Abstract
Four geographically distinct populations of the Spring Peeper, a North American frog,
were examined for phylogenetic relationships. 100 base pairs of the mitochondrial
cytochrome b gene from the frogs were sequenced and analyzed for mutations and crossreferenced with data on the mutation rate of the gene. The data suggested that the
Southern Ontario and Northern Minnesota populations split off from a common ancestor
well before the North-western Ontario and Nova Scotia populations. Genetic
diversification of the species may have been prompted by climatic change during the
Pleistocene.
Introduction
Genetic evidence present in mitochondria has been used to determine the evolutionary
relationships among different species. Colbourne et al (1998) documented the
phylogenetic relationship of the microcrustacean Daphnia by examining the ND5
mitochondrial gene. In their work, they were able to determine the evolutionary lineage
of the species.
Other segments of mitochondrial DNA have been used to provide similar rationale for
determining phylogenetic relationships. Huang et al (2000) examined the almost
complete cytochrome b gene (1,036 nucleotides) to construct a well-documented
phylogeny for the North American Field Cricket.
Although the Spring Peeper has been well studied on aspects of its mating call (Zimmitti,
1999) as well as a variety of other behavioural and physiological traits, there exists a
deficit in the literature on the movement of populations of the Spring Peeper. This report
uses the well-established technique of examining the cytochrome b gene to construct a
cladistic tree showing the diversification of four geographically distinct populations.
Methods
Microchondrial DNA was isolated from the Spring Peeper’s liver cells. 100 nucleotides
were sequenced from cytochrome b DNA using the Sanger dideoxynucleotide sequencing
method followed by gel electrophoresis. The gel was autoradiographed and examined for
nucleotide differences. The percentage of genetic differences between each of the four
populations of Spring Peeper were calculated and a table showing the probable time of
divergence was constructed using information on the mutation rate of the cytochrome b
gene, estimated at 1% per 500,000 years.
Results
The history of the phylogenetic diversification of the Spring Peeper is illustrated in
Figure 1. As is shown, the Northern Minnesota and Southern Ontario populations split
off well before the Nova Scotia and North-western Ontario populations.
Northern
Minnesota SP
Sourthern
Ontario SP
Nova
Scotia SP
North-western
Ontario SP
Timeline
Present
0.5 MYA (1% mutation)
1.0 MYA (2% mutation)
1.5 MYA (3% mutation)
2.0 MYA (4% mutation)
Common
ancestor
Figure 1. Phylogenetic tree of the Spring Peeper (SP) population geography based on the
mutation rates in the mitochondrial cytochrome b gene.
Discussion
It was found that 2 million years ago all four populations of Spring Peepers (SP) shared a
common ancestor. The N. Minnesota SP diverged from this common ancestor first (2
MYA), shortly after the S. Ontario SP (1.5MYA) then one million years later the Nova
Scotia SP from the N.W. Ontario SP. All of these diversions are thought to have
developed during Pleistocene that spans a time period from 1.8 million to 11,000 years
ago.
During the Pleistocene, glaciers advanced and retreated along North America, and
perhaps this could have lead to a geographic barrier separating the Spring Peeper
populations. In work done by Hundertmark et al phylogeographic associations of moose
were mapped out by evidence of genetic variation, and the authors believed this
geographic diversion to be a result of glacial movements in the most recent ice age.
Hundertmark et al also believed climate change affected on the movement of the moose,
and maybe this too affected the geographic diversion of the Spring Peeper.
With changes in climate, this can also change the habitat for disease. It may be possible
that infection rates of diseases that could affect the Spring Peeper including
Ichthyophonus-like organisms (Mikaelian et al, 2000) and lethal North American
ranaviruses (Marsh et al, 2002) could have concentrated in different geographic areas in
response to the climate changes during the Pleistocene effectively separating the
movement of Spring Peepers.
Literature Cited
Colbourne J.K., Crease T.J., Weider L.J., Hebert P.D., Dufresne F. and A. Hobaek. 1998.
Phylogenetics and evolution of a circumarctic species complex (Cladocera Daphnia
pulex). Biological Journal of the Linnean Society. 65 (3): 347-365.
Huang Y., Ortí G., Sutherlin M., Duhachek A., and A. Zera. 2000. Phylogenetic
Relationships of North American Field Crickets Inferred from Mitochondrial DNA Data.
Molecular Phylogenetics and Evolution. 17 (1): 48-57.
Hundertmark K.J., Shields G.F., Udina I.G., Bowyer R.T., Danilkin A.A. and C.C.
Schwartz. 2002. Mitochondrial Phylogeography of Moose (Alces alces): Late Pleistocene
Divergence and Population Expansion. Molecular Phylogenetics and Evolution. 22 (3):
375-387.
Marsh I.B., Whittington R.J., Rourke O.B., Hyatt A.D. and O. Chisholm. 2002. Rapid
differentiation of Australian, European and American ranaviruses based on variation in
major capsid protein gene sequence. Mol Cell Probes. 16 (2): 137-151.
Mikaelian I., Ouellet M., Pauli B., Rodrigue J., Harshbarger J.C. and D.M. Green. 2000.
Ichthyophonus-like infection in wild amphibians from Quebec, Canada. Dis Aquat Organ
40 (3): 195-201.
Zimmitti S.J. 1999. Individual variation in morphological, physiological, and biochemical
features associated with calling in spring peepers (Pseudacris crucifer). Physiol Biochem
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Phylogeographic analysis of the Spring
Peeper (Pseudacris crucifer) using
mitochondrial DNA
Written by John Stephenson (410-1306)
Prepared for Margarida Lopes
Department of Biology, Queen’s University, Kingston, Ontario
Friday May 31, 2002. Biology 101.