Divergence of larval morphology between Drosophila

sechellia and its sibling species caused by

cis-regulatory evolution of ovo兾shaven-baby
Élio Sucena* and David L. Stern
Laboratory for Development and Evolution, University Museum of Zoology, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ,
United Kingdom

Edited by Eric H. Davidson, California Institute of Technology, Pasadena, CA, and approved March 1, 2000 (received for review January 5, 2000)

We report an extreme morphological difference between Drosoph- varies dramatically between species of the Drosophila virilis
ila sechellia and related species of the pattern of hairs on first- group, we examined larvae from species of the D. melanogaster
instar larvae. On the dorsum of most species, the posterior region species subgroup for variation in hair patterning. Dickinson et al.
of the anterior compartment of most segments is covered by a found that in most species of the D. virilis group, most body
carpet of fine hairs. In D. sechellia, these hairs have been lost and segments possess three rows of robust denticles and a large lawn
replaced with naked cuticle. Genetic mapping experiments and of fine hairs, a pattern similar to that found in D. melanogaster.
interspecific complementation tests indicate that this difference is Four species, however, produce only the robust denticle belts,
caused, in its entirety, by evolution at the ovo兾shaven-baby locus. with naked cuticle replacing the lawn of fine hairs. We have
The pattern of expression of the ovo兾shaven-baby transcript is discovered that D. sechellia displays a similar phenotype, with
correlated with this morphological change. The altered dorsal each segment containing several rows of robust denticles, and a
cuticle pattern is probably caused by evolution of the cis-regula- large region of naked cuticle. All other species of the D.
tory region of ovo兾shaven-baby in the D. sechellia lineage. melanogaster species group possess a lawn of fine hairs instead of
naked cuticle. Here we document this variation and present
genetic evidence that this difference between D. sechellia and its
M ost recent studies of evolutionary developmental biology
have focused on establishing correlations between mac-
roevolutionary changes in morphology and alterations in gene
close relatives is caused in its entirety by evolution at the
ovo兾shaven-baby (ovo兾svb) locus.
expression patterns (1–4). Although such studies suggest possi-
ble modes of developmental change underlying phenotypic Materials and Methods
evolution (and have additional uses in phylogeny reconstruction Fly Stocks. Flies were maintained at 25°C on standard cornmeal
and determination of homology), distant taxonomic compari- agar. Wild-type lines of D. mauritiana (0241.5 and 0241.6) and
sons provide limited insight into how development evolves in D. sechellia (0248.2, 0248.3, 0248.4, 0248.5, and 0248.15) were
natural populations. In particular, such comparisons do not obtained from the Species Stock Center (Bowling Green, OH).
identify the individual mutations altering developmental pro- Stocks of D. melanogaster (Oregon-R) and D. simulans (Tsim-
cesses that were initially exposed to natural selection. This link bazaza and y1w1f 2) were obtained from the Ashburner lab
is required to connect population processes to evolutionary (University of Cambridge). The stock w1svb1兾FM7 was provided
patterns and is best addressed by determining the mutations by the Nüsslein–Volhard lab (Tübingen, Germany). We gener-
causing phenotypic evolution within and between closely related ated the recombinant y1w1svb1. Deficiency kit DK1, a collection
species. of 41 deficiencies covering most of the X chromosome, as well
Classic evolutionary theory predicts that interspecific differ- as svb2兾FM7, Df(1)cho2, Df(1)RC40, Df(1)HC244, Df(1)bi-
ences result from the accumulation of multiple mutations, each DL1, Df(1)bi-D2, and Df(1)bi-DL2, were obtained from the
of small effect (5), although more recent theoretical develop- Bloomington Stock Center.
ments have argued for a distribution of effects at a smaller
number of loci accounting for most variation (6). Most obser- Cuticle Preparations and Microscopy. First-instar larvae were
vations of genetic differences between Drosophila species are mounted as described (18) for microscopic examination of
broadly consistent with this latter expectation. For example, cuticle phenotypes. Dorsal hair patterns were imaged from
evolution at multiple loci underlies differences in the shape of standard cuticle preparations on a confocal microscope (Leica
the male genital arch (7, 8), differences in the acoustic mating SP) (larval cuticles autofluoresce when exposed to the Argon
signal (9), and the causes of male sterility (10, 11) between laser).
Drosophila simulans and Drosophila mauritiana. Likewise, D.
sechellia’s resistance to the toxic morinda fruit has a polygenic Crosses. Interspecific crosses were performed after maintaining
basis (12). Divergence in male secondary sexual traits between virgin females with females of the opposite species for several
the Hawaiian drosophilids, Drosophila heteroneura and Drosoph- days (19). Females of the second species, whose wings had been
ila silvestris, is also caused by evolution at multiple loci (13). In clipped for identification, were removed before adding males.
contrast, the intraspecific sitter兾rover behavioral polymorphism Eggs or larvae were collected from apple juice plates (18). D.
of Drosophila melanogaster is caused by variation at a single locus sechellia females laid eggs on normal medium supplemented with
(14). In addition, a surprising number of traits in populations octanoic acid (Sigma) (240 ␮l per 100 g) (20).
exposed to recent strong artificial selection have provided
evidence for evolution through changes in one or few genes of PCR Cloning. Genomic DNA was prepared by standard techniques
large effect [e.g., insecticide resistance (15) and maize evolution (21). PCR primers were designed for the D. melanogaster ovo兾svb
(16)]. Despite these examples of large single gene effects, it
remains unclear how often interspecific differences are gener-
ated by the evolution of one or few loci. This paper was submitted directly (Track II) to the PNAS office.
Inspired by the discovery of Dickinson and coworkers (17) Abbreviations: n.s., no significant difference; ovo/svb, ovo/shaven-baby.
that the pattern of hairs on the dorsum of the first-instar larva *To whom reprint requests should be addressed. E-mail: [email protected].

