Download The butterfly Danaus chrysippus (L.) in East Africa

The butterfly Danaus chrysippus (L.) in East
Afiica: polymorphism and morph-ratio clines
within a complex, extensive and dynamic hybrid
zone
DAVID A.S. SMITH F.L.S.
.Natural History Museum, Eton College, Wndsor, Berkshire S1246EW
DENIS,!I OWEN F.L.S.
School of Biological & Molecular Sciences, Oxfbrd Hrookes Universib, Oxford OX3 OBP
IAN J. GORDON F.L.S.
k$epeo Prqject, PO. Box 57, Kilzji, I2qa
NINIAN K. LOWIS
PO. Box 49538, Nairobi, Kerya
Samples of‘the polymorphic buttcrfly Dunnus c.hys$qus arc analysed from six well separated sitrs
in East Africa. Morph-ratio clines are described for four diallelic genes A, B, C: and I., each o f
which influences the visual phenotype. Each of the four clines has a diircrcnt orientation,
consistent with an hypothesis that the polymorphisrri originated from hybridization between a
ii~irnbcrofpolytypic denies which have at various times undergone range expansion. Allopatric
suhsprciation in isolated Pleistocene rcfugia is postulated. Thc phenotype of each geographical
race is shared with one o f the moiphs within the hybrid zonr; other sympatrically miiintained
polymorphic forms arc normally confined to the hybrid zoiic. Wright’s isolation-by-distance
model best explains the prcsent disrribu~iorio f p i c frequencies. Morph-ratios difler significantly
bctwecn the sexes and are sornctiinrs associated with hctcroxygote excess; garnctic and genotypic
disequilihria are general throughout the region and suggest the clines are maintained by strong
natural selection. Seasonal cycling of phenotype frequency is believrd to result from extrnsive
misgrgratory rriovenients rat her than natural selection. Female-biased sex-ratio, which is also
seasonal, and Haldaric rule cffects, result from hybrid breakdown when genetically distinct
drmcs meet and interhreed. Oscillating sex-ratios and frequency ofcolour genes are functionally
(:orrespondciice to: I>r 1). A. S. Smith.
0024-4082/97/05005 I
+ 28 $25 00/O/zj0(10073
51
0 1997 The Linnean Sotircy of 1,mdon
L). A. S. SMI'I'H
52
B'TAL.
linked b y negative feedback. The polymorphism owcs its origin t o allnpatric evolution but is
now mairitaincd sympatrically.
0 l9!17 'I'h1.innean So<irly 1~i'I.i~ndnn
ADI)I'FIONAL KEY WOl<DS:-allop;itry
migration
-
mimicry
~
Pleistocene
~
hcterozygotr excess linkagr disequilibrium
polytypism sex-ratio - wbspeciation.
CONTENTS
I ntroductioti . . . . . . . . . . . . . . . . . . . . . . .
Ivlrthods . . . . . . . . . . . . . . . . . . . . . . . .
'I'he pietical t)asis of polytypisrn and pnlyiiiorphisrn in I). c / i y $ p u . y
. . .
Corriparison of phenotype frequencies at Dar es Salaam, Nairobi and Kaiiipala
Comparison of phenotype fi-eyuciicies among all sitcs . . . . . . . .
'l'hc proposed geographical races of 1). chys$/)us in Africa . . . . . . .
Comparison of rnorph-ratios txtwceii sexes . . . . . . . . . . . .
(;amctic and gznotypic discquilihria . . . . . . . . . . . . . .
Hctcrozygntp cxccss at Nairobi and Dar c s Salaam . . . . . . . . .
Seasonal cycling of sex- and iiiorpti-ratios . . . . . . . . . . . .
Srasnnal variation at Nairobi . . . . . . . . . . . . . . .
Scasonal variation at Dar rs S;ilaani . . . . . . . . . . . .
Discussion
. . . . . . . . . . . . . . . . . . . . . . .
hliniicry . . . . . . . . . . . . . . . . . . . . . .
Hybridization Letwtwi races . . . . . . . . . . . . . . .
Acknowledgements
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Rcfcrrnces . . . . . . . . . . . . . . . . . . . . . . .
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5ti
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ti4
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ti9
72
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73
76
7 (i
The idcas which stimulated this paper can be traced back to a meeting of the
three senior authors in the field in Uganda in 1991 (Smith et nl., 1993). All of us
had been studying various aspects of the ecological genetics of Dnnaus ch:h?y.rippusand
its mimics for many years. Despite the fact that we had published extensivrly, in
numcrous papers over a period of some 25 years, large bodies of' data had languished
in files, either put aside for other work or pigcon-holed because their significance
was not hilly grasped. As we had all lived and worked in East Africa, in different
places and at dilferent times, our data, which were mostly confined to the environs
of the capital cities of Dar es Salaam, Kampala and Nairobi, respectively, had been
collected for difkrent reasons; it was only after joint appraisal, and discussing all
thc data in a regional context, that we became convinced that general statements
could and should be made. Through exchange of ideas and speculation we detected
a number of common themes emerging from our disparate data sets and decided
that a regional synthesis should be attempted. 'this paper represents such an attempt.
The most recent data were acquired as a result of a planned programme to fill in
some of the more obvious gaps in the geo,graphical coverage.
'The predominant population parameters identificd are: ( 1) the existence of four
morph-ratio clines for genes controlling the visual phenotype, each with a different
orientation, within the East African region and beyond; (2) the prevalence of
disturbed sex-ratios throughout the region (Owen & Chanter, 1968; Smith, 1975~1,
1976a; Gordon, 1984)(3)gametic (linkage)disequilibria (Smith, 1 9 8 4 some involving
unlinked genes and thus indicating strong natural selection; (4) unequal morph
frequencies in the two sexes caused by genotypic disequilibria (Smith, 1980, Smith
el al., 1993); (5) unorthodox segregations for colour pattern due to probable nontransmission of chromosomes in both sexes (Smith et nl., 1993); (6) abrupt seasonal
changes in both sex-ratio arid morph-ratio which are probably attributable to cyclic
migration and weather patterns (Smith & Owen, 1997); (7) pervasive heterozygotc
excess and occasional heterozygotc dcficiency; (8) the highest average heterozygosity
.of all danaine samples tested for allozyme variation by Kitching (1985); (9) prezygotic ethological isolation in the form of assortative mate selection (Smith, 1984;
Gordon, 1984). We think that the conjunction of all these phenomena strongly
suggests the operation of both intra- and inter-genomic conflict and consequent
hybrid breakdown. While parapatric and sympatric models might be appropriate
at some levcls in the system, the latter certainly in its current maintenance, the
widespread occurrence and high frequciicy of thelygeny and possible Haldane rule
effects firmly indicate allopatric evolution as the proximate phenomenon. We further
suggest that incipient speciation has occurred with post-zygotic isolation between
two karyotypes, probably by vicariarice during the Pleistocene. Thus, the present
‘populations’ are in reality mixturcs of a number of denies whose various relationships
lie somewhere along a spectrum from geographical races (subspecies) through
semispecies towards full species.
We have frequently remarked (Owen & Chanter, 1968; Smith, 197613, 1980;
Gordon, 1984; Smith et al., 1993; Owen & Smith, 1
) that D. chyippus, and its
Mullcrian co-mimics Acraea encedon and A. encedatza constitute a unique. assemblage
ofpolymorphic Batesian models throughout central and eastern Africa, a geographical
area roughly the size o f western Europe. D. ch?ysz$pus is Tor example quite unlike
the several Heliconius species, also models, in which polymorphism is generally
confined to narrow hybrid tension zones (Mallet, 1986; Mallet & Barton, 1989a).
Even within the species D.chpsippus itself, which has a vast geographical range over
tropical and sub-tropical Africa, Asia, Australia and almost every suitable island in
the Atlantic, Indian and western Pacific Oceans (Ackery & Vane-Wright, 1984), the
polymorphisms in East A k a are exceptional in their extent: visual polymorphism
in other parts of its range, such as Malaysia (personal obscrvation), is absent from
all but small contact areas between parapatric races. Within the subgenus Anosia, to
which D. chrys$/v~sbelongs along with the two similar Neotropical species, D. gil$pus
and U. eresirnus (possibly three species if the dubious specific status of U . plexnure is
confirmed (Ackery & Vane-Wright, 1984)), geographical polytypism is a general
feature. However polymorphism in both the New World species is always confined
to narrow contact zones. Indeed, this generalization can bc extended to the two
other Damus subgenera, Danaux (xen~uslricto) and Salnturu, which with Anoxia, comprise
the genus Danaus (sensu lato).
