Comparing Methodologies for Stomatal Analyses in the Context of Elevated Modern CO₂.

Area of Science:

• Paleobotany
• Paleoclimatology
• Plant Physiology

Background:

• Leaf stomata regulate gas exchange (CO2 and water) and their morphology varies over geologic time.
• Stomatal traits are influenced by environmental factors, particularly atmospheric CO2 concentrations.
• Stomatal parameters are used to reconstruct past CO2 levels, often employing gas-exchange models and plant carbon isotope data.

Purpose of the Study:

• To compare two methodologies (corrosive and non-destructive) for preparing leaf samples for paleo-CO2 studies.
• To assess the comparability of stomatal parameter measurements between the two methods across different leaf types (scale-leaved and broad-leaved).
• To evaluate the impact of methodological differences on paleo-CO2 reconstructions using gas-exchange models.

Main Methods:

• Collected scale-leaved and broad-leaved specimens from herbaria with known atmospheric CO2 levels.
• Prepared leaf samples using a corrosive (clearing) method and a non-destructive (impression) method.
• Measured stomatal parameters, including stomatal density and guard cell width (GCW), for both preparation methods.
• Applied gas-exchange models to reconstruct past CO2 levels using the measured stomatal parameters.

Main Results:

• Stomatal density measurements were comparable between the corrosive and non-destructive methods.
• Stomatal size measurements, specifically guard cell width (GCW), were not comparable between the two methods.
• Reconstructed CO2 levels using stomatal impressions (due to inaccurate GCW) significantly exceeded measured CO2 for modern plants.
• This bias in CO2 reconstruction was observed in both coniferous and angiosperm leaves.

Conclusions:

• The non-destructive stomatal impression method yields inaccurate guard cell width measurements compared to the corrosive clearing method.
• Inaccurate stomatal size measurements from impressions introduce significant bias when reconstructing paleo-CO2 levels using gas-exchange models.
• For accurate paleo-CO2 reconstructions, it is recommended to use chemically cleared leaves rather than stomatal impressions.