The extant plant genus Isoetes (Isoetaceae; lycophyte, quillwort) is important from an evolutionary point of view. Species of this heterosporous genus are small herbs (up to 50 centimeters) and exhibit some morphological, anatomical and embryological features of their Paleozoic arborescent lycopsid ancestors. The species Isoetes pantii produces three kinds of microspores (monolete, alete and trilete) and two types of trilete megaspores in one and the same heterosporangium. We attempt to associate these unusual functional megaspores with various Paleozoic spores described mainly from Devonian barinophytaleans such as Omniastrobus dawsonii, Barinophyton richardsonii, B. citrulliforme and Protobarinophyton pennsylvanicum. These have two kinds of spores in a sporangium and provide the first palynological evidence of heterospory at 405 Ma. The germination of microspores and megaspores and production of gametophytes within the heterosporangia of I. pantii corresponds with that of some of its Paleozoic ancestors. Retention of megaspores within heterosporangia and their germination in situ offers evidence that I. pantii exhibits the probable route of evolution of the seed habit. These observations support the hypothesis that a typical heterosporangium was the cradle for the evolution of heterospory.