Copyright ? 2007 by the Genetics Society of America
Quantitative Trait Loci Associated with Photoperiodic Response and Stage
of Diapause in the Pitcher-Plant Mosquito, Wyeomyia smithii
Derrick Mathias,1Lucien Jacky,2William E. Bradshaw3and Christina M. Holzapfel
Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon 97403-5289
Manuscript received November 22, 2006
Accepted for publication February 9, 2007
A wide variety of temperate animals rely on length of day (photoperiodism) to anticipate and prepare
for changing seasons by regulating the timing of development, reproduction, dormancy, and migration.
Although the molecular basis of circadian rhythms regulating daily activities is well defined, the molecular
basis for the photoperiodic regulation of seasonal activities is largely unknown. We use geographic
variation in the photoperiodic control of diapause in the pitcher-plant mosquito Wyeomyia smithii to create
the first QTL map of photoperiodism in any animal. For critical photoperiod (CPP), we detect QTL that
are unique, a QTL that is sex linked, QTL that overlap with QTL for stage of diapause (SOD), and a QTL
that interacts epistatically with the circadian rhythm gene, timeless. Results presented here confirm earlier
studies concluding that CPP is under directional selection over the climatic gradient of North America
and that the evolution of CPP is genetically correlated with SOD. Despite epistasis between timeless and a
QTL for CPP, timeless is not located within any detectable QTL, indicating that it plays an ancillary role in
the evolution of photoperiodism in W. smithii. Finally, we highlight one region of the genome that
includes loci contributing to CPP, SOD, and hormonal regulation of development.
seasonal events and a wide variety of organisms use day
length (photoperiod) to time their development, re-
production, dormancy, and migration. Concordance
between individual photoperiodic response and local
climate is an essential component of fitness for animals
living in the temperate zone (Bradshaw et al. 2004),
and modification of photoperiodic response is an im-
portant adaptation of animals during range expansion
(Danilevskii 1965; Cooke 1977; Tauber et al. 1986;
Danks 1987; Lounibos et al. 2003) or when confronted
with rapid climate change (Bradshaw and Holzapfel
2001a, 2006). Photoperiodism provides an ecologi-
cally relevant, highly heritable trait whose adaptive sig-
nificance in a temperate seasonal environment is not
questioned. Length of the favorable or growing season
in North America decreases with increasing latitude
(Bradshaw 1976). Consequently, the optimal time to
enter diapause advances to an earlier day in the year
HE annual change in day length at temperate lati-
tudes provides a highly reliable indicator of future
and the day length used to switch from active devel-
opment to diapause [hereafter, the critical photope-
riod (CPP)] is positively correlated with latitude and
altitude among a wide variety of temperate arthropods
(Danilevskii 1965; Taylorand Spalding 1986; Danks
1987). The consistent genetic change in photoperiodic
response over geographic and climatic gradients provides
one of the most robust examples of repeated adaptive
evolution in nature.
Although much progress has been made in identi-
fying the genetic components of the circadian clock
regulating daily activities, the molecular basis of the
photoperiodic timer regulating seasonal activities is
conspicuously absent from the literature. For insects,
the model system for circadian rhythmicity has been
Drosophila melanogaster; the Canton-S strain of D. mela-
nogaster is photoperiodic for ovarian diapause but only
over a very narrow range of temperatures, and its dia-
pausing status must be determined destructively from
dissection of the ovaries. Hence, while ‘‘D. melanogaster,
with its unrivalled genetic background has provided a
foundation of uncovering the molecular basis of the
circadian mechanism, ... it is probably less useful as a
ine species with a much more robust [photoperiodic]
response’’ (Saunders 2002, p. 481). Toward the goal of
understanding the molecular basis of photoperiodism
and its adaptive evolution, we developed QTL maps for
Wyeomyia smithii, using a southern (31?N) and a north-
ern (57?N) population recently collected from nature.
Sequence data from this article have been deposited with the EMBL/
GenBank data libraries under accession nos. EF094841–EF094856 and
1Present address: Kenya Medical Research Institute, Centre for Vector
Biology and Control Research, CDC Section, P.O. Box 1578, Kisumu,
2Present address: Department of Pharmacology, University of California,
Irvine, CA 92697.
3Corresponding author: Center for Ecology and Evolutionary Biology,
5289 University of Oregon, Eugene, Oregon 97403-5289.
Genetics 176: 391–402 (May 2007)
W. smithii lays its eggs and completes its preadult
development only within the water-filled leaves of the
carnivorous purple pitcher plant Sarracenia purpurea,
where it undergoes a larval diapause that is initiated,
range of temperatures. Southern populations (#36?N)
diapause primarily in the fourth larval instar, while north-
ern populations ($40?N) diapause primarily in the
third larval instar (Bradshaw and Lounibos 1977). The
earlier stage of diapause in northern populations pro-
vides them with a fail-safe opportunity to enter an addi-
tional fourth instar diapause in response to uncertain
vernal environments (Lounibos and Bradshaw 1975).
