Supporting Information

Wirta et al. 10.1073/pnas.1316990111 SI Text our success levels from field-caught samples may be considered 1. Performance and Validation of the Molecular Methods. Molecular high by comparison. analysis of linkages. With PCR-primers designed to se- lectively amplify the DNA of the order but not the parasitoid, 2. Incidence of Multiple Host and Parasitoid Sequences from the Same we successfully amplified and sequenced a host from 21.9% of 457 Individual. Incidence of multiple host sequences in the same parasitoid caught as adults. However, the rate of sequence re- individual. Our MAPL-AP analyses revealed evidence of two different covery was significantly lower for species of braconids than host species in seven individuals of parasitoid wasps (all family ichneumonids and tachinids [Braconidae, n = 34 (2.9%); ). In a single individual of Aoplus groenlandicus,both Ichneumonidae, n = 346 (24.0%); and , n = 77 Entephria polata and Sympistis nigrita were found; in Buathra labo- (20.8%); χ2 = 8.09, df = 2, P = 0.02). Interestingly, seven rator,bothGynaephora groenlandica and Euxoa adumbrata were ichneumonid specimens yielded sequences from two different found; in two Cryptus arcticus specimens, G. groenlandica was host species, as recovered from two separate body parts or found with Syngrapha parilis in one and S. nigrita in the other; from two different pieces of the abdomen (SI Text, section 2). For in Mesochorus n. sp., both G. groenlandica and S. nigrita were found; eight parasitoid species represented by only a few individuals andintwoPimpla sodalis specimens, G. groenlandica and S. parilis in our sample, no host could be identified through molecular were found in one and E. polata and S. nigrita were found in the analysis of parasitoid linkages–adult parasitoid (MAPL-AP) other. In each case, these different host sequences were recovered (Table S1). from two separate body parts or from two different pieces of the MAPL-host larvae. The selective amplification and sequencing of parasitoid abdomen analyzed as separate samples. For each of these parasitoid DNA from host tissue yielded a parasitoid sequence cases, one of the host sequences likely represents the host that was from 20.9% of 1,195 hosts examined. Of these host larvae, 6.1% fed upon by the parasitoid as a larva, whereas the other sequence were parasitized by an ichneumonid, 12.3% by a braconid, and was acquired from a host that was oviposited upon by the adult 2.4% by a tachinid. The level of parasitism detected varied parasitoids. In three of the seven cases, one host sequence was considerably among host species, ranging from 0% to 44% among unequivocally detected in a tissue sample consisting of the ovipos- species represented by more than one individual. In 2 of 12 host itor only, suggesting acquisition of DNA during oviposition or species included in this study no parasitoids were discovered, while probing a potential host. In the other four cases, oviposition likely due to small sample size (Table S1). emerges as a possible source of the secondary host sequence, al- Recovery of parasitoid sequences from visibly parasitized host larvae. though host feeding by the adult parasitoid cannot be ruled out. When using MAPL-host larvae (HL), the true level of parasit- Incidence of multiple parasitoid sequences in the same host individual. In ism in the sample is unknown (this contrasts with the situation for our MAPL-HL analyses, sequences of two separate parasitoid MAPL-AP, where each parasitoid must have fed on a host as taxa were discovered in 11 host individuals. In most cases (n = 7), a larva). To verify if our MAPL-HL protocol accurately detects the co-occurring taxa were primary parasitoids, whereas in 4 parasitoids when present, we analyzed a subset of host individuals cases, one of the taxa was a secondary parasitoid Mesochorus n. where the presence of a parasitoid larva was detected during sp. In all 11 cases, one of the parasitoids was a member of the sample preparation. Of 30 such samples, 80.0% yielded a para- family Braconidae whereas the other belonged to the Ichneu- sitoid sequence. This suggests that the true level of parasitism is monidae. The co-occurring parasitoids were Cotesia spp. and slightly higher than observed by MAPL-HL in the current study, Mesochorus n. sp. in Boloria chariclea, two cases of Microplitis a discrepancy potentially related to the specificity of the primers lugubris and Mesochorus n. sp. in B. chariclea, Cotesia spp. and used (SI Text, section 6). Nonetheless, the parasitism rate esti- Hyposoter frigidus in richardsoni, M. lugubris and H. frigidus mated by MAPL-HL (20.9%) was still higher than that estimated in another Polia richardsoni, M. lugubris and Mesochorus n. sp. in by rearing (16%; ref. 1). S. nigrita, two cases of M. lugubris and Campoletis horstmanni Recovery of host sequences from wild-caught versus reared parasitoid in S. nigrita, M. lugubris and H. frigidus in another S. nigrita, individuals. To verify that MAPL-AP correctly identifies the host M. lugubris and C. horstmanni in S. parilis,andM. lugubris and use of adult parasitoids, we compared a set of 422 wild-caught H. frigidus in S. parilis (the first listed parasitoid is the braconid individuals to a set of 35 parasitoids reared from known hosts. and the second is the ichneumonid). Within these sets, a host sequence was successfully recovered from The incidence of multiple parasitoids in the same hosts (mul- 22.5% of the wild-caught individuals, compared with 17% of the tiparasitism) might have been underestimated by the methods that reared individuals. Hence, the recovery rate was similar among we used. As we used Sanger sequencing (3), and sequenced only materials (Fisher’s exact test, P = 0.50). one PCR product, we might have failed to detect co-occurrence Overall, our success in detecting the host sequence from adult of two confamilial parasitoids in the same host individual. In parasitoids was comparable to that of Rougerie et al. (2), who future studies, additional sequencing of PCR products by next applied a method similar to MAPL-AP to reared parasitoid generation techniques may improve quantification of cases of specimens. Targeting adults of 3 parasitoid species in 2 families, multiparasitism. Rougerie et al. (2) detected a host in 24% of individuals, whereas our detection rate of 20.9% applies to a wider range of 3. Estimations of Link Richness and Sample Coverage. Methods. No parasitoid species (20 species from 2 wasp families and 1 finite sample will reveal all species or all interactions in a food web. family). Ultimately, the success rate in discovering remains of To examine how well our samples and methods describe the set of host DNA will be constrained by the limited amounts of DNA trophic links in the –parasitoid community at Zack- remaining in the gut of the parasitoid after metamorphosis, as well enberg, we used methods originally developed for the estimation as by the degradation of this DNA during both larval development of species richness to estimate the total number of different tro- and adult life span before collection (discussed by ref. 2). As phic links in our target community. For this, we used two non- Rougerie et al. (2) sampled their parasitoids directly after emer- parametric estimators: Chao1 (4, 5) and the abundance-based gence (thus minimizing the degradation of DNA after emergence), coverage estimator (ACE) (6, 7). Chao1 provides an estimate of

