The Genetic Basis of Naturally Occurring Pollen Color Dimorphisms in Nigella degenii (Ranunculaceae)
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Journal of Heredity 2005:96(5):550–556 ª The American Genetic Association. 2005. All rights reserved.
doi:10.1093/jhered/esi098 For permissions, please email: journals.permissions@oupjournals.org.
Advance Access publication August 31, 2005
The Genetic Basis of Naturally Occurring
Pollen Color Dimorphisms in Nigella
degenii (Ranunculaceae)
S. ANDERSSON AND T. H. JORGENSEN
From the Department of Ecology, Section of Plant Ecology and Systematics, Sölvegatan 37, Lund University, SE-22362
Lund, Sweden.
Address correspondence to Stefan Andersson at the address above, or e-mail: Stefan.Andersson@ekol.lu.se.
Abstract
Nigella degenii ssp. barbro and ssp. jenny differ from related taxa in being dimorphic for pollen color, with some plants having
dark pollen and others light pollen. In this study we performed experimental crosses to determine whether the difference in
pollen color is governed by few or many loci and whether the two subspecies utilize the same gene to control pollen color.
Patterns of segregation in crosses between morphs show that dark pollen is dominant over light pollen and that a single major
gene is responsible for most of the variation in pollen color. Consequently it should be relatively easy for pollen color dimor-
phisms to establish and spread in these subspecies. Aberrant segregation ratios were attributed to genetic factors that reduced
the expression of the allele conferring dark pollen or processes that sorted between color morphs during seed development.
Crosses between dark pollen plants from different subspecies showed signs of complementation in the F2 generation, but the
frequency of the light morph was too low to support a model involving complementary action of recessive alleles at two
separate loci. Based on this and other observations, we hypothesize that the pollen color difference is controlled by the same
major locus in the two subspecies.
Flower color polymorphism—the co-occurrence of two or dimorphism in two geographically and morphologically dis-
more distinct flower color phenotypes within the same tinct subspecies of Nigella degenii Vier., ssp. barbro Strid and
population—is a conspicuous feature of many plants, partic- ssp. jenny Strid. This dimorphism is expressed at the sporo-
ularly in species whose flowers typically contain blue, red, or phytic level, with some individuals producing pale or dark
purple anthocyanin pigments (Warren and Mackenzie 2001). yellow (henceforth ‘‘light’’) pollen and others violet (‘‘dark’’)
Such polymorphisms may be selectively neutral or a conse- pollen. The proportion of plants with dark pollen shows ex-
quence of balancing selection forces involving not only pol- tensive variation among populations, ranging from 0 to 0.93
linators (Brown and Clegg 1984; Mogford 1974), but also in ssp. barbro and from 0.02 to 0.71 in ssp. jenny (Jorgensen
pleiotropic relationships between the expression of floral TH and Andersson S, unpublished observations). Related
anthocyanins and related compounds that influence photo- species have light pollen (Strid 1970), indicating that the di-
protection, stress tolerance, disease defense, and herbivory morphism, or more strictly the dark pollen type, is derived
resistance (Clegg and Durbin 2000; Koes et al. 1994; Levin within N. degenii.
