Digitisation
We digitised images using geometric morphometrics to create
two-dimensional anatomical co-ordinates (reference points, called
‘landmarks’) that characterized body shape. First, lateral-view
photographs were uniformly orientated (head at the top) and prepared for
landmarking in Microsoft Paint. This involved drawing a vertical line
from the eye to the base of the tail (a standard measure of daphnid
length) and then two perpendicular lines, one at the midpoint of the
original line and another intersecting the rostrum. These lines provided
a clear method to capture the approximate location of the dorsal and
ventral midpoints, as well as the region on the back of the neck where
defences were induced, consistently between different images (Fig. 2A).
This process identified a total of six landmarks for each photo
including (1) the centre of the eye (near the top of the head), (2) the
neckteeth defence (or the corresponding area in the controls), (3) the
base of the tail, (4) the dorsal (back) midpoint, (5) the ventral
(belly) midpoint and (6) the rostrum (snout). These landmarks were
motivated by a method developed for linear dimensions in two species ofDaphnia , D. dentifera and D. mendotae and were
selected to capture key aspects of the defence, head and body shape.
Images were then digitised by applying landmarks using thegeomorph package v4.0.5 in R v4.2.1 . Before these images were
analysed in R, they were standardised using generalised Procrustes
superimposition from the geomorph package v4.0.5 . This process
aligned the images to account for differences in the size, position and
orientation of the original specimen so that shape could be compared in
a meaningful way. The resulting Procrustes shape coordinates (Fig. 2D)
were used in the subsequent analyses.
The amount of human (intra-observer) error in selecting landmarks was
measured using two sets of identical photographs. The same landmarks
were applied to 30 photographs of either the defended or undefended
morph, and the standard error of each landmark was measured from the
Procrustes shape coordinates.