Cuticle analysis, also known as fossil cuticle analysis and cuticular analysis, is an archaeobotanical method that uses plant cuticles to reconstruct the vegetation of past grassy environments. Cuticles comprise the protective layer of the skin, or epidermis, of leaves and blades of grass. They are made of cutin, a resilient substance that can preserve the shapes of underlying cells, a quality that aids in the identification of plants that are otherwise no longer visible in the archaeological record. This can inform archaeobotanists on the floral makeup of a past environment, even when surviving remains from the plants are limited. Plant cuticles have also been incorporated into other areas of archaeobotanical research based on their susceptibility to environmental factors such as p levels and stresses such as water deficit and sodium chloride exposure. Such research can help to reconstruct past environments and identify ecological events.
Method
There is no one universal method to cuticle analysis. Rather, it is the shared principle on which the applications are based which underpins the methodology—namely, that a well-preserved plant cuticle can, through the use of microscopy, yield information regarding the nature of the plant from which it originated, including its species and the environmental stresses acting upon it. Depending on the desired outcome, both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) can be used, the main difference being that while SEM can provide information regarding the outer characteristics of an organism, TEM can be used to show details of the inner structure. In SEM approaches, latex or silicone casts may be used to recreate epidermal and cuticular features in imperfectly preserved samples. Atomic force microscopy (AFM) can also be used as a complementary method to provide high-resolution topographic imaging at submicron scale. If the desired outcome is identification of the plant, the image created by one or a combination of these microscopy methods can then be compared to existing data, regarding both the impressions left in the cuticle of the underlying cell structure and the properties of the cuticle itself.
Cuticle extraction methods
Depending on preservation conditions, a method of extraction for the fossil cuticle is required before the analysis can take place. One such method for separating cuticles from a rock matrix is acid maceration, which involves soaking the sample in agents such as dilute hydrogen peroxide or hydrochloric and hydrofluoric acid (known as the HCI/HF protocol) to break down the matrix. However, this process is destructive and, if possible, avoided. Cuticle transfer techniques are generally preferred, as these methods preserve the physiognomy and morphology of the sample, regardless of how fragmented the specimen may be. Among the most practical and effective of these techniques is the polyester overlay transposition method, developed by Kouwenberg et al., which is both straightforward and non-destructive: the cuticle, either already accessible on the adaxial surface or exposed using cellophane tape from the abaxial surface, is pressed against the adhesive side of the polyester overlay and removed.
Applications
The most straightforward application of fossil cuticle analysis is in the identification of the plants which comprised a past environment. This is because the cutin layer preserves some of the defining characteristics of the plant's underlying cell structure, allowing it to be identified by experts at a microscopic level. Taxonomical differences in the epidermises of the two species Pinus sylvestris L. and Pinus uncinata Ramond ex DC., for example, can often be observed from cuticle analysis, meaning the plants can still be reliably identified and distinguished from one another in cases where other methods such as pollen analysis are not possible. In broader studies of past flora, this method can be expanded to not only inform researchers of the plant species present but also of patterns and trends underlying this distribution. In 2003, for example, cuticle analysis was used in a multi-proxy study to reconstruct changes in vegetation during the Late Pleistocene and Holocene in Kenya, with particular regard to the proportion of plants following the C4 photosynthetic pathway, and more specifically the NADP-ME C4 sub-pathway. There are, however, a number of other ways that such data can be used, including the following: