ORIGINAL ARTICLE
doi:10.1111/j.1558-5646.2007.00011.x
NATURAL VARIATION FOR A HYBRID
INCOMPATIBILITY BETWEEN TWO SPECIES
OF MIMULUS
Andrea L. Sweigart,1,2,4 Amanda R. Mason,3 and John H. Willis1
1 Department
2 E-mail:
3 North
of Biology, Duke University, Durham, North Carolina 27708
[email protected]
Carolina School of Science and Mathematics, Department of Biology, 1219 Broad St., Durham, North Carolina
27705
Received July 10, 2006
Accepted September 22, 2006
Understanding the process by which hybrid incompatibility alleles become established in natural populations remains a major
challenge to evolutionary biology. Previously, we discovered a two-locus Dobzhansky–Muller incompatibility that causes severe
hybrid male sterility between two inbred lines of the incompletely isolated wildflower species, Mimulus guttatus and M. nasutus.
An interspecific cross between these two inbred lines revealed that the M. guttatus (IM62) allele at hybrid male sterility 1 (hms1)
acts dominantly in combination with recessive M. nasutus (SF5) alleles at hybrid male sterility 2 (hms2) to cause nearly complete
hybrid male sterility. In this report, we extend these genetic analyses to investigate intraspecific variation for the hms1–hms2 incompatibility in natural populations of M. nasutus and M. guttatus, performing a series of interspecific crosses between individuals
collected from a variety of geographic locales. Our results suggest that hms2 incompatibility alleles are common and geographically
widespread within M. nasutus, but absent or rare in M. guttatus. In contrast, the hms1 locus is polymorphic within M. guttatus
and the incompatibility allele appears to be extremely geographically restricted. We found evidence for the presence of the hms1
incompatibility allele in only two M. guttatus populations that exist within a few kilometers of each other. The restricted distribution of the hms1 incompatibility allele might currently limit the potential for the hms1–hms2 incompatibility to act as a species
barrier between sympatric populations of M. guttatus and M. nasutus. Extensive sampling within a single M. guttatus population
revealed that the hms1 locus is polymorphic and that the incompatibility allele appears to segregate at intermediate frequency, a
pattern that is consistent with either genetic drift or natural selection.
KEY WORDS:
Dobzhansky–Muller incompatibility, hybrid incompatibility, Mimulus, natural variation, speciation.
Postzygotic reproductive isolation typically evolves when diverging populations accumulate different alleles at multiple loci
that are incompatible when brought together in hybrid genomes;
negative epistasis between the heterospecific alleles renders hybrids inviable or sterile (this scenario is commonly referred to
as the Dobzhansky–Muller model; Bateson 1909; Dobzhansky
1937; Muller 1942). Recent studies have described in detail the
genetic architecture of hybrid incompatibility (e.g. Harushima
4Present address: Department of Biology, University of Rochester,
Rochester, New York 14627.
141
et al. 2001, 2002; Tao et al. 2003a, b; Presgraves 2003; Moyle
and Graham 2005; Sweigart et al. 2006), and several have even
identified the genes that cause hybrid inviability and sterility
(Whittbrodt et al. 1989; Ting et al. 1998; Barbash et al. 2003;
Presgraves et al. 2003; Brideau et al. 2006). However, a major
remaining challenge is to understand how hybrid incompatibility
alleles become established in natural populations. Classical crossing experiments in animals and plants have often demonstrated
that incompletely isolated species are variable for the severity of
inviability or sterility when hybridized (e.g., Stebbins 1958; Vickery 1978; Patterson and Stone 1952; Christie and Macnair 1987;
2007 The Author(s)
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A. L. SWEIGART ET AL.
Wade and Johnson 1994; Reed and Markow 2004; Kopp and Frank
2005; Shuker et al. 2005). But despite such widespread documentation of within-species variability for barriers to interspecific
crossing, to our knowledge only a single study has directly examined standing genetic variation at loci underlying particular hybrid
incompatibilities (Christie and Macnair 1987).
Identifying natural variation at hybrid incompatibility loci is
a first step toward investigating at least three classic questions
in speciation genetics. First, when incompletely isolated species
come into contact, what is the potential for a Dobzhansky–Muller
incompatibility to act as a barrier to interspecific gene flow? In
hybridizing species, the fate of a particular incompatibility allele
presumably depends on both its selective advantage within pure
species and its deleterious effects in hybrids. If interspecific gene
flow is common, natural selection might be expected to eliminate
derived incompatibility alleles in favor of ancestral compatible
alleles (Noor et al. 2001). Second, how does population structure influence the potential for a particular hybrid incompatibility
allele to become an effective species barrier? Just as population
structure might affect the spread of beneficial alleles that underlie phenotypic traits (e.g., Morjan and Rieseberg 2004), degree
of substructuring may also dictate whether a particular incompatibility allele becomes locally or widely distributed. For many
species, population structure is likely to play an important role
in the accumulation of hybrid incompatibility alleles, although it
may be less relevant in species such as Drosophila melanogaster,
for which world-wide estimates of population structure are relatively low (Baudry et al. 2004; Haddrill et al. 2005). Third, do
hybrid incompatibility alleles have some adaptive value within
pure species or do they instead accumulate by random genetic
drift? Positive selection appears to have driven rapid sequence
divergence in the hybrid incompatibility genes OdsH, Hmr, Lhr,
and Nup96 of the D. melanogaster species group (Ting et al. 1998;
Barbash et al. 2003; Presgraves et al. 2003; Brideau et al. 2006).
In species that are polymorphic, it should be possible to combine
a molecular population genetics approach with field experiments
to examine the role of natural selection in the evolution of hybrid
incompatibility.
