Study System and testing for premating barriers to gene flow
In previous genetic work, Schoville et al. (2012) revealed that there are two major genetic lineages within the N. ingens complex associated with the allopatric species N. ingens and N. riversi , but morphologically and genetically intermediate populations suggested some gene flow. The morphologically intermediate populations were readily distinguished by color pattern, as they were dull green, whereas N. riversi was bright metallic green and N. ingenswas matte black. Here we use the same convention to group individuals into three morphotypes. In order to understand whether these morphotypes are capable of genetic exchange, we used a simple mating experiment to assess premating isolation among the three morphotypes (N. riversi , intermediate, and N. ingens ). Reduced copulation across the three morphotypes would indicate limited interspecific gene flow. In this experiment, samples from three populations (sites 1, 8, and 27;Figure 1 , Table 2 ) were chosen to represent N. riversi , the intermediate morphotype, and N. ingens , respectively. However, due to conservation concerns regarding the small local population sizes of beetles in the field, our experiment was constrained by sample size to 10-20 beetles per population. All beetles were collected in July 2018, maintained at ~5oC in a refrigerator with a mealworm diet prior to experiments, and all the trials were conducted within a week following field collection. For each trial, one male and one female were placed within a large yogurt cup (10 cm in diameter and 4 cm in depth) for 30 minutes. All experimental combinations of morphotypes were conducted (for both males and females), including experiments within each morphotype as controls, to determine the rate of successful copulation. A successful copulation was defined as the male mounting the female, with direct observation of his genitalia penetrating the female. Unfortunately, subsequent attempts to rear the progeny from the crosses failed in the lab.