A standardized protocol and basic ecological findings to include charophytes in functional trait studies

Background: and Scope. Charophytes comprise key benthic macroalgae that play multiple ecological roles in freshwater. Despite this, they are excluded from community-level functional trait research, leading to an underestimation of functional diversity of charophyte-dominated ecosystems. This study aims to develop a standardized protocol for measuring leaf functional traits for charophytes, and to provide a first functional comparison between charophytes and common vascular macrophyte life forms. Methods: Functional units were identified that perform the leaf function and reflect the modular growth of charophytes (whorls, branchlets, and internodes). Leaf (branchlet) area, specific leaf area, leaf dry matter content, chlorophylls, carotenoids, phosphorus, nitrogen and carbon content were measured on 9 charophyte species. The protocol was validated by comparing traits between charophytes and 5 macrophyte groups (lemnids, nymphaeids, submerged-broad-leaf, submerged-narrow-leaf, and utricularids), using a multidimensional niche approach. Key results: Trait measurements were comparable between charophytes and vascular macrophytes. Charophytes' functional traits showed alignment with ecological expectations, such as increased specific leaf area and reduced dry matter content along the expected growth depth. Overall, charophytes showed high intraspecific variability, attributable to their ability to grow across wide gradients of depth and light. Charophyte functional niche overlapped mostly with submerged narrow-leaved plants but maintained >40% niche uniqueness. Conclusions: Charophytes expand the functional space occupied by macrophytes, contributing to understanding the overall functional diversity of aquatic plants. Following research should apply the protocol to all genera of the family Characeae and investigate the environmental drivers of trait variability.