Genetica (2009) 137:165–171
DOI 10.1007/s10709-009-9381-z

High genetic differentiation between an Africanand a non-African strain of Drosophila simulans revealed
by segregation distortion and reduced crossover frequency
Haruki Tatsuta Æ Toshiyuki Takano-Shimizu

Received: 21 October 2008 / Accepted: 23 June 2009 / Published online: 10 July 2009Ó Springer Science+Business Media B.V. 2009

Abstract Drosophila simulans strains originating from
Madagascar and nearby islands in the Indian Ocean often
differ from those elsewhere in the number of sex comb teeth
and the degree of morphological anomaly in hybrids with D.
melanogaster. Here, we report a strong segregation distortion in the F1 intercross between two D. simulans strains
originating from Madagascar and the US, possibly at both
the gametic and zygotic levels. Strong bias against alleles ofthe Madagascar strain was observed for all ten marker locidistributed over the entire second chromosome in the F1intercross, but only a few showed a weak distortion in theisogenic backgrounds of either strains. Significant deviations of genotype frequencies from Hardy–Weinberg proportions were consistently observed for the second

chromosome. By contrast, the X and third chromosomes did
not show any strong segregation distortion. Crossover frequency on the second chromosome was uniformly reduced
in isogenic backgrounds whereas the map lengths in the F1intercross were comparable to or larger than that of thestandard D. melanogaster map. We discuss these findings inrelation to previous studies on other traits and interspecificdifferences between D. mauritiana, which is endemic to
Mauritius Island, and D. simulans.
Keywords Drosophila simulans  Segregation distortion 
Genetic map  Worldwide colonization  Introgression

Introduction
H. Tatsuta  T. Takano-Shimizu (&)
Department of Population Genetics, National Instituteof Genetics, Mishima, Shizuoka 411-8540, Japane-mail: totakano@lab.nig.ac.jp
T. Takano-Shimizu
Department of Genetics, Graduate University for Advanced
Studies (SOKENDAI), Mishima, Shizuoka 411-8540, Japan
T. Takano-Shimizu
Department of Biosystems Science, Graduate University for
Advanced Studies (SOKENDAI), Hayama, Kanagawa 240-0193,
Japan
T. Takano-Shimizu
Department of Biological Sciences, Graduate School of Science,
The University of Tokyo, Tokyo 113-0033, Japan
Present Address:
H. Tatsuta
Laboratory of Subtropical Zoology, Graduate School of
Agriculture, University of the Ryukyus, Okinawa 903-0213,
Japan

A widespread species is likely to have greater genetic
variation than a narrowly distributed species and may
represent the first stage of speciation (Mayr 1942). An
important but unresolved problem is what type of genetic
variation results from population expansion. This variation
can subsequently be the basis of adaptive evolution andspeciation (Lewontin 1974). A recently expanded species
may be particularly useful for tackling the problem.
Three Drosophila species in the simulans clade, D. simulans, D. mauritiana and D. sechellia, diverged from theirclose relative D. melanogaster about five million years ago
and then diverged from one another about one million years
ago (Tamura et al. 2004). D. simulans and D. melanogasterare widespread cosmopolitan species whereas D. mauritiana and D. sechellia are endemic to islands in the Indian
Ocean. It is hypothesized that, in late Pleistocene, these two
cosmopolitan species were restricted to the Afrotropicalregions; D. melanogaster inhabited west and equatorial
Africa, and D. simulans inhabited the east coast and Indian

