Orb-weaving spiders possess three types of aqueous silk glue genes with pervasive variation in repeat structure, organization, and length.

The evolutionary success of orb-web spiders has been attributed to the use of a liquid glue produced by the aggregate glands that coats the web's capture threads to secure prey. Remarkably, the primary protein components within these glue droplets are derived from the same family of genes, spidroins, that encode silk fibers. The complete gene sequence of the aggregate spidroins, AgSp1 and AgSp2, have been described in a few species, but the molecular variation across species is not well characterized. Here, we provide a comparative analysis of AgSp genetic diversity among thirteen orb-web and one cobweb species. We find that AgSp1 and AgSp2 are ancestral to the Araneoidea and identify a third gene, AgSp3, that originated from a duplication of AgSp2. While all spidroins are repetitive, the three AgSp genes are characterized by more complex gene structure and patterns of repeat homogenization than other spidroins. AgSp1 genes, the highest expressed copy, are the longest spidroins described to date and encode two primary repeat types that differ in their abundance of glycine-proline motifs and serine residues. Most AgSp1 gene copies contain both repeat types in discrete blocks but there is substantial variation in the relative proportion of each type across gene copies. AgSp2 is distinguished by the sporadic occurrence of unusually large simplified repeats, while AgSp3 exhibits abundant interspecific diversity. Across all AgSp genes, we identify widespread variation in molecular features with functional significance such as charged residues and amino acid motifs common in fiber-encoding genes and associated with post-translational modifications.