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

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

Species delineation of Eurasian ground squirrels by refined single linkage (RESL) analysis showed on the gene tree.

<p>Square brackets indicate putative species recognized by RESL. Barcode Index Numbers (BIN) assigned to each putative species are given as BOLD:XXXXXXX. Species split by RESL are red colored, while merged species are blue colored. ML tree (HKY+I+G model) of 657 bp COI fragment. Bootstrap values above 50 are indicated. The nodes with multiple specimens were collapsed to a triangle, with the horizontal depth indicating the level of intraspecific divergence. Individuals with introgressed mtDNA are not included into the tree.</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

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

Frequency of the introgressed mtDNA haplotypes in four ground squirrel species as revealed by DNA barcoding.

<p>Frequency of the introgressed mtDNA haplotypes in four ground squirrel species as revealed by DNA barcoding.</p

Identification success for the best 100 bp and 50 bp mini-barcodes identified by sliding windows analysis in Spider.

<p>* position of the first nucleotide of the given window in the alignment.</p><p>Identification success for the best 100 bp and 50 bp mini-barcodes identified by sliding windows analysis in Spider.</p

Species delineation of Eurasian ground squirrels by Poisson tree processes (PTP) on the ML tree (HKY+I+G model; 657 bp of COI).

<p>Bayesian support values for delimited species are indicated;intensity of red color reflects the strength of support. The nodes with multiple specimens were collapsed to a triangle, with the horizontal depth indicating the level of intraspecific divergence. Individuals with introgressed mtDNAare not included into the tree.</p

Intraspecific genetic variation of Eurasian Ground Squirrels.

<p>n/N – sample size/number of haplotypes; <i>h</i> ± SD – haplotype diversity ± standard deviation; <i>π</i> ± SD—nucleotide diversity (per site) ± standard deviation; Mean intra-sp ± SE – mean intraspecies p-distance ± standard error; Max intra-sp – maximum intraspecies p-distance; Distance to NS ± SE – <i>p</i>-distance to a nearest species ± standard error (based on 1000 bootstrap replications).</p><p>Intraspecific genetic variation of Eurasian Ground Squirrels.</p