The Four Forces of Evolution Affect a population, lead to genetic variation.

Evolution at its simplest level is a change in the frequency of an allele in a population Population genetics: describe the patterns of genetic variation and understand the evolutionary processes that have shaped that variation. Changes in allele frequencies evolution Individuals pass their alleles on to their offspring The individuals that survive and reproduce or who reproduce the most, are those with alleles and allelic combinations that best adapt them to their environment (adaptation).

The enzyme aldehyde dehydrogenase (ALDH2*1) helps in the breakdown of alcohol. People with variant form of this enzyme (ALDH2*2) feel unwell and experience flushing when they drink alcohol, and are less likely to become alcoholics. ALDH2*2 more common in Chinese and Koreans (and Thais), allele not present in Germans, Finns, Swedish Lapps or Papua New Guineans. In the absence of natural selection, mutation, migration and genetic drift, and if there is random mating, allele and genotype frequencies in a population will remain the same generation after generation. Mutation, genetic drift and natural selection can cause isolated populations to diverge from one another. Migration retards this divergence. History Darwin (1809-1882): emphasized the importance of variation and heredity in the evolutionary process. Mendel (1823-1884): provided the mechanism for heredity. G. H. Hardy & W. Weinberg laid cornerstone of modern population genetics in 1908. R. A. Fisher, J. B. S. Haldene & S. Wright examined the theoretical consequences of Mendelian inheritance in natural populations (statisticians). T. Dobzhansky (USA): Genetic variation in natural populations and speciation E. B. Ford, P. M. Sheppard and H. B. D. Kettlewell (UK): Genetic polymorphisms in natural populations (e.g. Industrial Melanism). HIV Example Global Pathogens o Black Death (bubonic plague) in Europe (1346) 28 million lives/ 40% o Spanish Flu (Influenza A, H1N1), global, 1918-19. Killed 50-80 million. Biggest single killer ever to hit humans. 3-4 every century. o HIV has infected 60 million people and a third have already died. HIV prevalence is highest in Africa. Followed by South & SE Asia. Low everywhere else. AIDS = 5% of all deaths worldwide. 90 million deaths by 2020 i.e. soon to be biggest killer. HIV is a good example to study the principles of evolution: o Accumulates genetic variation rapidly: error prone reverse transcriptase

o Strong Selective agents: immune system and anti-viral drugs. o These lead to changes in allele frequencies. HIVs gp120 protein binds to the CD4 and CCR5 co-receptor on the host cell. Viral RNA is converted to DNA. Viral DNA is integrated into the hosts genome (retrovirus). HIV targets immune cells (T Cells). No defence for other diseases/viruses etc. Anti-retroviral Drugs o Reverse transcriptase inhibitors: stop RNA DNA AZT is a Thymine with an N3 and not an OH group. When a reverse transcriptase adds AZT instead of T to a growing copy of HIV DNA, synthesis stops. HIV Populations evolve resistance to AZT. Higher concentrations of AZT required to curtail the replication of viruses. o Protease inhibitors: stop the processing of proteins to package the virus (virus takes part of the membrane with them). o Fusion inhibitors: prevent the virus from entering the cell o Integrase inhibitors: block HIV inserting DNA into the host genome. Why the drugs dont work: o The reverse transcriptase is error prone, which results in genetic variation in the virus population. o Some mutations in the active site of reverse transcriptase make this enzyme less likely to add AZT instead of T (i.e. phenotypic variation). o Variation due to mutation, inheritance and differences in survival due to AZT selection, results in a change in the composition of the population over time. Molecular phylogenetics: HOW did HIV enter the human population? o A phylogenetic tree (via DNA sequencing) of HIV suggests that there a 2 entries of HIV (HIV-1 & HIV-2) into the human population. Chimp populations have SIV which is a similar virus to HIV but with more variation. The molecular clock: WHEN did HIV enter the human population? o The ancestor (most recent common ancestor) of the HIV-1 virus was dated back to 1930, therefore HIV-1 was not spread through contaminated polio vaccine in the 1950s. Detecting human resistance: WHERE did the alleles that protect some people come from? The CCR5-32 allele confers resistance to HIV. Resistance common in populations with variation e.g. Scandinavia/Western/Northern Europe. How can we control HIVs evolution of resistance to drugs? The CCR5-32 allele that confers resistance to HIV exists at different frequencies in different human populations. How can we measure the frequency of this allele? o The CCR5 locus. 32 because missing 32 base pairs (deleted section). Therefore the two alleles are of different sizes. CCR5-+ = 350 bp long, CCR5-32 = 318 bp long. How do we determine the CCR5 genotype? o Isolate DNA Amplify the target DNA sequence using PCR Distinguish the different size products (alleles) using gel electrophoresis. o PCR agarose gel electrophoresis final product UV visualisation = 3-4 hours

o Shorter allele moves further (from ve +ve) in gel electrophoresis Diversity of CCR5 genotypes in Human populations o See allele (32) as heterozygote in low populations rather than homozygotes. The diversity of the CCR5 genotypes in the Icelandic population o # of people sampled = 102 o # of alleles sampled = 204 (diploid) o Freq. of genotypes homozygote Heterozygote Homozygote +/+ = .735 +/- = .235 -/- = .029 75 24 3 o Frequency of alleles F(+) = 174/204 = .853 and f(-) = 30/204 = .147 o Testing for H-W equilibrium X2 value = .493. Using X2 table, the probability = .483 (df=1) There is no statistical difference at the 5% level (0.05) The CCR5 locus is in H-W equilibrium in the Icelandic Population AIDS is primarily a disease of developing countries In developing countries heterosexual sex is the primary mode of HIV transmission Will the AIDS epidemic cause the frequency of the CCR5-32 allele to increase in the human population? Depends on 1. The relative fitness of the genotypes 2. The relative frequencies of the alleles in the population Where did the CCR5-32 allele come from? (Why is it common only in Europe?) o This question was addressed by measuring linkage disequilibrium between CCR5 locus and two other loci located nearby on the same xsome. o The nearly loci are short tandem repeat sites called GAAT and AFMB o GAAT has 3 alleles and AFMB has 4 alleles o GAAT and AFMB are noncoding sequences, and they appear to have no effect on fitness. o Almost all xsomes carrying CCR5-32 allele also carry allele 197 at the GAAT locus and allele 215 at the AFMB locus. How did the linkage disequilibrium between CCR5 and its neighbour arise? o 3 possibilities: multilocus selection, genetic drift and population admixture Multilocus selection is unlikely because both GAAT and AFMB are noncoding loci and their alleles appear to be selectively neutral Population admixture is also an unlikely candidate, because it would require a source population in which the frequency of the 32 allele is much higher than it is in Europe, and no such population exists. That leaves genetic drift as the only explanation. At some time in the past, the European population had only one allele for CCR5 (so chromosomes were the haplotype CCR5+ - GAAT197 - AFMB215) and then a mutation occurred that created the 32 allele

