New insights into the evolution of spider silk proteins illuminated by long-read transcriptomes

Abstract Spider silk has long fascinated scientists because of its exceptional mechanical properties, yet the evolutionary origins and diversification of spider silk proteins (spidroins) remain incompletely understood. By analyzing long-read spidroin transcripts from spiders representing key evolutionary lineages, we identify two ancestral proteins present in basal spiders: an alanine–serine-rich (AS-type) protein and a glycine–serine-rich (GS-type) protein. These ancestral proteins likely served as primary evolutionary templates for the diversification of modern spider silks. We show that the AS-type ancestral spidroin remained relatively conserved and ultimately gave rise to the specialized tubuliform spidroin (TuSp) used in eggcase silk. In contrast, the GS-type protein underwent extensive functional radiation, evolving into differentiated minor ampullate spidroins (MiSp) through the acquisition of distinct terminal domains and β-sheet–associated structural innovations. Our results further suggest that major ampullate spidroins (MaSp) may have originated from MiSp, with the GS motif representing an evolutionarily favorable genetic substrate for the emergence of high-performance silk proteins. Finally, we propose a revised evolutionary trajectory for flagelliform spidroins (Flag), suggesting that they were co-opted as components of ampullate silk during functional degeneration in modern non-web-weaving RTA clade spiders. Together, these findings provide a high-resolution framework for understanding the genetic innovations that drove the diversification of spider silk.