Appendometer: A system for simultaneous, high-throughput morphometry of Drosophila legs and wings

O_LIThe inheritance of many different organismal features is correlated, as is their evolution, sug-gesting that we need to understand the pattern and causes of those correlations to understand evolution. Unfortunately, we generally lack the ability to rapidly and accurately measure large numbers of traits, making it difficult to describe the patterns of trait relationships or for-mulate hypothesis about the causes of their entanglement. We have previously developed a system to make high-dimensional measurements of Drosophilid fly wings in live specimens. Here, we report the extension of this approach to rapidly assess the dimensions of the distal leg segments, femur, tibia, and tarsi. Using the system, we describe the covariance of the wing and leg morphology and evaluate the relative rates of evolution of legs and wings. C_LIO_LIWe use two simple suction devices to immobilize and position legs and wings of an anaes-thetized fly for imaging, then take a single image incorporating both appendages. We em-ployed a machine learning method to measure leg segment lengths, which should be broadly applicable across diverse taxa. Experienced users can image the legs and wings of a fly every two minutes, with outlier detection and correction taking approximately 40 seconds. To demonstrate the usefulness of these methods, we measured the legs and wings of over 4,000 specimens from 43 different Drosophilid taxa. We estimated the rate of wing and leg evolu-tion using a phylogenetic mixed model. C_LIO_LIRepeatabilities of leg segments lengths averaged over 80%. The rate of evolution of wing and leg sizes are similar, but the rate of wing proportion evolution is 1.6 times as high as that of leg proportions due to strong allometric changes in wing shape. Within-species variation in leg proportions is highly correlated with the rate of leg proportion evolution, as is true for wings. Relative lengths of leg segments showed a strong pattern of negative correlations be-tween the lengths of the tarsal segments and of the femurs and tibias, while all other segment correlations were positive. This pattern was repeated in the rate estimates, suggesting that se-lection favors tradeoffs between tarsi and the remainder of the leg. C_LIO_LIOur simple system for imaging and measuring legs and wings simultaneously has high throughput and repeatability. It is readily applicable to a wide variety of winged insects and other traits, including wings, that could be imaged. Applied to Drosophila, our morphometric system enlarges the ability to study inheritance, pleiotropy, and evolution in this important model taxon. C_LI