In W. smithii, the heritability of CPP increases from 0.15
in southern populations to 0.70 in northern popula-
tions (Bradshaw and Holzapfel 2001b) and CPP in-
(Bradshaw and Holzapfel 2001a). Within popula-
tions polymorphic for stage of larval diapause (SOD),
SOD is negatively genetically correlated with CPP
and the daily expression of the circadian rhythm gene,
timeless, differs between diapausing instars (Mathias
et al. 2005). Within northern populations of W. smithii,
the expression of timeless is inversely correlated with cri-
tical photoperiod (Mathias et al. 2005). Nonetheless,
critical photoperiod is not correlated with either the pe-
toperiodism (Bradshaw et al. 2006). Estimates from
line crosses for the minimum number of effective factors
underlying genetic differences in critical photoperiod
between populations range from 5 to 20 and involve
dominance and epistasis (Hard et al. 1992, 1993; Lair
are related through pleiotropy, and that there are
correlated evolutionary trends in CPP, SOD, and timeless
expression. Consequently, we developed QTL maps of
critical photoperiod and stage of diapause from F2hy-
in both characters, using timeless as well as other genetic
markers to construct the underlying linkage map.
We sought to address four main questions: First, are
therefore potentially capable of independent evolu-
tion? Second, are there QTL for CPP that do overlap
with QTL for SOD and may therefore include pleiotro-
pic genes responsiblefor their genetic covariationwithin
that involve dominance and epistasis that may account
for these nonadditive genetic differences in CPP be-
tween southern and northern populations? Fourth, is
timeless included within or does it interact epistatically
with QTL for CPP?
MATERIALS AND METHODS
Generation of the F2mapping population: To derive an F2
mapping population, a single pair of adults was mated in the
The parental pair was descended from wild-caught individuals
collected from Florida (FL) and Alberta (AL), Canada (Table
1). A full-sib family from FL was used to generate a partially
inbred line with reduced heterozygosity. The AB population
wasforced through abottleneckof 30individuals. Becausethe
AB locality is at the extreme edge of W. smithii’s range where
heterozygosity is low (Armbruster et al. 1998), we did not fur-
ther inbreed this population. A single FL$ 3 AB# cross
produced 19 F1offspring that were allowed to mate en masse
to generate the F2mapping population. Both parents were
established geographic variation in W. smithii (Bradshaw and
Lounibos 1977), the FL female diapaused in the fourth instar
and had a short CPP of 13.4 hr; the AB male diapaused in the
third instar and had a long critical photoperiod of 17.4 hr
CPP and SOD in the F2generation: F2larvae were reared
from day of hatch under diapause-inducing, short-day con-
ditions (light:dark ¼ 8:16 at 21?) and scored for stage of
were able tosynchronize the F2indiapause and thenplace the
synchronized F2on increasing day lengths to stimulate se-
quential development according to their individual critical
length of 13.25 hr that increased 3 min/day. Upon pupation,
the length of the previous day was recorded as an individual’s
critical photoperiod, along with its sex and stage of diapause
(Table 1B). Each pupa was transferred to a 1.5-ml centrifuge
tube and stored at ?70?.
DNA extraction: Genomic DNA from the experimental
animals, as well as that used to develop molecular markers
from stock populations, was extracted with a DNeasy tissue kit
(QIAGEN, Chatsworth, CA) following the protocol for insects
in Appendix G of the QIAGEN manual. DNA was eluted in
the Genomiphi genomic DNA amplification kit (GE Health-
displacing DNA polymerase from bacteriophage Phi29 to
amplify small quantities of genomic DNA, which can then be
fication using gene-specific primers. Following the manufac-
turer’s protocol, 2 ml of genomic DNA per individual was
amplified in a reaction volume of 20 ml and then diluted with
sterile water to a total volume of 100 ml. The remaining DNA
from the DNeasy extraction was stored at ?70?, while the am-
Gene-based markers: Partial sequences of 23 genes pre-
viously isolated in W. smithii were screened for polymorphisms
in the FL and AB parents. Fragments were amplified via PCR,
cloned using a TA Topo cloning kit (Invitrogen, San Diego),
and sequenced on a capillary sequencer. Sequences were
http:/ /prodes.toulouse.inra.fr/multalin/multalin.html) and
searched by eye for polymorphisms. Once found, sequence
differences among the parents were confirmed by restriction
endonuclease digestion where possible (i.e., a restriction site
found in one sequence was absent in the other due to one or
more base-pair differences within the restriction site).
