in a “flood of data,” as David Hibbett, a mycologist at Clark University, and his colleagues wrote in 2009. That influx exposed the sheer vastness of dark fungi. Today, Hibbett says, “our understanding of the richness of fungal diversity is really being enlarged with these dark organisms.”
Every year researchers stumble on some 2,000 new fungi via the standard route, spotting them in nature or under a microscope. Yet a single eDNA study can register 10 times more dark fungi than that. As often as not, the fragments are among the most abundant DNA samples in their ecosystem. “I don’t think I ever saw an environmental sequencing study with less than 30 percent unknowns,” Nilsson says, and the ratio is typically much higher. Sometimes only a minority of DNA sequences can be classified at any meaningful taxonomic level, narrowing them from a kingdom (in this case, fungi) to a phylum and then to a class, and so on down to a species.
Hellbound and Down 
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