As we have just seen, substitutions in a protein-coding gene can be either synonymous or nonsynonymous, depending on whether they change the resulting amino acid sequence of the protein. The relative rates of synonymous and nonsynonymous substitutions are expected to differ in regions of genes that are evolving neutrally, under positive selection for change, or staying unchanged under purifying selection:

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By comparing the gene sequences that encode homologous proteins from many species, scientists can determine the history and timing of synonymous and nonsynonymous substitutions. This information can be mapped on a phylogenetic tree, as we saw in Chapter 21. Regions of genes that are evolving under neutral, purifying, or positive selection can be identified by comparing the nature and rates of substitutions across the phylogenetic tree.

By examining genome sequences for synonymous and nonsynonymous substitutions, biologists have determined which amino acid positions of the surface proteins of influenza viruses are evolving under positive selection (to escape immune detection in their hosts). This information allows researchers to select viruses from current influenza epidemics that are the most likely to escape human immune detection. These viruses are the ones that are most likely to give rise to the next flu epidemic, so they are the best candidates to use in vaccine production. As we discussed in the opening story, production of successful flu vaccines has greatly lowered the impact of flu epidemics in recent decades.

A study of the evolution of lysozyme illustrates how and why particular amino acid positions might be under different modes of selection (Figure 23.6). The enzyme lysozyme (see Figure 3.9) is found in almost all animals. It is produced in the tears, saliva, and milk of mammals and in the albumen (whites) of bird eggs. Lysozyme digests the cell walls of bacteria, rupturing and killing them. As a result, it plays an important role as a first line of defense against invading bacteria. Most animals defend themselves against bacteria by digesting them, which is probably why most animals have lysozyme. Some animals also use lysozyme in the digestion of food.

Among mammals, a mode of digestion called foregut fermentation has evolved twice. In mammals with this mode of digestion, the foregut—the posterior esophagus or the stomach—has been converted into a chamber in which bacteria break down ingested plant matter by fermentation. Foregut fermenters can extract nutrients from the otherwise indigestible cellulose that makes up a large proportion of plant tissue. Foregut fermentation evolved independently in ruminants (a group of hoofed mammals that includes cattle) and in certain leaf-eating monkeys, such as langurs. Caro-Beth Stewart knew that these evolutionary events were independent because both langurs and ruminants have close relatives that are not foregut fermenters.

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In both foregut-fermenting lineages, lysozyme has been modified to play a new, nondefensive role. This lysozyme ruptures some of the bacteria that live in the foregut, releasing nutrients metabolized by the bacteria, which the mammal then absorbs. How many changes in the lysozyme molecule were needed to allow it to perform this function amid the digestive enzymes and acidic conditions of the mammalian foregut? To answer this question, Stewart and her colleagues compared the lysozyme-coding sequences in foregut fermenters with those in several of their nonfermenting relatives. They determined which amino acids differed and which were shared among the species, as well as the rates of synonymous and nonsynonymous substitutions in lysozyme genes across the evolutionary history of the sampled species.

For many of the amino acid positions of lysozyme, the rate of synonymous substitutions in the corresponding gene sequence was much higher than the rate of nonsynonymous substitutions. This observation indicates that many of the amino acids that make up lysozyme are evolving under purifying selection. In other words, there is selection against change in the protein at these positions, and the observed amino acids must therefore be critical for lysozyme function. At other positions, several different amino acids function equally well, and the corresponding gene sequences had similar rates of synonymous and nonsynonymous substitutions. The most striking finding was that amino acid replacements in lysozyme happened at a much higher rate in the lineage leading to langurs than in any other primate lineage. The high rate of nonsynonymous substitutions in the langur lysozyme gene shows that lysozyme went through a period of rapid change in adapting to the foregut of langurs. Moreover, the lysozymes of langurs and cattle share five unique amino acid replacements, all of which lie on the surface of the lysozyme molecule, well away from the enzyme’s active site. Two of these shared replacements involve changes from arginine to lysine, which makes the proteins more resistant to attack by the stomach enzyme pepsin. By understanding the functional significance of amino acid replacements, molecular evolutionists can explain the observed changes in amino acid sequences in terms of changes in the functioning of the protein.

A large body of fossil, morphological, and molecular evidence shows that langurs and ruminants do not share a recent common ancestor. Yet langur and ruminant lysozymes share several amino acids that neither mammal shares with the lysozymes of its own closer relatives. The lysozymes of these two mammals have undergone convergent evolution at some amino acid positions despite their very different ancestry. The amino acids they share give these lysozymes the ability to lyse the bacteria that ferment plant material in the foregut.

The hoatzin, an unusual leaf-eating South American bird and the only known avian foregut fermenter, offers another remarkable example of the convergent evolution of lysozyme. Many birds have an enlarged esophageal chamber called a crop. The crop of the hoatzin contains lysozyme and bacteria and acts as a fermenting chamber. Many of the amino acid replacements that occurred in the adaptation of hoatzin crop lysozyme are identical to those that evolved in ruminants and langurs. Thus even though the hoatzin and foregut-fermenting mammals have not shared a common ancestor in hundreds of millions of years, they have all evolved similar adaptations in their lysozymes that enable them to recover nutrients from their fermenting bacteria.