Here we examine natural variation for a genetically wellcharacterized hybrid incompatibility between two incompletely
isolated species of yellow monkeyflower, the highly selffertilizing M. nasutus and the predominantly outcrossing M.
guttatus. In a previous study, we discovered that a simple
Dobzhansky–Muller incompatibility can cause nearly complete
hybrid male sterility and partial female sterility between two inbred lines of M. nasutus and M. guttatus (Sweigart et al. 2006).
In a series of crosses (summarized in Fig. 1A), we demonstrated
that hybrids between the inbred lines SF5 (M. nasutus) and IM62
(M. guttatus) are highly male sterile if they carry incompatible heterospecific alleles at two loci, hybrid male sterility 1 and 2 (hms1
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EVOLUTION JANUARY 2007
and hms2). As predicted by the Dobzhansky–Muller model, the
two inbred parental lines carry complementary alleles at hms1 and
hms2: SF5 is compatible at the hms1 locus and incompatible at
hms2, whereas IM62 is incompatible at hms1 and compatible at
hms2. Hybrids are highly male sterile when they carry at least
one dominant incompatibility allele at hms1 (from IM62) and
two recessive incompatibility alleles at hms2 (from SF5). However, there is some evidence to suggest that natural populations of
M. guttatus and M. nasutus might not be fixed for the hms1–hms2
hybrid incompatibility. The M. guttatus species complex, which
includes both species, is highly polymorphic, exhibiting tremendous phenotypic diversity (Pennell 1951) and extensive molecular
variation (Sweigart and Willis 2003). In addition, the pattern of
hybrid male sterility observed between SF5 and IM62 is certainly
not typical of every M. nasutus–M. guttatus cross. Depending on
the particular strain used, F 1 hybrids between M. nasutus and M.
guttatus range from fully fertile to completely sterile (Vickery
1978), and patterns of hybrid male sterility in experimental F 2
populations also vary considerably (N. Martin unpubl. results).
Even within M. guttatus, there is widespread polymorphism for
two separate hybrid incompatibilities that cause lethality between
populations (Christie and Macnair 1987).
The aim of the present study is to investigate the extent of intraspecific variation for the hms1–hms2 incompatibility in natural
populations of M. nasutus and M. guttatus. However, because we
do not yet know the genes that underlie the hms1 and hms2 incompatibility loci, we cannot simply screen individuals for nucleotide
sequence variation that causes hybrid sterility. Instead, we take a
genetic approach, crossing tester strains, for which hms1 and hms2
genotypes are known, to a diverse set of M. nasutus and M. guttatus individuals collected from throughout the species’ ranges.
Importantly, our approach is not limited to measuring phenotypic
variation for hybrid male sterility (as were previous studies of
Mimulus hybrid incompatibility; e.g., Vickery 1978), which might
be caused by intraspecific differences in loci other than hms1 or
hms2. The advantage of this system is that we can also collect
genotypic information for hybrid incompatibility loci; hms1 and
hms2 are each mapped to a small chromosomal region between a
pair of flanking molecular markers (Sweigart et al. 2006). We can
use these linked markers to infer hms1 and hms2 genotypes, and
then determine if the hybrid incompatibility loci are associated
with variation in hybrid sterility. This study represents a first step
toward an empirical population genetics approach to understanding the evolutionary dynamics of hybrid incompatibility.
Materials and Methods
STUDY SYSTEM, MIMULUS LINES, AND
POPULATION SAMPLING
The Mimulus guttatus species complex (section Simiolus) consists of several closely related, potentially interfertile taxa. Natural
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VARIATION FOR MIMULUS HYBRID INCOMPATIBILITY
Figure 1.
A schematic of the genetic basis for the hms1–hms2 incompatibility in M. nasutus and M. guttatus. (A) An interspecific cross
between inbred lines of M. nasutus (SF5) and M. guttatus (IM62) reveals the two-locus hybrid incompatibility. Hybrids are highly male
sterile if they carry at least one copy of a dominant incompatibility allele (I) at the hms1 locus in combination with two copies of a recessive
incompatibility allele (i) at the hms2 locus. Parental inbred lines carry complementary alleles at these loci: SF5 is compatible at hms1 (cc)
and incompatible at hms2 (ii), whereas IM62 is incompatible at hms1 (II) and compatible at hms2 (CC). Roughly one fourth of the progeny
from an F 1 -backcross to SF5 (BC 1 ) carries the hms1–hms2 incompatibility (Ic; ii) and is male sterile (ms). The remaining three fourths of the
BC 1 progeny lack the incompatible genotype at one or both of the loci and are at least partialy male fertile (mf). By repeatedly selecting
on male sterility and backcrossing to SF5, we formed an RSB 3 population (three generations of recurrent selection with backcrossing) that
is approximately one-half male sterile (hms1–hms2 genotype: Ic; ii) and one-half male fertile (hms1–hms2 genotype: cc; ii). (B) Histograms
of pollen viability (proportion viable pollen grains per individual) for each hms1–hms2 genotypic class in the BC 1 -M. nasutus (N = 99)
and RSB 3 (N = 35) populations (see Sweigart et al. 2006). In the BC 1 population, there is considerable overlap in pollen viability among
genotypic classes. In the RSB 3 population, there are two discrete male fertility classes, which are perfectly associated with hms1 genotype.
Dominant alleles are given in uppercase and recessive alleles are in lowercase. For all crosses, the female parent is listed first.
populations of the predominantly outcrossing M. guttatus and the
highly self-fertilizing M. nasutus are abundant throughout western North America, although the range of M. nasutus is more
restricted. The two species most often exist in allopatry, but
sympatric populations are common in some geographic regions.
