Methods are further described in Lasker & Calderon (submitted), Asynchronous reproductive cycles among colonies of the Caribbean octocoral Antillogorgia americana.

All samples were collected from colonies, greater than 1 meter (m) in height, at 5-7 m depth on a shallow reef, locally referred to as Tiantupo, in the San Blas Islands, Panama (9° 32' 55" N, 78° 57' 30"W). Samples were obtained on scuba working from small boats in the vicinity of the Smithsonian Tropical Research Institute's field station in the San Blas Islands. To establish the reproductive status of colonies, 11 colonies were marked and mapped, and approximately 15 centimeters (cm) of tissue from an arbitrarily selected branch along with its associated branchlets was collected from each of the marked colonies from July 1990 through August 1991, with the exception of November 1990 and July 1991. Samples were fixed in 5% formalin and later rinsed and transferred to 70% ethanol. Each sample had a central branch with at least 10 branchlets. A total of 10 polyps were examined from the 10th branchlet, counting down from the tip. Polyps were sampled starting 1 cm above the base of the branchlet, extended no further than within 2 cm of the branchlet tip. Polyps were arbitrarily selected, never sampling adjacent polyps. Polyps were dissected open using fine forceps and a scalpel and examined at 25X (Wild M-5 dissecting scope) for the presence of gonad. A. americana is gonochoric (Yoshioka, 1979), and the sex was determined for those samples with gonads large enough to differentiate spermaries and eggs (≥ 160 micrometers (µm)). The eggs or spermaries were counted and the diameter of each was measured using an eyepiece micrometer. Spermaries are seldom perfect spheres, and an "average" diameter was chosen and measured for each. The volume of each egg or spermary was calculated from the measurement of the diameter assuming they were spheres, and the average egg or spermary size was calculated for each polyp. In figures depicting reproductive status, values for November 1990 and July 1991 were interpolated from the months before and after the missing sample. Statistical analyses of the number and sizes of gonads were conducted using SPSS (v.29).

Variation in reproductive output among branches within a colony was examined for a single large (>1m height) fecund female colony collected in January 1991. Five large branches each with at least 10 branchlets were collected. Analyses were then conducted on polyps from 4 branchlets from each of the branches. Counting down from the tip, branchlets at positions 1 and 10 were sampled, one from the right side of the branchlet and one from the left at each position. From each of those branchlets 20 polyps were sampled, 10 from 1 cm above the base and 10 from 1 cm below the tip. Polyps were arbitrarily selected, dissected and numbers and sizes of eggs enumerated and compared as described above.

918002_v1_a_americana_branch_variation.csv