Copyright 0 1987 by the Genetics Society of America
Genetics of Male and Female Sterility in Hybrids of
Drosophila pseudoobscura and D. persimilis
H. Allen Orr’
Department of Zoology, University of Maryland, College Park, Maryland 20742
Manuscript received January 9, 1987
Revised copy accepted May 6, 1987
ABSTRACT
The genetic basis of male and female sterility in hybrids of Drosophila pseudoobscura-Drosophila
persimilis was studied using backcross analysis. Previous studies indirectly assessed male fertility by
measuring testis size; these studies concluded that male sterility results from an X chromosomeautosome imbalance. By directly scoring for the production of motile sperm, male sterility is shown
to be largely due to an incompatibility between genes on the X and Y chromosomes of these two
species. These species have diverged at a minimum of nine loci affecting hybrid male fertility.
Semisterilityof hybrid females appears to result from an X chromosome-cytoplasminteraction; the X
chromosome thus has the largest effect on sterility in both male and female hybrids. This is apparently
the first analysis of the genetic basis of female sterility, or of sterility/inviabilityaffecting both sexes,
in an animal hybridization.
S
TUDY of the genetic basis of reproductive isolation can clarify the process of speciation. For
example, such analysis can reveal whether isolation
between closely related species results from divergence at one or a few major loci as suggested by
GOULD(1 980), or if many genes are involved. Second,
such studies can determine whether some chromosomes tend to diverge faster than others and thus play
a larger role in speciation. WU and BECKENBACH
(1983) and CHARLESWORTH,
COYNEand BARTON
(1987), for example, note that the X chromosome
often plays a larger role in hybrid sterility and inviability than the autosomes; the latter authors propose
a population genetic explanation of this pattern.
Furthermore, analysis of species differences can
help resolve the genetic basis of “Haldane’s rule,”
which states that “When in the F1 offspring of 2
different animal races one sex is absent, rare, or
sterile, that sex is the heterozygous [heterogametic]
sex” (HALDANE,
1922). This rule holds in taxa where
males (e.g., dipterans and mammals) and where females (e.g., birds and lepidopterans) are the heterogametic sex. An important goal of speciation genetics
has been determination of the genetic basis of HALDANE’S rule. Two explanations have been offered.
First, DOBZHANSKY
(1 937a) proposed that heterogametic hybrids suffer from an X-autosomal imbalance: while homogametic hybrids have one X chromosome and a haploid set of autosomes from each
species, heterogametic hybrids lack an X chromosome
that is “compatible” with one set of autosomes. There
’ Current address: Department of Biology, University of Chicago, 1103
Fast 57th Street, Chicago, Illinois 60637.
Genetics 1 1 6 555-563 (August, 1987)
are several variations of this hypothesis (for example,
1937b), but the most widely accepted
DOBZHANSKY
argues that the incompatibility results from a breakdown of normal epistasis, i.e., the usual interaction of
conspecific X chromosome and autosomal gene products cannot occur in heterogametic hybrids. Alternatively, several workers (HALDANE1932; CURTIS,
1980; COYNE1985) have
LANGLEY
and TREWERN
suggested that heterogametic sterility and/or inviability may result from an incompatibility between loci
on the X and Y chromosomes of the parental species.
Y-linked fertility genes are known in several species of
Drosophila (HESS1975).
Although examples of X-autosomal (DOBZHANSKY
1974), X-Y (COYNE1985) and Y-autosomal (VIGNEAULT and ZOUROS 1986) hybrid incompatibilities
have been found in Drosophila, the X-autosomal imbalance explanation remains the most popular (e.g.,
LEWONTIN1974; STANSFIELD1979). Much of this
(1936) classic
popularity derives from DOBZHANSKY’S
study of male sterility in hybrids of the sibling species
D. pseudoobscura and D. persimilis. D. pseudoobscura
[“race A” in DOBZHANSKY
(1936)l occurs in the western United States and in Central America; D. persimilis
(“race B”) is found in the western United States, where
it is sympatric with D. pseudoobscura (DOBZHANSKY
and EPLING1944). DOBZHANSKY
(1936) argued that
the fertility of male hybrids of these species increases
with the number of autosomes that are conspecific
with the X chromosome. He thus concluded that sterility is “determined by interactions of factors located
in the X chromosome with factors located in the
1936, p. 133) and that “the
autosomes” (DOBZHANSKY
556
H. A. Orr
Y is not connected with hybrid sterility” (DOBZHANSKY
1937a, p. 281). Here I present the results of an
experimental reanalysis of D. pseudoobscura-D, persimilis hybrid sterility.
