Review

Clines in clock genes: fine-tuningcircadian rhythms to the environmentCharalambos P. Kyriacou1, Alexandre A. Peixoto2, Federica Sandrelli3, Rodolfo Costa3

and Eran Tauber1

1 Department of Genetics, University of Leicester, Leicester LE1 7RH, UK2 Laboratorio de Biologia Molecular de Insetos, IOC, Fundacao Oswaldo Cruz, Rio de Janeiro CEP 21040-900, Brazil3 Department of Biology, University of Padova, 35131 Padova, Italy

Glossary

Balancing selection: Selection for more than one allele at a locus at frequencies

too high to be caused by recurrent mutation.

Circadian photosensitivity: The sensitivity of the circadian clock to light, which

is usually studied by giving a brief light pulse to an organism that is free-

running in darkness. A light pulse early at night gives a phase delay (usually of

a few hours) in the circadian cycle, whereas a pulse late at night generates an

advance. The size of the advances and delays provides a measure of the light

sensitivity of the clock. As an example, in D. melanogaster, ls-tim gives smaller

phase delays and advances than s-tim [51].

Clines: A gradual change in a character over the geographical range of a

species or population. Latitudinal clines are often associated with correlated

changes in environmental variables.

Darwinian fitness: The capacity of an individual to reproduce, which can be

measured as the relative proportion of that individual’s genes in the genes of

succeeding generations.

Directional selection: Selection for a phenotype at an extreme of the

phenotypic range, or in the case of a specific allele, selection for that allele

over alternative alleles because it determines a more extreme phenotype.

Effective population size: The average number of individuals in a population

that contribute to the gene pool of succeeding generations.

Entrain: An organism placed in an environmental cycle, for example 12 h of

light, 12 h of dark (LD12:12), will display an ‘entrained’ circadian rhythm to the

imposed 24-h oscillation, which usually differs from its ‘free-running’ pattern.

Free-running: An organism that is entrained to a light-dark cycle and then

placed in constant conditions (for animals, usually constant darkness, DD) will

display its own endogenous free-running rhythm with a period of �24 h.

Resonance: The free-running period of most organisms is close to 24 h,

thereby ‘resonating’ with the environmental cycle.

Selection coefficient: A relative measure of the rate of transmission of a given

allele compared with another allele, usually the wild-type.

Temperature compensation: The ability of the circadian clock to keep a stable

The dissection of the circadian clock into its molecularcomponents represents the most striking and well-stu-died example of a gene regulatory network underlying acomplex behavioural trait. By contrast, the evolutionaryanalysis of the clock has developed more slowly. Herewe review studies that have surveyed intraspecific clockgene variation over large geographical areas and havediscovered latitudinal clines in gene frequencies. Suchspatial patterns traditionally suggest that natural selec-tion shapes genetic variation, but it is equally possiblethat population history, or a mixture of demography andselection, could contribute to the clines. We discuss howpopulation genetics, together with functional assays,can illuminate these possible cases of natural selectionin Drosophila clock genes.

Evolution of the circadian clockThe genetic analysis of the endogenous circadian, 24-hclock represents a spectacular example of the moleculardissection of a gene regulatory network underlying a com-plex behavioural trait [1]. Recent transcriptome [2] andproteome studies [3] in several model organisms haveshown how a large proportion of both ‘omes’ cycles inabundance with 24-h periods, revealing that most bio-logical processeshave a rhythmic component. This reflectsthe four billion year evolution of life on a rotating planetwith its cycles of day and night and warmth and cold.Phylogenetic analysis of clock genes in Cyanobacteria, oneof the earliest photosynthetic organisms, reveals that thekaiC gene might have originated �3000 million years ago(Mya) [4]. The molecular analysis of clocks proceeds at anunprecedented pace, with most studies focusing on identi-fying new clock genes, dissecting clock gene networks andinvestigating how the clock works at molecular, bio-chemical and physiological levels [5–8] (see Figure I inBox 1).

How has the circadian clock evolved? The differentmodel organisms studied by circadian biologists providethe immediate comparative perspective to clock research,and this is buttressed by further studies in non-modelorganisms. For example, in bees and butterflies, clockgenes not only promote circadian functions but are alsoinvolved in related processes such as the development ofsocial behaviour and navigation, respectively [9,10].Furthermore, studies of intraspecific clock gene variation

Corresponding author: Kyriacou, C.P. (cpk@leicester.ac.uk).

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can reveal whether natural selection is involved inmaintaining these polymorphisms. [11]. Within this con-text, geographical surveys of genetic variation are useful,particularly if an association with latitude is observed[12,13]. Such ‘clines’ (see Glossary) indicate that environ-mental factors might shape the genetic variation at thatparticular locus, although it can become difficult to disen-tangle the effects of selection from population history [14–16]. This review focuses on the geographical variation inclock genes, particularly in Drosophila melanogaster,where we are beginning to understand the evolutionaryand functional implications of such spatial patterns.

