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A new study suggests the first published chromosome count for the monarch butterfly (right) was actually based on cells from the similar-looking common tiger butterfly (left).

(Left to right): bushton3/iStockphoto; Fabrice-Chanson/iStockphoto

Monarch miscalculation: Has a scientific error about the butterflies persisted for more than 40 years?

A few years ago, Christopher Hamm was reading up on monarch butterflies when he noticed something peculiar. All of the scientific articles that mentioned the number of the insect’s chromosomes—30, it seemed—referenced a 2004 paper, which in turn cited a 1975 paper. But when Hamm, then a postdoc at the University of Kansas in Lawrence, did a genetic analysis of his own, he found that his monarchs only had 28 chromosomes, suggesting that an error has pervaded the literature for more than 40 years. Another twist, however, was just around the corner.

Hamm suspected a mistake when he read the original 1975 paper. The authors, biologists N. Nageswara Rao and A. S. Murty at Andhra University in Visakhapatnam, India, had studied what they claimed was an Indian monarch butterfly in their work. But there’s a problem: Monarchs are nearly exclusively a North American species. “It’s implied they just went outside their building and collected some butterflies,” Hamm says. “I immediately thought, ‘Monarch butterflies in India? Really?’”

Sure monarchs are master travelers, with the longest-known seasonal migration of any insect. And it’s not uncommon for a few to get blown off course to Australia, the Philippines, the United Kingdom, and a handful of other places from time to time. But ending up as far away as India seemed like a stretch. Hamm, now a data scientist at Monsanto in Woodland, California, also knew that taxonomists since Carl Linnaeus have struggled to distinguish species in Lepidoptera, the order of insects to which monarchs belong. For example, the monarch (Danaus plexippus) and a similar-looking butterfly known as the common tiger butterfly (D. genutia) were thought to be the same for more than a century until they were reclassified as separate species in 1954. And guess what: D. genutia lives in India.

Hamm thinks that Rao and Murty, perhaps not knowing about the reclassification, netted bugs they assumed were monarchs but were actually common tiger butterflies. Back in the lab, they performed a technique known as a chromosome squash—squeezing the butterflies’ cells between thin films of glass until individual chromosomes are visible under a microscope—counted to 30, and published the results. Then, in 2004, Brazilian zoologist Keith Brown Jr. cited the work in his own research exploring the evolutionary history of butterflies; he never suspected that Rao and Murty might have been working with a misidentified species. Brown’s paper has been cited a dozen times since, and the idea that monarchs have 30 chromosomes is now well established in the literature.

Murty has since died—though his name lives on in a namesake flatworm, Pseudodiplodiscoides murtyi—and Rao could not be located to confirm the theory. Still, it’s a plausible explanation, says Krushnamegh Kunte, a biologist at the National Centre for Biological Sciences in Bengaluru, India, who studies butterfly genetics. “Unfortunately, history has a strong influence in taxonomy,” he says. “Many Indian taxonomists continued to erroneously refer to the Indian populations of Danaus genutia as Danaus plexippus.”

Hamm performed his own chromosome squash with six juvenile monarchs—real ones given to him by Kansas-based Monarch Watch, a network of scientists, teachers, and volunteers that supports research on the butterfly. Earlier this month, he reported his count of 28 chromosomes on the bioRxiv preprint server, an online repository where scientists publish work before it has been peer reviewed.

A zoomed-in view of a monarch butterfly's chromosomes.

Christopher Hamm

Case closed, right? Not quite. A paper published a few days later on bioRxiv by some of Hamm’s former colleagues at the University of Kansas claims to have found, like Rao and Murty, 30 chromosomes in monarchs. “Previously, an observation of N=30 chromosomes was reported only for males (Nageswara-Rao and Murty 1975),” the authors write. “Our current analysis confirms the same chromosome number not only in males but also in females.” The authors of that paper declined to comment on Hamm’s findings.

Hamm doubts that he miscounted the chromosomes in six different samples, but he says there’s a chance he and his former colleagues are both right. Lepidoptera genetics is notorious for the fact that chromosome counts can vary between populations of the same species and occasionally even within cells from the same individual, he explains.

“I am glad that other researchers are skeptical and want to build on my minor contribution,” Hamm says. “There could be some interesting biology going on.”

Kunte admits it won’t exactly shake up the field of monarch research to revise the species’s chromosome count; a few genetic studies might need to be reconsidered.

The larger point is that it’s important to correct the historical record, says Akito Kawahara, a butterfly researcher at the Florida Museum of Natural History in Gainesville. The work underscores a common complaint that all too often in genetic research, taxonomists are left out of the equation, he says. As a result, genetic studies are vulnerable to species misidentifications like this one.

“These kinds of things do happen with closely related species,” he says. “Twenty-eight versus 30 chromosomes doesn’t really have any impact on the conservation of the species or our understanding of it, but the next time someone makes a mistake like this, it could be with something important.”