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Ocean islands. They expanded their distribution recently,
probably within the last 10,000 years (Lachaise et al. 1988).
The spread of D. simulans out of Africa possibly occurred
more recently than that of D. melanogaster (Lachaise et al.
1988), which could explain the lower geographic differentiation of allozyme variation and quantitative traits in D.
simulans than in D. melanogaster (Hyytia et al. 1985; Singhet al. 1987; Morton et al. 2004).
All four species are morphologically very similar to one
another and differ only in male genitalia and a few othertraits. The secondary sexual character, the sex comb, is sucha trait. The sex comb is a specific row of enlarged bristles on
the foreleg of males. D. simulans, D. sechellia and D. melanogaster have a similar number of teeth in the sex comb
(average 10.3 per foreleg in D. simulans) whereas D. mauritiana has a significantly larger number of teeth (average
13.9 per foreleg; True et al. 1997). On the other hand, we
observed substantial variation in this trait among D. simulans strains (Tatsuta and Takano-Shimizu 2006). A strain
originating from a female collected in Madagascar (hereinafter ‘‘Tananarive’’) had a large sex-comb-tooth number(average 12.3 per foreleg). We then conducted quantitative
trait loci (QTL) mapping experiments for this trait using the
Tananarive strain and another D. simulans strain with asmaller tooth size (Sim-3 which originates from the US;
Tatsuta and Takano-Shimizu 2006). Three QTLs weresuccessfully mapped on the third chromosome. Interestingly, two of these QTLs are concordant with the locationsof the QTLs responsible for the difference in sex-comb tooth
number between D. simulans and D. mauritiana.
Bristle defects in interspecific hybrids with D. melanogaster also reveal hidden genetic variation both between
species and within D. simulans. Most simulans strains lose
many bristles in the hybrids, but strains from Madagascar
and the Mascarene and Seychelles islands showed reduced
anomalies. Moreover, such anomalies were not found in
hybrids of D. mauritiana and D. melanogaster (Takano
1998). Mapping analyses of genes responsible for thebristle loss phenotype identified a single major QTL on the
X chromosome that accounts for most of the difference
between the Tananarive strain with a low number ofmissing bristles and a strain with a high number (TakanoShimizu 2000).
In this paper, we further report on two features thatcharacterize the same two strains of D. simulans, Tananarive and Sim-3, namely segregation ratio distortion andreduction of crossover frequency in hybrids. Because we
hardly expect a common genetic basis underlying thesefour phenotypes, these strains are concluded to be highly
divergent in many ways. These differences may be either
because of changes during worldwide colonization or
because of genetic introgression in the Madagascar–
Mascarene region.

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Genetica (2009) 137:165–171

Materials and methods
We used two inbred strains of D. simulans, Sim-3 (G20;
Raleigh, North Carolina, USA) and Tananarive (G20;
Madagascar), established by sib-mating for 20 generations,which are the same strains in Tatsuta and Takano-Shimizu(2006). We obtained the following F1 intercrosses withthese two strains through two reciprocal parental crosses:
females from crosses between Sim-3 females and Tananarive males 9 males from the same crosses (abbreviated as S/T 9 S/T cross); the same F1 females 9 males
from crosses between Tananarive females and Sim-3 males(S/T 9 T/S cross); T/S 9 S/T cross; and T/S 9 T/S cross. A
total of 200 male progeny were sampled in approximately
equal numbers from each of the F1 intercrosses and typed
for markers, as described below. For the second chromosome, we also typed males from heterozygote crosses in theisogenic genetic background of Sim-3 (IIs cross; n = 212)and in that of Tananarive (IIt cross; n = 194). Figure 1
illustrates the mating scheme.
We typed five molecular markers on the X chromosome(sgg, G01498, Dm1865, Dm0478, and run), ten on thesecond chromosome (dpp, Pgk, ninaC, prd, Ddc, eve, vg,sli, Pcl, and twi), and twelve on the third chromosome (ve,h, Eip71CD, 5-HT2, Antp, ninaE, Mst87F, hb, Ald, Mlc1,
janA, and Ef1a100E). The genotyping methods aredescribed in detail in Tatsuta and Takano-Shimizu (2006).
Because no crossover was observed between 5-HT2 andAntp, they were treated as a single marker. Map distanceswere calculated with the map function described in Fosset al. (1993; with m = 4).