The profoundly illogical nomenclature applied to the ‘races’ and ‘morphs’ of U.
r,.h?ysippusin Africa, which has remained intact far too long, goes back to a thoroughly
obfuscatory paper by Talbot (1943),who failed to appreciate the distinction between
polymorphism and polytypism: in the light of knowledge acquired over the past 30
years we aim in the near future to revise the nomenclature for the African races or
subspecies of D. chlysippus. ‘l’hc failure to appreciate the possible hybrid origin of
the East African populations, frorn once allopatric geographical races which may
have expanded their ranges, overlapped and interbred, has meant that varietal
names such as aegyptius, alcippus, liboria and dorippus have been used indiscriminately,
rcferring both to moriomorphic and allopatric races arid also to sympatric morphs
within polymorphic populations. The prime example of this confusion is the African
taxon aqyptius Schreber, 1 759, to which all our samples traditionally ticlorig, and
the niorph aegyptiu.r witliiii it; both are ill-dcfined entitics and both usages of thc
name ciqgptiu~'are misleading without further explanation. Despite being guilty of
using the namc nqpyphs in many previous publications we now regard it as invalid.
Our evidence now suggcsts that the D. ch?y.vippu.r populations of East Africa
comprisc a complex of several, probably as many as five orice allopatric taxa, which
have converged upon each other arid coalesced in quite rcccnt times following the
rcmoval or barriers to dispersal. These barriers consisted of lowland and montane
forest during wet phases ( z interglacials) and of much extended sand desert during
arid phases ( z glacials).Extensive habitat changes resulted from both cyclical climatic
change over the 1.8 Myr of the Pleistocene (Morcau, 1963; Flenley, 1979; Hamilton,
1982; Williams & Faure, 1980; Deacon & Idancaster, 1988; Goudic, 1992) and, in
the Holoccnc through to historic times, deforestation and ticsertification resulting
kom human activity (Roberts, 1989). Possibly the most notable ecological characteristic of D.chys$pus at the present time is its almost universal association in the
tropics arid sub-tropics with human disturbance. Indeed, farms, shambas, plantations
and gardens are the places where the highest densities are invariably found aiid
most of our field data were gathered in such areas. This species, like D.plexippus
and many other members of the genus (Brower, 1995), can justly be considered a
culture-follower: both its range and abundance must have grown exponentially in
thc wake of escalating human population growth and its rnassivc impact on the
tropical environmcnt,
RIE'I'HODS
'The butterflies wcrc collected randomly with a nct in the field if sufficiently
numerous, or otherwise as eggs which were raised to adults in the laboratory ('l'able
1). The formcr mcthod always results in male bias (Owcn & Chanter, 1968) whereas
thc latter accurately reflects thc secondary sex ratio. 'The sampling sites are shown
in Figure 1. All the samples from Kenya and Uganda were scored by DASS and
LIFO together t o ensure consistency. 'The Dar es Salaam samples were scored by
TIASS in the fielcl.
'Ihc African genotypes and phenotypes of I). chrys$pus are listed in Table 2 and
[he four main phenotypes shown in Fig. 2 (colour plates in Kothschilct el ul.
( 1 1)75), Smith ( 1 980) and Owen arid Smith ( 1993)).Maps showing the approximate
distrihutiori within Africa of forms chysiplus ( zz aegyptius), dorippus, alcippus and ulbinus
arc given in Owen and Chanter (1968), Owcn (1971), Pierre (1973) arid Rothschild
rt al. (1975). Asian forms including liborin are mapped by Mnrishita (1985).
At lcast three arid possibly four loci control thc switches between the allopatric
races and sympatric morphs arid their inheritance is well known (Owen & Chanter,
1968; Clarke et a/., 1973; Smith, 1975b, 1980).
The A locus controls hindwing colour, the A allele giving a uniformly cdoured
(orange or hrown) wing while the aa genotype has a large central white arca.
I\hasha
K arnpala
I.ake hlaqxli
Nairobi
O/M
12/91-1/92
Xiiirrilii
Nairobi
4/93 & 9/93
I /Mi
I /w
I /9.5
Nairiili
2/8!l
Nairobi
4/87
5/87
7-8/87
11/88
12/93
2-1 2/72
! 12/73
1-12/74
I 12/75
Nairobi
Yairdii
Nairobi
( hl ana
Dar es Salaam
Dar rs Salaam
I)ar es Salaam
Dar es Salaam
ti1
!07
36
I01
I .69
NKI.
a(lults
!.03
I),\SS, DFO,
adults
IJG, AhiO
NKI.
IJG
NU.
IVKL
22
5
H
(i
20
7
I8
52
54
12
119
I .22
0.10
0.15
0.14
I0
5!J
I I2
0.70
0.53
0.63
0.M
2.64
I .8C1
1.29
1.50
1.53
32
20
21
37
537
?If3
14
299
77-1
923
!)!)!I
1383
I1)02
(i53
0.17
adults
%Ss
'ggS
adults
!JC
[JG
qgs
ITG
eggs
IJG
eggs
eggs
!JG
CggS
NKL
IIASS
adults
adults
DASS
adults
DIiSS
DASS,J.U,
;I(lults
adults
DMP
~
~
~~
-
~hllectiirs:NKI.=N.K. I.owis, iJC =I;]. Gordon, DASS=D.X.S. Smith, DFO =D.F. Owrn, 2.\h'fO=,\.lf.Owiny,
.J;\A=J.A. Allen, DhfP=D.?rI. Prnrson: all the huttrrflirs wrrr s(.orrd Cir ptwiiotypr antl genotype b y DASS Br
DFO t i i g c t l i ~ ~#. Srx-ratio = n i a l d l r m & s * kIehotls: 'adultj' = buttrrflies collectrd in thr firld, 'eggs' =eggs
(mainly) and some l a n a r collrrtrd in t!ir firld and raisrd t o atlult it1 the litlioi-iitoty. FW'=forcwing, HM'=
hindwing. Approxiniatc y(igraphirnl rangru of the prtqxisc'd rriotiornorphic and allopatric races are: (1) (orangr)
thlyJz$pus; klcditerranean Africa from the C a n q Islands to Egypt, through Arabia to India antl China; (2) oki,f$u\;
Sub-Saharan West Africa from SrnPgaI to Chat1 Basin; (3) d f m p p q Somdiii, Ethiiq,ia aiid iiorttiri-ri Ktriya; (4)
Ixown f h y z / J / ~ u \ ( = w p p / / u \ ) ; ( ~ d i ~ i i t(,: i i t i ~ o , wrstrrn and uouthcrn Zaire, Angola, Namibia; (5) liboria ( %orZenti$);
South Africa, hlopmhiqur, Zimbabwe, Zambia, hlalawi and islands in thr Indian Orran inrlritling Matlagast.ar,
hlauritius and Rt.nniiin. 1\11 five of the original allop&r Girrns also riccui- i l l the Ilybrid zone which covers soutltcrri
Sudan, southern Kenya, Central Aliicari Republic, Uganda, 'Tanzania, eastern Zaire, Rwanda and Burundi. Hyhrid
forms are rarr nutsidr this arra.
Heterozygotes vary from having a reduced white patch, through a few white scales
to no white. Race akippus, which inhabits Sub-Saharan West Africa from Senegal
to the Chad Basin, is monomorphic for the an ,qenotype. A similar form recurs in
West Malaysia south of Periarig Island where it is also monomorphic (personal
observation) and in Sumatra. All othrr races excepting f. gelderi from Sulawesi, which
has curious white wedge-shaped splashes on the discal area of the hindwing, arc
without white on the hindwings apart from marginal spotting. Aa heterozygotes with
visible white are named alcippoides or weak alcipfm.
The ground colour of both wings is governed by the €3 locus. B- genotypes are
coloured nutbrown and the hh genotype is tawny orange. Many of the variable Bb
heterozygotes are detectable having orange on the anal margin of the forewing and/
or much of the hindwing. From southern Tanzania and Zaire southwards (chrys$pus
arid liboriu) populations are niononiorphic for brown. Race dorippus in Somalia and
northern Kenya and the nominotypical subspecies chrysippus in North Africa are
monornorphic for orange; nlc$pus is polyrnorphic at the B locus.
The C locus controls forewing pattern. The C allclc produces a forewing which
is uniformly coloured orange/brown excepting a narrow black border. The cc
genotype has a forewing in which the apical half is black traversed by a row of
white spots, the latter usually detectable on the forewing underside of Cc heterozygotes
Figurc 1. Map of’ East Africa showing thc location of‘ thc sampling arcas in Uganda and Kcnya. An
atlditional sarq)lr was taken at Dar cs Salaam, Tanzania, 320 krn soilth of Mo1nt)asa.
(form tran.rien.s). In dorzppus the C; allelc is at fixation. All othcr phcnotypcs arc
monomorphic for cc.
Thc I, gene controls the switch between the liborin and cArysippus phenotypes: the
former has large and fused white spots in the forewing subapical area (broad-banded)
and a diagnostic white spot in the submarginal area of forewing space 2 (Cu? on
the Comstock system). Genotype LL is liboria and 11 i.s chry.c.$pus. A s with thc othcr
loci, most heterozygotes have an intermediate phenotype (weak liboria). Srnith &
Owen (1997) were unable to establish the L gene as a separate locus or its linkage
group; it is possible but unlikely that liboria is determined by a third allele C? at the
c locus.
