Morphometric and spectral signatures reveal strong structure and localized gray zones in Microgramma species delimitation

BACKGROUND AND AIMS: Recognizing lineages is a central challenge in plant systematics, making it essential to explore multiple analytical tools. In this context, this study investigates how frond shape can assist in discriminating against lineages within the Scaly clade of Microgramma (Polypodiaceae), and tests whether the integration of multiple lines of evidence enables a more consistent recognition of lineages than exclusively macromorphological approaches. METHODS: We analyzed 271 specimens representing eight species, using Elliptical Fourier Analysis (EFA) to quantify frond shape, followed by multivariate statistical tests (PCA, MANOVA, LDA). Evolutionary relationships between spectral and morphometric data were assessed through phylogenetic generalized least squares (PGLS) regressions and phylogenetic partial least squares (Phylo-PLS) analyses. RESULTS: Dimorphic species exhibited higher discrimination capacity (average accuracy of 80-83%). Fertile and combined fronds yielded the highest accuracy values. Morphologically similar species, such as M. reptans and M. tobagensis, showed considerable overlap, whereas M. percussa achieved the best performance (average accuracy of 80%). Morphometric-spectral covariation yielded R2 = 0.72 (P = 0.003) when sterile and fertile fronds were combined, although PGLS analyses detected no significant phylogenetic signal. The association differed between frond types and is interpreted here as exploratory evidence of morpho-spectral covariation. CONCLUSIONS: Outline morphometry combined with infrared spectroscopy within a phylogenetic framework improves lineage discrimination, although overlap zones persist, reflecting complex evolutionary processes. Our study highlights the potential of integrative systematics to elucidate species boundaries in groups with high morphological disparity, as well as the need for broad sampling and multi-evidence approaches in future systematic reviews.