Protocol

Author

Dana Royer

OVERVIEW

This protocol outlines how to quantify leaf shape and area using Photoshop and ImageJ, with particular advice regarding leaf margins and a standard definition of how to define lobes vs. teeth.

BACKGROUND

The sizes and shapes (physiognomy) of fossil leaves are frequently applied as proxies for paleoclimatic and paleoecological variables. Many physiognomic variables are closely related to: mean annual temperature (MAT), mean annual precipitation (MAP), leaf mass per area and nitrogen content, making these features very useful in the quantification of relationships between physiognomy and many leaf ecological variables.

Digital leaf physiognomy is a recently developed technique which allows for sophisticated measurements of leaf shape, size, margin characteristics etc. to a higher degree of accuracy than was previously possible.

MATERIALS/EQUIPMENT

• Digital camera or scanner
• Software: Photoshop and ImageJ

UNITS, TERMS, DEFINITIONS

Lobe

PROCEDURE

Let’s go DiLPing!

Use a digital camera or scanner to capture leaf images. Ensure you include a scale or scaled object in each image. While the image quality for most flatbed scanners is adequate, they typically introduce small shadows that require a large amount of post-processing, especially for leaves with small teeth. To eliminate shadows with a digital camera, use black velvet as a background or a light box.

In Photoshop:

1. Make a copy of the leaf image and paste it into a new layer. Don’t touch the original background layer (for archiving purposes). (Note: ‘layers’ refer to the way in which Photoshop allows you to work in the x-y plane as well as build up ‘layers’ in the x-axis.)
2. Isolate, move, and clean-up petiole (if present)
3. Correct imperfections in margins, including any remaining shadows. Make sure there is sufficient contrast between the leaf and the background.
   If leaf has teeth:
   • Enlarge canvas size (~ x2)
   • Highlight leaf only, create new layer, and move new leaf to a clean part of the canvas
   • Select teethª  (polygonal lasso tool) and detach them from the leaf blade (for demonstrative image, see Fig. S3 – Appendix S1 Royer et al. (2005) American Journal of Botany)
   • Correct any imperfections in margins (again) in new toothless leaf
   • Crop image (if appropriate, to save file space)
4. Save file (-save as’…)

In Image-J:

Setting up Image-J for the first time:

• Analyze set measurements: Uncheck all checked boxes. Then check, in this order (yes, order will matter), area, circularity, perimeter, and Feret’s diameter.
• If you wish, you can change the color of lines that you draw in image-J (Edit options color)

To set scale:

1. Select line tool, make line on ruler
2. Under “Analyze”, select “Set scale…”
3. Enter “known distance” (units don’t matter). (Based on scaled object included in each image)

To select an area:

1. Select Image Type 8-bit, image goes grayscale (you only need to do this step once per image file)
2. Select Image Adjust Threshold, adjust scales so leaf goes red (this is the critical step; don’t worry about holes inside the shape)

Measure:

1. Select magic wand tool, click on red leaf
2. Select Analyze Measure (ctl+m); paste appropriate data into spreadsheet

ª Tooth selection rules

(reproduced from Appendix S1 Royer et al. (2005) American Journal of Botany)

• Lobe vs. tooth rule
  Definition of “lobe”: find the axis of symmetry of the indentation, and project a line (d) along this axis from the apex of the indentation to the midvein. Typically, the axis follows the trend of the feeder vein for the indentation. Project a perpendicular (p) from the apical sinus of the indentation to d. If the distance from the apex to p is greater than 0.25d, the feature is a lobe.
  It is possible that this definition may lead to some large indentations being selected as teeth, for example leaves with compound lobes (e.g., species of Crataegus).

Lobe vs. tooth rule – Fig S1 from Royer et al. 2005. Quercus rubra leaf illustrating the lobe vs. tooth rule. Line segments p and d are defined above in text. Scale bars = 1 cm.

For further definitions and similar rules (Differentiating secondary from primary teeth, Pinnate lobe rule, Lobe priority rule, Solitary tooth rule, Primary vein rule), refer to “APPENDIX S1. Revisions to tooth selection protocols of Huff et al. (2003).” of Royer et al. (2005). ”Am. J. Bot. 92(7): 1141-1151.

Additional tooth selection rules are included in Huff et al. (2003).

NOTES AND TROUBLESHOOTING TIPS

Recommended order for measuring:

1. petiole (only area needed)
2. full leaf (all data needed)
3. leaf without teeth (perimeter needed -internal perimeter’; also use area to compute tooth area (full area – toothless area = tooth area)
4. I count tooth number by hand in Photoshop

Note: feret diameter in Image-J is the longest distance between any two pixels along the shape perimeter. Sometimes this is called ‘major axis length’.

Example spreadsheet for data collection and analysis

Note: orange columns are the core data you need to paste in; all other columns are calculations.

Download File

LITERATURE REFERENCES

Royer, D.L., Wilf, P., Janesko, D.A., Kowalski, E.A., and Dilcher, D.L. (2005) Correlations of digital leaf physiognomy to climatic and ecological variables: potential proxies for the fossil record. American Journal of Botany 92(7): 1141-1151.

Additional leaf tooth selection rules are included in:

Huff, P.M., Wilf, P. and Azumah, E.J. (2003) Digital Future for Paleoclimate Estimation from Fossil Leaves Preliminary Results. PALAIOS 18: 266-274. 0883-1351/03/0018-0000

Other:

Royer, D.L. and Wilf, P. (2006) Why do toothed leaves correlate with cold climates Gas exchange at leaf margins provides new insights into a classic paleotemperature proxy. Int. J. Plant Sci. 167(1):11-18. 1058-5893/2006/16701-0002

SEARCH TERMS AND CLASSIFICATION

Similar protocol: Measuring leaf perimeter and leaf area