and Brack 1995; Schemske and Bierzychudek 2001; Warren Pollen color differences in other plants have been found
and Mackenzie 2001). Selection on floral color polymor- to involve a single-gene system with complete dominance for
phisms is most efficient when differences in flower color dark pollen (Gerats et al. 1985; Mehlenbacher and Smith
are controlled by one, or a few, major genes, when novel color 2002; Qiao et al. 1993; Wakelin et al. 2003), a pattern that
morphs are specified by dominant alleles, and when a species also applies to many petal color dimorphisms [for references,
can achieve the same polymorphism by different genetic see Levin and Brack (1995)]. However, it is still uncertain
mechanisms (Haldane 1924; Lande 1983; Macnair 1976). whether the single-gene model holds for N. degenii (Strid
While much attention has focused on corolla color poly- 1970) and whether the two subspecies of N. degenii utilize
morphisms, only a few studies have considered color poly- the same gene to control pollen color. In the present study
morphisms involving sexual organs such as pistils, stamens, we obtained data from extensive crossing experiments to
and pollen (Rafinski 1979; Wolfe 2001). One exception is address these questions. Following the detection of single-
Strid (1970), who documented a conspicuous pollen color gene inheritance in most of the crosses, we also examined
550Andersson and Jorgensen Pollen Color Dimorphisms in Nigella degenii
whether aberrant segregation ratios could be attributed to combinations. One cross (ML3 MD1) was performed
genetic background effects or processes that sorted between twice, using different parent plants from the same inbred
color morphs before flowering. lines. Flowers used as pollen recipients were emasculated
during the male phase to avoid contamination with self-
pollen and attempts were made to perform each cross re-
Materials and Methods ciprocally. Most crosses yielded progeny with dark pollen
Study System (indicating dominance for dark pollen), but a few families
were dimorphic. In these cases we restricted the subsequent
Nigella degenii (Ranunculaceae) is an annual, diploid (2n 5 12) self- or cross-pollinations to F1 plants with dark pollen to
species that occupies disturbed habitats (e.g., abandoned maximize the probability that the resulting F2 or BC prog-
fields, seashores, open patches in phrygana vegetation) in enies segregated for pollen color. In the autumn of 2003
the Cyclades (Greece), where four geographically restricted we planted F2 or BC seeds in separate pots and scored
subspecies have been recognized, including ssp. barbro the resulting progeny for pollen color. Crosses that yielded
(northwest Cyclades) and ssp. jenny (island of Syros). Flowers too few plants (due to low seed set, germination ability, or
of Nigella have a conspicuous perianth, differentiated into an survival rate) were excluded, which reduced the number
outer whorl of five white, petaloid sepals and an inner whorl of progenies for which both reciprocal families were avail-
of eight, stalked nectaries. The androecium comprises a vari- able. Seventy-two percent of the F2 and BC seeds (4428
able number of stamens, which shed their pollen as the fil- of 6075 sown seeds) resulted in plants that could be scored
aments curve outward during the male phase. The gynoecium for pollen color.
consists of up to five completely united follicles (each with As a second step, we extended the genetic analyses to
a separate style) and develops into a capsule with numerous intermorph crosses involving plants with no prior inbreeding
seeds. The self-compatible, insect-pollinated flowers are pro- history. These ‘‘outbred-plant crosses’’ were based on pairs of
tandrous and herkogamous, reducing within-flower selfing plants sampled from the same base population before the
(Strid 1969, 1970). N. degenii ssp. barbro and ssp. jenny differ inbreeding phase (see above). These crosses represented
in nectary color, sepal shape, fruit morphology, and various 11 parent combinations in the Mykonos population and
features related to the pollen color dimorphisms character- 9 parent combinations in the Syros population. Each cross
izing these subspecies (Strid 1970). was performed reciprocally and replicated up to six times
(using different pairs of flowers), and the resulting seeds were
Plant Material sown into plug trays to provide data on pollen color. Given
This study involves plants from one population of N. degenii the low frequency (approximately 14%) of the dark morph in
ssp. barbro (Mykonos, about 2.5 km north-northwest of the the base populations, most dark pollen parents were expected
town) and one population of N. degenii ssp. jenny (Syros, about to be heterozygous at loci controlling pollen color, that is, to
300 m south of Kini), both sampled in 1993 and maintained produce segregating progenies in crosses with the light
in an insect-free greenhouse for several generations by ran- morph. Seed set, quantified as the proportion of ovules that
dom outcrossing within populations. Both populations were developed into seeds, was determined for each replicate cross
dimorphic for pollen color, the proportion of plants with (fruit) to assess the potential for differential selection on
dark pollen being approximately 14% in the initial population color morphs during seed maturation (see below). Seventy-
samples (Andersson S, unpublished observations). eight percent of the sown seeds (2574 of 3315 seeds) resulted
Four generations of enforced self-pollination were used in plants that could be scored for pollen color.