Environmental challenges to the circadian clockThe molecular basis for the circadian clock is similarbetween flies and mice: several conserved positive andnegative regulatory molecules are shared, most of whichare rhythmically expressed and form a feedback loop (seeFigure I in Box 1) [6,8]. Obligatory delays in the feedback

�24-h period under different temperatures.

$ – see front matter � 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2007.12.003

Box 1. The molecular circadian clock in D. melanogaster

The D. melanogaster circadian clock is generated by a series of

negative and positive feedback loops (Figure I). The expression of

the negative transcriptional regulators TIMELESS (TIM) and

PERIOD (PER), which make up the PER–TIM loop, and CLOCKWORK

ORANGE (CWO) is mediated through binding of their E-boxes

(hexameric sequence CACGTG) by the positive transcription

factors CLOCK (CLK) and CYCLE (CYC), which form a heterodimer

[6,68]. The negative regulators feed back, after a time delay, to

inhibit CLK and CYC activity. These delays are imposed by the

kinases SHAGGY (SGG), casein kinase 2 (CK2) and DOUBLETIME

(DBT) [8] and by phosphatases including protein phosphatases 2A

(PP2A) and 1 (PP1), which modulate the stability of the negative

regulators [6,8,69]. A second feedback loop interlocks with the

PER–TIM loop; Clk negative regulation by VRILLE (VRI) and positive

regulation by PAR domain protein 1e (PDP1e) [6]. However, more

recent data have shed some doubt on the role of PDP1e in this

process [70].

Cytoplasmic PER and TIM levels start to rise at the beginning of

the night, and late at night PER and TIM translocate to the

nucleus where they negatively regulate (through CLOCK-CYCLE)

their own genes. At dawn, the blue-light circadian photoreceptor

cryptochrome (CRY) is activated and interacts with TIM. This

interaction stimulates TIM phosphorylation, which recruits the F-

box protein JETLAG (JET), a component of a SKP1-CULLIN1-F-

box (SCF)-E3 ubiquitin ligase, which polyubiquitylates TIM,

thereby marking it for degradation [8]. Under conditions of

constant darkness, TIM is degraded by the proteasome at

subjective dawn by a CRY-independent pathway. PER monomers

are strong transcriptional repressors, but in the absence of TIM,

PER is vulnerable to phosphorylation by DBT kinase, and

phosphorylated PER is recognized by the F-box protein SUPER-

NUMARY-LIMBS (SLIMB) and marked for proteasomal degrada-

tion [6,8]. The negative regulation of per and tim is now lifted,

and CLK–CYC begins the cycle again by binding the per and tim

E-boxes. In the brain, the PER and TIM proteins are expressed in

�75 neurons on each side of the brain, as well as in glia and

photoreceptors [71]. Of the two main groups of neurons, the

dorsal and the lateral neurons, the lateral neurons are indis-

pensable for rhythmic behavioural expression [72,73]. However,

many peripheral tissues of the fly are also rhythmic [74], and in

peripheral tissues, CRY seems to function as a clock component

and a photoreceptor [75].

Figure I. Simplified view of the molecular circadian clock in Drosophila. The

expression of negative regulators TIMELESS (TIM, blue), PERIOD (PER, red)

and CLOCKWORK ORANGE (CWO, orange) is mediated by positive

transcription factors CLOCK (CLK, pink) and CYCLE (CYC, purple) binding to

E-boxes (pale blue) on their promoters. The kinases SGG, CK2 and DBT and

phosphatases such as PP2A and PP1, which modulate the stabilities of the

negative regulators are also shown. The second negative feedback loop that

interlocks with the PER–TIM loop has VRILLE (VRI, green) negatively and PDP1e(brown) positively regulating Clock (but see Box 1 for the role of PDP1e). The

circadian photoreceptor CRY is shown in yellow.

Review Trends in Genetics Vol.24 No.3

mechanisms are induced by modulating the stability ofthese canonical clock proteins through kinases, phospha-tases and the cellular degradation machinery [8,17]. Theenvironmental inputs that ‘entrain’ the circadian clockinclude light, temperature, social stimuli and food avail-ability, but light is the most potent ‘Zeitgeber’ (time-giver)[1]. Consequently, latitude differences challenge the clockbecause of associated changes in temperature and photo-period. For example, in light–dark cycles, D. melanogasteradults show a morning and late-afternoon/evening burst ofmovement. Under hotter conditions, evening activity isdelayed, so flies avoid desiccation [18]. This is achievedby repressing the splicing of a 30 period (per) intron,resulting in a delayed increase in per transcript andPER protein levels (Box 1) and a retardation of eveninglocomotion (with a longer afternoon ‘siesta’) [18]. Wemighttherefore expect higher levels of intron retention and lesssplicing in the lower latitudes of both hemispheres, wheretemperature is generally greater. Such amechanismwouldallow the fly to adjust its behaviour in response to changingtemperature. Clock gene polymorphisms provide anothermechanism by which flies can adapt to different environ-ments. Here we discuss two well-understood examples ofpolymorphism in D. melanogaster.