Results
Segregation distortion
Segregation distortions, which are often brought about by
genetic incompatibilities, were examined in terms of allele
and genotype frequencies. There were two contrasts at theallele-frequency level. Although only two of sixteen Xchromosome and third-chromosome markers showed significant deviations from the expected 1:1 ratio (both at the
5% level), all the second-chromosome markers exhibitedstrong deficiency of Tananarive alleles in the pooled datafrom the F1 intercross (Table 1). In contrast with the F1intercross, there was no bias for either allele in IIs and IIt
crosses (Table 1). Thus, the allele-frequency distortion
strongly depended on both chromosome and genetic
background.
Because only two of twenty-six markers showed significant heterogeneities in allele frequencies among thefour types of F1 intercrosses (both at the 5% level), we

Genetica (2009) 137:165–171
Fig. 1 Mating scheme for the
F1 intercross (a) and thesecond-chromosome IIs cross in
the Sim-3 isogenic background(b). Short bars in circles
represent the second
chromosomes and long bars
represent the third
chromosomes. Sex and fourthchromosomes are omitted forclarity. We also provide genetic
constitution of X, second andthird chromosomes inparentheses, where Y indicates
the Y chromosome. The filled
and open bars indicate the Sim3 and Tananarive chromosomes,
respectively. See ‘‘Materials andmethods’’ for further details

167

(b)

(a)

X

X

X

X

F1 intercross

Select1

X

Select1(S/Y;S/T;S/S)

X

Select1(S/Y;S/T;S/S)(S/S;S/T;S/S)

IIs crosspooled the data. However, at six second-chromosome
markers we found slight, but significant, differences inallele frequencies between two types of females (females
from Sim-3 female 9 Tananarive male cross, abbreviatedas S/T females, vs. T/S females from the reciprocal cross;
all at the 5% level; data not shown). The segregation bias
against Tananarive alleles was stronger in S/T 9 S/T and S/
T 9 T/S F1 intercrosses (crosses involving S/T females)than in T/S 9 S/T and T/S 9 T/S crosses (those involving
T/S females); the average frequency of Tananarive alleleson the second chromosome was 0.31 in the former and 0.40
in the latter. On the other hand, the same frequency was
0.38 in S/T 9 S/T and T/S 9 S/T crosses and 0.34 in S/
T 9 T/S and T/S 9 T/S crosses; no statistically significant
difference was found between these two types of crosses(S/T vs. T/S males), except in one case (eve at the 5%
level). In S/T 9 S/T and S/T 9 T/S crosses, the averagefrequencies of SS, ST, and TT genotypes were 0.44, 0.50,
and 0.06, respectively. These results can be explained by
selective elimination or destruction of gametes carrying
Tananarive alleles, particularly in S/T females.
Moreover, genotype frequencies at seven second-chromosome markers significantly deviated from Hardy–
Weinberg proportions (Table 1), where there was a significant excess of heterozygotes at the expense of both TT

and SS homozygotes (Table 1). The same tendency wasseen in isogenic backgrounds, particularly in the IIs cross.
The genotype frequency bias may be because of the lower
viability of homozygotes.
Genetic maps of D. simulans X, second, and third
chromosomes
Genetic maps of all the major D. simulans chromosomes
were constructed on the basis of 26 molecular markers in
the F1 intercross (Fig. 2). The total map length of the X
chromosome (63.7 cM) was the same as that of the standard map of D. melanogaster and slightly shorter than thatof D. simulans in Takano-Shimizu (2000; 75.9 cM). Thisdifference is likely to be because of the smaller number ofgenetic markers in this study; only five markers were usedin this analysis, whereas nine were used in Takano-Shimizu(2000). Indeed, the map length for the latter is reduced to67.8 cM when only five markers are used. Despite thesmall number of markers, the map length of the third
chromosome (128.3 cM) was larger than that of the standard map of D. melanogaster (101.8 cM).
The total length of the second chromosome in the F1intercross was 93.8 cM, which was almost the same as thatof D. melanogaster (96 cM; Fig. 2). On the other hand, the

