DNA barcoding the native flowering plants and conifers of Wales

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification

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Ability of the DNA barcode markers <i>rbcL</i> and <i>matK</i> to discriminate the Welsh flora.

<p>Discrimination (%) at species, genus and family level for <i>rbcL</i>, <i>matK</i> and both markers combined using monophyletic groups in Neighbour-Joining trees (Tree), BLASTn searches (BLAST) and barcode gap analysis using pairwise (Barcode gap pairwise) and multiple alignments (Barcode gap multiple). Species level discrimination for monophyletic groups in Neighbour-Joining trees is divided into bootstrap support values of ‘any’, >50% and >70% based on 1000 bootstrap replicates. Discrimination uses 808 species for which multiple individuals were DNA barcoded for both <i>rbcL</i> and <i>matK.</i> Species with single sequences were included in the analyses as sources of discrimination failure. For a complete list of which species can be discriminated using the different methods see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037945#pone.0037945.s006" target="_blank">Dataset S3</a>.</p

Recoverability of the orders of flowering plants and conifers found within Wales using herbarium and fresh material.

<p>Recoverability (%) of <i>rbcL</i> and <i>matK</i> across the 34 orders of seed plants found within the Welsh flora. Results are based on 3637 herbarium and 635 freshly collected specimens. White cells denote orders for which fresh specimens were not collected.</p

Species discrimination for the orders of flowering plants and conifers found within Wales.

<p>Species discrimination (%) for <i>rbcL</i>, <i>matK</i> and both combined across the 34 orders of flowering plants and conifers found within the Welsh flora. Discrimination is assessed using three methods; barcode gap using multiple alignments (Barcode gap), monophyletic groups in Neighbour-Joining trees (Tree) and BLASTn searches (BLAST). To allow for comparison across the markers and methods 808 species for which multiple individuals were sequenced for both <i>rbcL</i> and <i>matK</i> were used, but species with single sequences were included as a source of discrimination failure. The number of species per order in the Welsh flora (out of the 808) is shown in brackets next to the order name. Pearson correlation coefficients and associated p-values for the relationship between the number of species per order and % species discrimination success are shown.</p

Sequence quality of DNA barcodes from herbarium and fresh material for <i>rbcL</i> and <i>matK.</i>

<p>For a complete list of quality values for each DNA barcode see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037945#pone.0037945.s005" target="_blank">Dataset S2</a>. For CBOL PWG criteria see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037945#s4" target="_blank">Methods and Analysis</a>.</p

Effect of herbarium specimen age on recoverability (%).

<p>Using 3607 herbarium specimens ranging in age from 1899–2008, samples were divided into 11 age classes and Spearman rank correlation used to test for a relationship between age class and recoverability (%) using the DNA barcode loci, <i>rbcL</i> and <i>matK.</i></p