Specimen collection and preservation for barcoding
Extensive literature resources are available on the techniques for collecting and preserving mammal specimens, and this web site does not intend to duplicate them. It is assumed that prospective contributors are familiar with the methods of mammal collecting and preservation. DNA can be extracted from both dry (study skins and skeletal material) and ethanol-stored specimens, although some forms of preservation require complicated DNA extraction protocols. Moreover, DNA degradation can occur, especially if the specimen has been pre-treated using DNA-unfriendly protocols (e.g., formalin fixation, skin tanning, and arsenic treatment) or exposed to light, high temperatures and humidity. Hence, it is preferred to deliberately collect and specifically preserve tissue intended for DNA extraction.
Preferred types of tissue samples
It should be kept in mind that many tissue collecting protocols (especially those for marine mammals) were designed to facilitate studies of chemical contaminants or other specialized tasks (e.g., allozyme analyses) and promote collecting tissue from organs which are not otherwise a good source for intact DNA, such as liver or kidneys. It is preferred that these organs be avoided when collecting tissue for DNA barcoding. Below are a few examples of tissue preservation techniques which have proven friendly for DNA barcoding analyses.
Muscle or gonad tissue in 95-100% ethanol. It is important to know that DNA quality of ethanol-preserved tissues is dependent on many factors, including ethanol acidity and concentration, storage temperature, exposure to light, sample age, as well as internal quality of the tissue itself. Ethanol is a good preservative of morphological integrity of the tissue, but it does not suppress enzymatic activity, which may be another factor adversely affecting DNA — hence the need to avoid enzymatically rich tissue, such as liver. Therefore it is preferred that ethanol-preserved tissue is stored at low temperatures (ideally, -20°C or lower). It is also important that tissue is minced thoroughly before fixation to allow quicker penetration and that the ratio of tissue to fixative volume is below 1:10. Change of fixative is recommended after an initial one week of storage.
Dry muscle sampled from carcasses intended for skeletal preparation using dermestid beetles. This tissue source proved to be very good in a case study involving several dozen samples. Tissues could be shipped for analysis without additional fixation. This protocol is especially advocated for museums that prepare skin, skull, and skeleton vouchers, but do not have a designated tissue collection.
Small aliquot of blood, gonad or brain tissue blotted onto a blotting card. This technique is still under evaluation and it is advisable to check with the campaign coordinator for details on using this form of tissue preservation.
It is well-known that provenance data and biological information are an integral part of any collection voucher specimen and constitute a large portion of its research value. Therefore it is critical that any data associated with each specimen used for populating the reference library of DNA barcodes are recorded and digitized. There are several vital data fields required by BOLD (i.e., detailed locality with coordinates and elevation, collection date, name(s) of collector(s), sex and age), however, there are additional pieces of information, recording which constitutes good practices in mammalogy. These include standard measurements, weight, reproductive data, and habitat. Field collecting of mammals usually also produces ancillary information on mammal community structure, parasite infestations, habitat preferences, etc., which may pose significant synecological value. Although collecting these data falls outside the scope of mammalian DNA barcoding per se, prospective donors are encouraged to consult specialized literature on how to obtain and log this valuable information. The mammalian barcoding campaign has a set of standard protocols for detailed data capture (in both digital and analog format) which can be distributed among interested participants, upon request.
Because of their important economical, conservation, and biomedical status, mammals are among the most highly regulated living organisms when it comes to collecting, deposition, and international transfer. It is imperative that any prospective contributor to the mammal barcoding campaign is well aware of the national, international, and institutional regulations pertaining to the collection and shipping of mammal specimens and any derivative biomaterials and conducts his/her operations in a compliant manner. This particularly concerns locally or globally endangered species and those listed under the Convention of International Trade of Endangered Species (CITES). It is preferred that any institution hosting or facilitating such collecting activities has a nationally recognised collection and is registered with the national CITES authority. Regulations differ drastically from nation to nation, and it is important that they are followed by all campaign participants as they apply to their research activities. The Biodiversity Institute of Ontario is a CITES-registered institution and will gladly assist in obtaining Canadian import documentation for samples intended for processing at the core sequencing facility at Guelph, Ontario.