A multidimensional view of fronds reveals phenotypic structuring and delimitation problems

Abstract

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. 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. 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 significant overlap, whereas M. percussa achieved the best performance (average accuracy of 80%). Morphometric-spectral integration showed a strong correlation (R² = 0.72; P = 0.003), and both the combined datasets (spectra and outline) and the individual datasets of spectral and shape features revealed a high phylogenetic signal (λ = 1–0.84), indicating partial coevolution between frond shape, chemical composition, and the evolutionary history of the group. 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.