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Dr. Walter Salzburger The Neutral Theory Polymorphism [Greek: poly=many, morph=form] images: www.wikipedia.com, www.clcbio.com, www.telmeds.org The Neutral Theory | 2 The Neutral Theory | 3 Polymorphism ! ...can be defined as the existence of two or more forms within the same population (or the same species) ! the persistence of polymorphism within a species (just as evolutionary change between species) can be explained by: ! natural selection ! drift The Neutral Theory | 4 Genetic Drift ! random changes in gene frequencies in a population computer simulation of the Wright-Fisher model of random genetic drift (from: Hartl, 1997) The Neutral Theory | 5 The Neutral Theory ! The Neutral Theory of Molecular Evolution was formalized in the 1960s by M. Kimura ! The Neutral Theory deals with the relative importance of natural selection and random drift in molecular evolution Motoo K imura (1924-1 994) ! The Neutral Theory became - in modifications - a null model of molecular evolution The Neutral Theory | 6 The Neutral Theory ! ...states that the majority of molecular evolution is driven by neutral drift (which does not mean that the majority of mutations are neutral!) ! Natural selection is needed to explain adaptation. Yet, evolution at the DNA level is - according to the “neutralists” - primarily non-adaptive ! opposite view: molecular evolution is driven by natural selection The Neutral Theory | 7 selectionist neutralist frequency of mutations - 0 pan-neutralist frequency of mutations + - 0 frequency of mutations + - 0 + selection coefficient selection coefficient of a mutation: + mutation increases in frequency - mutation is eliminated 0 gene frequencies drift The Neutral Theory | 8 The Neutral Theory: evidence ! The rate of molecular evolution and the degree of polymorphism is too high to by explained by natural selection ! The constancy of molecular evolution (“molecular clock”) seems to be inconsistent with natural selection ! Functionally less constrained parts evolve at higher rate (which is the opposite of what a purely selectionist’s view would predict) The Neutral Theory | 9 Species Number of loci P(%) H(%) Phlox cuspicata 16 11 1,2 Limulus polypemus 25 25 5,7 28-42 18 3,8 Gryllus bimaculatus 25 58 6,3 Drosophila robusta 40 39 11 Bombus americanorum 12 0 0 Bufo americanus 14 26 11,7 Homo sapiens 71 28 6,7 Homarus americanus Nevo (1988) The Neutral Theory: evidence Variation measured as percentage of polymorphic loci (P) and average heterozygosity per individual (H) The Neutral Theory | 10 The Neutral Theory: evidence human/ kangaroo rabbit/ rodent 75 llama/ cow horse/ donkey goat/ cow 50 25 sheep/ cow 0 0 dog/ cow 25 pig/ cow human/ cow human/ rodent horse/ cow 50 75 Millions of years ago 100 125 Kimura (1993) Nucleotide substitutions 100 The Neutral Theory | 11 The Neutral Theory: problems ! Levels of heterozygosity are too constant between different species ! The molecular clock is not constant enough, and there are genes not following a clock-like behavior ! Genetic variation and evolutionary rates are not always related as predicted The Neutral Theory | 12 The Nearly Neutral Theory! ! ...is a modified version of the “purely” neutral theory (developed by Ohta in the 1990s) ! ...allows “nearly” neutral mutations such as slightly disadvantageous mutations. These behave neutrally in small populations but not in large ones (this is why genetic variation in large populations is somewhat smaller than predicted by the neutral theory) ! ...can in principle account of what is known about molecular evolution The Neutral Theory | 13 DNA sequence evolution non-coding segment (control region) T. moorii N. brichardi O. tanganicae! ATGAATGGTGATAAGATATTGATA ATGAGTGATAATGGAATAT-AATA ATGAATGATATTAAGATATTGATA ----*--*-**-***----**--- protein coding segment (ND2 gene) T. moorii N. brichardi O. tanganicae! ATTCTACTCTCCCTAGGAGGCCTC ATTCTCCTCTCACTGGGGGGCCTC ATTCTCCTTTCACTGGGAGGCCTC -----*--*--*--*--*------ The Neutral Theory | 14 DNA sequence evolution The Neutral Theory | 15 DNA sequence evolution non-synonymous substitution ...CCGCTCGTCAGCTAG... original DNA sequence Gly Glu Gln Ser Ile ...CCGCTCGTCAACTAG... Gly Glu Gln Leu Ile ...CCGCTTGTCAACTAG... Gly Glu Gln Leu Ile synonymous substitution The Neutral Theory | 16 dN/dS ratio ! dN/dS (Ka/Ks) provides information about the form of sequence evolution in a gene*: ! dN/dS < 1: purifying selection (non-synonymous sites are evolving slower than synonymous sites) ! dN/dS = 1: neutrality (non-synonymous and synonymous sites are evolving at equal rates) ! dN/dS > 1: positive selection (non-synonymous sites are evolving faster than synonymous sites; natural selection has acted to change the amino acid sequence) *dS is used as proxy for the neutral rate of molecular evolution The Neutral Theory | 17 Zhang et al. (2002) Pancreatic ribonuclease in colobine monkeys douc langur The Neutral Theory | 18 Pancreatic ribonuclease in colobine monkeys Ka = 0.0310 substitutions per non-synonymous sites Ks = 0.0077 substitutions per synonymous sites (and non-coding sites) Ka/Ks = 4.026 douc langur The Neutral Theory | 19 Zhang et al. (2002) Pancreatic ribonuclease in colobine monkeys douc langur adaptation to different pH in the colobine small intestine The Neutral Theory | 20 Pancreatic ribonuclease in leaf-eating monkeys douc langur Prud’homme & Carroll (2006) guereza The Neutral Theory | 21 Pancreatic ribonuclease in leaf-eating monkeys douc langur Zhang (2006) guereza The Neutral Theory | 22 Tests for positive selection ! HKA test (Hudson, Kreitman and Aguade 1987): ! compares the level of polymorphism in two nuclear loci to test for neutral versus adaptive sequence evolution ! McDonald-Kreitman (1991) test: ! uses fixed and polymorphic sites to test for neutral versus adaptive sequence evolution The Neutral Theory | 23 Tests for positive selection: McDonald-Kreitman species A Ind. Ind. Ind. Ind. 1 2 3 4 species B ATGACTTGAT ATGACTTRAT ATGACTTAAT ATGACTTAAT fixed site Ind. Ind. Ind. Ind. 1 2 3 4 ACGACTTGAT ACGACTTAAC ACGACTTGAT ACGACTTGAY polymorphic site The Neutral Theory | 24 Tests for positive selection: McDonald-Kreitman Alcohol dehydrogenase gene (adh) in Drosophila D. melanogaster (12) D. simulans (6) D. yakuba (24) The Neutral Theory | 25 Tests for positive selection: McDonald-Kreitman Differences Fixed sites Polymorphic sites Total synonymous SF = 17 SP = 42 SF + SP = 59 non-synonymous NF = 7 NP = 2 NF + NP = 9 SF + NF = 24 SP + NP = 44 68 Sum McDonald & Kreitman (1991) The Neutral Theory | 26 Tests for positive selection: McDonald-Kreitman NF/SF = 7/17 = 0.41 fixed non-synonymous/fixed synonymous sites NP/SP = 2/42 = 0.048 polymorphic non-synonymous/polymorphic synonymous sites NF/SF is significantly higher than NP/SP Fisher’s exact test: P = 0.006 McDonald & Kreitman (1991) The Neutral Theory | 27 Sliding Window Analysis ! Why? ! sequence evolution might not be the same over the entire locus sequence dN/dS 3 2 1 0 0 sequence length The Neutral Theory | 28 Sliding Window Analysis Demma et al. (2006) SIV envelope (env) protein The Neutral Theory | 29 Testing Neutral Evolution: Tajima’s D S... number of segregating sites is the amount of polymorphic nucleotide sites in the dataset !... average pairwise difference is the average of all pairwise differences in the dataset The Neutral Theory | 30 Testing Neutral Evolution: Tajima’s D segregating sites and average pairwise differences TaxonA TaxonB ATGGCAGTCT ACGGCAGTCT S=1 ! = 0.1 The Neutral Theory | 30 Testing Neutral Evolution: Tajima’s D segregating sites and average pairwise differences TaxonA TaxonB TaxonC ATGGCAGTCT ACGGCAGTCT ATGGCAGTTT S=2 ! = 0.13 The Neutral Theory | 30 Testing Neutral Evolution: Tajima’s D segregating sites and average pairwise differences TaxonA TaxonB TaxonC TaxonD ATGGCAGTCT ACGGCAGTCT ATGGCAGTTT ATGGCGGTCT S=3 ! = 0.15 The Neutral Theory | 31 Testing Neutral Evolution: Tajima’s D mutation rate " = 4N# genetic variation (theta) effective population size ...can be estimated using the proportion of segregating sites or the average pairwise differences The Neutral Theory | 32 Testing Neutral Evolution: Tajima’s D theta estimated from segregating sites D = " (!) - " (S) theta estimated from average genetic diversity The Neutral Theory | 33 Testing Neutral Evolution: Tajima’s D bottleneck, “selective sweep” neutral evolution D<0 balancing selection, sub-division D"0 D>0 The Neutral Theory | 34 Selective sweep & genetic hitch-hiking beneficial mutation “hitch-hikers” S1 !1 S1 > S 2 !1 >>> !2 S2 !2 The Neutral Theory | 35 nucleotide diversity (!) Selective sweep & genetic hitch-hiking distance (kb) beneficial allele