Wirta et al. www.pnas.org/cgi/content/short/1316990111 1of6 yet-undetected links based on the frequency of singletons provide a molecular toolset of prime utility in exploring the (trophic links revealed only once) and doubletons (trophic links structure of host–parasitoid food webs. revealed only twice) in the sample. ACE employs information on link types encountered at slightly higher frequencies. 4. Methodological Provisos. Using host larvae as the only in- Sample size variation not only influences the detection of in- formation source to describe trophic interactions may lead to dividual links by a particular method, but it also affects the links involving idiobiont parasitoids going undetected (see Results number of links jointly detected by multiple methods. Consider and Fig. 1). On the other hand, the use of adult parasitoids as the the case where a set of interactions is sampled by two methods, sole source of information might cause similar problems. As each with a small sample size. Under this scenario, one method DNA degrades in the gut of the parasitoid, individuals caught long will reveal a subset of all interactions, whereas the other method after their emergence may no longer carry host DNA of sufficient will detect another subset, with limited overlap between methods. quality to allow their identification. Finally, a sample of adult par- Although our sample sizes were reasonably large, they varied asitoids alone would not reveal parasitism levels suffered by the among methods (n = 457 for MAPL-AP, n = 1195 for MAPL- host, which is indeed an important dimension of interaction struc- HL, and n = 1420 for rearing). Naturally, more species will be ture. Thus, an adequate description of the interaction web can only detected by any methodological approach if sample size is in- be built on a combination of multiple sources of information. creased. To examine the complementarity of different methods Interpretation of trophic associations. Although the detection of para- while adjusting for differences in sample size, we applied the sitoid DNA in a living host larva (MAPL-HL) offers proof of an Chao1 estimator modified to derive the true number of species interspecific interaction, it is not sufficient proof of a feeding as- sociation that will lead to host death. Several studies (e.g., refs. shared among collection methods (7, 8). – In addition to estimating the number of trophic links in the 11 14) have indicated that host individuals can sometimes en- region, and their detectability by different methods, we also es- capsulate a developing parasitoid, enabling completion of their timated the proportion of links in the overall food web that re- own development. Likewise, host DNA detected by MAPL-AP main undetected in our samples (9). This fraction (i.e., the may in some cases derive from the ovipositor piercing a host while coverage deficit) is also the probability that the next trophic link injecting the eggs (SI Text, section 2), providing no proof that the examined represents a new interaction. Subtracting the coverage egg successfully completed its development. Host feeding by adult deficit from 1.0 yields the coverage per se, i.e., the proportion of female parasitoids (15, 16) may also result in either the detection of DNA from a potential, but not actual, host individual by links in the web that belong to the link types represented in the MAPL-AP, or in the detection of multiple host sequences in sample. All estimates were obtained with the Species Prediction a single parasitoid. As a consequence, the present study of food and Diversity Estimation program (10). web structure should be viewed as an analysis of the interaction Results. Taken at face value, methods MAPL-AP and MAPL-HL structure among hosts and parasitoids, rather than as a measure of yielded a comparable count of link types in the target community, host mortality. From this perspective, rearing may still offer the but the number was markedly lower for rearing (Table S3). only way to establish the successful development of parasitoids Despite the similarity in overall numbers, different links were within a particular host—again emphasizing the complementarity, revealed by each technique: from a total of 62 trophic links, 43 rather than the independence, of methods for revealing the in- links were only detected by one technique. When combined, the teraction structure within natural communities. methods revealed a far higher number of interactions than Factors causing differences in the detectability of individual links by detected by any individual technique (Table S3 and Fig. 1). Of all different techniques. In the Discussion, we have discussed some trophic interactions in the target food web, the interactions factors causing differences in the probability with which a spe- detected by molecular methods represent 88%, whereas rearing cific link is detected by a particular technique. Of these, some revealed only 34% (Figs. 1 and 2). relate to the lifestyle of the predator. For example, the trophic Differences among the methods persisted even when corrected associations between idiobionts and their hosts are more easily for differences in sample size. Estimates of the overall number of detected by MAPL-AP than MAPL-HL. Additional factors detectable links were twice as high for the combination of all causing potential variation in the frequency of individual links as methods as for any single technique (Table S3). Further support quantified by MAPL-HL versus MAPL-AP may relate to dif- for a major difference in the set of links detectable by each ferences in the preservation of host tissue and differences in method is shown by two patterns. First, estimates of sample rates of degradation of host DNA among parasitoid species. In coverage were high across methods (Table S3), suggesting that for addition, the duration of the pupal stage may vary among par- each method, the majority of common interactions detectable by asitoid species, adding variation to the amount and quality of this technique was revealed by our present sample. Second, DNA remaining in the gut of the adult parasitoid. estimates of the total number of links (detected and undetected) From a methodological perspective, the use of targeted primers shared among any two methods were low. A combination of can introduce a bias in the apparent frequency of a particular rearing and MAPL-AP will likely reveal only 6.5 shared links (with interaction. Even when primer-binding sites are conserved, the 95% confidence limits of 6.0–11.3 links), a combination of exact sequence of the target region differs slightly among taxa, rearing and MAPL-HL will reveal 16.8 shared links (CL 12.6– causing differences in annealing probabilities. This factor might, 51.6), and a combination of MAPL-AP and MAPL-HL will re- for example, provide an explanation for the lower success of host veal 23.5 shared links (CL 10.8–93.2). Thus, even in a large detection rates in Ichneumonidae than Braconidae (SI Text, sample, the detection of a specific link will typically be restricted section 1). Here, targeting regions conserved within the target to a single method. Such differences among methods likely re- group, but differing from nontargeted taxa, added challenges flect both biological and technical constraints associated with when aiming for short amplicons within the barcode region. One each approach (SI Text, section 4), causing individual trophic possible solution to detect different target taxa with similar associations to be revealed with different probabilities. probability could involve the move to several primer pairs, as Overall, the difference in the set of links detected by each method implemented in this study for MAPL-HL. However, even with indicates that the methods are complementary, not mutually this approach, not all parasitoid individuals were detected, re- exclusive, and that their joint use is required to detect all links in vealing room for further primer development. Another issue the food web. Nonetheless, we emphasize that the joint use of possibly leading to some undetected interactions is our use of MAPL-AP and MAPL-HL revealed nearly 90% of the trophic Sanger sequencing, which produces only one sequence result per links present in the Zackenberg food web. Hence, together they sample, ordinarily limiting recovery to one parasitoid per family.