Once a polymorphism was confirmed, restriction digests
were also used to verify homozygosity of alternate alleles in the
parents and to genotype all F2individuals. PCR primers were
392D. Mathias et al.
designed around polymorphisms so that, following a digest,
the three genotypes could be easily scored on an agarose gel:
homozygotes for one allele show a single uncut band, homo-
zygotes for the other allele show two shorter bands, and het-
digestion, polymorphic regions were amplified via PCR using
2.0 ml103 Taq polymerase buffer, 0.32 ml10 mm dNTPs, 0.4ml
template, 0.08 ml Taq DNA polymerase (5 units/ml), and 16.0 ml
sterile H2O for a final reaction volume of 20 ml. Reaction con-
ditions were 95? for 5 min plus 35 cycles of 95? for 30 sec, 60?
for 30 sec, and 72? for 30 sec, plus a final 72? extension for
5 min. To confirm a positive reaction, 5 ml of the PCR product
was electrophoresed on a 1% agarose gel. The remaining pro-
duct served as template for the restriction digest, all of which
Each digest proceeded for 8 hr at 37? with the exception of
those using BsmBI, which were performed at 55?.
An initial screen of parental genotypes for fragments of 23
loci that could be easily scored via restriction endonuclease
digestion (appendix). In addition, a 96-bp insertion/deletion
was found in an intron near the 39-end of locus Ws13043,
genes, numerous polymorphisms were found but were unus-
able due to heterozygosity in one of the two parents. All F2
individuals were genotyped for the nine loci with fixed poly-
morphisms, and only one marker departed from the expected
1:2:1 ratio for a codominant marker in an F2intercross (cutoff
of x2¼ 9.21 for a ¼ 0.01, 2 d.f.). However, transmission
distortion was only minor at this locus, as the genotypic ratio
fits expected values for a ¼ 0.001 (cutoff of x2¼ 13.82, 2 d.f.).
Furthermore, transmission distortion for codominant markers
is less of a concern compared to dominant markers since
confirmation of homozygosity is possible in each parent.
AFLPs: The AFLP technique generates numerous poly-
morphic markers in four steps: (1) digestion of genomic DNA
cutter; (2) ligation of oligonucleotide adapters to the DNA
fragments; (3) selective PCR amplification of fragment sets
using primers with a core sequence plus one to three arbitrary
nucleotides at the 39-end; and (4) gel electrophoresis to sep-
arate the amplified fragments. The major advantages of this
technique arethat itdoesnot requireaprioriknowledge about
DNA sequence and that it generates multiple polymorphic
markers with a single PCR reaction.
To find AFLPs in W. smithii, the protocol of Voset al. (1995)
for ‘‘complex genomes’’ was followed with minor changes and
the substitution of fluorescently labeled primers for [g-33P].
Briefly, 5 ml of genomic DNA was digested for 3 hr at 37? with
at 65?. Adaptor ligation was performed by incubating the
digested DNA for 3 hr at 37? with 12.5 pmol MseI adaptor, 1.25
pmol EcoRI adaptor, 1.25 mm ATP, and 0.5 unit T4, followed by
a 20-min incubation at 65? (for adaptor sequences, see Vos
et al. 1995). The restriction-ligation product was then diluted
5:1 with low TE.
Following ligation, the protocol required two PCR amplifi-
cations: the first (preamplification) used primers with a single
selective nucleotide at the 39-end, while the second (selective
amplification) used primers with three selective primers atthe
39-end. Each preamplification reaction was performed using
2.5 ml 103 Taq polymerase buffer, 1.5 ml 25 mm MgCl2, 0.5 ml
10 mm dNTPs (2.5 mm each), 1.0 ml 10 mm EcoRI 1 A primer,
1.0 ml 10 mm MseI 1 C primer, 0.1 ml Taq polymerase (5 units/
ml), and 13.4 ml sterile H2O for a final volume of 25 ml (see
Table 2 for the EcoRI and MseI core primer sequences). PCR
reaction conditions were those given in Vos et al. (1995) for
primers with a single selective nucleotide. The preamplifica-
tion product was diluted 5:1 with low TE, and 5 ml of the
dilution was used as template for the selective amplification
step. The PCR reaction mix was the same as for the previous
step with the exception of the primers and their concentra-
tions. For selective amplification, 0.5 pmol of a fluorescently
labeled EcoRI 1 3 nucleotide primer and 8 pMol of an un-
labeled MseI 1 3 nucleotide primer were used (see Table 2 for
the EcoRI and MseI core primer sequences and the selective
nucleotides used for each marker). PCR reaction conditions
were those given in Vos et al. (1995) for primers with three
After selective amplification, 10mlofloadingdyewas added
to all samples, which were then denatured for 3 min at 95?.
A total of 1.5 ml of each denatured sample was loaded on a
5.7% denaturing polyacrylamide gel (25 cm plates) and run at
1500 V, 20 mA, 40 W on a Li-cor 4200 sequencer. Gel images
were collected by the Li-cor software for 10 frames at a scan
speed of 4 and saved as TIFF files. Polymorphic AFLPs were
scored by eye and verified independently by at least two of the
authors using the program RFLPscan 3.0 (Scanalytics).
Initially, 16 primer combinations were screened with the
above protocol using DNA from the FL and AB stock
populations to identify the most promising primer sets based
on clarity, repeatability, and number of polymorphic bands.