This reanalysis seemed necessary for several reasons, First, DOBZHANSKY
(1933b, 1936) did not directly test the fertility of experimental males. Instead,
he measured their testis size, arguing that testis size
and fertility are strongly correlated. While it is true
that males with very small testes are sterile (DOBZHANSKY and POWELL1975), LANCEFIELD
(1929) had already shown that hybrid backcross males with testes
of normal size are sterile more often than males from
the pure species. Thus, the evidence for X-autosomal
incompatibility and against Y chromosome effects in
hybrid male sterility may be based upon data of questionable relevance. Here I measure male fertility by
scoring production of motile sperm. Also, DOBZHANSKY (1936) did not rigorously test the fertility of
hybrid females, which allows a powerful test of the Xautosomal imbalance theory: if F1 males are sterile
because they suffer from a greater X-autosomal imbalance than F1 females then any hybrid female having
an X-autosomal imbalance equivalent to F1 males
should be sterile. COYNE(1 985) tested this prediction
in the D. melanogaster group, and found that “unbalanced” hybrid females were perfectly fertile.
Analysis of female fertility can also clarify contradictory claims about D. pseudoobscura-D. persimilis F1
females. LANCEFIELD
(1929) described F1 females as
variously described F1
semisterile, but DOBZHANSKY
females as semisterile (DOBZHANSKY
1933a,b) or fertile (DOBZHANSKY
1937a). Although in either case D.
pseudoobscura-D. persimilis hybrid sterility provides a
rule (F, males are more
good example of HALDANE’S
affected than females), the reported semisterility of FI
females affords a rare opportunity to study the genetics of hybrid female sterility. Thus, one can assess
how similar the genetic bases of male and female
hybrid sterility are; does the X chromosome, for example, have a large effect in both cases? This information is useful in testing evolutionary explanations
of HALDANE’Srule and of the large role of the X
chromosome in speciation.
MATERIALS AND METHODS
T h e experimental design is similar to that of DOBZHANSKY
(1936):crosses are made between a D. pseudoobscura strain
carrying morphological markers and a wild-type D.persimilis
strain. T h e hybrid F1 females are backcrossed to one of the
parental species, producing offspring with different combinations of D.pseudoobscura and D. persimilis chromosomes.
T h e identity of these chromosomes is apparent from an
individual’s phenotype. By assessing the fertility of each
backcross phenotype, one can determine which combinations of chromosomes cause hybrid sterility.
Although the X, second, and third chromosomes of these
species differ by fixed inversions (TAN1935), some unde-
tected recombination between D.pseudoobscura and D. persimilis chromosomes occurs in F1 females when few mutant
markers are used. T h e result is an increase in variation in
fertility of a scored backcross class. This difficulty does not,
(1936)
however, invalidate this method. As DOBZHANSKY
argued, undetected recombination can change the mean
fertility of a phenotype but not the differences between
classes.
I made four backcrosses: two to each parental species.
Male and female fertility were studied in all backcross progeny. In the backcrosses to D. pseudoobscura, a D. pseudoobscura strain having each major chromosome marked with a
recessive mutation [yellow (y 1-59); glass (glII-83);orange
(or 111-0);incomplete (inc IV-0) (map locations from ANDERSON and NORMAN
(1977)l was crossed reciprocally to a wildtype strain of D. persimilis from Mather, California. T h e F,
females from each cross were backcrossed to the marked
strain, yielding 16 classes of offspring. As the initial cross
was reciprocal, the two backcrosses had different sources of
cytoplasm. All backcross males in this series carried the D.
pseudoobscura Y chromosome and all females carried at least
one D. pseudoobscura X chromosome. Every individual had
at least a haploid number of D.pseudoobscura autosomes.
I made backcrosses to D.persimilis to obtain hybrid flies
with a predominantly D. persimilis genetic background. T o
distinguish between females carrying one or both D. persimilis X chromosomes, I used a dominant X-linked mutation
in D. pseudoobscura. Thus, reciprocal crosses were made
between Pointed (Pt: 1-0) yellow (y: 1-59) D.pseudoobscura
and the D.persimilis Mather strain; the F1females from each
cross were backcrossed to D. persimilis. As no autosomes
were marked, this backcross produced only two classes of
males and females: those with homospecific or heterospecific
sex chromosomes.
I also tested the fertility of males and females from the
parental stocks and from the F I .
Male fertility was tested by the method of COYNE(1984).
