V.1 Molecular Evolution Charles F. Aquadro OUTLINE could be used to infer the date of a last common ancestor. 1. What is molecular evolution and why does it Molecular Evolution. Changes in the molecules of life occur? (DNA, RNA, and protein) over generations, for many 2. Origins of molecular evolution, the molecular reasons, including mutation, genetic drift, and nat- clock, and the neutral theory ural selection, resulting in different sequences of these 3. Predictions of the neutral theory for variation molecules in different descendant lineages. The study within and between species of molecular evolution is the study of the patterns 4. The impact of natural selection on molecular and process of change that result in these different variation and evolution sequences. 5. Biological insights from the study of molecular Mutation. Heritable change in genetic material, includ- evolution ing base substitutions, insertions, deletions, and re- 6. Conclusions arrangements; the ultimate source of new variation in populations. The molecules of life (DNA, RNA, and proteins) change Neutral Theory. Short for neutral mutation–random drift over evolutionary time. Much can be learned about evo- theory of molecular evolution, proposing that molec- lutionary process and biological function from the rates ular variation is equivalent in function (selectively and patterns of change in these molecules. The study of neutral), making genetic drift the main driver of these changes is the study of molecular evolution. This molecular genetic change in populations over time. chapter discusses why these molecules change, what can Positive Selection. New advantageous mutations, or be learned about pattern and process from these changes, changing environments, can present opportunities and how the changes in the molecules of life can be used for new, or currently existing, variants to now have to infer important past evolutionary events. a reproductive advantage. They thus relentlessly in- crease in frequency until they fix in the relevant pop- ulation. The selective pressure that leads to this fix- GLOSSARY ation is termed positive selection. Fixation. The population process in which, either by drift Purifying Selection. Selection against harmful (i.e., dele- or by natural selection, a new mutation increases in terious) mutations “purifies” the population of these frequency in a population until it replaces all other harmful variants. Such selection is due to constraint, variants and reaches a frequency of 100 percent. typically to maintain a specific important biological Molecular Clock. When the time at which organisms last function. shared a common ancestor is plotted over time (e.g., estimated time from the fossil record), a roughly lin- ear accumulation of genetic changes in DNA and the 1. WHAT IS MOLECULAR EVOLUTION AND encoded proteins is frequently observed. In 1965, the WHY DOES IT OCCUR? rough linearity of this accumulation of change moti- vated Emile Zuckerkandl and Linus Pauling to pro- The molecules of life (DNA, RNA, and proteins) are not pose that these data represent a sort of “molecular static. They change over evolutionary time, hence the clock” by which the amount of molecular divergence term molecular evolution. Some molecules evolve 368 Genes, Genomes, Phenotypes rapidly and some only very, very slowly. Their change is Harmful or deleterious mutations, or ones that re- due to the interplay between two fundamental evolu- duce reproductive output or success, often will not be tionary processes: mutation and fixation. Before dis- fixed but rather reduced in frequency or eliminated from cussing these two processes, it is important to note that populations. This is known as purifying selection, and it mutations come in two basic varieties: heritable muta- prevents the otherwise-inexorable, but very slow, march tions that occur in the germ line and are passed on in the of successive neutral mutation fixations over time in all genome of progeny, and somatic mutations that can finite populations. occur in the process of cell division during normal growth and development. For example, if the latter af- 2. ORIGINS OF MOLECULAR EVOLUTION, THE fect the control of certain cellular processes, they can MOLECULAR CLOCK, AND THE NEUTRAL THEORY lead to uncontrolled cell growth and cancer. Molecular evolutionary studies have traditionally focused only on Molecular evolution as a field of study originated sort of heritable genetic changes that accumulate within and accidentally, as biologists discovered how to determine between organisms. the sequence of proteins and started collecting data from Inherited mutations occur primarily during DNA diverse organisms in the 1950s and 1960s. Key early replication in the production of gametes and introduce data