The effects of cold stratification, light and seed clustering in petri dish on Ottelia alismoides seed germination were investigated. The seeds required light and an extended cold period in order to germinate, but neither treatment alone was effective. Seed germination significantly increased with length of the 4 degrees C cold stratification period. Freshly collected seeds failed to germinate while a 5-month period at 4 degrees C yielded 29 +/- 9% germination in the light, but none in the dark. Treatment with sodium nitroprusside, a nitric oxide source. failed to promote germination in the light or dark. Seeds of O. alismoides showed an unusual and significant positive response to aggregation. Germination in the light, after 5-month 4 degrees C cold stratification, was stimulated to almost five-fold in the dishes that were more densely sown with seed (20 seeds versus 200 seeds). Likewise, clustering seeds in dense aggregations stimulated germination significantly. Germination more than quadrupled with an increase from 1 to 50 seeds per cluster (200 seeds per dish), reaching a value of 72 +/- 4%. Linear regression analysis shows the correlation between seed cluster density (no. per cluster) and germination rate (%) was highly significant (R-2 = 0.85, P = 0.000). The extended cold stratification requirement is probably an over-wintering device. The mechanism of the density-dependent stimulation is unclear. (c) 2008 Elsevier B.V. All rights reserved.