Visualisation of calcium oxalate crystal macropatterns in plant leaves using an improved fast preparation method.

Leaves of the majority of plants contain calcium oxalate (CaOx) crystals or druses which often occur in spectacular distribution patterns. Numerous studies on CaOx in plant tissues across many different plant groups have been published, since it can be visualised readily under a light microscope (LM). However, there is surprisingly limited knowledge on the actual, precise distribution of CaOx in the leaves of quite ordinary plants such as common native and exotic trees. Traditional sample preparation for the documentation of the distribution of CaOx crystals in a given sample - including overall distribution - requires time-consuming clearing procedures. Here we present a refined fast preparation method to visualise the overall CaOx complement in a sample: The plant material is ashed and the ash viewed under the polarising microscope. This is a rapid method which overcomes many shortcomings of other methods and permits the visualisation of the entire CaOx content in most leaf samples. Pros and cons in comparison with the conventional clearing technique are discussed. Further aspects for CaOx investigations by micro-CT and scanning electron microscopy are discussed.

Traces of calcium oxalate biomineralization in fossil leaves from late Oligocene maar deposits from Germany.

Calcium oxalate (CaOx) is one of the most common bio-mineral in extant plants and is believed to serve a variety of functions such as calcium storage and herbivore defense. However, traces of CaOx crystals have rarely been identified in fossil plants, and they are primarily known from fossil gymnosperms, where empty cavities of former CaOx crystals or ghost crystals have been reported from leaf cuticles of some Late Cretaceous and Cenozoic conifers. Here we investigate fossil angiosperm leaves from the late Oligocene Rott Fossil Lagerstätte and report ghost crystals of various shapes, sizes and topology (distribution patterns), and cavities. These micromorphological structures of fossil leaves are compared to CaOx deposits in leaves of extant plants: globular structures in fossil leaves resemble CaOx druses (crystal aggregates) in fresh leaves in size and distribution; and angular or brick-shaped structures in the vascular system of fossil leaves closely resemble prismatic CaOx crystals in the vascular system of extant leaves in both size and topology. Chemically, CaOx druses have survived fossilization as cavities only, and were replaced by organic matter and ghost minerals containing Ca, Si, Al, S, and Fe. The identification of former CaOx remains in leaf fossils provides novel insights on the fate of plant bio-minerals during fossilization. More importantly, it provides an additional aspect of the ecophysiology of fossil plants thus improving the accuracy of palaeoecological reconstructions and can provide a broader perspective on the evolution of CaOx and their rule in plant ecology across geological timescales. Alternative interpretations of the fossil microstructures are discussed but ruled out.