2. Chapter 5 Rate and Patterns of Nucleotide Substitution 1 Chau-Ti Ting ctting@ntu.edu.tw Unless noted, the course materials are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Taiwan (CC BY-NC-SA 3.0)Attribution-NonCommercial-ShareAlike 3.0 Taiwan

3. § Rate of nucleotide substitution The rate of nucleotide substitution, r, is defined as he number of substitutions per site per year. The mean rate of substitution can be calculated by dividing the number of substitutions, K, between two homologous sequences by 2T, where T is the time of divergence between the two sequences. That is Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 100. Sinauer Associates, Inc. Sunderland, MA, USA. 2

4. Coding region Synonymous versus nonsynonymous The rate of nonsynonymous substitution is extremely variable among genes: Range: effective zero in actin   3.1x10 -9 in interferon  Extreme case – ubiquitin, which is completely conserved between human and fly and which differs among animal, plant, and fungi by only 2 or 3 out of 76 amino acid residues. In the vast majority of genes, the synonymous substitution rate greatly exceeds the nonsynonymous substitution rate. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 101. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 103. Sinauer Associates, Inc. Sunderland, MA, USA. 3

5. Transition versus transversion At fourfold degenerate sites it is possible to compare the rate of transitional substitution and transversional substitution, since both types of substitution are synonymous. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 104. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 105. Sinauer Associates, Inc. Sunderland, MA, USA. 4

6. National Taiwan University Chau-Ti Ting Exon Intron 5’5’ 3’3’ Flanking region GC box CAAT box TATA box Transcription initiation Initiation codon Stop codon AATAAA box Transcription termination Poly(A) site Promoter region GT AG Intron 19-27 bp upstream of the transcription startpoint TACTAAC box 30 bp upstream of the 3’ end of the intron GT-AG rule 5’5’ 3’3’ 5

7. Pseudogenes A pseudogene is a nongenic DNA segment that exhibits a high degree of similarity to a functional gene but which contains defects, such as nonsense and frameshift mutations, that prevent it from being expressed properly. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 28. Sinauer Associates, Inc. Sunderland, MA, USA. 6

8. Noncoding region In almost all genes, the substitution rates in the 5’ and 3’ UTR are lower than those at fourfold degenerate sites. The rate in pseudogenes is slightly higher than that in the other regions. Table 4.4 Numbers of nucleotide substitutions per site (K) between cow and goat globin genes Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 106. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 107. Sinauer Associates, Inc. Sunderland, MA, USA. 7

9. National Taiwan University Chau-Ti Ting Exon Intron 5’5’ 3’3’ Flanking region GC box CAAT box TATA box Transcription initiation Initiation codon Stop codon AATAAA box Transcription termination Poly(A) site Promoter region GT AG Intron 19-27 bp upstream of the transcription startpoint TACTAAC box 30 bp upstream of the 3’ end of the intron GT-AG rule 5’5’ 3’3’ 8

10. § Causes of Variation in Substitution Rates The rate of substitution is determined by two factors: (1)the rate of mutations (2)the probability of fixation of a mutation (Ch. 2) Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 108. Sinauer Associates, Inc. Sunderland, MA, USA. 9

11. Functional constraints Functional constraints or selective constraints defines that range of alternative nucleotides that is acceptable at a site without affecting negatively the function or structure if the gene or the gene product. Kimura has illustrate this principle by a simple model where v T is the total mutation rate by unit time, f 0 is the fraction of neutral or nearly neutral mutations and K is the substitution rate Whenever a particularly conservative sequence is found, researchers start looking for a specific function in this region Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 108. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 110. Sinauer Associates, Inc. Sunderland, MA, USA. 10

12. Patterns of substitution and replacement The pattern of nucleotide substitution is defined as the relative frequency with which a certain nucleotide changes into another during evolution. P ij = n ij nini Where n ij is the number of substitutions from i to j, and n i is the number of i nucleotides in the ancestral sequence Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 123. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 124. Sinauer Associates, Inc. Sunderland, MA, USA. A T G C National Taiwan University Chau-Ti Ting 11

13. Inferring substitutions on a specific lineage Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 125. Sinauer Associates, Inc. Sunderland, MA, USA. 12

14. 55 processed pseudogene sequences from human Pseudogenes are expected to become rich in A and T Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 126. Sinauer Associates, Inc. Sunderland, MA, USA. 13

15. Mutation maybe different between the two strands!! Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 127. Sinauer Associates, Inc. Sunderland, MA, USA. 14