?‘he B arid C loci are closely linked with 3.8’/0 recombination in males only
(Smith, 1975h, 1980) as expected in 1,epidoptcra (‘l‘urncr & Shcppard, 1975).
Previous sludies indicate that the A locus is riot or at most loosely linked with the
R/C: loci and that all thc thrcc or four colour pattcrii loci are autosomal ( o p ~ r ocit.).
<:OhIP..\RISON O F PHENOTYPE FREQJENCIES .\T DAR ES SATAAM, NATRORT ,ZND K/\hIPAT./\
Before analysing phenotype frequencies across all six sites sampled, which are
seasonally heterogeneous, we compare the frequencies at Nairobi, Dar es Salaam
and Kampala for a single season (December to February), the only months for
which we have striclly coniparable data Tor three sites based on large samples. ‘ilie
raw data (Table 3) show that dorippus (C-) is pre-dominant at both Dar es Salaam
arid Nairobi but that brown dorippus (B-C-),a form confined to the hybrid zone, is
much commoner at the former than the latter. At Kampala, dorippus is scarce and
the saniplc shows high frequencies for both ciirys$pus (A-cc) and akippus (aacc).
‘ 1 i ~ r . e2. Phenotypes, gellotypes and descriptions of D.chy.vippus in Africa
Ihrription
an
an
/ih i i
A;1
HH
/I
B- cc N
h l bh C C I/
;Iri LIB ( 1 II
11
!
,
I
.
EW patterned & narrow-bandrd, orarrcq-;
H W orange
Fb’as rhy$ppuJ, orange or brown;
H W with l a q r whitr patch
FM’unpattcrried, orangc; HW orange
M’as chpsippu.c hut brown; HW brown
pattrrnrd as chp$pus but broadliantlcd, l ~ r o w n ,spot in Cu,; HW browri
Hyllrid forms
FLY as dorzj~fius,orange or brown
HW as a/cip/iu\
as dorzppzu, orangc or brown. HW
with umall white patch or white
scaling along veins
F1.l‘ lirown. HW brown or somctinic.+ oriitijir
in Lib genotypes
F%’ as dorippus with faint ae+yfitiu\/
rby.\Z~ipu\patterning on underside,
orange or brown. H W oratqr o r 1)rown
alhzrru, I.an7,
nz!
FLV as alcipptt~,orange or brown. HM’
with small white patch or white
scaling akmg wins
I+L‘ pittcrn as liboria, orange. H W
orangr hbnnn
( % on’enfi3 hurivillius)
weak Izbona
.4-B- rc Ll
A bh
11
LI
orangr
EW as lzhonn hut with narrower
;
handing, small spot in C U ~orange
or Ixnwii. HW orange or brown
Comparing phenotype frequencies dominant: recessive (Table 4), the frequency
of aa phenotypes at Kampala is significantly higher than at the other two sites and
higher at Nairobi than Dar es Salaam. B- phenotypes are significantly predominant
at Dar es Salaam but do not differ between Nairobi and Kampala. The cc phenotypes
also show highly significant differences between Kampala on the one hand and Dar
and Nairobi on the other but not between the two latter. These data suggest a
north-south cline at the B locus and a broadly east-west cline for the A arid C genes.
An additional feature of these polymorphisms is that the frequency of phenolypes
at all three sitcs may differ significantly between the sexes (‘l’able 5). The morphratio for C locus phenotypes differs significantly between males and females at
Nairobi and that for both A and €3 locus phenotypes differs very significantly at
Kampala (Smith et a/., 1993). At Dar es Salaam no differences are apparent in the
samples for December to February but there are very significant difl’erences at both
B and C loci when the sexes are compared throughout the year (Table 8).
The practical implication of these sex differences is that true phenotype and gene
frequencies in the population can only be estimated if samples correctly reflect the
sex-ratio. While those samples based on the collection of eggs may be in this respect
reliable, those based on adults are virtually always male-biased (see Methods). Our
r). A. s. SMITH ETAL.
I:iSirure 2. ‘I’hc colour forms (f.) of flnnnris chys$pz,pzls: ( I ) f. chysippu.r (grnotype A clj; (2) r. ukippus
(grnotype) an rr; ( 3) f: doorippus (gcnotype A- C-); (4) f. nlbinus (genotypc aa C-). Black and whitc arcas
arc as shown; stippled areas are either hrown (genotype B-) or orang? (genowe bb). Gcnc A coritrols
1iiridwiIig colour, A - being orangc (or brown), an white with orangc (or brown) margin; grne U controls
ground colour on both wings, B- being brown, bb orange; gene C controls forewing pattcrri, C- being
unpattcrncd (dorzppzc.s/nlbinus), cc having a black apex with white hand (GhyYJ~pu~/alripl,u.c.).
Dominance
is incomplctc at all three loci in a large hut variablc proportion of hrterozygotes (for dctails see Smith,
197513; 1980.
inability to estimate accurately the sex-ratio is the reason why throughout this paper
we confine our analysis mainly to phenotypcs rather than gene frequencies.
COhlPAKlSON OF PHENOTYPE FREQ1JENC:IES AMONG ALL SITES
When comparing all six sites (Table 6) a few cavcats must be stated at the outset.
Only the Dar es Salaam sample, which consists entirely of adults, covcrs all months
of two successive ycars; with one exception, the Nairobi samples were collected as
eggs arid in seven different months of six diilerent years. The remaining four samples
were collected as adults in only one or two (different) months in two different years.
Furthermore, only three samples (Kampala, Nairobi and Dar es Salaam) are as
large as we would like (‘l’able 1).
‘l’hese precautionary remarks are important as we show latcr that morph frequencies vary, first, from year tu year at the same site; second, with season at those
I’( )LYMOKPHISM IN DANAIJ~CHlXYIPf’1J.Y
5I)
‘ I ~ L E3. Perccntage freqiirncy of phenotypes of D. chly”ippus (modal freqncncics in italics) captured as
adults or raised from eggs or l a n w collrctrd in the field in thr months Dccenihcr to Frhruary liom
three rexions of Last Africa
Dar cs Salaam
Male
Fcmalr
Total
Male
32.6
7.7
5.2
5.6
0.0
0.0
0.0
.i(i..i 50.9
53.7
89.7
74.2
76.1
0.0
5.8
2.8
‘3.8
2.li
7.1
6.5
2.8
14.4
8.5
Frrnale
9.3
10.2
1.1
2.7
I .!I
0.0
8.2
7.2
42.1
48.1
4.5.0
0.2
0.j
0.1
0.0
0.0
0.0
0.0
0.0
0.0
1.8
1.1
I .5
U.0
3.0
2.6
4.7
0.0
2.4
0.2
0.2
0.2
0.0
0.1
0.3
3.7
7.7
5.7
0.0
0.0
0.0
o.n
1 .8
1 .ti
46.7
24.0
35.5
547
I(i!l.5
548
Yo(;
267
39
I07
104
x2 (with Yatcs’ Correction) for comparisons of
phenotype i‘requmcies (dominant: recessive) at the A, €3 and C loci for
sarriplcs from 1)ar rs Salaarn, Nairobi and Kampala collected in the
inonths December to February (Table 3)
TMLE4. Values of
Dar es Salaam
I.ociis tested
Nairobi
~.
A
Xairoli
Nairobi
Nairobi
Kanip;~Ia
I<ampala
Kampala
K
c
A
B
c;
.5.3*
9.5.0***
~
2.811s
378.9***
60.3* **
114.3***
(i.2 11s
ti3!).7***
309.9***
‘Tm1.E 5, Values of 1’ (with Yatcs’ Correction) for comparisons of
phenotype frequency (dominant: recessive) betwcen the sexes (Tablc 3)
at Dar cs Salaam, n’airobi and Kampala in the months Dccemher t o
Fcbruary
Locus
Dar cs Salaarn
testid
~~
A
B
c
11s =not sigiiticant;
tailed.
Totd
_.
