Life-history traits of Drosophila melanogaster populations exhibiting early and late eclosion chronotypes

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1 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 DOI /s RESEARCH ARTICLE Open Access Life-history trits of Drosophil melnogster popultions exhiiting erly nd lte eclosion chronotypes K. L. Nikhil, Krtgi Rtn nd Vijy Kumr Shrm * Astrct Bckground: The hypothesis tht circdin clocks confer dptive dvntge to orgnisms hs een proposed sed on its uiquity cross lmost ll levels of complexity nd orgniztion of life-forms. This thought hs received considerle ttention, nd studies employing diverse strtegies hve ttempted to investigte it. However, only hndful of them hve exmined how selection for circdin clock controlled rhythmic ehviors influences lifehistory trits which re known to influence Drwinin fitness. The erly nd lte chronotypes re mongst the most widely studied circdin phenotypes; however, life-history trits ssocited with these chronotypes, nd their consequences on Drwinin fitness remin lrgely unexplored, primrily due to the lck of suitle model system. Here we studied severl life-history trits of Drosophil melnogster popultions tht were sujected to lortory selection for morning (erly) nd evening (lte) emergence. Results: We report tht the lte eclosion chronotypes evolved longer pre-dult durtion s compred to the erly eclosion chronotypes oth under light/drk (LD) nd constnt drk (DD) conditions, nd these differences pper to e medited y oth clock dependent nd independent mechnisms. Furthermore, longer pre-dult durtion in the lte chronotypes does not led to higher ody-mss t puprition or eclosion, ut the lte femles were significntly more fecund nd lived significntly shorter s compred to the erly femles. Conclusions: Coevolution of multiple life-history trits in response to selection on timing of eclosion highlights correltions of the genetic rchitecture governing timing of eclosion with tht of fitness components which suggests tht timing ecologiclly relevnt ehviors t specific time of the dy might confer dptive dvntge. Keywords: Circdin, Adptive significnce, Fitness, Lortory selection, Life-history evolution Bckground It is elieved tht circdin timekeeping mechnisms underlying rhythmic processes provide dptive dvntge to orgnisms [1 7], nd this hs prompted studies employing vriety of strtegies to exmine the dptive enefits of possessing functionl circdin clocks. Surgicl ltion of the mmmlin mster circdin clock - suprchismtic nucleus [8], nd genetic mnipultion of circdin clocks in fruit flies Drosophil melnogster [9] which re known to drive loss of rhythmicity in severl key circdin ehviors result in reduced * Correspondence: vshrm@jncsr.c.in Chronoiology Lortory, Evolutionry nd Orgnisml Biology Unit, Jwhrll Nehru Centre for Advnced Scientific Reserch, Jkkur, PO Box. 6436, Bnglore, Krntk , Indi survivorship [10 13]. Environmentlly induced or nturlly occurring circdin dysfunction hs lso een reported to reduce longevity in D. melnogster [14, 15]. Bever et l. [16, 17] reported tht D. melnogster strins crrying loss-of-function muttion in two core clock genes exhiit reduced reproductive output. In ddition, studies on orgnisms inhiting different ltitudes, s well s those living in constnt conditions reported lrge vrition in circdin phenotypes in ccordnce to their locl hitts, suggesting tht the underlying clocks my hve evolved s n dpttion to the presence or sence of cyclic environmentl conditions [6, 18 28]. Nevertheless, conclusions drwn from such studies re limited y the lck of dequte informtion out the ncestry, popultion size nd history of 2016 Nikhil et l. Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 2 of 14 the environmentl conditions pertining to the orgnism s ecology [6]. The eclosion wveform of D. melnogster comprises primry pek t dwn (under nturl conditions) or round night-dy trnsition (under lortory light/drk cycles) which grdully reduces through the dy with little or no eclosion occurring t night (Additionl file 1: Figure S1; [9, 29]). This restriction/gting of eclosion primrily round dwn is hypothesized to e n dpttion to void desicction of phrte dults y high temperture nd low humidity previling during the rest of the dy [3], prtly supported y the results of recent study [30]. Lortory selection pproch hs een previously dopted to study how circdin clocks evolve in response to selection for time/phse of eclosion. Selection for erly nd lte emerging strins of Drosophil pseudooscur nd moth Pectinophor gossypiell under LD12:12 (12 h of light nd drk cycles ech) resulted in the evolution of divergent phse of eclosion (4 h in D. pseudooscur nd 5 h in P. gossypiell) [31, 32]. As correlted response, the erly flies in oth studies evolved longer circdin clock period while the lte flies evolved shorter period. However, these studies suffered from some mjor shortcomings such s lck of popultion level repliction, detils of popultion ncestry nd selection protocols employed (popultion mintennce methodology, popultion size nd sex rtio) which re known to considerly modify the evolutionry trjectories in response to selection; nd thus might hve led to misinterprettion of the oserved responses to selection (reviewed in [6]). Although these studies suggest tht circdin clocks might hve evolved to ensure temporl order in ehvior nd physiology thus enhncing Drwinin fitness (reviewed in [6]), our understnding of how selection for timing of clock controlled ehviors influence life-history trits remins nominl. To explore the evolutionry trjectory of circdin clocks in response to selection for timing of eclosion, we initited long-term study on D. melnogster popultions y imposing selection for eclosion during erly morning nd lte evening hours, which is in contrst to the usul time of eclosion in this species. From set of 4 ncestrl control popultions we derived 8 popultions - 4 replicte erly popultions using flies tht eclose erly in the morning nd 4 replicte lte popultions using flies tht eclose lte in the evening (Additionl file 1: Figure S2; see mterils nd methods for detiled selection protocol). Consequently, the