Wirta et al. www.pnas.org/cgi/content/short/1316990111 2of6 As a result, cases of multiple parasitoids within a host were only and the entire individual (for host larvae) worked equally well detected for cases involving different families (SI Text, section 2). (SI Text, section 7), dissection is unnecessary. In this context, the use of next generation sequencing may offer Primers. To detect potentially degraded and/or low-yield host or a solution as all amplicons (representing one or multiple species) parasitoid DNA (35), we targeted primers at a short but variable are then sequenced separately. region of the CO1 gene. Using an alignment of full-length (658-bp) barcodes from all available host and parasitoid species in the study 5. Insights into the Species Composition and Life Histories of the area, we designed four primer sets targeting a 148-bp hypervariable Target Parasitoid Community. Sequences revealing species priorly region, allowing for species-level identification of all taxa included undiscovered in the area and/or cryptic variation. The assignment of in this study. Each primer set was chosen to include at least one species to sequences was conducted with the Barcode of Life Data primer binding to all of our potential host (MAPL-AP) or para- (BOLD) identification engine (17), using the option to search all sitoid (MAPL-HL) species but to none of the parasitoid (MAPL- barcode records in BOLD. In two cases, the match to all species AP) or host (MAPL-HL) species. Hence, for MAPL-AP, we de- with a DNA barcode from the area was lower than 98%. A se- signed one primer set [MAPL_LepF1_t1 (tgtaaaacgacggccagtCC- quence differing from Periscepsia stylata (Diptera: Tachinidae) CACTTTCATCTAATATTGC) and MAPL_LepR1_t1 (cag- by 5.4% was detected in B. chariclea, with no higher match for gaaacagctatgacAAATGCTGTAATTCCWACAGC)] specific any previously barcoded species, suggesting the occurrence of to the Lepidoptera of the study area. For MAPL-HL, we designed cryptic variation within P. stylata, or the existence of another three separate primer sets specific for the families Ichneumonidae, species of Periscepsia priorly undiscovered in Zackenberg. An- Braconidae, and Tachinidae, henceforth named as follows: other parasitoid sequence from the host S. parilis matched a MAPL-IchnF1_t1 (tgtaaaacgacggccagtCCTCCTTTATCTTTAA- specimen of Campoletis rostrata (: Ichneumoni- ATWTWAR) and MAPL_IchnR1_t1 (caggaaacagctatgacAGTA- dae)—a species found 400 km south of Zackenberg, but not AAATTGCTGTAATTWWAATTGA), MAPL-BracF1_t1 (tgta- previously detected in the study area. As there is substantial aaacgacggccagTCCTCCTTTATCTTTAATWTTRGG) and variation among sequences of C. rostrata, as well as within its MAPL_BracR1_t1 (caggaaacagctatgacAATAAAATAGCAGTA- congener C. horstmanni at Zackenberg, these two species may ATWAAWAYWGA), and MAPL-TachF1_t1 (tgtaaaacgacggcca- form a species complex where further work is needed to de- gtCCTCCTTTATCTTCAGTAATTGC) and MAPL_TachR1_t1 limit species boundaries. Hence, our DNA-based methods (caggaaacagctatgacTAATAAAGCTGTAATAACTACTGA). All proved useful in revealing the full species richness in this primers were tailed with a modified M13F (tgtaaaacgacggccag; area (18). tailing sequence shown above in lowercase) or M13R (cag- Application of MAPL-HL to host pupae. In addition to the larvae ex- gaaacagctatgac) sequence (22), which were subsequently used as amined, we tested MAPL-HL on a set of host pupae. Twenty the sequencing primer for all PCR products. individual pupae were collected in the wild, representing two PCR conditions for MAPL-AP and MAPL-HL. PCR conditions followed species (n = 19 pupae of G. groenlandica and n = 1 of standard Canadian Centre for DNA Barcoding (CCDB) proto- P. richardsoni). Among the pupae of G. groenlandica, we detected cols (36), with the exception of thermocycling, which depended three cases of parasitism by the wasp P. sodalis (Hymenoptera: on the primers used. All PCR reactions consisted of an initial hot Ichneumonidae), one case of parasitism