to establish five (partly) inbred lines for each color morph We employed log-likelihood G tests for goodness-of-fit
and population. Each line was derived from a single maternal (Sokal and Rohlf 1995) to compare observed morph ratios
plant in a base population and had reached (apparent) fixation with segregation ratios expected under the single-gene model.
for the intended color morph in the last generation (based on Individual G tests were carried out at a critical probability of
an examination of more than 20 progeny in each line). Lines a# 5 a/k, where a is the experiment-wise error rate (0.05,
were denoted as ML, MD, SL, or SD to indicate population 0.01, or 0.001) and k is the number of replicate progenies
origin (M for Mykonos, S for Syros) and pollen color (L for (tests) for the type of cross considered (Bonferroni adjust-
light pollen, D for dark pollen), and given a unique identifi- ment). A replicated goodness-of-fit test was carried out
cation number within each category (e.g., ML1, SD5). for each type of progeny in the inbred-line crosses (for cat-
egories, see Table 1) and for each population in the outbred-
plant crosses to evaluate the overall fit of the single-gene
Segregation Analyses
model (quantified as Gpooled) and to assess the heterogeneity
In 2001–2003, we established a series of F1, F2, and backcross in the proportion of morphs among replicate progenies
(BC) progenies from a number of reciprocal intermorph (quantified as GH).
crosses, each pair representing a distinct combination of
inbred lines from the same or different base populations.
Complementation Tests
These ‘‘inbred-line crosses’’ represented four parent line com-
binations in the Mykonos population, four parent line com- In addition to the segregation analyses, we performed a com-
binations in the Syros population, and six interpopulation plementation analysis by crossing dark pollen plants from
551Journal of Heredity 2005:96(5)
Table 1. Patterns of segregation in F2 and backcross (BC) progenies from intermorph crosses involving inbred plants from the same or
different populations
Parents F2/BC phenotypea
Type of progeny/population Male Female Dark Light Pred.b G
F2 Mykonos
ML4 MD4 122 46 3:1 0.50
MD4 ML4 159 42 3:1 1.88
ML1 MD5 90 37 3:1 1.12
MD5 ML1 121 49 3:1 1.28
ML3 MD1 119 40 3:1 0.00
MD1 ML3 124 33 3:1 1.38
ML3 MD1 83 47 3:1 7.95*
MD1 ML3 128 37 3:1 0.60
ML5 MD2 104 30 3:1 0.50
MD2 ML5 80 21 3:1 0.99
F2 Syros
SL1 SD10 30 7 3:1 0.78
SD10 SL1 1 74 3:1 195.13***
SL2 SD9 75 19 3:1 1.20
SD9 SL2 35 17 3:1 1.55
SL5 SD5 74 24 3:1 0.01
SD8 SL3 63 20 3:1 0.04
F2 Interpopulation
ML3 SD9 108 48 3:1 2.64
SD9 ML3 104 59 3:1 10.04*
MD5 SL3 118 30 3:1 1.85
SL3 MD5 137 33 3:1 2.99
MD1 SL5 131 38 3:1 0.58
SL5 MD1 119 28 3:1 2.95
MD2 SL1 111 34 3:1 0.19
SL1 MD2 74 14 3:1 4.28
ML4 SD8 120 63 3:1 8.08*
ML5 SD10 52 31 3:1 6.18
Backcrosses
MD1 ML3 ML3 99 85 1:1 1.07
MD4 ML4 ML4 42 25 1:1 4.36
ML4 MD4 ML4 34 35 1:1 0.01
ML3 SD9 ML3 18 33 1:1 4.48
SD10 SL1 SL1 19 33 1:1 3.82
SL1 SD10 SL1 9 12 1:1 0.43
MD5 SL3 SL3 83 93 1:1 0.57
SL5 MD1 SL5 18 11 1:1 1.71
SD9 ML3 ML3 51 58 1:1 0.45
ML5 MD2 MD2 56 0 1:0
ML4 MD4 MD4 94 0 1:0
MD4 ML4 MD4 52 0 1:0
ML3 SD9 SD9 17 0 1:0
MD1 SL5 MD1 15 0 1:0
SL5 MD1 MD1 33 0 1:0
a
Underlined values denote the excess morph in families showing a significant deviation from single-gene prediction.