were for hemoglobin and histones, proteins chosen new genetic variants into the population. For animals for their biomedical importance. The study of proteins and plants, the relevant genetic molecule is DNA. For was emphasized because of their clear functional rele- some viruses, the heritable molecule is RNA. Certain vance, and because methods were developed first for segments of DNA or RNA genomes are translated into sequencing these biological molecules. Comparison of proteins by the cells, and some changes lead to changes in sequence divergence among proteins revealed two im- the encoded protein sequence, leading to molecular evo- portant patterns: specifically, fibrinopeptides, hemoglo- lution at the amino acid level as well. Heritable muta- bin, and histones from various vertebrates with well- tions are largely considered to occur at random in time defined fossil records revealed that (1) different proteins and space and location across our genome, and at a rel- evolved at different rates, but (2) each protein seemed to atively constant rate, at least for many organisms (see accumulate changes at a surprisingly consistent rate, a chapter IV.2). pattern that led to the concept of a “molecular clock” The second process of molecular evolution is fixation, (figure 1). which is fundamentally the “population” phase leading The term molecular clock refers to both a mechanism to molecular evolutionary change. Most new mutations and a tool for evolutionary studies. As a mechanism, the are lost from populations as a result of chance (genetic presence of a roughly constant accumulation of change drift; see chapter IV.1), or because they are harmful led to the inference that chance, not local adaptation, is (deleterious). Mutations remain in populations because the cause of much of the observed molecular change. As of chance or because they increase the reproductive suc- a tool, the presence of a clock representing a molecule cess of offspring carrying them. Drift can lead to rapid also meant that if its rate could be calibrated with or- changes in frequencies of mutations in very small popu- ganisms of known age (e.g., from the fossil record), then lations, but is much less influential in large populations. observed sequence differences for organisms without a Ultimately, the outcome of drift alone is alwaysthe lossor fossil record could also be “dated.” fixation of every new mutation; in other words, every Until the mid-1960s, most evolutionary biologists mutation will eventually reach 0 or 100 percent fre- considered natural selection the primary determinant of quency. Fixation means that the new mutation now re- evolutionary change. Genetic drift was considered im- places all previous variants present (segregating) in the portant only in small populations; for example, drift population at a particular site in the genome. played an important role in Wright’s shifting balance Fixation can also be caused, or assisted, by natural theory, but not as a primary driving force in evolution, selection. Selection acting to directly increase a variant rather as a source of new combinations of alleles on frequency, as the result of an increased relative repro- which selection could act; thus, drift was rarely con- ductive success or survival of individuals carrying it, sidered, and most models focused on selection. is termed positive selection, or often simply adaptation. Several observations in the mid-1960s and early Such selection can cause rapid changes of allele frequen- 1970s challenged the dominance of selection as the driv- cies in populations, over tens of generations, versus mil- er of evolution. First, high levels of protein polymor- lions of generations by genetic drift alone in large pop- phism (i.e., variation within populations) were observed ulations. While the underlying mutation rate is thought in fruit flies, humans, and bacteria. Could all that var- not responsive to selective challenges, the fixation pro- iation within populations really be maintained by natu- cess is strongly influenced by selection. ral selection? Second, extrapolating from available data, Molecular Evolution 369 220 200 Mammals Birds/Reptiles Mammals/Reptiles Reptiles/Fish Carp/Lamprey Vertebrates/Insects abcde f g h i j 180 160 Fibrinopeptides 140 120 100 Hemoglobin 80 60 40 Cytochrome c (corrected amino acid changes per 100 residues) (corrected 20 m 0 0 100 200 300 400 500 600 700 800 900 1,000 Millions of years since divergence Figure 1. Molecular clocks. Rates of amino acid substitution in three proteins are from the same pair of organisms and time of divergence, proteins: fibrinopeptides, hemoglobin, and cytochrome c. The number the three proteins are evolving at very different rates (i.e., fibrino- of amino acid differences (per 100 residues,