Download Heterosis, epistasis and linkage disequilibrium in

Loitlogical Journalofthe Lznnean Society, 69 87-109 With 16 figures
June 1980
Heterosis, epistasis and linkage disequilibrium
in a wild population of the polymorphic butterfly
Danaus chrysippus (L.)
D. A. S. SMITH
Department of BioLogy, Eton CoLLege, Windsor,
Berks. SL4 6 E W , England
Arrepted fOr publication J u l ~1979
The colour polymorphism of the Danaus r h y i p p u s population at Dar es Salaam, East Africa, is
controlled at three major loci, each with two alleles. Two of the loci, one governing ground colour
and the other forewing pattern, are closely linked. Th e third locus, determining hindwing pattern,
assorts independently.
Thirty-eight broods raised from wild mated pairs, F1 and F2 generations gave 857 offspring of 23
genotypes (out of 27 possible). The forewing length, taken as an index of size, was investigated in
relation to the genotype. Heterosis is evident at all three loci. The two linked lori show epistatir
interaction of an unexperted kind : double heterozygotes are smaller than heterozygotes at only one
locus but larger than double homozygotes. The hrterotic effert at the third, unlinked locus is the
most pronounced and is additive to that at the other two. Heterosis is more marked in males than
females.
The possibility that body size has importance in connexion with sexual selection, food recources
and mimetic relationships is discussed.
Analysis of gene and chromosome frequenries in the wild parents of 61 broods suggests that
double heterozygotes for the two iinked loci may have heterozygous advantage. Seventy-eight per
c-ent of chromosomes are repulsion phase: thus, there is pronounced linkage disequilibrium which
must be maintained by selection as crossing over is almost 2%. In particular, the chromosome
carrying both dominant alleles in coupling is rare.
Consideration of the centres of distribution and present ranges of the alleles at all three loci
suggests that three geographiral races, aegyptzus, dorippus and alczppus, were isolated by forest
barriers, during wet periods in the Pleistocene, in south-west, north-east and north-west Africa
respectively. They have probably expanded their ranges in the post-glacial period to overlap and
interbreed in central and east Africa. Either heterozygous advantage o r seasonal (directional)
selection or a combination of both is responsible for the persistence of the polymorphism.
KEY WORDS: - polymorphism - genetirs - Danaus chryslppus - heterosis - epistasis - sexual
selection - mimicry - heterozygous advantage - linkage disequilibrium - subspecision Pleistocene.
CONTENTS
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Introdurtion
Materials and methods
. . . . . . . .
Breeding methods
. . . . . . . .
Statistical analysis
. . . . . . . .
The population sample
. . . . . . .
Scoring the genotypes
. . . . . . .
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0 1980 The Linnean Society of London
D. A. S . SMITH
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Breeding results
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Population genetics
. .
Randomness of mating
Sex ratio
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Hardy-Weinberg equilibrium
The A locus
. . . . .
Discussion
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Acknowledgements
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References
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Appendix
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INTRO DUCT10 N
The Old World queen butterfly, Dunuus chrysippus (L.1, is an exception to the
generalization that aposematic models belonging to Mullerian mimicry rings are
monomorphic (Fisher, 1930; Ford, 1971; Turner, 1977). In Uganda it
participates with Acrueu encedon L. and other acraeids in no less than four distinct,
sympatric Mullerian assemblages (Owen, 197 11, each with its attendant Batesian
members. These are by no means local phenomena, for at least two of the
Mullerian groupings are found throughout East Africa and Pierre (1974) has
demonstrated an impressive geographical correlation between the various comimetic forms of D. chvsippus and A . encedon. This situation is quite unlike that
pertaining for example, in the Neotropical genus Heliconius in which numerous
Mullerian rings are believed to have evolved in forest refuges, isolated during dry
periods in the Pleistocene. Most forms of Heliconius species are thus allopatric
races, sometimes giving rise to narrow hybrid zones where they meet (Turner,
1976). In contrast, the biogeography of the forms of D. chrysippus in Africa
suggests that race formation could have occurred in wet periods of the
Pleistocene (Smith, 1976a) when areas of dry grassland (the species does not
inhabit forest) in west, north-east and south-west Africa were isolated by
intervening lowland forest and montane vegetation (Moreau, 1963). Studies of
speciation and race formation in savannah birds (Moreau, 1966) support this
hypothesis. Subsequent contraction of the forests following both climatic change
and human activity has opened up vast new tracts of country to grassland species.
The races of D. chrysippus, having expanded out of their Quaternary refuges,
could have created the central and east African polymorphic populations by
admixture and hybridization. If this argument is correct, it is important to know
why the polymorphism has persisted and how it is maintained.
The mechanism most commonly postulated to maintain genetic
polymorphisms, particularly if alternatives are not readily demonstrable, is
heterosis or heterozygous advantage. In this paper, I examine the evidence from
two principal sources for heterosis in D. chrysippus: ( 1) broods reared from known
parents in the laboratory and (2) some aspects of the population genetics of the
wild population from which the breeding stock was taken.
Dobzhansky (1950, 1952) drew attention to the importance of distinguishing
two kinds of heterosis, namely, ‘euheterosis’ in whichfitness is improved in F1
hybrids and ‘luxuriance’ where the offspring are enhanced in a purely metric
sense. The former is now better known as ‘heterozygous advantage’ (Sheppard,
1967; Ford, 1971) or ‘overdominance’ (Wallace, 1970) while the latter is
synonymous with ‘hybrid vigour’. To distinguish the two phenomena, which
GENETICS OF POLYMORPHISM I N DANALJS
89
are quite different, I use respectively the terms ‘heterozygous advantage’ and
‘heterosis’.
By definition, heterozygous advantage occurs when the heterozygote exceeds
both homozygotes injtness. On the other hand, heterosis is defined as the amount
by which the mean of an F1 family exceeds, in a metric sense, its better parent
(Mather 8c Jinks, 1971). Here, I use the latter term to cover any segregation in
which the heterozygote d@ers significantly, for a metric character, either positively or
negatively, from both its parental genoQpes. The terms positive and negative heterosis
are used to distinguish the alternatives. The latter has been reported in wide
crosses between species or allopatric subspecies of Drosophila (Dobzhansky, 1950;
Wallace 8c Vetukhiv, 1955) but its occurrence in a monospecific, sympatric
population, as I show here for D. chrysifipus, is probably unusual and may
indicate its recent origin.
The basic genetics of the colour polymorphism in African D. chrysippus are
published (Owen 8c Chanter, 1968; Clarke, Sheppard & Smith, 1973; Smith,
1975a).The following facts are relevant to this paper (Figs 1-16).
The B locus has two alleles, B giving a brown ground colour and bb orange on
both wings. B is semi-dominant to dominant, heterozygotes being recognizable
in some cases; but BB and Bb butterflies cannot be reliably separated in 3 : 1
segregations. Forewing pattern is determined by the C locus, C giving a wing
uniformly coloured (orange or brown) except for a narrow black margin (form
dorippus Klug.) and cc a large black apical area traversed by a row of, sometimes
confluent, white apical spots (form aegvptius Schreber). Cc heterozygotes are
phenotypically close to CC butterflies but are often distinguishable by the row of
‘uegvptius’ subapical spots visible on the underside and occasionally the upper
side of the forewing (form transiens Suffert). The expressivity of c in heterozygotes
is modified by the B locus: 74.6% ( N = 173) of known B-Cc butterflies were f.
transiens compared with only 45% ( N = 129) of bbCc (unpublished data) and the
difference is significant (xf=26.2; P<O.OOl) with no sex difference. The B and C
loci are linked with a cross-over value of approximately 1.9% (Smith, 1975a,
1976b). As the known recombinants all resulted from cross-over in the male
parents, achiasmatic oogenesis is suspected as in other Lepidoptera
(Suomalainen, Cook 8c Turner, 1974; Turner 8c Sheppard, 1975).
The A locus has a recessive allele a giving, in the homozygous state, a large
white area on the hindwing (form alcippus Cramer when combined with the
aegvptius forewing and form albinus Lanz when combined with the dorippus
forewing). Au heterozygotes are often phenotypically distinct from both
homozygotes, having a small white patch o r a few white scales. The intermediate
phenotypes are named alcippoides Moore and semialbinus Strand. Dominance of A
is modified by both the B locus and sex (unpublished data). Of the known Aa
butterflies reared, the following proportions were intermediate phenotypes : Aabb
males, 77.8% (N=36); A d b females, 53.3% ( N = 3 0 ) ; AuB- males, 65.7% ( N = 70);
AuB- females, 28.1% (N=64). Thus, a significantly higher proportion of males
(xT=21.4; P(O.001) and of orange (x!=5.6; P(O.02) butterflies than offemales
or browns are intermediate. Clarke et al. (1973)found a modifying action of the B
locus opposite to my result in wide crosses between butterflies from Australia and
Sierra Leone. There is no evidence for linkage between the A locus and either the
B or C loci (Clarke et al., 1973; Smith, 1975a, and unpublished data):
independent assortment is assumed.
90
D. A. S . SMITH
GENETICS OF POLYMORPHISM IN DANAUS
91
MATERIALS AND METHODS
Breeding methods
The area of rough grassland at Dar es Salaam, Tanzania, from which all the
breeding stock was obtained, has been described before (Smith, 1976a).
D . chrysippus is not ideal for formal genetical studies. Laboratory reared males
are rarely able to mate until 7-10 days old because they lack the pheromone
necessary to elicit a female response to courtship (Seibt, Schneider 8c Eisner,
1972; Schneider et al., 1975) and mating will only occur in a spacious outdoor
flight cage. As females emerge from the pupa on average 1-2 days before their
brothers, they must be kept alive for 8-12 days to obtain sib matings. Therefore,
most broods were obtained either from wild mated females or laboratory reared
females mated to older wild males. As wild females were known from marking
studies to be polygamous, mainly fresh looking ones in copula were taken for
breeding. Parker (1970) showed that, in polygamous insects, most eggs are
fertilized by sperm from the most recent mating which, in the present context,
implies that they would be provided by the male observed in copula. The
segregations obtained suggest that multiple matings have not affected the
genetical interpretations (Smith, 1975a).
Eggs were obtained by sleeving individual females on a branch of the main
local foodplant, Calotropis gigantea (L.) Ait. (Asclepiadaceae). The broods were
then reared separately in indoor cages. The 38 broods included in the genetical
analysis comprised 857 butterflies.
Selection of the colour phenotypes of the wild female parents is unlikely to be
random with respect to the wild population but is not deliberately biased. All
healthy butterflies bred between September 1974 and May 1975 and fed as larvae
upon C. gigantea are included. Broods reared on other milkweeds are excluded as
t6e larvd-foodplant is an important source of phenotypic variation (Smith,
1978).
The forewing length, measured on the day of emergence after the wings had
fully expanded and dried out, is taken as an index of size. The genotypes are
known to the extent that they can be inferred directly from their phenotype
and/or that of their parent(s) and/or the segregations obtained. The dominant
phenotype classes at all three loci studied inevitably include both homozygotes
and heterozygotes in 3 : 1 segregations. Fortunately, the expressitivity of the
Figures 1 to 16. The colour forms of Danaus chryszppus occurring naturally around Dar es Salaam,
Tanzania. All the specimens illustrated were bred at D ar es Salaam. To p row, left to right: I , brown
dorippus 9,genotype A A B- C-. 2 , orange dompus 9 , genotype AQ bb CC (with some white scales on
hindwing veins). 3, orange dorippus 8,
genotype A A bb CC. 4, orange aeg~ptius8.genotype A- bb cc.
Second row: 5, brown aegyptius 9 , genotype A A B- cc. 6 , orange albinus 8,
genotype aa bb CC. I,
orange semi-albinus 8,genotype An bb C-. 8, orange alcippus 9 , genotype aa bb cc.
Third row: 9, brown a l t i p p ~8,
genotype QQ BB cc. 10, brown akippoides 8,
genotype Aa BB cc. 11,
brown albinus 6, genotype aa Bb Cc. 12, orange semi-albinus 9,Aa bb CC.
Bottom row: 13, orange aegyptius 3 with a little white scaling on the hindwing, genotype AQ bb cc.
This specimen is of the type named liboria Hulstaert in which the white sub-apical spots are enlarged
and confluent. This specimen (and no. 4 ) is more or less identical with the Asiatic race chrysippus L.
14, brown liborla 9 , genotype A A B- cc. Note the hold white spot in the brown area offorewing space
3 typical of this form. 15, brown liboria 8 (underside). 16, brown transiens (dorippus) (underside)
showing the “aegyptzuJ” sub-apical spots, genotype A A B- Cc.
Variation in the amount of white in AQ genotypes is seen by comparing nos 2 (white scarcely
visible), 7, 10, 12 and 13 (white scarcely visible). Magnification ofall specimens is x 6/7.
92
D. A. S. SMITH
recessive alleles in heterozygotes is fairly high for the A and C loci (see Introduction) and many heterozygotes can be classified on phenotypic grounds alone. At
the B locus, reliable separation of BB and Bb butterflies is impossible although
some heterozygotes are detectable. Consequently, the B- phenotype and
genotype classes are more heavily contaminated with heterozygotes than are
either the A- or C- classes. The impossibility of identifying all heterozygotes has
inevitably blunted the impact of the analysis.
Statistical analysis
The breeding data are analysed by Analysis of Variance to distinguish the
effects of the A, B and C loci, sex and their interactions on forewing length. The
data for both phenotypes (Table 1) and genotypes (Table 4) are far from
orthogonal so that a standard 4-way analysis for sex and the three loci, using all
the data, is impossible. Alternative methods are used (Snedecor & Cochran,
19671, two of which necessarily involve the sacrifice of some data.
First, I use unweighted class means (i.e. disregarding the size of and variance
within classes). Of the 27 genotypes possible, four are missing in both sexes and
an additional one in males. Thus, a single analysis including all A and B
genotypes is impossible. Secondly, a random sample of individuals from each
genotype is taken, using the maximum replication which the data will permit for
an orthogonal analysis. With the missing A/B genotypes, it is possible to analyse
only A with C or B with C. Finally, one-way analyses and calculation of least
significant differences (LSD) use all the data.
The population sample
Some aspects of the population genetics of D . chrysippus at Dar es Salaam can
be deduced from a larger sample of butterflies from which 151 broods were
obtained between July 1972 and August 1975. Thirty broods gave no adult
offspring due to virus disease ( 1 7 ) , parasitism (12) and ant predation (1). Of the
remaining 12 1, 36 are excluded because the female parent was not wild and 24
because the full genotype of both parents was unknown. There are several
reasons for the latter including small broods (9), male parent not seen (4),double
mating (1) and my failure to score the B locus" in early broods (10). Sixty-one
pairs remain for which a full B/C genotype can be deduced and 66 pairs can be
used for the A locus analysis.
Scoring the genotypes
Before presenting the results, the reader needs reassurance that the problems
involved in distinguishing genotypes, when heterozygotes are not always
phenotypically distinct from homozygotes, have been solved. In ten broods
where both parents were dorippus (CC or Cc) and there was no segregation for
aeoptius (cc) in the progeny, there is only one case where transiens (the Cc
phenotype) is absent from both parents and offspring. This brood is assumed to
* 1 first recognized the B locus in a backcross (brood 281 in February 1973, independently of Clarke el ~ l .
