Primers used for Sanger sequencing, NGS and in IAC.

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

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

Non-listed, non-filler species count (<i>ITS2</i> region with universal primers ITS_S2F/ITS3/ITS4) in herbal supplements tested.

<p>Non-listed, non-filler species count (<i>ITS2</i> region with universal primers ITS_S2F/ITS3/ITS4) in herbal supplements tested.</p

Listed DNA recovery.

<p>(A) Summary of target DNA detection with NGS, using custom <i>ITS</i> database for plant and fungi downloaded from Genbank. Coverage for <i>ITS2</i> is indicated by circle size. (B) Quality of read clusters (OTUs) for five supplements with significant amplification of target DNA. Size of the square reflects percentage of reads falling within a distance threshold of 0%, <0–2%, 2–5%, and <5% from the reference library sequences.</p

Contamination sources.

<p>Contamination sources.</p

Map of the study area showing collection localities for this study (see S1 Table for locality information).

<p>Map of the study area showing collection localities for this study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117201#pone.0117201.s001" target="_blank">S1 Table</a> for locality information).</p

ML tree (HKY+I+G model) for the 657 bp fragment of COI of all Eurasian ground squirrel species.

<p>Bootstrap values above 50 are indicated; asterisks represent bootstrap values of 100. The nodes with multiple specimens were collapsed to a triangle, with the horizontal depth indicating the level of intraspecific divergence. Numbers next to each species name indicate the sample size (not indicated when n = 1). * individuals with introgressed mtDNA are not included into this analysis.</p

Line-plot of the barcode gap for the Eurasian ground squirrels as generated by Spider [40].

<p>For each individual in the dataset, the grey lines represent the furthest intraspecific distance (bottom of line value), and the closest interspecific distance (top of line value). The red lines show where this relationship is reversed, and the closest non-conspecific is actually closer to the query than its nearest conspecific (i.e. no barcoding gap). Individuals with introgressed mtDNA are not included.</p

Implications of Hybridization, NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

<div><p>The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome <i>c</i> oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (<i>Spermophilus erythrogenys</i>, <i>S. pygmaeus</i>, <i>S. suslicus</i>, <i>Urocitellus undulatus</i>), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.</p></div