Dieter DEMSKE1*, Pavel TARASOV1 and Takeshi NAKAGAWA2 (December 2010)
1 Institute of Geological Sciences, Freie Universität Berlin, Germany
2 Department of Geography, University of Newcastle-upon-Tyne, UK
* Corresponding author: demske@zedat.fu-berlin.de
Introduction
The superb preservation of pollen, spores and other palynomorphs in the finely laminated sediments of Lake Suigetsu and the wish to appropriately document these fossil objects provided the incentive to enhance quantitative analysis – the count of pollen and spore types through time aiming at the reconstruction of palaeoenvironment and past climate – by a qualitative approach – i.e. the photographical record of individual fossil objects. This work is in progress, while here the first part is presented starting with pollen images representing members of the eudicotylonean group.
Materials and methods
The available material comprises sediment samples from deposits dated to the late Quaternary (“upper Laschamp section”), the late Last Glacial and the present interglacial (Holocene) as well as mixed test samples used for cross-calibration of counts from within the three above mentioned intervals. Preparation of the samples was accomplished using the Suigetsu standard procedure, which follows the method described in Nakagawa et al. (1998) with slight modifications. Samples are stained with safranine and kept refrigerated in glycerol. For analysis under the transmission light microscope, 10 μl of the palynomorph suspension were evenly spread under a 12x12 mm² cover glass.
The optical equipment comprised a Meiji microscope with lenses SPlan 40x/n.a. 0.65 Phaco, Plan 60x/n.a. 0.80 and Plan 100x/n.a. 1.25 as well as a digital camera mounted on the trinocular. The image capturing software provided 1280x1024 megapixel files in bitmap format (*.bmp) with a colour depth of 3x8 bit (RGB). Pixel-based calibration of the images, showing a scale bar of 100 μm/10, is related to a Carl Zeiss Jena object micrometer with a 1 mm/100 scale in correspondence to the different magnification rates of lenses.
Optical analysis and determination
Characteristic features of most objects are elucidated using the LO method (Erdtmann 1952), which is based on the principle that the limited microscopical depth of focus provides optical slices of three dimensional objects with brightness patterns related to structure and sculpturing (see Fig. 1 for details). On the photo plates series are shown as image sequences from high to low focus, subsequently revealing details about 1) the ornamentation with sculpture elements (e.g. echinae), exine surface pattern (e.g. scabra, perforations or surface of muri in reticulate types), inner exine structure (pattern and cross-section of columellae), 2) the apertures with margins, exoaperture, endoaperture and their features (e.g. scabrate colpus membranes or costae colpi), 3) the outline and exine stratification in equatorial or polar view. Determinations were mainly based on Beug (2004), Moore, Webb and Collinson (1991), Nakamura (1980a, 1980b), The Northwest European Pollen Flora I-IX (ed. by Punt et al., 1976) and Shimakura (1973). Determined taxa are grouped according to plant families in alphabetical order within the main systematic groups following Smith et al. (2006) and APG III (2009).
File handling and imaging
For appropriate handling of image data all saved files were named including 1) a running 4-digit object number followed by 2) a small letter (a, b, c etc.) marking the sequence of images through LO analysis (from high to low focus), 3) sample name and number, 4) magnification and numerical aperture of the lens, and 5) a provisional name of the determined object.
For documentation all original image files (bmp format) are stored in order of their file numbers. In a separate directory the files are systematically ordered according to plant families. Following a check of file names and quality the best images showing characteristic details were selected for assembling the photo plates.
For imaging with the computer the original files (BMP format, 1280x1024 MP, 3x8 bit RGB) were imported into the Adobe Photoshop CS4 software as individual layers. For optimum appearance and coordination of colours and brightness between images the Photoshop files (PSD format) were organized on a white background layer at a resolution of 300 dpi using the multi-layer format. Individual images were masked to appropriate size and enhanced by non-destructive adjustment layers including Levels (Tonwertkorrektur) and, only were appropriate, Exposure, Colour balance and Gradation Curve. Subsequently, black and white conversion (option maximum white) was applied to image and their adjustment layers, with figure numbers, scale bars (10 μm) and the text box (figure captions) being formatted as overlying layers. Finally the plates were exported to grey-scale files (TIFF format, 8 bit/256 levels of grey).
Plate format
The photo plates were conveniently formatted according to instructions required by the Elsevier journal Reviews of Palaeobotany and Palynology, i.e. with a size of 184 mm x 244 mm at 300 dpi and an RGB colour depth of 3x8 bit. A spacing of 2 mm was chosen to separate individual images between themselves and from plate edges. Scale bars are of 1 mm thickness with the text in 8 pt. Arial font, whereas numbering in 5 mm circles and the textbox content are written at 10 pt. Before adding the textbox the page format was extended to 210 mm x 297 mm (A4), with visible page margins of 15 mm.
The effective magnifications of images viewed at 300 dpi are dependent on the used lens and correspond to pixel-size calibration: 516x (with 10 μm in 61 pixel for 40x lens), 787x (with 10 μm in 93 pixel for 60x lens) and 1322x (with 10 μm in 156 pixel for 100x lens). In the plates only 10 μm scale bars are shown as magnification rates vary with zoom viewing.
Numbering of the plates starts with I within the systematic main group (see above), here the “Angiospermae – Rosidae (eudicots)”. Within plates the numbering of images follows the order of objects and the optical LO-sequence, with a scale bar appearing in the last (or only) image related to identical objects. Figure captions for the plates are kept in short, while details of the documentation are summarized in a separate table including data about corresponding sediment samples and file names.
References
APG III, 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III [THE ANGIOSPERM PHYLOGENY GROUP]. Botanical Journal of the Linnean Society 161, 105–121.
Beug, H.-J., 2004. Leitfaden der Pollenbestimmung: für Mitteleuropa und angrenzende Gebiete. Pfeil, München, 542 pp.
Erdtman, G., 1952. Pollen Morphology and Plant Taxonomy. Angiosperms. Almqvist and Wiksell, Stockholm, 539 pp.
Moore, P.D., Webb, J.A., Collinson, M.E., 1991. Pollen Analysis. 2nd ed. (reprint 1999). Blackwell Science, Oxford, 216 pp.
Nakagawa, T., Brugiapaglia, E., Digerfeldt, G., Reille, M., de Beaulieu, J.-L., Yasuda, Y., 1998. Densemedia separation as a more efficient pollen extraction method for use with organic sediment/deposit samples: comparison with the conventional method. Boreas 27, pp. 15-24.
Nakamura, J., 1980a. Diagnostic characters of pollen grains of Japan, Part I. Special Publications from the Osaka Museum of Natural History 13, pp. 1–91 (in Japanese with English abstract).
Nakamura, J., 1980b. Diagnostic characters of pollen grains of Japan, Part II. Special Publications from the Osaka Museum of Natural History 12, pp. 1–157 (in Japanese with English abstract).
Punt et al. (eds.), 1976. The Northwest European Pollen Flora, I-IX. Elsevier, Amsterdam.
Shimakura, M., 1973. Palynomorphs of Japaneses plants. Special Publications from the Osaka Museum of Natural History 5, pp. 1–60 (in Japanese with English abstract).
Smith, A.R., Pryer, K.M., Schuettpelz, E., Korall, P., Schneider, H., Wolf, P.G., 2006. A classification for extant ferns. Taxon 55 (3), 705–731.