Non-Destructive Sampling of Ancient Insect DNA

Philip Francis Thomsen,Kasper Munch,Richard N Holdaway,James Haile,M Thomas P Gilbert,Svetlana Kuzmina,Scott Elias,Duane G Froese,Eske Willerslev,Andrei Sher,Robert Desalle

doi:10.1371/journal.pone.0005048

Abstract

BackgroundA major challenge for ancient DNA (aDNA) studies on insect remains is that sampling procedures involve at least partial destruction of the specimens. A recent extraction protocol reveals the possibility of obtaining DNA from past insect remains without causing visual morphological damage. We test the applicability of this protocol on historic museum beetle specimens dating back to AD 1820 and on ancient beetle chitin remains from permafrost (permanently frozen soil) dating back more than 47,000 years. Finally, we test the possibility of obtaining ancient insect DNA directly from non-frozen sediments deposited 3280-1800 years ago - an alternative approach that also does not involve destruction of valuable material.Methodology/Principal FindingsThe success of the methodological approaches are tested by PCR and sequencing of COI and 16S mitochondrial DNA (mtDNA) fragments of 77–204 base pairs (-bp) in size using species-specific and general insect primers.Conclusion/SignificanceThe applied non-destructive DNA extraction method shows promising potential on insect museum specimens of historical age as far back as AD 1820, but less so on the ancient permafrost-preserved insect fossil remains tested, where DNA was obtained from samples up to ca. 26,000 years old. The non-frozen sediment DNA approach appears to have great potential for recording the former presence of insect taxa not normally preserved as macrofossils and opens new frontiers in research on ancient biodiversity.

Highlights

Most ancient genetic studies have focused on vertebrates, plants and to a lesser extent microbes revealing ancient DNA (aDNA) research as a powerful tool for testing hypotheses in biology [1,2]
The non-destructive DNA extraction procedure was tested on two types of samples: i) Twenty museum specimens of beetles collected between AD 1820 and AD 2006, and ii) fourteen beetle macrofossils from the late Pleistocene and late Pleistocene-early Holocene
All twenty specimens of museum beetles produced amplifiable and authentic COI mitochondrial DNA (mtDNA) sequences between 77–204 -bp in size. These were from the ground beetle Harpalus latus (Linnaeus, 1758), the pill beetle Byrrhus pilula (Linnaeus, 1758), the leaf beetle Chrysolina polita (Linnaeus, 1758) and two weevils, Otiorhynchus sulcatus (Fabricius, 1775) and Curculio pyrrhoceras Marsham, 1802 (Table 1)

Summary

Introduction

Most ancient genetic studies have focused on vertebrates, plants and to a lesser extent microbes revealing aDNA research as a powerful tool for testing hypotheses in biology [1,2]. A major constraint on the use of historical, and ancient, insect specimens in aDNA research is the destructive nature of the sampling procedure [15]. One potential solution is the application of an extraction protocol that uses digestion buffers designed to enable the recovery of DNA from insect remains without causing visual external morphological damage to the material [16]. This method has been used successfully on museum specimens of beetles collected between 1952 and 2002. We test the possibility of obtaining ancient insect DNA directly from non-frozen sediments deposited 3280-1800 years ago - an alternative approach that does not involve destruction of valuable material

Methods

Results

Conclusion