Restricted accessResearch articleFirst published online 2026-2
Selection for the Timing of Adult Emergence Leads to Evolution in the Molecular Circadian Clock and Its Light Input Pathway in Drosophila melanogaster Populations
The light input pathways and the molecular clock are tightly linked, with light serving as the most potent zeitgeber that entrains the clock to the external environment. Our present study focuses on the Drosophila melanogaster populations that have evolved with a precise circadian clock as a correlated response to selection for adult emergence in a narrow window of time over 335 generations. The results of our study showed that flies from populations selected for the timing of adult emergence sleep more during the night phase compared to controls. This sleep was even more enhanced when the light intensity was reduced to 1 lux under a 12 h light:12 h dark cycle. In addition, a significantly higher percentage of these flies exhibited free-running period rather than arrhythmicity compared to the control flies under constant light (1 lux). Moreover, the larvae from selected populations exhibited an increased preference toward darkness than light indicating that the effect of selection extends beyond the adult circadian light input pathway, influencing the innate circadian regulated photobehavior in larvae. We examined the transcript oscillation of the circadian photoreceptor cryptochrome (cry), along with the core clock genes period (per) and timeless (tim) in adult flies to explore the molecular basis of the evolved precise circadian clocks and to determine whether selection influences the circadian light input pathway. Flies from the selected population exhibited a phase advance in the transcript oscillation of per, tim, and cry, indicating that the molecular circadian clock and its light input pathway evolve as a correlated response to the selection for the timing of adult emergence in D. melanogaster populations.
AkpoghiranOStrichAKKohK (2025) Effects of sex, mating status, and genetic background on circadian behavior in. Drosophila. Front Neurosci18:1532868.
2.
AschoffJ (1981) Freerunning and entrained circadian rhythms. In: Aschoff. Boston (MA): Biological Rhythms. Springer.
3.
BaikLSRecinosYChevezJAAuDDHolmesTC (2019) Multiple phototransduction inputs integrate to mediate UV light–evoked avoidance/attraction behavior in drosophila. J Biol Rhythms34:391-400.
4.
BatscheletE (1981) Circular statistics in biology. San Diego (CA): Academic Press.
5.
BinghamCArbogastBGuillaumeGCLeeJKHalbergF (1993) Inferential statistical methods for estimating and comparing cosinor parameters. Chronobiologia20:565-581.
6.
BlauJYoungMW (1999) Cycling vrille expression is required for a functional Drosophila clock. Cell99:661-671.
7.
BrownEBTorresJBennickRARozzoVKerbsADiAngeloJRKeeneAC (2018) Variation in sleep and metabolic function is associated with latitude and average temperature in Drosophila melanogaster. Ecol Evol8:4084-4097.
8.
CheverudJM (1984) Quantitative genetics and developmental constraints on evolution by selection. J Theor Biol110:155-171.
9.
CusumanoPKlarsfeldAChélotEPicotMRichierBRouyerF (2009) PDF-modulated visual inputs and cryptochrome define diurnal behavior in Drosophila. Nat Neurosci12:1431-1437.
10.
DasAHolmesTCSheebaV (2016) DTRPA1 in non-circadian neurons modulates temperature-dependent rhythmic activity in Drosophila melanogaster. J Biol Rhythms31:272-288.
11.
EmersonKJBradshawWEHolzapfelCM (2008) Concordance of the circadian clock with the environment is necessary to maximize fitness in natural populations. Evolution62:979-983.
12.
EmeryPSoWVKanekoMHallJCRosbashM (1998) Cry, a Drosophila clock and light-regulated cryptochrome, is a major contributor to circadian rhythm resetting and photosensitivity. Cell95:669-679.
13.
EmeryPStanewskyRHelfrich-FörsterCEmery-LeMHallJCRosbashM (2000) Drosophila CRY is a deep brain circadian photoreceptor. Neuron26:493-504.
14.
Farca LunaAJvon EssenAMHJWidmerYFSprecherSG (2013) Light preference assay to study innate and circadian regulated photobehavior in Drosophila larvae. J Vis Exp74:50237.
15.
GohGVesterdorfKFullerABlacheDMaloneySK (2024) Optimal sampling interval for characterisation of the circadian rhythm of body temperature in homeothermic animals using periodogram and cosinor analysis. Ecol Evol14:e11243.
