1. = 2 = 8 = 0 = 14 Rose and Petals game = =

2. Assignment 1 Peer-reviews due today I will email everyone reviews of their draft proposals When you revise your proposal, please include a separate document that includes a point-by-point response to substantial comments raised in the reviews Assignment 2 Meetings to go over experimental design and analysis this week – if your group hasn’t organized a meeting with me, do that today Each group needs to prepare a ~12 minute presentation to the class for February 24. This presentation should: –Identify the specific question you’ll focus on –Convince the audience that the question hasn’t yet been comprehensively answered and that it is worth answering (what is its significance?) –Present your proposed experimental design. Include: a hypothesis; a prediction, the independent variables that you will manipulate (e.g., population size and mutation rate;) the dependent variable(s) that you will observe (e.g., rate of fitness increase); replication; etc.. Note that you will have to ‘operationalize’ (convert from abstract to something practical) your measure of rate of fitness increase – e.g., you might measure fitness change over the first 25,000 time units or time taken to evolve X number of logic functions. –Identify and justify any aspects of your design that are not straightforward – you might want to elicit feedback –Propose a strategy for analysis of experimental outcomes to answer your original question. You should aim to give critical feedback to the presenting group

3. To know what we need to know to conduct a study with the same goal as: de Visser et al. (1999) Diminishing returns from mutation supply in asexual populations. Science 283: 404- 406 o The experiment manipulates supply of beneficial mutations (population size, mutation rate and degree of initial adaptation) and measures the effect of these changes on the rate of adaptation o We need to know: (i) the basics of mutation and how it can be manipulated, (ii) how mutation supply is expected to influence adaptation, (iii) the basics of an evolution experiment, (iv) how the rate of adaptation can be measured, and (v) how the data that is generated can be analyzed to evaluate the hypothesis. How did the authors approach the design and analysis of the experiment? Are there any aspects of the study you think should be improved? If so, can you suggest how those improvement could be made? This week… Read by Thursday

4. Questions that motivate this paper We see high mutation rates in lots of microbes – especially in disease causing bacteria. Why?  Can we understand the circumstances under which high mutation rates provide an advantage (E.g., increases the rate of adaptation)?  What are the implications of pathogens having high mutation rates – does it make it easier for them to evolve drug resistance or the ability to cause disease?

5. de Visser et al. (1999) Science 284: 404-406 Observation: A high frequency of ‘mutators’ in many natural populations What is known…

6. Disease causing bacteria are often ‘mutators’ – why? Does it matter? Hall and Henderson-Begg (2006)

7. de Visser et al. (1999) Science 284: 404-406 Observation: A high frequency of ‘mutators’ in natural populations Inductive Conclusion: A possible explanation for high frequency of mutators Complications: Unknowns whose influence on the inductive conclusion should be examined Complication I: Genetic linkage complicates assignment of cause and effect Note that the Inductive Conclusion (probably true) and follow up presentation of complications (why it might not be true) effectively serves to: (i) identify the research question and (ii) identify gaps in our knowledge and why it is important they are filled

8. de Visser et al. (1999) Science 284: 404-406 Complications: Unknowns whose influence on the inductive conclusion should be examined Complication 2: Interference between competing mutations Predictions: 1. Increasing mutation supply will increase the rate of adaptation if a population has a limited supply of beneficial mutations 2. Increasing mutation supply will not increase the rate of adaptation of a population that is not limited by its supply of beneficial mutations 3. A fitter starting populations will have fewer beneficial available to it so will be mutation limited at a higher mutation supply than will a less fit population

9. Evolution by natural selection is the process that drives the origin and success of the great variety of species we see around us The basic requirements of evolution by natural selection are very simple: 1.Variation in fitness 2.Competition between variants 3.Differential production of offspring by variants 4.Inheritance of beneficial traits between parents and their offspring Without mutation we’d still be self-replicating molecules in a primordial soup! Why does mutation affect evolution? The ultimate source of variation is mutation! http://www.phidelity.com/photos/v/Artwork/Fractal+Flames/primordial_soup.jpg.html