The haplotype 32 - 197 - 215 was favoured by natural selection, it rose to a high frequency and carrying with it the neighbouring loci with the alleles 197 and 215 Since this haplotype first appeared, recombination and/or mutation has put 32 into other haplotypes (i.e. linkage disequilibrium is slowly breaking down) Based on rates of crossing-over and mutation it is estimated that the 32 allele first appeared roughly 700 years ago Why is 32 common only in Europe? o Either because the mutation has never occurred outside of Europe, or because when the mutation has occurred outside of Europe, it has not been favoured by natural selection o Only strong selection could have carried 32 from a frequency of virtually zero to 1020% in just 700 years. o Epidemic diseases: During the 14th century bubonic plague, Black Death, killed to of the population.

Genotype frequencies: describe the gene pool quantitatively. Allele Frequencies: genotypes breakdown to alleles when gametes are formed, and alleles, not genotypes, are passed from one generation to the next. Only alleles have continuity and evolution is change of allele frequencies in the gene pool. f(B) = p & f(b) = q. The allelic frequencies for a locus, like the genotypic frequencies = 1.0 i.e. p + q = 1.0 Allele and genotype frequencies in a population will remain the same unless some evolutionary process is operating to change them. Hardy-Weinberg Equilibrium In a population of sexually reproducing diploid organisms, allele, and genotype frequencies will remain constant (at equilibrium) unless some evolutionary process is operating to change them Assumptions 1. There is no natural selection 2. There is no mutation 3. There is no migration 4. Infinite population size (i.e. no genetic drift) 5. Random mating Example o Tracking new alleles through one generation o One locus with 2 alleles: A & a o Frequencies: A = 0.6 & a = 0.4; 0.6 + 0.4 = 1.0 Egg Sperm Zygote Probability A A AA .6 x .6 = .36 Homozygote AA = 0.36 A a Aa .6 x .4 = .24 Heterozygote Aa

a a

A a

aA Aa

.6 x .4 = .24 .4 x .4 = .16

= 0.48 Homozygote aa = 0.16

.36 + .48 +.16 = 1.0 Allele frequencies for the next generation = Allele frequencies do not change from one generation to the next. Hardy & Weinberg provided the mathematical proof using algebra. o Example o Let the frequency of the two alleles A & a be p & q. p + q = 1.0 o If we pick an egg at random the chance is p that it will have the genotype A. When a sperm fertilizes the egg, the chance is p that the sperm will have genotype A. o So, the probability we will witness the production of an AA zygote is p x p = p2 o Therefore random mating in the gene pool produces zygotes in the following proportions AA Aa aA aa 2 p Pq qp q2 i.e. p2 + 2pq + q2 =1.0 We have gone from the allele frequencies of the gene pool to genotypic frequencies among zygotes. o We can follow the alleles to the next generation: p2 + 2pq + q2 o For the frequency of the A allele in the new gene pool, AA will constitute p2 of the gene pool, and Aa will constitute 2pq of the gametes and half of these will carry the A allele. The total fraction of gametes carrying the allele A is: p2 + 2pq = p2 +pq o We can simplify p2 + pq by substituting (1 p) for q: p2 + pq = p2 + p(1-p) = p2 + p p2 =p Conclusions from the Hardy-Weinberg Principle 1. The allele frequencies in a population will not change, generation after generation 2. If the allele frequencies in a population are given by p & q the genotype frequencies will be given by p2, pq & q2. Relationship between genotypes and alleles in a population under H-W equilibrium 1. Heterozygote proportions are maximal when p = q = 0.5 2. Rare alleles are found more often in heterozygotes than in homozygotes H-W can be extended to include more than 2 alleles. o The allele frequencies must add to 1.0 e.g. q + p + r = 1.0 o Then the genotype frequencies are given by: p2 + q2 + r2 + 2pq + 2pr + 2qr = 1.0 and the allele frequencies do not change from generation to generation. Why use the H-W Principle? o This is a null model for how mendelian genes behave in a population o The H-W model is useful because it rests on a specific set of assumptions we made about an idealized population. o

By providing a set of explicit conditions under which evolution does not occur, the H-W can identify w\hen evolution is happening in a real population. How can we test whether a population is in H-W equilibrium? o Using genotype frequencies measured in a natural population (i.e. observed genotypes) we can calculate the allele frequencies p & q. o The allele frequencies can be used to calculate the expected genotype frequencies p2 + 2pq + q2 o The observed genotypes can be compared to the expected genotypes using a simple chisquare test. Difference H-W equilibrium. 2 = (O E)2/E o Differences between the observed and expected will occur by chance. To determine if the differences are of statistical significance, the deviation between the observed and the expected is tested using the chi-square test. o To assess the probability of obtaining a 2 value (= 0. 512) as great as this, we need to determine the degrees of freedom (df) and then look up the 2 value in a statistical table. o The df is the number of classes (3 genotypes) minus 1, minus the number of parameters (allele frequencies) that must be estimated from the data (1 since p + q = 1.0). Thus df = 3 -1 -1 = 1 o Using a 2 distribution, the probability (P) = .47 (> 0.05). There is no statistical difference at the 5% level.