The period Thr-Gly polymorphismAn uninterrupted stretch of alternating threonine-glycine(Thr-Gly) pairs is encoded within the per gene (Figure 1a).D. melanogaster natural populations in Europe and NorthAfrica differ in the length of Thr-Gly region: two majoralleles, per (Thr-Gly)17 and per (Thr-Gly)20, account for�90% of the variation, per (Thr-Gly)23 making up �8%,and per (Thr-Gly)14 at �1% (Figure 1b) [12]. Rare allelesencoding 15, 18, 21 and 24 Thr-Gly pairs constitute thefinal 1%, and each allele can be derived from another bysimple insertions and deletions, with all variants coales-cing back to a per (Thr-Gly)23 sequence [12,19]. In northernEurope, the frequency of per (Thr-Gly)20 increases,whereas that of per (Thr-Gly)17 decreases, generating arobust latitudinal cline (Figure 1b) [12]. A parallel cline inper (Thr-Gly)20 frequency is observed in Australia,although it is neither as steep nor as robust as in Europe.Furthermore, it also seems to be affected by yearly localgenetic drift or climatic changes (Figure 1b) [20–23].Nevertheless, the observation of similar clines in twocontinents suggests the action of selection rather thancoincidentally similar historical or migratory events. Dis-equilibrium pattern analysis, a technique for detectingselection among tightly linked loci, of European and NorthAfrican per sequences suggested that balancing selectionacts on the Thr-Gly region [24].

Functional analysis of Thr-Gly repeat length variationThe ability of the circadian oscillator to maintain 24-hperiods over a wide range of temperatures was examinedin a large sample ofD. melanogaster per (Thr-Gly) variantsfrom Europe and North Africa [25]. The northern per (Thr-Gly)20 allele provided the most thermally stable periods,with an endogenous period that barely deviated from�15–20 min less than 24 h (Figure 1c). The per (Thr-Gly)17allele provided a more precise 24-h free-running period

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Figure 1. The D. melanogaster period Thr-Gly repeat. The Thr-Gly repeat and the coevolving regions immediately flanking it are illustrated, together with a figure of the

clinal distribution of the (Thr-Gly)20 allele in Europe and Australia and a synopsis of the circadian temperature compensation profiles of the different European variants.

(a) Cartoon showing a per (Thr-Gly)20 allele, with each pink coloured cassette representing a Thr-Gly encoding hexamer. The green flanking regions 50 and 30 of the repeat

coevolve with the repeat length when different Drosophila species are compared [39,41]. The 50 region is depicted in two shades of green, each representing �30 residues.

Chimeric per transgenes have been created in which D. melanogaster 50 sequences are joined to the 30 sequences of D. pseudoobscura at different positions shown by the

coloured arrows. A join at the red arrow provides normal, wild-type rhythmic behaviour in flies, at the blue arrow, robust rhythms with highly temperature-sensitive periods

are observed, and at the green arrow, flies are nearly arrhythmic [41]. The 30 conserved sequence important for Doubletime (DBT) binding and phosphorylation, nuclear

translocation and repressor activity [46,47] is shown in pink. (b) Latitudinal variation of per (Thr-Gly)20 repeat length frequency in Australian (blue) and European (red)

natural populations collected and analyzed in the early 1990s [20,29]. The corresponding regression lines reveal that the frequency of per (Thr-Gly)20 increases in the colder,

higher latitudes of both continents. The arrow depicts the most dramatic European outlier population that was collected at high altitude (>1000 m) in Cyprus and which has

a frequency more representative of colder regions [12]. (c) Temperature compensation of the circadian period in different natural Thr-Gly length variants. The average

period obtained at 29 8C is subtracted from that obtained at 18 8C for each genotype. Variants from the more common Thr-Gly 14–17–20–23 series are shown in turquoise

and generate an approximately linear relationship between temperature compensation and Thr-Gly repeat length. The per (Thr-Gly)17 also has an endogenous period very

close to 24 h at high temperature and therefore, under these conditions, does not have to reset its clock every day [25]. At lower temperatures the period shortens

significantly; it is consequently best adapted to generally warmer regions of Europe [25]. The per (Thr-Gly)20 variant has the best temperature-compensated period and

would be favoured in more thermally variable regions of Europe. The very rare variants, per (Thr-Gly)15, per (Thr-Gly)21 and per (Thr-Gly)24 are shown in purple and have

generally poorer temperature compensation than the common variants [25].