16. Patterns of amino acid replacement Physicochemical distance are based on such properties of the amino acid as polarity, molecular weight, and chemical composition. Grantham’s distance (1974, Table 4.7) conserved replacementLeu  Ile d=5 radical replacement Trp  Cys d=215 typical amino acid leucins, isoleucin, glutamin, methionine Idiosyncratic amino acid cysteine, tryptophan, tyrosine, glycine Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 128. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 128. Sinauer Associates, Inc. Sunderland, MA, USA. 15

17. Wikipedia Dancojocjri 16

18. Patterns of amino acid replacement Physicochemical distance are based on such properties of the amino acid as polarity, molecular weight, and chemical composition. Grantham’s distance (1974, Table 4.7) conserved replacementLeu  Ile d=5 radical replacement Trp  Cys d=215 typical amino acid leucins, isoleucin, glutamin, methionine Idiosyncratic amino acid cysteine, tryptophan, tyrosine, glycine Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 128. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 128. Sinauer Associates, Inc. Sunderland, MA, USA. 17

19. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 130. Sinauer Associates, Inc. Sunderland, MA, USA. 18

20. Amino acid exchangeability Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 131. Sinauer Associates, Inc. Sunderland, MA, USA. 19

21. Molecular Clock In their comparative studies of hemoglobin and cytochrome c protein sequences from different species, Zucherkandel and Pauling (1962, 1965) and Margoliash (1963) first notice that the rates of amino acid replacement were approximately the same among various mammalian lineages. Zucherkandel and Pauling (1965) therefore proposed that for any given protein, the rate of molecular evolution is approximately constant over time in all lineage or, in other words, that there exists a molecular clock. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 139. Sinauer Associates, Inc. Sunderland, MA, USA. 20

22. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 140. Sinauer Associates, Inc. Sunderland, MA, USA. 21

23.  If a proteins evolve at a constant rates, they can be used to determine dates of species divergence and to reconstruct phylogeny relationships among organisms. Let us assume that the rate of nonsynonymous substitution for the  chain of hemoglobin is 0.56 x 10 -9 substitutions per site per year, and that  -globins from rat and human differ by 0.093 substitutions per site. Then, under the molecular clock hypothesis, the divergence time between the human and rat lineages is estimated to be approximately 0.93/(2 x 0.56 x 10 -9 )= 80 million years ago. Kimura (1983) – “For each protein, the rate of evolution in terms of amino acid substitution is approximately constant per year per site for various lineage, as long as the function is and tertiary structure of the molecule remain essentially unaltered.” Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 139. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 139. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Motoo Kimura 1983. The Neutral Theory of Molecular Evolution., p. 98. Cambridge University Press, Cambridge, USA. 22

24. Controversies and challenges Classical evolutionists argued against it because the suggestion of rate constancy did not sit well with the erratic tempo of evolution at the morphological and physiological levels. Based on molecular clock, the estimate divergence between humans and other African apes is only 5 million years. However, the then- prevailing view among paleontologists was that human and apes diverged at least 15 million years ago. Extreme high rate of amino acid replacement occurred following the gene duplication that gave rise to the  - and  -hemoglobins, and that the high rates were due to advantageous mutations that improved the function of these globin chains. Protein sequence evolution often proceeds much more rapidly at times of adaptive radiation than during periods in which no speciation occurred. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 141. Sinauer Associates, Inc. Sunderland, MA, USA. 23

25. Relative Rate Test The controversy over molecular clock hypothesis often involves disagreement on dates of species divergence. To avoid this problem, several test have been developed. Margoliash, Sarich and Wilson’s test Suppose that we want to compare the rates in lineage A and B. Then, we use a third species, C, as an outgroup reference. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 142. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 142. Sinauer Associates, Inc. Sunderland, MA, USA. 24

26. The number of substitutions between species A and B, K AB, is equal to the sum of substitutions that have occurred frin point O to the point A (K OA ) and from point O to the point B (K OB ). That is, K AB = K OA + K OB Similarly, K AC = K OA + K OC and K BC = K OB + K OC O ABC Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 142. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 143. Sinauer Associates, Inc. Sunderland, MA, USA. 25

27. Since K AB, K AC, and K BC can be direct estimated from the nucleotide sequences, we can easily solve the three equations to find the values of K OA, K OB, and K OC K OA = (K AC + K AB – K BC )/2 K OB = (K AB + K BC – K AC )/2 K OA = (K AC + K BC – K AB )/2 O ABC Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 142. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 143. Sinauer Associates, Inc. Sunderland, MA, USA. 26