(1973)whose paper was not then published. Many of the earlier broods could be interpreted retrospectively as
they had been preserved zn lolo.
GENETICS OF POLYMORPHISM IN DANAUS
93
Table 1. Mean forewing length (mm)with sample size (in parentheses), classified
by colour phenotype, males in roman type and females in italics
6 locus
C locus
A locus
A-
3-
C-
cc
CC
bh
C-
Aa
42.60 (42)
43.33 (6)
42.29 (17)
42.58 (65)
41.63 ( 3 0 )
4 2 . 0 0 (2)
42.50 (14)
41.91 ( 4 6 )
41.49 (49)
43.60 (20)
41.25 (53)
43.50 ( 4 )
41.85 (61)
40.21 (56)
40.81 ( I 13)
41.46 (11)
39.50 ( 4 )
40.94 ( 1 5 0 )
41.47 (70)
42.75 (24)
40.83 (126)
41.95 (20)
40.42 (26)
4 0 . 7 9 (43)
cc
41.50
(6)
41.89 (26)
CC
42.00
(1)
41.60 ( I S )
Total
Total
aa
41.98 (130)
41.76(105)
43.80 15)
(8)
45.00 (6)
41.88 (481
41.52 (79)
40.79 (68)
40.96 (119)
43.00
(1)
43.23 1131
433!
43.00
(3)
42.13 (32)
43.25
(3)
43.50 (2)
41.50
(4)
42.14
41.60 ( 5 )
42.00
(I)
41.62 (21)
(7)
41.58 (194)
43.40 (63)
41.88 (1421
41.97 (399)
41.11 (293)
4 2 . 4 1 (42)
40.75 ( 1 4 6 )
41.17 ( 4 8 1 )
result from a CC x CC cross. In the other nine crosses, known to be CC x Cc, the
heterozygous parent is identifiable from its phenotype in seven of them and in
the remaining two it is arbitrarily assigned, one to each sex.
The B locus presents greater problems. In 15 broods where both parents are
brown ( E B or Eb) and there are no orange (bb)segregants in the progeny, in every
case at least one of the parents or offpsring has orange on the hindwing, the
trailing edge of the forewing, or both. As this condition is always common in
backcross broods, where brown segregants must be Bb, such crosses are all
scored as BB x Bb. In 11 of these cases, all of which must be either Ec/bC x Bc/Bc
or B C / B c x Bc/bc as they are backcrosses at the C locus, neither parent has any
orange (some males were not seen) and the heterozygous one cannot, therefore,
be determined by inspection. However, in every case, the traces of orange
occurred in the dorippus but not the aegyptius offspring, proving the first
alternative to be correct. Thus, the dorippus parent is the carrier of the 6 allele in
all these broods.
In crosses between double heterozygotes, coupling x coupling and
repulsion x repulsion phase broods are easily distinguished as the former gives
an orange aegyptius class W b c ) in F1 (Smith, 1975a).Coupling x repulsion broods
are identified by the occurrence of orange transiens (bC/bc), which is rarely
produced by crossing-over in repulsion x repulsion crosses, and a lower than
expected proportion ofbrown transiens in F1. In the four crosses of this type, it is
impossible to match parental sex with linkage phase and they are arbitrarily
assigned, two to each sex, for the sex comparison. As the BC chromosome was
otherwise recorded only in males, it is possible that it does not occur in females.
These comments show that small errors are expected in the sex comparison
which do not affect the combined estimates of gene, chromosome or genotype
frequency.
94
D. A. S . SMITH
BREEDING RESULTS
T h e classification and analysis of phenotypes (Tables 1, 2) show that F is
significant for sex (males average 0.8 mm larger than females), the A and C loci
and for the interaction between the B and C loci. Examination of the phenotype
means (Table 3) shows that the effect at both A and C loci is due to the superior
size of the heterozygotes. AS the A/C interaction is not significant,' the separate
effects are likely to be additive. The nature of the significant B/C interaction is
unknown at this stage as BB and Bb butterflies are not distinguished. The use of
the two sexes to provide replication of phenotype classes is justified as their ranks
are significantly correlated (7=0.672; P < 0.001) by Kendall's method (Kendall,
1962).
Table 4 shows the reclassification of the butterflies using the genetic evidence.
The 3-way variance analysis of a random sample for the A and C loci and sex
(Table 5 ) may suffer interference from the B locus but the risk is minimized by
random allocation of the available B genotypes among the different A/C
genotypes. Heterosis at the A and C loci and the sex difference are again
significant. The small mean square for A/C interaction supports the inference
above (Table 2) that the effect is additive rather than epistatic. The first order
interactions are not significant but the significant value for the second order
Table 2. Three-way analysis of variance for the A, B and C loci, using the cell
mean values in Table 1
Source of
variation
Sums of
squares
Degrees of
freedom
Mean
square
Replicates (sexes)
A locus
B locus
c locus
A/B interaction
A/C interaction
B/C interaction
A/B/C interaction
Error
Total
3.560
20.53 1
1.047
4.157
1.155
2.340
5.912
3.903
6.288
46.892
I
2
1
2
2
4
2
4
17
35
3.560
10.266
1.047
2.079
0.578
0.585
1.956
0.976
0.370
F ratio
9.62.'
21.15***
2.85
5.62'
1.56
1.58
5.29.
2.64
" P <0.05, * ' P < O . O l , **'P<O.OOl and insubsequent tables.
Table 3. Forewing length (mm) at the A, B and C loci for the sexes combined,
averaged over the cell means in Table 1
Locus
A
C
B
Phenotypes
Homozygous
dominant
Heterozygote
Homozygous
recessive
4 1.46
42.11
43.13
42.47
4 1.62
4 I .64
41.90
42.24
GENETICS OF POLYMORPHISM IN D A N A U S
95
interaction undoubtedly reflects the fact that that double heterozygote AaCc
shows marked heterosis in the male but not in the female.
Table 6 summarizes three separate analyses for sex and the B and C loci.
Interference from the A locus is minimized either by random selection from the
A genotypes available (3-way analysis) or by using the cell means (Table 41, both
unweighted and weighted over the A genotypes. There is fair agreement between
the three methods: the C locus is significant in each case and the B locus, the B/C
interaction and sex in two of the three. The results suggest that there is heterosis at
both B and C loci with epistatic interaction between them.
Table 4. Mean forewing length (mm)with sample size (in parentheses), classified
by genotype, males in roman type and females in italics
B locus
C locus
A locus
Aa
A-
~
B-
Bb
~~~~
-
(3)
41.00 (1)
42.00 (2)
-
41.00
40.20
cc
41.89 (18)
41.86 (14)
43.32 (25)
43.21 (24)
-
42.72 (43)
42.71 (38)
cc
41.04 (48)
40.69 (87)
42.88 (33)
41.84 (32)
C-
43 06 (16)
41.09 (11)
43.33 (6)
4200 ( I )
-
43 14 (22)
41 1 7 (12)
Cr
41.68
40.54
42.70
4136
(28)
43.58 (12)
42.86 (7)
41 90 (76)
40 69 (70)
4280 ( 5 )
-
42.02 (1 16)
40 80 (103)
42 70 (10)
4 1 81 (16)
C-
CC
bb
Total
aa
41.00
39.00
(3)
(26)
(10)
(11)
41.46
38.25
(11)
(4)
(4)
(5)
41.75 (92)
40.91 (123)
C-
39.95 (20)
39.96 (23)
43.10 (10)
43.43 (7)
41.88 (501
40.80 (70)
41.55 (80)
40.79 (100)
Cr
41.44
(9)
42.05 (38)
44.71 ( 7 )
43.86 (7)
43.00
43.00
(1)
(3)
42.88 (17)
42.38 (48)
cc
42.00
(1)
41.60 (15)
43.50 (2)
41.60 ( 5 )
41.50
42.00
(4)
(I)
42.14
(7)
41.62 (21)
41.46 (153)
40.99 (228)
43.26 (96)
42.61 (90)
41.85 (142)
40.73 (148)
42.05 (391)
41.22 (466)
Total
Table 5. Three-way analysis of variance for the A and C loci and sex with five
replicates per treatment ( N = 9 0 )
Source of
variation
Replicates
A locus
C locus
Sex
A/C interaction
A/sex interaction
C/sex interaction
AICIsex interaction
Error
Total
Sums of
squares
6.51
70.56
31.62
9.34
6.84
12.29
2.02
28.44
169.49
337.12
Degrees of
freedom
Mean
square
4
2
1.63
35.28
15.81
9.34
1.7 I
2
1
4
2
2
4
68
89
6.15
1.01
7.11
2.49
-
F ratio
0.65
14.16"
6.35".
3.75'
0.69
2.47
0.41
2.85*
-
Table 6. Variance analyses for the B and C loci and sex
Source of
variation
Replications
B locus
c locus
Sex
B/C interaction
B/sex interaction
C/sex interaction
B/C/sex interaction
Error
Total
2-way analyses?
3-way analysis
Unweighted means
Degrees
of
freedom
Surnsof
squares
Mean
square
F
ratio
3
2
2
1
4
2
2
4
51
71
0.78
17.03
21.86
0.50
7.89
1.75
I .08
3.66
133.22
187.77
0.26
8.52
10.93
0.50
1.97
0.88
0.54
0.92
2.61
0.10
3.26'
4.19.
0.19
0.76
(1
2
2
1
0.34
-
0.21
0.35
-
-
t In the 2-way analyses, the sexes are the replicates.
Degrees
of
freedom
Sumsof
squares
Mean
square
3.14
0.54
2.68
3.14
4.93
3.14
0.27
1.54
3.14
1.23
-
-
-
8
17
1.17
12.46
0.15
4
-
Weighted means
F
ratio
21.50
1.86
9.16**
21.50"'
8.45.'
-
Sumsof
squares
Mean
square
2.23
2.26
2.80
2.23
3.69
2.23
1.13
1.40
2.23
0.92
-
-
1.37
12.34
-
0.17
-
F
ratio
13.00)
6.60'
8.16'
13.00*'
5.39'
-
-
P
?
?
GENETICS OF POLYMORPHISM I N D A N A U S
97
Finally, one-way variance analyses make use of all the genotype data. At the A
locus (Table 71, the heterozygote is very significantly larger than both
homozygotes in both sexes and positive heterosis is beyond dispute.
The B/C interaction indicated in Table 6 is analysed in Table 8. One-way
analysis of variance for the nine genotypes, based on all the data for the
combined sexes, shows that there are highly significant size differences.
Individual comparisons are made by the LSD method, which involves in this case
calculating a parameter No to correct for unequal sample sizes (Snedecor. &
Cochran, 1967). The comparisons made are between the heterozygote (at one or
both loci) and the two homozygotes in each of the eight arrays, three rows
(R1-3),three columns (Cl-3) and two diagonals (D1-2), i.e. 16 comparisons in
all.
Examination of rows shows that, when the B locus does not segregate, there is
positive heterosis at the C locus in R1 and R3 and negative heterosis in R2, the
Table 7. One-way analysis of variance for A locus genotypes
F ratio
R (mm)
and [ N )for genotypes
Sax
d
P
A-
Aa
aa
41.46(153)
40.99 1228)
43.26 (96)
42.6 1 190)
41.85 1142)
40.73 (148)
29.69 * * *
28.26***
Table 8 . Mean forewing length (mm) (in heavy type) and sample sizes (in
parenthesis) for the B/C genotypes (sexes combined) which would segregate in F2
from monohybrid crosses at the B locus (columns), the C locus (rows) and
dihybrid crosses in coupling and repulsion (diagonals)
Pairntal genotypes, crosses giving F2 results and array number (C, D or R )
BC/bc x
BC/bc
D1
BC/Bc x
BC/Bc
BC/bC x
BC/bC
BC/bC x
Bc/bc
Bdbc x
Bdbc
Bc/bC x
Bc/bC
c1
c2
c3
D2
B B CC
BB Cc
B B cr
40.556
42.716
41.270
R1
(9)
(81)
(215)
BC/Bc x
bC/bc
Bb CC
Bb Cc
Bb cc
42.441
41.438
42.154
***
***
cc < c c > cc
R1
**
(I
CC > cc < cc
R2
R2
(34)
(219)
(26)
bC/bc x
bC/bc
bb CC
bb Cc
bb cc
41.128
42.508
41.750
cc < Cc> cc
R3
( 1 80)
(65)
(28)
R3
*** **
***
*** * * *
***
.**
Bc/Bc =Bc/bC
=bC/bC
B B < B b > bb
B B > Bb < bb
D2
c1
C2
BC/BC
tf
B B < Bb=bb
= bc/bc
cs
~
< BC/bc
D1
~~
~~
Type of heternsis (ifany),level of probability (asterisked)and array
One-way variance analysis for the table gives F=8.428, d.f. 8/848, P<O.OOl. Critical LSD levels are 0.598 mm
f n r P < 0 . 0 5 ( ' ) , 0 . 7 8 6 m m f o r P < 0 . 0 1(**)and1.004mmforP<0.001(***).
98
D . A. S. SMITH
only array with the double heterozygote. The column results (C locus not
segregating) are similar. C1 shows positive heterosis, C3 has the heterozygote
superior to the dominant homozygote only but there is again negative heterosis
in the C2 array which includes double heterozygotes. These results show that, in
monohybrid crosses, 7/8 comparisons of single locus heterozygotes with their
homozygous segregants are positive whereas 4/4 comparisons between two locus
heterozygotes and their single locus segregants are negative. Comparing the
distribution of negative and positive differences between the two types of cross by
Fisher’s Exact Test gives P= 0.0000 1.
Examination of the dihybrid crosses on the two diagonals shows no heterosis.
On D 1 (coupling x coupling crosses), the homozygous dominant for both loci is
markedly inferior, as it is to all other genotypes, but the homozygous double
recessive is not. On D2 (repulsion x repulsion crosses), the three genotypes are
very similar in size and it is the only array in which no significant differences
occur (positive heterosis occurs in males only).
Comparison by the Exact Test of the 11/12 significant differences (in both
directions) in the monohybrid crosses with only 1/4 in dihybrid crosses gives
P= 0.027. Finally, comparing positive and negative significant deviations
between all arrays producing single locus heterozygotes ( R l , R3, C1, C3) with
those giving double heterozygotes (R2, C2, D1, D2) gives P = O . O l by the Exact
Test.
Table 9. Mean forewing length in F1 from different types of cross and in F2
assuming random mating among the F1 genotypes
Type of cross
I
I1
111
V
Deviation
from meant
F1
F2
41.26
42.94.
42.10
+1.18
+0.34
41.24
40.89
40.97
41.25
41.61
41.53
4 1.44
42.16*
42.72+
41.44
42.26.
42.63.