b
Observed frequencies were compared with segregation ratios predicted under a single-gene model with complete dominance for dark pollen (Pred.).
different populations (derived from the same inbred lines as populations. Comparisons of observed and predicted fre-
were used in the intermorph crosses) and then searching for quencies were carried out with a G test.
plants with light pollen in the F2 generation. Under the
assumption of Mendelian segregation at two unlinked loci,
Selection Analyses
1/16 of the F2 progeny were expected to be homozygous
recessive at both loci, that is, to express the light phenotype. There is great potential for selective processes to alter
Such ‘‘complementation’’ would indicate that the dark pollen progeny segregation ratios when only a few ovules develop
type is conditioned by different dominant genes in the two into germinable seeds or when many seedlings die before
552Andersson and Jorgensen Pollen Color Dimorphisms in Nigella degenii
flowering. To evaluate the role of selection during seed mat- Table 2. Morph frequencies in segregating F1 progenies from
uration, we assessed the relationship between the seed set of intermorph crosses involving noninbred plants from the same
each flower in the outbred-plant experiment and the corre- population
sponding segregation ratio in the progeny generation F1 phenotypea
(expressed as a fraction of the dark morph). Progeny segre- Reciprocal
gation ratios were subjected to analysis of covariance Population Cross familyb Dark Light G
(ANCOVA) involving: (1) ‘‘parent combination,’’ a group Mykonos
variable that provided a control for genetic background 1 1 30 32 0.07
effects, (2) seed set (covariate), and (3) the interaction be- 1 2 5 10 1.70
tween parent combination and seed set. A similar approach 2 1 30 7 15.40**
2 2 28 25 0.17
was used to examine the potential for selection between ger- 3 1 30 27 0.16
mination and flowering, as determined by the proportion of 3 2 27 29 0.07
seeds that developed into flowering plants. In this case it was 4 1 31 24 0.89
also possible to analyze data from the inbred-line crosses 4 2 22 21 0.02
(using different BC or F2 categories as groups; see Table 1). 5 1 42 30 2.01
The residuals from the ANCOVAs were approximately 5 2 31 46 2.94
normally distributed, hence no transformation was necessary. 6 1 12 59 33.91***
6 2 20 33 3.22
Initial analyses of data from outbred-plant crosses revealed 7 1 31 35 0.24
a nonsignificant difference between the two source popula- 7 2 40 34 0.49
tions (F 5 0.09, P 5 .773), so this factor was excluded from 8 1 37 28 1.25
the final analysis. 8 2 32 33 0.02
Syros
1 1 31 42 1.66
1 2 29 38 1.21
Results 2 1 42 38 0.20
Patterns of Segregation in Inbred-Line Crosses 2 2 39 37 0.05
3 1 33 41 0.87
The 15 intermorph crosses produced F1 progenies in one or 3 2 6 24 11.57**
both directions (as indicated by the presence of reciprocal or 4 1 21 26 0.53
unidirectional F2 families in Table 1). Based on data pooled 4 2 23 27 0.32
across families, 95% of the F1 progeny (106 of 111 plants) 5 1 33 35 0.06
5 2 26 44 4.68
produced dark pollen. The five light pollen plants repre- 6 1 33 28 0.41
sented two dimorphic F1 families from crosses within the 6 2 37 42 0.32
Syros population: SD10 (male) SL1 (female) and SD9 7 1 35 16 7.25
(male) SL2 (female). Backcrosses to the dark pollen morph 7 2 30 49 4.62
always yielded progeny with dark pollen (Table 1), confirming 8 1 40 31 1.14
the dominant nature of this phenotype. 8 2 30 42 2.01
9 1 37 25 2.34
Morph frequencies in segregating F2 or BC progenies gen-
9 2 29 19 2.10
erally conformed to the 3:1 or 1:1 ratio predicted under the
single-gene model, regardless of whether the two parent plants a
Observed frequencies were compared with the 1:1 segregation ratio pre-
represented the same or different populations (Table 1). There dicted under a single-gene model with complete dominance for dark pollen.
