Fossil avian eggshell preserves ancient DNA

Abstract

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.

Ancient DNA reveals extreme egg morphology and nesting behavior in New Zealand’s extinct moa

“…These samples were recovered from a range of sites including archaeological middens, rock-shelters and caves, dunes, as well as alluvial deposits. Eggs from archaeological sites have been shown to range in age from 400 to 700 y BP (16). For example, moa midden material from large sites at Rakaia Mouth, Wakanui, Waitaki Mouth (Oamaru), and Wairau Bar dated from 481 ± 51 y BP to 638 ± 44 y BP.…”

“…Moa samples from natural sites typically ranged in age from approximately 1,300 to 19,000 y BP. For example, moa bones recovered from Makirikiri and Pyramid Valley swamps and Honeycomb Hill cave (Oparara) dated from 1,296 ± 28 y BP to 18,901 ± 192 y BP (2,6,8,16).…”

True single-molecule DNA sequencing of a pleistocene horse bone

“…Oxidative derivatives of pyrimidines, abasic sites, and intermolecular cross-links have been shown to preclude molecular cloning, restricting its efficiency to exceptional cases only Lindahl 1993), unless end-repair reactions with DNA polymerase and PNK activities were performed (Noonan et al 2005(Noonan et al , 2006. While still sensitive to polymerase blocking lesions, such as abasic sites, single-strand breaks and intermolecular cross-links, PCR has opened access to a wide variety of aDNA templates coming from different preservation environments (e.g., Antarctic coast [de Bruyn et al 2009] or Balearic islands [Ramirez et al 2009]), source material (e.g., bones or egg shells [Oskam et al 2010]) and time periods (10,000-500,000 yr ago) (Willerslev et al 2004). However, another type of damage, namely miscoding lesions, promotes nucleotide misincorporation during PCR amplification of DNA fragments extracted from ancient organisms , the most important consisting of the deamination of cytosine into uracil, which leads to G/C to A/T misincorporation (Hansen et al 2001).…”

Biologically and diagenetically derived peptide modifications in moa collagens

“…Moa remains are of special interest because in addition to bone, diverse elements (i.e. feathers, bones, mummified remains, egg shells) are attributed to them [18][19][20]. Because of the availability of exceptionally preserved specimens, DNA recovered from various moa specimens has been used to differentiate species [20,21] and estimate rates of DNA degradation [22].…”