41.33
41.53
41.84
4 1.33
41.93
42.08
-0.32
+0.40
-0.43
-0.23
+0.96
+0.08
-0.32
+0.50
+0.87
-0.43
+0.17
+0.32
42.66
42.21
42.40
42.44
42.45
42.49
41.81
42.03
41.84
41.72
42.05
41.93
41.76
41.76
41.82
41.70
42.02
41.91
+0.05
+0.27
+0.08
-0.04
+0.29
+O. 1 7
0.00
0.00
+0.06
-0.06
+0.26
+0.15
x bC/bc
x Bdbc
42.72
42.16
42.63
42.26
41.84
41.52
42.08
4 1.93
41.84
41.52
42.08
4 1.93
+0.08
-0.24
+0.32
+0.17
+0.08
-0.24
+0.32
+O. 1 7
BC/bc x BC/bc
BdbC x Bc/bC
BC/bc x BdbC
4 1.44
41.44
41.44
4 1.33
41.33
42.44’
41.33
4 1.33
41.76
-0.43
-0.43
+0.68
-0.43
-0.43
A A x aa
BC/BC
BC/BC
BC/BC
BdBc
Bc/Bc
bC/bC
x bdbc
bC/bC
x Bc/Bc
x bC/bC
X bc/bc
x bc/bc
X
BC/Bc
BC/bC
BC/bC
BC/bC
x bC/bc
BC/Bc
BC/bC
bC/bc
Bc/bc
x BC/Bc
x BC/bC
x Bc/bc
x bC/bc
x BC/Bc
B d b c x bC/bc
BC/Bc x Bc/bc
IV
ic for forewing length (mm)
Parental
F1
F2
genotypes
genotypes
genotypes
0.00
’ Heterosis statistically significant. t Mean of all classes (sexes combined) is 41.76 mm. A wild population
mean, weighted according to the frequency of genotypes, would be substantially less.
GENETICS OF POLYMORPHISM IN DANAC’S
99
The major conclusions from the data in Table 8 are as follows : ( 1 ) Single locus
heterozygotes show positive heterosis compared with the homozygotes from
which they segregate. ( 2 ) Double heterozygotes show negative heterosis
compared with all single locus heterozygotes in monohybrid crosses but no
heterosis in dihybrid crosses. (3)The double heterozygotes in repulsion produce
an F2 close to the overall mean with minimal variance: stabilizing selection for
body size will thus favour the BdbC genotype.
The average effect of parental genotype on size in the F1 and F2 (sexes
combined) are given in Table 9. In homotypic crosses between homozygotes
(groups 1-11] there is always heterosis in F1 except when double heterozygotes
are produced (crosses BC/BC x bdbc and Bc/Bc x bC/bC. Assuming random mating
among the F1, there is regression to the mean in F2. Heterogametic (group 111)
and homogametic (group IV)crosses between single heterozygotes, the genotypes
showing maximum heterosis in F1, show regression to the mean over one or two
generations. Homogametic coupling and repulsion crosses (group V) both
produce an F1 averaging slightly less than the parents on account of the
homozygous segregants. The coupling x repulsion cross produces a 1 : 1 : 1 : I
segregation for all four single heterozygotes (BC/Bc, Bdbc, BC/bC and bC/bc) with
strong heterosis in F1 followed by sharp regression to the mean in F2. These
results show that epistasis between the B and C loci produces a balanced double
heterozygote, intermediate in size between the smaller double homozygotes and
the larger single heterozygotes (Table 10). Although there are sex differences, the
genotypes are significantly correlated by rank (7 =0.667 ; P=0.013).
I t is important to realize that the heterotic effects at all three loci are probably
greater than the analysis shows as each dominant class contains a proportion of
heterozygotes which must reduce any difference between the means of the two
classes. Thus, all the inferences are likely to err in the direction of caution.
Table 10. Rankings for forewing length (mm)of the ten B/C genotypes in the two
sexes
Genotype
BUbC
bC/bc
EUBc
Ec/bc
bc/hc
Bc/bC
BC/bc
BdEc
bC/bC
BC/BC
Mean of all genotypes
Males
Mean forewing
length
Rank
Females
Mean forewing
length
43.14
42.88
42.12
42.70
42.14
1
2
3
4
5
41.17
42.38
42.7 1
41.81
4 1.62
42.02
6
40.80
41.75
4 1.55
41.00
42.05
1
8
9
40.91
40.79
40.20
4 1.22
-
Rank
POPULATION GENETICS
Randomness $mating
There are ten different B/C genotypes of which nine occurred in wild male and
seven in wild female parents. Thus, 100 different pair combinations are possible
D . A. S. SMITH
100
of which 31 occurred in my sample of 61 wild mated pairs giving offspring.
Expected frequencies of the various pairings were calculated in a detailed matrix
from the genotype frequencies within each sex. They suggest that mating is
random. Much the commonest pairings, as expected from the high frequencies
of the genotypes, are Bc/bC x Bc/bC ( 10) and BdbC male x B d B c female (10).Of the
remaining 29 pair combinations, none has above three occurrences. As most
expectations are small, the null hypothesis that mating is random can be tested
only by combining data. The following results of 2 x 2 xz tests were obtained for
the B and C loci, both of which support random mating: brown (B-) v. orange
( b b ) ; ~ f = l . 0l ;. 3 > P > 0 . 2 : dorippus(C-) u. aegyptius (cc);x:=1.5; 0 . 3 > P > 0 . 2 .
Sex
ratio
At the B locus, the two alleles are of equal frequency in the sexes Cx!=0.4;
0.7 > P > 0.5) but at the C locus the C allele is more frequent in males (48.4%) than
females (32.8%).Therefore, male dorippus are more frequent (82%)than female
(57.4%)(x:= 7.6; P > 0.01). It follows that mating frequencies are significantly
different from expectation on a null hypothesis that the genes have equal
frequency in the two sexes (Table 11).The departure from expectation is main1
due to the heterotypic pairings which make the largest contribution to x .
Although mating is random, as there is a surplus of dorippus males and mgyptius
females unable to find mates of their own kind, hybridization and thus gene
exchange between the lines, is an enforced consequence of the sex-ratio within
each morph. This result does not invalidate earlier findings of non-random
mating and sexual selection (Smith, 1973a, 1975~)
as these are strictly seasonal
phenomena.
Furthermore, it is clear from Table 12 that there is a genetic component to the
sex-ratio imbalance. In backcross broods at the C locus, the sex-ratio is
J
Table 1 1 . Mating frequencies at the C locus tested against the null hypothesis that
allele frequencies are identical in the two sexes
Male genotype
Ccc
19
12.9
2.904
CC31
Female genotype
Pairs observed
Pairs expected
Contributions to,$
29.6
0.063
cc
cc
C4
12.9
6.125
CC
7
5.6
0.350
Totals
-
61
61
9.4429
(3 d.f.)
Table 12. Comparison of the sex ratios obtained from three types of cross
Number
of broods
lot
4
10tt
Parental genotyues
Offspring
d
9
6
9
N
X2
Bc/bC
BdBc
BdbC
BdBc
Bc/bC
Bc/bC
39
65
99
72
58
65
111
9.81 1"
0.398
7.049"
123
164
t There is heterogeneity, 4 of the broods being either all-female o r significantly biased to females.
tt There is heterogeneity, 3 of the broods being significantly biased to males and the others normal.
GENETICS OF. POLYMORPHISM IN D A N A ( / S
101
significantly biassed towards females if the female parent is aegyptius (Bc/Bc) but
not if it is dorippus ( B d b C ) ; on the other hand, the dihybrid crosses give a
significant excess of males. Thus, the female parental genotype affects the sexratio (Smith, 1975b, 1976b1, the genetic control of which will be discussed in
another paper. As there is seasonal selection which favours dorippus in the dry
season and aegyptius in the wet (Smith, 1975b), the sex-ratio also changes
dramatically being dominated by males in the former and females in the latter
season. Comparing the BdbC male x B d B c female with the dihybrid crosses
(Table 12), which together comprised 33% of the wild matings giving progeny, it
is easy to see how a seasonal shift in the selective value of the two female
genotypes will automatically alter the sex-ratio.