erly 1-4 nd the lte 1-4 popultions evolved significntly higher morning nd evening eclosion respectively reltive to the control 1-4 popultions, nd exhiited severl properties nlogous to the well-known morning/erly nd evening/lte chronotypes in humns. Similr to the erly nd the lte humn chronotypes [33 35], the erly nd the lte Drosophil popultions evolved shorter nd longer clock periods respectively with the control popultions exhiiting intermedite period [36], nd lso exhiited diverged photic phse response curves (PRCs) for oth eclosion [36] nd ctivity/rest rhythms [37]. These results indicte tht circdin clocks of the two sets of popultions entrin differently to LD cycles, or in other words, they re differentilly sensitive/interct differentilly with LD cycles. This is corroorted y the results of previous study which reported tht the erly popultions re sensitive to light primrily in the evening while the lte popultions re sensitive to light primrily in the morning [38]. Collectively, these studies suggest tht divergent coevolution of circdin clocks in the erly nd the lte popultions might medite differentil interction/entrinment to regulte time of eclosion. In the present study, we used the erly nd the lte popultions to exmine genetic correltions etween mechnisms tht underlie eclosion t specific time of the dy nd tht of vrious pre-dult (egg-to-puprium nd egg-to-dult durtion, egg-to-puprium nd egg-todult survivorship, nd pupril dry-weight) s well s dult life-history trits (dry-weight t eclosion, fecundity, pre- nd post-fecundity ssy dry-weight, nd longevity). As discussed erlier, the erly nd the lte eclosion chronotypes hve een shown to e ssocited with different circdin clock period nd differentil entrinment to LD cycles, nd pre-dult trits such s egg-todult durtion is known to e correlted with circdin clock period. Therefore, to ssess the reltive contriution of circdin clock period nd differentil entrinment to LD cycles in driving life-history trit differences etween the erly nd the lte popultions, we performed some of our experiments under oth LD12:12 s well s constnt drkness (DD). The rtionle eing tht if differences in life-history trits etween the erly nd the lte popultions re solely determined y circdin clock period s cn e oserved under DD when the clock is not under the influence of LD cycles, such differences would either decrese or cese to exist ecuse clock period of ll the popultions would e held t 24 h in LD 12:12 y virtue of entrinment [8]. Persistence of differences etween popultions under oth light regimes would imply tht the oserved life-history trit differences re lso driven y clock independent mechnisms. As mentioned erlier, since D. melnogster eclose predominntly during dwn, eclosion t other times of the dy is considered to e mldptive (Additionl file 1: Figure S1; [3]). If this is true, then the proportion of individuls which normlly eclose erly in the morning in the control popultions might lso differ in terms of fitness from those tht eclose lte in the evening. To test for such possiility, one genertion efore the ssys

3 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 3 of 14 we derived 8 dditionl popultions from the controls 4 popultions comprising individuls emerging erly in the morning, referred to s the erly-control, nd similrly, 4 popultions comprising individuls emerging lte in the evening, referred to s the lte-control. Also, the erly-control nd the lte-control popultions re likely to revel whether the oserved differences in fitness mesures etween the erly nd the lte popultions (if ny) re indeed evolved responses to the selection imposed on the timing of eclosion, or re merely environment-driven. We report tht the lte popultions hve evolved significntly longer medin egg-to-puprium durtion leding to longer egg-to-dult durtion, re more fecund round dy 11 post-emergence which is the usul dy for egg collection s per the selection protocol (see mterils nd methods), nd lso exhiit reduced medin longevity s compred to the erly popultions, wheres the erly-control nd the lte-control popultions did not differ in the foresid life-history trits thus suggesting tht the oserved differences etween the selected popultions (erly nd lte) re evolutionry responses to selection for timing of eclosion. Also, even though the erly popultions differed significntly from the lte popultions, they were similr to the control popultions for most of the trits ssyed, the possile resons for which re discussed lter. Results Egg-to-puprium durtion ANOVA on medin egg-to-puprium durtion showed sttisticlly significnt effect of popultion, light regime nd popultion light regime interction (Tle 1). Across light regime comprisons reveled tht egg-to-puprium Tle 1 Summry of results of ANOVA on () medin egg-to-puprium durtion, () rc-sine squre root trnsformed egg-to-puprium survivorship, (c) medin egg-to-dult durtion, (d) rc-sine squre root trnsformed egg-to-dult survivorship, (e) dry-weight t puprition, nd (f) dry-weight t eclosion vlues of ll popultions under LD12:12 nd DD light regimes. Summry of results of ANOVA on (g) verge eggs lid/femle, (h) dry-weight t pre- nd post fecundity ssy stges, (i) log trnsformed fecundity per unit dry-weight loss, nd (j) medin longevity of virgin mles nd femles of ll popultions under LD12:12 Trit Effect df MS F p () medin egg-topuprium durtion popultion < light regime < popultion light regime () egg-to-puprium survivorship popultion light regime popultion light regime (c) medin egg-to-dult durtion popultion < light regime < popultion light regime (d) egg-to-dult survivorship popultion light regime popultion light regime (e) dry-weight t puprition popultion light regime popultion light regime (f) dry-weight t eclosion popultion light regime popultion light regime (g) eggs lid/femle popultion < (h) pre- nd post-fecundity dry-weight popultion stge < popultion stge < (i) fecundity/unit dry-weight loss popultion < (j) longevity popultion < sex popultion sex