by the wasp Hyposoter start of 94 °C for 2 min and a final extension step of 72 °C for deichmanni (Hymenoptera: Ichneumonidae), and three cases 5 min. For MAPL_LepF1_t1 + MAPL_LepR1_t1, thermocycling of parasitism by the fly Exorista thula (Diptera: Tachinidae). Of consisted of 60 cycles of 94 °C for 40 s, 51 °C for 40 s, and 72 °C these, the trophic links between G. groenlandica and P. sodalis, for 1 min. For MAPL_IchnF1_t1 + MAPL_IchnR1_t1, thermo- and between G. groenlandica and E. thula, were priorly not known cycling consisted of 5 cycles of 94 °C for 40 s, 45 °C for 40 s, in the food web of Zackenberg. Hence, the explicit targeting of and 72 °C for 1 min followed by 40 cycles of 94 °C for 40 s, 48 °C cryptic stages in the life cycle by molecular techniques revealed for 40 s, and 72 °C for 1 min. For MAPL_BracF1_t1 + further trophic interactions. MAPL_BracR1_t1, thermocycling consisted of 5 cycles of 94 °C for 40 s, 48 °C for 40 s, and 72 °C for 1 min followed by 40 cycles 6. Genetic Analyses. A key feature of the present methods is the of 94 °C for 40 s, 51 °C for 40 s, and 72 °C for 1 min. For targeting of primers to taxon-specific sequence motifs. By gen- MAPL_TachF1_t1 + MAPL_TachR1_t1, thermocycling con- erating order- and family-specific primers, we were able to detect sisted of 5 cycles of 94 °C for 40 s, 50 °C for 40 s, and 72 °C for and discriminate hosts and parasitoids, identifying all sequences 1 min followed by 40 cycles of 94 °C for 40 s, 52 °C for 40 s, and to the species level. A closely related approach is the design of 72 °C for 1 min. The verification of PCR products, sequencing, blocking primers to prevent the amplification of selected taxa in and sequence editing followed standard CCDB protocols (36). trophic interactions (19, 20). Further developments include To avoid contamination during genetic analyses, DNA extraction amplification and sequencing of all interacting species together, and pre-PCR preparations were conducted in laboratories sepa- using next-generation sequencing (21). All of these methods rate from where the post-PCR products were handled. Negative avoid a severe limitation of prior molecular techniques which controls were included in all PCR and sequencing reactions, and were based on species-specific primers (e.g., refs. 22–24), the they always failed to generate an amplicon or sequence. need for a priori knowledge of all potential host taxa (25). By targeting specific orders and families, our approach makes it 7. Tissue Sampling. Tissue sampling for MAPL-AP. To identify the best possible to dissect interactions within the same kingdom, phylum, body part to recover host DNA for MAPL-AP, adult parasitoids and class (for applications to interactions between plants and ani- were dissected under a microscope using sterile equipment. mals,seerefs.26–29; and between mammals and , see refs. The head, thorax (mesosoma in parasitic Hymenoptera), ab- 30–32). Although the separation of predator and prey tissues before domen (metasoma), and ovipositor were separated. From each amplification would permit the use of universal primers in studies tissue subsample, DNA was extracted using a standard glass- of even closely related species (e.g., refs. 33 and 34), the laborious fiber protocol (37). dissection involved will likely preclude the wide-scale application Among parasitoid wasps, the metasoma (n = 366, 18.0%) of this approach to larger ecological surveys. In our investigation proved the best body part for DNA recovery [compared with the of taxon-specific primers, we first tested the use of several body head (n = 138, 5.8%), mesosoma (n = 137, 3.4%), and ovipositor parts of parasitoids and tissue pieces cut from host larvae for (n = 219, 4.6%)]. Seven wasps which each yielded two separate amplification (SI Text, section 7). As our trials showed that DNA Lepidoptera sequences are omitted from this analysis (SI Text, extraction from a single selected body part (for parasitoids) section 2). Among tachinids, the head yielded significantly more

