Absence of Ancient DNA in Sub-Fossil Insect Inclusions Preserved in ‘Anthropocene’ Colombian Copal

Abstract

Insects preserved in copal, the sub-fossilized resin precursor of amber, have potential value in molecular ecological studies of recently-extinct species and of extant species that have never been collected as living specimens. The objective of the work reported in this paper was therefore to determine if ancient DNA is present in insects preserved in copal. We prepared DNA libraries from two stingless bees (Apidae: Meliponini: Trigonisca ameliae) preserved in ‘Anthropocene’ Colombian copal, dated to ‘post-Bomb’ and 10,612±62 cal yr BP, respectively, and obtained sequence reads using the GS Junior 454 System. Read numbers were low, but were significantly higher for DNA extracts prepared from crushed insects compared with extracts obtained by a non-destructive method. The younger specimen yielded sequence reads up to 535 nucleotides in length, but searches of these sequences against the nucleotide database revealed very few significant matches. None of these hits was to stingless bees though one read of 97 nucleotides aligned with two non-contiguous segments of the mitochondrial cytochrome oxidase subunit I gene of the East Asia bumblebee Bombus hypocrita. The most significant hit was for 452 nucleotides of a 470-nucleotide read that aligned with part of the genome of the root-nodulating bacterium Bradyrhizobium japonicum. The other significant hits were to proteobacteria and an actinomycete. Searches directed specifically at Apidae nucleotide sequences only gave short and insignificant alignments. All of the reads from the older specimen appeared to be artefacts. We were therefore unable to obtain any convincing evidence for the preservation of ancient DNA in either of the two copal inclusions that we studied, and conclude that DNA is not preserved in this type of material. Our results raise further doubts about claims of DNA extraction from fossil insects in amber, many millions of years older than copal.

Production and preservation of resins – past and present

“…Penney et al . () showed that DNA from insect inclusions does not even survive in copal, which is immature amber, so that DNA preserved on geological timescales (millions of years) is extremely unlikely. Organelles and cell membranes have been reported from amber (Koller, Schmitt & Tischendorf, ), but even resistant macromolecules like lignin are significantly degraded (Stankiewicz et al, ).…”

Thermostable and promiscuous Precambrian proteins

“…In fact, the ancestral resurrection exercise of Benner and coworkers anticipated the existence of nondigestive ribonucleases with interesting and 'unusual' activities, of which examples are currently known (Pizzo and D'Alessio, 2007). Certainly, a 'direct' validation of these reconstruction/resurrection efforts is hardly possible, as preservation of sequenceable protein or DNA in millions-of-years-old fossils is expected to be extremely uncommon (Schweitzer, 2011;Greenwalt et al, 2013;Penney et al, 2013). Nevertheless, the study of Benner and coworkers on ribonucleases (Jermann et al, 1995) and several other analogous experimental analyses (for reviews, see Benner et al, 2007;Harms and Thornton, 2010) show that although the reconstruction of ancestral sequences is unavoidably uncertain to some extent, it is to a significant degree validated at the phenotypic level by the fact that the properties of the proteins resurrected in the laboratory are typically robust and consistent with the ancestral properties expected from coherent evolutionary narratives.…”

The current and future applications of ancient DNA in Quaternary science

“…The DNA sequences obtained from the dinosaur bone fragments were shown, soon after the initial publication, to have resulted from contamination with modern human DNA (Zischler et al, 1995). Attempts to replicate the early reports of DNA from amber specimens have been unsuccessful and indicate that the environment within amber might not be particularly conducive to DNA preservation (Austin et al, 1997;Penney et al, 2013). Other reports of million-year-old DNA, such as that from the Magnolia fossil, are now similarly ascribed to modern contamination.…”