Abstract
Homology is a crucial concept that should be considered while conducting a comparative analysis between organisms. In particular, in the subtribe Ligeriinae, the nectar guide pattern is associated with high diversity in petal shapes and sizes. This largely limits researchers to exclusively examining the interspecific variation in nectar guide patterns on the developmentally homologous region. Thus, to solve this problem, we proposed an approach for defining a homologous region of interest (ROI) that aligns the petal image between specimens based on petal contours and vasculatures. We identified petal contours and vasculatures from the fresh petal image and its histological image through image processing. The homologous ROI was subsequently obtained by applying geometric transformation to the contour and vasculature. The qualification and quantification of nectar guide patterns were subsequently performed based on the homologous ROI. Four patterning modes, namely vascular, random, distal, and proximal, were defined for the qualitative analysis of nectar guide patterns. In the quantitative analysis, principal component (PC) analysis was applied to homologous ROIs, and the PC score of each specimen served as the trait values of nectar guide patterns. The results of the two analyses coincided, and both showed significant associations between nectar guide patterns and pollination types. The proximal mode (corresponding to PC1) and distal mode (corresponding to PC2) together showed the strongest association with pollination types. Species exhibiting the hummingbird and bee pollination types tended to recruit the distal and proximal modes, respectively. Our study conducted a comparative analysis of nectar guide patterns on the developmentally homologous region and provided a comprehensive view of the variation in the nectar guide patterns of Ligeriinae.