~
31.3
tiainpala
hlalc
Total
Frrnalr
~-
30.9
Nairohi
N;iirohi
tiampila
~~
0.048 I1C
2.250 11s
1.148 11s
f’= 0.142 ns
9:
9.!)72* *
0.032 11s
8.148**
1.738*
0.060 ns
* O.O.j>r>O.OI; ** 0.01> ~ O . ~ l l i 3l ; Fisher’s Exact ‘l’cst (two-
211
D. .\I
(,(I
?AHI.E
S SMII‘H ETAL
6. Prrccntage frcqurncies (modal frcqurncies in italics) of phenotypes in polymorphic field
populations of D.chysiplus at six locations in East Africa
Phrt1otypes
ri
82
(C;riirrt)ilws)
I
Sampling arras
-~
~.
hl
N
G
1)
- -~
~~
0.0
0.0
15.0
8.0
3.9
92.3
2.I
2.8
7.5.0
77.I
92.P
44.2
55.7
8.5
0.0
5.9
11.1)
20.5
13.4
45.0
0.0
4.5
0.0
1.5
I .0
0.(1
2.5
0.0
0.0
0.3
(ilhi~u\,orarigr
1 .o
2.-1
5.0
2.5
3.9
0.9
bh C-)
d i i , h p u ~ , Imwn
I !).6
5.7
0.0
I.(J
0.0
0.2
7.2
35.5
2.5
1.1
0.0
0.0
darippii.,r, tir(iwt1
(11- H-
c-)
dOfl&m\, orilllgr
(ii bb (,’-)
( h y $ p u I , brou n
I1 l C )
rhyippuc, oraiipr
1.4- hb 1.0
nlhinii \ ,
(a0
brown
n- c-)
((/(I
(<I(/
B- CC)
alri/i/>ut,oritngr
(no lib cc)
v
97
21 1
40
828
31338 *
51
1 = Ishasha, R = Kampala, hl= Lake hfagadi, N = Nairobi, G = Galana, 1) = Ilar es Salaam.
prriotl~Januiiry1974 to Srptrmtwt- I ! J i S oiily.
* S;lmpIr
covers thr
sitcs from which we have several samples; and third, with sex at four of the six sites.
Moreover, the two wet and two dry seaso~isand the timing, intensity and duration
of the north-east and south-east trades, vary from year to year. Further, the method
of data collection (eggs or adults) influences the result since the former gives a morc
accurate estimate of thc sex-ratio and, as we have seen (Table 5), rnorph-ratios and
sex-ratios are not independent. Despite these problems, the magnitude of many of
thc d i h e n c e s in phcnotype frequency between sites is such that we belicvc they
must hc informative. We do however feel that fornial statistical tests on such
heterogeneous data are inappropriate; neither do we claim that some of thc smaller
diffcrcnces shown in Table 6 would be unaffccted by larger and more comprehensive
samples collected by a uniform method.
In ‘l’able 7 we compare the frequencies of the three recessive phenotypes across
all sites. The ua (mainly alcippu~)phenotype decreases in frequency from west to east
(Kampala Dar) with a steep fall between Kampala and Nairobi. Howevcr there is
also a north-south component as its frequency is considerably lower in the Western
Rift at Ishasha, than at Kampala 300 km to the north-east. We know from museum
collcctions (Owen & Chanter, 1968; Smith et al., 1993) that the a allele is a t fixation
in West Africa from Senegal to Camcroun and at high but declining frequency
across north-central Africa through the Central African Republic to southern Sudan
and Ethiopia; it does not however penetrate south of the rain hrest barrier which
runs from Gabon in the west to Uganda in thc east. ’I’husacross our sampling area,
clines of‘ diminishing frequency of n radiate from Kampala approximately northwest to south-east to the Indian Ocean and north-east to south-west towards the
Western Rift.
The b allele decreases steeply in a north-south direction (Kampala to Dar es
Salaam) and east-west (Galana to Ishasha). ‘The direction of the cline is probably
TBLE 7. Frequencies of rcccssive phcnotypes a t three gene loci, gerictic distances, physical distances
a n d co-ordinates of samples nf 11. chyyszjipu.~from six localitics in East Africa, ranked in order west to
rast
Distance
froin sample
below
Frequenries of recessive
phenotyprs (per cent)
.
Locations
Ishasha
(Lake Edward)
Kampala
Lake Magadi
Nairobi
Galana
J h r es Salaam
aa
11h
cc
28.9
23.7
95.9
97
43.6
85.8
82.5
85.2
96.1
46.6
94.8
2.5
12.1
0.0*
112.3
21 1
10.0
4.6
3.9
1.4
R = Rogers' Genetic Distance.
frequrnry ( c 0.15).
Co-ordiriates
_.
~
R
km
Latitude
Longitiide
0.260
300
00" 12's
29" 57'E
0.431
00' 18"
01" 53's
0.079
0.248
465
88
300
440
-
-
32"
36"
36"
38"
39"
n
40
845
51
3838
o.mo
* Sample includes seven tmnsim
010
in's
03" 04's
06" 51's
34%
17'E
~Y'E
42'E
IG'h
which indicate that the c allele is prcscrit at low
north-east to south-west at around 90" to the n cline. The very high frequency of
bb phenotypes at Galana is probably in part a seasonal effect. Perusal of museum
material (Smith et nl., 1988) supports this interpretation: the B allele is at fixation to
the south of Tanzania on both sides of the continent and the b allele is at or ncar
fixation in the north-east (Egypt, northern Sudan, Ethiopia and Somalia). In Wcst
Africa the locus is polymorphic.
The cc phenotype decreases from west to east with a sharp drop between Kampala
and Nairobi, possibly in the region of the Eastern (Great) Rift Valley. Here too
there is also a north-south componmt shown in comparisons between Kampala,
Nairobi and Dar: the r allele increases in frequency to the west arid south of Nairobi
(the low values for Magadi and Galana are probably seasonal effects). Although not
covered by our data, the G allele also increases to thc north of Nairobi (Smith, 1980;
Smith et aL, 1988), through Sudan and into E,gypt where it reaches fixation. T o the
north-east of Nairobi its frequency falls to zero in Somalia. The distribution of the
c allele is thus more complex than the other two with clines of increasing frequency
radiating from Nairobi (or eastern Kenya) in all directions except north-eastwards.
THE PROPOSED GKOGUPHICAL RACES OF D (;HRYSIPPlIS IN AFRICA
The clines identified suggest that several distinct cc allopatric forms (hereinafter
called races) may have entered the East African region. The one from the north,
which is found across North Africa from the Canary Islands to Arabia, is orange
(A-bbcc) and we believe indistinguishable from the nominotypical Asian race c/~lysz$pus;
it is predominant at Kampala, comprises about half the chrysippus (cc) phenotypes at
Nairobi and is rare at Dar es Salaam. Its distribution within East Africa supports
our contention that this race has an Asian origin; it probably entered East Africa
from the north via the Nile Valley during a dry phase of the Quarternary or
Holocene.
The brown chrysippus-like race ( U B c c )is monomorphic in the region from Gabon
south through the Congo Republic, western Zaire and Angola to Namibia though
62
D.
r\.
S. SMI'L'H E? AL.
it is also common in our area, especially in southern 'I'anzania. While the description
seems to correspond to form a e , p i u s Schreher 1759, the origin of the type material
is uncertain (Ackery et al., 1995) and we use the name brown chlysipPus for this
distinctive race. Its frequency increases towards the south arid west of our area; it
is most frequent at Ishasha but also common at Dar.
A third race which is similar to ch?ysippu.sand brown (AABBccLL)is widely known
as liborin Hulstaert 1931 ( % om'pntis Aurivillius 1909 which should probably have
Iiriority). We have recently shown (Smith & Owen, 1997) that it is genetically distinct
from brown chlysippus (AABBccllj and is marked phenotypically by the broader band
of subapical spots on the forewing (Talbot, 1943), the fusion of the spots in spaces
4 (M.J arid 5 (M2) and a characteristic submarginal white spot in space 2 (Cu2)
which is present in no other African race. 'I'his subspecies occurs from southern
'I'anzania through Malawi, Zambia, Zimbabwe and Mopmbique to South Africa.
It also occurs (in orange form) on Madagascar arid all suitable islands in the Indian
Ocean; the spot in space 2 is at high frequency in D. chlysippus from Malaysia
(pcrsonal observation) and may indicate the Asian origin of liboriu. It appears to be
rniSTatory and is scasorially frequent at Dar es Salaam (Smith & Owen, 1997) but
rare at the other locations included in this survey.
Race dorz&us (A-66CQ probably originatcd in the region where it is now monomorphic, the Somali Arid and northern Kenya, whence it spread south to 'l'anzania,
east to Kampala and north-east into Arabia and beyond. Brown dorappus (A-B-C-)
which is abundant at Dar es Salaam, results from crosscs between dorippus and
brown chpy.sippusor liboria and is found only within the hybrid zone.
Race a1cipru.r (aaB-cc or aabbcc) probably evolved in West Afirica. Hybridisation
with dortppus in East Afi-ica produces the form albinus (a&-C- or aabbC-) which is
confined to the hybrid zone.
The Kogers' Distances (Rogers, 1 '372), calculated from phenotype frequencies
('l'ahle 6), show that all the comparisons except Magacli-Nairobi and Nairohi-Galana
are substantially different. There is a particularly steep cline, based mainly on the
A and C loci, between Kampala and Magadi, which is likely to bc stepped in the
region 01' the Great Rift Valley. l'hc genetic distance of 0.260 between Kampala
and Ishasha is due almost entirely to clines for the A and B loci while the value of
0.248 between Galana and Dar es Salaam is predicated by steep clines for the B
and C loci. Thus each cline involves a dircrent pair of the three loci used in this
study. Without further samples we car1 do no more than speculate on the shape of
thc clincs, whether stepped or smooth, arid their precise orientation. However, the
considerable changcs in gene frequency recorded across all three clines are consistent
with a hypothesis that they originated by vicariance in refi.igia ('lurner, 1971). As
with dines in other unpalatable tiutterflies such as Heliconius, they arc probably now
maintaiiied by frequency-dependent selection against rare forms (Mallet & Singer,
1987; Endler, 1977) or, since many heterozygotes are relatively poor mimics, by
hcterozygote disadvantage (Owen & Chanter, 1968; Barton, 1979). We have also
shown that the polymorphisms in Kampala are unstable (Smith et al., 1993) which
implies that the clines have the potential to move (Benson, 1982; Mallet & Barton,
1989b; Mallet et nl., 1990).
