1. Mitochondrial Point Mutations and Evolution: A Comparative Study
Plethodon stormi
Mention what mitochondria do…
Plethodon elongatus
Stephanie Weitz
Mentor: Dr. Dee Denver
Department of Zoology
Plethodon asupak
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2. General Background on Western 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
Talk about drift in small populations
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3. Evolution Darwinian Theory: Neutral Theory of Molecular Evolution:
Species adapt to environment by natural selection
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
Briefly define mol clock hyp

4.
Hypothesis
The mitochondrial genomes of these three Plethodons have large amounts of non-coding sequences that experience faster rates of evolution than protein-coding sequences.
Muller et al. 2006

5. 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
*Rates are relative to each other, with P.el being a benchmark of sorts since previous research suggests that P.s and P.a descended from P.el
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6. Methods Designed primers Obtained tissue samples DNA extraction
Perform DNA
sequence alignments
MtDNA sequencing
PCR-amplify
Talk about how PCR method changed
Data analysis in MEGA 4.1 and DNAsp 4.1

7. 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
Since these are two different aa, this could have a negative effect on the fitness of the animal. If a mutated individual is less fit than the rest of the population, it’s going to be selected against and no evolution will occur.

8. Molecular Clock Equation
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

9. 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
Trouble primers: ND6R, Glutamic Acid (F+R), Phe. Was able to still obtain seq due to long frags from other primer sites. For P. el used seq from NCBI to complete align.

10. Objective I Results
Ks must be the best measure of TMRCA. Predict Knc to be like Ks, but looks more like Ka. Indicates that there is purifying selection occuring
Highest rates of evolution occurred at silent sites
Lowest rates of evolution occurred at replacement sites
Rate of evolution intermediate for Knc

11. Objective II Results 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

12. 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

13. Future Research
Amplify entire mitochondrial genomes for all three species
Find divergence times within species

14. 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