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

Franklin Alongi,Robert E Keane,Kristin Legg,Matt Lavin,Andrew J Hansen,David Laufenberg

doi:10.3390/f10121060

Abstract

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.

Highlights

Whitebark pine, Pinus albicaulis Engelm., is a candidate species for the Endangered Species Act threatened and endangered list that plays a keystone role in Rocky Mountain coniferous forests and elsewhere in western North America [1]
The matK coding sequences of limber and whitebark pine (GenBank accessions MN542807–MN542818; Supplemental Table S1) differed at eight nucleotide sites out of the approximately 1630 total sites. One of these involved a BsmAI restriction site that cleaved the whitebark pine matK amplicon into two fragments, about 623 and 1000 base pairs. This BsmAI restriction site found in whitebark pine occurred in most white pine species but was absent from the white pine group in just limber pine and its closest relatives, Pinus ayacahuite Ehreng. ex Schlecht. and P. strobiformis Engelm. (Appendix A)
The environmental conditions of pollen fossilization would have to be favorable to DNA preservation but given that chloroplasts are inherited paternally in gymnosperms [32], isolation of chloroplast DNA from fossil pollen would be amenable to the approach we detail in our study

Summary

Introduction

Pinus albicaulis Engelm., is a candidate species for the Endangered Species Act threatened and endangered list that plays a keystone role in Rocky Mountain coniferous forests and elsewhere in western North America [1]. Understanding the population responses of whitebark pine to climate change is of high interest to forest managers, especially in the northern Rocky Mountain. Forests 2019, 10, 1060 region encompassing Yellowstone National Park [2] This is because whitebark pine is ecologically important and its nutrient-rich seeds represent an important food source for grizzly bears and other animals. The issue is that whitebark pine populations can be sympatric with the similar five-needle limber pine, Pinus flexilis James, and the geographic ranges of these two pines are highly overlapping in the northern Rocky Mountains, Great Basin, and Southern Sierra Nevada of North America [4]. Limber and whitebark pine differ primarily by seed cone and secondarily by pollen cone morphology (Figure 1)

Methods

Results

Discussion

Conclusion