Statistical Analysis of Computer-Generated Measurements from Manually Outlined Pollen Perforations

Abstract

Abstract Quantitative pollen-sculpture variation can provide systematically useful information. To test manual-outlining techniques used with automated measurement of pollen-sculpture features, we conducted several experiments in which perforations were traced with felt-tip pen onto thin sheets of plastic. In each experiment, seven individuals made five tracings of the same pattern. Tracings were then digitized and perforations measured using image-analysis software. Three test patterns were used: (1) an SEM micrograph with “real” pollen perforations; (2) a computer-generated test pattern that simulated very small pollen perforations; and (3) a computer-generated test pattern that simulated very large 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 perforations. Tracings were statistically compared using the coefficient of variation and an analysis of variance. Tracings of computer-generated 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 individuals usually will be statistically significant, even with the sharpest possible perforations; (3) tracing with opaque ink is more accurate than with transparent ink; (4) both precision and accuracy increase with perforation size; and (5) deviation of a single-tracing measurement from the parametric value usually will be much less than five percent. These experiments demonstrated that manual tracing of pollen perforations prior to image analysis can produce repeatable measurements accurate to two significant figures.