Recovery of mitochondrial and nuclear DNA for systematic studies of Naucoridae (Heteroptera): an assessment of preservation techniques and specimen age

Abstract

AbstractSpecimens of Pelocoris femoratus (Palisot de Beauvois) (Heteroptera: Naucoridae) preserved in different ways were analyzed to determine which methods of preservation and specimen age lend themselves to the acquisition of PCR amplifiable nuclear and mitochondrial DNA, as a preliminary study to a phylogenetic analysis of the family. Specifically, total DNA was extracted from museum specimens that were either freshly collected, recently preserved in 100% ethanol, or of four age classes (1 to 45-year old) of specimens that were either pinned or preserved in 75% ethanol. Following DNA extraction, the appropriate length of COI mtDNA and nuclear 28s rDNA were amplified using PCR and sequenced. The data revealed that continuous degradation of DNA occurred in ethanol preserved specimens to the point that 30-year old specimens yielded no detectable DNA in spectrometric analysis. However, after an initial, substantial decline in yield, dry-pinned specimens were fairly stable in DNA yield through at least 25 years of specimen age. Both COI and 28s genes were successfully amplified from DNA extracted from pinned (≤ 25 years) or 75% ethanol preserved specimens (≤ 20 years) housed at room temperature in the dark. However, for specimens preserved for longer than 30 years, DNA recovery was less reliable. Our data suggest that it is possible to recover amplifiable DNA from pinned (≤ 25 years) or 75% ethanol preserved specimens (≤ 20 years) for use in phylogenetic analysis when fresh specimens are not available.