Quantitative Pollen-sculpture variation can provide systematically use
ful information. To test manual-outlining techniques used with automat
ed measurement of pollen-sculpture features, we conducted several expe
riments in which perforations were traced with felt-tip pen onto thin
sheets of plastic. In each experiment, seven individuals made five tra
cings of the same pattern. Tracings were then digitized and perforatio
ns measured using image-analysis software. Three test patterns were us
ed: (1) an SEM micrograph with ''real'' pollen perforations; (2) a com
puter-generated test pattern that simulated very small pollen perforat
ions; and (3) a computer-generated test pattern that simulated very la
rge pollen perforations. Experiments examined the effect on precision
and accuracy of using: (1) opaque-black versus transparent-red ink; (2
) small versus large perforations; and (3) fuzzy- versus sharp-edged p
erforations. Tracings were statistically compared using the coefficien
t of variation and an analysis of variance. Tracings of computer-gener
ated test patterns also were compared directly with the digitized test
pattern. Results indicated that: (1) variation between tracings by a
single individual will usually be very small; (2) variation between in
dividuals usually will be statistically significant, even with the sha
rpest possible perforations; (3) tracing with opaque ink is more accur
ate than with transparent ink; (4) both precision and accuracy increas
e with perforation size; and (5) deviation of a single-tracing measure
ment from the parametric value usually will be much less than five per
cent. These experiments demonstrated that manual tracing of pollen per
forations prior to image analysis can produce repeatable measurements
accurate to two significant figures.