Morphological scaling relationships between the sizes of individual traits and the body captures the characteristic shape of a species, and the evolution of scaling is the primary mechanism of morphological diversification. However, we have almost no knowledge of the genetic variation of scaling, which is critical if we are to understand how scaling evolves. Here we explore the genetics of population morphological scaling relationships – the scaling relationship fit to multiple genetically-distinct individuals in a population – by describing the distribution of individual scaling relationships – the genotype-specific scaling relationships that are unseen or cryptic. These individual scaling relationships harbor the genetic variation that determines relative trait growth within individuals, and theoretical studies suggest that their distribution dictates how the population scaling relationship will respond to selection. Using variation in nutrition to generate size variation within 194 isogenic lineages of Drosophila melanogaster, we reveal extensive variation in the slopes of the wing-body and leg-body scaling relationships among genotypes. This genetic variation reflects variation in the nutritionally-induced size plasticity of the wing, leg, and body. Surprisingly, we find that variation in the slope of individual scaling relationships primarily results from variation in nutritionally-induced plasticity of body size, not leg or wing size. These data allow us to predict how different selection regimes affect scaling in Drosophila and are the first step in identifying the genetic targets of such selection. More generally, our approach provides a framework for understanding the genetic variation of scaling, an important prerequisite to explaining how selection changes scaling and morphology.

Fly Stocks

All flies used in this study came from The Drosophila Genome Resource Panel (DGRP), a library of ~200 fully sequenced inbred isogenic Drosophila lineages that originated from a single outbred population (Mackay et al., 2012) collected from Raleigh, NC, USA. Flies were maintained on standard cornmeal molasses medium (Frankino et al., 2019) and maintained on a 12:12 light cycle at 22˚C and 75% humidity.

Starvation treatment

Drosophila egg collection, rearing, and phenotyping followed our established protocols (Stillwell et al., 2011, 2016; Frankino et al., 2019). For each DGRP lineage, females oviposited for three days. At 24h, 48h and 72h, eggs were collected, divided into lots of 50 and placed into vials containing 10ml of standard cornmeal molasses medium. This generated three age cohorts of flies (D0, D1 and D2, respectively). When third instar larvae from D0 began to pupariate, larvae from all cohorts were removed from the food and placed into empty food vials with a wet cotton plug to provide moisture. Pupae were removed from these vials and transferred to individual 1.5ml Eppendorf tubes, each with a small hole in the lid, to complete development to adulthood. Larvae in the D0 cohort were starved for between 0-24h before pupariation, larvae in the D1 cohort were starved for between 24-48h before pupariation, and larvae in the D3 cohort were starved for between 48–72h before pupariation. Because larvae stop feeding ~24h before pupariation (Testa et al., 2013), D0 larvae were essentially allowed to feed ad libitum and more-or-less achieved full adult body size. In contrast, D1 and D2 larvae were starved before larval wandering, reducing adult size depending on their size at initiation of starvation. Across all cohorts, our starvation treatment therefore generated nutritionally-induced variation in body size. Flies were collected in nine temporal blocks, with five lineages repeated across all blocks as a control.

Body and Trait Size Measurement 

Body and trait size were measured using established protocols (Shingleton et al., 2009; Stillwell et al., 2011). Briefly, Drosophila adults were dissected, and their right wing and right first leg were mounted in dimethyl hydantoin formaldehyde (DMHF). Pupal area (a proxy for body size; (Stillwell et al., 2016), wing area, and femur length (a proxy for leg length; (Shingleton et al., 2009) were measured across the full range of body size for ~50 individuals per sex per lineage; Figure 2). All traits were measured via semi-automated custom software (Metamorph, Molecular Devices LLC) that captures images from a digital camera-equipped microscope (Leica DM6000B, Leica Microsystems Inc). Femur length was squared to put it in the same dimension as wing and pupal area, and all measurements were log-transformed to ensure scale invariance across traits of different sizes.

All the data were analyzed using R and freely available R packages.