Computational protocol: Charcoal morphometry for paleoecological analysis: The effects of fuel type and transportation on morphological parameters1

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[…] Specimens of 26 plant materials () were obtained from 14 species, consisting of two pteridophytes, eight conifers, two grasses, and two other angiosperms—one weedy and one arborescent. Species were selected with the aim of including a broad range of physical forms and are weighted toward those with a long geological record, because sedimentary charcoal may be found dating back to the Paleozoic era (). Conifers were of particular interest to us, because of their long geological history and because they were not included in the experiments of . In most cases, both foliage and stems or branches were sampled. Native species were sampled from locations in southwestern England and northern Wales, and exotic species from the botanical collection at the University of Exeter. Specimens were dried to a constant weight at 50°C before samples were removed. Samples generally consisted of 1-cm lengths of stems, twigs, or long narrow leaves, or 1 × 1-cm squares of broad leaves. The morphology of the specimen determined the exact size and shape of the samples removed. These are given in detail in Appendix S1.Samples were tightly wrapped in aluminum foil and placed in batches of eight in 75-mL stainless steel crucibles, which were then filled with clean mineral sand of grain size ≤500 µm to exclude oxygen. The crucibles were placed in the center of a Carbolite GLM3 furnace (Carbolite Ltd., Hope Valley, United Kingdom) at 550°C for 20 min, during which time the temperature remained within the range 547–553°C, before being removed from the furnace to cool to room temperature. This produced samples of pure charcoal (), with no material left uncharred, and with only very slight ash production at the edges of some samples. Noncharcoalified material may have remained at the center of some woody samples.Each charcoal sample (mass 0.0008–0.1068 g; mean = 0.0191 g; σ = 0.0253 g) was placed in a 40-mL polypropylene tube (30 × 70 mm) with a polyethylene screw-cap. Approximately 10 g (9.71–10.36 g; mean = 9.96 g; σ = 0.10 g) of silicate gravel (mass 0.07–1.02 g; mean = 0.33 g; s = 0.17 g) was added, and the tube filled with tap water (). Sample tubes were affixed to an electric motor, at 10 cm from the axis of rotation and aligned tangential to the direction of rotation, and turned over at 47 revolutions per minute for periods of between one and eight hours. The speed of rotation is arbitrary, but low enough to avoid any inertial displacement of the contents of the tube. Samples were sieved at 125 μm, and the gravel removed. The charcoal particles retained on the sieve were dispersed in water in 55-mm Petri dishes and left at room temperature for the water to evaporate ().The particles were then imaged using a dissecting microscope and Tucsen ISH500 digital camera with View version imaging software (Xintu Photonics Co. Ltd., Fuzhou, Fujian, China). Where the original woody particle remained largely intact, this was removed prior to imaging. An area of 16 cm2 was photographed as a series of 16 overlapping images, using transmitted light, and the images saved in tagged image file format (TIFF).Images were processed using ImageJ 1.47t (; ). Each image consisted of a 1 × 1-cm square, and adjacent areas overlapping with other images from that sample. Most images contained some areas in which particle morphology was obscured, either by the density of the particles causing them to touch or overlap, or in some cases due to other material being present in the sample, or due to faults with the image itself. A region of interest, in which no distorted particle images were apparent, was therefore defined within each 1 × 1-cm square, and the remainder of the image deleted. The edited images were converted to 8-bit grayscale, and then binarized using the default IsoData algorithm (), adjusting the maximum threshold value manually to distinguish the charcoal particles, and with the minimum threshold value set at 0. A series of shape descriptors were generated for all the resulting particle images. These included projected area, Feret diameter (defined as the longest straight line obtainable within the particle image), circularity, and aspect ratio.Circularity was calculated according to the following formula, which results in values between 0 and 1, where 1 is a perfect circle and 0 an infinitely elongated polygon.Circularity=4π×AreaPerimeter2Aspect ratio was calculated as the ratio of the major and minor axes of the best-fitting ellipse.Aspect Ratio=major axisminor axisParticles of less than 315 μm2 or greater than 1,000,000 μm2 were excluded from the analysis. The lower limit serves to remove data derived from images of between one and nine pixels, from which meaningful information is unlikely to be obtained even for the most basic parameter of area (). It is also likely that images of this size would not have been easily visible during selection and thresholding, and they may not represent actual charcoal particles. The upper limit, which approximately coincides with the distinction between mesocharcoal and macrocharcoal as defined by , is essentially arbitrary. Particles at the high end of the size distribution were not present in sufficient numbers to produce statistically meaningful data, and their morphology may largely reflect the size and shape of the original sample cut, rather than the effects of internal structure and breakdown regime with which we are concerned.All statistical tests were conducted using SPSS version 21.0 (). […]

Pipeline specifications

Software tools ImageJ, ISODATA
Application Microscopic phenotype analysis