How much genetic variation exists within natural populations? At first glance, it seems that members of a well-adapted population should be highly heterozygous because the most favourable allele at each locus has become fixed. Most populations contain a high degree of heterzygosity This built-in genetic diversity is concealed, so to speak because it is not necessary apparent in the phenotype. Fisher-Wright Equation o = 4Ne Increase population size = increase in variation. The bigger the bucket, the more it can hold A phenotype with no genetic variation will not respond to selection; if there is genetic variation, the phenotype will change over a few generations o E.g. Wild wolves Artificial Selection Domestic dogs. Techniques for measuring genetic variation: proteins and DNA sequences o Allozyme variation (Protein Electrophoresis) Proteins consist of amino acid sequences Some AA carry a positive or negative charge. Overall the protein has a net charge = the sum of AA

AA substituents (DNA mutations) change the net charge, and hence change the rate of migration of a protein in an electric field. 1. Dissect tissues (sample from wild population) 2. Homogenize 3. Centrifuge, collect supernatant 4. Electrophorese 5. Stain gel slice 6. Score population (look at homozygous, heterozygous) Faster alleles move further Single band on gel = homozygous; 2 alleles overlap/move the same 2 bands = heterozygous o Microsatellite DNA Microsatellites have become the most widely used DNA marker in modern population genetics. 1.5% of genome = protein content 3% made up of simple repeats 50% parasitic DNA These DNA markers vary in their number of tandem repeats (i.e. size differences) Tandem repeats of a short sequence motif of one to six nucleotides E.g. TTTTTT or CACACACACA o Polymerase Chain Reaction (PCR) Exponential amplification DNA sequence variation o The bottom line of genetic variation o Sample coding and non-coding genetic variation, and functional as well as silent changes o DNA sequence data can be used to infer genealogical trees o Two main kinds: Direct DNA sequencing and DNA length differences DNA sequencing The same principle as PCR, but only 1 primer, and some of the free nucleotides are fluorescently labelled and modified to terminate synthesis of the DNA strand. Randomly terminates at every nucleotide position of the DNA strand Record colour: refers to which nucleotide sequence terminated at Electrophoresis laser processor chromatogram

What happens to H-W when individuals dont survive at equal rates and have equal reproductive success? Requirements for natural selection o There must be phenotypic variation among individuals o These variations must be heritable (at least to some degree)

These variations must cause differences in reproductive success (# of offspring you can produce determines your success) The nature of natural selection o Acts on individuals, but its consequences occur in populations. (You dont evolve, a population does). o Acts on phenotypes, but evolution consists of changes in allele frequencies. o Populations, not individuals, evolve. Evolution is a statistical change.

Natural Selection In most cases mutations will be deleterious and so lower fitness. These mutations will be removed fairly quickly from the population by purifying or negative selection. At other times a new mutation might have a higher fitness than all others in the population and it is able to multiply more quickly. (NB. Other alleles in the population are not necessarily deleterious). This is called positive selection, and will eventually lead to fixation of the selectively favoured allele in the population o Example of Natural Selection Native organisms appear to be adapting to the presence of an invader. Strong evidence of adaptive changes (reduction in gape size & a steady increase in body length) in Australian native predators (red bellied black snake & common tree snake) as a result of the invasion of toxic cane toads. Changes in allele frequencies Alleles B1 = .6 & B2 = .4 Genotypes B1B1 B1B2 B2B2 .36 .48 .16 1.0 36 48 16 100 zygotes Selection: all B1B1 individuals survive, 75% of B1B2 individuals survive, and 50% of B2B2 individuals survive. 36, 36, 8 (n now is 80) Alleles: B1 = 0.675 & B2 = .325 Example of Selection of an Allele Alcohol dehydrogenase (Adh): breaks down ethanol, a poison from rotting fruit Two alleles at the Adh locus AdhF & AdhS (fast vs. slow alleles). Two populations of flies: (1) with food spiked with ethanol & (2) with non-spiked food. In the tx, the AdhF genotype increased in frequency The alcohol dehydrogenase extracted from the AdhF individuals breaks down ethanol at twice the rate of AdhS individuals. The frequencies of the two Adh alleles in Australian populations of fruit flies AdhS allele is generally at higher frequencies at lower lattitudes (closer to equator).

AdhF allele is at higher frequency at higher lattitudes. The patter is similar in Europe & North America The significance of the pattern is unclear, although it may relate to the fact that AdhS is more stable at higher temperatures. Changes in genotype frequencies Alleles: B1 = .5 & B2 = .5 Genotypes: B1B1 = .25; B1B2 = .5; B2B2 = .25 i.e. 25, 50, 25 (100 zygotes) Selection: 60% of the B1B1 individuals survive; all of B1B2 survive; and 60% of B2B2 survive. 15, 50, 15 .1875, .625, .1875 Alleles B1 = .5 & B2 = .5

Spongy brain diseases CJD, Kuru in PNG, Scrapie in sheep, & Mad Cow Disease are all Spongiform Diseases Caused by rouge prion proteins (PrP) punching holes in brain tissue and the result is neurological deterioration. The tissue develops a sponge-like appearance. (PrP-sen = normal with alpha helices; PrP-res = disease causing with beta sheets). 3 ways for an individual to end up with a prion disease. 1. Eating tissue infected with PrP-res 2. An inherited mutation in the gene that codes for PrP-res 3. PrP-res forms spontaneously Example of selection of a genotype o Kuru is a fatal neurological disorder know only from an epidemic that struck the Fore people in PNG during the last century. Misfolded proteins kill neural cells. o Kuru means shivering, trembling; it describes the initial symptoms of the disease o 1000s of Fore people gave themselves Kuru by eating the bodies of relatives who had died of the disease. Fore funeral rituals: Fore cooked and ate their dead relatives. A practice typically carried out by the Fore women and children who lived apart from men. This explains why men were rarely infected, and why cases appeared within families. o At the height of the epidemic in the late 1950s kuru was killing about 1% of the Fore population each year. Since then cannibalism has stopped and the epidemic has gradually waned. o Kuru can have a long incubation time. The people who come down with kuru today are in their 50s and 60s, which means that they have been harbouring the disease ever since they ate infected tissue as young children. The prion protein locus (PrP) o An individuals genotype at the PrP gene on chromosome 20 influences his or her susceptibility to spongiform diseases (e.g. variants of CJD)

