2.1 Sample collections and DNA extraction
Samples of the mealybugs were collected in the native range of H. pungens sensu lato in South America (n= 80) and in the recently invaded areas of Puerto Rico and the continental United States (n= 93). Mealybugs were collected on the host plants reported as part of the diet of H. pungens sensu lato (Cactaceae, n= 89; Amaranthaceae, n= 69; and Portulacaceae, n= 25). In the native range, mealybugs were collected in northern and northwestern Argentina (n= 21 on Cactaceae and n= 18 on Amaranthaceae), along the Atlantic coast of Brazil (n= 23 on Cactaceae; n= 11 on Amaranthaceae and n=4 on Portulacaceae), and in western Paraguay (n= 3 on Cactaceae). We also included samples collected in the non-native range in Puerto Rico (n= 42 on Cactaceae; 24 on Amaranthaceae and n= 11 on Portulacaceae), southeastern United States (n= 11 on Amaranthaceae), and in southwestern United States (n= 5 on Cactaceae). Additionally, we included samples from southeastern Australia (n= 5 on Cactaceae), where H. pungens sensu lato from Argentina was introduced as a biological control agent against cactus weeds in the 1970s (McFadyen, 2012). All individuals were preserved in 100% ethanol and stored in a freezer until DNA extraction. Information concerning sampling localities, geographic coordinates, and host plants species are presented in Figure 1 and Table S1.
Genomic DNA was extracted using entire bodies of adult female mealybugs using Qiagen DNeasy Blood & Tissue Kit according to manufacturer’s instructions (Valencia, CA, USA), adding 2 µL of RNAse A after the lysis step. DNA was quantified using Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY, USA), and quality was assessed in a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA).