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Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

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

Alice Dalla Vecchia¹, Rossano Bolpagni¹, Mattia Martin Azzella²

¹Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy.

Annals of Botany

|April 16, 2026

Summary

Charophytes, crucial freshwater macroalgae, were studied for functional traits. This research establishes a protocol and reveals charophytes expand aquatic plant functional diversity, with unique niches overlapping submerged species.

Keywords:

Chara spp Nitella spp Nitellopsis obtusa Characeae freshwater ecosystems functional niche functional strategies leaf traits multidimensional macrophyte comparison

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Area of Science:

Ecology
Botany
Aquatic Plant Biology

Background:

Charophytes are ecologically important freshwater benthic macroalgae.
They are often excluded from functional trait research, underestimating ecosystem diversity.
A standardized protocol for charophyte functional traits is needed.

Purpose of the Study:

To develop a standardized protocol for measuring charophyte leaf functional traits.
To compare charophyte functional traits with common vascular macrophyte life forms.
To assess the contribution of charophytes to aquatic plant functional diversity.

Main Methods:

Identified functional units in charophytes (whorls, branchlets, internodes).
Measured leaf area, specific leaf area, dry matter content, chlorophylls, carotenoids, and nutrient content (N, P, C) for 9 charophyte species.
Validated the protocol by comparing charophyte traits with 5 macrophyte groups using a multidimensional niche approach.

Main Results:

Trait measurements were comparable between charophytes and vascular macrophytes.
Charophyte functional traits aligned with ecological expectations regarding depth and light gradients.
Charophytes exhibited high intraspecific variability and occupied a unique functional niche, overlapping with submerged narrow-leaved plants.

Conclusions:

Charophytes significantly expand the functional diversity of aquatic plants.
The developed protocol enables better integration of charophytes into ecosystem functional assessments.
Future research should apply the protocol to Characeae genera and explore trait variability drivers.