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A Keyhole Limpet specimen.
Keyhole limpets are sea snails that, despite their small size, offered a great opportunity to four Hamilton research students in Professor Patrick Reynolds’ lab. Part of the Diodora genus, these snails sit at the base of the main branch of the gastropod (snail) tree of life. As such, they provide an interesting perspective for tracking snail evolution, Reynolds said.
Pat Reynolds lab - Diodora
Student reserachers in Pat Reynolds' lab study the Diodora. Photo: Zack Stanek

“I wanted to study evolution,” said Erin Ebert ’22, whose senior thesis focused on studying the tiny life form. “Marine creatures like the keyhole limpets are particularly interesting to study because marine organisms are very ancient.”

“It means we’re either looking at a very rare, understudied species or possibly even an undiscovered species, though that’s less likely.”

Ebert, along with fellow thesis students Reilee Gunsher ’21 and Eamon Gibbons ’21 and research assistant Maddie Vavra ’23, began their research last January. At the time, their goal was to perform species identification for the keyhole limpet specimens that Reynolds had acquired through the Florida Museum of Natural History (FMNH). The specimens had been preliminarily identified based on their morphology, or physical traits, but the students had to confirm these identifications genetically through a process known as DNA barcoding. In doing so, they also retrieved genetic data necessary to create a well-supported phylogeny, or hypothesis for the evolutionary relationships among the species.

While other papers have been published on Diodora phylogeny, the FMNH’s collection of keyhole limpets spanned a wide geographical range — from the Red Sea to Hawaii and all the way to Australia and Singapore. In fact, the collection included species from the Pacific and Indian oceans that have yet to be featured in published research, Reynolds said.   

Diodora
Diodora - Photo (BY-NC-SA 3.0) 2005 California Academy of Sciences

Last spring, the students successfully identified 55 of the 70 specimens sent by the FMNH. Using this information along with clues gleaned from the snails’ geographical distribution, Gunsher and Gibbons wrote thesis papers on their evolutionary relationships and the possible anthropogenic influence on the Diodora distribution. 

Ebert and Vavra continued the work this fall on the remaining 15 unidentified specimens. They focused on adjusting the conditions for PCR (polymerase chain reaction) since this step  is key in successful DNA barcoding. By looking at the scientific literature, the pair and Reynolds found that a higher PCR annealing temperature resulted in greater success for some species. 

The increase in temperature did yield better results. However, despite having strong DNA sequences for the specimens, there were still some that didn’t match strongly with any other specimens on GenBank, the National Institute of Health genetic sequence database.

“This means there’s a good chance that the species isn’t in GenBank, which is pretty cool,” Vavra said. “It means we’re either looking at a very rare, understudied species or possibly even an undiscovered species, though that’s less likely.”

Ultimately, the DNA data retrieved from the specimens suggest that previous hypotheses published on the evolutionary tree of Diodora may be incorrect, Reynolds said, though more work still needs to be done.

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