In a phylogenetic study of arbuscular mycorrhizal fungal species in Acaulospora (Acaulosporaceae, Glomeromycota) we discovered that species classified in genus Kuklospora, a supposed sister clade of Acaulospora, did not partition as a monophyletic clade. Species in these two genera can be distinguished only by the position of the spore relative to a precursor structure, the sporiferous saccule, as either within (entrophosporoid) or laterally (acaulosporoid) on the saccule subtending hypha. Subsequent spore differentiation follows identical patterns and organization. Molecular phylogeny reconstructed from nrLSU gene sequences, together with developmental data, support the hypothesis that the entrophosporoid mode of spore formation evolved many times and thus represents a convergent trait of little phylogenetic significance. Therefore genus Kuklospora is rejected as a valid monophyletic group and it is integrated taxonomically into genus Acaulospora. Thus Acaulospora colombiana and Acaulospora kentinensis are erected as new combinations (formerly Kuklospora colombiana and Kuklospora kentinensis). Mode of spore formation is demoted from a genus-specific character to one that is included with other traits to define Acaulospora species. In addition we describe a new AM fungal species, Acaulospora colliculosa (Acaulosporaceae), that originated from a tallgrass prairie in North America. Field-collected spores of A. colliculosa are small (<100 μm diam), hyaline or subhyaline to pale yellow and form via entrophosporoid development based on structure and organization of cicatrices and attached hyphae. Each spore consists of a bilayered spore wall and two bilayered inner walls. A germination orb likely forms after the completion of spore development to initiate germination, but this structure was not observed. A character distinguishing A. colliculosa from other Acaulospora species is hyaline to subhyaline hemispherical protuberances on the surface of the outer spore wall layer. A phylogeny reconstructed from partial nrLSU gene sequences unambiguously placed A. colliculosa in the Acaulospora clade.