A Dual Targeted/Untargeted LC--MS/MS Protocol Uncovers Macroevolutionary and Ontogenetic Dynamics in Aristolochia Metabolomes

Coevolutionary plant–herbivore interactions are frequently mediated by phytochemicals shaped by antagonistic selection. Although some plants produce conserved defenses, macroevolutionary comparisons remain scarce. Furthermore, Optimal Defense Theory posits that plants allocate toxic chemicals to specific tissues to maximize fitness. Plants of the magnoliid “birthwort” family Aristolochiaceae produce aristolochic acids (AAs), potent xenobiotic toxins that cause urolethial cancer in humans and nephrotoxicity and renal failure in cattle. Interestingly, butterflies of the tribe \textit{Troidini} are sole herbivore on \textit{Aristolochia} plants and resist, tolerate, and sequester AAs. While \textit{Aristolochia} produce several forms of AAs, the butterflies sequester and tolerate only AA-I and small amounts of AA-II. However, we currently lack an updated metabolomic profiling protocol to characterize AA abundance and variation. Here, we leverage members of the large genus \textit{Aristolochia}, which serve as specialist host plants for the North American Genus of \textit{Troidini} butterflies, \textit{Battus philenor}, to understand macroevolutionary and ontogenetic stage-specific variation in AA production in response to herbivory. To this end, we developed a Liquid Chromatography Mass Spectrometry (LC–MS/MS) protocol for targeted and untargeted metabolomics to investigate phytochemical variation across multiple \textit{Aristolochia} species (the first to our knowledge). Our results demonstrate that: 1) LC–MS/MS recovered four major previously known dominant forms of AAs and detected a putative novel AA-II isomer in a single species, \textit{A. erecta}; 2) targeted profiling revealed remarkably conserved AA composition across species, with higher production of AA-I across all species; 3) in \textit{A. californica}, ontogenetic stage-dependent AA-I allocation aligned with Optimal Defense Theory through increased allocation to tissues with higher fitness costs; and 4) untargeted metabolomics showed extensive interspecific divergence in secondary metabolites alongside modest ontogenetic variation. Together, this novel LC–MS/MS framework demonstrates that combined macroevolutionary and ontogenetic selective pressures drive phytochemical evolution in \textit{Aristolochia} in response to herbivory by B. philenor.