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at 29 8C, but the period shortened significantly in coolertemperatures. Corresponding transgenic flies carrying persequences that differed only in per Thr-Gly repeat lengthshowed the same effects, ruling out any contribution fromgenetic variation linked to the repeat [25]. Thus, the‘temperature compensated’ per (Thr-Gly)20 allele is prob-ably better adapted in the colder and more thermallyvariable higher latitudes, whereas per (Thr-Gly)17 is suitedto the warmerMediterranean region: a potential balancingselection scenario that is consistent with the statisticalanalysis of Thr-Gly sequences [24].

Is a period that ‘resonates’ closely with the environmen-tal cycle of 24 h more adaptive? Probably, because theorganism does not have to expend energy in resetting itsclock every day. Research in Cyanobacteria (which unlikeDrosophila has a rapid generation time that lends itselfreadily to multi-generational Darwinian competitive fit-ness experiments) supports this view. Short or long periodclock mutants outcompeted the wild-type organisms inartificial short or long period environments, respectively,but in a 24-h world, the wild-type predominated, revealinga clear and unambiguous association between circadianresonance and Darwinian fitness [26]. Can small changesin temperature compensation of different natural Thr-Glyvariants, allied to the corresponding ‘resonance’ of eachvariant, provide enough selective pressure to change genefrequencies? D. melanogaster has an effective populationsize of �106, so any genotype that has a positive selectioncoefficient>1/106 is ‘visible’ to natural selection and will be

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favoured [27,28]. Consequently, tiny selective coefficientsthat might be impossible to measure in Drosophila, are,nonetheless, evolutionarily significant.

Several other observations support a thermal expla-nation for the pattern of Thr-Gly variation. First, from theEuropean fly populations that were sampled, one wascollected at high altitude (>1000 m) [29]. The Thr-Glyfrequencies in this sample showed a colder, more ‘north-ern’ profile, asmight be predicted [29] (Figure 1b). Second,in ‘Evolution Canyon’ on Mt. Carmel (Israel), the north-ern-facing ‘European’ slope is much colder than thesouthern-facing slope, and the ratio of per (Thr-Gly)17 toper (Thr-Gly)20 is significantly reversed comparedwith thehotter southern slope, again supporting the thermal hy-pothesis [30]. Finally, in Europe and North Africa, the per(Thr-Gly)20 cline is accompanied by an opposite cline in theMediterranean per (Thr-Gly)17 [12], whereas in Australia,the trend is toward a greater number of Thr-Gly allelesand higher heterozygosity in the tropical lower latitudes[20]. This occurs, in part, because those rare Thr-Glylength variants found in Europe show generally higherfrequencies in northern, tropical Australia [20], eventhough they show poorer temperature compensation[25] (Figure 1c); perhaps the relaxed thermal selectionof the tropics ismore forgiving than theEuropean climate?

Drosophila melanogaster originated in Africa and prob-ably invaded Europe after the last glaciation �15 000years ago and Australia in the past hundred years(Figure 2) [31–34]. Therefore, the Australian Thr-Gly cline

Figure 2. Drosophila melanogaster colonization of Europe, Asia and the New Worlds from Africa. Ancestral populations are shown in red and orange, ancient populations

in green and more recent dispersal in the past few centuries in blue. White areas show regions that are either inhospitable to D. melanogaster or areas where flies have not

been reported. D. melanogaster probably invaded Europe at the end of the last glaciation, �15 000 years ago, and have spread to the Americas and to Australia since the

colonization by Europeans in the past few centuries. Flies in the Caribbean islands have genetic variation that seems to have originated from West Africa, most likely

through the slave trade. Figure redrawn from David and Capy (1998) [31].

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is much younger than the European cline, suggesting thatselection on the Thr-Gly polymorphism is not as strong ason loci such as alcohol dehydrogensase (Adh) or esterase-6(Est6) that show robust Australian clines [35,36]. Popu-lations from ‘ancestral’ east Africa show enormous vari-ation in Thr-Gly length, and their sequences do notconform to the major European classes of 14–17–20–23repeats [20]. Nevertheless, all Thr-Gly sequences coalesceback to the same per (Thr-Gly)23 allele present in the threecontinents. This variation in sub-Saharan Thr-Gly allelesmight also reflect the relaxation of thermal selection inthese tropical environments allied to a large ancestralpopulation size. As with many different fly genes, a con-siderable bottleneck and loss of variation occurred duringthe colonization of Europe and the New Worlds (Figure 3)[14,15,37,38].