4 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 4 of 14 durtion for ll the popultions ws significntly longer (y 8.4 h or 7 %) in LD12:12 s compred to tht in DD thus highlighting the delying effect of LD cycles on egg-topuprium durtion (Fig. 1-c; Additionl file 1: Figure S3; Additionl file 1: Tle S1). In LD12:12, the lte popultions hd significntly longer (y 6.5 h or 5.4 %) egg-to-puprium durtion ( h) s compred to ll other popultions (erly = h, erly-control = h, control = h nd lte-control = h) while tht for the remining four sets of popultions did not differ mong ech other (Fig. 1, c; Additionl file 1: Figure S3; Additionl file 1: Tle S1). In DD, the lte popultions took significntly longer (y 5 h or 3.6 %) to puprite ( h) s compred to the erly ( h) nd the control ( h) popultions ut did not differ from the erly-control ( h) nd the lte-control ( h) popultions, wheres none of the other sets of popultions differed mong ech other (Fig. 1, c; Additionl file 1: Figure S3; Additionl file 1: Tle S1). Egg-to-puprium survivorship ANOVA on egg-to-puprium survivorship reveled tht the effect of popultion, light regime nd popultion light regime interction ws sttisticlly not significnt (Tle 1), indicting tht the popultions did not differ in their egg-to-puprium survivorship oth within nd cross light regimes. The verge egg-to-puprium survivorship cross popultions ws ± 1.24 % (men ± SD) in LD12:12 (erly = %, erly-control = %, control = %, ltecontrol = % nd lte = %) nd ± 1.72 % (men ± SD) in DD (erly = %, erly-control = %, control = %, lte-control = % nd lte = %; Fig. 1d; Additionl file 1: Tle S2). Egg-to-dult durtion ANOVA on medin egg-to-dult durtion reveled sttisticlly significnt effect of popultion, light regime nd popultion light regime interction (Tle 1c). As oserved for egg-to-puprium durtion, the egg-to-dult durtion in LD12:12 ws lso significntly longer (y 16 h or 7.5 %) for ll the popultions s compred to tht in DD (Fig. 2-c; Additionl file 1: Figure S4). In LD12:12, egg-to-dult durtion of the lte popultion ( h) ws significntly longer (y 4 h or 1.9 %) thn ll popultions (erly = h, erly-control = h nd lte-control = h) except the control ( h) popultions wheres tht of ll other popultions (erly, erly-control, control nd lte-control) did not differ significntly mong ech other (Fig. 2, c; Additionl file 1: Figure S4; Additionl file 1: Tle S1). % puprition () time from eggcollection (h) % puprition () time from eggcollection (h) medin egg-topuprium durtion (h) (c) c c,d c popultion % egg-to-puprium survivorship (d) LD DD LD DD c,d d popultion Fig. 1 Percentge puprition s function of time (in hours) from egg-collection in LD12:12 nd DD for the erly (E), the erly-control (EC), the control (C), the lte-control (LC) nd the lte (L) popultions. The lck nd white rs t the ottom represent night nd dy respectively. c Medin egg-to-puprium durtion (time tken for 50 % of totl puprition events) of ll popultions in LD12:12 nd DD, nd d Percentge egg-to-puprium survivorship vlues in LD12:12 nd DD. Error rs for pnels c nd d indicte 95 % confidence intervls clculted y method of Tukey s HSD. Brs shring sme letters do not differ sttisticlly while those with different letters re significntly different from ech other

5 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 5 of 14 % eclosion () time from eggcollection (h) % eclosion () time from eggcollection (h) medin egg-todult durtion (h) (c) c c, c popultion % egg-to-dult survivorship (d) LD DD LD DD c d popultion Fig. 2 Percentge of flies eclosing s function of time (in hours) from egg-collection in LD12:12 nd DD for the erly (E), the erly-control (EC), the control (C), the lte-control (LC) nd the lte (L) popultions. The lck nd white rs t the ottom represent night nd dy respectively. c Medin egg-to-dult durtion of ll popultions in LD12:12 nd DD, nd d Percentge egg-to-dult survivorship vlues of ll popultions in LD12:12 nd DD. All other detils re sme s in Fig. 1 Under DD, the lte popultions exhiited significntly longer (y 8 h or 3.5 %) egg-to-dult durtion ( h) s compred to ll other popultions (erly = h, erly-control = h nd ltecontrol = h) except the controls ( h; Fig. 2, c; Additionl file 1: Figure S4; Additionl file 1: Tle S1). Egg-to-dult survivorship ANOVA on egg-to-dult survivorship reveled sttisticlly significnt effect of light regime nd popultion light regime interction ut not of popultion (Tle 1d). However, post hoc multiple comprisons using Tukey s HSD did not revel ny sttisticlly significnt difference in eggto-dult survivorship either cross LD12:12 (erly = %, erly-control = %, control = %, ltecontrol = % nd lte = %) nd DD (erly = %, erly-control = %, control = %, ltecontrol = % nd lte = %) light regimes or cross popultions within light regime (Fig. 2d; Additionl file 1: Tle S2). Dry-weight Since the lte popultions exhiited significntly longer egg-to-puprium nd egg-to-dult durtion, we further tested if this lengthening of pre-dult developmentl durtion trnslted to higher dry-weight t puprition nd eclosion. ANOVA on pupl dry-weight reveled sttisticlly significnt effect of popultion nd light regime ut not of popultion light regime interction (Tle 1e). In ccordnce with the difference in egg-to-puprium durtion etween light regimes, the pupl dry-weight ws found to e significntly higher (on n verge y 6.3 %) in LD12:12 (erly = μg, erly-control = μg, control = μg, lte-control = μg nd lte = μg; Fig. 3; Additionl file 1: Tle S3) s compred to tht in DD (erly = μg, erly-control = μg, control = μg, lte-control = μg nd lte = μg; Fig. 3; Additionl file 1: Tle S3) wheres no difference ws oserved etween popultions within either of the light regimes. ANOVA on dry-weight t eclosion reported sttisticlly significnt effect of light regime ut not of popultion or popultion light regime interction (Tle 1f). In ccordnce with egg-to-dult durtion differences cross light regimes, dry-weight t eclosion ws found to e significntly higher (on n verge y 4.35 %) in LD12:12 (erly = μg, erly-control = μg, control = μg, lte-control = μg nd lte = μg; Fig. 3; Additionl file 1: Tle S3) s compred to tht in DD (erly = μg, erly-control = μg, control = μg, lte-control = μg nd lte = μg; Fig. 3; Additionl file 1: Tle S3) wheres the popultions did not differ mong ech other in either of the light regimes.

