Verification of Results

Since we cannot rule out that samples get contaminated on the basis of experimental setup, it is important to assess the authenticity using empirical tests. An independent line of evidence for authenticity of ancient DNA results is the application of relative rate analyses. One such approach — the evolutionary rate test — is an empirical test that exploits the temporal difference between related modern sequences and the very old DNA claims. The method infers the timing of the divergence between the ancient sequence and the modern sequences, by assuming a molecular clock and applying a published substitution rate for the particular gene. This approach can fail if the published rate of evolution is not correct for the taxa in question or the sequence in question. Furthermore, very old divergences may also be obtained if the ancient sequence is from a previously unknown modern contaminant (Hebsgaard et al. 2005).

A more solid approach is the relative rates test. Essentially, it examines if the relative distance between an outgroup and the ancient sequence is significantly different from the distance between the same outgroup and a modern sequence that is closely related to that of the ancient sequence (Fig. 4.2). A more vigorous approach is the relative rate analysis. This method estimates a likelihood function of the substitution per site, using database sequences of the most closely related

koa A

koa A

Fig. 4.2 The relative rates test uses an outgroup sequence, C, which is known to branch off before either sequence A or B. O is the common ancestor sequence of A and B. KOA is the relative substitution rate between O and A, and KOB is the relative substitution rate between O and B. Whether the genetic distance between O and A is significantly different from the distance between O and B can be evaluated by comparing KOA and KOB, which are calculated using the equations enclosed in the box

Fig. 4.2 The relative rates test uses an outgroup sequence, C, which is known to branch off before either sequence A or B. O is the common ancestor sequence of A and B. KOA is the relative substitution rate between O and A, and KOB is the relative substitution rate between O and B. Whether the genetic distance between O and A is significantly different from the distance between O and B can be evaluated by comparing KOA and KOB, which are calculated using the equations enclosed in the box sequences. This is then translated using published substitution rates to an estimate of age using a molecular clock (Willerslev et al. 2007).

Compared to the evolutionary rate test, the relative rate test is independent of an accurate calibration date and substitution rate. But the above molecular dating and relative rates tests all assume that substitutions accumulate in a clock-like manner. More important is the rate at which the DNA evolves; as mentioned before, these rates are different for different individuals but they are also different for different genes. This means that the rate can be very different for slow-evolving genes and fast-evolving genes. For example, for 14 published insect COI genes the published evolutionary rates vary between 0.3 x 10-8 and 9 x 10-8 substitutions per year (Morgan-Richards et al. 2001).

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