Septins are important components of the cytoskeleton that are highly conserved 
in eukaryotes and play major roles in cytokinesis, patterning, and many 
developmental processes. Septins form heteropolymers which assemble into 
higher-order structures including rings, filaments, and gauzes. In contrast to 
actin filaments and microtubules, the molecular mechanism by which septins 
assemble is not well-understood. Here, we report that in the filamentous fungus 
Aspergillus nidulans, four core septins form heteropolymeric complexes. AspE, a 
fifth septin lacking in unicellular yeasts, interacts with only one of the core 
septins, and only during multicellular growth. AspE is required for proper 
localization of three of the core septins, and requires this same subset of core 
septins for its own unique cortical localization. The ΔaspE mutant lacks 
developmentally-specific septin higher-order structures and shows reduced spore 
production and slow growth with low temperatures and osmotic stress. Our results 
show that at least two distinct septin heteropolymer populations co-exist in A. 
nidulans, and that while AspE is not a subunit of either heteropolymer, it is 
required for assembly of septin higher-order structures found in multicellular 
development.