Amber Insects: DNA Preserved?

Amber is a polymerized form of tree resin that was produced by trees as a protection against disease agents and insect pests. The resin hardened and, sometimes, captured insects, seeds, feathers, microorganisms, plants, spiders, and even small vertebrates that got stuck in the sticky exudate. The hardened resin was preserved in the earth for millions of years, especially in regions where it was deposited in dense, wet sediments such as clay or sand that formed in the bottom of an ancient lagoon or river delta. For thousands of years, people have collected amber. Many people use amber as a gem, but scientists find amber a magnificent way to identify ancient organisms.

Amber can be found in a variety of sites around the world. The composition, color, clarity, and other properties of amber vary according to age, conditions of burial and type of tree that produced the resin. The oldest amber is from the Carboniferous (360–285 million years ago, mya) and can be found in the United Kingdom and in Montana in the USA. Permian amber is 185–145 million years old and found most often in Russia. Triassic amber (245–215 mya) can be found in Austria, and Jurassic amber (215–145 mya) is found in Denmark. Cretaceous amber (65–140 mya) is found in many locations around the world and represents the time when dinosaurs reigned and flowering plants evolved along with a variety of insects. For example, the rich amber deposits in central New Jersey in the USA are from the Turonion period of the Upper Cretaceous, about 92 mya. Other Cretaceous- period amber is found in North Russia and Japan. Baltic amber is found in the Baltic sea where amber has been collected and made into decorative objects for at least 13,000 years. In the Dominican Republic, amber deposits 23–30 million years old are found in rock layers. Dominican amber is particularly rich in insect inclusions. This amber was formed from the resin of an extinct tree in the legume family. Tertiary amber deposits are found in several locations around the world and are from 1.6 to 65 million years old. Tertiary deposits in the USA are found in Arkansas.

Insect DNA in Amber?

The ability to amplify dinosaur DNA from insects preserved in amber in the film Jurassic Park captured the imagination of the public. Subsequently, the PCR was used to amplify DNA fragments from insects preserved in ancient amber, but these results have been controversial, as have been the results from amplifying dinosaur DNA.

Why the controversy? Is amber a special form of preservative that allows DNA to persist for unusually long periods of time (millions of years)? Amber entombs insect specimens completely, aſter which they completely dehydrate so the tissue is effectively mummified. Terpenoids, which are major constituents of amber, could inhibit microbial decay. Certainly, preservation of amberembedded insects seems to be exceptional and insect tissues in amber appear comparable in quality to the tissues of the frozen wooly mammoth (which is “only” 50,000 years old). But is the DNA in these tissues preserved?

DNA has been extracted from a variety of insects in amber, including a fossil termite Mastotermes electrodominicus estimated to be 25–30 million years old, a 120- to 130-million year old conifer-feeding weevil (Coleoptera: Nemonychidae) and a 25- to 40-million year old bee. These are extraordinary ages for DNA!

The DNA sequences obtained from all amberpreserved insects meet several, but not all, criteria of authenticity; the fossil DNA sequences “make phylogenetic sense” and DNA has been isolated from a number of specimens in several cases (although the weevil example was derived from a single specimen). However, the extraction and amplification of fossil DNA sequences from amber-preserved insects has yet to be reproduced in independent laboratories, despite multiple attempts to do so, which has cast doubt on the authenticity of the reports.

One of the most controversial claims involved the isolation of a “living” bacterium from the abdomen of amber-entombed bee. Bacterial DNA from a 25-million-year-old bee was obtained and sequenced and a bacterial spore was reported to be revived, cultured, and identified. The classification of the bacterium is controversial because the bacterium could have come from a currently undescribed species of the Bacillus sphaericus complex. The modern B. sphaericus complex is incompletely known, so the “new” sequence obtained could be that of a modern, but previously unidentified, bacterium because this group of bacteria often is isolated from the soil.

Other claims of amplifying ancient DNA have been disproved. For example, the mitochondrial cytochrome b sequence of an 80-million-year-old dinosaur from the Upper Cretaceous in Utah was later discovered to be, most probably, of human origin. Likewise, a 20-million-year-old magnolia leaf produced sequences that were similar to those of modern magnolias. The authenticity of the magnolia sequences were cast into doubt because they were exposed to water and oxygen during preservation and DNA is especially vulnerable to degradation under such conditions.

The most common ancient DNA analyzed is usually mitochondrial DNA because it is so abundant; however, this abundance makes it easy to contaminate the ancient sample with modern mtDNA. The amplification of ancient DNA remains highly controversial because the technical difficulties are great.

DNA is a chemically unstable molecule that decays spontaneously, mainly through hydrolysis and oxidation. Hydrolysis causes deamination of the nucleotide bases and cleavage of base-sugar bonds, creating baseless sites. Deamination of cytosine to uracil and depurination (loss of purines adenine and guanine) are two types of hydrolytic damage. Baseless sites weaken the DNA, causing breaks that fragment the DNA into smaller and smaller pieces. Oxidation leads to chemical modification of bases and destruction of the ring structure of base and sugar residues. As a result, it is almost always impossible to obtain long amplification products from ancient DNA.

PCR products from ancient DNA often are “scrambled.” This is due to the phenomenon called “jumping PCR,” which occurs when the DNA polymerase reaches a template position which carries either a lesion or a strand break that stops the polymerase. The partially extended primer can anneal to another template fragment in the next cycle and be extended up to another damaged site. Thus, in vitro recombination can take place until the whole stretch encompassed by the two primers is synthesized and the amplification enters the exponential part of the PCR. This phenomenon makes it essential that cloning and sequencing of multiple clones be carried out to eliminate this form of error in interpretation.

Most archeological and paleontological specimens contain DNA from exogenous sources such as bacteria and fungi, as well as contaminating DNA from contemporary humans. Aspects of burial conditions seem to be important in DNA preservation, especially low temperature during burial. The oldest DNA sequences reported, and confirmed in other laboratories, come from the remains of a wooly mammoth found in the Siberian permafrost; these sequences are “only” 50,000 years old rather than millions of years old.

Theoretical calculations and empirical observations suggest DNA should only be able to survive, in a highly fragmented and chemically modified form, for 50,000–100,000 years. Because only tiny amounts of DNA usually can be extracted from an archeological specimen, stringent precautions and multiple controls are required to avoid accidental contamination with modern DNA.

A methodology to deal with ancient specimens has been proposed that includes careful selection of well-preserved specimens, choice of tissue samples that are likely to have best DNA preservation, and surface sterilization to eliminate surface contamination. The operations should be carried out in a laboratory dedicated to work on ancient specimens and work on ancient DNA should be separated from that on modern DNA. Most importantly, multiple negative controls should be performed during DNA extraction and PCR set up, although a lack of positives in the negative controls is not definitive proof of authentic ancient DNA. Another crucial step is the authentication of the results. Putatively ancient DNA sequences should be obtained from different extractions of the same sample and from different tissue samples from different specimens. The ultimate test of authenticity should be independent replication in two separate laboratories. So far, this type of replication has not been achieved for DNA from amber-preserved arthropod specimens.