DOI: 10.5061/dryad.9zw3r22gg
title: Census and phenotype data supporting Drosophila adaptive tracking

description: Direct observation of evolution in response to natural environmental
 change can resolve fundamental questions about adaptation including its
 pace, temporal dynamics, and underlying phenotypic and genomic
 architecture. We tracked evolution of fitness-associated phenotypes and
 allele frequencies genome-wide in ten replicate field populations of
 Drosophila melanogaster over ten generations from summer to late fall.
 Adaptation was evident over each sampling interval (1-4 generations) with
 exceptionally rapid phenotypic adaptation and large allele frequency
 shifts at many independent loci. The direction and basis of the adaptive
 response shifted repeatedly over time, consistent with the action of
 strong and rapidly fluctuating selection. Overall, we find clear
 phenotypic and genomic evidence of adaptive tracking occurring
 contemporaneously with environmental change, demonstrating the temporally
 dynamic nature of adaptation.
description: Taken from SI of Rudman et al. 2022, Science
 Establishment of experimental populations  To
 examine the pace, magnitude, parallelism, and genomic architecture of
 adaptation in response to a temporally variable environment we created a
 genetically diverse founder population that was seeded into each outdoor
 replicate. This outbred founder population was constructed from 80 fully
 sequenced Drosophila melanogaster inbred lines to facilitate the use of
 haplotype inference to attain high effective sequencing coverage. These
 inbred lines were derived from wild-caught individuals collected June 1,
 2012 from Linvilla Orchards, Media PA USA (1). Each line was subsequently
 inbred for 20 generations by full-sib mating during which time they were
 maintained at 25 ˚C with a 16:8 photoperiod and fed ‘Spradling Cornmeal
 Recipe’ media. Then, 30-50 individuals from each line were pooled for
 whole genome sequencing. Sequencing and variant calling were performed as
 described in (2), with the addition that genomic DNA from certain lines
 was resequenced on an Illumina HiSeq X to increase coverage to a minimum
 of 10x for all lines. Mapped and de-duplicated bam files from all original
 and resequencing runs can be found on SRA under BioProject PRJNA722305
 (Table S1). To initiate the baseline population in this experiment, we
 combined 10 males and 10 females from each of the 80 lines into large
 cages in May 2014. We allowed 4 generations of unmanipulated recombination
 and population expansion to facilitate recombination between lines before
 using 500 males and 500 female flies to found each of 10 field cages.
 Inbred lines have many deleterious alleles; purifying selection against
 deleterious alleles fixed during inbreeding was likely strong during lab
 outcrossing, and potentially, the early field phase of the
 experiment. Each field cage is a 2m x 2m x 2m mesh
 enclosure around a dwarf peach tree located outdoors (Philadelphia, PA)
 and features a natural insect and microbial community. The ground was
 fresh soil with clover planted as ground cover in each cage. The only food
 source and egg-laying substrate was 400ml of Drosophila media (‘Spradling
 cornmeal recipe’) contained in 900cm3 aluminum loaf pans that were added
 every second day for the duration of the experiment (July 13th - November
 7th, 2014). After two days, each loaf pan was covered with a screen mesh
 lid and eggs were allowed to develop until eclosion ceased. Loaf pans of
 media within experimental cages were protected from rain and direct sun on
 shelving units oriented away from direct sunlight.
 Measurement of population size and evolution of fitness
 associated phenotypes  Census size of adults was
 estimated in each replicate over the course of the experiment by
 photographing an equal amount of the surface area (approximately 2.5%) of
 the ceiling in each cage at dusk (12 total census estimates per cage).
 This protocol did not disturb flies perched on the ceiling. The number of
 adult D. melanogaster in each of 8 standardized photographs in each
 estimate for each cage was counted and multiplied by 40 to correct for
 total surface area and obtain census estimates. Egg production was
 estimated every second day by counting the eggs present on a 1/24th
 portion of the exposed surface of the media. When adult population sizes
 were large and the weather was warm the density of eggs on the media was
 considerably higher than optimal lab conditions. To
 assess the rate and direction of phenotypic evolution over the course of
 the experiment we collected ~2500 eggs from each cage, brought them to the
 laboratory, and reared them for an additional 2 generations in a common
 garden (25˚C, 16L:8D) while maintaining population sizes at ~2500
 individuals. Fitness-associated phenotypes were measured on density and
 age-controlled replicates in the F3 generation. Fecundity was measured as
 the total number of eggs laid by a group of five females, counted each day
 for a period of three days, with twenty replicate vials for each cage at
 each time. Egg length was measured using a microscope and image processing
 software (3) on at least 15 eggs (average of 27) from each cage at each
 time point. Larval development rate was tracked as the time from when eggs
 were laid until pupation in three replicate vials from each cage at each
 time point with 30 eggs in each vial. Starvation tolerance was measured as
 time to starvation in three replicate vials containing moist cotton (1.5
 ml water) (following (4)) and 10 female flies with three replicates for
 each cage at each time point. Desiccation tolerance was measured as time
 to death in desiccation chambers containing 10 female flies with three
 replicates for each cage at each time point (4). Chill coma recovery was
 measured as the time it took for flies buried in ice and placed in a 4℃
 incubator for 2h to resume an upright stance at 25℃ (1). This was measured
 using groups of 10 female flies for each cage at each time point that had
 been allowed at least 24hrs to recover from light CO2 anesthetic. We also
 attempted to measure evolution in heat knockdown. However, the assay
 temperature we used for the founder population, a stressor that caused 50%
 of flies to knockdown by 12 minutes, was not sufficiently hot to cause
 knockdown by the second sample period. Thus, although we cannot quantify
 it, heat tolerance evolved rapidly. We assayed each of the remaining
 phenotypes in the founding population (founder assays failed for fecundity
 and egg size) and at four times during the experiment: day 11 (7/25/14),
 day 38 (8/19/14), day 61 (9/11/14), and day 90 (10/10/14). Census and
 phenotypic evolution data have been uploaded to Dryad. 
 Calculation of evolutionary rates and statistical analysis of
 phenotypic data to test for evolutionary parallelism 
 We calculated evolutionary rates in Haldanes by dividing the
 trait change over each interval by the pooled standard deviation and then
 by the number of generations elapsed (5,6). We calculated the rate of
 adaptation as the parallel change across replicates. To do so we took the
 average trait change across all 10 replicates and calculated a single rate
 in Haldanes. Haldanes were calculated for all six phenotypes for each
 experimental interval and over the whole experiment. We compared the rates
 of evolution measured in our experiment to those from a meta-analysis of
 evolutionary rates from field populations that focused on contemporary
 evolution (less than 200 generations) (7). The meta-analysis was focused
 on phenotypic change, which includes both genetic and environmental
 (plastic) effects, as few prior studies used common garden experiments to
 measure the rate of evolution.  To test for parallel
 phenotypic evolution in each of the six phenotypes we carried out separate
 linear mixed effect models (e.g. lme(phenotype measured ~ time,
 random=~1|cage/time)) and tested for significance using an anova (nlme and
 R respectively). For phenotypic responses that were rate calculations
 (i.e., development, starvation, chill coma recovery, and desiccation
 tolerance) a GLM was used to find the time at which 50% of individuals
 completed the assay (i.e., pupated, starved, recovered, desiccated) in
 each technical replicate. These values were used in the subsequent
 analysis.
description: Please see the README file. Additional information is in  SI of
 the paper.