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Genetica (2009) 137:165–171

Table 1 Segregation frequency
X chromosome
Marker

F1 intercross
T:S allleles

2nd ChromosomeaMarker

F1 intercross
T:S allleles

3rd Chromosome
a

IIs

b

c

IIt

Marker

F1 intercrossave

199:201

TT:TS:SS genotypes
(HW)
sgg

91:106dpp

G01498

95:102

PgK

Dm1865

83:115*ninaC

Dm0478

85:112

prd

157:243***

210:214

193:195

20:117:63**

44:122:46*

41:111:42*(30.8:95.4:73.8)(52.0:106.0:54.0)(48.0:97.0:49.0)

157:243***
20:117:63**

210:214
42:126:44**

192:196
41:110:43(30.8:95.4:73.8)(52.0:106.0:54.0)(47.5:97.0:49.5)

154:246***

209:215

193:195

20:114:66**

42:125:45**

41:111:42*(29.6:94.7:75.6)(51.5:106.0:54.5)(48.0:97.0:49.0)

138:262***

209:215

190:198

19:100:81

43:123:46*

39:112:43*(23.8:90.4:85.8)(51.5:106.0:54.5)(46.5:97.0:50.5)

138:262***

208:216

186:202

18:102:80

42:124:46*

38:110:46

34:117:49*(23.8:90.4:85.8)(51.0:106.0:55.0)(44.6:96.8:52.6)(42.8:99.4:57.8)

135:265***

216:208

188:200

17:101:82

45:126:41**

40:108:46(22. 8:89.4:87.8)
137:263***(55.0:106.0:51.0)
213:211(45.5:96.9:51.5)
188:200

16:105:79*

41:131:40***

39:110:45

47:105:48(49.5:100.0:50.5)
h198:202
46:106:48(49.0:100.0:51.0)

Eip71CD

184:214
37:110:52(42.5:98.9:57.5)

5-HT2

182:218

Antp

run

89:108

Ddc

eve

vg

sli

Pcl

twi

34:114:52*(41.4:99.2:59.4)
ninaE

Mst87F

185:215

188:21240:108:52

hb(44.2:99.6:56.2)
187:213
39:109:52(23.5:90.1:86.5)(53.5:106.0:52.5)(45.5:96.9:51.5)

135:265***

212:212

190:198(43.7:99.6:56.7)

16:103:81*

39:134:39***

38:114:42*(22.8:89.4:87.8)(53.0:106.0:53.0)(46.5:97.0:50.5)

138:262***

215:209

192:196

16:106:78*

42:131:39***

39:114:41*(23.8:90.4:85.8)(54.5:106.0:51.5)(47.5:97.0:49.5)

137:263***

220:204

187:201

14:109:77**

44:132:36***

37:113:44*

40:105:54(23.5:90.1:86.5)(57.1:105.8:49.1)(45.1:96.9:52.1)(43.0:99.0:57.0)

Ald

178:222*39:100:61(39.6:98.8:61.6)

Mlc1

180:218
40:100:59(40.7:98.6:59.7)

janA

Ef1a100E

185:213

187:211
41:105:53(43.9:99.1:55.9)

Significance of deviations of allele frequencies from a 1:1 ratio and of genotype frequencies from Hardy–Weinberg (HW) proportions (provided
in parentheses) was tested by the G-test with Williams’s corrections (Sokal and Rohlf 1995)* P \ 0.05, ** P \ 0.01, *** P \ 0.001
aF1 intercross: S/T; S/T; S/T females 9 S/Y; S/T; S/T or T/Y; S/T; S/T males, where genetic constitution of X, second, and third chromosomes isindicated, and Y stands for Y chromosomebc

IIs cross: S/S; S/T; S/S 9 S/Y; S/T; S/S
IIt cross: T/T; S/T; T/T 9 T/Y; S/T; T/T

map lengths of the second chromosome were 68.0 cM in
the Sim-3 isogenic background (IIs cross) and 65.0 cM in
the Tananarive background (IIt cross; Fig. 2). These