4530 – 4534 兩 PNAS 兩 April 25, 2000 兩 vol. 97 兩 no. 9

second exon (22): 5⬘-ATTGCCTCCGTTTTATGAGA and 5⬘-
TGCTCCAGTAAATGATCGGT. PCR was carried out with
standard reagents (Boehringer–Mannheim), and the amplifica-
tion products were cloned by using the pGEM-T Easy kit
(Promega).

In Situ Hybridization. In situ hybridization to whole mount embryos

was performed essentially as described (23). RNA digoxigenin
probes were prepared by using the Megascript Kit (Ambion,
Austin, TX). Antisense and sense probes were synthesized from
the cloned second exon fragment of D. sechellia.

Results
All species of the D. melanogaster species subgroup, except D.
sechellia, possess a dorsal pattern of denticles and hairs similar
to that described previously for D. melanogaster (24–26). The
precise pattern of hairs and denticles varies between segments
(25), and the following description focuses on the abdominal
segments. The most anterior cells of the anterior compartment
produce naked cuticle. More posteriorly, there are two to three
rows of short and thick denticles and then six to eight rows of fine
hairs (Fig. 1). On the lateral surface of the larvae, fine hairs are
also found in the same anterior–posterior domain of each
segment as the dorsal hairs (not shown). In the posterior
compartment, cells of the anterior row secrete naked cuticle, and
cells of the posterior row produce large thick denticles (Fig. 1).
The dorsal cuticle of D. sechellia first-instar larvae differs from
the above description primarily by the absence of the lawn of fine
hairs both dorsally (Fig. 1) and laterally (not shown). We have
noticed other minor variations in trichome patterning between
species of the D. melanogaster species subgroup, but we have not
characterized these in detail. Phylogenetic analysis suggests that

EVOLUTION
the naked cuticle phenotype has evolved within the D. sechellia
lineage (Fig. 1).
We performed a series of genetic crosses that together indicate
that this phenotypic difference is caused by evolution at a single
locus on the X chromosome. First, crosses between D. simulans
females and D. sechellia males produced larvae with a ‘‘simulans-
like’’ phenotype (n ⫽ 14), indicating that the D. simulans allele