6 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 6 of 14 dry-weight (µg) () puprium dry-weight (µg) popultion () dult LD DD LD DD popultion Fig. 3 Dry-weight per individul t puprition, nd eclosion for the erly (E), the erly-control (EC), the control (C), the lte-control (LC) nd the lte (L) popultions in LD12:12 nd DD light regimes. All other detils re sme s in Fig Fecundity ANOVA on verge fecundity dt reveled sttisticlly significnt effect of popultion (Tle 1g). Fecundity of the lte popultions (10.80 eggs/fly) ws significntly higher (y 32 %) s compred to tht of the other popultions (erly = 7.32 eggs/fly, erly-control = 7.74 eggs/fly, control = 7.01 eggs/fly nd lte-control = 7.68 eggs/fly), wheres none of the other popultions differed significntly mong ech other (Fig. 4; Additionl file 1: Tle S4). Pre- nd post-fecundity ssy dry-weights ANOVA on femle dry-weight mesurements t pre- nd post-fecundity ssy stges showed sttisticlly significnt effect of stge (pre/post-fecundity ssy) nd popultion stge interction ut not of popultion (Tle 1h). Post hoc multiple comprisons reveled tht post-fecundity ssy dry-weight of ll the popultions ws reduced y out μg (22 %; Fig. 4) s compred to the prefecundity ssy dry-weight. Pre-fecundity ssy dry-weight did not differ sttisticlly etween popultions (erly = μg, erly-control = μg, control = μg, lte-control = μg nd lte = μg; Tle 1h; Additionl file 1: Tle S4) ut post-fecundity ssy dryweight of the lte popultions ( μg) ws significntly lower (y 17 μg or ~10 %) s compred to tht of ll other popultions (erly = μg, erly-control = μg, eggs lid/femle fecundity/dry-weight lost (eggs/µg) () (c) popultion popultion dry-weight (µg) dry-weight lost (µg) () (d) pre-fec. popultion post-fec. c numer of eggs Fig. 4 Averge fecundity per femle on dy 11 (post-eclosion), dry-weight per femle t pre- nd post-fecundity stges, nd c fecundity per unit dry-weight loss (difference in pre- nd post-fecundity dry-weight) for the erly (E), the erly-control (EC), the control (C), the lte-control (LC) nd the lte (L) popultions in LD12:12. Error rs indicte 95 % confidence intervls clculted y method of Tukey s HSD. d Person correltion of verge fecundity/ femle with tht of dry-weight lost during the fecundity ssy (r = +0.75, p < ). The dt points for correltion were otined y pooling vlues cross ll 20 popultions. The dotted line indictes 95 % confidence intervl. Fecundity nd dry-weight mesurements ssys were performed only in LD12:12. Brs shring sme letters do not differ sttisticlly while those with different letters re significntly different from ech other

7 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 7 of 14 control = μg nd lte-control = μg; Fig. 4; Tle 1h; Additionl file 1: Tle S4). Fecundity per unit loss in dry-weight When normlized y the dry-weight lost (difference in pre- nd post-fecundity ssy dry-weight), fecundity per unit dry-weight lost did not differ sttisticlly cross popultions (erly = 0.15 eggs/μg, erly-control = 0.16 eggs/μg, control = 0.17 eggs/μg, lte-control = 0.15 eggs/ μg nd lte = 0.15 eggs/μg; Fig. 4c; Tle 1i), suggesting tht lthough the lte popultions were more fecund they lose more dry-weight due to the higher numer of eggs lid. As n dditionl confirmtion of this, we performed liner correltion etween egg output nd dryweight loss y pooling dt from ll the popultions, nd found tht the two were significntly positively correlted (r = +0.75, p < ; Fig. 4d). Longevity ANOVA on medin longevity reported sttisticlly significnt effect of popultion, sex nd popultion sex interction (Tle 1j). With the exception of the lte popultions where individuls of oth the sexes hd n verge medin longevity of ± dys (men ± SD), the verge femle longevity of ll the other popultions (erly = 47.5 dys, erly-control = dys, control = dys nd lte-control = dys; Fig. 5; Additionl file 1: Tle S4) ws ~7 % higher thn tht of the mles (erly = dys, erly-control = dys, control = dys nd lte-control = dys; Fig. 5; Additionl file 1: Tle S4). Within sex comprisons reveled tht femles of the lte popultions exhiited significntly shorter (~12 %) medin longevity s compred to femles of the other popultions with the exception of the lte-control femles which did not differ sttisticlly from the lte popultions. The medin mle longevity ws not oserved to differ sttisticlly cross popultions (Fig. 5; Additionl file 1: Tle S4). Discussion We oserve tht the lte popultions evolved longer egg-to-puprium nd egg-to-dult durtion s compred to the erly popultions thus highlighting n ssocition etween eclosion chronotype nd pre-dult developmentl durtion (Figs. 1-c, 2-c). Under LD12:12, the difference in medin egg-to-puprium durtion etween the lte nd the erly popultions is ~7 h, which is reduced to ~3 h t eclosion (Figs. 1c, 2c). One possile reson for this might e genotype dependent effect of light on pupl development s lso suggested y the medin eggto-purium durtion of the lte popultions which differs from ll other popultions under LD12:12 ut not DD (Fig. 1c). Alterntively, under LD cycles, timing of eclosion is known to e governed y circdin clock component s well s clock independent msking response to lights-on s discussed with respect to the sme popultions in previous study [39]. Thus, in ddition to the clock determined time of eclosion, % survivorship () mles ge (dys) % survivorship () femles ge (dys) medin longevity (dys) (c) mle femle Fig. 5 Survivorship curves of virgin mles nd virgin femles of the erly (E), the erly-control (EC), the control (C), the lte-control (LC) nd the lte (L) popultions in LD12:12. c Medin longevity (time tken for 50 % of individuls to die) of virgin mles nd femles of ll popultions in LD12:12. All other detils re sme s in Fig. 1

8 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 8 of 14 msking response to light, which results in n dditionl urst of eclosion immeditely following lights-on might hve reduced the medin pre-dult durtion. This is further supported y our oservtion under DD tht ~4 h difference in egg-to-puprium durtion etween the lte nd the erly popultions increses to ~7 h t eclosion (Figs. 1c, 2c). Thus, the oserved reduction in medin egg-to-dult durtion under LD12:12 my e result of the comintion of oth () rtefct of msking response to lights-on which is clerly sent under DD nd () differentil effect of light on pupl development, which remins to e ddressed further. In ddition to the divergent eclosion chronotypes, the erly nd the lte popultions hve lso evolved shorter nd longer clock periods differing y 40 min [36, 39] which suggests correltion etween emergence chronotype nd circdin clock period. Such correltions hve een reported erlier in the melon fly, Bctrocer cucurite [40, 41], nd etween clock period nd egg-to-dult durtion in fruit flies D. melnogster [42 44], suggesting tht clock period differences influence pre-dult developmentl rtes. In DD, egg-to-puprium durtion of the lte popultions ws h (4.9 dys) nd egg-to-dult durtion ws h (9.4 dys) s opposed to h (4.7 dys) nd h (9.1 dys) respectively of the erly popultions. If the pre-dult durtion of the erly nd the lte popultions ws entirely driven y circdin clock period difference, under DD the erly nd the lte popultions would drift prt y 0.66 h (40 min) every dy, nd consequently the two popultions would exhiit 3.12 h difference in egg-to-puprium durtion (in 4.7 dys which is equl to the time tken y the erly popultion to puprite) nd 6.01 h difference in egg-to-dult durtion (in 9.12 dys) which is considerly smller thn tht oserved empiriclly (Figs. 1, c; 2, c). Since eggs for the egg-to-puprium nd egg-to-dult durtion ssys were collected from ll popultions t the sme time (thus were ge mtched), the oserved differences in pre-dult durtion etween the erly nd the lte popultions re unlikely to e due to the differences in the ge of eggs. Moreover, the time of egg-collection or the ge of eggs does not lter the difference in egg-todult durtion etween the erly nd the lte popultions [45]. Tken together these results suggest tht difference in pre-dult developmentl rtes of the erly nd the lte popultions is not entirely circdin clock driven, nd my lso involve clock independent mechnisms which might drive differentil interction with LD cycles (significnt popultion light regime interction reported in Tle 1, c). Furthermore, light medited enhncement in the predult developmentl rtes is pprent s oth egg-topuprium nd egg-to-dult durtion of ll the popultions ws % longer in LD12:12 s compred to DD (Figs. 1-c, 2-c; Additionl file 1: Figure S3, Additionl file 1: Figure S4; Additionl file 1: Tle S1). While effect of light on pre-dult durtion hs een documented erlier [46, 47], precise mechnisms underlying such effects remins to e explored. The timing of eclosion in Drosophil is elieved to depend upon numer of fctors including the developmentl stte of the fly, the phse nd period of circdin rhythm, hormonl cscde, nd environmentl condition [48, 49]. The LD cycles interct with the circdin clock controlled gte of eclosion such tht even if flies hve completed development, they re llowed to eclose only during certin time of the dy nd not merely in ccordnce with their developmentl stte, nd consequently the time of eclosion is delyed in LD12:12 s compred to DD [47 50]. Additionlly, the time of eclosion on given dy is lso function of the circdin clock period such tht individuls with shorter period eclose erlier thn those with longer clock period [51]. This further supports the notion tht pre-dult development in D. melnogster is proly medited y the interction of circdin clocks with LD cycles. Differences in pre-dult developmentl rtes of the erly nd the lte popultions do not seem to influence their egg-to-puprium nd egg-to-dult survivorship; nor do the light regime medited differences ffect pre-dult survivorship (Figs. 1d, 2d; Additionl file 1: Tle S2). This might e due to the mgnitude of difference in egg-topuprium or egg-to-dult durtion etween the popultions not eing lrge enough to influence egg-topuprium nd egg-to-dult survivorship. Although the lte popultions hve evolved longer predult developmentl durtion, their ody-weight t puprition nd t eclosion did not differ from tht of ll the other popultions (Fig. 3; Additionl file 1: Tle S3). However, dry-weight of pupri nd dults were found to e significntly higher for ll the popultions in LD12:12 s compred to DD (Fig. 3; Additionl file 1: Tle S3) which is not surprising s egg-to-puprium nd egg-todult durtion is significntly enhnced in LD12:12 s compred to DD (Figs. 1, 2). Coevolution of pre-dult life-history trits in response to selection for timing of eclosion is intuitive, s chnges in pre-dult stges cn directly ffect the time course nd wveform of eclosion. It would e interesting to know whether selection for eclosion t different time of the dy lso led to correlted chnges in dult life-history trits tht my not necessrily influence eclosion time ut would highlight the underlying genetic correltion. In this regrd, we oserved tht the lte popultions exhiited significntly higher fecundity s compred to ll other popultions (Fig. 4; Additionl file 1: Tle S4). In D. melnogster, pre-dult developmentl durtion is known to e correlted with fecundity, nd delyed development is ssocited with higher dry-weight, nd in turn with higher

9 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 9 of 14 fecundity [52 54]. This does not pper to e the cse in the lte popultions since neither their dry-weight t eclosion nor pre-fecundity ssy dry-weight differed from the other popultions (Fig. 4; Additionl file 1: Tle S4). However, post-fecundity ssy dry-weight of the lte popultions ws significntly lower s compred to tht of the other popultions (Fig. 4; Additionl file 1: Tle S4), ut when normlized y the loss of dry-weight (difference in pre- nd post-fecundity ssy dry-weight), fecundity per unit dry-weight lost ws similr for ll popultions (Fig. 4c). Therefore, significnt reduction in postfecundity ssy dry-weight in the lte popultions ppers to e consequence of higher numer of eggs lid s lso sustntited y significnt correltion oserved etween the numer of eggs lid nd dry-weight lost (Fig. 4d). Therefore, contrry to the well-known positive correltion etween pre-dult developmentl durtion, dry-weight nd fecundity, our results suggest tht the oserved higher fecundity in the lte popultions is not due to higher dry-weight ttined ecuse of the dely in the timing of eclosion ut my e due to other mechnisms such s pleiotropy or muttion ccumultion [55]. Alterntively, higher fecundity in the lte popultions might hve evolved s n rtefct of the nture of selection protocol employed. For instnce, to ensure tht the numer of dults in ll popultions is ~1200, every genertion we collect lrger numer of eggs for the lte popultions followed y reltively smller numer of eggs for the erly popultions s compred to the control popultions (see mterils nd methods) which is 24 vils per replicte popultion for the erly popultions, 48 vils for the lte popultions s opposed to 16 vils for the control popultions with ech vil housing pproximtely 300 eggs. Therefore, the numer of eggs collected from the lte popultions (~14400 eggs) is pproximtely twice tht of the erly (~7200 eggs) nd thrice tht of the control (~4800 eggs) popultions. This might hve led to n indvertent selection for higher fecundity in the lte popultions, nd lso, possily s consequence of higher effective popultion size (N e )thelte popultions might experience reltively lower extent of inreeding depression followed y the erly popultions, with the control popultions experiencing highest degree of inreeding depression. If this were to e true, then the erly popultions would lso e expected to exhiit higher fecundity s compred to the control popultions, ut tht does not