o o

There are two common alleles of the gene, which differ in the AA specified for position 129 of the encoded protein: Met vs Val The genotype frequencies in the Caucasian population: 39% Met/Met, 50% Met/Val & 11% Val/Val. To date every vCJD victim in the Caucasian population has been the genotype Met/Met. Is there a similar pattern of genetic variation for resistance to kuku among the Fore? PrP Allele Frequencies Female Fore Observed allele freq. (n =140) Met: = .48; Val = .52 Expected genotype freq: Met/Met = .23; Met/Val = .5; Val/Val = .27 Test for H-W proportions: Observed: M/M = .22; M/V = .51; V/V = .26 The allele and genotype frequencies among these unexposed females conformed to conclusion 2 of the H-W analysis (hence, no evolution/selection). Secondly, the same analysis was conducted on a sample of 30 older Fore women who had participated in mortuary feasts, but had never contracted kuru. H-W for older women. Expected: Met/Met = .23; Met/Val = .5; Val/Val = .27 Observed: M/M = .13; M/V = .77; V/V = .1 The X2 = 8.55 and P < 0.0034, there is a significant difference between the observed and expected, so the null hypothesis H-W equilibrium was rejected (hence, evidence for evolution/selection). There is striking excess of heterozygotes, and a corresponding deficit of homozygotes among older Fore women. The most plausible explanation is that the homozygotes individuals are susceptible to kuru whereas the heterozygotes are resistant.

Fitness Values and Selection Coefficients A selection coefficient (s) is a measure of the relative intensity of selection on a genotype and is 1 w. The value of s can range from 0 1.0. In some cases s can be represented as a negative or positive. A positive selection coefficient means natural selection favours the variant; negative means it is selected against; zero means it is neutral. Genotype A1A1 A1A2 A2A2 Adults 16 10 20 Offspring 128 40 40 Average offspring 128/16 = 8 40/10 = 4 40/20 = 2 W (fitness) 8/8 = 1 4/8 = .5 2/8 = .25

S (selectn coefficient) Defining Fitness -

11=0

1 .5 = .5

1 - .25 = .75

Fitness is represented as w and is the reproductive success of a genotype. E.g. If A1A1 genotype on average produces 8 offspring A1A2 genotype on average produces 4 offspring A2A2 genotype on average produces 2 offspring Then A1A1 highest reproductive output so fitness = 1.0 (w11) A1A2 fitness = .5 (w12) A2A2 fitness = .25 (w22)

How does fitness (w) fit with p & q? p2w11 + 2pqw12 + q2w22 = w (mean fitness in the population)

Fitness values 1. w11 = w12 = w22 = 1 No selection 2. w11 = w12 < 1 & w22 = 1 Against the dominant allele (directional) 3. w22 < 1 & w11 = w12 = 1 Against the recessive allele (directional) 4. w11 < w12 < 1 and w22 = 1 Heterozygote intermediate (directional) 5. w11 < w22 < 1 & w12 = 1 Heterozygote favoured (stabilising) 6. w12 < w11, w22 = 1 Homozygotes favoured (disruptive) - Selection can have a wide range of effects o Eliminates genetic variation, but it can also maintain genetic variation o Changes allele frequencies, but it can also prevent allele frequencies from changing o Produces genetic divergence between population, but it can also maintain genetic uniformity. Which of these effects occurs depends primarily of the relative fitness of the genotypes and on the relative frequencies of the alleles in the population. - Strong selection can quickly change allele frequencies - Different Patterns of Selection o Selection on Recessive and Dominant Alleles Selection is most potent when it is acting on common recessive alleles (and rare dominant alleles)

Selection on Heterozygotes and Homozygotes Selection can act to maintain two alleles at a stable equilibrium When heterozygotes have inferior fitness, one allele tends to go to fixation while the other is lost. However, different populations may lose different alleles. o Frequency-dependent Selection Selection can maintain two alleles in a population if each allele is advantageous when it is rare. Addictive effect: The heterozy has a mean phenotype exactly intermediate between the 2 homozys. For example, if the mean enzyme levels for the genotypes FF, FS & SS at the Adh locus are 100, 95 & 90 units of activity, respectively. Dominant cant go to fixation because recessive allele is hidden in heterozygote Selection of Heterozygotes o Heterozy advantage: balancing selection, overdominance or Heterosis o This is an equilibrium of allele frequencies when the heterozygote (Aa) has the highest fitness o Maintains genetic variation in a population (i.e. both alleles) o E.g. Sickle-Cell Anaemia in the human population. Sickle-Cell Anaemia o Common in West Africa & Mediterranean o Recessive genetic disorder (ss) o Abnormal haemoglobin molecules: instead of remaining soluble in the cytoplasm, they aggregate to form long fibers that deform the RBC. o The deformed cells (sickle shaped) clot small blood vessels and reduce oxygen flow to tissues. Untreated it can be lethal. o Sickle cells are fragile. WBC consume the remains o Sickle cell haemoglobin is distinguished by a single AA substitution (GAG glutamic acid to GTG valine) from normal haemoglobin (H). HH normal RBCs; Hs mild form of sickle cell anaemia; ss severe anaemia o However, if heterozygotes (Hs) are infected by the malaria causing protozoan, the cells are broken down more rapidly than normal homozygotes (HH). o Heterozygote advantage arises from a balance of opposing selective factors (i.e. anaemia & malaria). Heterozygote advantage (overdominance) maintains variation Heterozygote disadvantage (underdominance) eliminates variation; different populations may lose different alleles.

Frequency-dependent selection Selection can also maintain two alleles in a population is each allele is advantageous when it is rare. o Yellow and purple colour polymorphism exists in populations if European Elderflower orchids. They are pollinated by newly emerged nave bumblebees, but the bees are always disappointed because these plants are rewardless; they have no nectar.

How can 2 distinct deceptive advertisements persist together in Elderflower orchid populations? Bumblebees tend to alternate between colours If a bee visits a purple colour first and finds no rewards, it looks next in a yellow Bumblebees tend to visit equal numbers of yellow and purple flowers, orchids with the less common of the 2 colours receive more visits per plant If more pollinate visits translates into higher reproductive success, then the rare colour has a reproductive advantage Selection by bumblebees favours yellow until it becomes too common, then it favours purple.