A male was scored as fertile if he had any motile sperm; a
male with no motile sperm was scored as sterile. T h e fertility
of a male genotype was calculated as the percentage of
individuals with at least one motile sperm. Though this
method ignores subtle differences in motility, the sterility
effects of chromosomes are often sufficiently large to allow
detection with this all-or-none approach (see RESULTS below).
Female fertility was tested by placing an individual female
in a 2-dram vial with one wild-type male from each parental
species. After 7 days, the vials were scored for the presence
of larvae. If no larvae were found, the female’s reproductive
tract was dissected in insect Ringer’s solution and her seminal receptacle/spermathecae scored for motile sperm under
a compound microscope. If motile sperm were found, she
was scored as sterile. When no motile sperm were found
(this was rare), the female was considered unmated and not
counted in the calculation of the fertility of a genotype.
Females that died before producing offspring were also not
counted in the fertility calculation. The vials which housed
“sterile” females were retained for 7 days and then reexamined. If larvae were found the female was reclassified as
fertile. Female fertility was calculated as the percentage of
mated females who produced larvae.
T o facilitate interspecific mating, crosses were kept at
18” for 2 days (KOOPMAN1950) and then transferred to
22”. Fertility data were analyzed with G (log likelihood
ratio) tests using the FUNCAT procedure (SAS Institute,
Inc.). This procedure measures the effect of a chromosome
substitution by comparing all genotypes that differ in this
substitution. T h e effect of a chromosome is thus reported
557
Genetics of Hybrid Sterility
X/Y 2 3 4
TABLE 1
94/98
Fertility of males and females from stocks and hybrid FI
27/32
Genotype
Males
D.pseudoobscura (J gl or inc)
D.pseudoobscura (Pt y)
D.persimilis (Mather)
F, (D.pseudoobscura cytoplasm)
F, (D.persimilis cytoplasm)
Females
D. pseudoobscura (J gl or inc)
D.pseudoobscura (Pt y)
D.persimilis (Mather)
F, (D.
pseudoobscura cytoplasm)
F1 (D.persimillis cytoplasm)
Percent
fertile
Total
99.5
94.0
99.5
0.0
0.0
200
200
200
200
100
98.6
100.0
96.7
71.6
96.0
74
25
30
102
50
37/43
17/35
34/45
29/38
26/92
31/54
0/40
0/6 1
0184
as a single G statistic. Several pairwise heterogeneity G tests
were also performed.
1 /75
11121
RESULTS
O / 104
Male fertility
Backcross to D. pseudoobscura: Pure species males
are highly fertile, and FL males are completely sterile
1929; DOBZHAN(Table l ) , as expected (LANCEFIELD
SKY 1936). Figure 1 shows male fertility data from the
backcross of F1 females having D.pseudoobscura cytoplasm to D. pseudoobscura [the backcross classes in
figures are numbered to match those of Figures 1 and
2 in DOBZHANSKY
(1936)l.
There is an obvious genetic basis to male sterility:
males having a D. pseudoobscura X chromosome are
usually fertile, while males having a D. persimilis X
chromosome are almost always sterile. Although there
was some variation in testis size, most males with a D.
persimilis X chromosome had atrophied testes with no
visible sperm, as reported by DOBZHANSKY
( 1 936).
T h e X chromosome has the largest effect on fertility
(G1= 161.6, P < 0.0001). To assess the effect of the
autosomes, I compared only classes 1-8 because males
in classes 9-16 are sterile and give no further information on autosomes. Each autosome significantly
affects fertility (11: GI = 44.8, P < 0.0001; 111: G1 =
13.7, P < 0.0005; IV: G1 = 4.9, P < 0.05). As the X
chromosome of the sterile males is potentially incompatible with the Y chromosome and the cytoplasm, the
basis of sterility cannot be determined from this backcross alone.
The X chromosome also has the largest effect on
fertility among males produced in the backcross of
the reciprocal F1 females (D.persimilis cytoplasm) to
D.pseudoobscura (Figure 2; G1 = 74.1, P < 0.0001).
As the cross of D.persimilis females to y gl o r inc males
goes very poorly, the small sample sizes allow only a
qualitative test of cytoplasm involvement. However,
the similarity between Figures 1 and 2 shows that the
effect of cytoplasm on fertility is, at best, very small:
01 128
I
20
40
.
60
11143
d
80
100
X WITH MOTILE SPERM
FIGURE1 .-Graph of hybrid male fertility from backcross of F1
females (D.pseudoobscura cytoplasm) to D. pseudoobscura. D.pseudoobscura chromosomes are shown as white and D.persimilis chromosomes as black. Fraction at end of each bar shows number of
males with motile sperm over number of males scored. Total N =i
1 193 males.
even with cytoplasm derived from D.persimilis, males
having a D.pseudoobscura X chromosome are largely
fertile while males with a D.persimilis X chromosome
are almost always sterile.
