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 <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 Talk about drift in small populations <http://www.bioone.org/perlserv/?request=display-figures&name=i0018-0831-61-2-158-f01>

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 http://www.biology-blog.com/images/blogs/12-2007/charles-darwin-8221.jpg http://www.philo5.com/images/VraisPenseurs/KimuraMotoo200.jpg

www.ccc.columbia.edu/Mitochondrial_Diseases/mito/round 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

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 <http://www.californiaherps.com/salamanders/pages/p.asupak.html>

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

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. http://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

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.

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

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

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