28. The time has passed since species A and B last shared a common ancestor is by definition equal for both lineage. Thus, according to the molecular clock hypothesis, K OA and K OB should be equal, i.e., K OA – K OB = 0. K OA – K OB = (K AC + K AB – K BC )/2 – (K AB + K BC – K AC )/2 = K AC – K BC = d where d is the difference in branch length between the two lineages leading from O to species A and B. O ABC Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 143. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 143. Sinauer Associates, Inc. Sunderland, MA, USA. 27

29. Nearly equal rates in mouse and rats Using relative rate test, K AC –K BC values for both synonymous and nonsynonymous are not significantly different from 0. Therefore, the null hypothesis of equal substitution rates in mice and rats cannot be rejected. The results are also consistent with the neutral mutation hypothesis. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 146. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 146. Sinauer Associates, Inc. Sunderland, MA, USA. 28

30. Tajima’s 1D (one-degree of freedom) method We start with three aligned nucleotide sequences, 1, 2, and 3. Let n ijk be the obsevered number of sites where sequences 1, 2, and 3 have nucleotide i, j, and k, respectively, where i, j, and k can be nucleotides A,G, C, or T. If sequence 3 is the out group, then the expectation of n ijk should be equal to that of n jik, i.e., E(n ijk ) = E(n jik ) Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 144. Sinauer Associates, Inc. Sunderland, MA, USA. 29

31. This equality holds regardless of the substitution model or the pattern of variation in substitution rates among sites. We define m 1 as follows: m 1 =  n ijj = n AGG + n ACC + n ATT + n GAA + n GCC + n GTT + n CAA + n CGG + n CTT + n TAA + n TGG + n TCC Similarly, we define m 2 as m 2 =  n jij = n AGA + n ACA + n ATA + n GAG + n GCG + n GTG + n CAC + n CGC + n CTC + n TAT + n TGT + n TCT Note that only sites in which exactly two types of nucleotides exist in the three sequences are used in this analysis. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 144. Sinauer Associates, Inc. Sunderland, MA, USA. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 145. Sinauer Associates, Inc. Sunderland, MA, USA. 30

32. When sequence 3 is outgroup, the expectation of m 1 is equal to that of m 2 under molecular clock: E(m 1 ) = E(m 2 ) The equality can be tested by using  2 with one degree of freedom, namely,  2 = (m 1 – m 2 )2/( m 1 + m 2 ) Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 145. Sinauer Associates, Inc. Sunderland, MA, USA. 31

33. Source: Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 147. Sinauer Associates, Inc. Sunderland, MA, USA. 32

34. WorkLicensingAuthor/SourcePage “The rate of nucleotide substitution, r, is defined as he number of substitutions per site per year. … K, between two homologous sequences by 2T, where T is the time of divergence between the two sequences. That is “ “Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 100. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P2 “The rate of nonsynonymous … human and fly and which differs among animal, plant, and fungi by only 2 or 3 out of 76 amino acid residues.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 101. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P3 “In the vast majority of genes, the synonymous substitution rate greatly exceeds the nonsynonymous substitution rate.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 103. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P3 “At fourfold degenerate sites it is possible to compare the rate of transitional substitution …, since both types of substitution are synonymous.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 104. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P4 33 Copyright Declaration

35. WorkLicensingAuthor/SourcePage Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 105. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P4 National Taiwan University Chau-Ti Ting P5, P8 “A pseudogene is a nongenic DNA segment that exhibits a high degree of similarity to a functional gene but which contains defects, such as nonsense and frameshift mutations, that prevent it from being expressed properly.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 28. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P6 “In almost all genes, the substitution rates in the 5’ and 3’ UTR are lower than those at … The rate in pseudogenes is slightly higher than that in the other regions.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 106. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P7 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 107. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P7 34

36. WorkLicensingAuthor/SourcePage “The rate of substitution is determined by two factors:(1) the rate of mutations(2)the probability of fixation of a mutation (Ch. 2)“ Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 108. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P9 “Functional constraints or selective constraints defines … nearly neutral mutations and K is the substitution rate” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 108. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P10 “Whenever a particularly conservative sequence is found, researchers start looking for a specific function in this region “ Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 110. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P10 “The pattern of nucleotide substitution is defined as the relative frequency with which a certain nucleotide changes into another during evolution. “ Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 123. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P11 35

37. WorkLicensingAuthor/SourcePage “Where n ij is the number of substitutions from i to j, and n i is the number of i nucleotides in the ancestral sequence “ Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 124. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P11 National Taiwan University Chau-Ti Ting P11 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 125. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P12 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 126. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P13 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 127. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P14 36

