Genetica per Scienze Naturali a.a prof S. Presciuttini Evolution in a glass Experimental work with bacteria, eukaryotic micro-organisms and very small animals can tell us much about the occurrence and properties of mutations, including beneficial mutations. Over the last fifty years or so beneficial mutations have been observed to occur in a number of studies Experimental work with bacteria, eukaryotic micro-organisms and very small animals can tell us much about the occurrence and properties of mutations, including beneficial mutations. Over the last fifty years or so beneficial mutations have been observed to occur in a number of studies Most of these experiments were done in a continuous culture system called a chemostat. Most of these experiments were done in a continuous culture system called a chemostat. A chemostat consists of a bottle in which the organisms grow. Growth medium (i.e. food) is continuously pumped into the bottle and waste products, residual medium and organisms flow out. The contents of the bottle are well mixed so that each critter in the chemostat has an equal chance of getting at each bit of food. Factors that affect the growth of the organisms such as temperature are controlled, sometimes quite rigourously. Several variations of chemostats have been developed. A chemostat consists of a bottle in which the organisms grow. Growth medium (i.e. food) is continuously pumped into the bottle and waste products, residual medium and organisms flow out. The contents of the bottle are well mixed so that each critter in the chemostat has an equal chance of getting at each bit of food. Factors that affect the growth of the organisms such as temperature are controlled, sometimes quite rigourously. Several variations of chemostats have been developed.

Genetica per Scienze Naturali a.a prof S. Presciuttini The chemostat Schematic diagram of a chemostat, a device for the continuous culture of bacteria. The chemostat relieves the environmental conditions that restrict growth by continuously supplying nutrients to cells and removing waste substances and spent cells from the culture medium Schematic diagram of a chemostat, a device for the continuous culture of bacteria. The chemostat relieves the environmental conditions that restrict growth by continuously supplying nutrients to cells and removing waste substances and spent cells from the culture medium Continuous culture, in a device called a chemostat, can be used to maintain a bacterial population at a constant density, a situation that is, in many ways, more similar to bacterial growth in natural environments. In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once growth is initiated, fresh medium is continuously supplied from the reservoir. The volume of fluid in the growth chamber is maintained at a constant level by some sort of overflow drain. Fresh medium is allowed to enter into the growth chamber at a rate that limits the growth of the bacteria. The bacteria grow (cells are formed) at the same rate that bacterial cells (and spent medium) are removed by the overflow. The rate of addition of the fresh medium determines the rate of growth because the fresh medium always contains a limiting amount of an essential nutrient.

Genetica per Scienze Naturali a.a prof S. Presciuttini Fluctuations of mutant strains In an early study, resistance to a phage was used as a marker to follow the appearance of some mutations in a chemostat culture. In an early study, resistance to a phage was used as a marker to follow the appearance of some mutations in a chemostat culture. Novick and Szilard grew E. coli in a chemostat at a steady-state density of about 3 × 10 8 cells per ml. Periodically they assayed cells sampled from the chemostat for resistance to infection by bacteriophage T5 and calculated the density of T5 resistant cells in the culture. Novick and Szilard grew E. coli in a chemostat at a steady-state density of about 3 × 10 8 cells per ml. Periodically they assayed cells sampled from the chemostat for resistance to infection by bacteriophage T5 and calculated the density of T5 resistant cells in the culture. At no time was phage T5 present in the chemostat nor had the cells in the chemostat been exposed to phage T5. They found that there was always a fraction of cells in the culture that was resistant to T5. At no time was phage T5 present in the chemostat nor had the cells in the chemostat been exposed to phage T5. They found that there was always a fraction of cells in the culture that was resistant to T5. The density of resistant cells fluctuated betweeen 10 2 and 10 3 per ml. The density of resistant cells fluctuated betweeen 10 2 and 10 3 per ml. The increases and decreases reflect the occurrence of mutations within strains in the chemostat. The initial increase in the frequency of resistant cells occurs because a mutation occurs within a T5 resistant strain that makes it (and its descendents) the fastest growing cells in the culture. As long as this strain remains the fastest growing one its representation in the population will increase. Eventually different favorable mutation occurs in a cell that is sensitive to T5 that makes it (and its descendents) the fastest growing cells in the culture. This causes the frequency of T5 resistance to decline. Later a different mutation occurs in a T5 resistant strain that makes it the fastest growing strain. Its frequency increases, and so on. The increases and decreases reflect the occurrence of mutations within strains in the chemostat. The initial increase in the frequency of resistant cells occurs because a mutation occurs within a T5 resistant strain that makes it (and its descendents) the fastest growing cells in the culture. As long as this strain remains the fastest growing one its representation in the population will increase. Eventually different favorable mutation occurs in a cell that is sensitive to T5 that makes it (and its descendents) the fastest growing cells in the culture. This causes the frequency of T5 resistance to decline. Later a different mutation occurs in a T5 resistant strain that makes it the fastest growing strain. Its frequency increases, and so on.