Wirta et al. www.pnas.org/cgi/content/short/1316990111 3of6 identifiable sequences (n = 56, 25.0%) than other body parts set of the larvae (n = 816) were then analyzed as whole speci- [thorax (n = 56, 0%) and abdomen (n = 74, 8.1%)]. mens or cut into two or four subsections each (depending on Overall, variation in the ability to detect host DNA among size) so that each specimen or subsection could be placed into different body parts may be attributable to biological differences. either a 1.1- or 1.5-mL tube. Each sample was dried overnight at As tachinids emerge from their puparium by inflating a balloon- 56 °C and individually ground in a TissueLyser (Qiagen GmbH). like ptilinum from behind the face, the frequent detection of host Tissue samples were disrupted with 3-mm tungsten carbide DNA on fly heads may reflect host tissue caught between folds of beads (Qiagen GmbH) at 30 Hz for 1 min or via manual grinding the ’ exoskeleton. Indeed, both Peleteria aenea and Peleteria with a sterile disposable pestle. After drying, 100–300 μL lysis popeli pupate within the host exoskeleton (38). For parasitic buffer was added (depending on the original amount of tissue) to wasps, remnants of host tissue appeared to be primarily retained fully cover the sample. The samples were then lysed overnight at in the gut, so the metasoma proved the best source of DNA. 56 °C and 50 μL of the resultant lysate was transferred to a 0.2- These findings call for caution in selecting the body part used mL well in a 96-well plate. Subsequent DNA extraction followed to detect trophic interactions. They also highlight the need for standard protocols (37). After DNA extraction all samples were considering the biology of the target taxa. treated in the same manner. Tissue sampling for MAPL-HL. The volume of the tissue sample, and No difference in the recovery of parasitoid DNA was observed the ratio between host and parasitoid tissue in the sample, might between DNA extracts from whole host larvae and smaller pieces affect the detection of parasitoids through MAPL-HL. To ex- (sliced larvae, n = 379, 18.2%; whole larvae, n = 816, 22.2%). This amine the strength of these effects, a set of lepidopteran larvae congruence identifies the whole specimen extraction technique as were dissected with sterile equipment into small pieces (n = 379), the method of choice, given its savings of costs and labor (yielding with each sample placed into a 0.2-mL well of a 96-well plate. mostly one sample per individual instead of multiple samples per From each sample, DNA was extracted as above (37). Another individual and bypassing the need for dissection).

