Materials and Methods:

Study Sites and Sample Collection:

Four populations of chicozapote were chosen for the study. Two were located in El Eden Ecological Reserve, a privately owned reserve 20 km west of Cancun, Mexico, in the northeast of the Yucatan peninsula. The first of these was a forest population, and the second, a swamp population approximately 2-3 km from the forest. The third and fourth populations were located at Chunchucmil and Hocaba, Mexico, which are in the northwest of the Yucatan peninsula near Merida. The Chunchucmil population was located in an inundated swamp, and the Hocaba population consisted of cultivated trees in the home gardens of the residents of the town.

At the El Eden sites (one and two), 30 samples were collected along 2 km trails through the forest and swamp. At Chunchucmil (site three), samples were taken from 10 individuals along a prehistoric roadway. At Hocaba (site four), samples were taken from 10 individuals in 6 different gardens. Approximately 2 g of leaves from each individual was collected and placed into plastic bags containing fine grain silica gel. The gel desiccated the leaf samples, preserving the DNA until the samples could be returned to the laboratory at UC, Riverside. The selection of these four sites allowed us to study variation in habitat type (i.e. swamp vs. forest vs. cultivated), as well as geographic variation (El Eden [East] vs. Chunchucmil and Hocaba [West]).

Genomic DNA extraction:

Genomic DNA from leaves was isolated with a modified cTAB extraction protocol (Saghai-Maroof et al. 1984). For each sample, 0.4 g of desiccated leaf material was ground to a fine powder with a mortar and pestle in liquid nitrogen. While the powder was still frozen, 2 ml of extraction buffer [100 mmol/L Tris-HCl (pH 8), 2% w:v mixed alkyltrimethyl-ammonium bromide, 1.4 mol/L NaCl, 20 mmol/L EDTA (pH 8), 4% w:v PVP-40, 0.1% w:v ascorbic acid, 0.1% w:v DIECA (diethyldithiocarbamic acid), 1% v:v 2-mercaptoethanol] was added and the resulting mixture was ground for 30 sec. The mixture was transferred to 12 ml polypropylene tubes, mixed and incubated for 30 min at 60?C. The tubes were then returned to room temperature and extracted twice with chloroform/octanol (24:1). DNA was precipitated with -20?C isopropanol and centrifuged. The supernatant was poured off and the DNA was washed with 2 ml of 76% ETOH/0.2 M sodium acetate for 20 min. After centrifugation and removal of supernatant, DNA was washed with 1 ml of 76% ETOH/10mM ammonium acetate for 1 min, followed by air-drying. The resulting DNA pellets were transferred to microcentrifuge tubes and suspended in 50 ul of TE [10 mmol/L Tris-HCl (pH 8), 1 mmol/L EDTA (pH 8)]. Tests showed that the DNA samples were contaminated with RNA, so 1 ul of RNAse (1 mg/ml) was added to each sample and incubated at 38?C for 1 hr. The volume in each tube was brought to 500 ul with ddH2O, extracted once with phenol/chloroform and extracted once with chloroform/octanol (24:1). DNA was precipitated with ice-cold ethanol and 0.3M sodium acetate. The resulting pellets were washed with 70% ethanol, air dried, and re-suspended in 50 ul of TE. DNA concentrations were measured with a Hoefer (San Francisco, CA) TKO100 Mini-Fluorometer, following manufacturer's instructions. Prior to use for PCR, samples were diluted to a DNA concentration of 5 ng/ul.

Polymerase chain reaction (PCR)

PCR reactions (25ul final volume) contained 25 ng DNA, 100 uM each of dATP, dCTP, dGTP, and dTTP, 200 uM primer, 1X reaction buffer, 2 mM MgCl₂ and 0.65 units of Taq polymerase (Gibco BRL, Gaithersburg, MD). Each reaction was overlaid with 30 ul of mineral oil to prevent evaporation. The random primers were obtained from Operon Technologies Inc., Alameda, CA. For enzymatic amplification, samples were subjected to 45 cycles of: 1 min at 94?C, 1 min at 38?C, 30 sec at 54?C, and 2 min at 72?C. After the final cycle, samples were incubated at 72?C for 13 min, then held at 4?C until analysis. Fragments generated by amplification were separated according to size on 2% agarose gels, stained with ethidium bromide and visualized and photographed under ultraviolet light.

Bulking of DNA and Screening of Bulks:

To create the DNA bulks for each population, 50 ul of 5 ng/ul of DNA from every individual were combined into a single microcentrifuge tube. The tubes were briefly mixed and held at 4 ?C overnight to allow the DNA to evenly disperse throughout the solution. Bulked DNAs were amplified with eighty random primers to find primers that revealed inter-population variation.

When a primer was found that showed a polymorphism between samples, DNA samples of every individual in each population were amplified using that primer, in order to score population variation.

Data analysis:

Individuals were scored for polymorphic loci as either present (1) or absent (0). Pairwise comparisons between all individuals were calculated from the data using the simple matching coefficient. Trees were constructed using unweighted pair group method analysis (UPGMA) and neighbor-joining (Saitou and Nei 1987). Trees were also constructed using only individuals from the El Eden populations (sites one and two). All analyses were conducted using the MEGA package (Kumar et al. 1993). To further characterize any genetic variation between the swamp and forest populations, Fisher's exact test was performed for each locus using data from sites one and two. Results from individual loci were combined using Fisher's combined probability to give a one-tailed chi-square value for 2n degrees of freedom (n=number of loci). Finally, the distance-data matrix generated by the MEGA program was used to calculate the mean distance and standard error for forest x forest, swamp x swamp and forest x swamp comparisons for the El Eden populations (sites one and two).