Hardy- Weinberg equilibrium
When the B and C genotypes are examined together (Table 131, the double
heterozygotes exceed expectation and all other genotypes except bdbc are rarer
than predicted (x:= 14.4; P<O.O5). On the other hand, the heterozygotes at
neither the B nor the C locus alone are in significant excess. This suggests the
possibility of heterozygous advantage dependent on epistatic interaction between
the two loci.
As expected, there is a significant sex difference in chromosome frequencies
(Table 14): the BC and bC chromosomes are commoner in males and the Bc and
bc chromosomes in females (xi=8.4; P < 0.05). The excess of repulsion (77.5%)
over coupling chromosomes is very highly significant (xi= 82.3 ; P < 0.00 11,
Table 13. Goodness of fit to Hardy-Weinberg ratios for B/C genotypes in 6 1 wild
mated pairs
Geriotype
BC/BC
BC/Bc
BC/bC
BC/bc
BdbC
Number
observed
expected
0
2
2
50
6
4.10
o'21
3.65
3;:;;
1
1
X2
1.970
8.123
Genotype
Number
obselved
expected
I7
Bc/Bc
Bc/bc
bC/bC
bC/bc
bc/bc
Total
14
12
I3
6
122
20.51
18.41
16.25
16.39
4.13
122.00
X*
0.601
1.056
1.112
0.701
0.84 7
14.410'
Chi-omosome frequencies: EC=0.041, Ec=0.4 10, bC=0.365, bc=0.184.
Gene frequencies: B=0.451, b=0.549, C=0.406. c=0.594.
Table 14. Number of coupling and repulsion phase chromosomes in a sample of
80 male and 64 female wild butterflies
Chromosome
Males
observed
BC
BC
bC
bc
Totals
11
59
64
26
160
~~~~~
xt3,for the sex difference=8.397;
Females
expected
7.2
65.0
57.8
30.0
160.0
~~
Totals
observed
expected
2
58
40
28
I28
5.8
52.0
46.2
24.0
128.0
~
P<0.05 by Brandt & Snedecor's formula.
13
117
104
54
288
D. A. S . SMITH
I02
indicating gametic excess (Turner, 1967 1 or linkage disequilibrium. From the
data in Table 14, the gametic determinant D is -0.142 (maximum_+0.25,
equilibrium zero). D’ (Lewontin, 1964) is - 0.7 76 (maximum k 1, equilibrium
zero) (see Appendix). Linkage disequilibrium of this magnitude must be
maintained by both restricted recombination (close linkage) and strong selection.
In particular, there must be powerful selection against the coupling
chromosomes as 1.9% recombination in each generation is sufficient, in the
absence of selection, to establish linkage equilibrium quite rapidly (Lewontin 8c
Kojima, 1960; Bodmer 8c Parsons, 1962; Arunachalam 8c Owen, 197 11, especially
as there are 12 overlapping generations a year.
The A Locus
Accurate scoring of the A alleles is impossible in field specimens. If traces of
white are visible on the hindwing, the a allele is known to be present in the
heterozygous state but absence of white does not imply the reverse. Moreover,
traces of white scaling, which can be scored with confidence in freshly emerged
butterflies, would be undetectable in even slightly worn wild specimens.
Variation in the expressivity of a in heterozygotes, due both to sex and the
modifying action of the B alleles, further complicates the task.
In the 66 wild matings from which five or more offspring were obtained, one
Aa parent is inferred if white scaling is detectable on either parent or any of the
offspring. The data in Table 15 do not indicate heterozygous advantage: indeed
the observed values are a good fit with the Hardy-Weinberg expectations.
However, the frequency of aa butterflies in the sample is approximately three
times their known field frequency (1.2%) and the fit is, therefore, probably
fortuitous.
Table 15. Goodness of fit to Hardy-Weinberg ratio for A locus genotypes in 66
wild mated pairs
Genotype
Observed ( N )
Expected ( N )
X2
AA
110
107.4
0.063
Aa
aa
Totals
18
23.3
4
1.3
132
132.0
0.338
0.275
DISCUSS1 ON
Danaus chrysippus is probably the most abundant and widespread of all tropical
butterflies. It also supports the greatest array of mimics, both Batesian and
Mullerian, of any butterfly: the list in Africa alone embraces, on a conservative
estimate, some 30 species from the families Nymphalidae (12), Acraeidae
(numerous), Hypsidae (several), Papilionidae (1) and Lycaenidae (1 (Rothschild,
Von Euw, Reichstein, Smith & Pierre, 1975: pl. 1). It is not unusual to find half a
dozen co-mimics flying together. Although the combination of unconcealed lifestyle and aposematic colouration have convinced most observers that D .
chryszppus is distasteful to predators, hard evidence was until recently somewhat
scanty. Swynnerton (1915) was the first to observe birds vomit after swallowing
African Queens. However, the chemical weaponry, which is the mainstay of the
species’ defensive system, is a comparatively recent discovery.
GENETICS OF POLYMORPHISM IN DANAUS
103
Two distinct classes of deterrent plant products are known to be stored by the
adult insects : (1) cardiac glycosides (cardenolides),which are well known for their
cardioactive, emetic and noxious properties, are sequestered by larvae from their
foodplants, mainly members of the Asclepiadaceae (milkweeds), and
subsequently stored in all stages of the life-cycle (Rothschild et al., 1975; Brower,
Edmunds 8c Moffitt, 1975); (2) pyrrolizidine alkaloids are ingested, mainly by
males, from the dead or dying parts of plants from several families including the
Boraginaceae, Compositae and Leguminosae. These alkaloids, which are potent
hepatotoxins and often lethal to domestic stock (Bull, Culvenor & Dick, 1968),
are also known to be distasteful to some predators when stored in their prey (T.
Eisner, in lztt.). Male danaids of many species metabolize the alkaloids into a
component of the aphrodisiac pheromone, without which courtship is
unsuccessful (Schneider, 1975; Schneider et al., 1975; Meinwald et al., 1974):
however, Edgar, Cockrum & Frahn (1976)found that adult D.chrysippus, of both
sexes but principally males, store pyrrolizidine alkaloids unaltered in their
tissues. This means that despite the fact that D. chrysippus larvae may feed on
milkweeds not containing cardenolides (in some populations predominantly
so-Brower et al., 19751, adult males might, nevertheless, be distasteful due to
their alkaloid content (Boppri., 1978). In East Africa, a high proportion of both
sexes may contain deterrent amounts of cardenolide (Rothschild et al., 1975)and
the males are presumably doubly protected by the presence of the alkaloid in
addition. Overall, there are no longer grounds for doubting that D . chrysippus is a
well protected species, even in areas such as West Africa, where cardenolide
storage is often dispensed with.
The colour polymorphisms of D.chrysippus in East Africa have been described
by Owen & Chanter (1968) and Smith (197313, 1975a, 1976a). That a widely
mimicked and proven unpalatable species should also be highly polymorphic
constitutes something of a puzzle. The function of the aposematic life-style is
surely to convey to experienced predators an image easily perceived from afar
and instantly associated with an unpleasant experience; and one which is,
moreover, frequently reinforced to the mutual benefit of predators and prey.
While accepting that avian predators may generalize images (Duncan &
Sheppard, 1965), economy in their education is probably jeopardized if they
must learn to avoid as many as eight distinct phenotypes (excluding intermediates) such as occur in D.ch7ysippus around Dar es Salaam (Figs 1-16). The
rare phenotypes are likely to be particularly at risk as few predators will have
experienced them.
