Nonlethal Age Estimation of Three Threatened Fish Species Using DNA Methylation: Australian Lungfish, Murray Cod and Mary River Cod

Age-based demography is fundamental to management of wild fish populations. Age estimates for individuals can determine rates of change in key life-history parameters such as length, maturity, mortality and fecundity. These age-based characteristics are critical for population viability analysis in endangered species and for developing sustainable harvest strategies. For teleost fish, age has traditionally been determined by counting increments formed in calcified structures such as otoliths. However, the collection of otoliths is lethal and therefore undesirable for threatened species. At a molecular level, age can be predicted by measuring DNA methylation. Here, we use previously identified age-associated sites of DNA methylation in zebrafish (Danio rerio) to develop two epigenetic clocks for three threatened freshwater fish species. One epigenetic clock was developed for the Australian lungfish (Neoceratodus forsteri) and the second for the Murray cod (Maccullochella peelii) and Mary River cod (Maccullochella mariensis). Age estimation models were calibrated using either known-age individuals, ages derived from otoliths or bomb radiocarbon dating of scales. We demonstrate a high Pearson's correlation between the chronological and predicted age in both the Lungfish clock (cor = .98) and Maccullochella clock (cor = .92). The median absolute error rate for both epigenetic clocks was also low (Lungfish = 0.86 years; Maccullochella = 0.34 years). This study demonstrates the transferability of DNA methylation sites for age prediction between highly phylogenetically divergent fish species. Given the method is nonlethal and suited to automation, age prediction by DNA methylation has the potential to improve fisheries and other wildlife management settings.