Taxonomic reinterpretation of morphological characters in Acaulosporaceae based on developmental patterns

Sequences in the development of subcellular organization in soil-borne spor es of Acaulospora laevis, A. spinosa and Entrophospora colombiana were anal yzed to define discrete morphological characters informative in taxonomic a nd phylogenetic analyses of arbuscular mycorrhizal fungi in the family Acau losporaceae, Glomales. A sporiferous saccule initially forms terminally on a sporogenous hypha, followed by spore formation. Spore ontogenesis proceed s in a linear series of discontinuous stages that are identical in all thre e species studied, with each stage recognized by addition of a new subcellu lar structure from the spore cytoplasm. The juvenile spore wall is a single hyaline layer continuous with the wall of the sporiferous saccule hypha, f ollowed by differentiation of a pigmented spore wall layer with thin adhere nt sublayers (or laminae) which varies phenotypically among species. A thir d spore wall layer then is differentiated in spores of Acaulospora, which v aries phenotypically between species. Once the spore wall is completely dif ferentiated and spores have ceased expansion, species in both genera sequen tially form two discrete and separate bilayered flexible hyaline inner wall s. Layers of the first inner wall are thin (< 1 mu m) and adherent. The inn er layer of the second flexible wall increasingly reacts in Melzer's reagen t as it matures in E. colombiana, indicating the layers are formed sequenti ally rather than concurrently.,After both inner walls are fully differentia ted, a "germination orb" is synthesized, although this structure rarely is observed in newly harvested spores from pot cultures. Structures formed in each stage of spore differentiation vary in the extent to which they are co nserved among species, indicating different levels of taxonomic resolution. All layers of the spore wall and process-related features such as spore si ze define species-level variation, and flexible inner walls resolve taxa no t yet recognized in glomalean classification. Ontogenesis in Acaulosporacea e consists of both shared and unique stages relative to developmental stage s in other glomalean families, Glomaceae and Gigasporaceae. However, early stages are more similar to those of Glomaceae, suggesting closer relatednes s to that family.