SPECIAL FEATURE
is completely dominant to the D. sechellia allele. No intermediate
phenotypes were observed (we have not succeeded in crossing
flies in the opposite direction).
Hybrid D. sechellia兾D. simulans females were backcrossed to
both parental species. Because recombination could occur
within these hybrid females, we could estimate the number of
evolved genes. The backcross to D. sechellia males produced a
ratio of simulans-like to sechellia-like larvae that did not deviate
significantly from a 1:1 ratio expected from the segregation of a
single locus [21 simulans-like: 19 sechellia-like, ␹2 ⫽ 0.10, no
significant difference (n.s.)]. The backcross to D. simulans males
produced a ratio of larvae that did not deviate significantly from
a 1:3 ratio expected of a single sex-linked locus (52 simulans-like:
14 sechellia-like, ␹2 ⫽ 0.51, n.s.).
Crosses between D. melanogaster females and D. sechellia
males produced only ‘‘melanogaster-like’’ F1 larvae (n ⫽ 15),
indicating that the D. melanogaster allele is also dominant to the
D. sechellia allele [the offspring of this cross are sterile (27),
preventing F2 mapping].
Given the dominance of the D. melanogaster allele to the D. Fig. 1. The phylogenetic distribution of dorsal hair patterns for five mem-
sechellia allele, we attempted to locate the gene by performing bers of the D. melanogaster species group. A phylogeny of these species is
interspecific complementation tests by using a standard set of shown (Left, modified from ref. 36). Confocal micrographs are shown for
chromosome deficiencies covering approximately 80% of the D. abdominal segments 1 and 2 for each species. A cartoon of the pattern of hairs
is shown beside each micrograph. In the cartoons, cells in the anterior and
melanogaster X chromosome. In this test, the dominant D.
posterior compartment of the segment are shown as white and gray rectan-
melanogaster allele was hypothesized to be removed by one or gles, respectively. The relative positions of the three types of cuticular pro-
several overlapping deficiencies, which would thereby reveal the jections, short denticles, fine hairs, and large denticles are illustrated (see
recessive D. sechellia allele in a hybrid. Only Df(1)JC70 produced text). Anterior is up. The dorsal hair patterns for the remaining members of the
a proportion of ‘‘sechellia-like’’ larvae consistent with the defi- group (Drosophila erecta, Drosophila orena, and Drosophila teissieri) are
ciency having uncovered the evolved gene. Complementation similar to Drosophila yakuba (not shown).

