DOI: 10.5061/dryad.vq83bk42z
title: Data from: Circadian plasticity evolves through regulatory changes in a neuropeptide gene

description: Many organisms, including cosmopolitan drosophilids, show circadian
 plasticity, varying their activity with changing dawn–dusk intervals[1].
 How this behaviour evolves is unclear. Here we compare Drosophila
 melanogaster with Drosophila sechellia, an equatorial, ecological
 specialist that experiences minimal photoperiod variation, to investigate
 the mechanistic basis of circadian plasticity evolution[2]. D. sechellia
 has lost the ability to delay its evening activity peak time under long
 photoperiods. Screening of circadian mutants in D. melanogaster/D.
 sechellia hybrids identifies a contribution of the neuropeptide
 pigment-dispersing factor (Pdf) to this loss. Pdf exhibits
 species-specific temporal expression, due in part to cis-regulatory
 divergence. RNA interference and rescue experiments in D. melanogaster
 using species-specific Pdf regulatory sequences demonstrate that
 modulation of this neuropeptide’s expression impacts the degree of
 behavioural plasticity. The Pdf regulatory region exhibits signals of
 selection in D. sechellia and across populations of D. melanogaster from
 different latitudes. We provide evidence that plasticity confers a
 selective advantage for D. melanogaster at elevated latitude, whereas D.
 sechellia probably suffers fitness costs through reduced copulation
 success outside its range. Our findings highlight this neuropeptide gene
 as a hotspot locus for circadian plasticity evolution that might have
 contributed to both D. melanogaster’s global distribution and D.
 sechellia’s specialization.
description: Confocal images were obtained using the Zeiss microscopy ZEN 2.3
 SP1 software. Drosophila Activity Monitor data were collected using the
 DAM System software, version 311X. All image analysis was performed in
 Fiji running v2.9.0 of ImageJ. RNA spots were detected using the RS-FISH
 macro, and Scholl analysis was performed using the neuroanatomy plug-in.
 Sequences were analyzed and visualised using SnapGene (www.snapgene.com)
 running MUSCLE (v3.8.1551), and Jalview (v2.11.2), and using the R package
 Phangorn (v2.11.2). Sequence alignments were perfomed using Samtools
 (v1.19.2), and tests of neutrality were performed using the DNA sequence
 polymorphism software (v6). Drosophila Activity Monitor data were analysed
 using Rethomics in R (v3.6.3). All R code used for data analysis are
 available at: github.com/mshahandeh/circ_plasticity. No statistical
 methods were used to predetermine sample size. For behavioural
 experiments, we aimed for a sample size of 25-30 individuals, as
 significant differences were easily detected at these sizes. In the case
 of our hybrid screen, where hybrid flies were harder to make, we aimed for
 15 individuals per strain but due to the strong reproductive isolation
 between species, some genotypes were difficult to cross to <em>D.
 sechellia</em>, resulting in a lower sample size. For image
 analyses, we obtained images from 5 brains from each strain per treatment
 because this allowed for parallel processing of multiple genotypes and
 time points. Additionally, quantifications of these stainings were largely
 consistent, and significant differences were apparent at this sample size.
 All behavioural experiments were collected over at least two technical
 replicates with corresponding controls to ensure reproducibility, with the
 exception of those shown in Fig. 1c, where we replicated only two
 photoperiod treatments: 12:12 h LD and 16:8 h LD, because we continued the
 use of these specific photoperiods throughout the work. All brain
 dissections and stainings for a given experiment were performed in
 parallel to ensure comparability between time points and fixed microscope
 settings were used within each experiment. Two time-points of each
 histological experiments (Pdf smFISH, Pdf immunofluorescence, and Scholl
 analysis of s-LNv axonal projections) were replicated once in order to
 ensure the overall patterns of expression were the same. We did not
 randomise within experiments, and instead ran all genotypes in parallel
 for all experiments, except for the hybrid screening, where we measured
 the behaviour of hybrids as and when crosses produced offspring, with at
 least one replicate run in parallel with corresponding controls (data
 collection for hybrid screening took &gt;2 years). Quantifications for
 stainings and collection of behavioural data were performed blind to
 treatment. 
