In-depth phenotyping reveals unexpected floral trait variation in Mimulus cardinalis across a range-wide latitudinal gradient

Background & Aims: Pollinators play a crucial role in the evolution and diversification of flowering plants. Intraspecific variation in floral traits occurs frequently and likely reflects variable biotic and abiotic selection pressures. The hummingbird-pollinated monkeyflower Mimulus cardinalis occurs across western North America, where geographic mosaics of selection have likely influenced its natural history, due to this wide species range. Processes driving early stages of divergence and speciation remain poorly understood, though theory predicts that trait divergence will be most likely at range edges. Within M. cardinalis, which is usually red-flowered and hummingbird-pollinated, two independent shifts to yellow flowers have occurred at the northern and southern range edges, including a population found on Cedros Island, off the coast of Baja California, Mexico. Methods: This study characterises the local climatic variables of five accessions of M. cardinalis derived from locations across a latitudinal geographic gradient. Highly integrative methods in metabolomics and morphological analyses were used to characterise a suite of pollinator-relevant floral traits across the range of M. cardinalis species variation. We use transcriptomics and whole genome sequencing to study underlying genetic variation in two yellow-coloured, range-edge accessions. Key Results: This study uncovers high levels of variation in morphology, nectar properties, pigmentation and scent profile across geographically diverse Mimulus cardinalis accessions, in addition to profiling how these accessions are perceived by potential pollinators. We find high levels of phenotypic variation across M. cardinalis, particularly in the biochemistry of pigment and scent, where inflorescences that appear to be the same shade of red have completely different anthocyanin profiles. Floral trait-underlying genetic differences between different M. cardinalis lines were also investigated, revealing potential mechanisms underlying floral diversification. Conclusions: This work highlights the importance of interplay between floral trait diversification, pollinator perception and climate. This work also highlights the importance of considering suites of floral traits in a quantitative fashion when studying intraspecific variation in relation to species range. This study also provides genetic insight into changes in these traits and provides many future directions to study the early stages of pollinator-mediated trait differentiation across a species range.