was a close fit to single-gene predictions after pooling b
The number specifies the color morph of the maternal parent (1 5 dark,
data across progenies within the same F2 or BC category 2 5 light).
(Gpooled , 1, P . .05), the only exception being the F2 Syros
group (Gpooled 5 29.3, P , .01). The latter result was almost
entirely due to the extreme excess of light pollen plants in the family was always associated with normal Mendelian segre-
family SD10 (male) SL1 (female) (1 dark versus 74 lights), as gation in the reciprocal family (when available, Table 1).
shown by the drastic reduction in the G value after excluding The replicated cross in the Mykonos population (ML3
this F2 family from the analysis (Gpooled 5 0.2, P . .05). This MD1) showed a weak, though significant, unilateral bias in
procedure also reduced the heterogeneity among replicate one replicate but not in the other.
families within the F2 Syros group (GH 5 3.3, P . .05 versus
GH 5 169.4, P , .001 for the full dataset). Remaining back-
Patterns of Segregation in Outbred-Plant Crosses
cross and F2 categories showed moderate among-family
variation (F2 Mykonos: GH 5 16.1, P . .05; F2 between pop- Three intermorph crosses, all involving Mykonos plants, pro-
ulations: GH 5 39.0, P , .001; backcrosses: GH 5 16.7, P , duced progeny with only dark pollen, presumably because the
.05), with three significant deviations from single-gene predic- dark parent was homozygous dominant at the inferred major
tions, each involving a slight excess of the light morph (Table 1). effect locus. Morph frequencies in dimorphic progenies
Incidents of deviant segregation were unilateral: a signif- showed a close agreement with the 1:1 ratio predicted under
icant deviation from the predicted Mendelian ratio in one the single-gene model (Table 2), especially when data where
553Journal of Heredity 2005:96(5)
pooled over families (Mykonos: Gpooled 5 0.68, P . .05;
Syros: Gpooled 5 2.16, P . .05). Nevertheless, there was sig-
nificant heterogeneity in the proportion of morphs among
replicate families (Mykonos: GH 5 61.88, P , .001; Syros:
GH 5 39.18, P , .001), with three families showing (unilat-
eral) distortion of morph frequency, one involving a signifi-
cant excess of the dark type and two involving a significant
excess of the light type (Table 2).
Complementation Tests
Crosses between dark pollen plants from different popula-
tions yielded two plants with light pollen in the F2 generation:
1 of 70 plants in the cross MD5 SD5 and 1 of 120 plants in
the cross MD2 SD9. A third cross produced plants with
only dark pollen (MD4 SD5, 90 plants). The overall fre-
quency of the light morph (2 of 280 plants) was significantly
lower than the frequency expected under a model involving
complementary action of recessive alleles at two separate
(unlinked) loci (17.5 plants; Gpooled 5 24.67, P , .001).
Figure 1. The relationship between the proportion of the
dark morph (morph ratio) and the proportion of ovules
Selection Analyses developing into seeds (seed set) in the fruit giving rise to each
Pollinated flowers varied greatly in the proportion of ovules progeny family. The insert shows the original scatter plot and
that matured into seeds (range 47–100% for outbred-plant the main figure illustrates regression lines for progenies from
crosses) and there was extensive among-family variation in different parent combinations.
the proportion of sown seeds that resulted in flowering plants
(range 40–92% for inbred-line crosses and 21–100% for
basis of naturally occurring pollen color dimorphisms in
outbred-plant crosses). The segregation ratio in the progeny
N. degenii, a species in which the dark pollen morph repre-
generation showed no relationship with seed set (F 5 0.49,
sents the derived rather than the ancestral condition.
