Combined EST and proteomic analysis identifies rapidly evolving seminal fluid proteins in Heliconius butterflies

Seminal fluid proteins (SFPs) directly influence a wide range of reproductive processes, including fertilization, sperm storage, egg production, and immune response. Like many other reproductive proteins, the molecular evolution of SFPs is generally characterized by rapid and frequently adaptive evolution. However, the evolutionary processes underlying this often-documented pattern have not yet been confidently determined. A robust understanding of the processes governing SFP evolution will ultimately require identifying SFPs and characterizing their evolution in many different taxa, often where only limited genomic resources are available. Here, we report the first comprehensive molecular genetic and evolutionary analysis of SFPs conducted in Lepidoptera (moths and butterflies). We have identified 51 novel SFPs from two species of Heliconius butterflies (Heliconius erato and Heliconius melpomene) by combining "indirect" bioinformatic and expression analyses of expressed sequence tags from male accessory gland and wing tissues with "direct" proteomic analyses of spermatophores. Proteomic analyses identified fewer SFPs than the indirect criteria but gave consistent results. Of 51 SFPs, 40 were identified in both species but fewer than half could be functionally annotated via similarity searches (Blast, IPRscan, etc.). The majority of annotated Heliconius SFPs were predicted to be chymotrypsins. Comparisons of Heliconius SFPs with those from fruit fly, mosquito, honeybee, and cricket suggest that gene turnover is high among these proteins and that SFPs are rarely conserved across insect orders. Pairwise estimates of evolutionary rates between SFPs and nonreproductive proteins show that, on average, Heliconius SFPs are evolving rapidly. At least one of these SFPs is evolving adaptively (dN/dS > 1), implicating a role for positive selection in this rapid evolution. This work establishes a strong precedent for future research on the causes and consequences of reproductive protein evolution in the Lepidoptera. Butterflies and moths have an extremely rich history of organismal research, which will provide an informative ecological context for further molecular evolutionary investigations.