Student Research Project
Relationships among Holarctic hylid frogs: Insights from mitochondrial DNA sequences

Student
Candace J. Riehl
Major: Pre-medicine
Future Plans: Medical school

Professors
Linda Maxson, Associate Vice-Chancellor for Academic Affairs, The University of Tennessee, Knoxville

Carla Ann Hass, Instructor, Department of Biology, Pennsylvania State University, University Park

For the last 20 years, my research has focused on using the information encoded in macromolecules, proteins, and nucleic acids to elucidate relationships among and within many groups of amphibians, frogs in particular. Candy Riehl, an undergraduate at Penn State, has worked with me for the past three years. Under the supervision of Dr. Carla Hass, another researcher in the lab, and me, Candy has used molecular techniques to investigate relationships among the Holarctic hylid frogs. These frogs occur across North America, Europe, and Asia; the approximately three dozen species are placed in three genera: Acris (cricket frogs), Hyla (tree frogs), and Pseudacris (chorus frogs). These frogs have been studied by researchers in many areas of biology, including speciation, ecology, and physiology. Knowing the relationships among the members of this group will allow scientists to better understand the results of those studies.

Although the phylogenetic relationships within this group have been examined using both morphological and molecular data, questions remain about the relationships among the genera and among the species in those genera. For example, the spring peeper has been placed in Hyla based upon morphological data, but some molecular and behavioral data suggest that it is a Pseudacris. Candy sequenced part of a mitochondrial gene coding for ribosomal RNA (the 16S gene) to provide a new molecular data set to address this and other questions about the relationships within the Holarctic hylid frogs.

We extracted DNA from 30 species of frogs representing the three genera. Candy amplified a portion of the 16S gene using the polymerase chain reaction (PCR). She then performed dideoxy nucleotide sequencing with a radiolabeled nucleotide and exposed the sequencing gel to x-ray film to recover the sequences. Candy aligned the sequences from all the species to ensure that the same sites on the DNA molecules were being compared. These sequences were added to sequences of a second mitochondrial gene (the 12S) from the same species. Using data from both genes, we constructed phylogenetic trees showing the relationships among all these species.

These DNA sequence data support the placement of the spring peeper (species crucifer) in the genus Pseudacris. It was incorrectly placed in Hyla because of morphological convergence due to its arboreal habits. These data also place the little grass frog (species ocularis) in the genus Pseudacris. Due to its unique morphology, the result of its small size and resulting loss of many characteristics, this species had been placed in its own genus (Limnaoedus). We were not able to determine the relationships among the three genera in this group. Candy's work is part of an ongoing project to examine the relationships among the Holarctic hylids. We now are sequencing a nuclear gene, serum albumin, for the same species to add to the data that Candy has collected.

Return to Main Page

Copyright © The McGraw-Hill Companies, inc. Permission required for reproduction or display.