These data also suggest that X-autosomal effects are
not the main cause of sterility: although there is significant heterogeneity among autosomal classes of
males with conspecific X and Y chromosomes (D.pseudoobscura cytoplasm results: G7 = 131.8, P < 0.001;
D. persimilis cytoplasm results: G7 = 17.3, P < 0.05),
males with heterospecific X and Y chromosomes are
sterile regardless of their autosomal genotype. There
are no significant interactions between the X chromosome and an autosome on either cytoplasmic background (although the near complete sterility of males
with a D. persimilis X chromosome clearly makes detection of such effects unlikely).
It may not be surprising that all heterospecific X-Y
classes are sterile since all such classes have an X autosomal balance worse than or equivalent to that of
F1 males, who are known to be sterile. However, one
can distinguish between the X-autosomal and X - Y theories by comparing classes 8 and 9 (Figures 1 and 2).
These are the only classes with equal X-autosomal
incompatibilities (both classes have a haploid set of
558
H. A. Orr
X/Y 2
3 4
0000
0
13/13
0002 0 0 0 0
819
1
0
0
0
0010,
3 0 0 0 0
0100
4 0 000
5 0011
8/10
8/14
15/18
n ono
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11/13
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00
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0
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12 0 0 0 0
-mu=
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1111
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n o o o
0/47
Fertility of males from both reciprocal backcrosses to D.
persimilis males
Genotvnr
Percent
fertile
Total
~
D.pseudoobscura cytoplasm
+ (XPeJPer)
Pt (XJp-)
D.persimilis cytoplasm
+ (XPeJPer)
Pt (XLJOer)
40.2
0.0
29.9
0.0
107
43
174
38
autosomes that are incompatible with the X chromosome), but different X-Y compatibilities. In class 8 the
X and Y chromosomes are homospecific, while in class
9 they are heterospecific. If hybrid F1 male sterility is
due to X-autosomal imbalance, these classes should be
equally sterile. However, on both cytoplasms, class 8
is much more fertile than class 9 (D. pseudoobscura
cytoplasm: G I = 89.2, P < 0.0001; D. persimilis cytoplasm: GI = 21.0, P < 0.0001). Indeed, class 8 males
are often fertile despite the fact that they have an F1like X-autosomal genotype. This suggests that sterility
arises primarily from an X-Y, not X-autosomal, incompatibility. A more direct test of the Y chromosome is
described below.
Backcross to D. persimilis: Table 2 gives the results
of the backcross of FI females (D. pseudoobscura and
D. persimilis cytoplasms) to D. persimilis. All the males
produced in these backcrosses have a D. persimilis Y
chromosome and at least a haploid set of D. persimilis
autosomes, which are unmarked. Again, the X chromosome has a highly significant effect on fertility
regardless of cytoplasm: males with a D. persimilis X
chromosome are often fertile while those with a D.
pseudoobscura X chromosome are always sterile (D.
pseudoobscura cytoplasm: G1 = 35.5, P < 0.0001: D.
persimilis cytoplasm: G I = 23.9, P C 0.0001). As the
autosomes cannot be resolved, one cannot distinguish
between X-Y and X-autosomal effects on male fertility
in these backcrosses.
It should be noted that the fertility difference between homo- and heterospecific X-Y males is not due
to pleiotropic effects of the mutant markers on a
hybrid background: in the backcross to D. pseudoobscura, the fertile males are those with more markers,
while in the backcross to D. persimilis they are those
with fewer markers. Nor is the fertility difference an
artifact of viability differences between homo- and
heterospecific X-Y males: the fertile (homospecific XY) males are less numerous in the backcross to D.
pseudoobscura, but are more abundant than the sterile
(heterospecific X-Y) males in the backcross to D. persimilis. Fertility is correlated with X-Y genotype, not
with mutant markers or viability.
A direct test of the Y chromosome: The previous
analyses cannot test the effect of the Y chromosome
because all males produced in a backcross carry the
same Y chromosome. T o directly test the role of the
Y chromosome in hybrid sterility, backcross males of
y phenotype (Figure 1, class 8) were crossed to D.
persimilis Mather females, producing sons with a D.
persimilis X chromosome and a D. pseudoobscura Y
chromosome. Although the different autosomal
classes are indistinguishable in these sons, we expect
that, on average, 3 of 4 of their autosomes are from
D. persimilis. These males thus have a greater Xautosomal compatibility than any heterospecific X-Y
males produced in the above backcrosses. As a comparison, D. pseudoobscura y g l or inc males were crossed
to D. persimilis females and the F1 females backcrossed
to D. persimilis. The sons produced here also have an
X chromosome, 3 of 4 of their autosomes, and a Y
chromosome from D. persimilis.