10. Mutations are changes in an organisms genetic material (usually DNA/RNA) atgtatcatcatcatcat met tyr his atgtaacatcatcatcat nonsense met stop his … atgtaccatcatcatcat synonymous/sile nt met tyr his … atgtgtcatcatcatcat non- synonymous/misse nse met cys his … mutation t atgatcatcatcatcat indel – frameshift (here a deletion) met thr … We can classify mutations in several different ways (e.g., the effect they have on a protein, the type of change to the DNA sequence) Here we are mostly concerned about the effect of a mutation on the fitness of an organism – does a mutation increase (beneficial), decrease (deleterious) or have no effect (neutral) on fitness

11. The basis of mutational change: mechanisms of mutations - spontaneous Spontaneous mutations: changes in DNA sequence that happen without any external physical or chemical cause DNA polymerase incorporates the wrong nucleotide into a growing DNA strand about every 10 4 – 10 5 base pairs (more later) Transitions can occur when rare tautomeric forms (= alternative structures) of bases are produced – these forms pair with a base different from the original E.g., the usual keto (RC(=O)R’) form of guanine can sometimes convert to an enol (RC(OH)R’) form that pairs with thymine (instead of cytosine)

12. Sanders Fig. 12.11 (edited) Original matched base pair The basis of mutational change: mechanisms of mutations - spontaneous

13. Mutations can also be induced by external agents (“mutagenic” chemicals, UV, radiation, etc.) Mutation – induced

14. The basis of mutational change: repairing mutations If mutations are constantly introduced into DNA, how do organisms remain reasonably ‘error-free’ from generation to generation? Most biological molecules are replaced if they become damaged, new molecules will take their place – DNA is not replaced, it needs to be repaired There are many biological mechanisms that act to repair DNA

15. The basis of mutational change: repairing mutations How important is error repair? By itself, DNA polymerase incorporates a wrong nucleotide every 10 4 -10 5 base pairs Many DNA polymerases have an intrinsic ‘proofreading’ ability (3’-5’ exonuclease) that allows them to remove mispaired bases Proofreading increases DNA polymerase fidelity to one wrong nucleotide per 10 6 -10 7 base pairs (i.e. a 100-fold improvement) https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Polymerase_Figure2.jpg

16. The basis of mutational change: repairing mutations DNA polymerase makes ~1 mutation per 10 6 -10 7 base pairs but actual mutation rates are much lower than this – usually ~1 mutation per 10 9 -10 10 base pairs – error correction systems account for this ~1000-fold improvement mutation rate (changes from parent to offspring per base pair) Bioessays 2000 each point shows an experimental estimate of the mutation rate for a given species actual mutation rates are much lower than expected from DNA polymerase alone in E. coli we expect about 1 mutation per 500 cell divisions; in humans we expect about 1 mutation every generation (although the mutation rate per base pair is similar, humans have larger genomes) RNA viruses have no proofreading or error correction This difference is due to error correction This difference is due to polymerase proofreading

17. Repairing mutations – methyl mismatch repair (MMR) Several repair pathways recognize damaged bases and direct removal and repair What if the mutation is a mismatch of two normal bases (e.g. G-T, A-C)? How can the cell determine which base is the error and which base is supposed to be present? The mismatch repair (MMR) system allows this to be accomplished…it can distinguish the original from the new base – and remove the new base In E. coli and many other bacteria: Genomes contain ‘GATC’ motifs ~256 bases apart (4 4 ) The ‘A’ in these motifs is methylated by the enzyme Dam (DNA adenine methyltransferase) There is a lag of a few minutes after a strand is synthesized before methylation occurs – during this time only the old strand is methylated

18. Repairing mutations – methyl mismatch repair (MMR) The crucial condition for distinguishing new and old DNA strands is that the Dam enzyme takes some time to find and methylate GATC sites – when only one strand is methylated this can be recognized as the newly synthesized strand The mismatch repair systems starts with MutS recognizing a mismatch and binding to the DNA helix MutS recruits other enzymes including MutH, which binds to a nearby ‘GATC’ site to distinguish the new and old DNA strands The new strand is removed between MutS and MutH – this gap is then repaired If a gene involved in MMR is non- functional, fewer errors will be fixed and the mutation rate will be increased (a “mutator”) for a few minutes after a GATC site is replicated only the old strand will be methylated (hemimethylated) – this fact is used by the MMR system to distinguish the new and old strand