Mutation Mutation can change allele frequencies. o E.g. linear increase in the frequency of fhuA mutants in a culture of E. Coli, due to recurrent mutation. The mutants are detected by resistance to the bacteriophage T5. The mutation rate is estimated as the slope, in this case 7.2 x 10-8 per generation initially, and 6.6 x 10-7 per generation after the addition of caffeine. DNA damage; how do mutations arise? 1. Errors during DNA replication 2. Spontatneous DNA damage 3. Induced DNA damage o Cells have several DNA repair systems but every so often a mistake is a made E.g. Certain parts of the DNA molecule are prone to spontaneous damage by water (hydrolytic attack) and free radicals (oxidative damage). Very unstable in the presence of oxygen and water. There are 2 types of DNA changes: Transitions and Transversions o Transition type changes (AG & CT; purinepurine & pypy) are far more common than transversion type changes (AC, AT, GC & GT) because the physical changes to the structure of the nucleotide are more simple. There is lots of DNA damage per lecture o E.g. depurination, depyrimidation, deamination (modification of the C base), singlestranded breaks, pyrimidine dimers (T-T) in skin (noon sun) & single-strand breaks from background ionising radiations. Sunburn o Exposure to UV light (from the sun, tanning lamps etc) causes adjacent thymine molecules to pair with each other. This damage halts the replication and transcription process, and can result in cell death. Forms a T-T dimer, rather than hydrogen bonding with A on other strand. Types of mutations o Silent mutation (synonymous): redundancy in the genetic code. More than one possible set of nucleotides code for the same AA. E.g. GTC (Val) GTA (Val)

Replacement mutation (non-synonymous): change in AA E.g. GTC (Val) TTC (Phe) o Nonsense mutation: AA stop codon E.g. AAG (Lys) TAG (stop) o Some mutations are neutral (e.g. synonymous mutations) and others can affect the function of the DNA sequence. Mutation changes in Allele frequencies o Alleles: A = .9; a = .1 o Genotypes: AA = .81; Aa = .18; aa = .01 = 1.0 o Mutation: converts 1 of every 10000 copies (0.0001) of allele A into a new copy of a each generation. The frequency of A in the next generation is p = p - p, and the frequency of a is q = q + p, where is the mutation rate. o Alleles: A = 0.9 (0.0001)(0.9) = 0.89991; a = 0.1 + (0.0001)(0.9) = 0.10009 Over very long periods of time, mutation can eventually produce appreciable changes in allele frequencies. o E.g. = 0.0001 (1 per 10000 per generation). Over 1000 generations, frequency of A has gone from 90% to 81%. Mutation-Selection Balance o In combination with selection, mutation becomes a potent evolutionary force o Most mutations are deleterious and selection acts to eliminate them from a population o Deleterious mutations can persist, however because they are continually created anew o When the rate of elimination by selection equals the rate at which new copies are being created by mutation, the frequency of the allele is at equilibrium o If an allele is recessive, its equilibrium frequency is given by q = (/s). Where is the mutation rate, and s is the selection coefficient. If s is small (the allele is only mildy deleterious) and is high, then the equilibrium frequency of the allele will be relatively high. If s is large (the allele is highly deleterious) and is low, then the equilibrium frequency of the allele will be low. Mutation-Selection Balance example: Spinal Muscular Atrophy o Weakness and wasting of the muscles that control voluntary movement o Caused by deletions in a locus called telomeric survival motor neuron gene (telSMN) located on chromosome 5 o The secong most common lethal autosomal recessive diseasenin Caucasions after cystic fibrosis o Collectively the loss-of-function alleles of telSMN have a frequency of about 0.01 and s has been estimated at 0.9 o With such strong selection, how to these alleles persist at a frequency in 1 in 100. o q = (/s). q = .01, s = 0.9 then = .9x10-4 mutations per telSMN allele per generation. o Analysis of 340 individuals with SMA later showed that 7 affected individuals carried new mutations that were not present in either parent. o The estimated mutation rate for these recessive alleles is 1.1 x 10-4

Are the alleles that cause cystic fibrosis maintained by a balance between mutation and selection? o Cystic fibrosis is caused by recessive loss-of-function mutations in a locus on chromosome 7 that encodes a protein called the cystic fibrosis transmembrance conductance regulator (CFTR) o CFTR is a cell surface protein expressed in the mucus membrane lining the intestines and lungs o CFTRs key function is to enable cells of the lung lining to ingest and destroy Pseudomonas bacteria o These bacteria cause chronic lung infections and lead to severe lung damage o Until recently, few affected individuals survived to reproductive age; those that do survive are often infertile. o The alleles that cause cystic fibrosis have a collective frequency of about 0.02 among people with European ancestry. o If we assume s is 1, then would have to be 4 x 10-4 o But the actual mutation rate is about 6.7 x 10-7 o Therefore a steady supply of mutations cannot explain the maintenance of cystic fibrosis alleles at 0.02. (Why is there such a high freq? It is NOT the mutation rate). o Hypothesis: cystic fibrosis heterozygotes are resistant to typhoid fever. Salmonella typhi bacteria infiltrate the gut using CFTR proteins as entry points. The freq of the allele, among CF mutations, in the generations that follow a typhoid fever outbreak increases with the severity of the outbreak.

Migration Migration, in an evolutionary sense, is the movement of alleles between populations (gene flow). The mechanisms range from occasional long-distance dispersal of juvenile animals to the transport of pollen, seeds, or spores by wind, water or animals The actual amount of migration varies enormously among species and depends on the mobility of individuals or propagules. Mainland-island (source sink). Large population (e.g. mainland) 1-way migration to islands (little migration back). o Migration changes allele frequencies o Alleles (island): A1 = 1.0 & A2 = 0.0 (mainland only has this allele). o Genotypes: A1A1 = 1; A1A2 = 0.0; A2A2 = 0.0 o Migration: 800 zygotes, develop to adults. The mainland population is fixed for A2 and 200 individuals migrate to the island (i.e. now 80% = island individuals & 20% = mainland individuals). o Genotypes: A1A1 = .8; A1A2 = 0.0; A2A2 = 0.2; heterozygote deficiency is an early sign of recent migration. o Alleles: A1 = .8; A2 = .2