Owen & Chanter ( 1968) attribute the polymorphism to the disadvantage
attached to a model which supports an excessive load of Batesian (edible)mimics.
They argue that mutations which alter the appearance of the model, to an extent
that weakens mimetic resemblance, may be beneficial to the possessor by
allowing it to escape (in the evolutionary sense) from its mimics. Thus, selection
pressure favouring diversity may lead to a stable polymorphism maintained by
frequency-dependent selection. A second possibility is the acquisition of
heterozygous advantage, allowing the mutant alleles to increase in frequency to
an extent not wholly determined by the pressures of mimicry and predation. This
explanation is favoured by Ford (197 1) without facts to support it. Thirdly, the
mutants might increase through other advantages, such as superior adaptation to
part of a diverse habitat or to a particular seasonal climate, and become in time
7
104
D. A. S SMITH
sufficiently common to be established as distinct models. There is strong
evidence for seasonal changes in the selective value of forms uegyptius and dorippus
in Tanzania (Smith, 1975b). Other possibilities include frequency-dependent
selection in response to Miillerian mimics (Smith, 1976a), and density-dependent
selection (Smith, 1975b,c). Should Ford’s suggestion prove correct, none of the
other possibilities is thereby invalidated: on the contrary, the stability of the
polymorphism is guaranteed.
The study of size, which is, in D. chrysippus, at least partly a pleiotropic effect of
the major colour genes, provides some important clues to the nature of the
polymorphism. Assuming that size in a butterfly is not selectively neutral, there
must be selection for an optimum size, or perhaps for several optima, which
might differ between sexes and phenotypes, from season to season or within
parts of a diverse habitat. The breeding results show that mean size does indeed
vary with sex and phenotype but the possibility of seasonal or habitat variation
has not been studied. Environmental factors which might select for size in the
African Queen are to some extent speculative as none has been positively
identified.
It is generally easier to visualize or demonstrate factors favouring large size
than small (Cook & O’Donald, 197 1). Factors conducive to large size in females
are enhanced reproductive output and heightened visual attractiveness to males
(Crane, 1955, 1957). Large males may achieve, through faster flight, a
competitive edge in seeking out mates. In aposematic species especially, large size
improves the individual’s chances of being recognized by an experienced
predator at a distance sufficient to prevent its being molested. It is noteworthy
that the male is the larger sex in most danaids as it is arguably the more generally
distasteful one (Edgar et al., 1976). In populations not storing cardenolides,
where males may be protected mainly by pyrrolizidine alkaloids, females are, in
effect, potentially their Batesian mimics.
On the other hand, small genotypes are preadapted to food shortage which is
experienced by butterflies in the tropical dry season when both larval foodplant
and nectar sources for adults are relatively scarce. Also, Rothschild (1971) has
pointed out that the less distasteful species of a pair of Mullerian mimics is
expected to be the smaller. At Dar es Salaam, both the uegyptius and dorippus
forms of D . chrysippus are respective co-mimics of forms acedon and dairu of
Acrueu encedon, a smaller species which is thought to be well protected by a
noxious secretion of yellow fluid, containing HCN, released from a thoracic
gland. As female D. chrysippus may contain neither cardenolides nor alkaloids,
the species as a whole is possibly the less effectively protected of the pair, a factor
which could favour small size, particularly in females, where they are syrnpatric.
The breeding results show that the mean size of BdbC males is almost identical
with the overall mean although in Bc/bC females it is somewhat (but not
significantly) below (Table 10). The excess of heterozygotes in this multilocus
system might indicate heterozygous advantage if there is also stabilizing selection
for size as seems likely to be the case. Stabilizing selection would also embrace the
genotypes produced from the commonest crosses, BdbC x BdbC and BdbC
male x BdBc female (Tables 8,9). The three genotypes from a dihybrid
repulsion x repulsion cross, Bc/Bc, BdbC and bC/bC, are of almost identical size in
females although in males the double heterozygote shows significant heterosis. In
contrast, a dihybrid cross between coupling phases produces unbalanced
GENETICS OF POLYMORPHISM I N D A N A U S
105
progeny including the two extreme genotypes, B U B C (small) and W b c (large).
Assuming no position effect (differences of viability or phenotype between
coupling and repulsion phases), the repulsion phase will be selected for its more
balanced progeny, thus creating linkage disequilibrium. In other words, the
breeding results show that the two pairings found to have the highest frequencies
in the wild population both produce progenies with an array of genotypes giving
a good approximation to the population mean size and a low brood variance.
Inbreeding between double homozygotes will always depress the mean whereas
all types of outcross will produce a heterotic F1 followed by regression to the
mean under random mating in F2. Protogyny and wide scattering of the eggs by
laying females both tend to enforce out-breeding. Thus, the genetic architecture
of the wild population is superbly adapted to the maintenance of the average,
balanced phenotype, in the manner predicted for two linked loci by Mather
(1973). Moreover, the flow of variability from the potential to the free state
(Mather, 1973) is stemmed by the restriction of recombination, which produces
coupling chromosomes, to about 4% in males and probably zero in females.
The B/C polymorphism in D. chvsippus gives impressive support in general
terms to the model for two loci proposed by Turner (1967). In particular, it
agrees with his prediction that close linkage, with epistasis and linkage disequilibrium, would be expected. On the other hand, the African Queen
population at Dar es Salaam has an extraordinarily complex population genetics
which is known to include many factors invariably omitted from non-verbal
models in the interest of tractable mathematics. Such factors include at least the
following (Smith, 1973a,b, 1975a,b,c, 1976a,b): ( 1 ) overlapping generations; (2)
seasonal non-random mating and sexual selection, affecting both sexes; (3)
deviant sex and morph ratios, probably caused by meiotic drive in the female; (4)
cyclic selection acting on both colour and sex-ratio polymorphisms and possibly
density-dependent ; ( 5 ) possible frequency-dependent selection in response to
both Batesian and Mullerian mimics; (6) different selective values between sexes
within morphs (present paper), suggested by sex differences in gene,
chromosome and genotype frequencies, recombination values, expressivity of
the a gene in heterozygotes, size and heterotic effects; ( 7 ) pleiotropy for size and
colour; ( 8 ) interaction between the linked complex and unlinked loci (e.g. the A
locus). Any of the factors 4-8 alone could underpin a genetic polymorphism and
their relative importance in D.chlysippus is impossible to assess at present. Factors
2-3 are probably parts of the machinery involved in the adjustment of equilibria.
The very marked heterosis at the A locus in both sexes (Table 7 ) is not easily
accounted for. Analysis of the wild parental genotypes (Table 16) gives no
evidence for heterozygous advantage. Moreover, evidence obtained from reared
broods is conflicting. In five monohybrid crosses (broods 60-62, &2,99), which
gave 65 A- and 13 aa offspring, the departure from the expected 3: l,ratio is not
significant Cx!= 2.9; 0.1 > P > 0.05 with no heterogeneity). However, if the Aclass is corrected for expressivity (only 24 being Aa phenotypes), the segregation
is AA 15: Aa 50: aa 13, a significant deviation to the heterozygote from the
expected 1 :2: 1 ratio (xi=6.3; P < 0.05). On the other hand, three backcross
broods (6, 7, 93) gave 36 Aa and 40 ~1 offspring, a result very close to 1: 1
(x:=O.2; 0 . 9 > P > 0.8). Therefore, it is impossible to draw any conclusion from
these results without postulating another interacting factor to account for the
difference between the monohybrid and backcrosses.
