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Significance The venom of the Australian funnel-web spider is one of the most complex chemical arsenals in the natural world, comprising thousands of peptide toxins. These toxins have a diverse range of pharmacological activities and vary in size from short (3 to 4 kDa) to long (8 to 9 kDa). It is unclear how spiders evolved such complex venoms and whether there is an evolutionary relationship between short and long toxins. Here, we introduce a “structural venomics” approach to show that the venom of Australian funnel-web spiders evolved primarily by duplication and elaboration of a single ancestral knottin gene; short toxins are simple knottins whereas most long toxins are either highly elaborated single-domain knottins or double-knot toxins created by intragene duplications.

Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene