Biologically speaking, nearly every species on Earth has two opposite sexes, male and female. But with some fungi and other microbes, sex can be a lot more complicated. Some members of Cryptococcus, a family of fungus linked to human disease, can have tens of thousands of different mating types.

In a study appearing early online Aug. 11 in PLOS Biology, Duke researchers have mapped the evolutionary turning point that transformed the pathogenic form of Cryptococcus from an organism of many sexes to one with only two. They found that during evolution, a reshuffling of DNA known as translocation brought together separate chunks of sex-determining genes onto a single chromosome, essentially mimicking the human X or Y chromosome.

Surprisingly, they've shown that these crucial translocations occurred at the centromeres, the twisty ties that hold together chromosomes at the center of an x-shaped pair. These regions of the chromosome are so dense that they were once thought to be removed from recombination.

"Recombination at the centromere doesn't have to happen frequently, it just has to happen often enough that it punctuates the evolution of the organism," said Joseph Heitman, MD, PhD, senior study author and professor and chair of molecular genetics and microbiology at Duke University School of Medicine. "With each translocation, the genome is altered again and again, until you have evolved an entirely new species."

Scientists have been studying the evolution of sex chromosomes for more than a century. In the 1960's, Japanese-American geneticist and evolutionary biologist Susumu Ohno proposed a theory in which the genes determining sex first arose at various spots scattered across the entire genome, but over time were "captured" on the sex chromosomes. In humans, those chromosomes go by the familiar X and Y; in birds, they are known as Z and W; in moss, they are called U and V.

Regardless of the name or species, Heitman contends that some universal principles could govern the evolution of all sex chromosomes. He and an international team of researchers focused on the last common ancestor of the human pathogen Cryptococcus neoformans and its nearest sibling species, a non-pathogen called Cryptococcus amylolentus.

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In C. amylolentus, dozens of genes at two different locations on the chromosomes control what's called a tetrapolar, or four-part, mating system. At one location or locus known as P/R, genes encode pheromones and pheromone receptors that help the fungus recognize compatible mating types. At the other locus, called HD, genes govern the development of sexual structures and reproductive spores.

The researchers sequenced the entire genome of C. amylolentus, mapping the location of all the genes as well as the centromeres on each of the organism's 14 chromosomes.

They found that the genomes had undergone quite a bit of rearrangement since the two species shared a common ancestor, at least 50 million years ago. For example, chromosome 1 of C. neoformans contained pieces of four different chromosomes from C. amylolentus, providing evidence of multiple translocations, some within the centromere.

"That was very surprising. The dogma has been that recombination is repressed in centromeric regions," said Sheng Sun, PhD, lead study author and assistant research professor at Duke University School of Medicine.

In the 1980's, a seminal paper by Duke colleague Tom Petes demonstrated recombination could occur across the centromeres in Saccharomyces cerevisiae, but some attributed the finding to a quirk of the favored model organism with its tiny point centromeres. But since then, other studies have emerged suggesting that the phenomenon was wider spread.

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In this study, the researchers showed that in Cryptococcus amylolentus, the ancestral state, the P/R locus resided on chromosome 10 and the HD locus on chromosome 11. But in Cryptococcus neoformans, the evolved state, those loci ended up in one place. According to their model, multiple translocations deposited the two sex determinants on the same chromosome, with a centromere in between. Subsequent rearrangements put P/R and HD next to each other. The result was an organism with a bipolar mating system, much like the male and female sexes that embody most species.

"In any kind of model like this, you are thinking about what could have been the organization in the last common ancestor, which is now extinct so you can't know definitively," said Heitman. "But in each of these lineages, there are multiple evolutionary events that have occurred, and you can use genomics to turn back the hands of time and deduce the trajectory."

Heitman says their study suggests that other researchers should actively look for translocations, both in the expected locations as well as within centromeres. These chromosomal rearrangements are a common cause of birth defects and cancer in humans.

He and his colleagues are currently investigating whether similar translocations occur in the evolution of sex chromosomes in other fungal families, such as Ustilago and Malassezia.

The study was an international collaborative effort, involving key contributions from Vikas Yadav and Kaustuv Sanyal at the Jawarharlal Nehru Center for Advanced Scientific Research in Bangalore, India; Christina Cuomo at the Broad Institute in Cambridge, Massachusetts; Minou Nowrousian at Ruhr University in Bochum, Germany; Teun Boekhout at CBS in the Netherlands; and Jean-Luc Souciet, Betina Porcel and Patrick Wincker at Genoscope in France.

The research was funded by the National Institutes of Health/National Institute of Allergy and Infectious Diseases (R37 MERIT award AI39115-20 and R01 grant AI50113-13), National Human Genome Research Institute (U54HG003067) and the German Research Foundation (NO407/7-1).