Prezygotic barriers to interspecific crossing include species differences in floral morphology, flowering phenology, and pollenpistil interactions (Kiang and Hamrick 1978; Ritland and Ritland 1989; Dole 1992; Diaz and Macnair 1999; Martin and Willis
2007). Despite such barriers, hybrids are often observed where
M. nasutus and M. guttatus exist in sympatry (Vickery 1964,
1978; Kiang and Hamrick 1978; Ritland 1991; Fenster and Ritland 1992), and there is evidence for introgression at nuclear
loci in some areas of their shared range (Sweigart and Willis
2003). Postzygotic reproductive isolation is also common; however, as noted above, each species exhibits genetic variation for
the severity of hybrid incompatibility when crossed to each other
(Vickery 1978).
This study used the same inbred parental lines that have been
used previously (Fishman and Willis 2001; Sweigart et al. 2006).
The M. nasutus inbred line (SF5) originated from the Sherar’s
Falls population in central Oregon and has been maintained in
the greenhouse for more than 10 generations by autonomous selffertilization. The M. guttatus inbred line (IM62), derived from the
Iron Mountain (IM) population in the Oregon western Cascades,
was formed by more than six generations of selfing with single
seed descent. The Sherar’s Falls and IM populations are allopatric
and are separated by a distance of roughly 120 km.
To characterize patterns of variation in the hms1–hms2 incompatibility within and among populations of M. nasutus and
M. guttatus, we crossed individuals that were collected from
locales throughout the species’ ranges (Table 1, Fig. 2). For
EVOLUTION JANUARY 2007
143
A. L. SWEIGART ET AL.
Table 1.
Geographic locations of Mimulus populations studied.
Species
Population code
Location1
M. guttatus
IM
ICS
CP
EBT
BR
CGR
BLY
GTR
GDP
GCC
MED
CLR
SF
M12
NCL
BRI
KIN
FGC
Iron Mountain, Hwy. 20, Linn Co., OR
Iron Mountain-Cone Peak saddle, Hwy. 20, Linn Co., OR
Cone Peak, Hwy. 20, Linn Co., OR
Echo Basin Trail, Hwy. 20, Linn Co., OR
Browder Ridge, Hwy. 20, Linn Co., OR
Cougar Reservoir, Lane Co., OR
Bailey Hill Rd. and Lorane Hwy, Lane Co., OR
Hwy. 128 and Berryessa-Knoxville Rd., Napa Co., CA
Don Pedro Vista Point, Hwy. 120, Tuolumne Co., CA
Chinese Camp, Tuolumne Co., CA
Hwy. 120 and Jacksonville Rd. jct., Tuolumne Co., CA
Columbia River, Klickitat Co., WA
Sherar’s Falls, Tygh Valley, Wasco Co., OR
Deer Creek Rd. ml. 12, Tehama Co., CA
Cherry Lake Rd., off Hwy. 120, Tuolumne Co., CA
Bridal Veil Falls, Yosemite Nat’l Park, Mariposa Co., CA
Hwy. 180 near King’s Canyon, Fresno Co., CA
Fern Glen Canyon, Grand Canyon, AZ
M. nasutus
N C1 2
N C2 2
N C3 2
12
1
1
1
3
3
1
1
1
1
7
1
1
1
1
1
1
1
1
1
1
1
1 Population locations are listed from north to south for each species.
2 Number of individuals tested per population for cross 1, 2, and 3.
M. guttatus, we sampled one to three individuals from each of 11
populations. For M. nasutus, we sampled a single individual from
each of six populations. To examine within-population variation
in hms1, we sampled 12 individuals from the IM population. Sam-
ples originated either as field-collected seeds or as plants that were
propagated and selfed in the greenhouse to produce seeds. Some of
the IM lines were inbred in the greenhouse for several generations.
All plants were grown using conditions described in Sweigart et
al. (2006) in the Duke University greenhouses.
ASSESSMENT OF MALE FERTILITY AND HMS1–HMS2
GENOTYPE
Figure 2. Geographic locations of sampled populations of M. nasutus (open circles) and M. guttatus (closed circles) in western
North America. Note that one M. nasutus population sampled from
Arizona (FGC) is not pictured on this map.
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EVOLUTION JANUARY 2007
We measured male fertility in terms of the proportion of viable
pollen grains per flower. For each plant, we collected all anthers from one or two flowers, suspended the pollen from each
flower separately in 60 microliters of aniline blue-lactophenol
stain (Kearns and Inouye 1993), and visualized pollen grains on a
slide using a compound microscope. To estimate pollen viability
for each flower, we determined the proportion of viable pollen
grains in a sample of 100 that was haphazardly selected. Viable
pollen grains appear spherical and darkly stained, whereas inviable pollen grains are misshapen and remain unstained. In cases
for which pollen from two flowers was measured, our estimate of
male fertility was an average of the proportion of viable pollen
grains for the two flowers.
Because hybrid male sterility in other crosses might be caused
by distinct incompatibilities, we collected genotypic information
for hms1 and hms2 to directly examine the effects of these two
loci on hybrid male fertility. Genomic DNA was isolated from bud
tissue using a modified hexadecyl trimethyl-ammonium bromide
(CTAB) chloroform extraction (Kelly and Willis 1998). To infer
VARIATION FOR MIMULUS HYBRID INCOMPATIBILITY
the hms1 and hms2 genotypes of hybrid progeny generated from
experimental crosses, we determined the genotypes of flanking
markers (hms1: MgSTS22 and MgSTS426, hms2: MgSTS104 and
MgSTS599; see Sweigart et al. 2006), and excluded individuals
with crossovers between either pair of flanking markers (based on
expected frequency of double crossovers between flanking markers, genotyping error rates for hms1 and hms2 were 0.014 and
0.011, respectively). Although the MgSTS markers were themselves polymorphic within species, they were still diagnostic in all
genetic crosses (i.e., polymorphic between crossed individuals).