seem to e the cse. Therefore, it is unlikely tht this resoning cn ccount for the evolution of higher fecundity in the lte popultions even though it cnnot e entirely disregrded. However, the possiility of such scenrio cn lso e clrified y cross experiment etween the erly nd the lte popultions. Additionlly, given tht fecundity in Drosophil is not constnt cross lifespn, the difference in fecundity etween the popultions oserved on dys post-eclosion might lso vry cross different ges. For instnce, in light of the results from previous study [55], it is lso possile tht the erly popultions which exhiit significntly lower fecundity on dys might otherwise exhiit higher fecundity t n erlier stge nd vice vers for the lte popultions. However, since the fly popultions used in the current study re mintined on non-overlpping 21 dy genertion cycle (see mterils nd methods) wherein eggs lid only on the dy 11 of dulthood re used for the next genertion, only eggs lid round this dy determines the popultions fitness in this regime. Therefore, fecundity during other lifestges is irrelevnt under the currently discussed regimen ut nevertheless will e interesting to exmine. Further, we found tht femles of the lte popultions live significntly shorter s compred to those from the erly nd the control popultions, while no difference in longevity ws oserved etween mles (Fig. 5-c). The reduction in longevity of the lte femles s compred to the erly nd the control femles ws consistently oserved in four replicte popultions mintined under similr environmentl conditions. In light of fecundity nd dry-weight results, the oserved reduction in longevity of the lte femles ppers to represent the clssic trde-off etween fecundity nd dult lifespn due to the ntgonistic pleiotropic effects of the underlying genes [56 58]. However, since the results presented here re on virgins, the oserved reduction in longevity cnnot e explined entirely y such trde-off. Therefore, even though higher reproductive output my hve evolved s n rtefct of the selection protocol, reduced longevity in the lte femles s compred to the erly nd the control femles my hve evolved s correlted response to selection for lte evening eclosion nd not directly s consequence of higher fecundity. Interestingly, we lso oserve tht the reduced longevity in the femles of the lte popultions is primrily consequence of erly deth (round dys 20-40) nd not during the lter lifestges, nd similr trend is lso oserved in the mles. However, the possile reson for such oservtions remins to e explored. Thus, we report tht selection for lte evening eclosion in fruit flies D. melnogster is ssocited with the coevolution of severl life-history trits in the lte popultions, while no difference ws oserved etween the erly, the erly-control nd the lte-control popultions. Such correltions etween chronotypes/circdin clocks nd life-history trits hve een reported erlier. Notly, Ydv nd Shrm [59, 60] demonstrted tht selection for fster pre-dult development leds to the coevolution of shorter clock period, nd tht the fster developing popultions evolve reduced dry-weight, ody size, fecundity, strvtion nd desicction resistnce, nd longevity. Similrly, in seprte study on the melon fly

10 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 10 of 14 Bctrocer cucurite, selection for egg-to-dult durtion resulted in the coevolution of divergent phse of ctivity/rest nd mting rhythms [40]. Most differences oserved in our study, however, correspond to the lte popultions reltive to the control popultions, while very little difference ws oserved etween the erly nd the control popultions. Also, most of the life-history trits ssyed in the lte popultions differed y smll mgnitude vrying from 2-10 % s compred to the erly nd the control popultions. This is not surprising considering the lrger time difference etween the selection window of the lte popultions from the eclosion pek of the control popultions, nd proximity of the selection window of the erly popultions from the eclosion pek of the control popultions (see mterils nd methods). Since evening eclosion is not predominntly seen in the control popultions, the lte popultions would experience much stronger selection pressure s compred to the erly popultions which in turn might drive fster coevolution of life-history trits. In summry, selection for lte evening eclosion leds to lengthening of pre-dult durtion without ny increse in ody-weight t eclosion, incresed fecundity ssocited with greter post-fecundity ssy dry-weight loss nd reduced virgin femle longevity. The oserved life-history trits in the lte popultions eing evolved responses to selection is further supported y our oservtion on the erly-control nd the lte-control popultions. Tht life-history trits of the erly-control nd the lte-control popultions did not differ significntly from ech other ut were different from tht of the erly nd the lte popultions (in most cses) suggests tht the oserved life-history trit differences etween the erly nd the lte popultions re evolutionry response to the imposed selection nd re not merely environmentlly driven. Furthermore, lthough the pre-dult nd dult life-history trits studied here re known to e highly correlted, enhnced fecundity in the lte popultions does not seem to e consequence of higher iomss ttined y lengthening of egg-to-dult durtion. Thus the differences in dult trits do not seem to e ssocited with pre-dult trit differences nd pper to e driven y independent mechnisms tht might hve evolved s consequence of selection. Conclusions Thus, in contrst to studies which demonstrted the effect of direct mnipultion of circdin clock on fitness spects (see introduction), we report coevolution of life-history trits in independently evolved replicte popultions of D. melnogster exhiiting erly nd lte eclosion chronotypes, suggesting tht the genetic rchitecture underlying eclosion t specific times of the dy (eclosion chronotypes) is geneticlly correlted with severl life-history trits, nd these correltions pper to encompss oth circdin clock-dependent nd clock-independent mechnisms. Thus the extent of circdin clocks influence in the oserved trit differences, nd the underlying genetic rchitecture remins to e explored. Methods Experimentl popultions nd ssy conditions Additionl file 1: Figure S2 presents schemtic of the selection protocol employed to generte the erly nd the lte popultions from the control popultions. Popultions selected for erly morning nd lte evening eclosion comprised four replictes ech of the erly i nd the lte j (i = j = 1-4) initited from four replictes of the control k (k = 1-4) whose ncestry detils re provided elsewhere [36]. Briefly, the erly nd the lte popultions with given suscript were derived from the control popultion with the sme suscript, nd therefore shre common ncestry. For exmple, the erly 1 nd the lte 1 popultions were initited from