Epigenetic estimation of age in humpback whales

Abstract

Age is a fundamental aspect of animal ecology, but is difficult to determine in many species. Humpback whales exemplify this as they have a lifespan comparable to humans, mature sexually as early as 4 years and have no reliable visual age indicators after their first year. Current methods for estimating humpback age cannot be applied to all individuals and populations. Assays for human age have recently been developed based on age-induced changes in DNA methylation of specific genes. We used information on age-associated DNA methylation in human and mouse genes to identify homologous gene regions in humpbacks. Humpback skin samples were obtained from individuals with a known year of birth and employed to calibrate relationships between cytosine methylation and age. Seven of 37 cytosines assayed for methylation level in humpback skin had significant age-related profiles. The three most age-informative cytosine markers were selected for a humpback epigenetic age assay. The assay has an R(2) of 0.787 (P = 3.04e-16) and predicts age from skin samples with a standard deviation of 2.991 years. The epigenetic method correctly determined which of parent-offspring pairs is the parent in more than 93% of cases. To demonstrate the potential of this technique, we constructed the first modern age profile of humpback whales off eastern Australia and compared the results to population structure 5 decades earlier. This is the first epigenetic age estimation method for a wild animal species and the approach we took for developing it can be applied to many other nonmodel organisms.

Keywords: DNA methylation; age; cetacean; epigenetics; population.

Figure 1

Regressions of CpG methylation and age at sites selected for the HEAA. CpG methylation was measured at each site by a PyroMark assay in N = 45 whales. Females are shown by a green circle and males by blue triangles. CpG sites shown were as follows: (A) TET2_CpG+31, (B) CDKN2A_CpG+297 and (C) GRIA2_CpG+202.

Figure 2

Accuracy and precision of the HEAA. (A) Multiple linear regressions for predicted ages of N = 45 whales from measurement of CpG methylation at three CpG sites. 95% confidence limits of the placement of the regression line are shown. (B) Results of ‘Leave One Out Cross Validation’ (LOOCV) analysis. The estimated ages of every whale in the ‘calibration’ population when the predictive model is based on data for the other N = 44 whales are plotted. 95% confidence limits for age prediction are shown.

Figure 3

Age estimates generated by the HEAA for east Australian humpback whales. (A) Population age distribution estimated with the HEAA for N = 63 noncalf whales samples near Evans Head. Ages are grouped into categories of 4 years. The mean observed age of 10.01 years was used for estimation of the negative exponential distribution of age shown in (B). Whales with an estimated age of <2 years are indicated in green and were not included in this comparison.

Figure 4

Population age profiles for humpback whales from east coast Australia. Ten profiles for each year from 1952 to 1962 were produced from ear plug growth layer measurement. The HEAA was used to estimate the profile for 2009.