An Economical Approach to Distinguish Genetically Needles of Limber from Whitebark Pine

Whitebark pine is difficult to distinguish from limber pine when seed cones are not present. This is often the case because of young stand age, growth at environmental extremes, or harvesting by vertebrate species. Developing an economical genetic identification tool that distinguishes non-cone-bearing limber from whitebark pine, therefore, could aid many kinds of research on these species. Phylogenetic studies involving limber and whitebark pine suggest that chloroplast DNA sequences differ between these species. We therefore wanted to identify chloroplast loci that could differentiate limber from whitebark pine trees by taking an economical approach involving restriction-site analysis. We generated chloroplast DNA barcode sequences sampled from limber and whitebark pine trees that we identified using attached seed cones. We searched for nucleotide differences associated with restriction endonuclease recognition sites. Our analyses revealed that matK and the psbA-trnH spacer each readily amplified and harbored multiple DNA-sequence differences between limber and whitebark pine. The matK coding sequence of whitebark pine has a BsmAI restriction site not found in limber pine. The psbA-trnH spacer of limber pine has two PsiI restriction sites, neither of which is found in whitebark pine. DNA-sequence and restriction-site analysis of the psbA-trnH spacer from 111 trees showed complete congruence between visually and genetically identified limber (n = 68) and whitebark (n = 43) pine trees. We conclude that restriction site analysis of the chloroplast psbA-trnH spacer and matK involves both minimal technical expertize and research funds. These findings should be of value to foresters interested in species identification and distribution modeling, as well as the analysis of fossil pine pollen, given that gymnosperms transmit chloroplast DNA paternally.