Interspecific analysis of Thr-Gly regionsThere is considerable variation in length of the Thr-Glyregion amongDrosophila species. [39]. InD. pseudoobscura,the basic Thr-Gly repeat has been replaced with�35 copiesof a related degenerate pentapeptide repeat, increasing thelength of the repetitive region to �200 residues comparedwith D. melanogaster’s �50 [40]. Statistical analyses ofrepeat region length variation among 20 Drosophilid andnon-Drosophilid flies suggested that repeat length coe-volved with coding changes in the immediate 50 and 30

flanking nonrepetitive regions [39,40]. This was confirmedexperimentally by using interspecific chimeric per trans-genes between D. melanogaster and D. pseudoobscura(Figure 1a). Depending on the position of the boundarybetween the two species’ coding regions, it is possible toproduce both completely arrhythmic transformants

and robustly rhythmic transformants with extremelytemperature-sensitive periods (Figure 1a) [41]. These inter-specific results may explain why changes in Thr-Gly lengthwithinD.melanogaster gently alter temperature responses,because here, these length changes are not associated withany compensating flanking alterations [25].

A similar argument has been proposed to explain thetemperature compensation of the three major D. simulansThr-Gly variants, persim (Thr-Gly)23, persim (Thr-Gly)24 andpersim (Thr-Gly)25, which each have a different flankinghaplotype [42,43]. In Europe, these three variants aredistributed in the ratio 2:2:1, respectively, but not in aclinal pattern [42]. The temperature compensation of per-

sim (Thr-Gly)25 is more stable than that of the other var-iants, but the period of persim (Thr-Gly)25 is �45 minshorter than 24 h, in contrast to the other variants whoseperiod ‘resonates’ better with 24 h but is more tempera-ture-sensitive [43]. This provides another possible balan-cing selection scenario in which the persim (Thr-Gly)25might have the advantage of a better compensated clockbut the disadvantage of a period offset from 24 h. Neutral-ity tests revealed a balancing selection signature in thisregion [42]. Intriguingly, a natural D. simulans chimericvariant from Africa, carrying the per (Thr-Gly)24 repeatwith a per (Thr-Gly)23 flanking haplotype, was found tosegregate with a complex temperature-sensitive longperiod phenotype [44]. This phenotype mapped to gene(s)on the X chromosome and was modified by factor(s) on theautosomes [44]. Thus, disturbing the usual linkage dise-quilibrium between the repeat and its immediate flankingregion in D. simulans provided a ‘sensitized’ circadianbackground, reminiscent of the interspecific chimericgenes described above [41].

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Figure 3. The timeless polymorphism. The 50 tim sequence in which the G insertion that gives rise to the natural ls-tim variant is illustratedn, together with the spatial

patterns of ls-tim in Europe. The diapause of ls-tim and s-tim females from southern and northern European populations are shown, as is the strength of the molecular

interactions between L- and S-TIM with CRYPTOCROME in yeast. (a) The ls-tim sequence is shown in blue, ATG start codons in red, and the G insertion (black) is indicated

by an arrow [50]. Both L-TIM1421 and S-TIM1398 isoforms are produced [51]. In the s-tim sequence (green), the G deletion is denoted by an asterisk and hence a premature

stop codon, TGA (red), is produced, potentially generating a 19-residue peptide, S-TIM19 (bold, italics) from the upstream ATG, hence the major product from s-tim is the

shorter S-TIM1398 isoform. The amino acid sequences are shown beneath the gene sequences. (b) The latitudinal cline in ls-tim frequency (blue) in Europe, from

southeastern Italy to Sweden. The two outlier populations are from Crete and Haifa, Israel. If the ls-tim frequencies are plotted as an overland distance from southeastern

Italy, a linear relationship is generated. Redrawn from Tauber et al., (2007) [13]. (c) Diapause incidence of ls-tim (blue) and s-tim (green) females at different photoperiods at

13 8C from a southern Italian (South) and a Dutch (North) population. The y-axis represents the proportion of females (arcsin transformed, degrees) in diapause. Adapted,

with permission, from Tauber et al., (2007) [13]. (d) In light conditions, CRY interacts more strongly with S-TIM (note darker blue colonies in plate assay) than L-TIM in the

yeast two hybrid system [51]. (Figure provided by F. Sandrelli).

