To begin, open ImageJ software and set the parameters for automatic measurements. Navigate to Analyze and select Set Measurements. Then select the options for area, perimeter, and Feret's diameter.
Now open the prepared fossil leaf image by selecting File and Open. Use the straight line tool to zoom in on the scale bar and draw the longest straight line possible across it. To set the scale of the image, click Analyze and Set Scale.
Enter the measured length of the scale bar in centimeters and click OK.In the data entry spreadsheet, mark the leaf as zero for toothed or one for entire. Also measure the petiole width if present And record the data in the data entry spreadsheet. Then select Image, Type, and 8-bit to convert the leaf image to black and white.
Navigate to Image, Adjust, and Threshold to begin thresholding the image. Using the slider bar, adjust the threshold until the interior of the leaf turns red and is distinctly separate from the background. Zoom in on sections of the margin to ensure accuracy.
To measure the leaf prepared for leaf area and shape measurements with the petiole removed, use the wand tool and click on the interior of the leaf to outline it in yellow, ensuring the outline correctly represents the leaf shape. Make measurements using Analyze and Measure or using the designated keyboard shortcut and record the data in the spreadsheet. For a half-leaf analysis, select the segmented line tool in the line tool and trace the entire length of the artificial middle perimeter.
Then click Analyze and Measure or use the shortcut to measure the length of the artificial middle perimeter. Record area, perimeter, Feret, minimum Feret, and the length of the artificial middle perimeter. To adjust the measurements for a half-leaf in the data entry spreadsheet, multiply the area and minimum Feret by two and calculate the blade perimeter using the given formula.
Measure the area of the cut-out petiole if present and record its area. To measure the raw blade, use the wand tool to select the interior of the blade outlined in yellow. Proceed with measurements by clicking Analyze and Measure or using the keyboard shortcut.
In the data entry spreadsheet, record the raw blade's area and perimeter in the designated fields. To measure the internal raw blade, select the interior of the blade and outline the entire leaf in yellow. Make measurements by clicking Analyze and Measure or using the shortcut.
Record the area and perimeter of the internal raw blade in the data entry spreadsheet under the respective fields. Clear the threshold to see the leaf clearly by clicking Reset in the Threshold box or using Edit and Undo to remove modifications like the black and white conversion. Then use the segmented line tool to trace the full length of the cut perimeter.
Measure the traced perimeter by clicking Analyze and Measure or using the shortcut. To count the number of primary teeth, right-click the point tool first to select the multi-point tool. Click on each primary tooth to number it.
To remove a point selected by mistake, press the Alt key or Command or CMD or Option while clicking the point. Record the final number under primary teeth in the data entry spreadsheet. Click Edit, Selection, and Select None to clear the multi-point tool counts and annotations.
Then, to count the total number of teeth, right-click the point tool and select the multi-point tool. Click on each tooth, including primary and subsidiary, to number it. Both McAbee H1 and H2 sites showed leaf physiognomy that fits well within the calibration dataset's range, indicating reliability in the derived paleoclimate estimates.
The reconstructed mean annual temperature and mean annual precipitation for McAbee H1 and H2 suggested a temperate seasonal biome.