Four combinations were chosen and used to genotype the two
parents and all F2individuals for 77 polymorphic markers.
a spreadsheet as 0’s or 1’s for absent or present, respectively,
and then converted into Mapmaker 3.0 format for dominant
markers. A segregation ratio was then calculated for each
AFLP and a chi-square test (cutoff of x2¼ 6.64 for a ¼ 0.01,
1 d.f.) was used to test goodness of fit to the 3:1 Mendelian
were included in the linkage map (appendix).
Linkage map construction: The F2mapping population
consisted of 264 individuals genotyped for 45 markers. Ini-
tially, the markers were separated into two groups of over-
other with the 9 codominant markers. Each set was then used
to produce two separate linkage maps using Mapmaker 3.0
(Lander et al. 1987). The codominant markers on both maps
provided landmarks so that the two could be merged into one
with positions biased by linkage phase, which in turn can lead
to the misordering of closely linked markers of the opposite
sort each subset of AFLP plus codominant markers into likely
linkage groups (LGs) using the ‘‘group’’ command with the
Kosambi mapping function (Kosambi 1944) (two-point link-
age criteria: minimum LOD 6.0, maximum distance between
markers of 30 cM). Marker order and position was then es-
timatedusing the‘‘compare’’command andthenrefined with
the ‘‘ripple’’ command. The complete set of markers was then
maps as guides.
Homology with other mosquitoes: To find orthologs of W.
smithii genes in other mosquitoes, we used the TBLASTX al-
gorithm to search the Anopheles gambiae and Aedes aegypti
genomes in the Ensembl database (Birney et al. 2004; http:/ /
www.ensembl.org/index.html). We used the default parame-
ters for the program, which compares a translated DNA query
with a translated DNA database. In cases with more than one
corresponding ortholog. For all nine genes, there were
QTL Map of Photoperiodic Response 393
substantial decreases in both E-values and BLAST scores
between the first and second matches, suggesting that each
is a single-copy gene in both An. gambiae and Ae. aegypti.
Mapping the sex locus: In mosquitoes of the subfamily
Culicinae (which includes W. smithii), males are heterozygous
(Mm) and females are homozygous recessive (mm) (Gilchrist
and Haldane 1947). The sex-determining locus in W. smithii
was mapped by performing a chi-square test for sex ratio and
marker genotype. The expectation is that the three possible
genotypes of each gene-based marker and the two possible
genotypes of each AFLP will have a 1:1 sex ratio. The point of
maximal departure from this expectation approximates the
position of the sex locus (Wilcox 1995).
Composite interval mapping: QTL underlying geographic
variation in both CPP and SOD were mapped in the F2
generation using composite interval mapping (CIM) (Zeng
1994) via Windows QTLCartographer version 2.5 (Wanget al.
stage of diapause is categorical with two possible states in W.
smithii. Although CIM was developed to analyze continuous
characters, it also works for categorical traits that have an
underlying polygenic basis (McIntyre et al. 2001). In essence,
CIM combines interval mapping (Lander et al. 1987) with
marker interval, while using specific markers as cofactors to
account statistically for QTL outside the test interval. The
likelihood-ratio (LR) test statistic is ?2 ln(L0/L1), where L0/
L1is the ratio of the likelihood of the null hypothesis (i.e., no
QTL in the test interval) to the likelihood of the alternative
hypothesis (i.e., a QTL present in the test interval) (Basten
etal.2004). Two parametersaffecting QTLdetectionwithCIM
are (1) number of marker cofactors in the multiple regression
and (2) size of the exclusion window flanking the test interval.
The number of marker cofactors is left to the user’s discretion
with a default value of 5. Increasing cofactor number may
map. The other key parameter, window size, is also left to the
user’s discretion (default value of 10 cM). Essentially, this
parameter removes from the analysis any marker cofactor
located within the specified window flanking the test interval.
Thus, if the window size is broad, closely linked markers with
large effects are not taken into account and may therefore
inflate the likelihood ratio of a given interval. Conversely,
making the window size too narrow may eliminate or diminish
a true QTL signal.
Both linkage and genetic background are factors that must
be considered, given that W. smithii has only three pairs of
chromosomes and that epistasis contributes to geographic
variation in CPP in this species (Hard et al. 1992; Lair et al.
1997). We varied the number of conditioning markers from
a compromise between the marker number and window size
that minimized the effects of linkage, while not eliminating
thebackground effect of non-QTL regions. Ultimately, weused
regression) as cofactors with an exclusion window of 2.5 cM.
