Lab for reconstructing atmospheric CO2 from leaf stomata

Objectives:

1. Characterize the stomatal frequency of modern and fossil leaves in order to estimate the concentration of atmospheric CO2 in which the plants grew.
2. Think about how best to manage noisy data sets.

Hypotheses to test:

1. Do stomata faithfully record the level of atmospheric CO2?
2. Do other environmental factors impact stomatal distribution?

CO2 is key for understanding Earth's climate system. Knowledge of CO2 in Earth's past is important not only for understanding the functioning of the Earth at that time, but—for times in Earth's past that were warmer than today—where we may be headed in our near future. Proxies are an important way for reconstructing CO2. This lab focuses on one of these proxies: the inverse relationship between CO2 and stomatal frequency.

No specialized prior knowledge is required. Relevant content covered in the lab is introduced in the companion powerpoint.

The lab has been run for a major-level course at Wesleyan University for 20 years. A single 3-hour block should be sufficient, although students may need to finish the thought questions (#7-9) on their own time.

Groups of up to three are recommended.

There are two versions of the lab, depending on whether or not the instructor has access to microscopes; the "no microscopes" version is also online-friendly.

The powerpoint provides background for some of the topics covered in the lab.

Leaf stomata - CO2 cuticle lab (microscope version) (Microsoft Word 2007 (.docx) 586kB Jul1 24)
Leaf stomata - CO2 cuticle lab (no microscope version) (Microsoft Word 2007 (.docx) 534kB Jul1 24)
Introduction to stomatal and reconstructing CO2 (PowerPoint 2007 (.pptx) 1.6MB Jul1 24)
Leaf stomata - CO2 cuticle lab (microscope version) Instructor version (Microsoft Word 2007 (.docx) 1.1MB Jul1 24) 
Leaf stomata - CO2 cuticle lab (no microscope version) Instructor version (Microsoft Word 2007 (.docx) 543kB Jul1 24)

We recommend starting with the content covered in the companion powerpoint file.

"with microscopes" version: you will likely need to modify the content, for example based on your choice of living species and the lens selection of your microscopes. We recommend collecting two groups of leaves so that students can test for differences between groups. This could be sun vs. shade leaves on the same tree, or two different trees from different environments. In the current lab, this is what the "label" refers to. Students can collect and prepare leaves as well. This is not described in the lab and you would need to budget additional time to do it. Clearing leaves often takes hours to days. In many taxa, it can be difficult to count the number of epidermal cells with 100% accuracy; keep this in mind when grading. It is also an opportunity to talk about the scientific method, etc. (for example, how do you count partial cells along the edge of the field-of-view?). Once you have graded the lab, it is useful to report back to your class the class-mean stomatal index and class-mean CO₂ concentration. The class mean CO₂ is usually close to the correct concentration, even if some individual estimates are way off.

"no microscopes" version: each group will analyze two leaf images. We recommend that each group analyze an older leaf and a more recent leaf, so that they can test for potential differences based on the CO₂ concentration. This requires that you look at the Cuticle Database in advance to select leaves. In ginkgo, it can be difficult to count the number of epidermal cells with 100% accuracy; keep this in mind when grading. It is also an opportunity to talk about the scientific method, etc. (for example, how do you count partial cells along the edge of the field-of-view?).

Student answers to questions can be collected and graded, or self-graded and discussed.

Good references for background information on using stomata to reconstruct CO₂:
McElwain and Steinthorsdottir (2017)

Supplement in Hönisch et al. (2023)