The population dynamics of the budworm can be mediated by insect parasitoids, (wasps (Braconidae [5], Ichneumonidae [6], Chalcidoidea [7]) and flies (Tachinidae [8])) operating at two trophic levels (primary parasitoid and secondary (or hyper-) parasitoid). This array of just over 100 primary and secondary parasitoids that prey upon the budworm, and competing Lepidoptera [5] can decrease the magnitude of budworm outbreaks [9] and are therefore critical components of this complex food web. From a community ecological perspective, the parasitoids within the SBW food web can canalize energy and nutrient flow dependent on whether they attack many hosts (a generalist) or a small number of (or single) host species (a specialist) in the ecosystem. Currently, the majority of the parasitoids in the SBW food web are considered to be generalists [9]. A thorough understanding of the SBW food web is then contingent on the efficient and accurate identification of the individual species within that food web (who is who, and who eats whom). Currently, this identification involves highly specialized taxonomic expertise [5,6,7,8,10] and expensive long-term rearing programs [9]. This diversity- enforced bottleneck is not unique to this system but is a global phenomenon. Consider the magnitude of the insect diversity problem. Nearly J of all animal species are insects; we expect that up to J of all insects are parasitoids and, furthermore, it is within this enormous block of life that we are most exposed to the taxonomic impediment [11]. While only 10% of all insect species are described [12] – identified parasitoid diversity may be as low as 1% [13,14]. Thus, there is a grave need for accelerated identifications within these diverse and econom- ically important groups. The majority of the parasitoids in the SBW food web are currently considered to be generalists [9]. However during comparable studies of parasitoid diversity in tropical food webs, the iterative process of barcoding a parasitoid fauna associated with rearing records and permanent collections increased the estimates of host-specialization and drastically reduced the frequency of the generalist strategy [15,16,17]. Specifically, the majority of the morphologically cryptic, presumably polyphagous species dissolved into monophagous species groups supported by ecology, and both mitochondrial and nuclear genetic divergences. In this study, we were interested in asking if applying the iterative [15] process of DNA barcoding to the parasitoid individuals within the temperate SBW food web would produce the same fraction of newly-revealed cryptic species as in tropical food webs. In the Area de Conservacion de Guanacaste in Costa Rica, approximately 25% of the ‘named’ species involved in parasitizing larval Lepidoptera were in fact genetically divergent – and are now considered to be different species [18]. We predicted that some proportion of the parasitoid fauna in the SBW food web would also be revealed as morphologically cryptic, but genetically distinct – but that this rate would be less than was uncovered in the tropics. If this prediction was supported, how would such an increase in diversity affect food web structure? The SBW is an exceptional dataset in food web ecology in that it has been collected in multiple locations through time, has been accessioned in a fashion that is amenable to the recovery of multiple fragments of DNA (both nuclear and mitochondrial), and it has been measured in space and time for not just diversity, but for species abundances as well [9]. Thus, by inserting a barcoding component into the quantified SBW food web we are in the exceptional position of asking whether identifying units using barcoding affects the structure of a food web that varies in space, time and abundance. Historically, food web ecology has suffered from a misunder- standing of rules and principles because of problems with Cryptic Species in a Food Web resolution – not just ‘‘who eats whom’’ but ‘‘who is who’’ – in node selection [19,20]. To properly understand connectivity and energy flow in ecological communities, a pragmatic and repeatable resolution of taxa or nodes is required. Molecular markers have had a long history of being used to identify ‘who is who’, and have also recently been used in several instances to help identify ‘who eats whom’ [21]. For instance, Garros et al [22], used DNA barcodes to identify blood meals of malarial mosquitoes. Clare et al [23], used DNA barcodes generated from fragments retrieved from bat guano to help construct the diet of a generalist top predator. Gariepy et al [24] used multiplex PCR to estimate levels of parasitism and parasitoid species composition. Hardy et al [25] used 16S DNA sequences and microarrays to delineate carbon flow in an Australian riverine system. Corse et al [26], used group specific primers to amplify DNA from the diet of freshwater cyprinid species. Locke et al [27] used DNA barcodes and nuclear sequences to identify cryptic host and tissue specialization of Diplostomoidea (Platyhelminthes: Digenea) parasitizing freshwater fishes in Canada. Kaartinen et al [28] were the first to use CO1 DNA barcodes and ITS2 sequences to test species memberships and connections in a food web derived from leaf-mining Lepidoptera and gall-inducing Hymenoptera occurring on Quercus robor in northern Europe. Our molecular ecological characterization of a food web is unique in that it involves a very diverse system, based on outbreaking host species of enormous economic importance, where all species are from a food web that has already been characterized in abundance, space and time [9], and that the molecular comparisons made include the standardized DNA barcode region – thereby permitting direct comparison to other systems. We are therefore able to compare barcode-based analyses of food web structure to previous analyses where nodes (species) were identified using principally morphological methods. Specif- ically, we were able to ask whether the ‘bird feeder effect’ (that fluctuations in budworm density will cause diversity cascades such that more higher order parasitoids will occur at higher SBW densities [9]) is amplified, reduced or not affected when the units of higher order diversity are enumerated using DNA barcodes, nuclear genes, host records and morphology rather than morphological identifications alone. The erection of a species hypothesis within a morphologically cryptic taxon based on DNA barcodes ought to be supported by additional, independent nuclear marker(s) [16,17,29,30]. Even with small sample sizes, uncovering a matching split between two independent loci by chance is low [31]. We used several rDNA loci (ITS1, ITS2 and 28S-D2). Some have suggested that the presence of compensatory base pair changes (CBCs) in the secondary structure of the ITS2 region can be used as a proxy to identify sexually incompatible pairs [32,33]. CBCs occur when both nucleotides of a paired site mutate but the pairing remains stable. If CBCs do correlate with (or cause) sexual incompatibility – they could be a molecular, ‘holy grail’ [34] for identifying species as defined by the biological species concept [35]. We tested whether the units of food web diversity were differentiated differently using morphology, barcodes and ITS2 CBCs for a subset of the food web diversity. Finally, we considered the importance and ramifications of species concepts on food web node identification whether determined using morphology, genetic information or ecology in either an integrative or a separate fashion. We make specific recommendations regarding the erection of ‘species hypotheses’, the importance of recognizing both Type I and II errors in formulating and testing these hypotheses, and the likelihood that barcodes will solve the species problem. PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e14424