We found that barcoding our quantified insect food web resulted in an approximate 41% increase in the number of nodes within the web and thus the connectence of the web was reduced. However, previous conclusions regarding the basic structure of the web in different forest plots were not significantly altered by identifying nodes via molecular tools rather than by strictly morphological ones. Results From 1983 to 1993 there were 12, 292 parasitoid specimens, from 98 species collected at three collection sites in New Brunswick, Canada. From this dataset, 1,710 specimens were sampled for barcoding between 2007 and 2009. Of these, CO1 fragments were generated for 1,492 specimens (12.1% of the total collected and 87.3% of the specimens extracted), 28S for 573 specimens (38.4% barcoded), ITS2 for 80 specimens (5.4% barcoded) and ITS2 for 93 specimens (6.2% barcoded). In one case (Mesopolobus verditer - EE-13510-86 P1) we amplified the CO1 of a bacterial endosymbiont rather than of the insect host. Specimens from 11 genera within five families of parasitoid wasps displayed a characteristic 6 bp deletion in CO1 in the 155th and 156th amino acids of the barcoding region (Chalcididae (Conura), Encyrtidae (Copidosoma), Eulophidae (Aprostocetus, Baryscapus, Elacher- tus, Elasmus, Euplectrus and Pediobius), Perilampidae (Perilampus) and Pteromalidae (Mesopolobus, Pteromalus)). This six base pair deletion occurs within the third internal loop, (likely at the meeting with the fifth membrane-spanning helix), is in frame, has no anomalous amino acid variation following the deletion and occurs within all GenBank sequences from this family. Agarose gels made of the CO1 amplification contained no anomalous secondary bands, and there was no evident systematic heteroplasmy within the trace files. Thus we consider that these gene fragments represent true mitochondrial products and not pseudogenes. Specimens from the genus Copidosoma (Encyrtidae; Encyrtinae) were also characterized by a 1 base pair deletion that, if unrecognized, would place the alignment out of frame and result in stop codons, and is likely a pseudogene or NUMT [36]. Interestingly, specimens from each provisional species displayed this deletion. While there were no corresponding divergences within the 28S, there were three ITS2 groups. Further work is clearly required on this species and for the purposes of this food web analysis, we considered this species to be two. We used the putative pseudogene as the CO1 markers for this species in these analyses. Barcoding the reared specimens revealed 32 individual insect specimens (2.1% of barcoded total) which had apparently been misidentified morphologically (Figure S1). The majority of the species barcoded produced CO1 barcodes that displayed little or no intra-specific variation (Figure 1). There were 22 cases where a named species (or morphospecies; out of the 91 named species barcoded (Table S1), 24%) was revealed to contain deep mitochondrial divergences that were suggestive of multiple species existing within the currently acknowledged name (Figure 2, Figure S1). These cryptic provisional species were distributed amongst all trophic levels (Figure 1) and decreased the connectence of the food web (Figure 3). Specifically, delineating species via barcodes caused an increase in diversity (Nodes (N) increased from 110 to 156 (an increase of 41% from nodes calculated based on morphology as in [9]) and links (L) from 336 – to 449) while the connectence (Measured as L/N2) was reduced from 0.03 to 0.02. Barcoding resulted in the re-evaluation of 10 polyphagous generalist species into 30 provisional species including more monophagous host-specialists (Table S2). While sample sizes are small for some of these comparisons, 16 (53.3%) of these new species were host-specialists while 14 (46.7%) remain generalists. Cryptic Species in a Food Web From the newly recognized provisional species, (possibly representing 64 cryptic species) we selected cases to amplify a ribosomal DNA marker (ITS2 (n = 36 of the provisional species) or 28S-D2 (n = 40 of the provisional species) to test for congruence of the divergence within the species named. (GenBank accessions for all sequences analyzed here are included in Table S1).For those species where we generated ITS2 data (n = 39), we found that 87.2% of the CO1 groups tested also displayed ITS2 variation. In 5 cases there was no ITS2 variation in corroborate to CO1variation, and in one case, there was evident ITS2 variation within a CO1 invariant set. In addition to these ITS2 sequence based comparisons, we also made pairwise comparisons for CBCs. In only two cases (5.1% of those species examined with ITS2), were provisional species identified by sequence variation in CO1 and ITS2 – supported by the presence of CBCs (Table S3). For those species where we generated 28S data (n = 45), 88.9% of the CO1 groups we tested also displayed D2 variation. In 5 cases there was no D2 variation that corroborated the CO1vari- ation (Table S3). GenBank accessions for D2 sequences analyzed here: HQ025168-HQ025800. See Table S3 for a comparison of provisional species splits across CO1 and the rDNA loci. One of the principle predictions of the ‘‘bird feeder effect’’ (that parasitoid diversity should track SBW abundance) was tested when parasitoid diversity was determined using barcode provisional species (molecular operational taxonomic units (MOTU) [37]) or the phylogenetic diversity (PD) [38] contained within a barcode neighbor-joining tree. We found that measuring the diversity of the parasitoid community using either measure of barcode diversity did not affect the prediction of the model – namely diversity increased with increasing budworm density (Figure 4) and that these effects were more evident in the more heterogeneous environments. Discussion SBW Food Web Diversity Barcoding the SBW food web resulted in an increased appreciation of diversity across all trophic levels (Figures 1, 2). Species presumed to be single biological units were revealed by examination of genetic divergences for both single mitochondrial and multiple nuclear loci to be multiple units. These deep genetic divergences were often, but not always, coincident with different host records. The proportion of cryptic diversity uncovered was greater in smaller taxa (Eulophidae, Pteromalidae), than in larger taxa (Tortricidae, Tachinidae, Ichneumonidae). The importance of body size to the proper characterization of a food web is well known [39,40,41,42], and if smaller organisms are more likely to contain cryptic diversity – then the relative importance of body size may be even more challenging to quantify if it is particularly confounded by problems of species resolution and identification amongst the smallest size classes. For instance, theory predicts that small body size individuals ought to be lower in the food web [41]. In our web, the highest order parasitoids are often amongst the smallest individuals – often containing a large preponderance of cryptic diversity. If different aspects of food web structure are prone to an effect of body size then there are likely to be theoretical repercussions when web resolution is determined via such a standardized approach as DNA barcodes. We found it very interesting how the rate of discovery of cryptic species here in the temperate Acadian forest is so similar (24% of presumed single species decayed into multiple provisional species following barcoding) to the rate of cryptic parasitoid species revealed in a tropical environment [25% –15]. Indeed, similar findings have been recently reported from northern Europe where 31% of species designations in leaf-mining Lepidoptera and gall- PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e14424