This direct genetic approach allowed us to follow the inheritance of
all hms1- and hms2-containing chromosomal fragments. Marker
loci were amplified using standard touchdown PCR conditions
(annealing temperatures incremented from 62◦ C to 52◦ C for the
first 10 cycles and then an additional 30 cycles at 52◦ C). All marker
genotyping was performed by sizing PCR-amplified DNA fragments with an incorporated 5# fluorescent-labeled primer on an
ABI 3700 automated capillary sequencer (Applied Biosystems,
Foster City, CA). Marker genotypes were assigned automatically
using the program GeneMapper (Applied Biosystems) and then
verified by eye.
EXPERIMENTAL CROSSES
Overview of the hms1–hms2 incompatibility in
inbred lines
To test for the presence of hms1 and hms2 incompatibility
alleles in a variety of M. nasutus and M. guttatus individuals, we
used crosses between them and tester plants that carried known
genotypes for each locus. To explain the rationale of our crossing scheme, we first need to review the basis for the hms1–hms2
incompatibility in inbred lines of M. nasutus and M. guttatus.
Although it is clear that hms1 and hms2 are the major factors
responsible for SF5-IM62 hybrid male sterility, their phenotypic
effects depend somewhat on additional small-effect genetic factors in the genetic background (Sweigart et al. 2006). For example, consider the expression of male sterility in early- versus advanced-generation backcross hybrids of SF5 and IM62.
Roughly one-fourth of the progeny of an F 1 -backcross to SF5
(BC 1 ; Fig. 1A, B) carries the incompatible genotype (in agreement with the Mendelian expectation), and indeed hybrid male
sterility is most severe in this genotypic class. However, some
BC 1 hybrids with the incompatibility are only partially male sterile, and there is phenotypic overlap among hms1–hms2 genotypic
classes (Fig. 1B, Sweigart et al. 2006). In contrast, when the incompatible hms1 allele (from IM62) is in a nearly isogenic SF5
genetic background, its effect on hybrid male sterility is complete
and discrete. By repeatedly selecting on male sterility and backcrossing to the SF5 parent, we formed a population that we refer to
as RSB 3 (3 generations of recurrent selection with backcrossing;
Fig. 1A) in which half of the individuals are heterozygous for an
hms1-containing introgression, and therefore are completely male
sterile (Fig. 1B). Taken together, these crossing results suggest that
additional small-effect factors from SF5 are required for complete
hybrid male sterility (Sweigart et al. 2006). Nevertheless, despite
modest differences in the penetrance of hms1 and hms2 incompatibility alleles in the BC 1 versus RSB 3 hybrids, the effects of
these loci on male fertility in both experimental populations are
demonstrable and clear. An ANOVA among BC 1 hybrids shows
highly significant male fertility effects of hms1, hms2, and the
genetic interaction between the two loci (Sweigart et al. 2006).
Therefore, our strategy was to test for the presence of a dominant hms1 incompatibility allele in any M. guttatus individual by
performing a BC 1 to SF5. Similarly, we tested for the presence of
hms2 incompatibility alleles in other individuals by crossing them
to a male sterile RSB 3 plant, which carries the incompatible hms1
allele.
Characterizing variation for the hms1–hms2
incompatibility within M. nasutus
Our previous genetic analyses determined that the M. nasutus inbred line SF5 carries compatible alleles at hms1 and incompatible
alleles at hms2. To test whether other M. nasutus also carry hybrid
incompatibility alleles, we crossed a male sterile RSB 3 individual to six different M. nasutus plants (Fig. 3A, cross 1). Because
M. nasutus is highly selfing, most loci are likely homozygous.
Therefore, if a particular M. nasutus individual carries alleles that
are incompatible with the IM62 hms1 allele (which is contained
in a heterozygous introgression in the male sterile RSB 3 ), approximately half of its progeny should segregate for the hybrid
incompatibility, and be completely male sterile. For each of the
six crosses, we measured pollen viability and determined hms1
genotype for approximately 20 to 30 progeny. We then performed
an ANOVA for each cross to determine the effect of hms1 genotype
on male fertility. Note that this cross cannot determine whether
the M. nasutus component of the hybrid incompatibility maps to
the hms2 locus.
Characterizing variation for the hms1–hms2
incompatibility within M. guttatus
Our previous study demonstrated that the M. guttatus inbred line
IM62 carries incompatible alleles at hms1 and compatible alleles
at hms2. To examine intraspecific variation for the hms1–hms2
incompatibility in M. guttatus, we used the original BC 1 crossing
scheme (see Fig. 1A) and substituted various M. guttatus plants
for the IM62 inbred lines. Note that the majority of the M. guttatus individuals used in our experimental crosses were not highly
inbred (i.e., they were derived from seed that was wild-collected
or generated from a single generation of selfing), and therefore
were potentially heterozygous for hms1 and hms2 loci. In any interspecific cross, we have only sampled one of the two M. guttatus
alleles by using a single F 1 hybrid.
EVOLUTION JANUARY 2007
145
A. L. SWEIGART ET AL.
First, we tested whether 25 different M. guttatus individuals
carry incompatible alleles at hms1 by backcrossing SF5-M. guttatus F 1 hybrids to SF5 (Fig. 3B, cross 2). If the M. guttatus parent
contributes a dominant incompatibility allele at hms1, roughly a
fourth of the backcross progeny—those with the hms1–hms2 incompatibility genotype—is expected to be highly male sterile (as
is the case when IM62 is used as a parent). If instead the M. guttatus
parent does not contribute a dominant hybrid incompatibility allele at hms1, genotypes at hms1 and hms2 should not affect male
fertility. For each of the 25 crosses, we measured pollen viability
and determined hms1 and hms2 genotypes for approximately 20
to 30 progeny. We then performed an ANOVA for each cross to
assess the contribution of hms1 and hms2 to variation in hybrid
male fertility.
