Well-preserved subfossil bones of Adélie penguins, Pygoscelis adeliae, underlie existing and abandoned nesting colonies in Antarctica. These bones, dating back to more than 7000 years before the present, harbor some of the best-preserved ancient DNA yet discovered. From 96 radiocarbon-aged bones, we report large numbers of mitochondrial haplotypes, some of which appear to be extinct, given the 380 living birds sampled. We demonstrate DNA sequence evolution through time and estimate the rate of evolution of the hypervariable region I using a Markov chain Monte Carlo integration and a least-squares regression analysis. Our calculated rates of evolution are approximately two to seven times higher than previous indirect phylogenetic estimates.

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent.

Ancient DNA studies have typically used multi-copy mitochondrial DNA sequences. This is largely because single-locus nuclear genes have been difficult to recover from sub-fossil material, restricting the scope of ancient DNA research. Here, we have isolated single-locus nuclear DNA markers to assign the sex of 115 extinct moa and, in combination with a mitochondrial DNA phylogeny, tested competing hypotheses about the specific status of moa taxa. Moa were large ratite birds that showed extreme size variation both within and among species. For some taxa, this large variation was hypothesized to represent sexual dimorphism, while for others it was argued to reflect the existence of different species. Our results show that moa were characterized by extreme reverse sexual dimorphism and as a result we have been able to clarify the number of moa species. For example, we show that the three recognized 'species' of Dinornis comprised only two monophyletic groups and that two of these 'species' comprised individuals of one sex only. This study also illustrates that single-locus nuclear DNA sequences can be consistently recovered from ancient material.

Using entire modern and ancient mitochondrial genomes of Adélie penguins (Pygoscelis adeliae) that are up to 44000 years old, we show that the rates of evolution of the mitochondrial genome are two to six times greater than those estimated from phylogenetic comparisons. Although the rate of evolution at constrained sites, including nonsynonymous positions and RNAs, varies more than twofold with time (between shallow and deep nodes), the rate of evolution at synonymous sites remains the same. The time-independent neutral evolutionary rates reported here would be useful for the study of recent evolutionary events.