The black scavenger fly Sepsis thoracica exhibits polyphenic development resulting in alternate small black and large amber male morphs. Although the behavior, ecology, and physiology of both morphs are being scrutinized, the evolutionary origins of the nutritional polyphenism remain poorly understood. I here use a comparative approach to study variation in the degree of melanization of the forefemur —a secondary sexual trait. Melanization showed nutritional plasticity in all species and character mapping suggests polyphenic development to represent the ancestral character state that was lost repeatedly. That is, interspecific variation among the studied species is mainly caused by the loss and not the gain of polyphenic development. Coevolution between male melanization and mating system differences further implicates sexual selection in the evolution of male melanization. These findings highlight the usefulness of comparative and natural history data in shedding new light on the evolution of phenotypic variation.

All measurements were taken from animals generated by a resource manipulation experiment described in Rohner and Blanckenhorn (2018; doi: 10.1086/700096) with the exception of S. fulgens, which was reared at a later stage but under identical laboratory conditions. In brief, eggs were collected from outbred laboratory cultures (200-300 individuals) and haphazardly distributed among plastic containers with varying amounts of homogenized cow dung, and subsequently incubated all offspring at constant 18°C. Upon adult eclosion of all individuals, 30-50 individuals of all sizes per species and sex were selected for morphometric measurements.

The right front legs were removed from the thorax and mounted on a glass slide using Euparal and imaged using a Leica DFC490 camera mounted on a Leica MZ12 microscope. All images were taken in one sitting under standardized light conditions and camera settings to keep coloration comparable across specimens. Images were then analyzed using the custom ImageJ script from Busso and Blanckenhorn (2017; doi: 10.1111/een.12413). This script first measures the number of pixels of the forefemur and then quantifies how many of them fall below a certain brightness threshold in the YUV color space. Pixels with a V-value (i.e. brightness) higher than 163 were defined as melanic, and the proportion of these melanic pixels in the full femur estimates the overall degree of melanism (i.e. coloration), the measure used for further analysis and listed in the excel file deposited here. A modified version of the original ImageJ script provided by Juan Pablo Busso is attached as an ImaheJ Macro (.ijm).