1. Roslin T, Wirta H, Hopkins T, Hardwick B, Várkonyi G (2013) Indirect interactions in 21. Piñol J, San Andrés V, Clare EL, Symondson WOC (2014) A pragmatic approach to the the High Arctic. PLoS ONE 8(6):e67367. analysis of diets of generalist predators: The use of next-generation sequencing with 2. Rougerie R, et al. (2011) Molecular analysis of parasitoid linkages (MAPL): Gut no blocking primers. Mol Ecol Resour 14(1):18–26. contents of adult parasitoid wasps reveal larval host. Mol Ecol 20(1):179–186. 22. Agustí N, et al. (2003) Collembola as alternative prey sustaining spiders in arable 3. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating ecosystems: Prey detection within predators using molecular markers. Mol Ecol inhibitors. Proc Natl Acad Sci USA 74(12):5463–5467. 12(12):3467–3475. 4. Chao A (1984) Non-parametric estimation of the number of classes in a population. 23. Ratcliffe ST, Robertson HM, Jones CJ, Bollero GA, Weinzierl RA (2002) Assessment Scand J Stat 11(4):265–270. of parasitism of house fly and stable fly (Diptera: Muscidae) pupae by pteromalid 5. Chao A (2005) Species Estimation and Applications (Wiley, New York). (Hymenoptera: Pteromalidae) parasitoids using a polymerase chain reaction assay. 6. Chao A, Lee S-M (1992) Estimating the number of classes via sample coverage. JAm J Med Entomol 39(1):52–60. Stat Assoc 87(417):210–217. 24. Traugott M, Zangerl P, Juen A, Schallhart N, Pfiffner L (2006) Detecting key 7. Chao A, Shen T-J, Hwang WH (2006) Application of Laplace’s boundary-mode parasitoids of lepidopteran pests by multiplex PCR. Biol Control 39(1):39–46. approximations to estimate species and shared species richness. Austral and New Zeal 25. King RA, Read DS, Traugott M, Symondson WOC (2008) Molecular analysis of predation: J Statist 48(2):117–128. A review of best practice for DNA-based approaches. Mol Ecol 17(4):947–963. 8. Chao A, Hwang W-H, Chen Y-C, Kuo C-Y (2000) Estimating the number of shared 26. Jurado-Rivera JA, Vogler AP, Reid CAM, Petitpierre E, Gómez-Zurita J (2009) DNA species in two communities. Statist Sinica 10(1):227–246. barcoding -host plant associations. Proc Biol Sci 276(1657):639–648. 9. Chao A, Jost L (2012) Coverage-based rarefaction and extrapolation: Standardizing 27. Raye G, et al. (2011) New insights on diet variability revealed by DNA barcoding and high- samples by completeness rather than size. Ecology 93(12):2533–2547. throughput pyrosequencing: Chamois diet in autumn as a case study. Ecol Res 26(2):265–276. 10. Chao A, Shen T-J (2010) Program SPADE (Species Prediction and Diversity Estimation). 28. Soininen EM, et al. (2009) Analysing diet of small herbivores: The efficiency of DNA Available at http://chao.stat.nthu.edu.tw/software/SPADE/SPADE_UserGuide.pdf. Ac- barcoding coupled with high-throughput pyrosequencing for deciphering the cessed May 5, 2013. composition of complex plant mixtures. Front Zool 6:16. 11. Kapranas A, Hardy ICW, Morse JG, Luck RF (2011) Parasitoid developmental mortality 29. Valentini A, et al. (2009) New perspectives in diet analysis based on DNA barcoding in the field: Patterns, causes and consequences for sex ratio and virginity. J Anim Ecol and parallel pyrosequencing: The trnL approach. Mol Ecol Resour 9(1):51–60. 80(1):192–203. 30. Alberdi A, Garin I, Aizpurua O, Aihartza J (2012) The foraging ecology of the 12. Kraaijeveld AR, Godfray HC (2009) Evolution of host resistance and parasitoid mountain long-eared bat Plecotus macrobullaris revealed with DNA mini-barcodes. counter-resistance. Adv Parasitol 70:257–280. PLoS ONE 7(4):e35692. 13. Hochberg ME (1997) Hide or fight? The competitive evolution of concealment and 31. Bohmann K, et al. (2011) Molecular diet analysis of two african free-tailed bats encapsulation in parasitoid-host associations. Oikos 80(2):342–352. (Molossidae) using high throughput sequencing. PLoS ONE 6(6):e21441. 14. Strand MR, Pech LL (1995) Immunological basis for compatibility in parasitoid-host 32. Calvignac-Spencer S, et al. (2013) Carrion fly-derived DNA as a tool for comprehensive and relationships. Annu Rev Entomol 40:31–56. cost-effective assessment of mammalian biodiversity. Mol Ecol 22(4):915–924. 15. Godfray HCJ (1994) Parasitoids: Behavioral and Evolutionary Ecology (Princeton Univ 33. Clare EL, Fraser EE, Braid HE, Fenton MB, Hebert PDN (2009) Species on the menu of Press, Princeton), p 488. a generalist predator, the eastern red bat (Lasiurus borealis): Using a molecular 16. Quicke DLJ (1997) Parasitic wasps (Chapman & Hall, London). approach to detect prey. Mol Ecol 18(11):2532–2542. 17. Ratnasingham S, Hebert PDN (2007) bold: The Barcode of Life Data System (. ). Mol 34. Hrcek J, Miller SE, Quicke DLJ, Smith MA (2011) Molecular detection of trophic links Ecol Notes 7(3):355–364, http://www.barcodinglife.org. in a complex insect host-parasitoid food web. Mol Ecol Resour 11(5):786–794. 18. Várkonyi G, Roslin T (2013) Freezing cold yet diverse: Dissecting a high-Arctic parasitoid 35. Greenstone MH, Rowley DL, Weber DC, Payton ME, Hawthorne DJ (2007) Feeding mode community associated with Lepidoptera hosts. Can Entomol 145(2):193–218. and prey detectability half-lives in molecular gut-content analysis: An example with 19. Leray M, Agudelo N, Mills SC, Meyer CP (2013) Effectiveness of annealing blocking two predators of the Colorado potato beetle. Bull Entomol Res 97(2):201–209. primers versus restriction enzymes for characterization of generalist diets: 36. Smith MA, Poyarkov NA, Jr., Hebert PDN (2008) DNA BARCODING: CO1 DNA barcoding Unexpected prey revealed in the gut contents of two coral reef fish species. PLoS amphibians: Take the chance, meet the challenge. Mol Ecol Resour 8(2):235–246. ONE 8(4):e58076. 37. Ivanova NV, deWaard J, Hebert PDN (2006) An inexpensive, automation-friendly 20. Vestheim H, Jarman SN (2008) Blocking primers to enhance PCR amplification of rare protocol for recovering high-quality DNA. Mol Ecol Notes 6(4):998–1002. sequences in mixed samples—a case study on prey DNA in Antarctic krill stomachs. 38. Bystrowski C (2010) A few interesting new host records for European Tachinidae. The Front Zool 5:12. Tachinid Times 23:8–9.