Second, we tested the possibility that some M. guttatus individuals that do not carry the incompatible hms1 allele may instead
carry incompatible alleles at hms2 (i.e., like SF5). This test was
performed by substituting 12 M. guttatus plants for the SF5 parent
in the original BC 1 crossing design. We backcrossed F 1 hybrids
between the 12 M. guttatus individuals and IM62 to the non-IM62
parent (Fig. 3B, cross 3). If a particular M. guttatus individual carries incompatible alleles at hms2, then roughly one-fourth of its
backcross progeny is expected to segregate for the hms1–hms2
incompatibility and be highly male sterile. We measured pollen
viability for approximately 20 to 30 progeny for each of the 12 M.
guttatus individuals tested.
Results
CHARACTERIZATION OF VARIATION FOR THE
HMS1–HMS2 INCOMPATIBILITY WITHIN M. NASUTUS
Figure 3.
Crossing design to characterize intraspecific variation
for the hms1–hms2 incompatibility in natural populations of M.
nasutus and M. guttatus. In a series of crosses, we substituted individuals from geographically diverse populations of M. nasutus
and M. guttatus for the original inbred lines (SF5, IM62) to examine the hms1–hms2 incompatibility (see text for additional details).
For each cross, the hypothesized genotypes appear in boxes. (A)
Cross 1: if a particular M. nasutus individual carries incompatibility alleles that interact with hms1, half of its progeny should be
male fertile (mf; compatible for hms1) and half should be male
sterile (ms; incompatible for hms1). (B) Cross 2: if other M. guttatus carry the incompatible hms1 allele, roughly one-fourth of
their BC 1 progeny should segregate for the hms1–hms2 incompatibility (Ic; ii) and be male sterile (ms). Cross 3: if the M. guttatus
parent carries hms2 incompatibility alleles, roughly one-fourth of
its IM62–BC 1 progeny should carry the hms1–hms2 incompatibility
(Ic; ii) and be male sterile (ms). Dominant alleles are given in uppercase and recessive alleles are in lowercase. For all crosses, the
female parent is listed first.
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EVOLUTION JANUARY 2007
To examine whether M. nasutus populations carry incompatibility alleles that can interact with the hms1 incompatibility allele to
cause hybrid male sterility, we crossed a male sterile RSB 3 plant to
six different M. nasutus individuals, each collected from a different
population (Fig. 3A, cross 1). For each of the six crosses, pollen viability of progeny was bimodally distributed into non-overlapping
groups: hybrid progeny that received the incompatible hms1 allele (from IM62) were always highly male sterile and progeny
carrying the compatible hms1 allele (from SF5) were at least partially male fertile (Table 2). This result suggests that the IM62
hms1 allele causes severe hybrid male sterility in combination
with any of the M. nasutus genetic backgrounds tested. In other
words, multiple M. nasutus individuals are functionally similar
to SF5 for hybrid incompatibility alleles that interact with hms1,
despite their considerable geographic distance from the Sherar’s
Falls population.
In contrast, M. nasutus individuals do not appear to be genetically homogeneous at other hybrid sterility loci: average pollen
viability of the male fertile progeny class varied depending on
VARIATION FOR MIMULUS HYBRID INCOMPATIBILITY
Table 2.
The IM62 hms1 allele is incompatible against every M. nasutus genetic background tested.
Individual1
N2
PV3 , incompatible hms1
PV4 , compatible hms1
F hms1 5
P5
SF5
CLR
M12
NCL
BRI
KIN
FGC
35
29
31
21
26
23
30
0.009 (0.004)
0.031 (0.016)
0.005 (0.019)
0.025 (0.013)
0.005 (0.018)
0.001 (0.025)
0.025 (0.022)
0.972 (0.004)
0.371 (0.014)
0.229 (0.010)
0.303 (0.010)
0.481 (0.017)
0.652 (0.019)
0.643 (0.021)
24768.54
254.009
104.721
268.659
370.272
424.626
403.953
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
1 Population of origin for the M. nasutus individual crossed to a male sterile RSB . SF5 is the parental M. nasutus line used to create the RSB population.
3
3
Aside from SF5, individuals are ordered in the table according to their geographic locations, arranged from north to south.
2 Number of progeny analyzed from the cross.
3 Least squares means of pollen viability for progeny that inherited the incompatible hms1 allele (derived from the IM62 parent). Standard errors are given
in parentheses.
4 Least squares means of pollen viability for progeny that inherited the compatible hms1 allele (derived from the SF5 parent). Standard errors are given in
parentheses.
5 Results of ANOVA to test the effect of hms1 genotype on pollen viability.
which of the six M. nasutus parents was crossed to the male sterile RSB 3 (Table 2). Pollen viability of the male fertile progeny
class was reduced by as much as 76% relative to progeny from
the RSB 3 –SF5 cross (i.e., compare male fertility in progeny of
SF5 versus M12 M. nasutus parents, Table 2). These results cannot be explained by differences in pollen viability among the M.
nasutus parents, which were all highly fertile (data not shown).