Sucena and Stern PNAS 兩 April 25, 2000 兩 vol. 97 兩 no. 9 兩 4531
 Fig. 3. Confocal micrographs of the first and second abdominal segments of
Fig. 2. Localization of the evolved gene by failure of complementation of X
D. sechellia (a) are similar to those of a hybrid between a D. melanogaster svb1
chromosome deficiencies. (a) The cytological regions covered by deficiencies
mutant and D. sechellia (b). In contrast, a hybrid of wild-type D. melanogaster
that produced viable larvae when crossed to D. sechellia males are shown as
and D. sechellia (c) displays a cuticular pattern similar to D. melanogaster (see
black boxes. Approximately 75% of the chromosome was screened success-
Fig. 1). The D. melanogaster svb1 mutations leads to complete loss of denticles
fully with deficiencies. In the original screen, only Df(1)JC70 produced larvae
and hairs on the dorsal surface (not shown) and loss of most denticles on the
with the D. sechellia hair pattern in the expected ratio (n ⫽ 57).[Some of the
ventral surface (d). [In some svb1兾D. sechellia larvae, small patches of hairs in
crosses yielded single larvae with a D. sechellia hair pattern. These larvae are
the middle of naked cuticle were occasionally observed (b). Such hairs were
likely the result of meiotic nondisjunction in the female parent generating
never observed in hybrid backcrosses and in crosses between svb deficiencies
nullo X eggs fertilized by X-bearing D. sechellia sperm, which in turn gener-
and D. sechellia, suggesting that svb1 is not a complete loss-of-function allele.]
ated XO embryos displaying the D. sechellia hair pattern. Nondisjunction is
Anterior is up.
elevated in stocks carrying balancer chromosomes and stocks with XXY fe-
males (37)]. (b) Further localization to the 4C15-E1 cytological region was
performed with overlapping deficiencies. Regions deleted by deficiencies are
indicated by bold horizontal lines with the name of the deficiency next to the melanogaster svb chromosome)—in ratios not significantly dif-
line. The continuation of a deficiency outside this region is shown by a dashed ferent from the expected 2:1:1 (svb1, 14:8:8, ␹2 ⫽ 0.13, n.s.; svb2,
line. Deficiencies producing larvae with a D. sechellia hair pattern in the 38:17:16, ␹2 ⫽ 0.38, n.s.). We determined subsequently that the
expected frequencies when crossed to D. sechellia males are indicated with a embryos exhibiting the svb-like phenotype were indeed males by
plus sign. Deficiencies producing only larvae with a hair pattern typical of D. crossing y1w1svb1 D. melanogaster heterozygous females to D.
melanogaster are indicated by a minus sign. The distal limit of the evolved sechellia males. All ‘‘svb-like’’ embryonic cuticles from this cross
gene is defined by the right breakpoint of Df(1)bi-DL2 (4C15-D1) and the left
breakpoint of Df(1) JC70 (4C15–16), and the proximal limit is defined by the
had yellow mouthparts (n ⫽ 7), and all ‘‘sechellia-like’’ larvae had
right breakpoint of Df(1)bi-D2 (4D7-E1). (c) Genes known to exist within this brown mouthparts (n ⫽ 16), confirming that the ‘‘svb-like’’
region are listed in their approximate cytological location. The genes cut up larvae were male and the ‘‘sechellia-like’’ larvae were female.
and ovo兾svb have been localized previously to the small regions, 4D1–3 and One explanation for these results is that mutations at ovo兾svb
4E1, respectively, and the three genes Protein tyrosine phosphatase 4E, generate a ‘‘sechellia-like’’ phenotype in any hybrid, because of
Protein phosphatase 2C1, and lethal(1)4Ea have been localized previously to hybrid incompatibilities. Crosses between D. melanogaster svb
4E1–2. The remaining genes shown have been localized to large regions that mutants and D. simulans males allow us to reject this possibility.
include 4D-E1 (see http:兾兾flybase.bio.indiana.edu for details).
In the cross between D. melanogaster y1w1svb1 females and D.
simulans males, we observed ‘‘melanogaster-like’’ and ‘‘svb-like’’
tests with six additional deficiencies confirmed the initial result phenotypes in the expected proportions, but never ‘‘sechellia-
and refined the cytological region to 4C15-E1 (Fig. 2). like’’ phenotypes. (One embryo was found with a trichome
Of the candidate genes in this region (Fig. 2c), one of them, pattern similar to D. sechellia. This embryo also possessed
ovo兾svb, had been described previously as a gene involved in extensive defects in the head, mouthparts, posterior spiracles,
denticle and hair differentiation (22, 28, 29). Mutations in this and Keilin’s organs, as well as polarity defects in the dorsal
gene remove most or all of the denticles and hairs, both ventrally denticles. We have not observed such pervasive defects corre-
and dorsally, on D. melanogaster larvae (Fig. 3d). lated with the ‘‘sechellia-like’’ phenotype in other crosses, and
We tested directly whether mutations of ovo兾svb failed to this is probably an unrelated phenomenon.) Finally, hatched
complement the D. sechellia phenotype. The cross of svb1兾FM7 larvae from crosses between Df(1)biD2, which removes the svb
and svb2兾FM7 females to D. sechellia males produced embryonic region, and D. simulans males never displayed a ‘‘sechellia-like’’
cuticle patterns of three types—‘‘melanogaster-like’’ (putatively, phenotype (n ⫽ 20).
embryos carrying the D. melanogaster balancer chromosome), We performed a mapping experiment to determine whether
‘‘sechellia-like’’ (putative female embryos carrying the D. sech- the evolved gene maps near ovo-svb. Female D. simulans carrying
ellia X chromosome and a D. melanogaster svb mutant chromo- an X chromosome marked with y1w1f 2 were crossed to D.
some), and ‘‘svb-like’’ (putative male embryos carrying the D. sechellia males, and the hybrid females were backcrossed to