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estimates were only 70% of the map length in the F1intercross. There were significant heterogeneities in distributions of the number of crossovers per chromosome

Genetica (2009) 137:165–171

X sgg

169
Dm1865*

G01498*

Dm0478*run
D. simulans (F1 intercross)

18.2

18.2

14.1

13.1

D. melanogaster
63.7

II dpp Pgk ninaC

prd

Ddc

eve

vg

sli

Pcl

twi
D. simulans (IIt)

0.5

9.8

11.4

8.8

8.2

5.2

9.3

3.1

8.8

D. simulans (IIs)
10.9

2.8

11.3

13.7

7.1

5.7

5.2 3.3

8.0

D. simulans (F1 intercross)
0.0

18.1

14.5

16.1

9.5

9.0

9.0

8.5

9.0

D. melanogaster
1.9

III

16.1

ve

23.0

h

8.9

4.1

Eip71CD

9.0

10.0

5-HT2/
Antp

ninaE

7.0

16.0

Mst87F

Ald Mlc1 janA Ef1α100E

hb

D. simulans
18.8

31.2

11.6

3.5

16.7

19.8

16.2

3.0 5.5

2.0(F1 intercross)D. melanogaster

26.3

15.5

5.5 0.5 6.0

12.4

25.1

3.5 4.0 3.0

Fig. 2 Genetic maps of D. simulans and D. melanogaster chromosomes. Markers used for genetic analysis are overlaid on eachchromosome with genetic map distances. Gray lines on the third
chromosome indicate the fixed inversion between D. simulans and D.

melanogaster. Three X-chromosome markers, G01498, Dm1865, andDm0478 (shown by asterisks) are markers of Berkeley Drosophila
Genome Project sequence tagged sites (STSs) of which there are no
genetic data for D. melanogasterbetween the F1 intercross and IIs (62 chromosomes with nocrossover, 95 with one crossover, and 43 with two or threeor four crossovers in the former; 96 with no crossover, 89
with one crossover, and 27 with two or three crossovers in
the latter; G0 = 10.9, df = 2, P \ 0.01) and between the
F1 intercross and IIt (88 with no crossover, 88 with one
crossover, and 18 with two or three crossovers in IIt;
G0 = 15.2, df = 2, P \ 0.001), but not between IIs and IIt(G0 = 1.4, df = 2, P [ 0.5).
Taken together, the map length is highly dependent on
the genetic background, and the background homozygosity
significantly shortens the map length of the second
chromosome.

1929; Ohnishi and Voelker 1979; True et al. 1996; TakanoShimizu 2000), the smaller maps in the isogenic backgrounds compared with that of D. melanogaster (Fig. 2)
were unusual. Additionally, crossover frequency is unlikely
to have been affected by structural variation because D.
simulans populations exhibit virtually no inversion polymorphism (Ashburner and Lemeunier 1976).
Recessive defects in trans-acting regulator(s) on the X
or the third chromosome is a likely cause of the reduction
in crossover frequencies in the two isogenic backgrounds.
Evidence strongly suggests large genetic variation incrossover frequencies in natural populations of Drosophila.
Screening of natural populations for meiotic mutants in
Drosophila has proven to be quite productive (Sandler
et al. 1968), and meiotic genes that affect crossover frequency in mutant conditions are mapped on the X and third
chromosomes and on the second chromosome (Baker andHall 1976; Wilson et al. 2008). In females homozygous at
mei-S282, for example, crossover frequency is reduced toabout half of the control (Sandler et al. 1968). Moreover,as in many quantitative characteristics (Falconer andMackay 1996), artificial selection for increased anddecreased crossover frequency has been successfully
achieved (Detlefsen and Roberts 1921; Chinnici 1971;
Kidwell 1972). Based on these results, it might not be