the control 1 popultion, nd similrly for the other three replictes. Since our study ims t exploring evolutionry trjectories of trits in popultion, the unit of iologicl repliction is popultion nd thus, the four popultions of ech selection type re iologicl replictes in ll our experiments. All 12 popultions (four replictes ech for erly, control nd lte) were mintined on 21 dy discrete genertion cycle, nd flies were housed in plexi-glss cges of dimension cm 3 with ~1200 individuls per cge (sex rtio ~1:1), nd were provided with d liitum nn-jggery (BJ) medium. The prentl popultions were provided with food supplemented with yest pste (to oost their fecundity) for three dys prior to egg collection, nd ~300 eggs were collected nd dispensed into ech culture vil (16 vils for control, 24 for erly nd 48 for lte popultions) contining ~6 ml BJ medium. From the initition of eclosion, which is generlly on dy 9 (t 25 C) post egg collection, flies tht eclosed erly in the morning etween Zeitgeer Time (ZT) (ZT00 nd ZT12 represents time of lights- ON nd lights-off respectively under LD12:12) for 3-4 consecutive dys were collected to form the erly popultions, while flies tht eclosed lte in the evening etween ZT09-13 formed the lte popultions. For the control popultions, flies were collected once every 24 h for the sme 3-4 dys nd thus, comprised individuls emerging throughout these 3-4 dys without ny selection imposed on timing of eclosion. The dys of initition nd termintion of fly collection within the respective selection windows ws kept constnt for ll popultions. In other words, if collection of flies for the erly popultions ws strted on dy x nd terminted on dy y, collection of flies for the control nd the lte popultions ws lso initited nd terminted on dys x

11 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 11 of 14 nd y respectively so s to ensure tht the popultions re selected only for eclosion t different gte/time of the dy nd to void ny unintended selection for fster nd slower pre-dult development. The implementtion of selection protocol nd regulr mintennce of popultions ws performed under LD12:12 with ~0.4 Wm -2 light intensity during the light phse, 25 ± 0.5 C temperture, nd 75 ± 5 % reltive humidity. In ddition to the four replicte popultions ech for the erly, the control nd the lte, we used four replictes ech for the two other popultions (erly-control nd lte-control; see Introduction). From the control popultions, flies emerging in the morning window (ZT21-01) were collected to form the erly-control popultions nd similrly, flies emerging in the evening window (ZT09-13) formed the lte-control popultions. This procedure ws implemented on ll four replictes of the control popultions for only one genertion prior to the ssys, nd therefore, unlike the erly nd the lte popultions, the erly-control nd the lte-control popultions were not sujected to ny long-term selection protocol. To minimize the effects of non-genetic inheritnce (reviewed in [61]) due to different selection regimes, ll popultions were sujected to one genertion of stndrdiztion with the mintennce protocol which is identicl to tht used for the control popultions. This ws chieved y relxing selection on timing of eclosion y collecting ll flies tht eclosed throughout the first 4 dys similr to tht for the control popultions while the popultion size ws mintined t ~1200 flies per replicte popultion. Since the primry purpose of using the erly-control nd the lte-control popultions ws to sses if the oserved differences etween the erly nd the lte popultions re evolved responses to selection s not merely environmentl in origin, these popultions were lso sujected to stndrdiztion y deriving them from the control popultions followed y relxtion of selection for one genertion s descried ove. All ssys descried in the present study were performed on the progeny of the stndrdized popultions t the 242 nd genertion (~14 yers) either in LD12:12 or DD, or oth, with light intensity, temperture nd humidity sme s tht for the mintennce of popultions. Fly hndling nd experiments in the drk were performed under dim red light (λ > 650 nm). Egg-to-puprium durtion ssy Egg-to-puprium durtion for ll the popultions ws ssyed under two light regimes LD12:12 nd DD. After hving provided yest pste supplemented medi for three dys, ll popultions were provided with medi pltes for 1 h s sustrte for oviposition. These pltes were then replced y fresh food medium pltes for the next 1 h. Eggs lid on these pltes were collected nd 30 eggs were dispensed into ech vil. A totl of 10 such vils were used per replicte popultion per light regime mking totl of 300 eggs per popultion per light regime. These vils were trnsferred to respective light regimes nd monitored for the first puprition event. After the first puprium ws oserved, vils were checked every two hours to count the numer of pupri formed therefter, nd the ssy ws terminted when no puprition event ws seen for 24 consecutive hours. It ws oserved tht smll proportion of lrve took reltively longer to puprite, thus rendering the egg-to-puprium durtion distriution right skewed (Fig. 1, ; Additionl file 1: Figure S3). Men egg-topuprium durtion cnnot e used s relile mesure for such distriutions [62], nd therefore, we used medin egg-to-puprium durtion (clculted s the time from egg collection for 50 % of totl puprition events in vil) for the sme. The medin egg-to-puprium durtion ws estimted for every replicte vil nd then verged cross vils to otin medin egg-to-puprium durtion for given replicte popultion. Egg-to-dult durtion ssy Egg collection protocol nd environmentl conditions for the egg-to-dult durtion ssy were identicl to eggto-puprium durtion ssy. After egg collection nd trnsfer to LD12:12 or DD, eclosion of the first dult fly ws monitored following which vils were sujected to two hourly checks to count the numer of flies tht eclosed therefter. The ssy ws terminted when no eclosion event ws oserved for 24 h. To fcilitte comprisons etween egg-to-puprium nd egg-to-dult durtion, we used medin durtions s mesure for nlysis. The procedure to estimte medin egg-to-dult durtion ws sme s tht descried for medin egg-topuprium durtion. Estimtion of egg-to-puprium nd egg-to-dult survivorship Egg-collection protocol nd environmentl conditions for the survivorship ssys were sme s tht for the egg-to-puprium nd egg-to-dult durtion ssy. Proportion of 30 eggs (totl numer of eggs dispensed per vil for the ssy) tht successfully puprited ws used s mesure for egg-to-puprium survivorship, while proportion of dults tht successfully eclosed ws used to estimte egg-to-dult survivorship. Individuls tht were stuck in the pupl cse nd died within the pup were considered to not hve eclosed successfully. Percentge survivorship ws clculted for every replicte vil nd then verged cross vils to otin verge survivorship per replicte popultion.