I06
D. A. S. SMITH
As all D. chrysippus south of the Sahara and west of Cameroun are homozygous
for a, the mutation probably occurred in West Africa and went to fixation. Yet
the a allele occurs at various lower frequencies, both eastwards across Africa to
the coast, and sporadically in Asia as far east as Sumatra: it has also spread
southwards, in an arc around the northern and eastern fringes of the Congo
basin, to Natal (distribution maps in Rothschild et ul., 1975: 2-4). Although I am
unable to prove heterozygous advantage at the A locus, it is the most likely
explanation for both the heterosis described here and the extensive distribution
of the a allele outside its heartland. Eventually, analysis of extensive field data
from Dar es Salaam may help to resolve this matter.
The substantial proportion of Aa butterflies which are of intermediate
phenotype are poor mimics. In Uganda, Owen & Chanter (1968) have shown that
they are less frequent in the field than predicted from rearing wild larvae, thus
suggesting selection against them. The white hind-winged forms of A . encedon
(forms alcippina and alcippina-dairu), which are Mullerian mimics of the D.
chrysippus aa genotypes, are common around Kampala in Uganda but absent
from Dar es Salaam, where the a allele is rare, and also from most parts of West
Africa where it is fixed. Consequently, Aa phenotypes may suffer disadvantage in
Uganda where they impair Miillerian mimicry with some forms of A . encedon, but
not in other areas, such as the Dar es Salaam region, where the only mimics of
the ua phenotypes are forms of Hypolimnas misippus L. (Smith, 1976a). The
intermediate D. chrysippus phenotype may have the advantage of escaping some
Batesian mimics although intermediate phenotypes are also present in H .
misippus.
Double homozygotes in coupling are rare (Table 14): no proven BC/BC
individual was recorded and bc/bc butterflies from Africa are rare in the field and
in museum collections. Through much of Asia, however, the species is
monomorphic for bc/bc (form=race chrysippttus L.). Examination of the collections
in the British Museum (Natural History) and the University Museum, Oxford,
suggests the b and C alleles have gone to fixation in north-east Africa, especially in
the Somali Arid. On the other hand, in west-central and south-west Africa,
southwards from the Bight of Biafra to the Namib desert (Gabon, Congo
Republic, west Zaire, Angola and Namibia), only the B and c alleles occur. The
causes of the gene biogeography at the present time must be sought in the
Pleistocene history of Africa.
Between 25,000 and 18,000 B.P., there is good evidence for a barrier of
montane vegetation which stretched more or less without a break from the
Ethiopian highlands to Cape Province and Angola, with a western arm reaching
across from Ruwenzori to Mount Cameroun. Both east and west of this barrier,
but particularly in the north-east (Somalia) and the extreme south-west (Namib
Desert), dry conditions seem to have held throughout the Pleistocene (Moreau,
1966). Furthermore, the late Pleistocene history of the Chad basin shows that,
from 22,000 until as recently as 8500 years ago, it was occupied by the vast Lake
Mega-Chad, which was as big as the Caspian, with a northern shore 400 miles
north of the present lake. A contemporary northward extension of the equatorial
rainbelt, by some 300 miles, over the ridge dividing the Congo and Chad basins,
would have produced a continuous belt of evergreen forest extending quite
possibly from the coast to the Tibesti mountains. As both montane and lowland
forest present an impenetrable barrier to D. chrysippus, the importance of these
GENETICS OF POLYMORPHISM IN DANAUS
107
recent and sweeping changes in climate and vegetation for the evolution of the
species can hardly be exaggerated. Moreover, although the primary cause of the
massive extension of grassland and desert in the last 5000 years has undoubtedly
been climatic, the unprecedented impoverishment of the vegetation from human
interference must have greatly accelerated the process. Thus, both the
biogeographical and genetic evidence point to the conclusion that the
polymorphism has originated since the Pleistocene, by hybridization between
three until recently allopatric races, alcippus (aa E- cc) to the west of the Chad
basin, dorippus ( A A bb CC) in the north-east and aegyPtius ( A A BB cc) in the south.
The establishment of heterozygous advantage could account for its persistence. I t
follows that neither Batesian nor Mullerian mimetic relationships are likely to
have had much to d o with the origin of the polymorphism; on the contrary, they
probably result from it. Subsequently, frequency-dependent, density-dependent
and cyclic selection have become important in fixing equilibria, which vary at
each locus throughout the east and central African region and, in the Dar es
Salaam area at least, with the season of the year.
A curious fact emphasized by Rothschild et al. (1975) is the dearth of species
mimicking D . chrysippus form alcippus in West Africa (or, indeed, anywhere else)
where it nevertheless retains the aposematic life-style. The same applies to some
extent to form dorippus which lacks Batesian mimics compared with form
aegyptius in East Africa. In Batesian mimics which are dimorphic ( H . misippus,
Pseudacraea poggei, Mimacraea murshalli, Papilio dardanus), having forms matching
both models, the one mimicking dorippus is always rarer, if present at all, even in
areas where its model is commoner as in Tanzania (Smith, 1976a). These facts
support the idea that the a and C alleles are recent mutations, good mimics of
which have in some cases not yet evolved and in others had insufficient time to
match the geographical range or frequency of their models.
The larger size of single locus heterozygotes compared with both homozygotes
and two locus heterozygotes shows that heterosis and heterozygous advantage (if
it occurs) must be distinct phenomena. The two locus heterozygotes, which are in
excess, exhibit either zero or negative heterosis with respect to their fellow
segregants. Thus, the multiple heterozygotes are of intermediate size as a result of
epistatic interaction between linked loci and additive effects between unlinked
ones and are the average and balanced phenotypes. As cyclic, directional
selection favours the bC chromosome in the dry season and Bc in the wet (Smith
1975b, 1976b, and unpublished data), the BclbC genotype is the reservoir of
potential variation, providing a steady output of Bc/Bc and bC/bC segregants
which are favoured in alternate seasons. The double heterozygote in repulsion is
possibly protected by heterozygous advantage although seasonal (directional)
selection for a dominant gene could alone produce an excess of heterozygotes.
The hybridization between two races monomorphic for Ec and bC, which is
probably post-Pleistocene in origin, would provide the array of genotypes on
which seasonal selection could act. The B and C loci may not initially have been
linked and the present rather close linkage is possibly due to selection.
ACKNOWLEDGEMENTS
I am grateful to Professor A. S. Msangi, Head of the Department of Zoology,
University of Dar es Salaam, for providing the facilities for this research.
D. A. S. SMITH
108
Professor G. C. Varley and Mr R. I. Vane-Wright have been most helpful in
allowing me access to the museum collections in their care.
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APPENDIX
To derive the basic parameters for a system of two loci, each with two alleles, I
follow Lewontin & Kojima (19601, Lewontin (1964) and Turner (1967). The
frequencies of the genes A , B, a, b are q A , q B , ya, 46. The frequencies of the
gametes AB, aB, Ab, ab are v, x, y, z. D , the gametic determinant, is defined as
D = v z - XY
or, the difference between the frequencies of the coupling and repulsion gametes.
If the frequency of each gamete is the product of the frequencies of the genes
composing it, then the alleles at the loci are distributed at random with respect to
each other. In other words, they are in linkage equilibrium and D=O. If D is
positive, the coupling gametes are in excess; if negative, there is an excess of
repulsion gametes. I f all the alleles have a frequency of 0.5, it is easy to show that
the maximum values of D are 0.25 (coupling chromosomes only) and -0.25
(repulsion chromosomes only).
The maximum value D can attain depends on the gene frequencies. The
parameter D’(Lewontin, 1964)is the value of D divided by the maximum value it
can attain at the observed gene frequencies. D’is zero at linkage equilibrium and
otherwise lies in the range f 1.