Wirta et al. www.pnas.org/cgi/content/short/1316990111 4of6 it tal. et Wirta www.pnas.org/cgi/content/short/1316990111

Table S1. Lepidopteran species and their parasitoids detected in the Zackenberg area, and trophic links between them as resolved by individual methods

Parasitoid species

Hymenoptera Diptera

Ichneumonidae Braconidae Tachinidae

Pimplinae Cryptinae Campopleginae Mesochorinae Metopiinae Horminae Microgastrinae Exoristinae Tachininae

Pimpla Aoplus Ichneumon Acrolyta Bathythrix Gelis Buathra Cryptus Cryptus Campoletis Campoletis Diadegma Hyposoter Hyposoter Mesochorus Exochus Hormius Cotesia Dolichogenidea Microplitis Protapanteles Exorista Perisceptia Peleteria sodalis groenlandicus discoensis glacialis longiceps maesticolor laborator arcticus leechi horstmanni rostrata majale deichmanni frigidus n. sp. pullatus moniliatus spp. sp. lugubris fulvipes thula stylata aenea

Lepidopteran (host) species n 34 26 14 6 1 14 34 150 5 4 0 6 21 11 10 10 13 3 0 18 0 2 0 74

Olethreutes inquietana 1/0 1/0/0 Stenoptilia islandica 0/1 0/0/1 Pyla fusca 10/8 0/0/2 Colias hecla 1/2 0/1/0 Boloria spp. all* 96/151 6/0/0 6/0/2 2/0/1 0/0/2 0/3/9 0/27/27 0/1/0 Boloria chariclea 65/0 6/0/0 6/0/0 2/0/0 0/3/0 0/17/0 0/1/0 Boloria polaris 14/0 0/4/0 Entephria polata 58/57 3/0/0 1/0/0 0/2/0 0/2/1 0/1/0 0/8/0 Entephria punctipes 0/2 Gynephora groenlandica 157/254 2/0/0 0/0/1 3/0/0 3/0/0 4/34/39 0/0/1 1/0/0 0/2/0 0/0/3 1/7/0 Syngrapha parilis 40/39 5/0/0 1/0/0 0/9/4 0/1/0 0/6/0 2/0/0 1/2/0 Sympistis nigrita 678/755 2/0/0 1/0/0 2/0/0 2/0/0 20/1/0 0/3/1 0/0/7 1/8/14 2/1/8 0/3/0 0/101/87 0/1/0 1/1/0 zetterstedtii Apamea zeta 43/45 1/0/0 1/0/0 4/0/0 0/3/0 14/16/2 Polia richardsoni 70/100 0/1/0 0/4/9 0/5/0 0/1/0 Euxoa adumbrata drewseni 41/6 4/0/0 10/0/0 0/1/0 0/2/1 0/2/0

The four levels of headings in the table represent, from top to bottom, Order, Family, Subfamily, and Species. Subfamilies are shown in phylogenetic order for Lepidoptera (1–3) and Hymenoptera (4, 5). Within subfamilies, genera are shown in alphabetical order. The total numbers of samples (n) available for each type of analyses are identified by column n (referring to the number of host larvae used for MAPL-HL and Rearings, respectively, as separated by a slash) and by the row of numbers below parasitoid species names (referring to the number of adult parasitoid individuals used for MAPL-AP). All other three-number cell entries with slashes in the table identify the number of times each (nonzero) host–parasitoid interaction was observed by the three different methods: MAPL-AP/MAPL-HL/Rearings (rearing data is from ref. 6). Overall, the following taxa were encountered in the region, where the following numbers correspond to the numbers in Fig. 1: 1, P. sodalis;2,A. groenlandicus;3,I. discoensis;4,Ichneumon lariae;5,Coelichneumonops occidentalis;6,A. glacialis;7,B. longiceps;8,G. maesticolor;9,B. laborator; 10, C. arcticus; 11, C. leechi; 12, Glypta arctica; 13, C. horstmanni; 14, C. rostrata; 15, D. majale; 16, H. deichmanni; 17, H. frigidus; 18, Cremastus tenebrosus; 19, Mesochorus undescribed species; 20, E. pullatus; 21, Meteorus arcticus; 22, Meteorus rubens; 23, H. moniliatus; 24, Cotesia spp.; 25, Dolichogenidea sp.; 26, M. lugubris; 27, P. fulvipes; 28, Elachertus fenestratus; 29, E. thula; 30, P. stylata; 31, P. aenea; 32, Rhigognostis senilella; 33, O. inquietana; 34, Olethreutes mengelana, 35, S. islandica; 36, P. fusca; 37, Gesneria centuriella; 38, Udea torvalis; 39, C. hecla; 40, Agriades glandon; 41, B. chariclea; 42, B. polaris; 43, Boloria spp.; 44, E. polata; 45, E. punctipes; 46, G. groenlandica; 47, S. parilis; 48, S. nigrita zetterstedtii; 49, A. zeta; 50, P. richardsoni; 51, Rhyacia quadrangula; 52, E. adumbrata drewseni. In addition to the rearing links shown in the above matrix, seven unidentified hymenopteran parasitoids were detected from S. nigrita and one from E. polata, as well as three unidentified tachinid parasitoids from G. groenlandica (6). *Boloria spp. all includes all interactions for B. chariclea, B. polaris and unidentified Boloria spp. together.