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Possible mechanisms for Thr-Gly circadianmodulationA PER-LACZ fusion protein (in which the central 230residue fragment containing the Thr-Gly repeat and itscoevolved regions was removed) is hypophosphorylatedand stable compared with a protein with an intact centralregion [45]. The key sequence within this fragment is ashort motif that binds Doubletime (DBT) kinase and isconserved among flies and mammals (Box 1; Figure 1a). Adeletion of this motif generates behaviourally arrhythmictransformants [46,47]. This sequence lies immediatelydownstream of the 30 coevolved region that flanks theThr-Gly repeat [41]; therefore, the phosphorylation,nuclear localization and repressor activities associatedwith this fragment might be modulated by modificationsto the Thr-Gly region (Figure 1a). In addition, Nuclearmagnetic resonance (NMR) studies of poly(Thr-Gly)peptides revealed that (Thr-Gly)3 has a b-turn

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conformation, as does D. pseudoobscura’s correspondingpentapeptide repeat [48,49]. This (Thr-Gly)3 conformation-al monomer is particularly interesting because the majorallelic European variants are in a (Thr-Gly)3 interval series(14–17–20–23) that have a linear relationship withtemperature compensation (Figure 1c). The rarer variantsnot included in this series have a poorer thermal response,which does not follow a predictable linear thermal pattern(Figure 1c) [25]. The major variants are not divisible by(Thr-Gly)3 because the degenerate Thr-Gly repeats oneither side of the uninterrupted repeat also contribute tothe b-turn conformation [40,48].

Natural polymorphisms in timeless

The 50 coding region of tim in D. melanogaster is charac-terized by a single nucleotide insertion (Figure 3a) [50].The insertion haplotype (ls-tim) has two in-frame initiationcodons, producing a long TIM isoform from the upstream

Review Trends in Genetics Vol.24 No.3

methionine (L-TIM1421) and a shorter isoform from thedownstream ATG. By contrast, the major products of thedeletion haplotype is the short isoform, S-TIM1398, and aputative truncated 19-residue peptide, S-TIM19, from theupstream ATG (Figure 3a) [51]. Natural EuropeanD. melanogaster populations show a latitudinal cline forthis polymorphism, with ls-tim being most frequent insoutheastern Italy, whereas s-tim prevails in northernEurope (Figure 3b) [13]. A battery of neutrality tests wereperformed on the tim polymorphism and on two intergenicregions�15- and 30-kb downstream. The null hypothesis ofneutrality was rejected for the tim polymorphic region, butnot for the two downstreamregions [13]. In addition, the lowlevel of genetic variation in the polymorphic region com-pared with the downstream regions suggested that the 50

polymorphism is driven by directional rather than balan-cing selection. Although a bottleneck might give a similarresult, we would expect it to affect all nearby sites equally,and this was not the case in the neighbouring 30 loci [13].

Phylogenetic analysis, rooted and calibrated with theorthologousD. simulans sequence, revealed that ls-timwasa recent mutation �10 000 years old [13] and thereforeyounger than the 15 000 years since the last glaciation,when D. melanogaster likely colonized Europe (Figure 2)[31–34]. This ‘modern’ view is reinforced because the ls-timmutation has not been identified in a survey of naturalpopulations from the ancestral homelands of sub-SaharanEast Africa [13]. However, if ls-tim is so recently derived,enough time might not have elapsed for new neutral poly-morphisms to become linked to ls-tim, a requirement forsome neutrality tests to detect the signature of balancingselection [16].

Functional implications of the tim polymorphism:circadian and photoperiodic phenotypesUnlike theperpolymorphism, the tim variants donot showany systematic differences in temperature compensation[13]. Because TIM is the light-sensitive clock molecule(Box 1), seasonal day length,which co-varieswith latitude,is a potential selective agent. In light pulse experiments,circadian photosensitivity is compromised in homozygousls-timflies, and this findingwas recapitulated using trans-formants that mimicked the natural variants (i.e. expres-sing only S-TIM or LS-TIM) [51]. Transformantsexpressing only L-TIM showed a more severe defect thanls-tim flies [51]. These transgenic experiments also ruledout the possibility of linkage disequilibrium contributingto the attenuated circadian photosensitivity phenotype, aswell as of any major role for S-TIM19 in these phenotypes.Changes in circadian photosensitivity have been docu-mented in D. auraria Japanese populations, where fliesfrom the northern islands (438N) have a reduced circadianlight response compared with those in the southernislands (348N) [52]. This cline was interpreted as beingan adaptation of the circadian system to the long summerday lengths that can disrupt circadian behaviour andphysiology in the higher latitudes [52]. For example, inLeicester (528N) on June 21–22, 2007, we measured theabsolute light-dark (LD) cycle as 19.5 h light, 4.5 h dark,whereas on the equator, the photoperiod would be LD12:12. It is therefore puzzling that ls-tim, whose circadian

photosensitivity is attenuated, predominates in southernrather than northern Europe [13].