We also know from inspection of museum material that all the alleles involved
in this study cxtcnd at low frequency far beyond the confines of East Africa. Both
introgrcssion and especially migration are probably involved (Smith & Owen, 1997).
As both paiimixia and discrete subpopulation models must be rejected for U.
POLYMORPHISM IN DANillJS CHRKYII‘PUS
‘rAm.F,
63
8. C:omparison of the frequcncics of dorninant (1)) and rrcrssive (R) phenotypes betwccn x x e s
in h r r samples of D. chyysiplus fro111 East Africa
bfak
Female
~.
Locatiun
LOCUS
D (%I)
R
(%I)
D (Yn)
R (%)
n
?,I,
~
Ishasha
A
B
c
Kampala
A
R
c
Nairobi
A
B
c
Dar cs Salaam
(1974-75 only)
A
B
c
78.7
67.2
3.3
44.9
6.5
4.7
96.6
9.7
94.4
98.5
51.5
79.4
21.3
32.8
96.7
55.I
93.5
95.3
:1.4
90.3
5.(i
1.5
48.5
20.6
58.3
91.7
5.6
f1H.Y
22.1
5.8
94.8
17.0
84.9
98.7
56.3
75.1
41.7
8.3
94.4
31.7
77.9
94.2
5.2
83.0
15.1
1.3
43.7
24.9
97
97
97
21 1
21 I
21 1
845
845
845
3838
3838
3838
3.63
6.19*
0.0003
10.82**
9.25 * *
0.002
0.99
7.03**
14.44 * * *
0.09
8.10**
9.83**
All ,y2 values are calculatrd with Yatrs’ Corrrrtiori
chrys$pus, the isolation-by-distance model (Wright, 1969) probably best explains the
genetic geography of this species.
COMPARISON OF MORPH-RATIOS BETWEEN SEXES
We have previously drawn attention (Smith, 1980; Smith et al., 1993) to a
phenomenon which on further enquiry now proves to be a general characteristic of
the polymorphisms of D. ch?ysz$pus in East Africa, namely that the morph-ratios
frequently, indeed usually, differ between the sexes. In the later paper (Smith et nl.,
1993) we said, “we predict that discquilibria of the types described hcrc will in time
be established as a general feature of all polymorphic East African populations of
D.chys$pu.c. hut we are awarc of the importance of replicating the Kampala and
Dar es Salaam results in a large random sample from at least one other area”. This
has now been accomplished at two furthcr places, Nairobi and Ishasha (Table 8),
both places with phenotype frequencies significantly different from either Kampala
or Dar es Salaam (Tables 6 & 7) and well separated geographically from them (Fig.
1).
At both Nairobi and Dar es Salaam the a allele is comparatively rare and no sex
differences for this locus can be detected. However, sex differences at both B and
C loci are very significant. At both places the bb genotype is more frequent in malcs
and the cc genotype in females. At Ishasha the hb genotypc only is significantly more
frequent in males, thus agreeing with all the other three sites. The sample is however
small (n= 97) and the sex difference at the A locus approaches significance ( K O . 1O),
though in the opposite direction to Kampala, i.e. the dominant phenotype being
more frequent in males.
We will show elsewhere (Smith et ul., in prep.) that the differences in gene
frequency between the sexes are explained, at least for the B and C loci, by
unorthodox segregations for these loci in both unisexual (all-female)broods, in which
all males die, and in same bisexual broods with partial sex-linkage. The very
D. A. S. SMITH ETAL.
(il
’IAIHX
9. Morph-ratio heterogcncity bctween the sexes and linkage discquilibriuni in 11. rhyys$pu.c from
four localities in ISast Africa
Locus
combination
Locality
C;twotypts in
significant cxc~”ss
tcstctl
Mali.
A/B
A - bh
Frmalr
f , ,h, r
heterogeneity
hrtween sexts
for linkagr
distquilibrium
~
lsll~lsll~l
aa
B-
1m 6 * *
h/C:
n/<:
..~
~~
0.21 11s
f = o.sn2 11s #
P=0.373
~
11s #
A - RA - rc
2 1.74***
n.n.5 11$
4/c
an hh
an cc
n- (-1
7.68* 0
.4/B
bb ( I
A- bb
10.94**5
9.08*
CI.CI3 11s
P=0.177ns#
I .cj5 IlS
.4/c
fl-
c-
I7.:46***
R/C
bb
(,‘-
I 6.83 * * *
?h,
2 2* * *
I t6.29***
liar es
Am
1lOIlC
SaImn1
A/C
‘4-
c-
6.95 11s
10.27*
7. I$)**
I .50 tlS
H/C:
bb
(.’-
Karripala
h/ H
n/c:
Nairobi
A - Ban bb
il- Iy‘
B-
cr
hb
IT
none
A - cc
Li- rc
bb cc
2H.44***
WkY96* **
1’ for linkagc discquilibriurri is tested in 2 x 2 txIJIcs using Y a t o ’ Correction for continuity. * O.O5>/30.0 I ,
** 0.0 I >130.01)I, *** D0.00 I . 5 d r q t t s of kredoni reduced to two due to low experted vahirs. # Fisher’s Exact
‘l‘cst. Low fiequciicy 01’ thc C,’ allele at Ishasha prccludcs testing the A/C: and H/C: combinations for lictt.rogc-c.ricity.
significant sex differences for A locus genotypes at Kampala suggest that this gene
may also be sex-linked in nlcippus as are the B and C genes at Nairobi and L)ar es
Salaam (Smith, 1976a; Smith et al., 1997). ‘The phenomenon has not however been
investigated genetically in Ugandan populations. As the B and C loci are closely
linked (Smith, 1975b), a similar efriect at both loci is not surprising. At Dar cs Salaam
and elsewhere the A locus was shown to be independent of the BC chromosome
(Clarke el al., 1973; Smith, 1975b): a translocation of the A gene to the BC
chromosome in Uganda is an alternative explanation for the Kampala data.
GAMETIC: AND GENOTYPIC DTSEQUTIdRRIA
There arc several typcs of non-random association (disequilibrium) which can
occur when combiriatioris of two alleles at two loci are examiried in the ten possible
genotypes (Weir, 1990). For two loci ,J and K, each with two alleles, <7,j and X; k,
excess over expectation of coupling (Jh-andjk) or repulsion (Jk andjh] combinations
constitutes gametic or linkage disequilibrium (the loci need not bc on thc samc
chromosome). The presence of linkage disequilibrium (D& can be tested for in our
data in 2 x 2 tables by i2,
using the observed frequencies of the four phenotypes
to generate random expectations under H,,. We are however unable to quantify I?)7h
as wc can neither assume Hardy-Weinberg Equilibrium (HWE) (in order to calculatc
gene frequencies), nor visually identify all heterozygotes, nor distinguish double
heterozygotes in coupling and rcpulsion phases.
Tests for the heterogeneity of genotypes between sexes (Table 9) show that,
in addition to gametic disequilibrium, two typcs of non-gametic or genotypic
disequilibrium occur in our samples, namely digenic disequilibrium (D7,k)and trigenic
POLYMORPHISM IN DRlrAUS CHRYSIPPUS
65
disequilibrium (DJJh,and DJKh).Both modes show up primarily as sex differences
whereas the several linkage disequilibria are independent of sex. However, as the
gene frequencies are not known, the presence of these disequilibria can be detected
but not quantificd.
Linkage disequilibrium is absent from both Uganda populations though nongametic disequilibria are very significantly apparent for all three combinations of
loci at Kampala and for thc A/R combination at Ishasha. Highly significant linkage
disequilibrium occurs for the tightly linked B and C loci at both Dar es Salaam (D’
(T,ewontin, 1964)= - 0.7 76 (Smith, 1980))and Nairobi; in both cases the repulsion
chromosomes Bc and bC are in excess. At Nairobi, the A and C loci also show
linkage disequilibrium for the coupling arrangements AC and ac while at Dar cs
Salaam the coupling arrangements AB and ab for the unlinked A and B loci exceed
expectation. Furthermore, either digenic or trigenic disequilibria occur for all locus
combinations at Nairobi and for the A/C and B/C combinations at Dar es Salaam.
