Post on 14-Dec-2015
Mitochondrial Point Mutations and Evolution: A Comparative Study
Plethodon elongatus
Plethodon stormi
Plethodon asupakStephanie Weitz
Mentor: Dr. Dee Denver
Department of Zoology<http://www.californiaherps.com/salamanders/images>
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
<http://www.bioone.org/perlserv/?request=display-figures&name=i0018-0831-61-2-158-f01>
Evolution
Darwinian Theory: 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
http://www.biology-blog.com/images/blogs/12-2007/charles-darwin-8221.jpg
http://www.philo5.com/images/VraisPenseurs/KimuraMotoo200.jpg
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
www.ccc.columbia.edu/Mitochondrial_Diseases/mito/round
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
DNA extraction Designed primers
PCR-amplifyPerform DNA
sequence alignments
Data analysis in MEGA 4.1 and DNAsp 4.1
MtDNA sequencing
Obtained tissue samples
Measuring Rates of Evolution
Ka: rate of substitution at amino acid-changing (replacement) codon positions
Ks: rate of substitution at silent codon positionsKnc: rate of substitution at any site in non-coding
regionshttp://evolution.berkeley.edu/evosite/evo101/images/codon_GCA.gif http://www.mun.ca/biology/scarr/MGA2-03-28_mtDNA_code.jpg
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
2K
TMRCA
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
Highest rates of evolution occurred at silent sites Lowest rates of evolution occurred at
replacement sites Rate of evolution intermediate for Knc
Rates of Evolution Between Pairwise Comparisons
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Ks Ka Knc
Ra
te
P. elongatus + P. stormi
P. elongatus + P. asupak
P. stormi + P. asupak
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
Time to the Most Recent Common Ancestor
0
0.05
0.1
0.15
0.2
0.25
P. elongatus + P.stormi
P. elongatus + P.asupak
P. stormi + P. asupak
Mil
lio
ns o
f Y
ears
Ks TMRCA
Ka TMRCA
Knc TMRCA
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