4532 兩 www.pnas.org Sucena and Stern

 dorsal hair patterning between D. sechellia and other species of
the D. melanogaster species subgroup. This change may be caused
by evolution of cis-regulatory transcriptional control of ovo兾svb.
The regulatory nature of the change is supported by several
observations. First, independent regulatory elements drive the
ovo and svb functions (22, 31, 32). The ovo function is required
for development of the female germline, and ovo mutants can be
rescued by genomic DNA spanning the known exons of ovo. The
svb function is required for the differentiation of denticles and
hairs in the larvae. Some svb mutants, including breakpoints 5⬘
of the known exons of ovo兾svb, are not rescued by the ovo
transgene, suggesting that regulatory elements 5⬘ of the known
exons are required for svb function (22). Second, the pattern of
naked cuticle and denticles in D. sechellia is not identical to the
svb loss-of-function phenotype (28) and instead appears to
represent the loss of a subset of the hairs lost in svb mutants
(Fig. 3). This suggests that the transition to the D. sechellia
phenotype was caused by cis-regulatory evolution of part of svb
function. Our analysis of the distribution of ovo兾svb transcripts
in embryos of D. melanogaster and D. sechellia (Fig. 4) suggests
that this change occurred at the level of transcriptional control
of ovo兾svb.
Modular enhancer architecture (33) allows mutations to
alter only one part of a gene’s function, without generating
multiple deleterious pleiotropic effects. In the case we have
documented, evolution of ovo兾svb resulted in an alteration in
the function of only part of ovo兾svb function, apparently
without pleiotropic effects on other aspects of trichome and
Fig. 4. The pattern of svb expression in D. melanogaster (a, c, and e) and D. hair patterning. Because the ovo function is also required for
sechellia (b, d, and f ) stage 17 embryos. Dorsal views are shown in a, c, and d, female germline differentiation, variation at this locus might
dorso–lateral in b, and lateral optical cross sections in e and f. The svb
be expected to affect also ovarian development. In fact,
transcript is detected in both species in rows corresponding to the dorsal stout
females of D. sechellia have fewer ovarioles than females of its

EVOLUTION
denticles (a– d and arrows in e and f ). svb transcript is detected at lower levels
in the rows giving rise to fine hairs in D. melanogaster (horizontal bars in c and sibling species. However, none of the genes generating this
e) and is not detected in the corresponding positions in D. sechellia (horizontal difference map near ovo兾svb (34). Therefore, evolution of the
bars in d and f ). The cells differentiating the ventral denticle belts express high cis-regulatory regions of patterning genes can result in rela-
levels of svb (arrowheads in b, e, and f ). Anterior is left in all images, and dorsal tively dramatic evolutionary transitions without potentially
is up in b, e, and f. deleterious pleiotropic consequences, even when such genes
play pleiotropic roles in development.
Models of adaptation suggest that single mutational events