Linkage disequilibrium indicates the operations of powerful selection and especially
so when the loci involved arc on diffrrent chromosomes (I,ewontin, 1974). The
genotypic disequilibria arid their association with sex result from unorthodox
se<gregationscaused by one or more of the following effects: karyotype polymorphism
and partial sex-linkage in one of the karytoypes (Smith et al., in prep.), male-specific
death at hatching or in young larvae and possible Haldane rule e
(Haldane, 1922). Smith et al. (in prep.) show that all these influences are apparent
in the hybrid zone at the three places where breeding has been done, Kampala,
are. interpreted as indicators of hybrid
Nairobi and Dar es Salaam. All three e
breakdown.
HETEROZYGOTE EXCESS AT NAIKC)BI AND D A MES SALAAM
A predictable consequence of the sex-ratio genetics of D.chrysippus at Nairobi and
Llar es Salaam and probably also at Kampala (Owen & Chanter, 1968)is heterozygote
excess (HE); this is a phenomenon distinct from heterozygote advantage (HA) and
demonstration of HE in a hybrid zone should not be automatically extrapolated to
an assumption of HA. The former is in this case an inevitable consequence of the
distorted sex-ratios and morph-ratios described above. Here we demonstrate for the
C locus that HE is usual at Nairobi and also occurs commonly but less consistently
at Dar es Salaam. We cannot demonstrate HE at the A and B loci as we have 110
reliable way of estimating the rxpcctrd number of heterozygotes but it would be
expected at these loci also.
‘ h e triangular co-ordinatc graphs (Fig. 3) show the fit of samples from Nairobi
(Fig. 3.4) and Dar es Salaam (Fig. 3B-D) to Hardy-Weinberg Equilibrium (HWE).
Points falling on the curve are in HWE, those above show HE, those below
Though
heterozygote deficiency (HD). As a test for deviation from HWE, we use
this is a more conservative test than Smith’s If Statistic (Smith, 1970), we feel that
the nature of our samples is such that a conservative test is required, despite the
increased risk of ‘l‘ype 2 crror. As not all Cc heterozygotes are phenotypically
detectable, we use a correction factor based on breeding results at Dar es Salaam
(Smith, 1980) which may therefore be subject to greater error when applied to
Nairobi, where expressivity of c in heterozygotes has not been directly measured.
x2.
D. A. S. SMITH E T A L .
66
A0..
A
0.8
\
n = 845
0.6
A0.4
0
1.0
0.8
0.6 cc 0.4
0.2
Figure 3. Triangular co-ordinatc plots showing thc fit to a Hardy-Weinberg cquilibriuin for thc
populations of I). chly.rij+?u.r sampled at Nairobi (A) and Dar es Salaam (B, 1973; C , 1974; I), 1975).
Points ahove the curve show heterozygote excess, points below a deficiency. Open symbols indicate
statistical significance (P<O.O5from 2 tests). Symbols give the month of sampling: large circle =
January, small circle = February, large square =March, small square =April, large triangle =May,
srriall triangle =.June, large inverted triangle =%July,small inverted triangle =August, large diamond =
September, small diaiiiond =October, large star = Novcmbcr, sniall star = Dcccmbcr.
The correction factor is obtained from the proportion of hnsiens in backcrosses at
the C locus in which all dorzppus ( + trunsien.$ offspring must be heterozygotes.
Hxpressivity of the c allele in Cc heterozygotes diflers between B- and bb offspring.
74.6 & 3.30/0 of B-Q progeny (n= 173) are trunsiens compared with 45.0 f4.4'/0 (n=
129) of hhCc progeny (Smith, 1980). Hence the observed number of Cc hctcroxygotes
in a sample is calculated as:
C Cc=(C brown trunsiens x 100/74.6)
+ (C orange trunsiens x
100/45)
Five of the six monthly samples from Nairobi (Fig. 3A) show significant HE. In
three months, February, July/August & Novenber, homozygous CC butterflies arc
shown as being entirely absent from our samples (they will in fact be present in the
population though too rare to detect). Despite likely error in calculating the
expressivity correction factor, HE must in reality be exceedingly high as it is
demonstrable in four of the samples fkom the frequency of trunszens alone. It is also
variable as shown by the January sample being in HWE and the others not. Although
January data for three years are amalgamated, this result is unlikely to be a Wahlund
=0.59; 0.8>
effect (Wahlund, 1928) as the samples are formally homogeneous
B 0 . 7 ) . January is the month when the frequency of females is maximal (88.72'Yo)
and is followed by maximal HE in February as predicted. As the monthly samples
from Nairobi were collected in different years, howcvcr, it is unsafe to deduce a
causal relationship between female excess and H E from these data alone. However
it is clear that both phenomena are evident through most of the year.
Only four of the 1973 Dar es Salaam samples (Fig. 3B) show HE: January, April,
May and June. The remainder are not significantly different from HWE. At Dar
the phenomenon is apparently seasonal and there is some concordance of HE with
rainy seasons. All these months coincide with or immediately follow peaks of female
frequency (Fig. 5A) suggesting a causal relationship.
In 1974 (Fig. 3C) six samples (February, March, April, June, July and December)
show H E and there is some replication, especially the association with wetter months,
compared with 1973. As in 1973, all the heterozygote peaks coincide with or
immediately follow peaks of female frequency. The 1973-74 results indicate a
probable causal link between HE and a female-biased sex ratio.
The 1975 data (Fig. 31)) show that HE was absent, a finding that cannot readily
be explained. The sex ratio data fbr 1975 do in fact differ from 1973 and 1974
(Fig. 3B): there is only one mode for female frequency in January followed by a
consistent slow fall to August; in 1973 and 1974 the curve is bimodal with peaks in
both January and May. While this d i k e r i c e might explain the absence of HE from
April through June or July, when it mainly occurs in 1973-74, it does not explain
why there is no peak or heterozygotes in January or February 1975.
= 3 1.97; P<0.00 1).
The 1975July sample is uniquely deficient in heternzygotes
'l'his last result is far too pronounced to be ignored as a possible statistical artefact.
The H D could be a Wahlund Effect, indicating an influx of migrants of different
genotype from the residents: although examining the other two loci (A & B) and
the frequency of lihoriu suggests that this is possible, we have no reliable wdy of
estimating A & B locus o r lihoria heterozygotes and the evidence is no more
compelling than for many other of the 33 months of sampling shown in Figure
3B-D.
Many of these samples are smaller than ideal for testing a fit to HWE and thereby
detecting H E or HD. However, as gene frequencies are subject to continuous and
at times sharp change, pooling of data over several months (zgcncrations) is almost
invariably misleading. In particular, pooling risks the creation of a spurious HD or
a reduced H E (Wahlund effect). For this reason we have largely avoided pooling
monthly data, with three exceptions-January at Nairobi (3 samples), July/August
at Nairobi (collection end July to early August) and October/Novcmber 1973 at
Dar es Salaam (monthly samples too small).
Thus although the single H D result at Dar es Salaam cannot be explained
satisfactorily, the overall picturc from Nairobi and through 1973-74 at Dar es
Salaam, shows that HE is the normal outcome of female biased sex-ratios. 'l'he
former generally follows the latter alter a short interval of 1-2 generations.
Determination of heterozygote frequency at Dar must involve several other known
balancing factors. H E will be generated riot only by a high frequency of thelygcnic
(x'
(x'
D. ,4.S. SMITH E7 AL.
68
(all-female) broods but also the availability in the previous generation of Cc males,
which have the highest mating success (Smith, 198 1). The genotype of these males
is mainly Bc/bC (brown dorippus) and they mate chauvinistically with females of the
same or similar phenotype (Smith, 1984). Many of the resulting progenies show
heterosis, in particular a shortage of CCoffspririg(Smith, 1975b),thereby contributing
to HE. O n the other hand, as mate choice at both the B and C loci, mainly exercised
by females, is predominantly assortative (Smith, 1984), in the absence of female
excess, this choice must produce HD. None of these balancing factors is precisely
quantifiable; they are moreover seasonally variable due to immigration and emigration and their magnitude at any time is dependent on continuously changing sexand morph-ratios (Fig. 5). HWE is therefore not to be expected other than in the
short term and is probably rather less common at Dar es Salaam than is indicated
by the intentionally conservative method we have used to test for it. Smith’s H
Statistic for these data suggests that most of the l)ar samples are not in HWE.
SEASONAI, CYCLING OF SEX- AND MORPH-RATIOS
Seasonal variation at Nairobi
The sex ratio measured as per cent female (Fig. 4A) is clearly unstable (i‘(5)=
90.4; P<O.OOl) varying between 88.6% in January and 50.8% in April. It is also
apparent however that a low sex-ratio is the normal state ( f = 68.4%) of the Nairobi
population. This is not surprising as 83% (n=41)of the randomly caught wild
females used to investigate the genetics of sex-ratio at Nairobi produced all-female
broods. ‘l’here is a hint of bimodality in the data with modes in January and May
(similar to Dar es Salaam) though this is uncertain as data are missing for five
months. As most of the monthly estimates of sex-ratio are derived from laboratory
rearing of eggs collected in the wild, they must provide a clearer picture of the true
sex-ratio than the Dar or Kampala data which are based on field collections of
adults. At both these places, field collections of adults show sex-ratios % 1:l and
yet all-female broods occur at high frequency (Owen & Chanter, 1968; Smith,
1975a) and the populations must in reality be remale-biased.
