Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb

Coevolutionary interactions are responsible for much of the Earth's biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein (NSP) and major allergen (MA). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes' importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

Automated summary

Coevolutionary interactions play a crucial role in Earth's biodiversity, and key innovations drive the speciation bursts of species involved. This study focused on the butterfly species Pieris brassicae and found that nitrile specifier protein (NSP) and major allergen (MA) genes are essential for survival on plants containing glucosinolates (GSLs), which are a key innovation in mustard-feeding butterflies. The expression differences of these genes correlated with detoxification performance, particularly when using natural host plants. Positive selection signatures were also detected for NSP and MA genes across Pieris species, highlighting their importance in recent coevolutionary interactions. This research emphasizes the necessity of including ecological relevant experiments to gain insights into coevolutionary dynamics.