ABOVE: Trichaptum abietinum growing on a pine log Dabao Sun Lu

Some common mushrooms likely have more than 17,000 sexes, researchers report March 31 in PLOS Genetics. The work could help us better understand the evolution of sexual reproduction as well as showcases the increasing power of genome sequencing.

While scientists have long suspected that certain species of fungi have thousands or even tens of thousands of biological sexes, the new research employed cutting edge genetic tools to confirm the extreme diversity of sexes in Trichaptum mushrooms.

“The pace of advances in DNA sequencing are just mind boggling,” says Joseph Heitman, a geneticist and infectious disease expert at Duke University who wasn’t involved in the study but has investigated fungal mating strategies before. “This kind of study would have been cost and time prohibitive even five years ago.”

Petri dish with white fungal growths on each half
Two compatible strains of Trichaptum fuscoviolaceum mating on a petri dish
Vilde Bruhn Kinneberg

Trichaptum is a group of woody, plate-like mushrooms (shelf fungi) that commonly grow on trees and fallen logs in cooler parts of the Northern Hemisphere. “I think they’re quite pretty, but not very showy,” says Inger Skrede, a fungal biologist at the University of Oslo and a coauthor of the paper. The fungi weren’t chosen for their looks, of course. The international team of researchers decided to examine species in this genus because they had been studied by scientists previously and were easy to grow in the lab.

In total, the scientists obtained 180 specimens from three Trichaptum species; some they collected themselves, while others were sent to them from colleagues around the world (“You can just ship it in regular mail,” says Skrede). From each fungal strain, they harvested spores, which were grown individually on agar plates for several weeks so that their DNA could be analyzed and they could be paired with growing spores from other specimens to see if they’d crossbreed.

Previous work had suggested that sex in these mushrooms was controlled by two regions of the genome, known as MATA and MATB, and that each of these regions had many different possible alleles. For potential mates to be compatible, both regions must be different from their prospective partner’s.

This diversity has hampered sequencing efforts. The many divergent alleles make primer design all but impossible, thwarting the use of less expensive, targeted sequencing methods. This hurdle means that researchers wanting to sequence these fungi would need to rely on so-called next generation, short-read genomic sequencing technologies—methods which, given the number of individuals and the depth of sequencing needed to ensure accuracy, have simply been too expensive.

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Now, though, lower costs of next generation technologies made sequencing so many individuals feasible.  And Skrede and her team were able to use newer, long-read sequencing to generate high-quality genomes that acted as scaffolds for accurate assemblies of the short reads. The combination of methods made it so the researchers could precisely pinpoint which parts of the MATA and MATB genetic regions were important for sex determination as well as count the number of different relevant variations in those two areas. Putting it all together, the team found that, contained within these unassuming shelf fungi, there could be a staggering 17,550 different combinations to choose from.

Microscopic image of joined hyphae
Mating in Trichaptum occurs where the hyphae of two growing spores meet, as seen in this microscopic image of T. abietinum. The small bumps that be seen on the side of some hyphae are known as clamps, which only occur after successful mating.
Dabao Sun Lu

Why any organism would need so much sexual variation remains an open question, but study author and University of Oslo geneticist David Peris suspects it has to do with the mushrooms’ sessile lifestyle: having to be different at two different gene regions makes it less likely for spores released from the same mushroom to successfully combine, thus lowering the odds of inbreeding.

Also, having so many variants to choose from makes it more likely any given neighbors will be sexually compatible—up to a 98 percent chance, says Peris—which could help the species survive in the long run. “When conditions are changing, you want to generate this genetic variation,” says Peris, as it can act as fodder for adaptation.

Duur Aanen, an evolutionary biologist at Wageningen University in the Netherlands, says the study is such a good example of balancing selection—a phenomenon where the evolutionary favoring of rare alleles leads to higher levels of heterozygosity—that he is considering using it in his population genetics classes.

However, Aanen, who was not involved in the study, also noted that there’s one big question none of the scientists who’ve looked at this system have been able to fully answer: While diversity may help, why push it to this extreme?  “If you have 100 alleles, you already have quite a lot of compatibility,” says Aanen. “What is the benefit of number 101?”