Specimen collection and preservation for barcoding
The conventional ways of preserving ant specimens include either pinning workers and reproductives or preservation of larval and adult stages in 70-99% ethanol.
Both pinned (dried) and ethanol preserved specimens perform well in DNA amplification - although the utility of ethanol preserved specimens is very related to how frequently the ethanol has been changed, the ambient storage temperature, and how packed the storage vial is. Very large barcode studies (>100K specimens) on Lepidoptera have shown that best results derive from specimens collected within the last 5 years, but it is possible to analyze much older material.
When preserved in ethanol, DNA can degrade due to acidification of ethanol through time. Collecting and preserving ants in higher concentration ethanol (95%) does slow DNA degradation. Ideally, ethanol should be changed a few days after the initial collection. This is particularly critical for larval specimens because of the large amount of water in their bodies. Other factors, such as temperature and exposure to sunlight can affect the life of DNA as well. Specimens should be kept in a refrigerator when possible.
All life-history stages can be barcoded - although preferences are to those specimens that can be identified to species-level. In order to measure the levels of genetic divergence within species, we plan to analyze multiple (5-10) individuals for each species. When choosing the particular individuals for DNA analysis, the widest range of intraspecific morphological differentiation from the widest geographic distribution should be selected.
While fresh materials are preferred for establishing the majority of a DNA barcode library, museum collections will serve as a critical resource. Although museum materials generally have lower success rate in DNA sequencing, they typically provide much more complete species coverage than new collection efforts. Additionally, freshly collected specimens can be examined against the type specimens that are deposited in various museums over the world using DNA sequences. A series of studies have shown that a very short fragment of COI sequences (~130 bps on the 5' terminus) can provide surprisingly good resolution (>95%) for species-level identification in Hymenotpera (Braconidae, Formicidae) and other insect groups. Because short DNA fragments are much easier to amplify in old specimens, museum materials, including type specimens, can be associated with fresh materials using these "mini-barcodes".
Ant specimens have been contributed from various sources (including amateur collectors and museums), to BIO since 2004. Researchers at BIO have also collected specimens at varied sites in the eastern half of North America ranging from the Northwest Territories to the southern USA. Many of the species encountered still need to have their identifications confirmed by specialists. Certainly, with the help from taxonomic specialists, the process of collecting reference barcodes for world ants will be greatly accelerated.