Under these parameters, the likelihood-ratio test statistic
was computed at every centimorgan across all marker inter-
vals. QTL significance thresholds for all parameter sets were
estimated by permutation tests. Briefly, trait data and marker
genotypes were permuted 1000 times and the maximum-
likelihood ratio statistic across all intervals was recorded for
each permutation. Likelihood statistics computed from the
original data that exceeded the 50th greatest likelihood-ratio
statistic from the permuted data were significant at the level of
a ¼ 0.05 under the null hypothesis (Churchill and Doerge
Estimation of QTL effects: Both additive (a) and domi-
nance (d) effects and the proportion of phenotypic variance
explained were estimated for each QTL using Windows QTL
Cartographer version 2.5 (Wang et al. 2006). Briefly, estimates
of a and d were obtained by maximum likelihood through an
expectation/conditional maximization algorithm (Meng and
Rubin 1993). The proportion of the variance explained by a
the null model, and s2
alternative model (Basten et al. 2004).
QTL sign test: To test for evidence that the parental pheno-
model of neutral evolution, the expectation is that an equal
number of antagonistic (i.e., plus and minus) alleles are re-
sponsible for the phenotypic difference. In contrast, direc-
tional selection should favor the accumulation of consistently
signed alleles, which in this case are plus alleles toward the
northern parent. To perform the test, we determined the con-
ditional probability of observing by chance n ‘‘plus’’ QTL of m
total, given the phenotypic difference (R) between parents.
Prior to calculating this probability, the additive effects were
fitted to a gamma distribution to approximate shape and scale
parameters required by the test. The threshold for heterozy-
program code downloaded from the website cited in Orr
models to evaluate interactions between all possible marker
log-linear models with frequency as the dependent variable
and the trait and marker genotypes as factors. Two models
were then computed: (1) a model including all factors, all
two-way interactions, and the three-way interaction and (2) a
model including all factors and all two-way interactions (i.e.,
was then used to determine the significance of the three-way
interaction term. Two markers were considered epistatic if the
log-linear model including the three-way interaction had a sig-
To account for multiple testing in the epistasis analysis, we
performed the Benjamini–Hochberg test (Benjamini and
Hochberg 1995; Pavlidis 2003), a post-hoc false discovery
rate with the expected value of Q, defined in the expression
Q ¼ V/(V 1 S), where V is the number of false rejections and
S is the number of correct rejections of the null hypothesis
(Sabatti et al. 2003). For all three traits, a significance level of
a ¼ 0.001 led to at most one false-positive result and was thus
set as the level of significance in this study. All ANOVAs and
Benjamini–Hochberg tests were performed using the statisti-
cal program R (R Development Core Team 2006).
Ais the variance of the residuals under the
Trait values for parents and F2 generation: The
individuals crossed in the parental generation differed
in CPP by 4 hr (Table 1A). In the F2generation, males
and larvae diapausing as third instars, respectively, had
longer CPPs than females (F1,262¼ 35.42, P , 0.0001)
and larvae diapausing as fourth instars (F1,262¼ 106.77,
P , 0.0001) (Table 1B).
Linkage map: The W. smithii FL 3 AB linkage map
consists of 45 marker loci spanning 286.9 cM on three
394 D. Mathias et al.
linkage groups (Figure 1). Average interval length or
marker spacing (s) was estimated at s ¼ 6.82 cM by
dividing the summed length of all linkage groups by the
number of intervals (Fishman et al. 2001). Genome
length (L) was estimated using two different methods.
For the first method, we assumed a random distribution
of markers and added 2s to the length of each linkage
group to account for the ends of chromosomes beyond
the terminal markers (Fishman et al. 2001). This ap-
proach yielded an estimate of L ¼ 327.2 cM. For the
second method, we multiplied the length of each link-
age group by the factor (m 1 1)/(m ? 1), where m is the
number of the markers for a specific linkage group
(Chakravarti et al. 1991). This approach yielded an
estimate of L ¼ 330.4 cM. Next, we calculated map
coverage from c ¼ 1 ? e?2dn/L(Fishman et al. 2001). Us-
ing L ¼ 330, we found that 93.5 and 99.6% of the
genome lies within 10 and 20 cM of a marker, respec-
tively. Thus, the proportion of the genome that is in-
cluded in our linkage map is well covered. Finally, we
approximated the relationship between linkage map
units and units of DNA sequence by dividing W. smithii’s
L ¼ 330 cM, yielding a minimum average of 2.58 Mb/cM.
Correspondence to other mosquitoes: By conven-
tion, chromosomes of culicine mosquitoes are named
according to length, with the shortest designated as
chromosome 1, the longest as chromosome 2, and the
intermediate as chromosome 3 (Clements 1992, p. 3).
Hence, numbers were assigned to the three linkage
groups according to the chromosome that each is most
likely to represent (Figure 1).
Position of the sex locus: Using a chi-square test, we
found that the sex locus is on linkage group 1. The sex
ratio of the two genotypes for each AFLP marker on this
linkage group departed significantly from the 1:1 ex-
2 and 3 departed from a 1:1 ratio. On linkage group 1,
the markers EAGCMCTA.c and EAGCMCTT.s have the
highest chi-square values, with the former having only
1 male and 49 female recessive homozygotes and the
latter having 0 male and 52 female recessive homozy-
the 2.3 cM region between these two markers.