The two groups of males thus have equivalent X autosomal balance and differ only in the species origin
of the Y chromosome and the “purity” of their cytoplasm (males with a D. pseudoobscura Y chromosome
have pure D. persimilis cytoplasm while those with a
D. persimilis Y chromosome inherit hybrid cytoplasm
ultimately derived from D. persimilis). Of the males
with a D. pseudoobscura Y chromosome 32.0% were
fertile ( n = 50), and of the males with a D. persimilis
Y chromosome 62.5% were fertile ( n = 72). This
559
Genetics of Hybrid Sterility
significant difference (Cl = 11.2, P < 0.001) is probably not due to cytoplasm: first, previous backcrosses
show no effect of cytoplasm on male fertility; second,
the cross with the best X-cytoplasm compatibility produces the most sterile males. Thus, substitution of the
Y chromosome appears to have a large effect on the
fertility of male hybrids. Moreover, this effect is in
the direction predicted by the X-Y incompatibility
theory. These results also show that autosomal genotype is not entirely irrelevant to fertility: heterospecific X-Y males with sufficiently good X-autosomal
compatibility are sometimes, but not usually, fertile.
This degree of compatibility is not possible in F1
males, however, who receive a haploid set of autosomes from each species.
Female fertility
Fl female fertility: Females from the parental species are highly fertile (Table 1). Hybrid F1 females
with D.pseudoobscura mothers, however, are semister(1929). Only 72% of
ile, as suggested by LANCEFIELD
these femles produced offspring (Table l), which is
significantly lower than either parental species (comparison by y gl or inc: GI = 28.3, P < 0.0001; to D.
persimilis: GI = 10.9; P < 0.001). Interestingly, F1
females from the reciprocal cross (Table 1) are as
fertile as those of the pure species (comparison by y gl
or inc: G1 = 0.87, P > 0.30; to D.persimilis: G1 = 0.02,
P > 0.85), indicating an effect of cytoplasm on female
fertility.
Backcross to D. pseudoobscuru: Backcross females
(D.pseudoobscura cytoplasm) are frequently sterile
(Figure 3). Moreover, female. sterility has “anobvious
genetic basis: females with two D. pseudoobscura X
chromosomes are usually fertile while those with one
D. pseudoobscura and one D.persimilis X chromosome
are usually sterile. T h e X chromosome has the largest
effect on fertility ( C , = 127.1, P C 0.0001). T h e
second chromosome also has a significant effect (G1 =
5.3, P < 0.05) while that of the third chromosome
approaches significance (Cl = 3.0, 0.05 < P < 0.10).
Figure 4 shows the fertility of females produced by
backcrossing the reciprocal F1 females (D.persimilis
cytoplasm) to D.pseudoobscura. T h e sample sizes are
small because, as noted above, the cross of D.persimilis
females to y gl o r inc males goes poorly. Nonetheless,
it is obvious from Figures 3 and 4 that the ultimate
source of cytoplasm does not dramatically affect backcross fertility: females with homospecific X chromosomes remain mostly fertile and those with heterospecific X chromosomes remain largely sterile. T h e X
chromosome again has the largest effect on fertility
(Cl = 52.9, P < 0.0001).
T h e large fertility difference between homo- and
heterospecific X chromosome females cannot be explained by X-autosomal imbalance. This theory predicts that backcross females with an F1 male-like gen-
x z 3 4
I 0000
47/47
noon
O O O I
29/30
ooon
3 O O I O
0000
37/37
1
O I O O
0000
27/29
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0000
37/38
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22/29
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4 1 /47
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I
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47/57
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I O 0 0
16 0000
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I
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9
o
o
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=
o
20
o
40
13/65
13/47
34/95
20/69
,~
60
.
80
25/66
50/109
100
%’ FERTILE
FIGURE3.-Fertility of hybrid females from backcross of F1
females (D.pseudoobscura cytoplasm) to D.pseudoobscura. Fraction
at end of each bar shows number of fertile females over number of
inseminated females. Total N = 861 females.
otype (one set of autosomes incompatible with both
sex chromosomes) should be sterile (COYNE1985).
