Methods: quantifying color

LEICA EX4 HD settings/guidelines for carapace and tergite color measurements:

• • photos are taken in completely dark room

  • 8x magnification

  • all upper microscope lights on, plate microscope light off

    • microscope light level 4/10 (4th light level of a range of 10)

  • specimen submerged in clean EtOH (70% or 95%)

  • white paper placed under specimen

  • photograph specimens dorsally

    • for prosomal photos, specimen is placed longitudinally with tip of chelicera at top of image field and 3 tergite at lower image field limit (as in image below)

    • for mesosomal photos, specimen is placed longitudinally with tergite 4-7 in image field (as in image below)

Addendum*

-Patella III

-flatten dorsoventrally

-light level 4/10

-16x magnification

Settings:

Live Video:

-auto: off

-Mode: 1024 x 768

-Exposure: 70ms

-Gain: 7.0x

-Brightness: 50%

Sharpen: 50

Color:

-auto: off

-center color diagram*

-Saturation: 100%

Levels:

-auto: off

-0/1.0/255

Output:

-Image size: 2048 x 1536

Measurements with ImageJ:

• • measurements set to 1) area, 2) standard deviation, 3) mean gray value

  • I use the polygon tool to create an inner outline of the segment to be measured

    • I chose inner as this is still likely to capture the necessary range of color on the specimen and will be less likely to show deviations due to white background, intersegmental membrane, or other segments.

  • *repeatability of both values from repeated photos and repeated measurements with the polygon tool indicated consistency using the aforementioned guidelines and settings. See data below