It appears that although M. nasutus is genetically uniform for incompatibility alleles that interact with hms1, the species harbors
substantial variation at additional incompatibility loci that cause
male sterility in crosses with IM62 and/or between populations of
M. nasutus.
CHARACTERIZATION OF VARIATION FOR THE
HMS1–HMS2 INCOMPATIBILITY WITHIN M. GUTTATUS
To determine whether the incompatible hms1 allele is present in
other M. guttatus populations, we crossed SF5 to different M.
guttatus individuals and then backcrossed F 1 hybrids to the SF5
parent (Fig. 3B, cross 2). Using this approach, we tested for the
presence of a dominant hms1 incompatibility allele in 25 M. guttatus individuals from 10 populations located throughout much of
the species’ range (Table 1, Fig. 2). In general, our sample sizes
from cross 2 were too small to detect with certainty the 3:1 ratio
of male fertile to sterile progeny that is expected if the M. guttatus parent contributes an hms1 incompatibility allele (see Fig. 3B,
cross 2). Moreover, in several crosses, very few progeny carried
the putative incompatible genotype due to segregation distortion
at (or linked to) the hms1 and hms2 loci. In other cases, although
the pattern of male fertility among cross 2 progeny approximated
a 3:1 ratio, variation in male fertility was unaffected by hms1 and
hms2 genotypes. Nevertheless, because we obtained both pheno-
typic and genotypic information for all cross 2 progeny, we were
still able to determine which M. guttatus individuals contributed
hms1 incompatibility alleles, a task that would have been impossible had we relied solely on phenotypic ratios.
Our crossing results suggest that the hms1 incompatibility
allele is extremely geographically restricted. We found evidence
for the presence of the hms1 incompatibility allele in only two
populations: IM, from which IM62 originated, and Echo Basin
Trail (EBT), a population located roughly 3 km away from IM
(Table 3). Aside from Echo Basin, we found no evidence for the
hms1 incompatibility allele in other populations located close to
IM; however, sampling within these populations (N = 1 to 3 individuals) and of the region was certainly not exhaustive.
Once we determined that the incompatible hms1 allele is not
geographically widespread, we sampled extensively from IM to
examine within-population variation for hms1. Remarkably, we
discovered that the hms1 locus is polymorphic within the IM population. Of the 12 IM M. guttatus individuals we tested, 6 appeared
to carry the hms1 incompatibility allele (Table 3). Among the backcross progeny of these six IM M. guttatus individuals (as well as
the individual collected from the EBT population), the effect of
hms1 genotype on male fertility was highly significant (Table 3),
similar to what was seen in the original SF5–IM62 interspecific
cross (see Methods and Sweigart et al. 2006). Moreover, those
backcross progeny that carried the hms1–hms2 incompatibility
genotype were often highly male sterile relative to other genotypes (Table 3). For two of these IM M. guttatus individuals (IM2
and IM14), extreme segregation distortion at hms1 among the
backcross progeny (toward heterozygous genotypes) prevented us
from testing for an association between hms1 genotype and male
fertility (Table 3). Nevertheless, reduced male fertility of IM2 and
EVOLUTION JANUARY 2007
147
A. L. SWEIGART ET AL.
Table 3.
Natural variation for the hms1–hms2 incompatibility in M. guttatus.
Individual1
IM2
IM14
IM18
IM693
IM712
IM1177
EBT12
GDP15
IM135
PV2 hms1: cc
PV2 hms1: Ic
F hms 1 3
hms2: ii
hms2: Ci
hms2: ii
hms2: Ci
–4
–4
0.488 (0.089, 3)
0.703 (0.079, 8)
0.909 (0.065, 8)
0.843 (0.098, 7)
0.874 (0.087, 4)
0.751 (0.083, 8)
0.746 (0.083, 6)
–4
0.626 (0.119, 1)
0.830 (0.054, 8)
0.833 (0.100, 5)
0.907 (0.061, 9)
0.622 (0.082, 10)
0.884 (0.087, 4)
0.750 (0.105, 5)
0.850 (0.098, 5)
0.213 (0.053, 6)
0.141 (0.035, 11)
0.137 (0.046, 11)
0.263 (0.112, 4)
0.238 (0.130, 2)
0.355 (0.098, 7)
0.197 (0.061, 8)
0.710 (0.105, 6)
0.923 (0.113, 3)
0.269 (0.046, 11)
0.301 (0.042, 8)
0.329 (0.031, 25)
0.556 (0.084, 7)
0.402 (0.061, 9)
0.513 (0.078, 11)
0.339 (0.066, 7)
0.758 (0.083, 8)
0.781 (0.074, 7)
–
–
52.102∗∗∗∗
14.390∗∗
48.367∗∗∗∗
11.086∗∗
64.692∗∗∗∗
0.030
0.342
1 M. guttatus individual used in the BC to SF5 (cross 2). Letters indicate population of origin (Table 1) and numbers refer to the individual.
1
2 Least squares means of pollen viability for each of the four hms1–hms2 genotypic classes: homozygous for SF5 alleles at hms1 (cc) and hms2 (ii),
homozygous for SF5 alleles at hms1 (cc) and heterozygous for hms2 (Ci), heterozygous at hms1 (Ic) and homozygous for SF5 alleles at hms2 (ii), and
heterozygous at hms1 (Ic)and hms2 (Ci). In parentheses are standard errors and number of progeny for each genotypic class.
3 Results of ANOVA to test the effect of hms1 genotype on pollen viability.
4 Indicates a genotypic class that is missing (N = 0) due to segregation distortion.
5 Representative M. guttatus individuals that appear to carry compatible hms1 alleles.
∗∗ P < 0.005, ∗∗∗∗ P < 0.0001.
IM14 cross 2 progeny (relative to the cross 2 progeny of other M.
guttatus) suggests that these individuals might have contributed
hms1 incompatibility alleles.
Next, we investigated whether M. guttatus individuals that
do not carry incompatible alleles at hms1 are instead incompatible at hms2 (i.e., hms1–hms2 genotype: cc; ii). We tested this
possibility by substituting 12 M. guttatus individuals collected
from geographically diverse populations for SF5 in the original
BC 1 design (Fig. 3B, cross 3). If the M. guttatus individual carries
incompatible alleles at hms2, then we expect that roughly a fourth
of the backcross progeny will be highly male sterile. Instead, we
observed that the M. guttatus–IM62 BC 1 progeny were generally
highly male fertile, and none was highly sterile (data not shown).