Yet, on both cytoplasms, these females (class 8) are
highly fertile. In fact, females with the worst X-autosoma1 balance (class 8) are significantly more fertile
than those females with the best X-autosomal balance
(class 9) (D.pseudoobscura cytoplasm: GI = 22.1, P <
0.0001; D.persimiliscytoplasm: Cl = 12.5, P < 0.001).
However, among females with heterospecific X
chromosomes there is some evidence of smaller Xautosomal effects: fertility is roughly “staggered” between autosomal classes in the manner expected from
X-autosomal imbalance (Figures 3 and 4). There are
significant interactions between the X and second
chromosomes (G1 = 19.0, P < 0.0001) and the X and
the fourth chromosomes (Cl = 5.3, P < 0.05) on D.
pseudoobscura cytoplasm.
Backcross to D. persimilis: About half of the females produced from the backcross of F1 females
(both cytoplasms) to D.persimilis are sterile (Table 3).
However, on both cytoplasms, there is no fertility
difference between females carrying both D.persimilis
X chromosomes and those with one D. persimilis and
pseudoobscura
one D.pseudoobscura X chromosome (D.
cytoplasm: G I = 0.52, P > 0.45; D. persimilis cytoplasm: GI = 0.21, P > 0.60). Backcross female sterility
H. A. Orr
560
DISCUSSION
X t 3 4
I 0000
0000
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0000
3 0010
0000
0100
0000
0011
0000
0101
,
0000
0110
0000
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0000
I000
16 0 0 0 0
15
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0000
I 0 1 0
14 0 0 0 0
1100
13 0 0 0 0
m
1/11
I O 1 1
12 0 0 0 0
1101
I I 0000
1I10
10 c 3 0 0 0
9 1111
13/34
I
2/24
0000
FIGURE4.-Fertility of hybrid females from backcross of F,
females (D.persimilis cytoplasm) to D . pseudoobscura. Total N = 298
females (small sample size reflects difficulty of hybridization).
TABLE 3
Fertility of females from both reciprocal backcrosses to D.
persimilis males
Genotype
D.pseudoobscura cytoplasm
+ (X,rX-x,3
Pt (XF&J
D.persimilis cytoplasm
+ (XFerXPer)
Pt (X,X”J
Percent
fertile
Total
77
57.1
50.9
57
47.0
50.0
134
106
is not simply determined by homo- us. heterospecificity of the X chromosomes.
While other interpretations are possible, the simplest genetic rule which accounts for backcross female
sterility is: sterility often results whenever a D. persimilis X chromosome occurs in a “foreign” or “hybrid”
cytoplasm. As all backcross females inherit cytoplasm
affected by a hybrid female genome (regardless of the
ultimate source of the cytoplasm), only those with two
D. pseudoobscura X chromosomes are highly fertile.
This accounts for the high fertility of such females on
both cytoplasmic backgrounds and the frequent sterility of all other genotypes. This rule also explains
the nonreciprocal semisterility to F1 females.
These results agree with those of DOBZHANSKY
(1936) in several ways. First, all four major chromosomes affect D. pseudoobscura-D. persimilis hybrid male
fertility; DOBZHANSKY
(1936) similarly showed that all
marked chromosome segments affect hybrid testis
size. Both analyses thus found the maximum number
of genetic differences between D. pseudoobscura and
D. persimilis that were detectable with the methods
used. Second, both analyses showed that the X chromosome has the largest effect on male sterility. LANCEFIELD (1929) showed that at least two loci on the left
arm of the X chromosome are involved in male sterility
(he measured testis size and fertility of some recombinants); WU and BECKENBACH
(1983) further demonstrated that at least three loci on the right arm of
the X chromosome affect D. pseudoobscura-D. persimilis
hybrid fertility. If these patterns do not differ between
strains, at least five X-linked and nine total loci (five
X-linked plus at least three autosomal loci plus at least
one Y-linked locus) are involved in hybrid male sterility.
However, these results suggest that hybrid F1 male
sterility is largely due to an incompatibility between
loci on the X and Y chromosomes of the two species,
not to an X-autosomal imbalance as suggested by
DOBZHANSKY
(1936). This is demonstrated by three
lines of evidence: (1) Among backcross males with
equivalent X-autosomal balance, those with homospecific X and Y chromosomes are fertile much more
often than those with heterospecific X-Y. (2) Substitution of the Y chromosome has a large effect on the
fertility of hybrid males in the direction predicted by
the X-Y incompatibility theory. (3) Hybrid females
having an X-autosomal imbalance as severe as that of
F, males are highly fertile. The difference between
these conclusions and those of DOBZHANSKY
(1933b,
1936) is almost certainly due to his use of testis size as
a measure of fertility.
