CLEAN CUTICLE OR CLEARED LEAF – FOSSIL PLANT LEAF PREPARATION METHODS FOR PCO2 RECONSTRUCTION

Fossil plant leaves are one of the most commonly used material for reconstructing palaeo-atmospheric CO₂ concentration (pCO₂). Traditionally, pCO₂ are reconstructed from fossil plant leaves either by their stable carbon isotopic signals or by Stomatal Frequencies (SFs) such as Stomatal Density (SD, the number of stomata in a unit size) and Stomatal Index (SI, the proportion of epidermal cells that are stomata). In recent years, several "leaf gas-exchange models" (such as the “Franks Model”) have been developed for pCO₂ reconstruction by combining isotopic data and stomatal apparatus features (SD, stomatal orifice length, guard cell width, etc.).

Stomatal data thus are essential for both the SF method and the “model” method. They can be obtained directly by epifluorescent microscopic imaging from some well-preserved fossil leaf compressions. However, for less well-preserved samples or leaves with uneven epidermal sculptures, this method is insufficient to obtain acurate stomatal data. Laboratory preparations to obtain clean cuticle (CC) or cleared leaf (CL) by chemial treatements are thus applied.

We tested CC and CL by using Metasequoia and Sequoia from the Early-Middle Miocene Hannuoba Fomation of Inner Mongolia, China. These close “relatives” of the Cupressaceae family from the same fossil location posess different types of cuticle (thin versus thick; uneven versus even), allowing us to compare the advantages and disadvantages of these two preparationss.

CC has only cuticular membrane remained, with all other leaf parts removed, wheras CL is obtained simply by making the fossil leaf transparent (cleared) with almost all parts of the leaf remained. CC is ideal for SF method as a clean and thin cuticular membrane allows easy calculation of cells. However, other stomatal measurements such as orifice length and guard cell width necessary for the “model” method are no longer preserved in it. CL is more difficult to observe under light microscope as it is thick, but faithfully preserves almost all anatomical details of the leaf; it can thus not only provide stomatal data for both the SF and the model methods, but also be used for other research purposes using leaf anatomy. In addition, as CL takes less steps of chemical treatements, it reduces costs and makes less damage on samples to achieve large pieces of fragments.