SPECIAL FEATURE
y1w1f 2 D. simulans males. Two of these recessive markers are causing dramatic phenotypic alterations are more likely to be
visible in the first-instar larvae; y1 produces yellow or light brown fixed by strong selection and at the beginning of a bout of
mouthparts, compared with the normal dark brown, and f 2 adaptation (6, 35). This contrasts with the traditional Darwin-
produces thinner and more sinuous ventral denticles, similar to ian view that large differences arise from the accumulation of
the phenotype of the D. melanogaster f 3N allele (30), as well as many small changes. Although our experiments indicate that
stouter lateral sensory bristles. The observed number of recom- a major difference in larval hair patterning is caused by
binants for each class did not deviate significantly from the evolution of a single gene, this large change may have resulted
expectation that the evolved gene maps to the ovo兾svb locus (n ⫽ from the accumulation of multiple mutations of smaller effect
66, ␹2 ⫽ 5.76, 7 degrees of freedom, n.s.). within the cis-regulatory region of ovo兾svb. In either case, the
Examination of the distribution of svb transcripts indicates results are surprising, but for different reasons. If this mor-
that cell-specific loss of svb transcription is correlated with the phological transition is caused by a single mutational change,
absence of hairs in D. sechellia (Fig. 4). The pattern of svb then mutations of relatively large effect must be recognized as
transcript in D. melanogaster is similar to that previously contributors to species differences. If, however, the transition
described (31). The svb transcript is detected at lower levels in is caused by multiple mutations at ovo兾svb, then we must
the cells that differentiate the fine hairs of the dorsum, in explain why all of the mutations occurred at a single locus and
contrast to the stronger expression detected in the rows of were not distributed among multiple loci. The latter result
more robust denticles on both the dorsum and ventrum (Fig. would support the idea that a limited number of genes may be
4 a, c, and e). In D. sechellia, the svb transcript is detected at available to generate evolution of at least some morphological
high levels in the cells forming the ventral denticle rows and features. Resolution of this problem requires experiments to
in other trichome-forming cells, including the dorsal denticle identify the individual mutations at ovo兾svb that have gener-
rows (Fig. 4 b, d, and f ). The svb transcript was not detected ated these differences. Given the complexity of the ovo兾svb
in the dorsal cells that differentiate naked cuticle (Fig. 4 b, d, locus and particularly the currently unknown structure of the
and f ). svb regulatory regions, these experiments will not be trivial,
but they are tractable in Drosophila, and they are currently
Discussion under way.
We have shown that a dramatic difference in larval morphology This study demonstrates further the power of using species
between sibling species is caused by evolution at a single locus. closely related to the ‘‘model’’ species, D. melanogaster, for
Complementation tests and mapping experiments indicate that studying problems in evolution and development. Only the
evolution at the ovo兾svb locus is responsible for the difference in powerful tools and vast knowledge of D. melanogaster allowed us

Sucena and Stern PNAS 兩 April 25, 2000 兩 vol. 97 兩 no. 9 兩 4533
to move quickly from observation of morphological differences For fly stocks, we thank John Roote at the Ashburner lab (Cambridge,
to study of a single gene. The D. melanogaster species group U.K.), the Nüsslein–Volhard lab, the Bloomington Stock Center, and the
displays a diversity of morphology, behavior, physiology, and life Species Stock Center. We thank A. Orr for suggesting a useful control
experiment and Christen Mirth, the editor, and anonymous referees for
history, and all of these traits are amenable to comparatively
useful comments on an earlier draft. This work was funded by a
thorough genetic analysis because of the phylogenetic proximity Programa Gulbenkian de Doutoramento em Biologia e Medicina and
of D. melanogaster. Further studies of the D. melanogaster species Praxis XXI Fellowship (Portugal) to E.S. and a Biotechnology and
group will generate central insights into the process, as well as Biological Sciences Research Council David Phillips Research Fellow-
the pattern, of developmental evolution. ship to D.L.S.