16.
HardinPE (2011) Molecular genetic analysis of circadian timekeeping in Drosophila. Adv Genet74:141-173.
17.
HardinPEHalltJCRosbashM (1990) Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels. Nature343:536-540.
18.
HendricksJCFinnSMPanckeriKAChavkinJWilliamsJASehgalAPackAI (2000) Rest in Drosophila is a sleep-like state. Neuron25:129-138.
19.
HillWG (1982) Predictions of response to artificial selection from new mutations. Genet Res40:255-278.
20.
HongSFSaundersDS (1994) Effects of constant light on the rhythm of adult locomotor activity in the blowfly, Calliphora vicina. Phys Entomol19:319-324.
21.
HornMMitesserOHovestadtTYoshiiTRiegerDHelfrich-FörsterC (2019) The circadian clock improves fitness in the fruit fly, Drosophila melanogaster. Front Physiol10:1374.
22.
HughesMEAbruzziKCAlladaRAnafiRArpatABAsherGBaldiPde BekkerCBell-PedersenDBlauJ, et al. (2017) Guidelines for genome-scale analysis of biological rhythms. J Biol Rhythms32:380-393.
JoyceMFalconioFABlackhurstLPrieto-GodinoLFrenchASGilestroGF (2024) Divergent evolution of sleep in Drosophila species. Nat Commun15:5091.
25.
KannanNNVazeKMSharmaVK (2012a) Clock accuracy and precision evolve as a consequence of selection for adult emergence in a narrow window of time in fruit flies Drosophila melanogaster. J Exp Biol215:3527-3534.
26.
KannanNNMukherjeeNSharmaVK (2012b) Robustness of circadian timing systems evolves in the fruit fly Drosophila melanogaster as a correlated response to selection for adult emergence in a narrow window of time. Chronobiol Int29:1312-1328.
27.
KannanNNVarmaVDeJSharmaVK (2012c) Stability of adult emergence and activity/rest rhythms in fruit flies Drosophila melanogaster under semi-natural condition. PLoS ONE7:e50379.
KonopkaRJPittendrighCOrrD (1989) Reciprocal behavior associated with altered homeostasis and photosensitivity of Drosophila clock mutants. J Neurogenet21:243-252.
30.
LandeR (1976) Natural selection and random genetic drift in phenotypic evolution. Evolution30:314-334.
31.
LearBCZhangLAlladaR (2009) The neuropeptide PDF acts directly on evening pacemaker neurons to regulate multiple features of circadian behavior. PLoS Biology7:e1000154.
32.
LinYHanMShimadaBWangLGiblerTMAmarakoneAAwadTAStormoGDVan GelderRNTaghertPH (2002) Influence of the period-dependent circadian clock on diurnal, circadian, and aperiodic gene expression in Drosophila melanogaster. Proc Natl Acad Sci U S A99(14):9562-956799.
33.
MarkBBustos-GonzálezLCascallaresGConejeraFEwerJ (2021) The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis. Proc Natl Acad Sci U S A118:e2023249118.
34.
MazzoniEODesplanCBlauJ (2005) Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response. Neuron45:293-300.
35.
MuradAEmery-LeMEmeryP (2007) A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila. Neuron53:689-701.
36.
NelsonWTongYLLeeJ-KHalbergF (1979) Methods for cosinor-rhythmometry. Chronobiologia6:305-323.
37.
NikhilKLAbhilashLSharmaVK (2016) Molecular correlates of circadian clocks in fruit fly Drosophila melanogaster populations exhibiting early and late emergence chronotypes. J Biol Rhythms31:125-141.
38.
OosthuizenMKBennettNC (2022) Clocks ticking in the dark: a review of biological rhythms in Subterranean African mole-rats. Front Ecol Evol10:878533.
39.
OuyangYAnderssonCRKondoTGoldenSSJohnsonCH (1998) Resonating circadian clocks enhance fitness in cyanobacteria. Proc Natl Acad Sci U S A95:8660-8664.
40.
PandaSHogeneschJBKaySA (2002) Circadian rhythms from flies to human. Nature417:329-335.
41.