P . .05; ANCOVA), but was affected by parent combination
Genetic data from N. degenii indicate a Mendelian basis
(F 5 2.19, P , .05) and by the parent combination–seed set
for most of the variation in pollen color: patterns of segre-
interaction (F 5 2.43, P , .01). As for the significant inter-
gation in crosses between the different color morphs gener-
action term, our data indicate large changes in the ranking of
ally conformed to segregation of two alleles at a single major
parent combinations between fruits with low and high seed
locus, regardless of whether the parents represented inbred
set (crossing regression lines) and a tendency for the most
or outbred genotypes from the same or from different pop-
extreme segregation ratios (within a given parent combina-
ulations (subspecies). Both populations showed dominance
tion) to represent the low seed set category (Figure 1).
for the dark pollen type; consequently there should be great
The progeny segregation ratio was independent of survival
potential for local selection pressures to act on a newly risen
rate and showed no significant interaction between survival
mutant with dark pollen. Given the existence of a major locus
rate and progeny type (inbred-line crosses) or parent combi-
controlling pollen color and the observed dominance rela-
nation (outbred-plant crosses) (F , 1.70, P . .19 in all cases;
tionship, it should be relatively easy for pollen color dimor-
ANCOVA).
phisms to establish and spread in N. degenii. The evolutionary
lability of pollen color is also manifested by the simple genetic
control of pollen color in Petunia hybrida (Gerats et al.
Discussion 1985), Helianthus annuus (Qiao et al. 1993), Corylus avellana
Knowledge of the genetic basis of phenotypic variation—for (Mehlenbacher and Smith 2002), and Eschscholzia californica
example, the number of genes involved and the dominance (Wakelin et al. 2003), even though no persistent pollen color
relationships among the alleles at these loci—is fundamental dimorphisms have evolved in these species.
when inferring the potential for selection to shape evolution- In a previous study of N. degenii, we used field data, com-
ary change in floral characters, including color polymor- mon garden experiments, and pollen competition experi-
phisms. To date, only a few investigations have explored ments to explore the potential role of pollinators and
the genetic control of pollen color, and these are restricted postpollination processes in exerting selection on pollen
to plant species whose pollen coats normally contain dark color dimorphisms (Jorgensen TH and Andersson S, unpub-
pigments and where the light morph results from mutations lished data). Our results show that pollinators can discrimi-
that prevent the synthesis of dark pigments (Gerats et al. nate between plants with different pollen color and that the
1985; Mehlenbacher and Smith 2002; Qiao et al. 1993; dark and light pollen morphs sometimes differ in fertilizing
Wakelin et al. 2003). In this study we examined the genetic or siring success; however, the relative morph fitness is
554Andersson and Jorgensen Pollen Color Dimorphisms in Nigella degenii
strongly determined by the type of pollinator (bumblebees alleles at linked loci. In this context we also note that previous
versus honeybees), pollination treatment (one- versus two- common garden experiments have demonstrated morph-
donor pollinations), and the particular combination of plants specific differences in vegetative survivorship, at least for
used as pollen and seed parents in the pollen competition plants of N. degenii ssp. barbro grown under stressful condi-
experiments. Pollen color has also been shown to influence tions (Jorgensen TH and Andersson S, unpublished data).
pollination success in Campanula americana: halictid bees were Indirect selection during embryonic or vegetative stages
more likely to discriminate against flowers without pollen extends the range of ecological factors that might influence
when they foraged in arrays of flowers with tan-colored pol- the spread and maintenance of pollen color dimorphisms,
len than in arrays of flowers with purple pollen (Lau and a hypothesis that also applies to flower color polymorphisms
Galloway 2004). Although these results suggest a role for in general (Clegg and Durbin 2000; Koes et al. 1994; Levin
selection in shaping evolutionary change in pollen color, and Brack 1995; Schemske and Bierzychudek 2001; Warren
there are no obvious between-species differences in habitat and Mackenzie 2001).
