Mitochondrial Point Mutations
and Evolution: A Comparative
Study
Plethodon stormi
Plethodon elongatus
Stephanie Weitz
Mentor: Dr. Dee Denver
Plethodon asupak
Department of Zoology
<http://www.californiaherps.com/salamanders/images>
General Background on Weste
Plethodons

Important distinction between three species is range:
• P. elongatus has large range in SW Oregon and NW California
• P. stormi is restricted to small pockets in Siskiyou mountains of
California
• P. asupak is found a few miles west of P. stormi in the
Siskiyous
<http://www.bioone.org/perlserv/?request=displayfigures&name=i0018-0831-61-2-158-f01>
Evolution
Darwinian Theory:
 Species adapt to environment
by natural selection



http://www.biology-blog.com/images/blogs/12-2007/charles-darwin8221.jpg
Neutral Theory of
Molecular Evolution:
Introduced by Motoo
Kimura in the late 1960s
At molecular level
evolution occurs via
random drift of neutral
mutations
Foundation of molecular
clock hypothesis
http://www.philo5.com/images/VraisPens
eurs/KimuraMotoo200.jpg
www.ccc.columbia.edu/Mitochondrial_Diseases/mito/round
Hypothesis
The mitochondrial genomes
of these three Plethodons
have large amounts of noncoding sequences that
experience faster rates of
evolution than proteincoding sequences.
Muller et al. 2006
Objectives
1. Compare rates of evolution between
three species using three different
measures
2. Use rates to calculate time to the most
recent common ancestor between
species
<http://www.californiaherps.com/salamanders/pages/p.
asupak.html>
Methods
Obtained tissue
samples
Perform DNA
sequence alignments
DNA extraction
MtDNA sequencing
Data analysis in MEGA 4.1 and
DNAsp 4.1
Designed primers
PCR-amplify
Measuring Rates of Evolution

Ka: rate of substitution at amino acid-changing
(replacement) codon positions
Ks: rate of substitution at silent codon positions
 Knc: rate of substitution at any site in non-coding

regions
http://evolution.berkeley.edu/evosite/evo101/i
mages/codon_GCA.gif
http://www.mun.ca/biology/scarr/MGA2-03-28_mtDNA_code.jpg
Molecular Clock Equation
K
TMRCA 
2



TMRCA=Time to the most recent common
ancestor (millions of years)
K= Rate of evolution (Ka, Ks, Knc)
µ= Mutation rate (humans=.95/base pair/Million
year)
 Assumptions necessary to use equation
Data

Not all primers worked in all species


P. asupak: only half of the primers worked
Used pairwise DNA sequence alignments to
calculate Ka, Ks, Knc
Objective I Results
Rates of Evolution Between Pairwise Comparisons
0.4
0.35
Rate
0.3
0.25
P. elongatus + P. stormi
0.2
P. elongatus + P. asupak
P. stormi + P. asupak
0.15
0.1
0.05
0
Ks



Ka
Knc
Highest rates of evolution occurred at silent sites
Lowest rates of evolution occurred at
replacement sites
Rate of evolution intermediate for Knc
Objective II Results
Time to the Most Recent Common Ancestor
Millions of Years
0.25
0.2
Ks TMRCA
0.15
Ka TMRCA
0.1
Knc TMRCA
0.05
0
P. elongatus + P.
stormi



P. elongatus + P.
asupak
P. stormi + P. asupak
Ka and Knc give comparable, more recent times
P. elongatus and P. stormi are the most closely
related
P. stormi and P. asupak are the most distantly
related
Conclusion
Highest rate of evolution occurred at
silent sites
 P. elongatus and P. stormi are the
most closely related. P. stormi and P.
asupak are the most distantly related
 Speciation occurred 250,000 years
ago

Future Research
Amplify entire mitochondrial genomes
for all three species
 Find divergence times within species

Acknowledgements





Denver lab: Dee, Dana, Sam,
Caroline, Ashley, Bobby, Peter,
Larry
Dr. Kevin Ahern
HHMI
Albert Lee, PharmD Candidate 2010
Dr. Stevan Arnold and Douglas
DeGross