DOBZHANSKY
(1933b, 1936) found that testis size
variation is unaffected by the Y chromosome. However, among males with heterospecific X and Y chromosomes (Figure 1 of this paper, classes 9-16), mean
testis size varies with autosomal genotype; those males
with better X-autosomal compatibility (e.g., class 9)
have larger testes than those with poorer compatibility
(e.g., class 16). While LANCEFIELD
(1929) had shown
that males with very small testes are always sterile,
DOBZHANSKY
(1936) further assumed that males with
slightly larger testes are proportionately more fertile.
He thus concluded that X-autosomal imbalance accounts not only for backcross male testis size, but also
for backcross male sterility. My results do not support
this assumption; heterospecific X-Y males are almost
always sterile regardless of autosomal genotype or
testis size.
Genetics of Hybrid Sterility
There are several additional problems with the use
of testis size as a measure of fertility. First, DOBZHANSKY’S ( 1 936) heterospecific X-Y class with the largest
testis size (class 9) is known to be sterile: this class is
genotypically identical to F1 males. If hybrid-like
males with the largest testes are completely sterile, it
is not clear what relevance further reductions in testis
size have to fertility: genotypes with even smaller
testes cannot be “more completely” sterile. T h e difficulty in using testis size as an indicator of fertility is
further demonstrated by the fact that hybrid F1 males
with D.pseudoobscura mothers possess testes of normal
1929;
size and yet are completely sterile (LANCEFIELD
DOBZHANSKY
1936). [DOBZHANSKY
(1933b) argued
this pattern results from a maternal effect which does
not act in backcross generations.] Additionally, DOBZHANSKY and BOCHE(1933) showed that although
hybrid testis size varies widely depending on which
strains are used, F1 males from all crosses are all
completely sterile. Fertility and testis size are not
sufficiently well correlated to allow use of one as an
indicator of the other.
Moreover, it appears that not even backcross male
testis size is adequately explained by X-autosomal imbalance. Examination of DOBZHANSKY’S
( 1936) Figure
1 (same backcross as Figure 1 of present paper) shows
that the mean testis size of class 8 (526.5 & 8.9 pm) is
over four times larger than that of class 9 ( 1 23.9 &
2.8 pm), despite the fact that these genotypes have
equal X-autosomal imbalance. As these classes differ
in both their X-Y and X-cytoplasmic relationships, it is
not obvious which incompatibility accounts for this
(1936) did not perform the
difference. DOBZHANSKY
backcross of F1 females (D.persimilis cytoplasm) to D.
pseudoobscura (Figure 2 of this paper) needed to distinguish unambiguously between these possibilities.
However, among males with equal X-autosomal imbalance, those with homospecific X chromosome and
cytoplasm have larger testes than those with heterospecific X-cytoplasm in all backcrosses [DOBZHANSKY
(1936): class 8 us. 9 (Tables 1-3); class 5 vs. 4 (Table
4)]. This suggests that DOBZHANSKY’S
testis size data
are confounded with X-cytoplasm effects.
LANCEFIELD
( 1 929) claimed that F1 hybrid females
are semisterile. Unfortunately, his tests of female fertility were confounded with mating ability (“sterile”
females were not checked for insemination). By scoring only mated females, I verify that F1 females with
D. pseudoobscura mothers are semi-sterile, although
the reciprocal females are fully fertile. DOBZHANSKY
and STURTEVANT
( 1935) and DOBZHANSKY
(1 936)
noted that backcross females are also frequently sterile. DOBZHANSKY
and STURTEVANT
(1935, pp. 569570) claimed, however, that female sterility has no
genetic basis, and results only from the general inviability of marked backcross females. My analysis shows,
56 1
however, that female sterility has a definite genetic
basis, with the X chromosome having the most dramatic effect. (Moreover, backcross females appear
fairly viable: in most backcrosses, over 80% of females
survived at least 7 days.) It is not yet clear whether
several X-linked loci are involved in female sterility;
this analysis is difficult because the two species differ
by two large inversions on the X chromosome that
prevent recovery of most recombinants. Mapping of
the X-linked loci causing hybrid male and female
sterility is nevertheless important as it could clarify
whether the same chromosome regions (and perhaps
loci) are involved in both sexes.