specificity or pollination biology that could explain why Populations of N. degenii ssp. barbro and ssp. jenny are geo-
N. degenii possess pollen color dimorphisms while other graphically isolated and have diverged for a wide variety of
Nigella species do not. vegetative and reproductive characters (Andersson 1997;
The few incidents of deviant segregation were distributed Strid 1970). For example, the pollen color dimorphism in
uniformly over the two study populations and generally in- ssp. jenny is expressed by both the pollen and anthers, whereas
volved an excess of the light pollen morph, the most extreme plants of ssp. barbro have dark anthers irrespective of pollen
being one of the F2 progenies from the Syros crosses (1 dark color. These observations not only suggest that N. degenii ssp.
versus 74 light). This and other, less extreme segregation dis- barbro and ssp. jenny have been genetically isolated for sub-
tortions were always unilateral: the detection of a non- stantial periods of time, but also raise the possibility that di-
Mendelian morph ratio was always associated with a normal morphic populations have evolved independently in the two
Mendelian ratio in the reciprocal family. In view of these find- subspecies. One testable prediction of this ‘‘multiple-origin
ings, and the close agreement with single-gene predictions in scenario’’ is that the production of dark pollen pigments is
a majority of the crosses, we hypothesize that some segregants controlled by different dominant genes in the two subspecies.
failed to produce dark pollen pigments despite being hetero- Our complementation crosses showed some evidence for
zygous or homozygous for the allele conferring dark pollen, segregation in the F2 generation, but the frequency of the
and that the magnitude of this penetrance-reducing effect was light morph was too low (less than 1%) to support a model
influenced by some nonnuclear (cytoplasmic) factor carried by involving complementary action of recessive alleles at two
some of the plants in the parent generation. However, it is independent loci. Based on this result and the excess of
difficult to explain why the replicated cross (ML3 MD1) the light pollen morph in some within-population crosses,
showed a unilateral bias in only one of the two sets of recip- we cannot exclude incomplete penetrance of dominant
rocal crosses; since each inbred line was derived from a single alleles—at the same locus—as the principal cause of the
female, one would expect all plants within a given line to have few light pollen plants that appeared in the complementation
the same cytoplasm and to express the same progeny morph crosses. Consequently our results provide little support for
ratio bias in replicate crosses with another line. Although more rejecting the hypothesis that the two subspecies of N. degenii
detailed genetic data are needed to confirm the role of cyto- utilize the same major locus to control differences in pollen
nuclear interactions, our observations clearly differ from the color. Further evaluation of the multiple-origin hypothesis
more strictly dominant expression of pollen color loci in other will benefit from the identification of pigments responsible
plants (Mehlenbacher and Smith 2002; Qiao et al. 1993; for light and dark pollen color in the two subspecies.
Wakelin et al. 2003; but see Gerats et al. 1985). The detection
of such heterogeneity accentuates the advantage of perform-
ing multiple crosses—involving several unrelated parents— Acknowledgments
before any broad generalizations are made regarding the
This work was supported by a fellowship from the Danish Natural Science
heritable basis of pollen color polymorphisms. Research Council (to T.H.J.) and grants from the Swedish Natural Science
Some of the aberrant segregation was probably mediated Research Council (to S.A.), the Julie von Müllens Foundation (to T.H.J.), the
by processes that sorted between morphs during the embry- Svend G. Fiedlers Foundation (to T.H.J.), and the Frimodt-Heineke Foun-
onic stages: progenies from outbred-plant crosses showed dation (to T.H.J.). Thanks to Olympia Tassopoulou and colleagues at the
greater deviations from Mendelian ratios when there was Department of Environment, Prefecture of Syros, for encouraging the pro-
an enhanced potential for postzygotic selection (low seed ject and to D. Richardson for comments on the manuscript.
set) than when little or no selection was possible (high seed
set). Interestingly, low seed set was not associated with a con-
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