A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF–atpH, and psbK–psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85–100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK–psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69–71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems.

The ability to discriminate between species using barcoding loci has proved more difficult in plants than animals, raising the possibility that plant species boundaries are less well defined. Here, we review a selection of published barcoding data sets to compare species discrimination in plants vs. animals. Although the use of different genetic markers, analytical methods and depths of taxon sampling may complicate comparisons, our results using common metrics demonstrate that the number of species supported as monophyletic using barcoding markers is higher in animals (> 90%) than plants (~70%), even after controlling for the amount of parsimony‐informative information per species. This suggests that more than a simple lack of variability limits species discrimination in plants. Both animal and plant species pairs have variable size gaps between intra‐ and interspecific genetic distances, but animal species tend to have larger gaps than plants, even in relatively densely sampled genera. An analysis of 12 plant genera suggests that hybridization contributes significantly to variation in genetic discontinuity in plants. Barcoding success may be improved in some plant groups by careful choice of markers and appropriate sampling; however, overall fine‐scale species discrimination in plants relative to animals may be inherently more difficult because of greater levels of gene‐tree paraphyly.

Stutter products are a common artifact in the PCR amplification of frequently used genetic markers that contain mononucleotide simple sequence repeats. Despite the importance of accurate determination of nucleotide sequence and...

Lodgepole pine (Pinus contorta Dougl.) exhibits pronounced morphological variation across its range, historically attributed to allopatric differentiation during the Wisconsin glaciation. However, whether genetic divergence aligns with morphological differentiation—a fundamental prediction of allopatric speciation theory—remains untested. We conducted a comprehensive phylogeographic analysis of chloroplast DNA (trnL intron and trnL/trnF spacer) and mitochondrial DNA (nad1 b/c intron) across 31 populations representing all four recognized subspecies to test hypotheses of refugial isolation and to evaluate the genetic basis of current taxonomic classification. Contrary to predictions of allopatric divergence, both organellar genomes showed striking genetic uniformity (π = 0.000178–0.000186; intersubspecific genetic distances: 1.06 × 10⁻⁴ to 3.96 × 10⁻⁴) with no phylogenetic structure corresponding to morphological boundaries. Significant negative neutrality test values (Tajima’s D = −2.26, p < 0.02; Fu and Li’s D* = −4.52, p < 0.02) suggest recent demographic expansion rather than equilibrium divergence. A distinctive 5 bp indel in coastal populations provides molecular evidence for a northern Pacific refugium, and its occurrence in interior populations is consistent with post-glacial pollen-mediated gene flow, though this directionality remains inferential pending nuclear genomic confirmation. These findings suggest that morphological divergence reflects rapid adaptive evolution in heterogeneous environments rather than deep phylogenetic divisions. This pattern exemplifies gene flow-selection balance, in which divergent selection maintains local adaptation despite extensive gene flow—supporting an ecotypic rather than a phylogenetic interpretation of intraspecific diversity. The persistence of morphological variation despite genetic homogeneity indicates strong selection on ecologically important traits, likely driven by variation in fire regimes, differential seed predation, and climate gradients. These results have critical implications for understanding adaptive evolution rates in widespread conifers and for developing conservation strategies that emphasize adaptive processes over taxonomic categories.