## When you can

Despite all the reasons why you shouldn't get too excited about the idea of the molecular clock (see the preceding section), in some cases, scientists can show that the accumulation of mutations is an excellent predictor of the time since two species diverged.

The key to using the molecular clock effectively is calibrating it. To do that, you must know the time at which some lineages diverged so that you can translate the amount of divergence between different lineages into time. Then you can use this information to estimate the divergence times of additional species.

### Performing experiments in the lab

For organisms that have generation times short enough to create divergent lineages in the laboratory, you can ask how fast mutations accumulate in these microorganisms over time. Then you can look at sequence variation that you know to be neutral — changes in the DNA that don't result in any changes in the protein. These changes pile up over time, based on the mutation rate. You measure how fast they pile up, and then you know the mutation rate — simple as that! (This strategy doesn't work for organisms that are much longer lived, of course.)

### Looking at ancient DNA and the fossil record

As researchers' biochemical techniques become more sophisticated, they've been able to retrieve DNA sequences from the distant (but quantifiable) past. By studying these ancient sequences, they've been able to put bounds on the rates of mutation accumulation between the date of the old sequence and the current time.

The fossil record also allows scientists to generate estimates of when different lineages diverged by noting the geological era in which the fossil was found. Then they can use these divergence times to calculate the rate at which mutations have accumulated in the lineages since the divergence.

Imagine you have three species: species 1, species 2, and species 3. You sequence all three, generate a phylogeny, and find that species 2 and 3 seem most closely related, and they're both related to species 1. Just by digging and finding fossils at different ages, you have a pretty good idea of when species 1 and 2 split. But you have absolutely no fossils telling you anything about the history of species 3, and you want know when species 3 split off from species 2.

By knowing when species 1 and 2 diverged, you can correlate the number of DNA differences between these two species and the amount of time since they diverged. You can then take this estimate and use it to translate the number of DNA differences between species 2 and 3 into the time since they diverged, even in the absence of any fossil record for species 3. And that's the molecular clock!

From the fossil record, scientists know the approximate times when many lineages arose. Date the rocks that a fossil is found in, and you pretty effectively date the fossil. Scientists know, for example, about when the mammal lineage split off from the rest of the tetrapods (four-limbed creatures, such as lizards, turtles, and birds). On a finer scale, they have a pretty good idea about when the hominid lineage diverged from the chimpanzee lineage.

Scientists can use the fossil record to generate divergence times for lineages for which there is a good fossil record, and then they can use the molecular clock to estimate divergence times for species for which a good fossil record isn't available.

### Examining biogeographic patterns

Biogeographic patterns can also generate estimates of divergence times. Take, for example, the fruit fly species (Drosophila) that lives in the Hawaiian islands. Because geologists have an excellent understanding of how the Hawaiian islands formed, they can date the islands accurately.

The Earth's crust has a thin spot, and as the Pacific plate moves across this hot spot, periodic eruptions have generated the chain of the Hawaiian islands. Because geologists can date exactly the age at which lava solidifies, they can figure out when the islands were formed.

In addition, the Hawaiian islands are extremely distant from other land masses. As a result, much of the biological diversity on the islands is a result of speci-ation events that happened in Hawaii. Most of the fruit fly species in Hawaii occurs nowhere else, for example. Hawaii is so far from anywhere else that the rate at which fruit fly speciation occurs on the islands far exceeds the rate at which non-Hawaiian fruit fly species could arrive. So although some fruit flies got to Hawaii initially from elsewhere, the original colonists have radiated into many of the species you find there today.

As new islands appear in the chain, flies from the neighboring island colonize them and, over time, diverge to become separate species. As explained in Chapter 8, this divergence is a consequence of the very reduced rates of genetic exchange between the islands. A fruit fly occasionally gets from one island to another, but this migration doesn't happen often enough to overwhelm the gradual divergence between the separated populations and their subsequent speciation.

Phylogenetic analysis (refer to Chapter 9) of the Hawaiian fruit fly yields a tree that matches geologists' understanding of the geological formation of the islands — and sure enough, the fly species on different islands each share a most recent common ancestor with the species on the next island over.

Usually, we don't know when in the past two lineages became geographically separated, but with the Hawaiian islands, we know exactly when the new islands popped up out of the ocean. We can combine the data about how genetically different two species are (which we get from the sequence of their DNA) with the length of time they've been separate species.

Not surprisingly, the longer two species have been separated, the more genetically different they will be, simply because changes add up over time. But what's most important about these Hawaiian flies is that, because we know the dates the islands appeared, we can tell that the amount of genetic difference is exactly correlated with the length of time since the species diverged. If one pair of species diverged twice as long ago as another pair, it has twice the amount of genetic differences.

This information tells us that the molecular clock can tick at a constant rate for long periods of time. Proving that (through studying the phylogeny of Hawaiian fruit flies and knowing the dates when the islands formed) makes us more comfortable with assuming that the rate of molecular evolution may be constant in other species as well.

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