The Wahlund Effect: A single bout of random mating will put the population back into HW equilibrium for genotype frequencies Alleles: A1 = .8; A2 = .2 Genotypes: A1A1 = .8; A1A2 = 0.0; A2A2 = 0.2 Expected genotypes: A1A1 = .64; A1A2 = .32; A2A2 = .04; o The frequency of the A1 allele in the island population after migration pI = mpM + (1-m)pI Where m is the proportion of migrants that make up the new island population. (Rate of migration). The frequency of A1 after migration is determined by the proportion of A1 alleles in the two groups that now make up the island population. The change in allelic frequencies from migration depends on two factors: p = m(pM pI) M = the proportion of migrations in the final population Pm - Pi = the difference in allelic frequency between the migrants and the residents. If Pm = Pi = 0 Populations must differ in their allelic frequencies for migration to affect the make-up of the gene pool. With continual migration Pm and Pi become increasingly similar and as a result, the change in allelic frequency due to migration decreases. Island model: complex rates of migration between islands Linear stepping stone model: A B C D Two dimensional stepping-stone model Metapopulation Power of dispersal varies between species Example of Migration o Water snakes of Lake Erie, Ohio. o Live on the mainland surrounding Lake Erie and on islands in the lake. o Banded, unbanded or intermediate. o On islands snakes bask on limestone rocks at the waters edge. o Very young unbanded snakes are more cryptic than banded vulnerable to predation. o Mark-recapture studies showed unbanded snakes have higher survival rates o However, why are the islands not all unbanded? o Every generation several banded snakes move from the mainland, contributing to the gene pool. o (Evolutionary forces opposing each other. Migration preventing the island population diverging from the mainland population). Migrationis a potent force of evolution In practice, migration is most important in preventing populations from diverging Gene flow between populations creates a larger single population increase the effective population size (Ne).

The introduction of new alleles into a population from another species (hybridization) or subspecies is called introgression.

Genetic Drift Genetic drift is random variation of allele frequencies from one generation to the next In populations of finite size, chance events in the form of sampling error in drawing gametes from the gene pool can cause evolution Selection is differential reproductive success that happens for a reason; genetic drift is differential reproductive success that just happens. Genetic drift is most important in small populations A model of genetic drift o Randomly select gametes to make 10 zygotes (with replacement)A1 = .6; A2 = .4; 100 gametes in the gene pool: o A1A1 = .6; A1A2 = .2; A2A2 = .2 o A1 = .7; A2 = .3 o Range of possible outcomes Overall probability that new frequency is less that .6 is ~ 40.5% Probability that new frequency is exactly .6 = 18% Overall probability the new frequency is greater than .6 ~41.5% Genetic drift and population size o Drift is fundamentally the result of population size o These graphs show a stimulation of drawing alleles from a gene pool, run three times o Drew out a total of 250 zygotes and recorded the frequency of allele A1 as it proceeded o At first the new frequency of A1 fluctuates considerably, in a unique trajectory for each run o As the number of zygotes made increases, the new frequency of A1 settles towards the expected value of .6 Sampling Error as a mechanism of Evolution: The Founder Effect o When a new population is founded by a small number of individuals, it is likely that chance alone will cause the allele frequencies in the new population to be different from those in the source population o New populations can be founded by new arrivals to an area, or by the suvivors of a larger pre-existing population (e.g. after a natural disaster) through a bottleneck. Founder Effect Example o Silvereyes are small songbirds native to mainland Australia and Tasmania o Five times in recent history, naturalists documented their migration to new islands. o Allelic diversity has declined along the Silvereyes route of travel o The decline from any one population to the next is so small as to be statistically undetectable o But because they were sequential, these declines in diversity add up o The Norfolk Island population harbours 60% of the allelic diversity present in Tasmania, and just over half that on the mainland.

The Random Fixation of Alleles and Loss of Heterozygousity o Under genetic drift, every population follows a unique evolutionary path o Genetic drift is rapid in small populations and slow in large populations o If genetic drift is the only evolutionary force at work, eventually one allele will drift to a frequency of 1 (to fixation) and all other alleles will be lost. o Alleles drift to fixation or loss, the frequency of heterozygotes in the population declines Effective Population Size (Ne) o The equivalent number of adults contributing to the next generation o If sexes are equal in number and all individuals have an equal probability of producing offspring, the effective population size equals the number of breeding adults in the population: The census population size (N). o However, when males and females are not present in equal numbers, the effective population size in: Ne = (4 x Nf x Nm) / (Nf + Nm) o Where Nf = # of breeding females & Nm = # of breeding males Effective Population Size (Ne) o Females, as a group, contribute half to all genes to the next generation and males, as a group, contribute the other half. o E.g. consider a population of 70 females and 2 males. They are not genetically equivalent. Each male contributes x - .25 of the genes to the next generation, whereas each female contributes x 1/70 = 0.007 of all genes. Ne = (4 x 70 x 2) / (70 + 2) = 7.8, or approximately 8 breeding adults. o Therefore in a population of 70 females and 2 males, genetic drift occurs as if the population have only 4 breeding females and 4 breeding males o Genetic drift will have a much great effect in the above population than in one with 72 breeding adults equally divided between males and females. A randomly mating population is called a panmictic population Non-random mating o Inbreeding: Mating between related individuals; that is, between individuals have a recent common ancestor. Decreases the frequency of heterozygotes and increases the frequency of homozygotes compared to expectations under H-W assumptions Most extreme example of inbreeding is self-fertilisation or selfing Alleles: A1 = .5; A2 = .5 Genotypes: A1A1 = .25; A1A2 = .5; A2A2 = 0.25 (250; 500; 250) Self-fertilization: A1A1 produce A1A1 offspring; A1A2 produce 1:2:1 offspring; A2A2 produce A2A2 offspring. Genotypes: .375; .25; .375 (375; 250; 375) Alleles: A1 = .5; A2 = .5 Coefficient of inbreeding (F) Inbreeding less extreme than self-fertilization can occur, for example, mating with close relatives.

