Improved non-destructive 2D and 3D X-ray imaging of leaf venation.

BACKGROUND: Leaf venation traits are important for many research fields such as systematics and evolutionary biology, plant physiology, climate change, and paleoecology. In spite of an increasing demand for vein trait data, studies are often still data-limited because the development of methods that allow rapid generation of large sets of vein data has lagged behind. Recently, non-destructive X-ray technology has proven useful as an alternative to traditional slow and destructive chemical-based methods. Non-destructive techniques more readily allow the use of herbarium specimens, which provide an invaluable but underexploited resource of vein data and related environmental information. The utility of 2D X-ray technology and microfocus X-ray computed tomography, however, has been compromised by insufficient image resolution. Here, we advanced X-ray technology by increasing image resolution and throughput without the application of contrast agents. RESULTS: For 2D contact microradiography, we developed a method which allowed us to achieve image resolutions of up to 7 µm, i.e. a 3.6-fold increase compared to the industrial standard (25 µm resolution). Vein tracing was further optimized with our image processing standards that were specifically adjusted for different types of leaf structure and the needs of higher imaging throughput. Based on a test dataset, in 91% of the samples the 7 µm approach led to a significant improvement in estimations of minor vein density compared to the industrial standard. Using microfocus X-ray computed tomography, very high-resolution images were obtained from a virtual 3D-2D transformation process, which was superior to that of 3D images. CONCLUSIONS: Our 2D X-ray method with a significantly improved resolution advances rapid non-destructive bulk scanning at a quality that in many cases is sufficient to determine key venation traits. Together with our high-resolution microfocus X-ray computed tomography method, both non-destructive approaches will help in vein trait data mining from museum collections, which provide an underexploited resource of historical and recent data on environmental and evolutionary change. In spite of the significant increase in effective image resolution, a combination of high-throughput and full visibility of the vein network (including the smallest veins and their connectivity) remains challenging, however.

Water supply and demand remain coordinated during breakdown of the global scaling relationship between leaf size and major vein density.

Vein networks that disobey the global scaling of major vein density with leaf size shed light on functional constraints of vein network formation in dicotyledons. Understanding their evolution, distribution and impact on vein-stomata-climate associations is an important contribution to our global view of vein network organization. Based on vein traits of 55 species of pantropical Ochnaceae, stomata and climatic niche data, and a dated molecular phylogeny, we unveil major structural shifts in vein networks through deep time, relationships between leaf size, vein and stomata traits, and their interplay with climate. Dense 2° veins, reduction of minor veins and the associated breakdown of vein-leaf size scaling evolved multiple times independently in Ochnaceae. In spite of the drastic changes in vein architecture in this venation type, vein and stomatal densities remain correlated. Our study demonstrates that shortening the major vein-stomata distance is economically not less advantageous than by increasing minor vein density, as illustrated by the same degree of coordination between vein and stomatal densities and the similar construction costs across networks with dense 2° veins and those with 'normally' spaced 2° veins.

Chemical, experimental, and morphological evidence for diagenetically altered melanin in exceptionally preserved fossils.

In living organisms, color patterns, behavior, and ecology are closely linked. Thus, detection of fossil pigments may permit inferences about important aspects of ancient animal ecology and evolution. Melanin-bearing melanosomes were suggested to preserve as organic residues in exceptionally preserved fossils, retaining distinct morphology that is associated with aspects of original color patterns. Nevertheless, these oblong and spherical structures have also been identified as fossilized bacteria. To date, chemical studies have not directly considered the effects of diagenesis on melanin preservation, and how this may influence its identification. Here we use time-of-flight secondary ion mass spectrometry to identify and chemically characterize melanin in a diverse sample of previously unstudied extant and fossil taxa, including fossils with notably different diagenetic histories and geologic ages. We document signatures consistent with melanin preservation in fossils ranging from feathers, to mammals, to amphibians. Using principal component analyses, we characterize putative mixtures of eumelanin and phaeomelanin in both fossil and extant samples. Surprisingly, both extant and fossil amphibians generally exhibit melanosomes with a mixed eumelanin/phaeomelanin composition rather than pure eumelanin, as assumed previously. We argue that experimental maturation of modern melanin samples replicates diagenetic chemical alteration of melanin observed in fossils. This refutes the hypothesis that such fossil microbodies could be bacteria, and demonstrates that melanin is widely responsible for the organic soft tissue outlines in vertebrates found at exceptional fossil localities, thus allowing for the reconstruction of certain aspects of original pigment patterns.