Despite the generally low frequency of aa phenotypes (Fig. 4B), there is significant
monthly heterogeneity (x2(5)= 12.1; 0.05>p>0.02).
Frequency of bb phenotypes (Fig. 4C) is always high at Nairobi, averaging 85.1 %,
but very heterogeneous
= 20.1; O.Ol>p>O.OOl),varying between 88.2% in April
and 65.4% in July/August. Frequencies of cc genotypes (Fig. 4D) tend to be negatively
correlated with those for bb, with a peak frequency of 2 1.2 per cent in July/August
12.4%); monthly heterogeneity is highly
and a low point of 3.5% in April (A?=
;
For both €3 and C loci, there is also a significant
significant ( ~ ‘ ( ~ , = 2 7 . 9P<O.OOl).
difference between the frequencies of genotypes in the two sexes (‘l’able 8). There
is, moreover, linkage disequilibrium for the closely linked B & C: loci at Nairobi
(Table 9) with the repulsion chromosomes Be and bC exceeding expectation: thus a
negative correlation between the b and c allele frequencies is expected.
Fluctuations in the frequencies of a, 6, and c alleles, but especially the last two,
are of such magnitude over a few months ( z generations) that natural selection can
he safely ruled out as the proximate causal agent. We suggest that large-scale
(xi(,)
POLYR4OKPHISM IN DRN,IlLS CHRTMPPUS
B
12
10
8
m
m
1 1
A
891
c
M
J
24
F A
D
70
65
J
F
A
H
N
R
Figure 4. Histograms showing the fiequcncics (per cent) of (A) females, and (B-C)) thc three homozygous
recessive genotypes aa (B), bb (C) and u (I)) at Nairobi. Symbols on thc x co-ordinate: J=January,
F = February, A =April, M = May, J/A=July-August, N =November; is the mean value of the six
nionthly samples.
movements of populations must be responsible for thesc changes (Smith & Owen,
1997).
Src-w.onal variation at Dar es Salaam
The data summarized in Fig. 5 are the results of an uninterrupted sampling
pro<grammeover 44 consecutive months on the campus of the University of Dar es
Salaam. The habitat is grassland with some trees and bush. The raw data for per
D. .I,S. SMITH ETAL.
B
Zsons
Monsoon
-
50
50
40
4o
v
0
0
0
2
20 g
a,
30
30
a,
c
c
C
I
a,
ati
-
20
L
a"
10
10
0
1972
1973
1974
1975
Figurc 5, Prcyuciicics @cI ccnt) for the rr genotype (0)
and females ( 0 ) in monthly sarriplrs or 11.
ch~yzppu,!tirorn Krbruary 19712 to Scpwtiibcr 1975 a~ D a r t's Salllaarn: (A) raw data; (R) thrm-month
moving avcrages. 'l'he approximate durations of wet scasuns ([tit. dashcd line indicating a pcriod which
is rsprcially variatilc) and thc two niunsouii regimes (SE= south-east, NI:= north-rast) a r r also shown.
ccnt fcmalr and per cent L L (mainly brown h y ~ z p p u sas akzppur is rare) arc shown
in Fig. 5A arid thrcc-month moving avcragcs in Fig. 5B.
The data for percent fymale (Fig. 5B) are bimodal with peaks in Dcccmher and
April (1973) and January and May (1974); 1972 is unclear as the April peak, which
is apparent in the raw data (Fig. 5A), disappears in the moving averagcs; 1975
FOLYhIORPHISM IN I,R'VAl'S LHRYSIPPl'S
71
appears trimodal (Fig. 5A) with an extra mode in February but is probably in reality
unimodal (Fig. 5B). In general terms there is a fair measure of replication when
comparing years; the major peak is in Dec,cmber to January, which usually coincides
with the short rains, and the lesser in April to May when the long rains invariably
occur.
Both wet seasons, but especially the former, are highly variable with respect both
to timing and amplitude; moreover the intervening months of February and March
are not infrequently also wet so that the two rainy seasons sometimes merge into a
single one lasting four to six months. It is unlikely that the rains per se are responsible
for the twice-yearly escdation of fernale bias hut quite possible that the monsoon
winds which bring the rain also bring in a race of different karyotype from the
residents, first from the north-east (short rains) and then from the south-east (long
rains). We have presented evidence for such migrations (Smith & Owen, 1997) and
for hybridization between karyotypes leading to low sex-ratios (Smith et al., in prep.).
High sex ratios (low female frequencies) occur consistently in September to
October with less marked lows for female frequency in February to March in some
years. The data are consistent with an hypothesis that thelygeny is maximal at those
times when the wind direction changes (south-east to north-east monsoon or viceversa) which ushers in new populations thus causing hybrid dysgenesis. 'l'he effect
is clearest in Fig. 5R (3-month moving averages)-every year the change from the
south-east to north-east monsoon coincides with a reversal of the trend in sex-ratio.
The data for the cc genotype are similarly cyclical with good replication over the
four years. The cc peak is in. June to July cvcry year and coincides broadly with the
middle of the south-cast monsoon regime; the timing of the trough is morc variable
but always falls in the period December to Fchruary, in other words approximating
to the mid-point in the duration of the north-east monsoon. Neither peaks nor
troughs correspond with those for female frequency; visual inspection of the graphs
suggests a negative feedback relationship between the two phenomena. The strongest
positive correlation between the two data sets (r=0.673; P< 0.001) is for female
frequency and per cent cc 5 months later (or 7 months earlier); testing female
frequency against cc frequency one month earlier gives the highest negative correlation
(r= - 0.718; P<O.OOl). These tesL rcsults strongly suggest that the two phenomena
are involved in a negative feedback effect over the annual cycle. On this hypothesis,
the two variables can be either independcnt or dependent at different times; thus
regression coefficients would be invalid.
We have reason to expect a causal link between the two data sets since at Dar
es Salaam (Smith, 1976a) the bcT or bc and Y chromosomes are transmitted nonrandomly to female offspring in thelygenic broods. Thus, following each peak of
females, there is an increase in the frequency of those genotypes carrying the bc or
bcT chromosome (bC/bc, &/be, bc/bc). At the same time, the frequency of cc males
arriving on the south-east monsoon from the south starts to rise. As the proportion of
homotypic matings between cc genotypes increases, reinforced by positive assortative
mating i.e. between like genotypes (Smith, 1984),the production of all-female broods,
which result from matings disassortative for karyotype, is expected to fall rapidly as
shown. ?'he raw data (Fig. 5A) show that the period of production of excess females
is short, generally lasting only one or two generations. It is likely that the death of
males is maximised by heterotypic mating, which occurs when demes dfiering in
karyotype mix as a consequence of a change in wind direction; suppression of
thelygeny then increases with the subsequent rise in homotypic mating.
72
D. A . S.SMI I’H E’l AL.
Reversal of thc decline in female frequency occurs every year in September to
Octobcr as the north-east monsoon begins to bring in durippus (C-) butterflies from
the north. The enforced disassortative matings, between the newly arrived C-males
and the surplus of resident ec females, through November to December, causc: hybrid
dysgenesis and an increased frequency of thelygenic broods. The female oK7pring
(mainly bC/bc and Rc/bc) must mate predominantly with inales which are B d b C or
bC/bc: it is these heterotypic matings which produce the H E observed at Dar es
Salaam at times whcn or soon after the wind direction changes. ‘lhese periods are
also marked by maximal admixture of resident arid immigrant populations, enforced
heterotypic mating and hybrid dysgenesis.
The annual cycle of the h allele (Smith & Owen, 1997) shows a pattern similar
to the c allele but out of phase with it by almost exactly six months; the former
pcaks in.Jariuary to February when the latter is niiriinial (Fig. 5R) and the latter in
June when the former rcaches its low-point. At Dar es Salaam the b allele is gcnerally
linked in repulsion to C (Smith, 1 980); its frequency therefore rises with the frequency
of dorippus (Bc/hC‘,hL’/bC‘ and bC/bc) over the six month period,June to January. In
the period Yet)ruary to April, dorippus individuals tend to niigratc north u<quinst the
wind and the frrcpency of bb genotypes declines steadily. Similar seasonal changes
probably occur at Nairobi (Fig. 4C:) but the bb peak is as expected 2-3 months later,
crcatccl by butterflies arriving from the south and passing through; the nadir of hti
frequency in July to August is also some two months latcr than in L)ar es Salaam,
presumably reflecting the emigration of many dari&~us southwards. We are short of
data fbr Nairobi in the second half of the year but thc rise of bb and fall of cc (Fig.
