MetabarcodingParasitoidwasps
All parasitoids, along with some visually similar Hymenoptera, were extracted from the samples, sorted according to their size (large vs. small), and counted. Samples of the same size class were usually combined for the two sampling dates for a given sapling, resulting in samples containing between 7 and 58 individuals (average 33). Sorting to size is recommended because in meta-barcoding the signal of some large-bodied specimens can overwhelm the signal of small-bodied specimens (Elbrecht et al. 2017). Additionally, tTo build a reference library of parasitoids that target caterpillars feeding on oak leaves, we pooled parasitoids reared from caterpillars collected during the springs of 2019 (Molleman et al. 2022) and 2021 (Molleman and Walczak 2024). These parasitoids were sorted into small and large-bodied species categories, and DNA metabarcoding was performed on these two samples. (2021). In summary, after removing the ethanol, samples were dried at in a 65 C in an oven overnight in 20ml glass vials and then homogenized using stainless steel beads within a FastPrep 96 instrument (MP Biomedicals). We estimate  tThe total mass of the samples to be between 200-700 mg of biomasswas not measured (probably between 200-700mg of biomass). DNA extraction was performed on all samples using a 90:10 solution of animal lysis buffer (buffer ATL, Qiagen DNEasy Tissue Kit with 10% proteinase K, Qiagen, Hilden, Germany) and Proteinase K. Lysis was carried out overnight at 56 C, followed by cooling to room temperature . in a total volume of 1mL. DNA extraction from 200L aliquots of the lysate was performed using the DNEasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions and the residual lysate was then discarded. From each sample, 5L of extracted genomic DNA was used for PCR amplification with Plant MyTAQ (Bioline, Luckenwalde, Germany) and high throughput sequencing (HTS) adapted mini-barcode primers targeting the mitochondrial CO1-5P region, following Leray et al. (2013). One PCR was performed for each sample. Amplification success and fragment length were assessed using gel electrophoresis. Clean-up of amplified DNA and Illumina Nextera XT (Illumina Inc., San Diego, CA, USA) indices ligations were performed following standard protocols. DNA concentrations were quantified using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA), and samples were combined into equimolar pools. The pooled DNA was purified using NGS magnetic beads (MagSi-NGSprep Plus Magtivio). HTS was performed on an Illumina MiSeq using v3 chemistry (2*300bp, 600 cycles, maximum of 25mio paired-end reads). Sequencing data were processed using the VSEARCH suite v2.9.1and Cutadapt K5. Quality filtering and chimera removal were performed, and the remaining sequences were clustered into operational taxonomic units (OTUs) at 97% identity. OTUs were then assigned to barcode index numbers (BINs) using the BOLD database (Ratnasingham and Hebert 2007). A cleaning step was applied to exclude low-abundance OTUs. Additionally, a separate BLAST search against the NCBI nucleotide database was conducted to validate BOLD results.