QTL for CPP: CIM detected nine QTL underlying
geographic variation in CPP, accounting for 61.7% of
2). Two QTL each account for .10% of the variance,
one located on linkage group 1 (QTL 1, 20.7%) and the
other on linkage group 2 (QTL 8, 11.5%). Of the re-
maining seven QTL, one is located on linkage group 1
Geographic and phenotypic data for parental and F2generations
PopulationLocation Latitude, longitudeCPP (hr)a
A. Parental generation
Sex Third instar Fourth instarCombined
B. F2generation: mean CPP 6 2 SE (n) according to sex and instar of diapause
15.63 6 0.17 (52)
15.79 6 0.14 (86)
15.73 6 0.11 (137)
14.81 6 0.12 (85)
15.26 6 0.15 (42)
14.96 6 0.10 (127)
15.12 6 0.12 (137)
15.62 6 0.11 (127)
15.36 6 0.08 (264)
aCPP, critical photoperiod determined with increasing day length as in Lair et al. (1997).
bSOD, stage of diapause determined at 30 days post-eclosion from larvae reared at 21? on diapause-inducing
short days (light:dark ¼ 8:16).
Figure 1.—Linkage map for W. smithii showing the linkage
groups (Lg) based on the gene-based and AFLP markers in
QTL Map of Photoperiodic Response395
(QTL 2, 6.5%), five on linkage group 2 (QTL 3–7, 1.5–
6.9%), and one on linkage group 3 (QTL 9, 4.8%). Ad-
ditive effects were generally positive, as 8 of 9 QTL were
toward the northern parent with a longer CPP. Orr’s
(1998) QTL sign test indicated that significant direc-
tional selection has acted on CPP (P ¼ 0.039).
Dominance effects were generally negative and to-
ward the southern parent with a shorter CPP (Table 2).
Dominance (d/a) was complete for QTL 6, accounting
for 3.4% of the variance in CPP, and lowest for QTL 5,
accounting for 1.5% of the variance; the two major QTL
(1 and 8) had intermediate levels of dominance.
QTL for SOD: CIM resolved four QTL for SOD,
accounting for 42.3% of the variation in SOD between
FL and AB (Figure 3, Table 2). Two QTL each account
for .10% of the variance, one located on linkage group
1 (QTL 1, 16.4%) and the other on linkage group 2
(QTL 3, 10.5%). Both of the remaining two QTL are
located onlinkagegroup2 (QTL 2,7.9%;QTL 4,7.5%)
and none is detected on linkage group 3. Additive ef-
fects were evenly split between positive toward the nor-
thern parent diapausing in the third instar and negative
toward the southern parent diapausing in the fourth
QTL for SOD because a minimum of six QTL are nec-
essary for the test to be valid. Dominance effects were
Dominance (d/a) was complete for QTL 2 and 3,
accounting for 18.4% of the variance in SOD; interme-
diate for QTL 4, accounting for 7.5% of the variance;
and virtually absent for QTL 1, accounting for 16.4% of
Epistasis for CPP: Fourteen interactions were signif-
icant using the false discovery rate procedure, which
estimates that no more than 1 of the 14 interactions is a
false positive (Table 3A). These 14 interactions fell into
separate linkage group (Figures 4 and 5). In linkage
group 1, the marker EAGCMCTA.o at 0 cM interacted
with three other markers, two of which spanned CPP
QTL 2 (Figure 2) and the sex locus (Figure 1). In link-
age group 2, the marker EACCMCTT.a at 108 cM inter-
acted with markers inCPPQTL5 (Ws5096),CPPQTL6
(EAGCMCTT.t), SOD QTL 3 (EACCMCTT.a), and with
and 3). In linkage group 3, timeless at 0 cM interacted
with a marker in CPP QTL 9 [l(1)G0156] (Figure 2).
Epistasis for SOD: Only one interaction was signifi-
cant using the false discovery rate criterion (Table 3B).
In linkage group 2, marker EACCMCTT.a at 108 cM
within SOD QTL 3 interacted with EACCMCTT.q at
88.7 cM within CPP QTL 2 (Figures 2 and 3).
To our knowledge, the QTL map in Figure 2 provides
the first such map of CPP in any animal.The underlying
linkage map (Figure 1) shows three linkage groups,
consistent with the haploid number of chromosomes in
W. smithii (Moeur and Istock 1982), and shows the sex
locus on the shortest chromosome, as in other mosqui-
toes (Clements 1992, p. 2). In Figures 2 and 3, we mark
nine QTL for CPP and four QTL for SOD. We note first
that this number may be an overestimate because CPP
QTL 1–2, 4–5, and 7–8 and SOD QTL 2–4 may each
constitute a single QTL; conversely, there are undoubt-
et al. 2004; Mackay 2004). Second, because of our sam-
ple size of 264 F2individuals, we are likely overestimat-
ing the magnitudeofQTL effects, the variation ofeffects
among QTL, and both the additive and dominance
effects in Table 2 (Beavis 1998). With these caveats in
that a complex genetic architecture underlies photope-
riodism in W. smithii (Bradshaw and Holzapfel 2000,
account for 29% of the phenotypic variation in CPP,
have no detectable overlap with QTL for SOD, and are
not involved in any of the significant epistatic interac-
with QTL for SOD, or contained markers with signifi-
cant epistatic interactions.
