MATERIAL AND METHODS

The fossils presented here were collected from exposed vertical banks of the Puerto Viejo river, on the northeastern edge of the La Selva Biological Station. The specific locality is called Site PV1 and was under clay-rich volcanic tephra up to 1.4 m thick overlain by 11.8 m of alluvium, colluvium, and modern soil. Radiocarbon analyses of wood from the deposit, and ⁴⁰Ar/³⁹Ar analysis of plagioclase in the tephra, indicate that the flora is Pleistocene in age (older than 46 ka but younger than 915 ka; Horn et al. 2003). Sally Horn and Robert Sanford collected fossils and bulk clay samples in August 1992 from which the fossils were isolated. The sedimentology and dating of this site is presented in Horn et al. (2003), along with initial macrofossil and microfossil determinations. The present physiognomy of the site is a tropical wet forest in the Atlantic lowlands, about 35 m above sea level.

The bulk clay samples were washed at the Paleobotany and Palynology Laboratory, Florida Museum of Natural History, University of Florida using commercial detergent to separate the leaves and remove debris. The isolated organic material was soaked in hydrofluoric acid (HF) to remove silicates, and then thoroughly washed with filtered water. Specimens include fragmentary leaves, cuticle, wood, fruits, and seeds. They were photographed with a Nikon 35 mm (Melville, New York, USA) and a Zeiss 35 mm camera (Thornwood, New York, USA). Specimens were also imaged using a Nikon Coolpix990 and Zeiss AxioCam camera. Square sections (1.0 cm x 1.0 cm) were cut from the middle of the leaves, and these sections were soaked in Jefferey's solution (50% Nitric acid/ 50% of 10% Chromic acid) to remove the mesophyll. An insect pin mounted in a wooden probe was used to tease apart the upper and lower cuticle, and then a small paint brush with 2-3 bristles was used to gently clean away the mesophyll. The cuticle then went through an alcohol dehydration series of 50%, 95%, and 100% ethanol, stained with 1% Safranin–O in 100% ethanol, then through a series of 100% ethanol, 10%Etoh/90%HemoDe (or Citrisolv), and then 100% HemoDe (or Citrisolv [Fisher Scientific, Pittsburgh, Pennsylvania]). The cuticle was then mounted in a drop of HSR (synthetic resin) in HemoDe (or Citrisolv) on a microscope slide, placed on a slide warmer with a lead weight over the cover slip, and later sealed with fingernail polish. Over 100 cuticle and mesofossil slides were prepared, and the cuticle of over 100 modern leaf species were also prepared and examined. All cuticle parameters were determined using the Zeiss KS400, Version 3.0, 1999 imaging system. Five different and randomized areas within the alveoles were measured for epidermal cell density (ED [n/mm²]) and stomatal density (SD [n/mm²]). The stomatal density was then calculated using the Stomatal Index formula of SI = [stomatal density/(stomatal density + epidermal cell density)] × 100 (Salisbury 1927).

The fossil material is numbered with the University of Florida acronym (UF) followed by the locality number and then the specimen number (e.g., UF18882–32685). Modern leaves used for comparisons are numbered in the University of Florida Modern Leaf Reference Collection according to a consecutive numbering system (e.g., UF5437). Modern leaf specimens were examined from the herbaria of the University of Florida (FLAS), Smithsonian Institution (SI), Missouri Botanical Garden (MO), and the Modern Reference Collection of the Paleobotany and Palynology Laboratory (prepared cuticle slides of nearly 5,000 species). All sorted and unsorted fossil samples are stored in the Florida Museum of Natural History Paleobotany/Palynology Collection. Systematic description and terminology follows Dilcher (1974) and Ellis et al. (2009), and classification follows APG III (2009) and Judd et al. (2002). Terminology for stomates is as follows: the term stomata includes stomatal aperture plus guard cells, while stomatal complex includes stomatal aperture plus guard cells plus subsidiary cells (Dilcher 1974; Ellis et al. 2009).