Genetica per Scienze Naturali a.a prof S. Presciuttini Neutral mutations It is important to note that in this environment sensitivity and resistance to infection by T5 is a neutral trait here. Because there is no T5 in the environment, resistance does not provide an advantage. It is important to note that in this environment sensitivity and resistance to infection by T5 is a neutral trait here. Because there is no T5 in the environment, resistance does not provide an advantage. But it doesn't seem to provide much disadvantage either. If it provided a disadvantage, the resistant cells would washout of the chemostat. In this environment, it is selectively neutral. But it doesn't seem to provide much disadvantage either. If it provided a disadvantage, the resistant cells would washout of the chemostat. In this environment, it is selectively neutral. Mutations in other genes cause some cells to have a higher growth rate. It is just a matter of whether these mutations occur first in resistant or sensitive cells that determines whether the frequency of T5 resistant cells increases or decreases. Mutations in other genes cause some cells to have a higher growth rate. It is just a matter of whether these mutations occur first in resistant or sensitive cells that determines whether the frequency of T5 resistant cells increases or decreases. It's a hitchhiking effect - the T5 resistance gene just goes along for the ride with the genes causing the fluctuations. It's a hitchhiking effect - the T5 resistance gene just goes along for the ride with the genes causing the fluctuations. Bacteria carrying neutral mutations constitute a fluctuating proportion of growing cultures. The fluctuations are attributed to periodic selection of fitter clones, with each successive sweep replacing less fit members of the population, including those with neutral mutations. The frequency of neutral mutations can also change in clonal populations as a consequence of hitchhiking with favorable mutations. Bacteria carrying neutral mutations constitute a fluctuating proportion of growing cultures. The fluctuations are attributed to periodic selection of fitter clones, with each successive sweep replacing less fit members of the population, including those with neutral mutations. The frequency of neutral mutations can also change in clonal populations as a consequence of hitchhiking with favorable mutations.

Genetica per Scienze Naturali a.a prof S. Presciuttini An example with yeast Paquin & Adams (1983) studied haploid and diploid populations of yeast to estimate the relative rate that beneficial mutations would arise in an asexual population of each type. Paquin & Adams (1983) studied haploid and diploid populations of yeast to estimate the relative rate that beneficial mutations would arise in an asexual population of each type. Populations were kept in a chemostat (a fairly constant environment) at a population size of about 5 billion. Initially, the population was started from a single clone (one genotype). Populations were kept in a chemostat (a fairly constant environment) at a population size of about 5 billion. Initially, the population was started from a single clone (one genotype). A neutral marker, canavanine resistance then increased in frequency due to mutation pressure alone (amino acid mutation rate = ), although the mutations always remained low in frequency (< ) during the hundreds of generations of the experiment. A neutral marker, canavanine resistance then increased in frequency due to mutation pressure alone (amino acid mutation rate = ), although the mutations always remained low in frequency (< ) during the hundreds of generations of the experiment. When a beneficial mutation occurred, it was most likely to arise in a canavanine sensitive cell. The beneficial mutation would then sweep through the population. Canavanine sensitivity would "hitch- hike" along, driving back down the frequency of canavanine resistance.

Genetica per Scienze Naturali a.a prof S. Presciuttini Interpreting mutant fluctuations This chart is an explanation of what happens in the chemostat This chart is an explanation of what happens in the chemostat

Genetica per Scienze Naturali a.a prof S. Presciuttini Genetic drift Fluctuations of mutant-clone frequencies in the chemostat are examples of the process known as genetic drift. Fluctuations of mutant-clone frequencies in the chemostat are examples of the process known as genetic drift. If a population is finite in size (as all populations are) and if a given pair of parents of a diploid species has only a small number of offspring, then, even in the absence of all selective forces, the frequency of a gene will not be exactly reproduced in the next generation, because of sampling error. If a population is finite in size (as all populations are) and if a given pair of parents of a diploid species has only a small number of offspring, then, even in the absence of all selective forces, the frequency of a gene will not be exactly reproduced in the next generation, because of sampling error.  If, in a population of 1000 individuals, the frequency of a is 0.5 in one generation, then it may by chance be or in the next generation because of the chance production of slightly more or slightly fewer progeny of each genotype. In the second generation, there is another sampling error based on the new gene frequency, so the frequency of a may go from to or back to This process of random fluctuation continues generation after generation, with no force pushing the frequency back to its initial state, because the population has no "genetic memory" of its state many generations ago. Each generation is an independent event.

