DOI: 10.5061/dryad.98sf7m0nd
title: Genetic variation of morphological scaling in Drosophila

description: 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.
description: Fly Stocks All flies used in this study came
 from The <em>Drosophila</em> Genome Resource Panel (DGRP), a
 library of ~200 fully sequenced inbred
 isogenic <em>Drosophila</em> 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
 <em>Drosophila</em> 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 <em>ad</em> <em>libitum</em> 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, <em>Drosophila</em> 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.
description: All the data were analyzed using <em>R </em>and
 freely available <em>R </em>packages.