Note on using nuclear 28S rDNA for sequencing ancient and strongly degraded insect DNA

Abstract

A protocol using insect specimens or parts thereof allows for sequencing of sections of nuclear 28S rDNA. In the present note it is demonstrated that this protocol can readily be applied to strongly degraded DNA (ancient, fixed or contaminated). Primers that are specifically designed to discriminate against human DNA but also other non‐arthropod species are tested on a range of species covering all insect groups (59 insect species from all 33 orders). Additionally, the samples represent a selection of various, mostly DNA‐degrading, preservation methods, including the most common fixatives used for morphological investigations and for long‐term storage in collections. Successful amplification was possible for all tested samples including ca. 200 year‐old dried museum specimens as well as for over 4000 year‐old fossil insects embedded in copal. When the NCBI database contained information on the tested species an unambiguous taxonomic discrimination was possible. This approach is based on a standardized protocol that guarantees easy application. This note presents primer pairs for 28S rDNA that can be a useful tool for ancient DNA (aDNA) research.

DNA from resin-embedded organisms: Past, present and future

“…However, modern techniques are more powerful and more sensitive. Recently, Büsse et al [51] reported successful amplification of aDNA from sub-fossil specimens, but limited information was given about extraction methods, the exclusion of contamination or aspects of reproducibility; therefore, the positive results obtained in that study should be viewed with caution. By contrast, the methodology discussed here is intended for future use as a guide and base for studies of specimens embedded in resin.…”

“…The two samples of ancient resin (sensu [52]) were analysed via accelerator mass spectrometry (standard-AMS) radiocarbon and dated to 790-700 years BP and 4030-3900 years BP. However, the positive results reported by Büsse et al [51] should be viewed with caution as the publication does not contain a detailed discussion of the authentication procedures employed. The greatest problem with many studies of insect aDNA is their lack of reproducibility [47], a requirement for scientific evidence.…”

“…Some success with DNA amplification has been reported with pinned museum and permafrost-preserved invertebrates [ 43 , 48 – 51 ], and some of these samples were older than the oldest specimens analysed by Penney et al [ 21 ]. These studies concluded that museum specimens could serve as a source of molecular information [ 48 – 50 ].…”

“…These studies concluded that museum specimens could serve as a source of molecular information [ 48 – 50 ]. Of all recent publications, the sole hypothetical success with aDNA extraction from sub-fossil specimens embedded in resin was reported by Büsse et al [ 51 ], who claimed to have successfully amplified aDNA from 61 species of 46 higher arthropod taxa from ca. 200-year-old dried museum specimens, as well as from two beetles embedded in sub-fossil resin.…”

A revised definition for copal and its significance for palaeontological and Anthropocene biodiversity-loss studies

“…New Zealand copal has been analysed by 14 C reaching an age of 2000-37,000 years BP, however these do not contain bioinclusions 43 . One piece of Colombian copal has been dated as 10,600 years BP 44 , and one analysed piece of Sulawesi copal (Celebes, Indonesia) obtained an age of 4000 years BP 45 . One important problem is the fact that for different copals from diverse regions no occurrences of stratigraphically well-dated (i.e., with index fossils) copals exist 30 .…”