Like many insects in temperate regions, Drosophilaanticipate the oncoming winter; under short photoperiodsand colder temperatures, females respond with a protec-tive physiological diapause, which includes an ovariandevelopmental arrest. Studies of different insect species,including Drosophilids, have, as expected, revealed thatpopulations from higher latitudes show enhanced levels ofdiapause [53]. D. melanogaster lines with high levels ofdiapause are more resistant to environmental stressessuch as cold or starvation and display enhancedfitness under these conditions [54–56]. Natural ls-timfemales (and their respective transgenics), show signifi-cantly higher diapause levels even under relatively longday lengths than do s-tim flies, reflecting again an atte-nuated photoresponse (Figure 3c) [13]. These results, likethose on circadian photosensitivity, do not fit with thespatial distribution of these alleles, because the southernls-tim variants would seem better adapted to northernconditions. However, when the diapause of northern andsouthern European populations is compared (irrespectiveof tim haplotype), the northern population has a generallyenhanced response (Figure 3c) [13], suggesting the possib-ility of at least a shallow cline in diapause in Europe tomatch one that has been reported in North America[55,57]. Thus, other loci, in addition to tim,might generatea European latitudinal cline for diapause, althoughthe geographic variation in tim would seemingly act inthe opposite direction. One candidate gene encodes theinsulin-regulated phosphoinositide-3 kinase, which isassociatedwith diapause incidence in natural North Amer-ican populations [58].

A molecular basis for the altered tim phenotypesTIM regulates the transduction of photic signals to thecircadian clock through its interaction with cryptochrome(CRY), the circadian blue-light receptor (see Figure I inBox 1) [1]. In a yeast two-hybrid assay, CRY interactedmorestrongly with S-TIM than L-TIM (Figure 3d) [51]. If thischange inTIM–CRYdynamics is reflected in vivo, ls-timfliesshould have more stable TIM products. Immunoblotanalysis showed this to be the case [51,59] and is buttressedby the observation that daytime TIM levels do not fall asexpected in L-TIM transformants [51]. Thus, the L-TIMisoform likely provides greater overall TIM stability andcauses the attenuated circadian and seasonal photore-sponses of the ls-tim genotype [13,51].

Solving the tim spatial puzzleThe discrepancy between the geographical distributionof tim alleles and the observed phenotypes can bereconciled with the results from the neutrality tests, whichrevealed that the tim polymorphism might be under direc-tional and not balancing selection, representing an evol-utionary transient for a young allele (ls-tim) presentlyincreasing in frequency [13]. That this new variant seemsto have enhanced cold and photoperiodic adaptation(earlier diapause and reduced circadian photosensitivity)explains why it was favoured by selection in a Europeanseasonal environment. In additional populations from

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Box 2. A clock gene clines in a non-model vertebrate

Repetitive sequences, as within per, are believed to represent a

major substrate for evolutionary change given their rapid mutation

rates [76]. The positive circadian regulator, Clock (CLK), contains a

poly-glutamine (polyQ) region that serves as a transcriptional

transactivation domain [77]. Variation in the length of the polyQ-

encoding repeat in Clk does not show a cline in Australian D.

melanogaster populations [23]. However, in birds, the polyQ repeat

length shows a latitudinal cline within European blue tit (Cyanistes

caeruleus) populations, with longer repeats found in higher

latitudes [78]. Patterns of length variation within and between

populations do not reflect those of neutral microsatellite markers,

suggesting the possibility of balancing selection, and the authors

speculate that this could be related to the seasonal migratory

polymorphism also observed in this species. The implication of this

study is that changes in polyQ length might alter the transactivation

potential of Clk, which in turn could alter the response of the

circadian mechanism to environmental inputs. However, any direct

relationship between these circadian phenotypes and polyQ varia-

tion in this vertebrate remains to be defined.

Review Trends in Genetics Vol.24 No.3

Crete and Israel, the frequency of ls-tim declined and thelatitudinal cline collapsed (Figure 3b) [13]. Correlating ls-tim frequencies with overland distances from southeast-ern Italy, where ls-tim frequency is highest, restores ahighly predictable linear relationship (Figure 3b). Thisfinding suggests that the ls-tim allele arose in south-eastern Italy and through directional selection is spread-ing outward in every direction, presumably because of theselection imposed by the European seasonal environment[13]. However, it is difficult to explain why, if the newallele emerged several thousand years ago, it has not yetbeen fixed in lower and higher latitudes? D. melanogasteris a human commensal; therefore, ls-tim might have beenmaintained in small isolated populations in southeasternItaly for hundreds or even thousands of years until thelate Bronze age (�3000 ya) and the development of viti-culture. Human migration would have spread the allele,albeit slowly, in every direction (Figure 2) [31,34]. Inaddition, in southern Europe, seasonal photoperiodicselection is expected to be less intense, inevitably slowingthe initial spread of the new allele.