Females in the F2hybrids had shorter CPPs than
males (Table 1B), likely due to linkage of the sex locus
Figure 2.—Composite interval map for critical photope-
riod. (Top) Likelihood ratios. (Bottom) The corresponding
R2for each of the nine QTL. Digenic epistatic interactions be-
tween markers are shown as brackets subtended by a dashed
line in the top graph. For clarity, individual markers from Fig-
ure 1 are shown only if they are mentioned in the results
and discussion. For the gene-based markers, both homozy-
gotes and the heterozygote were distinguishable.
396 D. Mathias et al.
with QTL 2 for CPP (Figure 2) because the female
grandparent came from the southern locality with the
short CPP (Table 1). In Drosophila littoralis, critical pho-
toperiod is inherited primarily as a single autosomal
Mendelian unit, but the X chromosome of a southern
population exhibited a recessive factor having some
influence on the expression of diapause (Lumme 1981,
p.243). Hybridsbetween D. lummei females and D. virilis
males with a white-eye marker and backcrossed with D.
virilis males showed that photoperiodic diapause was
controlled by a monofactorial unit on the X chromo-
some (Lumme and Kera ¨nen 1978).InD. triauraria, phe-
notypic frequencies among recombinant inbred lines
suggest that the difference in the photoperiodic re-
sponse between diapausing and nondiapausing strains
isdue to genes atthreeor four loci, at least one ofwhich
is located on the X chromosome (Kimura and Yoshida
1995). Photoperiodic control of diapause among spe-
cies of Drosophila therefore generally involves a less
complex genetic architecture than is found in W. smithii
but, like W. smithii, often involves sex-linked genes.
The latter half of the second chromosome from ?66
to 134 cM represents a region of overlap between QTL
between EACCMCTT.a and five other markers within
the QTL for CPP and SOD (Figures 2 and 3). The epis-
this region is important because in the genome of the
mosquito An. gambiae UbcD4 is tightly linked with puta-
tive orthologs of the Drosophila genes protein on ecdysone
Locations and effects of QTL for critical photoperiod and stage of diapause
QTLLinkage group Positiona(cM) LRb
aEach QTL position is the point on a linkage group where the likelihood ratio reaches the highest value after
crossing the significance threshold (a ¼ 0.05).
bThe likelihood-ratio test statistic for H0/HA: *P , 0.05, **P , 0.01, and ***P , 0.001.
cAdditive effect of the QTL in hours for CPP and for proportion of one larval instar for SOD. The sign in-
dicates whether the QTL effect is toward the northern (positive) or southern (negative) parent.
dDominance effect of the QTL: the sign indicates whether the QTL effect is toward the northern (positive) or
southern (negative) parent. The scale is in hours for CPP and instar stages for SOD.
eAbsolute value of the ratio of dominance to additive effects; a QTL is considered completely dominant when
jd/aj ¼ 1, partially dominant when jd/aj is between 0 and 1, additive whenjd/aj ¼ 0.
fProportion of the phenotypic variance explained by the QTL.
Figure 3.—Composite interval map for stage of diapause.
Top and bottom graphs are as in Figure 2. The dashed red
line plots QTL for critical photoperiod as a reference. Note
that the QTL for critical photoperiod are scaled down to fit
the lower values of the likelihood ratios for SOD and that only
the significant QTL for critical photoperiod are shown. The
digenic epistatic interaction in LG2 is shown as a bracket sub-
tended by a dashed line in the top graph.
QTL Map of Photoperiodic Response 397
et al. 1993; Andres et al. 1993). The implication is that
genes in this region of the second chromosome are in-
environment (photoperiod) with both active develop-
In the drosophilid fly Chymomyza costata, the nonpho-
toperiodic diapause strain has arrhythmic eclosion, does
Epistatic interactions between marker pairs affecting variation in critical photoperiod and stage of diapause
(cM) Marker 2
A. Critical photoperiod
B. Stage of diapause
Figure 4.—Epistatic interactions af-
fecting critical photoperiod (CPP) in
LG1 (top row) and LG2 (bottom two
rows). Note that the AFLP markers are
dominant so that individuals are scored
either as homozygous recessive or as
combined homozygous dominant plus
heterozygote. The first marker is indi-
cated on the horizontal axis, the second
marker at the top of each plot. Nonpar-
allel lines indicate epistatic interaction.