Genetica per Scienze Naturali a.a prof S. Presciuttini Extinction of genetic variability by genetic drift The final result of this random change in allelic frequency is that the population eventually drifts to p = 1 or p = 0. After this point, no further change is possible; the population has become homozygous. A different population, isolated from the first, also undergoes this random genetic drift, but it may become homozygous for allele A, whereas the first population has become homozygous for allele a. As time goes on, isolated populations diverge from each other, each losing heterozygosity. The variation originally present within populations now appears as variation among populations. The final result of this random change in allelic frequency is that the population eventually drifts to p = 1 or p = 0. After this point, no further change is possible; the population has become homozygous. A different population, isolated from the first, also undergoes this random genetic drift, but it may become homozygous for allele A, whereas the first population has become homozygous for allele a. As time goes on, isolated populations diverge from each other, each losing heterozygosity. The variation originally present within populations now appears as variation among populations. Computer simulation of genetic drift. The frequency of an allele (e.g., A in a system with A and a) is shown for five replicate populations over the course of 100 generations, with a population size (N) of 20. The effect of drift is inversely proportional to population size, a fundamental driving force in many evolutionary divergences

Genetica per Scienze Naturali a.a prof S. Presciuttini Genetic drift over evolutionary time The appearance, loss, and eventual incorporation of neutral mutations in the life of a population. If random genetic drift does not cause the loss of a new mutation, then it must eventually cause the entire population to become homozygous for the mutation. The appearance, loss, and eventual incorporation of neutral mutations in the life of a population. If random genetic drift does not cause the loss of a new mutation, then it must eventually cause the entire population to become homozygous for the mutation. At that point, the mutation has been fixed. At that point, the mutation has been fixed. In the figure, 10 mutations have arisen, of which 9 (light red at bottom of graph) increased slightly in frequency and then died out. Only the fourth mutation eventually spread into the population. In the figure, 10 mutations have arisen, of which 9 (light red at bottom of graph) increased slightly in frequency and then died out. Only the fourth mutation eventually spread into the population. Therefore, a steady substitution of one allele for another is expected to occur due to genetic drift alone

Genetica per Scienze Naturali a.a prof S. Presciuttini The probability of fixation of a neutral allele It has been proven by matematical analysis (and it is quite intuitive) that the probability of fixation u of any neutral allele a is equal to its frequency in the population: It has been proven by matematical analysis (and it is quite intuitive) that the probability of fixation u of any neutral allele a is equal to its frequency in the population: u = p a In a finite diploid population, p a takes discrete values only, starting from 1/(2N) in diploid species (when one copy only of allele a is present in the population), and incrementing at steps of 1/(2N). In a finite diploid population, p a takes discrete values only, starting from 1/(2N) in diploid species (when one copy only of allele a is present in the population), and incrementing at steps of 1/(2N). In other words, the initial frequency of a mutant allele is, by definition, p a = 1/(2N) In other words, the initial frequency of a mutant allele is, by definition, p a = 1/(2N) Thus, the probability of ultimate fixation of any new neutral mutation is equal to the reciprocal of twice the population size. Thus, the probability of ultimate fixation of any new neutral mutation is equal to the reciprocal of twice the population size.

Genetica per Scienze Naturali a.a prof S. Presciuttini The concept of gene substitution It is important to distinguish between "Mutation" and "Substitution" with respect to individuals and populations: It is important to distinguish between "Mutation" and "Substitution" with respect to individuals and populations: The rate of gene substitution (K) is defined as the number of mutants reaching fixation per unit time. The rate of gene substitution (K) is defined as the number of mutants reaching fixation per unit time. If neutral mutations occur at a rate of μ per gene per generation, then the number of mutants arising in a diploid population of size N is 2N μ mutant alleles per generation. Since the probability of fixation for each of these mutations is 1/(2N), we obtain the result that If neutral mutations occur at a rate of μ per gene per generation, then the number of mutants arising in a diploid population of size N is 2N μ mutant alleles per generation. Since the probability of fixation for each of these mutations is 1/(2N), we obtain the result that K = μ. Thus theoretically, if the mutation rate μ is constant over time, neutral alleles accumulate at a fixed rate independently of population size, and their rate of accumulation can be used as an evolutionary clock to measure divergence times. This is one of the fundamental tenants of molecular evolution. Thus theoretically, if the mutation rate μ is constant over time, neutral alleles accumulate at a fixed rate independently of population size, and their rate of accumulation can be used as an evolutionary clock to measure divergence times. This is one of the fundamental tenants of molecular evolution. This result can be intuitively understood by noting that, in a large population, the number of mutations arising every generation is high but the fixation probability of each mutation is low. In comparison, in a small population, the number of mutations arising every generation is low, but the fixation probability of each mutation is high. As a consequence, the rate of substitution for neutral mutations is independent of population size. This result can be intuitively understood by noting that, in a large population, the number of mutations arising every generation is high but the fixation probability of each mutation is low. In comparison, in a small population, the number of mutations arising every generation is low, but the fixation probability of each mutation is high. As a consequence, the rate of substitution for neutral mutations is independent of population size.