19. Adaptation describes how well an organism does in its environment – quantified as the relative number of its offspring that have offspring themselves How does mutation influence adaptation? Genetic mutations sometimes change the phenotype of an organism and sometimes these changes will increase fitness. Mutations and adaptation A mutation that causes an increase in the fitness of an individual is called ‘beneficial’ and will tend to increase in frequency NOTE: Especially in asexual organisms, simply observing that a mutation increases in frequency doesn’t mean that it is beneficial – WHY NOT? W Q = 1, W P = 1 + s (s = 0.01); generation 0 p:q = 1:1000 If you know the benefit of a mutation and its initial frequency, it is simple to predict how it will change (t = time; s = selective benefit; p and q different alleles)

20. de Visser et al. (1999) Science 284: 404-406 Observation: A high frequency of ‘mutators’ in natural populations Inductive conclusion: A possible explanation for high frequency of mutators Complications: Unknowns whose influence on the inductive conclusion should be examined Complication I: Genetic linkage complicates assignment of cause and effect

21. Complete genetic linkage in asexual organisms makes it hard to distinguish between a high rate of adaptation: 1.…being caused by mutators: Mutator alleles allow more beneficial mutations to be produced  as the beneficial mutations increase in frequency (causing a high rate of adaptation), so do the mutator mutations 2.…providing a background that benefits mutators: If beneficial mutations are actually quite common (as they would be if the rate of adaptation is high)  as beneficial mutations increase in frequency they ‘drag’ along any other mutations they are linked to on a genome, including mutator mutations Complication 1: Genetic linkage (cause or effect…) Sniegowski et al. Bioessays (2000) Mutator mutation Beneficial mutation time Mutator mutation Beneficial mutation or is it… 1. 2. Can’t an experimenter just look at the mutation dynamics to see whether a mutator or a beneficial mutation occurred first? Actually, this is hard (see last figure for intuition).

22. To test if mutator alleles can be selected, Chao and Cox mixed otherwise identical mutator (mutT) and non-mutator (mutT-) bacteria and followed the change in ratio of the two types as the population adapted to a new environment They found that mutators tended to outcompete non-mutators as long as they were initially present at a frequency above ~10 -4 Why does this finding support the hypothesis that mutators can cause an increase in the rate of adaptation? Is there any evidence that mutators can cause an increase in adaptation rate? Chao and Cox (1983) Evolution mutator wins if initially present above ~10 -4 mutator:non-mutator

23. de Visser et al. (1999) Science 284: 404-406 Complications: Unknowns whose influence on the inductive conclusion should be examined Complication 2: Interference between competing mutations Predictions: 1. Increasing mutation supply will increase the rate of adaptation if a population has a limited supply of beneficial mutations 2. Increasing mutation supply will not increase the rate of adaptation of a population that is not limited by its supply of beneficial mutations 3. A fitter starting populations will have fewer beneficial available to it so will be mutation limited at a higher mutation supply than will a less fit population

24. Crow and Kimura, 1965 (after Muller, 1932) Complication 2 & Prediction 1,2: competition between beneficial mutations *’A’, ‘B’ and ‘C’ indicate newly arising beneficial mutations Theory: o When the supply of new beneficial mutations is low, the population spends a lot of time waiting for new beneficial mutations to occur – the *waiting time* for mutations limits the rate of adaptation o When the supply of beneficial mutations is high, mutations interfere with one another and many go extinct – called clonal interference – the *fixation* of mutations limits adaptation (this is the “speed- limit” that de Visser talks about) Prediction: a decelerating (non-linear) relationship between mutation supply and rate of adaptation ? mutation supply rate of adaptation

25. de Visser et al. (1999) Science 284: 404-406 Complications: Unknowns whose influence on the inductive conclusion should be examined Complication 2: Interference between competing mutations Predictions: 1. Increasing mutation supply will increase the rate of adaptation if a population has a limited supply of beneficial mutations 2. Increasing mutation supply will not increase the rate of adaptation of a population that is not limited by its supply of beneficial mutations 3. A fitter starting populations will have fewer beneficial available to it so will be mutation limited at a higher mutation supply than will a less fit population

26. If the ratio of beneficial:deleterious mutations can change, the mutation supply that the “speed-limit” starts at might also change This ratio is not known – still, some models of adaptation allows to predict how it might change Prediction 3: the ‘speed limit’ depends on initial fitness trait 1 trait 2 starting phenotype optimum phenotype Fisher’s Geometric model of adaptation – one of very few models that try to predict the general underlying basis of adaptation

27. optimum phenotype trait 1 trait 2 starting phenotype Theory: The proportion of all mutations that are beneficial will decline as a population moves toward the phenotypic optimum Prediction: Increasing the mutation rate might have a bigger benefit for populations close to the optimum because these populations spend most of their time waiting for new beneficial mutations to occur If the ratio of beneficial:deleterious mutations can change, the mutation supply that the “speed-limit” starts at might also change This ratio is not known – still, some models of adaptation allows to predict how it might change Prediction 3: the ‘speed limit’ depends on initial fitness

28. How to test the three predictions Predictions: 1. Increasing mutation supply will increase the rate of adaptation if a population has a limited supply of beneficial mutations 2. Increasing mutation supply will not increase the rate of adaptation of a population that is not limited by its supply of beneficial mutations 3. A fitter starting populations will have fewer beneficial available to it so will be mutation limited at a higher mutation supply than will a less fit population Test: 1. Measure the rate of adaptation of a genotype evolved at increasing levels of mutation supply. Note: the prediction is qualitative – the authors don’t know exactly where mutation supply stops limiting adaptation, so start at very low mutation supply and increase to very high levels! 2. Repeat evolution starting with a second genotype that is better adapted (closer to the optimum phenotype) to the evolution environment. Note: the authors don’t know how much closer to the optimum phenotype this second genotype is – but they do know that it is closer!

29. Methods – how do you measure the rate of adaptation? Ancestor Ancestor split into populations evolved at different mutation supply (1000 generations) Compare fitness of evolved populations

30. Methods – how do you measure the rate of adaptation? Ancestor Why might it be important to evolve replicate populations at each mutation supply? In practice, mutation supply was manipulated by a combination of: 1.deleting the mutY or mutS genes, which prevents mismatch repair and increases mutation rate by ~3- and ~30-fold, respectively 2.Evolving populations at different population sizes Together, these manipulations allowed de Visser et al. to cover a range of mutation supply of ~1000-fold difference – hopefully enough to include the onset of the speed limit of adaptation Ancestor split into populations evolved at different mutation supply (1000 generations) Compare fitness of evolved populations

31. Results Ancestor poorly adapted – higher proportion of total mutations are beneficial Note: arbitrary time chosen to assess the rate of adaptation (like asking: “how fast does your car accelerate?” – which would depend on the distance you are observing it over) Ancestor well adapted – lower proportion of total mutations are beneficial – speed limit happens at a higher mutation supply Here’s the raw data – how do they fit the predictions? (What questions should you be asking yourself?)

32. Results Ancestor poorly adapted – higher proportion of total mutations are beneficial Note: arbitrary time chosen to assess the rate of adaptation (like asking: “how fast does your car accelerate?” – which would depend on the distance you are observing it over) Speed limit Ancestor well adapted – lower proportion of total mutations are beneficial – speed limit happens at a higher mutation supply The ‘best fit’ lines seem to support expectation of a speed limit…

33. Results Note: arbitrary time chosen to assess the rate of adaptation (like asking: “how fast does your car accelerate?” – which would depend on the distance you are observing it over) Speed limit The ‘best fit’ lines look good, but do they describe the data better than the null hypothesis? Just what is the null hypothesis, anyway? Ancestor poorly adapted – higher proportion of total mutations are beneficial Ancestor well adapted – lower proportion of total mutations are beneficial – speed limit happens at a higher mutation supply

34. Conclusions Null model (no speed limit = linear relationship between mutation supply and fitness increase) Alternative model: Speed limit modeled as a plateau in fitness Comparison of models – which one better explains the data

35. Conclusions