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.

Variation in TaqI-digested DNA of Sugar and Black Maples Is Independent of Taxon and Plant Origin

Morphological distinctions between sugar maples and black maples are not consistently evident, and molecular assessment of genetic diversity is lacking for these taxa. We examined restriction-site polymorphisms in the ndhA intron of the chloroplast DNA (cpDNA) in populations of sugar maples and black maples representing their zones of allopatry and sympatry in eastern North America. Restriction-site analysis of the ndhA intron after digestion with HinfI and Sau3AI yielded no polymorphisms. Restriction digestion of the ndhA intron with TaqI revealed two cpDNA haplotypes that were neither geographically localized nor taxon specific. Although testing additional accessions of sugar maples and black maples for cpDNA variation will further elucidate patterns of genetic variation, our initial results suggest that the taxa are either exchanging genes or share an ancestral cpDNA polymorphism.

Phylogenetic relationships and infraspecific variation in Canadian Arctic Poa based on chloroplast DNA restriction site data

Infraspecific variation and phylogenetic relationships of Canadian Arctic species of the genus Poa were studied based on chloroplast DNA (cpDNA) variation. Restriction site analysis of polymerase chain reaction amplified cpDNA was used to reexamine the status of infraspecific taxa, reconstruct phylogenetic relationships, and reexamine previous classification systems and hypotheses of relationships. Infraspecific variation was detected in three species, but only in Poa hartzii Gand. did it correspond to infraspecific taxa where recognition of subspecies ammophila at the species level is supported. Additional variation in P. hartzii ssp. hartzii is hypothesized to be the result of hybridization with Poa glauca in the High Arctic and subsequent introgression resulting in repeated transfer of P. glauca DNA. The variation in Poa pratensis L. had a geographical rather than taxonomic basis, and is hypothesized to correspond to indigenous arctic versus introduced extra-arctic populations. In P. glauca Vahl cpDNA variation was detected only in western Low Arctic and boreal populations and may represent greater variation where the species survived the Pleistocene glaciations. Cladistic parsimony analysis of cpDNA restriction site data mostly confirms recent infrageneric classification systems. Poa alpina L., along with the non-arctic Poa annua L. and Poa sect. Sylvestres, formed the basalmost clades. The remaining taxa group into two main clades: one consisting of Poa sects. Poa, Homalopoa, Madropoa and Diocopoa; the second, of Poa sects. Secundae, Pandemos, Abbreviatae and Stenopoa. Poa sect. Poa, comprising Poa arctica R. Br. and P. pratensis, is a strongly supported monophyletic group, not closely related to P. alpina. Poa hartzii is confirmed as a member of a paraphyletic or weakly supported P. sect. Secundae. Poa glauca and Poa abbreviata R. Br. are distinct members within a generally unresolved Poa. sect. Stenopoa-Abbreviatae complexKey words: Poa, Canadian arctic, chloroplast DNA, restriction site analysis, infraspecific variation, phylogeny.