12 Nikhil et l. BMC Evolutionry Biology (2016) 16:46 Pge 12 of 14 Dry-weight t puprition The protocol for egg collection nd susequent environmentl conditions for development under LD12:12 nd DD ws the sme s tht descried for egg-to-puprium durtion ssy. From the initition of the first puprition event, freshly formed pupri (P1 stge) were collected every 2 h nd frozen t -20 C. These pupri were lter sorted into 10 replicte groups with 5 pupri in ech group; dried t 70 C for 36 h fter which their dry weight ws ssyed. Dry-weight of ech group ws mesured t lest thrice to ccount for instrument error nd then normlized y the numer of pupri (n = 5). The dry-weight mesurements from 10 such groups were then verged to otin men puprium dryweight per replicte popultion. Dry-weight t eclosion The protocol for ssying dry-weight t eclosion ws the sme s tht for dry-weight t puprition ssy except tht insted of pupri freshly eclosed (within 2 h of eclosion) dult flies in LD12:12 or DD were used. Fecundity ssy The popultions used in the present study re mintined on 21 dy discrete genertion cycle where eggs for the next genertion re collected on dy 21 post egg collection (verge dult ge of 11 dys). Since only eggs lid round this dy would determine n individul s contriution to the gene pool for the next genertion nd consequently to its fitness, we estimted fecundity only under LD12:12 round dy 11 (post-eclosion) in the progeny of stndrdized popultions, which were collected in plexi-glss cges nd mintined under LD12:12 in mixed-sex groups similr to tht used for regulr mintennce of popultions. On dy 8 (verge dult ge), flies from plexi-glss cges were collected, seprted using mild cron-di-oxide nesthesi nd trnsferred into vils contining ~4 ml BJ medi for conditioning t density of 10 flies/vil (5 of ech sex). In prllel, dditionl sets of conditioning vils were set side from which flies for pre-fecundity dry-weight ssy were to e collected lter (descried in the following section). On dy 10, flies from the conditioning vils were sorted into single mle femle pirs nd trnsferred into 20 vils/popultion contining 1 ml BJ medium. After 24 h (dy 11), flies were trnsferred to fresh set of vils nd the sme ws repeted on dy 12. Averge numer of eggs lid per femle cross dys ws used s mesure of men fecundity/femle round dy 11. Only vils from which dt could e collected for ll three dys were used nd those in which either mle or femle died within the three dys were not used for dt nlysis. Estimtion of pre- nd post-fecundity ssy dry-weights To ssess pre-fecundity ssy dry-weight of femles, 20 femles (for every replicte popultion) from seprte sets of conditioning vils (which were not used for fecundity ssy) s descried in the preceding section were frozen t -20 C t the eginning of dy 10. Additionlly, t the end of the fecundity ssy (end of dy 12), femles used for the ssy were collected nd frozen. All flies were then dried t 70 C for 36 h, sorted into groups of 5 individuls ech nd weighed t lest thrice to estimte dry-weight/femle. Dry-weight mesurements were then verged cross groups to clculte men pre- nd postfecundity ssy dry-weight/femle/replicte popultion. Further, dry-weight loss during fecundity ssy ws estimted y clculting the difference in pre- nd postfecundity ssy dry-weight nd ws used to normlize the fecundity/femle vlues to clculte fecundity per unit dry-weight lost s n estimte for iomss to egg conversion rtio. However, this is under the ssumption tht the iomss lost is entirely converted to eggs lid which my not necessrily e the cse ut nevertheless cn e used s proxy for ssessment of for iomss-toegg conversion rtio. Longevity ssy Longevity of flies ws ssyed only in LD12:12 with environmentl conditions sme s descried previously. Freshly eclosed virgin mles nd femles were collected from the progeny of stndrdized popultions every 6 h over three consecutive dys. On the fourth dy, ll flies of given sex nd popultion were mixed nd rndomly distriuted in groups of 10 flies/vil/sex into 10 replicte vils contining ~4 ml BJ medi. Therefore, every replicte popultion comprised 20 vils in totl with 10 vils for ech sex nd ech vil housing 10 flies (verge ge of 2 dys). Therefter, flies were trnsferred to fresh BJ medi every 3rd dy nd longevity ws estimted y counting the numer of ded flies in ech vil every 24 h. The ssy ws continued until ll flies died. While cre ws tken to ensure no flies escped during trnsfers to fresh medi vils, few of them either escped or were crushed etween the cotton plug nd the vil, nd hence were not considered for clculting percentge survivorship for tht vil. Similr to egg-to-puprium nd egg-to-dult durtion, longevity distriution ws lso right-skewed nd therefore, we used medin longevity (time tken for the deth of 50 % of individuls in given vil) s the mesure of longevity. Sttisticl nlyses All mesures of pre-dult durtion, pre-dult survivorship, fecundity, dry-weight nd longevity were estimted for ech replicte vil nd then verged to otin men vlues for replicte popultions. These replicte mens