Allozyme analysis of a contact zone between two mtDNA haplotypes in Desmognathus ocoee
(Amphibia: Plethodontidae)
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Recent phylogeographic analyses of mitochondrial DNA (mtDNA) sequences by Yoke et al. have
revealed five distinct well supported clades within the salamander species traditionally called
Desmognathus ocoee Nicholls (Fig. 1 and 5). This information suggests that the organisms currently
recognized as D. ocoee may belong to more than one species. In the Blue Ridge Mountains of
northeastern Georgia, a contact zone between two mtDNA haplotypes has been identified.
Populations within this area vary notably in appearance, especially in regard to dorsal pattern and
coloration although little has been done to resolve the relationships among these populations. My
study sought to augment the mtDNA data with information from nuclear genes by comparing allele
frequencies at six allozyme loci among populations of D. ocoee at and around the contact zone.
Previous studies have found that allozyme frequencies tend to corroborate mitochondrial sequence
data, indicating similar patterns of geographic differentiation among populations. The mitochondrial gene sequenced, Cytochrome b (cyt b), has proven to be valuable when delineating caudate
species in previous publications (ie: Tilley et al., 2008). Independently, allozyme electrophoresis (Fig.
2) has been demonstrated to be an effective tool for elucidating species relationships.
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INTRODUCTION
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Noëlle K. J. Bittner and Stephen G. Tilley
Department of Biological Sciences, Smith College, Northampton, MA
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The origin of the discrepancy between the nuclear allozymes and mitochondrial nucleotide data
poses questions of both animal behavior and molecular evolution. The geographic division of
mitochondrial haplotypes could be linked to dispersal disparities between the sexes with migrating
males and static females within populations. Little is known about the distances desmognathine
salamanders disperse from their hatching sites. This study gives evidence for nuclear gene flow
among populations greater than 60 km apart suggesting movement throughout the area. The
discordance could also suggest very recent evolutionary divergence. Mitochondrial genes evolve at
a faster rate than many nuclear genes because they are maternally inherited and thus the effective
breeding size is smaller. A combination of behavioral and molecular explanations likely contribute
to the discrepancy between the mtDNA and nuclear information.
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CHEEK
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Individuals were collected from 21 populations across the contact zone of two clades
recognized by Yoke et al. in northeastern Georgia. Population sample sizes ranged from ten to 34
specimens; samples of fewer individuals were excluded from analysis. Dorsal and ventral
photographs were taken of all specimens pre-sacrifice then individuals were processed and
stomach, liver and ventral muscles were extracted for analysis. Tail tips were frozen for future
analysis and carcasses were preserved as voucher specimens. Standard methods of horizontal
starch gel electrophoresis (Murphy et al., 1990) were utilized to determine allele frequencies at six
allozyme systems: aspartate aminotransferase 1 (AAT1), glyceraldehydes-3-phosphase
dehydrogenase (G3PDH), isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, Fig. 2), lactate
dehydrogenase 2 (LDH2) and peptidase (PEP). Seventeen additional loci were demonstrated to be
essentially monomorphic during preliminary tests and were eliminated from further analysis. As a
control, local Desmognathus fuscus samples, which have proven to be monomorphic at all loci
(excluding G3PDH), were run on each gel. Allele frequencies were computed by Genepop
(Raymond and Rousset, 1995).
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MATERIALS AND METHODS
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The mtDNA data display a North-South shift between the two haplotypes, with two populations
containing both (Fig. 3G). The allozyme patterns do not correlate with this North-South trend
(Fig. 3A-F). While varying in frequency among populations, allozyme variants do not show major
frequency shifts where the mtDNA haplotypes replace each other. Different species of
Desmognathus typically do not share alleles at a few to several of the loci surveyed in this study. The
allozyme results suggest that the populations belong to a single species.
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RESULTS AND DISCUSSION
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FIGURE 1: Dorsal (L) and ventral (R) photographs of D. ocoee.
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FIGURE 4: Locations of collection sites in northeastern Georgia, USA.
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SOURCES
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FIGURE 5: D. ocoee beautifully displaying cheek coloration.
Photo Credit: S. G. Tilley
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Murphy, R.W., J.W. Sites Jr., D.G. Buth, and C.H. Haufler. 1990. Proteins I: Isozyme elec-trophoresis. Pp. 45-126. In D. M. Hillis and C. Moritz (Eds.), Molecular
Systematics. Sinauer Associates, Inc., Sunderland, Massachusetts.
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FIGURE 2: An example of a stained starch electrophoresis gel showing IDH1 (top) and IDH2 (bottom).
Different bands within these staining regions represent products (allozymes) of different alleles.
Three-banded patterns represent heterozygotes.
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Raymond M. and F. Rousset. 1995. GENEPOP (version 1.2) population genetic software for exact tests and ecumenicism. Journal of Heredity 86: 248–249
10 km
Tilley, S. G., Eriksen, R.L., and L.A. Katz. 2008. Systematics of dusky salamanders, Desmognathus (Caudata: Plethodontidae), in the mountain and Piedmont
regions of Virginia and North Carolina, USA. Zoological Journal of the Linnean Society 152: 115-130.
FIGURE 3A-H: Maps showing the collection sites with allele frequencies (A-F),
haplotypes (G), and cheek coloration (H) for each population.
Yoke, M. M., J. Bernardo, S.G. Tilley and J. W. Sites Jr. Phylogenetic history and species limits of the Ocoee salamanders Desmognathus ocoee (Amphibia,
Plethodontidae). In prep.