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