A new method to prepare clean cuticular membrane from fossil leaves with thin and fragile cuticles

Abstract

Leaf cuticle analysis has long been a powerful tool for fossil plant identification, systematics, and palaeoclimatological reconstruction. In recent decades the application of stomatal frequency data that are relied on precise calculation of stomata on plant fossil cuticles to reconstruct ancient atmospheric CO 2 concentration made the preparation of cuticular membrane with sufficient size a critical technique in palaeoclimatological research. However, for plants with originally thin and fragile cuticles, e.g., most deciduous plants, conventional techniques sometimes fail to obtain cuticular membranes with sufficient size, or sometimes unable to recover any. This has largely hampered the usage of fossil cuticle analysis in palaeobotanical and palaeoclimatological research. Here, we describe a new method using clear nail polish as a medium to "strengthen" the originally thin and fragile cuticles prior to maceration procedures. We demonstrate the method by using middle Eocene Metasequoia fossils that were notorious for the difficulty of recovering large-sized clean cuticular membranes due to their thin and fragile nature. Metasequoia, with well-documented and widely-distributed fossil records since the Late Cretaceous and with a living representative, M. glyptostroboides, as a comparative reference, has been widely used as a model genus for the study of evolution of plants, palaeoclimatological reconstruction, and plant adaptation to climate changes. But its deciduous habit produces thin cuticles and makes the preparation of clean cuticular membranes a tedious process. The new method successfully allows us to recover its delicate cuticular membranes with sufficient sizes for SEM observation and stomatal frequency analysis.fossil leaf cuticle, Metasequoia, new method, thin and fragile cuticle, SEM observation, stomatal frequency, CO 2 Citation:Wang L, Leng Q. A new method to prepare clean cuticular membrane from fossil leaves with thin and fragile cuticles.Terrestrial plant leaves are covered by cuticles which are composed of resistant chemicals of cutin and sometimes cutan [1][2][3][4]. Due to this layer of protection, plant leaf fossils are resistant to post-depositional decay, and sometimes are the only organs of ancient plants found in terrestrial sediments. As cuticles are tightly adhered to epidermal cells, many epidermal information including cell type (e.g., guard cells, subsidiary cells, ordinary epidermal cells), size, shape, orientation, and arrangement, can be obtained from cuticular membranes. Furthermore, as the barrier between the interior of plants and its external environment, epidermal cells and their cuticle coverage record unique information regarding plant's physiological responses to climate changes. All these make cuticle analysis one of the most widely used techniques in palaeobotanical and palaeoclimatological research. In addition, since the demonstration of the correlation between plant leaf stomata and the ambient CO 2 concentration [5], the past decades have witnessed a remarkable

Historical Distribution of Metasequoia Referenced to Paleoclimate Factors

“…Metasequoia as a "living fossil" is now ascribed to the sub fa mily of Sequoioideae (Cupressaceae) (Gadek et al 2000) and characterized as deciduous, monoecious tree; trunk is mo no podial with branches irregularly whorled; leaves are linear, flat, decussate, with basic parts twisted into two co lumns and apical parts obtuse; petioles are absent; stomata on the lower surface of leaves are generally parallel to the midrib; male inflorescence is racemose or paniculate; male cone is ovoid with a short pedicel; female cone is generally borne singly at the end of a leafy twig with a pedicel; seed scales are decussate; seeds are flat surrounded by wings (Liu et al 1996, Farjon 2010, Wang & Leng 2011.…”

Constraints on Atmospheric CO2 Obtained from Fossil Metasequoia for the Middle Eocene Polar Forests on Axel Heiberg Island, Nunavut, Canada

“…Following Eberle and Storer (1999) and Harrison et al (1999) many studies have adopted an age of "~45 Ma" for Napartulik (e.g., Jahren, 2007;Jahren et al, 2009;Jahren and Sternberg, 2008;Wang and Leng, 2011;Yang et al, 2005). This age is near the old end of all published age ranges for the site, and is therefore somewhat misleading.…”

“…However, obtaining cellular detail from the cuticle has proven difficult (Liu and Basinger, 2009;Wang and Leng, 2011;Royer and LePage, pers. comm., 2012) because it is thin and fragile.…”

“…The method presented here incorporates elements from both of these studies, but is simplified and appears to yield better cellular detail (seeFigure 3.1 for comparison). This method does not involve splitting the cuticle, but rather clearing the top of the leaf surface in order to view features of the cuticle with the reflected light technique of epifluorescence microscopy (compared with SEM used in bothBasinger, 2009 andWang andLeng, 2011).…”

“…Comparison of cuticle preparations from Napartulik Metasequoia using different methodologies. A. SEM image of cuticle prep presented inWang and Leng (2011), scale bar = 30µm. B. and E. Cleared leaf surfaces of Metasequoia from fossil forest layer C at Napartulik, obtained using method described here, scale bars = 50µm.…”