F = the probability that the 2 alleles in an individual are identical be descent. F is computed using a pedigree. Adding inbred genotype frequencies to H-W equation A1A1 p2 (1-F) +pF; A1A2 = 2pq (1-F); A2A2 = q2 (1-F) + qF F = 0, gives the original H-W ration, and F = .5 represents selfing in a diploid system. Inbreeding depression usually results from the exposure of deleterious alleles as homozygotes Inbreeding depression refers to the effects these alleles have on the average fitness of offspring in the population Some of the mechanisms evolved to avoid inbreeding: mate choice, selfincompatibility, and dispersal. Assortative mating (positive assortative matine): when two mating individuals are phenotypically more alike than two individuals chosen at random E.g. light and dark phases of the Artic skua mate assortatively Fruit flies from different geographic regions sometimes mate assortatively under lab conditions Plants that flower early in the season tend to pollinate and be pollinated by other early-flowering plants, and the same is true for late-flowering plants. Disassortative mating (negative assortative mating)L when mates are phenotypically less alike than two individuals chosen at random. E.g. rare in most animal populations Certain mating systems in plants and fungi are disassortative (e.g. left and right anthers & styles). Nonrandom mating does not, by itself, cause changes in allele frequencies (evolution), nonetheless it can have profound effects.

Evolution at multiple loci: linkage disequilibrium Evolution at Two Loci o So far we have considered evolution at one locus, we will now expand on this and consider two loci simultaneously o In a two locus system we are concerned with tracking both allele frequencies and chromosome frequencies. o In our example there can be 4 different chromosome genotypes: AB, Ab, aB and ab o The multilocus genotype of a chromosome or gamete is often referred to as its haplotype. If there is no site of recombination between them, then together they are haploid. o We could focus on any pair of loci in an organisms genome, but we will focus on a pair of loci located on the same chromosome. o Oue goal is to determine whether selection at the A locus will interfere with our H-W models to make predictions about evolution at the B locus.

The answer will be: sometimes depending on whether the loci are in linkage equilibrium or linkage disequilibrium o When a genotype at one locus (A with alleles A and a) is independent of genotypes at another locus (B with alleles B and b), the loci are in linkage equilibrium. o When a genotype at one locus is dependent on genotypes at another locus, the two loci are in linkage disequilibrium. o A pair of populations can have identical allele frequencies but different chromosome frequencies. o Another way to see the difference between 2 populations is to calculate the frequency of allele B on chromosomes carry allele A versus chromosomes carrying allele a. Population 1: Allele B on A chromosome: 12/15 = .8; Allele B on a chromosome: 8/10 = .8 Population 2: Allele B on A chromosome: 11/15 = .73; Allele B on a chromosome: 9/10 = .9 Allele B is more often found with allele a o Linkage equilibrium: knowing the genotype of the xsome at one locus is of no use at all in predicting the genotype at the other locus o Linkage disequilibrium: if we know the genotype of a xsome at one locus, it provides a clue about the genotype at the other. E.g. Allele B occurs with a frequency of 0.9, hence more often than B at a frequency of 0.73 with A. Linkage Equilibrium defined 1. The freq of B on xsomes carrying A is equal to the frequency of B on xsomes carrying a 2. The freq of any xsome haplotype can be calculated by multiplying the frequencies of the constituent alleles (e.g. frequency of AB xsomes can be calculated by multiplying f(A) allele and f(B) allele) 3. The coefficient of linkage disequilibrium (D) is equal to zero. D is calculated by the frequencies of xsomes fABfab fAbfaB = D o Maximum value of D = 0.25, when AB and ab are only xsomes present and each is at a frequency of .5. Minimum value of D = -.025, when Ab and aB are the only xsomes present and each is at a frequency of .5 What Creates Linkage Disequilibrium in a Population? o Three mechanisms can create linkage disequilibrium in a randomly-mating population Selection on multilocus genotypes Predators catch and eating every individual whose size is less than x (gets rid of genotypes with small #s) ??? Genetic Drift (a) Xsome frequencies in a population of finte size in which only one allele, A, is present at locus A. Both loci are in linkage equilibrium

(b) A mutation occurs on a single Ab xsome and converts allele A into allele a. This put the population into linkage disequilibrium because there is no xsome haplotype aB. However, in a population of infinite size (i.e. without drift) this mutation would not happen once, but with many times each generation, on both AB and Ab xsomes. At no point would aB be missing. In diagram (c) selection favours allele a over allele A, so that a increases in frequency and A decreases. This increases the degree of linkage disequilibrium between locus A and locus B. Population admixture (act of populations mixing) What Eliminates Linkage Disequilibrium from a population? o Sexual reproduction (meiosis with crossing over and outbreeding) reduces linkage disequilibrium o The union of gametes from unrelated parents brings together xsomes with different haplotypes. When the zygotes grow up to adulthood and themselves reproduce, crossing-over breaks up old combinations of alleles and creates new ones. o Recombination tends to randomize genotypes at one locus with respects to genotypes at another, decreasing and increasing the frequency of over- and under-represented haplotypes, respectively. Curves showing decline in linkage disequilibrium, according to the equation D = D(1 r), for different values of recombination (r). With r =.5, which corresponds to the free recombination of loci, the population reaches linkage equilibrium in less than 10 generations. The further apart a pair of loci are, the less likely it is that they will be in linkage disequilibrium. When an allele at a coding locus is in linkage disequilibrium with alleles at nearby neutral loci, we can infer that the coding allele is relatively young When a young allele is at high frequency, we can infer that is has recently been favoured by positive selection (CCR5 example). Linkage disequilibrium is a nonrandom association between genotypes at different loci Selection on one locus can alter allele frequencies at another locus Multilocus selection, genetic drift and population admixture create linkage disequilibrium, and sexual reproduction (recombination) reduces linkage disequilibrium Measurements of linkage disequilibrium are useful for inferring the history of alleles

Gene Genealogies Population geneticists traditionally relied upon analyses of gene frequencies to determine which evolutionary process has shaped the genetic structure of populations However, in the case of DNA sequence data genetic variation is much more usefully presented as gene genealogies