1. Averof, M. & Akam, M. (1995) Nature (London) 376, 420–423. 20. R’kha, S., Capy, P. & David, J. R. (1991) Proc. Natl. Acad. Sci. USA 88,
2. Carroll, S. B., Weatherbee, S. D. & Langeland, J. A. (1995) Nature (London) 1835–1839.
375, 58–61. 21. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A
3. Panganiban, G., Sebring, A., Nagy, L. & Carroll, S. (1995) Science 270, Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY).
1363–1366. 22. Mével-Ninio, M., Terracol, R. & Kafatos, F. C. (1991) EMBO J. 10, 2259–2266.
4. Gellon, G. & McGinnis, W. (1998) BioEssays 20, 116–125. 23. Tautz, D. & Pfeifle, C. (1989) Chromosoma 98, 81–85.
5. Fisher, R. A. (1930) The Genetical Theory of Natural Selection (Dover, NY), pp. 24. Martinez Arias, A. (1993) in The Development of Drosophila melanogaster, eds.
1–188. Bate, M. & Martinez-Arias, A. (Cold Spring Harbor Lab. Press, Plainview,
6. Orr, H. A. (1998) Evolution (Lawrence, KS) 52, 935–949. NY), pp. 517–608.
7. True, J. R., Liu, J., Stam, L. F., Zeng, Z.-B. & Laurie, C. C. (1997) Evolution 25. Kuhn, D. T., Sawyer, M., Ventimiglia, J. & Sprey, Th. E. (1992) Drosophila
(Lawrence, KS) 51, 816–832. Information Service 71, 218–222.
8. Zeng, Z., Liu, J., Stam, L. F., Kao, C., Mercer, J. M. & Laurie C. C. (2000) 26. Bokor, P. & DiNardo, S. (1996) Development (Cambridge, U.K.) 122, 1083–
Genetics 154, 299–310. 1092.
9. Pugh, A. R. & Ritchie, M. G. (1996) Heredity 77, 378–382.
27. Lemeunier, F. & Ashburner, M. (1976) Proc. R. Soc. London Ser. B 193,
10. Palopoli, M. F. & Wu, C.-I. (1994) Genetics 138, 329–341.
275–294.
11. Coyne, J. A. (1996) Genet. Res. 68, 211–220.
28. Wieschaus, E., Nusslein-Volhard, C. & Jurgens, G. (1984) Roux’s Arch. Dev.
12. Jones, C. D. (1998) Genetics 149, 1899–1908.
Biol. 193, 296–307.
13. Carson, H. L., Val, F. C. & Templeton, A. R. (1994) Proc. Natl. Acad. Sc. USA
29. Payre, F., Vincent, A. & Carreno, S. (1999) Nature (London) 400, 271–275.
91, 6315–6318.
14. Osborne, K. A., Robichon, A., Burgess, E., Butland, S., Shaw, R. A., Coulthard, 30. Dickinson, W. J. & Thatcher, J. W. (1997) Cell Motil. Cytoskeleton 38, 9–21.
A., Pereira, H. S., Greenspan, R. J. & Sokolowski, M. B. (1997) Science 277, 31. Mével-Ninio, M., Terracol, R., Salles, C., Vincent, A. & Payre, F. (1995) Mech.
834–836. Dev. 49, 83–95.
15. ffrench-Constant, R. H., Rocheleau, T. A., Steichen, J. C & Chalmers, A. E. 32. Mével-Ninio, M., Mariol, M.-C. & Gans, M. (1989) EMBO J. 8, 1549–1558.
(1993) Nature (London) 363, 449–451. 33. Arnone, M. I. & Davidson, E. H. (1997) Development (Cambridge, U.K.) 124,
16. Doebley, J., Stec, A. & Hubbard, L. (1997) Nature (London) 386, 485–488. 1851–1864.
17. Dickinson, W. J., Tang, Y., Schuske, K. & Akam, M. (1993) Evolution 34. Coyne, J. A., Rux, J. & David, J. R. (1991) Genet. Res. 57, 113–122.
(Lawrence, KS) 47, 1396–1406. 35. Lande, R. (1983) Heredity 50, 47–65.
18. Stern, D. L. & Sucena, E. (2000) in Drosophila: A Laboratory Manual, eds. 36. Harr, B., Weiss, S., David, J. R., Brem, G. & Schlötterer, C. (1998) Curr. Biol.
Ashburner, M., Hawley, S. & Sullivan, B. (Cold Spring Harbor Lab. Press, 8, 1183–1186.
Plainview, NY), pp. 601–615. 37. Ashburner, M. (1989) Drosophila: A Laboratory Handbook (Cold Spring
19. Coyne, J. A. & Charlesworth, B. (1997) Genetics 145, 1015–1030. Harbor Lab. Press, Plainview, NY).

4534 兩 www.pnas.org Sucena and Stern