Primers used for Sanger sequencing, NGS and in IAC.

<p>Primers used for Sanger sequencing, NGS and in IAC.</p

Percentage of taxonomic resolution by BLAST to family, genus and species.

<p>Taxonomic resolution for <i>rbcL</i>, <i>matK</i>, and ITS2 for 25 species-rich families.</p

Level of taxonomic resolution provided by <i>rbcL</i>, <i>matK</i> or ITS2 for 25 families.

<p>Level of taxonomic resolution provided by <i>rbcL</i>, <i>matK</i> or ITS2 for 25 families of vascular plant that are species-rich in Canada. The three colours show the proportion of species identified to a family (blue), genus (orange) or species (green) level.</p

Boxplots of MPD and MNTD for <i>rbcL</i>, <i>matK</i>, and ITS2.

<p>Boxplots comparing MPD and MNTD for the vascular plant families of Canada for <i>rbcL</i>, <i>matK</i>, and ITS2. Significance (p–adjusted < 0.005) is indicated with an asterisk(s).</p

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada

<div><p>Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (<i>rbcL</i>, <i>matK</i>, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for <i>rbcL</i> to 60% for ITS2 and 39% for <i>matK</i>. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for <i>rbcL</i> to 97% for ITS2 and 98% for <i>matK</i>. However, <i>matK</i> delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and <i>rbcL</i> (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with <i>rbcL</i> to 69% with <i>matK</i>. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for <i>rbcL</i> and 87% for <i>matK</i>. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest.</p></div

List of localities, corresponding terrestrial ecozones and biogeographic regions used to test the taxonomic resolution of <i>rbcL</i>, <i>matK</i>, and ITS2 libraries for the vascular plants of Canada.

<p>The number of species at each locale is in parentheses.</p

Coverage by barcode locus for the plant communities at 28 Canadian localities.

<p>The number of plant species present at each site is indicated in parentheses.</p

Four scenarios of PCR amplification of short <i>rbcL</i> fragment with IAC.

<p>(A) Trigonella-13: both amplicons (IAC and target) detected in both lysate dilutions. (B) Echinacea-1: both amplicons (IAC and target) detected only in diluted lysates. (C) Echinacea-3: IAC detected only in diluted lysates. (D) Ginkgo-8: only IAC detected for both lysate dilutions.</p

The mean MPD and MNTD for 25 species-rich families with the number of sampled species.

<p>The mean MPD and MNTD for 25 species-rich families with the number of sampled species.</p

Relative peak height for ten <i>Ginkgo</i> active compounds as inferred from Agilent Qualitative Analysis software.

<p>Whiskers indicate standard offsets (+SE) of the means; asterisks (*) indicate significant pairwise difference (p<0.05) between mean heights of HPLC-MS peaks for corresponding metabolites.</p