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.
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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.
Finding a full specimen can be difficult. Many fungi are microscopic, even unicellular, and a tiny sample of biological material may contain hundreds that are hard to isolate. Other species grow as visible mushrooms and rootlike mycelia, but they’re ephemeral and therefore easy to miss. As for raising the organisms in a lab culture, it can be arduous to provide the right conditions. A team of Czech microbiologists recently claimed that “in theory, nothing is impossible to culture,” but mycology is strapped for cash and personnel, so as a practical matter, our insight into dark fungi usually ends at DNA snippets.
What we do know isn’t trivial, however. As Rosling put it, “In the environmental DNA sequence, there’s so much more information than just the base pairs.” By looking for similarities with known species, mycologists can pinpoint the closest relatives of a dark fungus, and from that they can often infer a lot about its life cycle and ecological role. Still, there’s a limit to what someone can learn without a complete specimen, especially when there are no particularly close relatives. “If it’s something out in left field,” Hibbett says, “that’s very mysterious.”
Take the group of fungi whose DNA Rosling unearthed in grad school. Years later she was stunned to find one of them thriving in a long-forgotten culture from 1999. It turned out to be the first known member of a class called Archaeorhizomycetes, comprising hundreds of dark species that live in soils around the world. For reference, mammals are a class. “There are groups at that scale that we don’t know,” Rosling says with wonder.
In 2011 a British microbiologist named Meredith Jones discovered a possible new phylum, aptly named Cryptomycota, which was previously dark. (For reference, the class of mammals is a subgroup of the phylum Chordata.) Such discoveries are more than minor revisions to the tree of life. Besides adding an enormous branch on the fungal limb, Cryptomycota was a bombshell because it lacked the fibrous substance chitin, once considered a defining characteristic of all fungi. And since Rosling’s team made its discovery, Archaeorhizomycetes have been deemed potential keystone species. If they weren’t around—forming symbiotic relationships with plants, decomposing organic molecules into carbon and nitrogen that other organisms can utilize—whole ecosystems could collapse. Clearly we can’t read the full story in DNA, but it’s a starting point. Until researchers track down or culture dark fungi, they exist for us mainly as a great constellation of ITS sequences. Mycologists refer to those sequences as “barcodes” because they unambiguously link genetic material to a species. Of the 10 million barcodes in the worldwide DNA UNITE database, many are languishing under the label “unidentified.”
Hoping to inspire interest in these taxonomic orphans, Nilsson made a list of the “top 50 most wanted fungi,” representing the largest unidentified lineages for which there is DNA but no specimens. A couple have been defined, but he doesn’t feel the rest have received enough attention since he put out the warrants in 2016. “Everyone wants to produce papers, and here’s the chance,” he says. “All the data have been assembled for you. Go and have a look, and I’m sure you can find something.” Nilsson means one can do so not only digitally but also physically: UNITE entries include the geographic distribution of DNA sequences, so anyone can hunt for the corresponding species in the real world.
The list doesn’t necessarily cover the most economically or ecologically significant dark fungi, just groups of them that have yet to be classified at even the highest levels of taxonomy, such as phylum and class. Nevertheless, when pressed for possible benefits, Nilsson has no trouble obliging. The unexplored edges of fungal biology could, for example, yield valuable chemicals or medicines (penicillin and the organ-rejection treatment cyclosporine are derived from fungi, to name just a couple). “One or more of these species will prove super important to humans,” he says.
Most crucially, dark fungi are essential to the proper functioning of the biosphere. They’re thoroughly entwined with the rest of nature. To preserve them and all the intricate, life-supporting processes they underlie, researchers have to keep pinning them down one by one. “Capturing that diversity,” Rosling says, “can help us have a more informed perspective on conservation.”
As techniques for handling eDNA advance, mycologists are better positioned than ever to extract the dark secrets. High-throughput sequencing and single-cell genomics—which, as the name implies, can glean nearly an entire genome from a single cell—both offer dramatic improvements over traditional barcode sequencing. Combined with cutting-edge technology for sorting and culturing the legion cells in environmental samples, such methods could give a deeper view into the enigmatic world of dark fungi. “Just imagine all the cool chemistry and biology and ecology that are going on,” Nilsson says. “All these interactions, and we have absolutely no idea about them. That thought gives me energy.”