Call of the Cajun Chorus Frog

LSU Researchers Analyze Chorus Frog Biodiversity with 40-Year-Old LSU MNS Samples

In a new open access study published in Ecology and Evolution, LSU researchers Jeremy Brown and Eric Rittmeyer along with collaborators at Florida State University show that two species of chorus frogs now interbreed, or hybridize, across a much wider area of the Gulf Coast than they did just 30-40 years ago. Frog tissue samples frozen in LSU Museum of Natural Science’s (LSU MNS) Genetic Resource Collection were key to making this discovery.

 In the 1970’s Donald Gartside, then a faculty member at the LSU Health Sciences Center in New Orleans, began collecting and characterizing tiny singing frogs across Louisiana. He also contributed tissues from the frogs to a frozen tissue collection established by his colleague Herbert Dessauer.

What Gartside didn’t know is that he had an undiscovered species on his hands. These frogs were not Upland chorus frogs (Pseudacris feriarum) as he thought. And what Dessauer didn’t know is that the tissues he saved for genetic analysis would pay off for two prepared LSU researchers.

Gartside’s frogs today are known as Cajun chorus frogs
(P. fouquettei). Emily Lemmon, an associate professor in the Department of Biological Science at Florida State University, first described the species in her PhD dissertation. “Lemmon is essentially the world’s foremost expert on these frogs,” said Jeremy Brown, assistant professor in the LSU Department of Biological Sciences. “She studies the patterns of their speciation and hybridization across their range.” Lemmon and Brown were classmates in graduate school.

From Gartside’s work, Lemmon knew of a hybrid zone occurring along the Pearl River where the Cajun chorus frogs from Louisiana and Southern chorus frogs (P. nigrita) from other regions of the Gulf Coast meet. “I decided it would be interesting to compare how the structure of the hybrid zone had changed through time, so I went back to Gartside's original sites and started recording and sampling,” Lemmon said. But the zone where the two frog species interbreed and form hybrids was geographically too distant for Lemmon’s lab to study it regularly.

“She asked if we would be interested in working on studying that zone, and I was looking for an excuse to get outside,” Brown said. Brown studies computational biology, but he tries to spend time on field projects as much as he can. This project was a good excuse to do that, he said. “I just like catching things, being outside. It was also a great project for student involvement.”

Brown involved Eric Rittmeyer, then a graduate student at LSU, as well as several undergraduates who assisted with fieldwork. Brown’s group caught frogs in Louisiana, in Mississippi and within the hybrid zone. They sent the frogs to Lemmon’s lab for extraction of DNA and other genetic data collection. Kristin Engebretsen, at the time an undergraduate in Lemmon’s lab and the lead author on the paper, collected the genetic data and conducted many of the analyses.

“Being out in the field finding and observing reptiles and amphibians in their natural environment is really what drew me to biology,” Rittmeyer said. “Yet at the same time, it is challenging. There are the logistics, of course, such as obtaining permits, university travel forms and permission from landowners. And even once all this is accomplished, it can be a challenge to find the target organism.”

In the hybrid zone near the Pearl River, catching chorus frogs can be particularly difficult. Hybrid zones tend to have fewer individuals from both species, and the individuals who are in the zone are skittish. Brown and Rittmeyer would go out at night for fieldwork within the hybrid zone and only catch a few frogs on a good night. Eric turned out to be the expert catcher.

“There’s a certain stalking technique to catching these frogs,” Brown laughed. “You need a lot of patience and a really good ear. Eric was really good.”

“Even if you can hear a male calling, it can be a challenge to find exactly where he's hiding,” Rittmeyer said. Brown and Rittmeyer would drive around with their windows down on winter nights, listening for the male chorus frogs calls. When they heard calls, they would pull over and start searching.

“In hand, these two frog species are almost impossible to tell apart,” Brown said. “Even their calls are similar to the human ear. And they will stop calling when they sense you are around. So we would get as close as we could until they stopped calling, and then stand still until they started calling again. Then we would get a bit closer, and repeat that process until we were close enough to pinpoint which tuft of grass they were in.”

The researchers’ smartphones were valuable when it came to catching the frogs. Brown and his students would all be playing recordings of male chorus frogs from their phones as they stalked them around a pond, because when the frogs hear the calls of other males they call more frequently.

Much active research in evolutionary biology involves looking at the process of speciation, or how and when new species form or how and when different species converge. Sometimes barriers that have divided species in the past, such as rivers, disappear. If the individuals can successfully interbreed and produce competitive offspring, the zone of intersection between the two species, also called the hybrid zone, starts to widen. The different species may even become a single species again.

Brown and Lemmon wanted to investigate how Cajun chorus frogs and Southern chorus frogs have interbred over time and whether the two species might be converging due to changes in the landscape.
But the researchers quickly ran into a problem. To investigate this question, they needed historical data on how these two species interacted in the past.

Lemmon knew about the genetic data Gartside had collected on chorus frogs in Louisiana in the 1970’s, but she assumed the samples had been left in New Orleans and destroyed during hurricane Katrina.

“We were going to do the best we could to compare our modern genetic analyses to Gartside’s analyses of chorus frogs,” Brown said. “But Eric, who was a curatorial assistant at the museum [LSUMNS], had a hunch that the tissues had been transferred to the collection at LSU.” LSUMNS has one of the largest, oldest and most actively used genetic resource collections in the country.

“Eric was able to track them down, which was a big surprise to us,” Brown said. “It really changed the nature of the study. Lemmon’s lab was able to extract new DNA from these 40-year old tissue samples. That’s the real value of these tissue collections. Although our ability to gather genetic data changes through time, as long as the tissues are there we are able to apply our newest methods.”

“We were so surprised because everyone, including Gartside, thought the samples had been lost,” Lemmon said. “This makes our study unique, in that we could do a comparison between samples over such a large time period.”

Brown’s group contributed computational analysis to the project based on the genetic data collected in Lemmon’s lab. They computed the shape of the zone where Cajun chorus frogs and Southern chorus frogs overlap and potentially interbred. In their paper, Brown and his colleagues describe how the shape of that zone has changed over time.

For chorus frogs, this zone of hybridization appears to be widening. “We found Southern chorus frog genes much further into Louisiana than Gartside did in 1976, and we found Cajun chorus frog genes much further east along the Gulf Coast,” Brown said. “We don’t exactly know why.”

One possibility is that individual Cajun and Southern chorus frogs disperse more than they used to, perhaps moving up and down man-made road-side ditches. Brown’s favorite hypothesis, which is most likely wrong, he laughs, is that strong hurricanes in the recent past have picked frogs up and dispersed them far into regions where they wouldn’t normally reside. But to answer the question definitively, the chorus frog research team needs additional data. By collecting more data on the different mating calls of the two frog species and the degree of female preference for males of the same species, the researchers could get a clearer idea of whether these species are starting to converge genetically or not, and to what extent.

“It is often assumed that hybrid zones are stable over time, yet only a handful of studies have sampled from two different time points to actually test this assumption,” Rittmeyer said.

“Fundamentally, our data is important for understanding what is going to happen with these species going forward,” Brown said.