ParanjpeDASharmaVK (2005) Evolution of temporal order in living organisms. J Circadian Rhythms3:7.
42.
PicotMCusumanoPKlarsfeldAUedaRRouyerF (2007) Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock. PLoS Biology5:2513-2521.
43.
PittendrighCSDaanS (1976) A functional analysis of circadian pacemakers in nocturnal rodents: I. The stability and lability of spontaneous frequency. J Comp Physiol106:223-252.
44.
PittendrighCSTakamuraT (1989) Latitudinal clines in the properties of a circadian pacemaker. J Biol Rhythms4:217-235.
45.
PrasadNGShakaradMAnithaDRajamaniMJoshiA (2001) Correlated responses to selection for faster development and early reproduction in Drosophila: the evolution of larval traits. Evolution55:1363-1372.
46.
RefinettiRLissenGCHalbergF (2005) Procedures for numerical analysis of circadian rhythms. Biol Rhythm Res38:275-325.
47.
RoseMR (1984) Laboratory evolution of postponed senescence in Drosophila melanogaster. Evolution38:1004-1010.
48.
Saint-CharlesAMichard-VanhéeCAlejevskiFChélotEBoivinARouyerF (2016) Four of the six Drosophila rhodopsin-expressing photoreceptors can mediate circadian entrainment in low light. J Comp Neurol524:2828-2844.
49.
SaundersDS (2002:) Insect clocks. 3rd ed. Amsterdam (The Netherlands): Elsevier.
50.
SawyerLAHennessyJMPeixotoAARosatoEParkinsonHCostaRKyriacouCP (1997) Natural variation in a Drosophila clock gene and temperature compensation. Science278:2117-2120.
51.
SchmittgenTDLivakKJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc3:1101-1108.
52.
SharmaVKChandrashekaranM (1999) Precision of a Mammalian circadian clock. Naturwissenschaften86:333-335.
53.
SheebaVFogleKJHolmesTC (2010) Persistence of morning anticipation behavior and high amplitude morning startle response following functional loss of small ventral lateral neurons in drosophila. PLoS ONE5:e11628.
54.
SrivastavaMVarmaVAbhilashLSharmaVKSheebaV (2019) Circadian clock properties and their relationships as a function of free-running period in Drosophila melanogaster. J Biol Rhythms34:231-248.
55.
StanewskyRKanekoMEmeryPBerettaBWager-SmithKKaySARosbashMHallJC (1998) The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell95(5):681-692.
56.
SvetecNZhaoLSaelaoPChiuJCBegunDJ (2015) Evidence that natural selection maintains genetic variation for sleep in Drosophila melanogaster evolutionary ecology and behaviour. BMC Evol Biol15:41.
57.
TakahashiJSHongHKKoCHMcDearmonEL (2008) The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet9:764-775.
58.
The MathWorks Inc (2024). MATLAB version: 9.13.0 (R2024a). Natick (MA): The MathWorks Inc. https://www.mathworks.com.
59.
TurelliM (1988) Phenotypic evolution, constant covariances, and the maintenance of additive variance. Evolution42:1342-1347. http://www.jstor.org/stable/2409017.
60.
Van GelderRNKrasnowMA (1996) A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression. EMBO J15:1625-1631.
61.
WheelerDAHamblen-CoyleMJDushayMSHallJC (1993). Behavior in light-dark cycles of Drosophila mutants that are arrhythmic, blind, or both. J Biol Rhythms8:67-94.
62.
YangLEderyI (2018) Parallel clinal variation in the mid-day siesta of Drosophila melanogaster. J Biol Rhythms33:75-87.
63.
YoshiiTHermann-LuiblCHelfrich-FörsterC (2015) Circadian light-input pathways in Drosophila. Commun Integr Biol9:e1102805.
64.
YuanQJoinerWJSehgalA (2006) A sleep-promoting role for the drosophila serotonin receptor 1A. Current Biology16:1051-1062.
65.
YuanQLinFZhengXSehgalA (2005) Serotonin modulates circadian entrainment in Drosophila. Neuron47:115-127.
66.
ZhangXSHillWG (2005) Predictions of patterns of response to artificial selection in lines derived from natural populations. Genetics169:411-425.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
