Proof of homothally of Pheopolykrikos hartmannii and details of cyst germination process

Resting cysts play crucial roles in the ecology of dinoflagellates,especially in wintering or surviving unfavorable conditions,seeding harmful algal blooms(HABs),and facilitating the geographic expansion.Encystment of dinoflagellates is tightly coupled with sexual reproduction in most cases,which can occur either through homothallism(self-fertilization)or heterothallism(intercrossing of+/-strains).The types of sexual reproduction have important ecological implications.The toxic and HAB-forming dinoflagellate,Pheopolykrikoides hartmannii has been previously reported to be heterothallic.Here,we provide visual confirmation of homothally of P.hartmannii and the first detailed visual recording of cyst germination based on the observations of a clonal isolate from Jiaozhou Bay,China.To document the homothallism,we first observed cell pairs in sexual mating,planozygotes with two longitudinal flagella,and cysts with typical morphology as described previously from the clonal culture.We then germinated a single cyst,established a new clonal culture from one of the two daughter cells after the first cell division of the germling(i.e.from the diploid germling to two haploid cells),and produced cysts again from the newly established clonal culture.For the observation of the germination time-series,we took micrographs and videos to show all germination processes,particularly with an interesting observation of the short amoeboid stage of the germling releasing from the archeopyle(~15 s),which was a landmark of the germination process and has not been reported elsewhere.This definitive evidence of homothallic sexuality and cyst production in P.hartmannii provides a new insight into the biology and ecology of the species,particularly a mechanism that may partly account for the population dynamics and ubiquitous distribution of the species.