Computational protocol: Sexual and Natural Selection Both Influence Male Genital Evolution

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Protocol publication

[…] Male genitalia were separated from the abdomen and soaked in a drop of 50∶50 glycerol and lactic acid for 60 minutes, which softens and clears the tissues. The genital arch is a delicate, paired structure that is prone to damage during dissection. Therefore, the intact genital arch (be it the left or right) was oriented consistently and mounted using Hoyer’s solution. In addition, one non-sexual trait, the left or right hind tibia was randomly selected and removed from the thorax and mounted using Hoyer’s solution also. Digital images of the genitalia and hind tibia were captured using a Leica M125 microscope with mounted camera that conveyed images to a PC. Two measures of the length of the hind tibia, (which was used as an index of body size) was measured using Image J. The repeatability of the measurement is high (r-value = 0.98; β = 1.01, n = 25).Geometric morphometric analysis was used to quantify the variation in the size and shape of the outline of the posterior and ventral lobe of the genital arch. Four points along the outline that could be located precisely across all specimens were applied as landmarks (type-two landmarks). Another 30 points, called sliding semilandmarks, were allowed to slide along the outline in a trajectory that minimizes shape changes between specimens and the Procrustes average of all the specimens (). The points (landmarks and semilandmarks) were digitized in TPSDIG 2.14 and the semilandmarks were identified by use of a ‘sliders file’ in TPSUTIL 1.46 . To eliminate non-shape variation, the digitized landmark data were normalized for position, orientation and scale (generalized least squares superimposition). Centroid size, the square root of landmarks from the centroid, was extracted and the data were reduced to a series of relative warp scores. Our 34 landmarks and semilandmarks yield 64 relative warp scores that explain progressively less variance. Beyond RWS 7, less than 2% of the variance in shape was distinctly explained, so we only interpret RWS 1–7 (). Changes in the shape of the posterior and ventral lobe of the genital arch were visualized as shape deformations of the thin plate spline. tpsRELW 1.46 was used for the superimposition, calculation of centroid and relative warp scores and thin-plate spline plot visualizations . We assessed the repeatability of digitization by digitizing two images of the same genitals twice (n = 25). Ordinary least squares regression (i.e. RW1 on RW1; RW2 on RW2 etc) revealed that we were able to digitize the genital arches consistently (r-values ranged from 0.95 to 0.64; β ranged from 1.06 to 0.79 in RW1–RW7) although, error in RW 1 and subsequent RWs accumulated so that the r-values were lower in RW7 (r-value = 0.64; β = 0.79). […]

Pipeline specifications

Software tools TpsDIG, TpsUtil, TpsRelw
Application Macroscope & basic digital camera imaging
Organisms Drosophila simulans