# Unveiling Heterospecific Pollen Deposition in Ranunculus Plants Along a Land‐Use Gradient Through DNA Metabarcoding

## TL;DR

This study uses DNA metabarcoding to show how land use and plant diversity affect the variety of pollen deposited on Ranunculus plants.

## Contribution

The study introduces DNA metabarcoding as a novel method to analyze plant-received heterospecific pollen from the plant's perspective.

## Key findings

- Intermediate plant species richness leads to the lowest heterospecific pollen diversity on Ranunculus stigmas.
- Low and high plant diversity landscapes show greater heterospecific pollen diversity due to pollinator behavior.
- DNA metabarcoding effectively reveals pollen transfer dynamics influenced by plant community composition.

## Abstract

Animal pollination, the transfer of pollen by animal agents, is essential for plant reproduction. Methods like microscopy and DNA metabarcoding have been used to investigate pollen transport and plant–pollinator interactions. DNA metabarcoding, in particular, is a reliable method to identify the origins of mixed pollen samples. Although it has mainly been used to study pollinators' dietary patterns, it does not provide insights from the plant's perspective, such as the type of viable pollen received. We aimed to explore the potential of DNA metabarcoding to analyse heterospecific pollen transfer to plants in semi‐natural and agricultural landscapes along a land‐use intensity gradient. We collected stigmas of three closely related Ranunculus species (
R. acris
,
R. bulbosus
and
R. repens
) from 20 grassland plots in Germany with varying land‐use intensities and flowering plant diversity and subjected them to internal transcribed spacer 2 (ITS2) metabarcoding. Our results revealed a nonlinear relationship between flowering plant species richness and heterospecific pollen richness on Ranunculus stigmas. The lowest heterospecific pollen diversity occurred in landscapes with intermediate plant species richness, whereas plots with low or high richness showed greater heterospecific pollen diversity. Reduced plant species richness, found mostly on intermediate and high LUI plots, forces pollinators to visit multiple plant species and thus increases heterospecific pollen transfer. Plots with intermediate plant species richness, on the contrary, likely provide a balanced mix of resources for pollinators, visiting multiple plant species within a foraging round and thus decreasing the amount of heterospecific pollen. Increased heterospecific pollen at high‐richness plots may result from competition in pollinator‐rich communities. Our results show that DNA metabarcoding is a powerful tool for assessing heterospecific pollen diversity, revealing that pollen transfer is heavily influenced by plant community composition. This approach provides novel insights into pollinator fidelity and potential pollination outcomes across diverse environments.

We used DNA metabarcoding to explore heterospecific pollen transfer to three Ranunculus species in semi‐natural and agricultural landscapes across a land‐use intensity gradient. Our results show that flowering plant diversity significantly influences the diversity of pollen deposited on plant stigmas, with intermediate plant species landscapes exhibiting the lowest pollen diversity. This study highlights the potential of DNA metabarcoding to assess pollen transfer dynamics and their ecological implications in varying landscapes.