Indeed, none of these 12 M. guttatus individuals we tested, including 7 individuals from IM, appeared to carry incompatible hms2
alleles.
Discussion
In this report, we have characterized intraspecific variation for a
Dobzhansky–Muller incompatibility between two closely related
species, M. nasutus and M. guttatus. Originally identified in a single interspecific cross between two inbred lines, the M. guttatus
(IM62) allele at hms1 acts dominantly in combination with recessive M. nasutus (SF5) alleles at hms2 to cause nearly complete
male sterility in affected hybrids (Sweigart et al. 2006). Our purpose here was to examine the extent of genetic variation within
species for the hybrid incompatibility. The results of our genetic
crosses provide evidence that the hms2 incompatibility allele is
148
EVOLUTION JANUARY 2007
widespread in M. nasutus and rare or absent in M. guttatus. In
contrast, the hms1 incompatibility allele appears to be extremely
geographically restricted within M. guttatus, and is even polymorphic in a single population. Below we discuss our findings and
their implications for investigating the evolutionary dynamics of
hybrid incompatibility.
We detected no variation among six M. nasutus individuals—
each sampled from a different population—in their reaction to
the incompatible hms1 allele. No matter which M. nasutus individual was crossed to the male sterile RSB 3 , any resulting
progeny that inherited the hms1 incompatibility allele were always completely male sterile (Table 2). Because the vast majority of genetic diversity in the highly selfing M. nasutus is partitioned among populations (Sweigart and Willis 2003), our sampling scheme likely maximized the potential to detect variation.
It appears that hms1-interacting hybrid incompatibility alleles are
extremely widespread within M. nasutus. The most parsimonious
explanation for this pattern is that many M. nasutus populations
carry recessive incompatibility alleles at the hms2 locus. However, because segregating genetic variation in cross 1 was limited
to the hms1 introgression, we were unable to map the interacting
loci. Therefore, it is formally possible that male sterility in cross
1 progeny is caused by epistasis between hms1 and a different,
dominant M. nasutus incompatibility allele, at the hms2 locus or
elsewhere in the genome. Because the progeny from cross 1 inherit
half of their genome from the RSB 3 parent (which is expected to
be homozygous for SF5 alleles at 93.75% of its genome, apart
from the region linked to hms1) and half from the experimental
M. nasutus parent, any hms1-interacting incompatibility allele at
VARIATION FOR MIMULUS HYBRID INCOMPATIBILITY
a locus other than hms2 would have to be dominant. However,
if the hybrid incompatibility is between dominant alleles, all F 1
hybrids between M. nasutus and IM62 should be completely male
sterile, but this result has never been observed in any M. nasutus–IM62 cross (including M. nasutus individuals from some of
the same populations sampled for this study; N. Martin unpubl.
results). Previous analyses of molecular variation point to a single
evolutionary origin for M. nasutus (Sweigart and Willis 2003).
Therefore, the establishment of the incompatible hms2 allele in
M. nasutus is likely to have preceded the expansion of this species
to its current geographic range.
In contrast, the hms1 incompatibility allele does not appear to be geographically widespread within M. guttatus. Our
cross 2 experiments seem to suggest that only a few M. guttatus
populations—all from a single geographic locale—might carry
dominant incompatibility alleles at the hms1 locus. Alternatively,
it is possible that M. guttatus is actually fixed for the incompatible allele at hms1, and that variation in male fertility among BC 1
populations is instead due to polymorphism at additional modifier loci that mask the phenotypic effects of hms1. However, this
latter scenario seems unlikely for two reasons. First, to account
for the fact that we often observe no effect of hms1 genotype on
BC 1 hybrid male fertility, it is necessary to invoke very strong effects for putative modifier loci. In fact, the modifiers would have
to eliminate the sterility effects of the incompatible hms1 allele.
Second, unless modifier loci are linked to hms1, independent assortment should ensure that some BC 1 hybrids will be completely
male sterile. (In the case of a single modifier locus, we would
expect an eighth of BC 1 hybrids to segregate for the hms1–hms2
incompatibility in the absence of the modifier allele.)
Does the hms1–hms2 incompatibility contribute to reproductive isolation between M. nasutus and M. guttatus? Because the
hms1 incompatibility allele is likely restricted to only a few M.
guttatus populations in the western Cascades of Oregon, its potential to act as a barrier between the two species might be limited.
Our results indicate that the incompatible hms1 allele is absent, or
at a relatively low frequency, in populations of California where
M. guttatus and M. nasutus co-exist and are known to hybridize
(e.g., GTR, GCC, GDP, MED; Sweigart and Willis 2003; N. Martin unpubl. results). Of course, it is possible that the incompatible hms1 allele is rare in these populations as a consequence of
interspecific hybridization; natural selection against male sterile
hybrids might have removed incompatibility alleles (Noor et al.
2001). However, the incompatible hms1 allele is also absent from
several Oregon populations (e.g., BLY, CGR, BR) that are currently geographically distant from any M. nasutus. Instead, we
consider it more likely that the hms1 incompatibility allele arose
relatively recently within M. guttatus and is currently restricted to
IM (where it was discovered) and nearby populations. Nevertheless, because we have not exhaustively sampled M. guttatus, which
densely populates much of western North America, we cannot be
certain that the hms1–hms2 incompatibility never acts as a barrier
to interspecific gene flow. Future sampling efforts will target M.
guttatus populations that co-exist with M. nasutus in regions of
central Oregon, Washington, and British Columbia.
