Cryptic Species in a Food Web Figure 4. Food-web diversity measured using DNA barcodes and budworm density. Here diversity was calculated as MOTU (A&B), or PD (C&D) and separate trends were plotted for the nearly homogenous (98% balsam fir), intermediate (77% balsam fir) and the most heterogeneous site (50% balsam fir) (C&D). The ‘bird feeder’’ effect predicts that the diversity of parasitoids will increase with the budworm density. As was found in [9], the slopes of these lines (in the 50% and 98% balsam fir plots) are significantly different from the hypothesis of 0 – supporting the bird feeder effect. As in [9], the data were de-trended to remove potential temporal autocorrelations but as this yielded consistent results, the original comparisons are illustrated here. doi:10.1371/journal.pone.0014424.g004 Ultimately testing these lineages will necessarily be contingent upon multiple loci; however the initial identification of these lineages using mtDNA is a critical and useful first step [52]. To examine the hypothesis that compensatory base changes (CBCs) specifically within the ITS2 rRNA secondary structure are an effective method for delineating cryptic species, we examined multiple comparisons within our insect food web. The CBC hypothesis for species identification is, by nature, comparative, and thus it is critical that the sequences being compared have sufficient variation so that compensatory muta- tions can be identified, but not so much variation that alignment is compromised [53]. Achieving this balance is a non-trivial problem. Our data suggest that the pipeline of identification is not yet mature for insects (at least from the families examined here) and while it may be eventually, without a larger group of secondary structure models for insects, one must depend on provisional estimates of folding and structure – and thus, at best, provisional estimates of CBCs. In addition, we uncovered CBCs in only a very small proportion of the provisionally identified (barcoding, host, rDNA sequence divergence) new species sequenced here. While CBCs undoubtedly exist between many sexually-isolated species – we did not find them here frequently enough to warrant their discussion as a ‘‘holy grail’’ for species identification. Our species are not the only ones where sequence data from ITS2 has been used to identify cryptic species – but where CBCs are absent. Van Veen et al [54] has been cited as a pioneering study in the use of molecular markers to resolve the identification of ‘difficult’ parasitoid species. In the four cryptic species of Alloxysta victrix that were examined – there are CBCs present between only one of 6 possible pairwise compar- isons (Table S4). Species, barcodes and concepts Species hypotheses should not be formulated in the absence of a species concept [55]. In this work, we use a species concept derived from the evolutionary species concept [56] and the general lineage concept of species [57], implemented using the phylogenetic species concept [58] where we seek to identify individual, independent-evolutionary lineages as species using operational criteria that include molecular (DNA sequence data), morpholog- ical and ecological information. A species concept tests an individual’s membership based upon different criteria (of which a barcode could be one). This depends on the a priori erection of a hypothesis; either 1) an individual is a member of species A (as in [59]), or 2) an individual is not a member of species A. In each case, the is different (Null 1 = separate species; Null 2 = same species). Historically, the stress has always been on the p value approach (significance) and considering Type 1 error (failure to reject a hypothesis when it is true). Emphasis has also been predominantly on the second hypothesis – the of ‘same species’ approach. If the cannot be rejected, Scenario 1 will increase species number while Scenario 2 will decrease species number. This leads to an underestimation of diversity and furthermore, it fosters an under PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e14424