Controlled crosses
We collected mountain pine beetle larvae (F0 generation) from infected lodgepole pine trees from Smokey River Lowlands near Grande Prairie (Alberta, Canada; 54°21.376’ N, 118°19.112’ W) and BURNCO Quarry near Canmore (Alberta, Canada; 51°04.026’ N, 115°17.237’ W) in October of 2014 (Supp. Fig. 1). These sites represent the two genetically distinct sub-populations previously identified within Canada (Batista et al., 2016; Janes et al., 2014; Samarasekera et al., 2012) and are referred to in this study as North and South, respectively. Collected larvae continued development in their natal bolts at 4°C until spring 2015. Next, we placed bolts in opaque emergence boxes at room temperature (approximately 22°C) and mature mountain pine beetles were collected daily for a maximum of 15 days. Freshly emerged mountain pine beetles were sexed using the auditory method described by Rosenberger et al. (2016) to minimize physical damage. We temporarily stored the emerged adults at 4°C until a sufficient number had been collected to begin crosses (maximum five days). Further details are provided in Trevoy et al. (2019).
We reared North x South crosses of the F0 generation in 55 cm bolts from three lodgepole pine, Pinus contorta Douglas ex Louden (Pinales: Pinaceae), trees felled in May 2015 at Nojack, Alberta (53°36.103’ N, 115°35.239’ W). Females were placed in 1.5 ml microcentrifuge tubes attached to bolts to encourage gallery construction within a specific bolt. Males from the opposite population were placed in the tube 48 h later. Females or males that failed to enter the bolt within 24 h were replaced with alternates from the same site of origin. We stored F0 crosses in a sealed room at approximately 22°C for six weeks, at which point we transferred them to emergence boxes. F1 progeny were collected over 15 days and physically sexed using the non-destructive auditory method. Mountain pine beetle adults have been known to establish secondary galleries in different trees (Janes et al., 2016). To ensure that F1 beetles were assigned to the correct gallery, and therefore the correct cross, each bolt was stripped and visually inspected. Any progeny from a bolt containing multiple galleries was discarded from further analysis.
We crossed F1 beetles with gallery siblings to produce highly related F2 individuals. Males tend to guard the entrance of a gallery and pack it with frass (Reid, 1962). Thus, after seven days, we peeled back the wood around each gallery to a depth of 5 cm and the male was removed for genotyping. In accordance with Amman (1972), females were expected to complete gallery construction within three weeks. Thus, we extracted females at a later date in a similar fashion to males. Bolts were placed in emergence boxes at approximately 22°C until additional F2 progeny were no longer recovered. All F2 individuals were stored at –20°C prior to DNA extraction after physical sexing, using the dimorphic seventh tergite (Safranyik & Carroll, 2006). Sexing using the dimorphic seventh tergite was reserved for F2 individuals because, while more accurate than the auditory method, it typically causes damage to the specimen. The crossing design and family summaries are illustrated in Supp. Fig. 1. Specimens have been vouchered at the E. H. Strickland Entomological Museum at the University of Alberta with UASM numbers 391988-391991.