SoHom and NoHom indicate southern
(FL) and northern (AB) homozygotes;
Het indicates a north–south heterozy-
gote. The error bars show 6 1 standard
398D. Mathias et al.
not enter diapause on short days, and has mutations at
the circadian rhythm genes period and timeless. The mu-
tation at the period locus renders flies behaviorally ar-
rhythmic but they remain normally photoperiodic if
they possess wild-type timeless. The double mutant is both
et al. 2003). Flesh flies (Sarcophaga bullata) that have ele-
vated expression of period and timeless are behaviorally
arrhythmic and nonresponsive to short days for the in-
duction of diapause (Goto et al. 2006). Finally, period null
mutants in the Canton-S strain of D. melanogaster are be-
haviorally arrhythmic but have a robust photoperiodic
response curve, albeit shifted toward shorter day lengths
(Saunders 1990). In combination, these results indicate
necessary for photoperiodic time measurement in flies,
an individual circadian clock gene, such as timeless, may
still have an ancillary effect on photoperiodism inde-
pendently of and incidentally to its role in circadian
In W. smithii, the expression of timeless covaries with
CPPgeographicallyandwithSOD within apolymorphic
not overlap with any QTL for SOD (Figures 2 and 3),
suggesting that any functional connection between time-
less with SOD is due to differences between the Alberta
and Florida populations in regulatory regions (e.g.,
transcription factors) located elsewhere in the genome
(Arnosti 2003; Wray et al. 2003) or within a QTL for
CPP on the third chromosome below our level of de-
tection. Nevertheless, timeless, or agene closely linked to
it, does have multiple epistatic interactions with other
markers on the third chromosome, including markers
within QTL 9 for CPP (Figures 2 and 5). These results
indicate that timeless itself plays no detectable role in the
evolution of SOD, but that timeless may play an ancillary
role in the evolution of photoperiodism in W. smithii.
In summary, we use geographic variation between
natural populations of W. smithii to illustrate the evolu-
tion of two physiological traits essential to fitness in a
seasonal environment: the photoperiodic timing of hi-
bernal diapause (CPP) and the developmental stage of
diapause itself (SOD). Significant positive, additive ef-
fects for CPP substantiate earlier conclusions that, in
latitudinal scale. The main region of overlap in QTL for
CPP and SOD also coincides with two developmental
genes activated by ecdysteroid, revealing a portion of
the W. smithii genome that is involved in active devel-
opment, diapause, and their photoperiodic regulation.
QTL for CPP are involved in a number of epistatic in-
in CPP between southern and northern populations
identified in earlier studies. Finally, the key circadian
in one of them, suggesting that timeless may play an in-
direct role in the evolution of the photoperiodic timer.
This genetic architecture underlying photoperiodism
not only has permitted diversification of W. smithii from
the Gulf of Mexico to northern Canada, but also has
enabled it to track recent rapid climate change.
We thank K. Emerson, M. Haley, P. Phillips, J. Postlethwait, and
M. Whitlock for useful discussion and for reading previous versions of
the manuscript; B. Kolaczkowski and K. Emerson for computational
support, and D. Houle and two anonymous reviewers for helpful com-
ments. We gratefully acknowledge support from National Institutes of
Health training grant to G. F. Sprague (23T2GM07413-26) and from
the National Science Foundation through a Doctoral Dissertation Im-
provement Grant for D.M. (IBN-0408154) and research grants DEB-
9806278, IBN-9814438, IOB-0415653, and IOB-0445710 to W.E.B.
Figure 5.—Epistatic interactions af-
fecting critical photoperiod in LG3.
All involve timeless, which is codomi-
nant. Abbreviations are as in Figure 4.
QTL Map of Photoperiodic Response 399
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Windows QTL Cartogra-
Communicating editor: D. Houle
Polymorphic markers used to distinguish FL and AB alleles in the F2generation
Primers (59 / 39)
A. Gene-based codominant markers
HinfI AB28 50.0
UbcD4EcoRVFL2 18 124.6
BccI AB35 22.6
AciI AB3 12 53.1
B. Dominant AFLP markers
QTL Map of Photoperiodic Response 401
aThe genes are named according to the putative ortholog from TBLASTX results of the W. smithii sequence
against the genome of D. melanogaster. The numbered genes are unannotated on FlyBase and the prefix Cg- has
been replaced by Ws- for W. smithii. The GenBank accession nos. for the nine genes, in descending order, are
EF094841–EF094856 and AY943312.
bThe restriction endonuclease used in genotyping (see materials and methods for details).
cThe polymorphism for Ws13043 is a 96-bp insertion/deletion. Genotyping in the F2was performed by scor-
ing length differences of PCR amplicons instead of by restriction endonuclease digestion.
dThe EcoRI and MseI core primer sequences for both the pre- and selective amplification steps of the AFLP
protocol are 59-GACTGCGTACCAATTC-39 and 59-GATGAGTCCTGAGTAA-39, respectively. The three bases af-
ter the letters E and M in each marker name denote the three arbitrary nucleotides added to the 39-end of the
core sequence for selective amplification. The lowercase letter at the end of the name distinguishes between
multiple markers generated by the same set of selective primers.
402 D. Mathias et al.