Does the hms1–hms2 incompatibility contribute to reproductive isolation between M. guttatus populations? The discovery that
hms1 varies within M. guttatus prompted us to investigate whether
the hms2 locus might also be polymorphic. In principle, M. guttatus individuals that carry compatible alleles at hms1 might be
incompatible for hms2 (i.e., identical to the SF5 genotype). If this
were the case, the hms1–hms2 incompatibility could potentially
cause hybrid sterility and limit gene flow between adjacent populations of M. guttatus. However, our genetic analyses found no
evidence for the presence of hms2 incompatibility alleles in any
M. guttatus; unlike the BC 1 hybrids of SF5 and IM62, all progeny
from cross 3 were highly male fertile. Therefore, the presence of
the hms1 incompatibility allele within the IM population is unlikely to result in any hybrid sterility with other local M. guttatus
populations.
Our finding that the hms1 locus is polymorphic within M.
guttatus is certainly not unprecedented. In the only other study to
examine standing genetic variation for hybrid incompatibility loci,
Christie and Macnair (1987) identified considerable variation for
two separate incompatibility systems that cause lethality between
populations of M. guttatus. In one of these systems, a locus that
confers copper tolerance is tightly linked to (or is itself) a hybrid
incompatibility locus (Macnair and Christie 1983). In some interpopulation crosses, the copper tolerance/incompatibility locus
causes lethality by interacting with a small number of additional
loci (Macnair and Christie 1983). Divergence at the copper tolerance locus is presumably adaptive; the tolerance allele is restricted
to and fixed within the Copperopolis population, which exists on a
copper mine tailing (Macnair 1983). In the second hybrid incompatibility system (referred to as the C7/U8 system), only two loci
interact to cause hybrid lethality between M. guttatus populations
(Christie and Macnair 1984). To characterize natural variation
for the two hybrid incompatibility systems, Macnair and Christie
(1987) performed extensive genetic analyses, crossing a number
of individuals from each of 21 M. guttatus populations to tester
strains. Their crosses identified four M. guttatus populations—
including one that is geographically proximate to Copperopolis—
that segregate for alleles incompatible with the copper tolerance
allele (Christie and Macnair 1987). Polymorphism within M. guttatus was also discovered for the C7/U8 incompatibility system
(Christie and Macnair 1984, 1987). In this two-locus hybrid lethality, incompatible alleles at each locus are restricted to mutually
exclusive populations that form two distinct geographic clusters.
Remarkably, of the 13 populations that carry incompatibility alleles at one of the two C7/U8 hybrid lethality loci, only one appeared
EVOLUTION JANUARY 2007
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A. L. SWEIGART ET AL.
to be not polymorphic (Christie and Macnair 1987). In addition,
incompatible alleles often segregated at intermediate frequencies
within these M. guttatus populations, in agreement with our findings for the hms1 locus in the IM population.
The picture that emerges from these studies is one of a highly
structured, genetically variable M. guttatus species complex, in
which it is not uncommon for Dobzhansky–Muller incompatibilities to reach appreciable frequencies, either between populations
or species. Of course, because of substantial population structure,
the long-term effects of hybrid incompatibility alleles such as
hms1 might be quite minor. As Vickery (1978) noted in his classic
investigation of cross compatibility within and between Mimulus
species, hybrid incompatibility appears “to constitute a common,
normal part of the gene pool of each member of the [M. guttatus]
complex.” In this study, we even discovered heterogeneity for
hybrid incompatibility factors among populations of the highly
self-fertilizing M. nasutus (albeit not for hms1 or hms2). Because
we assessed variation within M. nasutus by crossing individuals
to an RSB 3 tester (cross 1), we cannot be certain whether M. nasutus is polymorphic for incompatibility loci that interact with
SF5 or with dominant factors from IM62 (i.e., in addition to being
heterozygous at the hms1 locus, the RSB 3 plant is expected to be
heterozygous at 7.25% of its genome). If among-cross variation in
hybrid sterility is in fact due to hybrid incompatibilities between
M. nasutus populations, it does not appear to be a function of
isolation by distance. On the contrary, the M. nasutus individuals
that were most interfertile with the RSB 3 plant originated from
populations located at the greatest distances from Sherar’s Falls.
Of course, the question remains as to what is the maintaining variation for the hms1 locus within M. guttatus populations
of Oregon’s Cascades. Recent studies have almost exclusively
emphasized the role of positive selection in fixing hybrid incompatibility alleles (reviewed in Coyne and Orr 2004, but see Shuker
et al. 2005). Indeed, sexual selection and/or sexual conflict appear likely to have driven the evolution of hybrid male sterility in
Drosophila (Wu et al. 1996), and positive selection has also been
implicated in the divergence of the hybrid incompatibility genes
OdsH, Hmr, and Nup96 (Ting et al. 1998; Barbash et al. 2003;
Presgraves et al. 2003). However, because hms1 is not fixed, even
within local populations, it may not be a target for strong positive
selection. Instead, it is possible that hms1 variation is maintained
by balancing selection or is simply the result of genetic drift. Fortunately, because of this segregating variation for the hms1 locus,
it is possible to design field experiments to estimate potential selective effects of the incompatibility allele within M. guttatus populations. In addition, once we know which genes underlie hms1
and hms2, it will become possible to perform molecular population genetics tests to investigate the role of selection in shaping
the pattern of sequence variation at these hybrid incompatibility
loci.
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EVOLUTION JANUARY 2007
ACKNOWLEDGMENTS
We thank A. Bouck, A. Case, A. Cooley, L. Fishman, Y. W. Lee, D.
Lowery, M. Noor, M. Purugganan, M. Rausher, A. Sheck, M. Uyenoyama,
G. Wray, and K. Wright for helpful discussions about this project. We
are also grateful to A. Cooley, Y. W. Lee, M. Purugganan, M. Rausher,
M. Uyenoyama, D. Schoen, M. Streisfeld, G. Wray, C. Wu, and two
anonymous reviewers for helpful comments on a draft of this paper. This
research was supported by the National Science Foundation grants DEB0408098 to ALS, DEB-0075704 and EF FIBR-0328636 to JHW.
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