*2.1. Sample Collection*

All WCR individuals used in this research were populations from the US. The same individuals were used both for the genetic and morphometric analysis. WCR individuals were collected from South Dakota in the fields containing transgenic corn. Individuals adapted to crop rotation from Illinois were collected in fields with documented resistance. Non-resistant (susceptible) adults were obtained from the NCARL laboratory. The nonresistant laboratory population was originally collected in 1987 near the town of Trent, South Dakota, in Moody County. It has been in continuous rearing since that time without mixing with other collections. It is approximately one generation per year. The original beetles were selected in cornfields or on the edge of cornfields and the adult beetles were returned to the laboratory. The non-resistant colony is reared in soil on maize roots and the adult beetles are fed on an artificial diet. Attempts are being made to keep the rearing protocol "field-like" to keep it "wild" (Chad Nielson personal communication). According to Mikac et al. [45], there are minimal differences between rotation-resistant laboratory and field-collected populations, suggesting that the rearing system was not the main reason for the differences observed in their study. Therefore, we excluded the possibility that different conditions (field, laboratory rearing) may contribute to differences in wing shapes and sizes.

Individuals were placed in 95% ethanol pending genetic and morphometric analysis. WCR individuals used in this research were adapted to crop rotation, were non-resistant, and were collected from *Bt* corn expressing different toxins (Cry3Bb1, Cry34/35Ab1, Cry3Bb1, and Cry34/35Ab1) (Table 1).

**Table 1.** Number of WCR individuals used for geometric morphometric and SNPs analyses. *n* = sample size.


#### *2.2. DNA Extraction and SNPs Genotyping*

Before DNA extraction, hindwings from all individuals were removed for morphometric analysis. DNA was then extracted from the whole-body tissue of 29 adult WCR. DNA extractions were performed using the Qiagen DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer's protocol.

The DNA concentration for all samples was measured using spectrophotometer (BioSpec-nano Micro-volume) and adjusted to 50 ng/μ<sup>L</sup> prior to SNPs genotyping by Diversity Arrays Technology (DArT) [57,58]. After quality control, 29 samples were sent for genotyping. Genotyping was undertaken by Diversity Array Technology Pty Ltd. (DArT, Canberra, Australia) using the extracted WCR DNA. This method is based on methyl filtration and next-generation sequencing platforms [58]. The data we received were filtered for minor allele frequency (MAF) lower than 0.1 and also for missing data higher than 10%. Quality of SNP markers was determined by the parameters "reproducibility" and "call rate" [59]. Remaining SNPs were used for further analysis of genetic diversity and population structure.

#### *2.3. Geometric Morphometric Sample Preparation*

The adult WCRs (see Table 1) were investigated using geometric morphometric procedures and analyses based on hindwing venation undertaken. In total, 775 hindwings of WCR were analyzed. Left and right hindwings were removed from each individual and slide-mounted using the fixing agen<sup>t</sup> Euparal (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) based on standard methods [60]. Slide-mounted wings were photographed using a Canon PowerShot A640 digital camera (10-megapixel) on a trinocular mount of a Zeiss Stemi 2000-C Leica stereo-microscope and saved in JPEG format using the Carl Zeiss AxioVision Rel. 4.6. (Carl Zeiss Microscopy GmbH, München, Germany). Fourteen type 1 landmarks defined by vein junctions or vein terminations were used (Figure 1.) [47–49,61].

**Figure 1.** Representation of the 14 morphological landmarks identified on the hindwings of western corn rootworm [61].