Discussion
Crossover frequencies on the second chromosome strongly
depended on its genetic background; both the second-chromosome maps obtained in the isogenic Sim-3 and Tananarive backgrounds (IIs and IIt crosses) were significantly
smaller than that in the mixed background (F1 intercross).
The reduction in crossover frequencies was observed along
the entire second chromosome. Because the D. simulans map
is generally longer than the D. melanogaster map (Sturtevant

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Genetica (2009) 137:165–171

surprising that both Sim-3 and Tananarive carry recessive
mutant or mutants reducing crossover frequency. The
reduced crossover frequency may also be caused byincompatibility between the two genomes, recessive factor(s) on the X or third chromosome (or both) and dominant factor(s) on the second chromosome.
Together with previous findings, the features of the twosimulans strains can be summarized as follows.
1

2

3

4

The Tananarive strain, originating from Madagascar,
has large sex combs, which is comparable to that of D.
mauritiana (Tatsuta and Takano-Shimizu 2006).
Sim-3 loses many bristles in hybrids with D. melanogaster, but such anomalies are not found in Tananarivemelanogaster hybrids. Normal bristle development inhybrids with D. melanogaster is a feature specific tosimulans strains originating from Madagascar, theMascarene and Seychelles islands, and Kenya, whichis also shared by D. mauritiana (Takano 1998).
Strong segregation distortion of the second chromosome occurs in crosses between Tananarive and Sim-3,
possibly at both the gamete and zygote levels. Distortion at the allele-frequency level is less severe in
isogenic backgrounds of either strain and does notoccur for the X and third chromosomes.
Crossover frequency on the second chromosome is
reduced in isogenic backgrounds.

In short, the two strains diverged substantially in multiple ways and all major chromosomes are involved in the
differences. Interestingly, Tananarive shares two featuresthat characterize D. mauritiana: large sex combs and normal bristle development in hybrids with D. melanogaster.
Moreover, the same genes may be responsible for both theintraspecific variation within D. simulans and the interspecific difference between D. simulans and D. mauritiana
for sex-comb tooth number.
There are several possible explanations of these findings. The geographic origin of D. simulans is likely to betropical East Africa, Madagascar, and the Mascarene
islands (Lachaise et al. 1988; Dean and Ballard 2004). The
shared features of the Madagascar strains of D. simulans
and D. mauritiana are most parsimoniously explained asancestral traits or ancestral polymorphisms. In the former
case, all the differences between the Madagascar and US
strains of D. simulans represent changes during worldwide
colonization of D. simulans. Indeed, it seems that all
populations from the ancestral range of the species sharethe characteristic of normal bristle development in hybrids
with D. melanogaster (Takano 1998). Alternatively, thehigh divergence between the two strains could be caused
by genetic introgression of the D. mauritiana genome into
D. simulans in the Madagascar–Mascarene region. There iscompelling evidence for introgression of D. simulans

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cytoplasm into D. mauritiana (Solignac and Monnerot
1986; Satta and Takahata 1990; Rousset and Solignac
1995; Ballard 2000). In this scenario, the segregation distortion and reduced crossover frequency found in this study
represent not within-species, but between-species
incompatibility.
In summary, we found evidence that the Madagascar
and US strains of D. simulans are highly differentiatedbecause of the strong segregation distortion and reductionof crossover frequency, depending on chromosome andgenetic background. These two phenomena may be
attributable to changes during worldwide colonization ofthis species and represent the first sign of speciation.
Finally, these intraspecific changes are amenable to genetic
analysis and the findings pave the way toward identification of genes involved in segregation distortion and chromosome-wide control of recombination.
Acknowledgments We express our thanks to Yuriko Ishii fortechnical assistance. We also thank two anonymous reviewers forhelpful comments and for pointing out errors in an earlier version ofthis paper. This work was supported in part by the Yamada Science
Foundation (T.T.-S.), the Mitsubishi Foundation (T.T.-S.) and Grantsin-Aid for Scientific Research (C) (T.T.-S.).

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