However, although neutrality test results are consist-ent with directional selection, it is still possible that ls-tim is maintained by balancing selection for reasonsstated earlier. One might rationalize that the attenuatedcircadian photoresponsiveness and enhanced diapausemediated by tim variation could be favoured only in anarrow range of southern latitudes. For example, dia-pausing females go into spontaneous remission after afew weeks [60]. As ls-tim females move into diapauseunder relatively long days early in the season, anyspontaneous re-emergence might find them underunfavourable mid-winter conditions, particularly innorthern regions, with a consequently higher fitnesscost. Further south, in Crete or Israel, seasonal selectionmight be more relaxed, leading to reduced ls-tim fre-quencies. This contrived balancing scenario would elim-inate the need to propose a single ‘epicentre’ for wherethe new allele arose. One possibility for clarifying thebalancing versus directional hypotheses might be tosurvey tim variation in another continent. The coloniza-tion of North America by European immigrants, and theflies they brought with them over the last four centuries,suggests there is not a single geographical focus for thels-tim allele, as proposed for Europe, because ls-timwould have been repeatedly reintroduced (Figure 2)[34,61]. If 400 years is long enough for selection toinfluence ls-tim gene frequencies, under balancing selec-tion, a similar European-like distribution of the twovariants at similar latitudes might be observed in theUnited States and Canada. Under directional selection,we would expect a relatively smooth gradient withhigher levels of ls-tim in the northern parts of the con-tinent (Labrador, latitude �538N, assuming flies can befound there; Figure 2), with reduced frequencies insouthern Florida (latitude �258N), which is furthersouth than Greece or Israel.

Concluding remarksA tenet of modern evolution is that unfavourable newmutations are eliminated by selection, neutral mutations

130

can be maintained by drift, and adaptive mutations willspread through the population. The relatively new ls-timallele provides an example of the latter that has beenstudied at several biological levels [13,51]. Several out-standing issues remain; American diapausing flies seem tohave enhanced fitness under stressful environmental con-ditions [54–56], but this has not been shown specifically forEuropean ls-tim. Furthermore, the observation that ls-timis a young allele canmake interpretation of neutrality testsproblematic; therefore, functional data offer additional andimportant contributions to understanding the evolutionarybiology of this locus. Indeed, a recent review outlines thecomplexities of neutrality tests and advocates the use offunctional assays in verifying targets of selection [16].Nevertheless, examination of ls-tim spatial variation onother continents could be helpful in further illuminatingthe selective forces that might be at work here. Moregenerally, the involvement of circadian clock genes inseasonal phenomena has been an area of much speculationand controversy in the insect literature [62], whereas it isgenerally accepted inmammals [63] andArabidopsis thali-ana [64]. In D. melanogaster [13,51], and in a relatedDrosophilid, Chymomyza costata [65,66], it seems thattim plays a role in diapause, but whether tim affectsdiapause through its effects on the clock, rather thanexclusively on a shared light input to both circadian andphotoperiodic mechanisms, is an important question to beanswered. Similarly, a biochemical mechanism has yet tobe shown through which the Thr-Gly repeats affect naturalvariation in temperature compensation.

Nature provides us with long-term experiments whereall the complexities of dynamically changing environmentsare brought to bear on individual loci. Clines suggestselection, but simulating environmental conditions in anattempt to isolate any selective agent, as well as beingdifficult in the laboratory, is perhaps premature unlessindependent evidence for selection can be obtained.Neutrality tests, although sometimes difficult to interpret[16], can detect the signature of selection at the DNAsequence level and provide the evolutionary frameworkfor studying putative selective forces. Natural polymorph-isms also provide less severe phenotypes for genetic

Review Trends in Genetics Vol.24 No.3

analysis compared with mutagenesis-derived variants [67]and, whereas the latter is required to define the locus, theformer firmly places that locus in an evolutionary andecological context. Thus, the study of natural geographicalvariation in clock genes has added a novel dimension torhythm research, bringing into focus the selective value offine tuning the underlying circadian mechanism by thelocal environment. We envisage that, in the future,these types of geographical studies of clock gene variationmight be extended to bothmodel and non-model organisms(Box 2), in an attempt to learn more about their roles inanimal adaptation.

AcknowledgementsC.P.K. thanks the Royal Society for a Wolfson Research Merit Award andBBSRC and NERC for grant support. C.P.K. and R.C. acknowledgeEuropean Community grants under Frameworks 2, 4 and 6 (EUCLOCK018741) and Ministero dell’Universita e della Ricerca Scientifica eTecnologica (MURST)/British Council. R.C, also acknowledges a grantfrom the Italian Space Agency, DCMC grant. A.A.P. is supported by theHoward Hughes Medical Institute, Fiocruz and CNPq. E.T. acknowledgesa project grant from NERC.

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