38. WorkLicensingAuthor/SourcePage “Physicochemical distance are based on such properties of the amino acid as polarity, molecular weight, and chemical composition. “ Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 128. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P15, P17 Grantham’s distance (1974, Table 4.7) conserved replacementLeu  Ile d=5 Radical … Idiosyncratic amino acid cysteine, tryptophan, tyrosine, glycine Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 128. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P15, P17 Wikipedia Dancojocjri http://en.wikipedia.org/wiki/File:Amino_Acids.svg 2012/07/25 visited P16 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 130. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P18 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 131. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P19 37

39. WorkLicensingAuthor/SourcePage “In their comparative studies of hemoglobin and cytochrome … is approximately constant over time in all lineage or, in other words, that there exists a molecular clock.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 139. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P20 Wiki brian0918™ http://en.wikipedia.org/wiki/File:ADN_animation.gif 2012/02/21 visited P20 Open Clip Art Library fzap http://openclipart.org/detail/16605/clock-sportstudio-design-by-fzap 2012/02/21 visited P20 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 140. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P21 “If a proteins evolve at a constant rates, they can be used to determine dates of species divergence and to reconstruct phylogeny relationships among organisms.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 139. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P22 38

40. WorkLicensingAuthor/SourcePage “Let us assume that the rate of nonsynonymous substitution for the  chain of hemoglobin is … estimated to be approximately 0.93/(2 x 0.56 x 10 -9 )= 80 million years ago.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 139. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P22 “For each protein, the rate of evolution in terms of amino acid substitution is approximately constant per year per site for various lineage, as long as the function is and tertiary structure of the molecule remain essentially unaltered” Motoo Kimura 1983. The Neutral Theory of Molecular Evolution., p. 98. Cambridge University Press, Cambridge, USA. http://books.google.com.tw/books?id=olIoSumPevYC&printsec=frontcover&d q#v=onepage&q&f=false It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P22 “Classical evolutionists argued against it because the suggestion of rate constancy did not sit well with … adaptive radiation than during periods in which no speciation occurred” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 141. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P23 “The controversy over molecular clock hypothesis often involves disagreement on dates of species divergence. To avoid this problem, several test have been developed.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 142. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P24 39

41. 40 WorkLicensingAuthor/SourcePage “Margoliash, Sarich and Wilson’s test Suppose that we want to compare the rates in lineage A and B. Then, we use a third species, C, as an outgroup reference.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 142. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P24 “The number of substitutions between species A and B, K AB, is equal to the sum of substitutions that have occurred frin point O to the point A (K OA ) and from point O to the point B (K OB ). That is, ” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 142. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P25 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 143. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P25, P26, P27 “Since K AB, K AC, and K BC can be direct estimated from the nucleotide sequences, we can easily solve the three equations to find the values of K OA, K OB, and K OC ” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 142. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P26

42. 41 WorkLicensingAuthor/SourcePage “The time has passed since species A and B last shared a common ancestor is by definition equal for both lineage … between the two lineages leading from O to species A and B.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 143. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P27 “Using relative rate test, K AC –K BC values for both synonymous and … rates in mice and rats cannot be rejected. The results are also consistent with the neutral mutation hypothesis.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 146. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P28 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 146. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P28 “We start with three aligned nucleotide sequences, 1, 2, and 3. Let n ijk be the obsevered number of sites where sequences … expectation of n ijk should be equal to that of n jik, i.e.,” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 144. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P29

43. 42 WorkLicensingAuthor/SourcePage “This equality holds regardless of the substitution model or the pattern of variation in substitution rates among sites. … ” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 144. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P30 “Note that only sites in which exactly two types of nucleotides exist in the three sequences are used in this analysis.” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 144. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P30 “When sequence 3 is outgroup, the expectation of m 1 is equal to that of m 2 under molecular clock: …” Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 145. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P31 Dan Graur and Wen-Hsiung Li 2000. Fundamentals of Molecular Evolution., p. 147. Sinauer Associates, Inc. Sunderland, MA, USA. It is used subject to the fair use doctrine of: Taiwan Copyright Act Articles 52 & 65 The "Code of Best Practices in Fair Use for OpenCourseWare 2009 (http://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf)" by A Committee of Practitioners of OpenCourseWare in the U.S. The contents are based on Section 107 of the 1976 U.S. Copyright Acthttp://www.centerforsocialmedia.org/sites/default/files/10-305-OCW- Oct29.pdf P32