1. Heikkilä M, Kaila L, Mutanen M, Peña C, Wahlberg N (2012) Cretaceous origin and repeated tertiary diversification of the redefined butterflies. Proc Biol Sci 279(1731):1093–1099. 2. Kristensen NP, Scoble MJ, Karsholt O (2007) Lepidoptera phylogeny and systematics: The state of inventorying moth and butterfly diversity. Zootaxa 1668:699–747. 3. van Nieukerken EJ, et al. (2011) diversity: An outline of higher-level classification and survey of taxonomic richness: Order Lepidoptera. Zootaxa 3148:212–221. 4. Sharanowski BJ, Dowling APG, Sharkey MJ (2011) Molecular phylogenetics of Braconidae (Hymenoptera: Ichneumonoidea), based on multiple nuclear genes, and implications for classification. Syst Entomol 36(3):549–572. 5. Quicke DLJ, Laurenne NM, Fitton MG, Broad GR (2009) A thousand and one wasps: A 28S rDNA and morphological phylogeny of the Ichneumonidae (Insecta: Hymenoptera) with an investigation into alignment parameter space and elision. J Nat Hist 43(23-24):1305–1421. 6. Roslin T, Wirta H, Hopkins T, Hardwick B, Várkonyi G (2013) Indirect interactions in the High Arctic. PLoS ONE 8(6):e67367. 5of6 Table S2. Differences in food web structure among methods within , and among different food webs from separate parts of the world as resolved by a single method † Greenland*, Global*

Method Range Range Parameter Rearing MAPL-AP MAPL-HL Molecular All (within) Minimum Median Maximum (between) Ratio (w:b)

Linksobs 21 30 32 54 61

Linksunique 7201640 Connectance 0.036 0.051 0.054 0.092 0.104 0.068 0.024 0.043 0.115 0.091 0.7 Nestedness 0.243 0.289 0.491 1.220 2.242 1.999 0.329 1.321 8.124 7.795 0.3 Generality 0.677 0.968 1.032 1.742 1.968 1.291 1.069 1.235 1.778 0.709 1.8 Vulnerability 1.105 1.579 1.684 2.842 3.211 2.106 0.259 0.545 0.700 0.438 4.8 Linkage density 0.891 1.273 1.358 2.292 2.589 1.698 0.713 0.807 1.197 0.484 3.5

When reconstructed by different techniques, the structure of a single food web differs more than food webs reconstructed from separate parts of the world. Here we compare method-specific descriptors of network structure (Greenland) with descriptors of networks from different parts of the world including the continental United States, Hawaii, the United Kingdom, and Japan (Global). For Greenland, separate qualitative metrics for webs resolved by three individual methods (rearing, MAPL-AP, and MAPL-HL) and the combination of all three methods (All) are shown. For the specific methods used in this comparison, see Materials and Methods. Range (within) identifies absolute variation between metrics observed by different techniques, i.e., the difference between the smallest and the largest value of each metric as observed within our study area. For Global studies of food web structure, we show the Minimum, Median and Maximum value observed among previously published food webs of free-feeding insects and their parasitoids, with Range (between) reflecting the difference between the smallest and largest value observed among sites. Ratio (w:b) shows the ratio between Range (within) and Range(between), i.e., relative variation between food webs described by different techniques for the same site, and food webs described for different sites by the same technique.

Linksobserved refers to the number of different links empirically observed by the method(s) in question, whereas Linksunique indicates the subset of these which was detected uniquely by this method. In both cases, each entry refers to the number of different interaction types (not frequencies) detected by the method in question. Connectance, Nestedness, Generality, Vulnerability, and Linkage density describe features of the web frequently used to describe emergent web structure and species-specific linking structure (for details see Fig. 2). Note that for tractability, all metrics are given in their qualitative form (according to ref. 1), but that relative variation within versus between webs remains the same for quantitative estimates of generality, vulnerability, and linkage density. † To calculate these metrics, all Boloria specimens were considered as one taxon.

1. Bersier L-F, Banasek-Richter C, Cattin M-F (2002) Quantitative descriptors of food-web matrices. Ecology 83(9):2394–2407.

Table S3. Number of trophic links detected by each of three methods used to establish feeding associations Method

Parameter MAPL-AP MAPL-HL Rearing All methods

Linksobserved 30 32 21 61

Linksunique 20 16 7 Chao1 38.6 (32.2, 63.8) 42.1 (34.6, 71.7) 27.1 (22.2, 52.0) 74.7 (65.8, 105.9) ACE 41.4 (33.6, 65.6) 43.7 (35.9, 67.3) 29.6 (23.3, 53.4) 77.4 (67.7, 101.1) Sample coverage 0.85 0.96 0.97 0.97 Coverage deficit 0.15 0.04 0.03 0.03

Each entry refers to the number of different interaction types (not frequencies) detected by the method in

question. Linksobserved refers to the absolute number of links empirically observed by the respective method, whereas Linksunique indicates the subset of these which was not detected by any other method. Chao1 and ACE estimate total link numbers (detected plus undetected links, with 95% confidence limits in parentheses), whereas Sample coverage and Coverage deficit describe sample completeness (see SI Text, section 3 for details). In terms of trophic links, B. chariclea, B. polaris, and Boloria spp. were treated as one taxon in this table. A subset of 74 larvae was identified by DNA barcodes, thus allowing us to establish two species-specific links for MAPL-HL (B. chariclea–Cotesia spp. as well as B. polaris–Cotesia spp.). This approach added a count of one to the specific link number detected by MAPL-HL. From counts of links detected by rearing, 10 unidentifiable immature para- sitoids were omitted (1).

1. Várkonyi G, Roslin T (2013) Freezing cold yet diverse: Dissecting a High-Arctic parasitoid community associated with Lepidoptera hosts. Can Entomol 145(2):193–218.

Wirta et al. www.pnas.org/cgi/content/short/1316990111 6of6