Although the X chromosome is the largest chromosome in D. pseudoobscura and D. persimilis (DOBZHANSKY 1936), its effect on male and female sterility
is clearly disproportionate to its size. While the X
chromosome frequently plays a major role in hybrid
F1 male-only sterility or inviability (WU and BECKENBACH 1983; NAVEIRA
and FONTDEVILA
1986; CHARLESWORTH, COYNEand BARTON,1987), this work is
the first to test the genetic basis of hybrid female
sterility. T h e genetic basis of hybrid female inviability
has been analyzed in only two studies; these examined
rule (only F1 females afexceptions to HALDANE’S
fected). In both cases [ D . montanu X D. americana
texana (PATTERSON
and GRIFFEN1944); D. mulleri x
D.aldrichi-2 (CROW1942)] the X chromosome has the
greatest effect.
While it is possible that the importance of the X
chromosome in all three analyzed cases of hybrid F1
female effects is a coincidence, this seems unlikely.
CHARLESWORTH,
COYNEand BARTON(1987) have
recently shown that alleles causing hybrid sterility or
inviability tend to cluster on the X chromosome only
if they are partially recessive ( h < 0.5) and selectively
favored in the heterogametic sex o r in both sexes
during their evolution. Thus, the large role of the X
chromosome in female hybrid sterility/inviability can
be explained only if the alleles responsible frequently
( 1 ) behave as partial recessives on their normal genetic
background, but as partial o r complete dominants in
hybrid females (because a partially recessive X-linked
allele cannot have a substantial effect in heterozygous
F1 hybrid females), and (2) have pleiotropic (and often
favorable) effects in males during their evolution. We
do not know how often such pleiotropy and changes
of dominance occur. Thus, it is not clear how well this
theory explains the role of the X chromosome in
hybrid female sterility/inviability and therefore the
rapid divergence of the X chromosome in speciation.
Further experiments of this type are needed to
determine if X-Y incompatibility is a frequent cause of
heterogametic sterility/inviability in species hybrids.
COYNE( 1 985) has shown that X-Y incompatibility is
important in hybrids in the D. melanogaster group.
562
H. A. Orr
ZOUROS (1986) suggests, however, that “incompatibilities between sex chromosomes and autosomes leading
to male sterility are at least as likely to mark the
beginnings of speciation as are X / Y incompatibilities.”
As support, he notes that the D. mojavensis X chromosome is compatibile with the D.arizonensis Y chromosome and that the D.pseudoobscura USA X chromosome is compatible with the D. pseudoobscura Bogota Y chromosome. However, to determine when
each type of incompatibility (X-Y or sex chromosomeautosome) arises in speciation one must test for both
types in a number of species pairs. One can infer
which incompatibility appears first during divergence
only by observing which incompatibility often occurs
without the other present; similarly, one can conclude
that the two incompatibilities are equally likely to
mark early speciation only if X-Y and sex chromosomeautosome incompatibilities usually appear together or
appear alone equally often. Unfortunately, X-Y incompatibility was not tested in the cases cited by Z O W R O ~
(1986): both D.mojavensis-D. arizonensis and D.pseudoobscura USA-Bogota crosses produce sterile F1
males in only one direction of the hybridization. In
both cases, only the reciprocal combinations of X and
Y chromosomes mentioned by ZOWROS (1986)-combinations that were not adequately tested-could possibly be incompatible given the direction of FI male
sterility. Thus, these cases provide little information
about the order of appearance of X-Y and sex chromosome-autosome incompatibilities.
To determine how early in speciation hybrid female
semisterility and divergence of the Y chromosome
occur, it is desirable to analyze “species” that are even
more closely related than D. pseudoobscura and D.
persimilis. Thus, I have begun analysis of sterility in
hybrids between the North American populations of
D.pseudoobscura and the isolate population found near
Bogota, Columbia. As noted, F1 males produced in
one direction of this cross (Bogota mothers) are sterile;
males produced in the reciprocal cross are fully fertile
(PRAKASH
1972; DOBZHANSKY
1974). The USA and
Bogota populations are separated by a genetic distance
(NEI’Smeasure) of only 0.194, compared to a distance
and
of 0.30 for D.pseudoobscura-D. persimilis (AYALA
DOBZHANSKY
1974). Combined with the present analysis, the USA-Bogota study will allow comparison of
the genetics of hybrid sterility in two independent
speciation events in the same lineage.
I thank JERRY COYNEfor his advice and assistance throughout
for technical assistance, and JEFFREY
this study, JEFFREY BEECHAM
POWELL
for providing the D . persimilis strain. B. CHARLESWORTH,
J. COYNE
and S. ORZACK
provided valuable comments and criticism.
This work was supported by grants GM-32221 from the National
Institutes of Health, and BSR-83-18558 from the National Science
Foundation to J. COYNE.
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Communicating editor: C. C. LAURIE