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).