description: # Circadian plasticity evolves via regulatory changes in a neuropeptide
 gene README * /DAM: All files used for analysis of Drosophila activity
 monitoring (DAM) are contained within this folder. * /DAM_metadata_files
 contains metadata files listing time, date, genotype, sex, status of flies
 recorded * /DAM_monitor_files contains the raw data produced from the DAM
 system software * /DATA: contains excel files with all data
 quantifications required for analyses, organized by Figure (details for
 each figure organized by panel below) * /Images: contains all .czi
 confocal Z-stack images taken (specific quantifications can be found in
 the /Data folder for each image panel) * /R: contains all R code required
 to reproduce the analyses ### Specific metadata, xcel, and image files for
 each figure, organized by panel, are provided below: Figure1- data
 visualisation and analysis code in Rethomics.R; DAM metadata or excel data
 files listed below c: Dmel+Dsec_all_1212LD.csv, metadata_14-10LD.csv,
 metadata_18-6LD.csv, metadata_18-6LD.csv. metadata_20-4LD.csv &
 Epeak_all_photoperiods.csv\ d: Dmel+Dsec_all_1212LD.csv\ e:
 metadataLD2DD.csv\ f: Dmel_Dsec_MPeak_1212LD.csv Figure2 - data
 visualisation and analysis code in Rethomics.R; DAM metadata, or excel
 data files listed below d: metadata_KODFs+PDF-168LD.csv\ e:
 Pdf_DeltaME.csv\ f and g: metadata_KODFs+PDF-1212LD.csv Figure3- data
 visualisation and analysis code in Rethomics.R; DAM metadata, or excel
 data files listed below a: Dmel- MS370_4R.czi; Dsec- MS373_4R700.czi\ b:
 Mpeak_RNAspots.xlsx, example images highlighted within c: Images MS50_3L
 and MS51_2L; quantifications: s-LNV_terminals_PDF.xlsx d:
 s-LNV_cellbodies_re_PDF.xlsx, example images highlighted within e:
 Scholl_ZT2&ZT14_12hLD.xlsx, example images highlighted within f: Top-
 371_3L.czi; Bottom- 372_1L.czi g: BT412_413_Epeak_12hLD.xlsx, example
 images highlighted within h: BT412_413_Epeak_168hLD.xlsx, example images
 highlighted within i: BT412_413_Mpeak_12hLD.xlsx, example images
 highlighted within Figure4- data visualisation and analysis code in
 Rethomics.R; or excel data files listed below a:
 metadata_PDFRNAi_12hLD.csv, metadata_PDFRNAi_168hLD.csv b-c:
 DeltaME_RNAi.csv d: metadata_PDFRNAi_12hLD.csv e:
 metadata_PDFswap-12hLD.csv, metadata_PDFswap-168hLD.csv f-g:
 Epeak_attP2pdf01_rescue.csv h: metadata_PDFswap-12hLD.csv Figure5- a:
 DmelDsimDsecDmau_enhancers_woutgroups.fasta b:
 Dmel&Dsec&Dsim_meme-motif-locations.svg c-d:
 neuropeptide_enhancersPDF+control.xlsx e:
 Dsec_pop_Pdfenhancers_aligned.fasta, AstC_aligned.fasta,
 sNPF_aligned.fasta f: Longevity_168hLD_rep1.xlsx g:
 2h_copulation_photoperiods.xlsx EDFigure1 data visualisation and analysis
 code in Rethomics.R; DAM metadata or excel data files listed below a:
 Epeak_all_photoperiods.csv b: metadata: Dmel+Dsec_all_1212LD.csv,
 metadata_14-10LD.csv, metadata_18-6LD.csv, metadata_18-6LD.csv.
 metadata_20-4LD.csv c: metadata_12-12LD_females.csv,
 metadata_16-8LD_females.csv, Epeak_females_12hLD.csv,
 Epeak_females_168hLD.csv d: metadata_12-12LD_females.csv e-f:
 metadataDD.csv EDFigure2- data visualisation and analysis code in
 Rethomics.R; DAM metadata or excel data files listed below a:
 metadata_16-8LD_replicate.csv ,Epeak_replicate_168hLD.csv\ b:
 metadata_12-12LD_replicate.csv EDFigure3- data visualisation and analysis
 code in Rethomics.R; DAM metadata or excel data files listed below a,c,e:
 metadata_KODFs+PDF-168LD.csv b: DeltaME_Dsec07.csv d: DeltaME_Dsec28.csv
 f: DeltaME_w1118.csv EDFigure4- data visualisation and analysis code in
 Rethomics.R; DAM metadata or excel data files listed below a-f:
 metadata_KODFs+PDF-1212LD.csv EDFigure5- Dmel_Dsim_Dsec_CDS_EDF5.fasta
 EDFigure6- data visualisation and analysis code in Rethomics.R, example
 images highlighted within data files below a: smFISH_RS_12hLD_Epeak.xlsx\
 b: smFISH_RS_168hLD_Epeak.xlsx\ c: Dmel_Dsec_Epeak_12hLD.xlsx\ d:
 Dmel_Dsec_Epeak_168hLD.xlsx EDFigure7- data visualisation and analysis
 code in Rethomics.R; RNAi_smFISH_63x.xlsx, example images highlighted
 within EDFigure8 (also in Figure 5)- data visualisation and analysis code
 in Rethomics.R a: Longevity_168hLD_rep1.csv b: Longevity_168hLD_rep2.csv
 c: 3d_copulation_photoperiods.xlsx EDFigure9- data visualisation and
 analysis code in Rethomics.R; DAM metadata or excel data files listed
 below a: metadata-CS[W]_16-8.csv b: DeltaME_CS[W].csv c-d:
 metadata-CS[W]_12-12.csv