Nov 13: Vividly colored fish dwelling at the bottom of shallow Midwestern streams may help biologists understand evolutionary mechanisms by which species remain distinct despite similarities and crossed paths. Orangethroat and rainbow darters regularly interbreed where their ranges overlap, yet — millions of years after they first diverged — they remain separate species.

“A big focus of our lab is speciation: the fundamental evolutionary process by which one lineage splits into two distinct species,” said Rachel Moran, PhD, Assistant Professor of Ecology and Evolution at the University of Chicago. “Observing these species that hybridize in the wild was puzzling. How can you have two different species that persistently coexist if they're interbreeding?”

A new study published in Nature Communications helps explain that puzzle. By sequencing whole genomes from multiple darter species, UChicago researchers found that closely related darters use entirely different chromosomes to determine whether offspring develop as male or female. These rapid “sex chromosome turnovers,” combined with subtle conflicts between the genes housed in the cell nucleus and those in the mitochondria, appear to create a genetic barrier that keeps hybridizing species from combining into one. The findings offer a glimpse into a mechanism of speciation that evolutionary biologists have long speculated about but rarely documented.

“Speciation is very important for generating biological diversity,” Moran said. “Understanding its mechanisms also helps us infer how we can maintain that diversity, which is important in a changing world.”

Unexpected hybridization results

The project grew out of an earlier observation in Moran's lab: when orangethroat (Etheostoma spectabile) and rainbow (E. caeruleum) darters were crossed in the lab, their second-generation hybrid offspring were overwhelmingly male. Purebred crosses, by contrast, produced the expected roughly 50-50 sex ratio.

Crucially, hybrid clutches were not smaller. The “missing” females weren't dying as embryos.

“It seemed like genetically female individuals were phenotypically male in these hybrid crosses,” said Moran, the study’s senior author. “So, we thought there must be something happening with their sex-determining regions.”

Different chromosomes, same function

To find out, first author Wynne Radcliffe, a graduate student in Moran's lab, ran genome-wide association studies comparing males and females of multiple darter species. 

“Fish are weird,” Radcliffe explained. “At one point on the ancient tree of life, there was a second whole-genome duplication in bony fishes, so their genomes are already very crazy and full of repeats. They have a lot of different sex-determining systems.” 

Even knowing that fish genomes harbor many quirks, the results of Radcliffe’s analyses were striking. All three species use an XY system, similar to humans, in which males carry one copy each of two different sex chromosomes. But which chromosome plays the role of "X" and "Y" has shuffled repeatedly across the darter family tree. In orangethroat darters, the genetic signal for sex appeared on chromosome 9, but in rainbow darters, it was on chromosome 23. A third hybridizing species, E. radiosum, used chromosome 22. Such rapid turnover has rarely been documented across a large, closely related group of fish, particularly one that hybridizes as freely as darters do.

The team's analyses suggest these turnovers happened while the species were geographically isolated, before they came back into contact in overlapping streams. By the time the fish met again, their sex-determining machinery had already diverged enough that hybrids suffered — a built-in block to prevent the two species merging back into one.

When mitochondria and the nucleus disagree

The contents of new sex chromosomes held intrigue of their own. The regions most resistant to gene flow between species are peppered with nuclear genes whose protein products physically interact with proteins encoded by the mitochondrial genome — the cell's energy-producing machinery, which is inherited only from the mother.

When hybrids inherit a mitochondrial genome from one species and mismatched nuclear partners from the other, those molecular machines may not fit together properly, potentially disrupting cellular metabolism.

“Scientists have thought about mitonuclear incompatibilities and sex-linked incompatibilities as important separately, but we've very rarely considered how they may interact,” Moran said. “But because we found a lot of these mitochondrial-associated nuclear genes near sex-determining regions that have low recombination, they can't co-evolve with the mitochondrial genome as easily. That could favor the sex chromosome switching over to a different spot to restore recombination.”

In other words, sex chromosome turnover may act as an evolutionary “escape hatch,” relieving genetic conflicts between the nucleus and mitochondria — and, as a side effect, generating incompatibilities that keep species apart.

Drawing big implications from observing small fish

Darters are one of the most diverse vertebrate groups in North America, but they remain understudied at the genomic level. As part of her doctoral dissertation work, Radcliffe plans to map sex chromosomes across many more species in the group and hopes to eventually use CRISPR to pinpoint the exact genes flipping the sex switch.

"Understanding how species evolve and diverge is really important when we're thinking about how we want to maintain species diversification and prevent loss of species," she said.

“Support for a role of sex chromosome turnover and mitonuclear conflict in reproductive isolation in darters” was published in Nature Communications in October 2026. Co-authors are Wynne Radcliffe, Mysia Dye, Daemin Kim, Pavitra Muralidhar and Rachel Moran.