A gene genealogy describes the ancestral relationships of the sequences sampled from population. As we continue backwards in the time the # of lineages is reduced by one at each coalescence, creating nodes in the tree. This backwards pruning of lineages gives rise to a bifurcating genealogy However, recombination can complicate a genealogical analysis as it means that different portions of the gene sequence have different genealogical histories The coalescent process o The branching structure of a genealogy is influenced by natural selection and genetic drift o If we study gene genealogies carefully, particularly the different branching events, we can say something about what processes have given populations their present-day structure. o Coalescent theory tells us what genealogies of neutral alleles are expected to look like if populations have different demographic histories (i.e. population size and structure) o Genetic drift: changes in population size affect the number of lineages; in a expanding or large population more lineages will persist o Natural selection (non-neutral): fit alleles will produce more copies of themselves, so producing more branches of that lineage Case Study: The population genetics of human origins o There are two evolutionary scenarios concerning the origin of (anatomically) modern human populations: 1. Multiregional Model: Different regional populations (races) of Homo sapiens evolved independently from their ancestor H. erectus, long ago in different parts of the old world. a. Neanderthals are thought to be ancestors of modern Europeans 2. out-of-Africa Model: H. sapiens arose only once, and relatively recently, in Africa a. Neanderthals contributed little to the Human European gene pool, and were replaced by invading humans who originated in Africa o Mitochondrial Eve mtDNA is maternally inherited and typically does not undergo recombination The deep divergence of African lineages indicates an old population: supports out of Africa hypothesis (more genetic variation accumulated in Africa) Sequences from Asians, aboriginal Aussies, Europeans and New Guineas generally are scattered throughout branch B of the gene tree The African woman (called Eve) who was the Most Recent Common Ancestor (MRCA) of all modern human mtDNAs lived ~170,000 years ago, based on a molecular clock estimate. The Hierarchical Nature of Trees o Macro-scale -> micro-scale (e.g. pedigree)

Evolutionary Trees o The evolutionary history of a group of species is called its phylogeny o A phylogenetic tree describes the pattern, and in some cases the timing, of events that occurred as species diversified o It records the sequence that lineages appeared and documents which taxa are more closely related. Parsimony Analysis of DNA Sequence Data o (Cow + Deer + (Whale + Hippo)) (Whale + Hippo + Pig + Peccary) The Molecular Clock o It is thought that some types of DNA sequences evolve at a constant rate, giving rise to a molecular clock o Clock-like evolution many enable us to estimate times of species divergence simply y comparing their gene sequences o This does not mean that all genes and proteins evolve at the same rate, ad a long-term average rate does not exclude the possibility of shirt-term rate fluctuations. o The tick of the clock is estimated by measuring the genetic distance between species and dividing it by the time their ancestors first appeared in the fossil record. This gives a maximum estimate of the rate, because they could have diverged before the time we see them in the fossil record. o For deep phylogenies the gene needs to encode a product with essential functions and thus be subject to strong stabilising selection. The small-subunit ribosomal RNA: in all organisms their function is to translate the genetic code (this is universal) A gene genealogy for the Influenza A (flu) virus o The host species is indicated for each viral strain was isolated, and the year o How do flu viruses cause global epidemics? Human strains and bird strains simultaneously infect a pig, swap genes and later move from pigs to people o Also provides a forecasting method for the next flu virus and helps make your next flu shot.

Selection Can be a major force driving allele frequency change, and leads to adaptation. Some individuals are more successful at surviving and reproducing than others. Selection on alleles and genotypes Recessive & dominant Homozygotes and heterozygotes Frequency-dependent selection Types of selection: directional, stabilizing & disruptive W is for FITNESS Alcohol soaked fruit for Adh flies Heterozygous Fore women can eat their dead relatives

Bumblebees alternate between flowers; if youre rare youre a success

Migration Gene flow from other populations can alter allele frequencies Depends on the frequency in the source population and the fraction of migrants The force can retard the divergence of populations Genes for camouflage water snakes arent fixed because of migration

Mutation Creates new alleles in a gene pool and segregation and independent assortment shuffles them into new combinations (Variation of phenotypes). Can create new alleles in a gene pool or be recurrent mutations Typically a weak force Mutation-selection balance Spinal Muscular Atrophy Cystic Fibrosis and typhoid

Genetic Drift Causes random changes in allele frequency especially in small populations Random changes in allele frequency (gamete sampling error) Dependent on the effective population size (Ne). Non-random mating (inbreeding) reduces the number of heterozygotes but does not change allele frequencies. Silvereyes bottleneck as they spread through NZ

Change in allele frequencies (=evolution) Selection: Can be a strong force and cause the widest range of outcomes (w and initial allele frequency) Mutation: Weak force, often mixed with selection & drift Migration: Strongest when the difference btw pops is great Random drift: Chance effects greatest in small populations Nonrandom mating: inbreeding eliminates heterozygotes. Inbreeding depression is when hidden deleterious recessive alleles are exposed as homozygotes. Linkage disequilibrium: indicates young alleles (before recombination breaks them up) or selection of haplotypes Gene Genealogies: the shape of the tree is the result of historical evolutionary forces (eg. pop size, or selection) HIV: selection of the 32-CCR5 allele Fruit flies: selection on the AdhF allele

Kuru (killer prion proteins): selection on a genotype Sickle cell anaemia: heterozygote advantage Elderflower orchids: frequency-dependent selection Spinal muscular atrophy: mutation-selection balance Cystic fibrosis: heterozygote advantage Water snakes: migration Silvereyes: random genetic drift (founder effect) 32-CCR5: linkage disequilibrium Human evolution: gene genealogies In the past, humans, like all mammals, lost the ability to digest lactose after weaning. The lactase gene gets "switched off", and they became "lactose-intolerant." Today, most of the world's population is lactose-intolerant. However, certain human populations/cultures (those with history of diary farming) have mutations that allow them to have "lactose persistence" - that is, mutations that cause the enzyme to remain "switched on" throughout adult life. Where have all the fish gone? Today, fishing is the dominant source of mortality in most commercially exploited fish stocks. Evolutionary theory predicts that increased mortality typically selects for fish that mature younger and smaller Conservation Genetics: NZ Hector Dolphin (~7000): Striking degree of differentiation among females in the four regions (estimated to be less than one female migrant per generation) Microsatellite DNA show low diversity and strong genetic differentiation of the North Island population, but considerably weaker genetic differentiation among the three South Island populations Population genetic data was the basis for finding the distinct NI population and naming it a new sub-species (Mauis dolphin)

The enzyme aldehyde dehydrogenase (ALDH2) helps in the breakdown of alcohol People with a variant form of this enzyme (ALDH2*2) feel unwell and experience flushing when they drink alcohol, and are less likely to become alcoholics Explain how the four forces of evolution might give rise to this pattern of genetic variation among humans?