192 
Psyche 
[Vol. 95 
Materials and Methods 
Foundress associations of V. pergandei were collected from two 
sites, “Main” and “Granite” (2 km apart) immediately south of the 
southeast corner of South Mountain Park, Phoenix, AZ during 
February-March 1988. Foundress associations of A. versicolor 
were collected from a site in North Scottsdale, AZ (described in 
Rissing et al. 1986) in September 1987. In each case, existence of a 
single characteristic mound of freshly excavated soil indicated a 
single foundress association. Live co-foundresses were air expressed 
immediately to Michigan State University where they were frozen at 
-80° C and stored until electrophoresed. 
Electrophoretic methods. We prepared frozen ants for electro- 
phoresis by grinding them individually in an extraction buffer at 
4°C. We removed the gasters of V. pergandei queens before grind- 
ing in a pH 7.0, 0.1 M tris buffer (with 40 mg EDTA, 20 mg NAD, 
10 mg NADP and 250 p\ beta-mercaptoethanol per 100 ml: Buffer 
1) or in an unbuffered detergent solution (with 100 p\ Triton-X, 10 
mg NADP and 100 p\ beta-mercaptoethanol per 100 ml: Buffer 2). 
Buffer 2 gave superior results for esterases but was no better, and in 
some cases worse, than Buffer 1 for other enzymes. We ground 
whole A. versicolor in buffer 1. For each ant we adjusted the 
amount of buffer from 10 to 100 p\ to give an approximately equal 
ratio of buffer to ant tissue. 
We applied extracts from 12 ants (ca. 1 p\ from each) to thin-layer 
cellulose acetate plates (Titan III: Helena Laboratories, Beaumont, 
TX). Plates were soaked for at least 30 min in a running buffer 
before sample application; we used the same buffer for the electro- 
phoretic run. We used cellulose acetate running buffers “A”, “B”, 
“C”, “D”, and “I” of Richardson et. al. (1986); no single buffer gave 
good resolution for all enzymes tested. Run durations ranged 15-35 
minutes, under constant voltage (200-300 V); durations and vol- 
tages were adjusted to optimize separation for each enzyme that 
showed clear activity. Combinations of running buffer, voltage and 
time giving best results are noted below. All electrophoresis was 
done at 4°C. 
To visualize the allozymes we used enzyme-specific stains (Harris 
and Hopkinson 1978, Richardson et al. 1986), mixed 1:1 with 1.5% 
agar solution and poured onto the plates. When sufficient stain 
intensity was reached, we rinsed off the agar layer and soaked the 
