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.