Conifer Needle Epidermal Imprints for Stomatal Size and Density Measurements

Stomata play a central role in regulating plant water loss and gas exchange, making functional trait data on stomatal density and size increasingly relevant in the context of climate change. Expanding comparative datasets for these traits requires simple, low-cost methods that can be applied consistently across species and environments. However, while simple stomatal imprint techniques using nail varnish and adhesive tape are widely established for angiosperms, their application to conifer needles is challenging. Stomata in conifers are typically sunken within epistomatal chambers and occluded by densely meshed wax tubules, which can prevent accurate replication of stomatal complexes in epidermal imprints and thereby limit reliable measurement of stomatal size. In addition, stomata are often heterogeneously distributed across needle surfaces and may occur on multiple surfaces depending on species, requiring tailored sampling approaches for accurate density estimation. Consequently, reliable measurement of stomatal traits in conifers would require chemical processing and advanced microscopy, making these methods time-intensive, expensive and less suitable for large-scale comparative studies. Therefore, we lack an approach for conifers that generates high-quality trait data while remaining fast, simple, and comparable to established angiosperm methods. Here, we present a straightforward protocol for preparing stomatal imprints in conifer needles based on standard methods for angiosperm species but including simple preprocessing steps to improve imprint consistency and quality. We further provide guidance for identifying, imaging, and quantifying stomata while accounting for their species-specific spatial distribution on needle surfaces. The protocol enables reliable measurements of stomatal size and density using basic laboratory equipment and standard light microscopy. We demonstrate its applicability across 12 northern hemisphere conifer species, highlighting its suitability for comparative trait-based studies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Support Protocol 1: Sampling conifer needles for stomatal traits Basic Protocol 1: Epidermal imprints using nail varnish Basic Protocol 2: Measurement of stomatal density and size.