4D) Ixtwcen J ~ l yto Auqust and November probably indicates immigration of
dor$pu.s (hC/b-) from the north over the intervening period.
DISCUSSION
The polymorphism of D.chg~ys$pu.r in East Africa has been frequently remarked
upon as an enigma. T h e species is assumed to be well protected. Although it is a
much less consisten1 storer of cardiac glycosides than the American monarch
(liothschild et a/.,1975; Brower et ul., 1975, 1978), 6 2 male D.ch?ysipj~u.rfrom Dar
cs Salaarn, from which hair-pencils and wing pouches had been reniovccl before
testing, contained on average 3.9’/0 pyrrolizidine alkaloid (PA) per butterfly by dry
weight (J.A. Edgar, in litt. to DASS). PAS are known to confer defensive properties
on storers (Rrown, 1984). L). chysippus has numerous presumed Batesian mimics in
thc area including Hypalimnas misippus (L.), Pseudacrueu pugqei Dewitz, f. pogei and 1:
ca?pent~ri,Mimacraea mnrshnlli Trimen, 1: murshulli and f. doherpi, Pap& durdunus Brown
1.. truphonius, Euriphene in’s Aurivillius and several other credible mimics. It is also
involved in a Mullerian mimicry cycle with two Acru~uspecies, A. encedun (L.) and A.
encrdana Pierre (Owen, 197 1; Owen et nl., 1994) arid arguably with several day-flying
agaristid moths (e.g. Wcymeriu uthene Weymer). If.misippus (Owen, 197 1; Smith, 1973,
1976b) and A. enceduna (Owcn el al., 1994) have forms which mimic all four basic
forms (Fig. 2) o f the model.
In terms of Batesian mimicry theory, sympatric polymorphism in the model is
paradoxical since the system can only function smoothly if predators are initially
educated, through an unpleasant experience, to eschew the noxious model so that
they can subsequently bc duped into avoiding the edible mimic (Brower, 1958a, b;
Hrower & Brower, 1964; Brower et al., 1968). An economical education, which is
in fact almost universal, involves propagation by the model of unambiguous and
indelible signals (e.g. nasty taste or smell combined with aposematic coloration) to
any presumptive prcdator. Proliferation of signals is expected to result in more
models being attacked in error and it is for this reason that the D. chrysippus
polymorphism has eluded understanding. Smith (1979) showed that the rarer of two
phenotypes of D.ch?y.rippus suKered significantly more attacks, presumably because
predators had Sewer learning opportunities from encounters with it. Furthermore,
in the same study femalc, H. mis$pus (nialcs are non-mimetic) were less beak-marked
(3.2"/0, n=94) than D.ch?ys$j~us(7.3%, n = 1595). This result suggests that the mimic
is more often consumed after capture while the model is liable to be rejected and
allowed to escape.
In tropical Asia and Australia 11. ch?ysz$pus is polytypic but monomorphic within
each subspecies as are the closely related D.gil$pus and D. eresirnus in the Neotropical
region (Ackery & Vane-Wright, 1984). Narrow hybrid tension zones in, for example,
Heliconius are usual where geographical races meet (Turner, 1972; Mallet, 1986;
Mallet & Barton, 1989a), but polymorphism is rare outside the confines of these
zones. Throughout the three butterfly subfamilies Danainae, Heliconiinae (including
the Acraeinae: Harvey, 1991) and Ithomiinae and the moth families Arctiidae,
Agaristidae, Ctcnuchidae and Zygacnidac, all members of which are distasteful and
aposematic, monomorphism is the almost invariable rule. The unusual case of
polymorphism in ,?'jgaenn ephialtes L. in Central Europe probably results from
hybridization of geoLqaphicalraces (Turner, 197 1). D.ch?ysipPus, A. encedon and A.
encedana, which comprise a Miillerian mimicry ring in East Africa, are three further
exceptions to the rule; hence, there may be a common explanation for all these
polymorphisms. The occurrence of hybrid breakdown in the form of disturbed sex
ratios in the three African species (Owen, 1970; Owen & Chanter, 1968, 1969,
1971; Owen & Smith, 1991; Gordon, 1984; Smith, 1975a, 1976a, 1980; Smith et
al., 1993) is then either a remarkable co-incidence or a vital clue to its interpretation.
Hybridization between races
Our analysis of the sex-ratio genetics of D.chrysippus at Nairobi and Dar es Salaam
suggests that two semisprcies, possibly differing in karyotype and in cytotype, occur
in both areas (Smith et al., in prep). In both reciprocal heterotypic crosses, the
progenies are thclygcnic, all males dying at or soon after hatching. Thelygcnic
females usually themselves produce thelygenic progenies though switches from
amphogeny to thelygeny arid vice-versa are frequent; switches are controlled by the
male genome. O n reversion from a thclygenic line to amphogeny, the progeny is
always of the same karyotype as the stock from which the thelygenic line originated.
Thus, though hybrid females are very common, the two semispecies are effectively
isolated by a post-zygotic mechanism which ensures continuity of cytoplasm and Ylinked genes down the female line. Paternal X-linked genes always pass to male
eggs which die (Smith et al., in prep).
One of the semispecies carries the B and CI loci on the X and Y chromosomes
74
D A. S. SMITH E T AI.
(partial sex-linkage) while the other has independent assortment for the B/C genes
and sex. As there has been extensive recombination, through both independent
assortment and crossing-over in homokaryotic males (the female is achiasmatic in
Lepidoptera (Suomalainen et a/., 1974; Turner & Sheppard, 197S)), it is difficult to
relate the two karyotypes to the subspecific phenotypes. However, the bcY chromosome is, with only rare exceptions, transmitted by hybrid thelygenic females to
their daughters at Dar. Thus it is probable that the sex-linked race is Asian chvysippus
with the female genotype bcX/bcY.
One consequence of hybridisation between karyotypes, and of partial sex-linkage
of the B/C genes in one of them, is the production of significantly different
frequencies of phenotypes in the two sexes as shown in Tables 3, (S), 8 and 9.
Frequencies of B-(brown) and bb (orange) phenotypes differ between the sexes at
four sites (Table 8), browns being commoner among females in each case. ‘lhe
difference is small ( ~ 5 %at) Dar es Salaam but is still highly significant. At the C
locus, C- (dor$$~us)males and cc (chysippus) females exceed expectation at Nairobi
and Dar es Salaam: at the two Ugandan sites the frequency of the C allele is too
low to demonstrate a difference on the small sample sizes. Unexpectedly, however,
the A- and aa phenotypes show a highly significant sex difference at Kampala and
approach significance (0.1>p>0.05) at Ishasha. At both Nairobi and Dar es Salaam,
the a allele is too rare to demonstrate any difference without huge sample sizes. The
Kampala result is especially interesting as the A locus has independent segregation
from both B/C and sex chromosomes in previous studies (Clarke et aL, 1973; Smith,
1975b). The result for the Kampala sample suggests that the A gene might either
be sex-linked or subject to restricted disjunction. Smith et al. (in prep) provide
evidence for Y-linkage (or restricted disjunction) of the A locus (with B/C) in females
from Nairobi and Dar es Salaam which suggests a polymorphism for an A-Y
translocation. Our data (Table 8) suggest that the linkage arrangements in Kampala
could be predominantly aX and AT.
The inevitable consequence of the sex dfierences in phenotype frequency is
heterozygote excess as shown in Figure 3.
The clines which have been demonstrated here may be interpreted as rcsulting
from range expansion by once allopatric semispecies or geographical races. There
is good evidence for post-zygotic isolation between two karyotypes; one, with sexlinkage for the B/C genes is probably chgs$pus of Asian origin, the other, in which
the B/C genes assort independently of sex, probably includes several African races,
brown chlysippus, atc$pus and dorippus; crosses among these phenotypes at Dar es
Salaam showed independent assortment for A, on the one hand, and R/C, on the
other, with no sex-linkage (Smith, 197513).
The two karyotypes do not necessarily differ in chromosome number; all Asian
(ch?ysippus)and Madagascar (liboriu)karyotypes are given as n = 30 (Srivasta Ilr Gupta,
1961; Gupta, 1964; de Lesse, 1972; Saitoh & Kumagai, 1974) while the only
mainland African result from Senegal also gives n=30 (de Lesse & Condamin,
1962). Male bias in some individual amphogenic broods and collectively (Owen &
Chanter, 1968; Smith, 1980), suggest that a Haldane rule effect (Haldane, 1922)
may also be widespread, indicating that hybridization between races of allopatric
origin which share the same karyotype is also somewhat discordant. Evidence for
further karyotype variation is suggested by the genetics of the A gene which certainly
assorts independently in most cases but is apparently sometimes linked to the B/C
POLYMORPHISM IN DRNAIJS CHRYSIPPUS
75
loci and/or sex chromosomes. Taken together, these findings support our interpretation that a widespread and complex hybrid zone is the explanation for the
east African polymorphisms.
The unusual size of the hybrid zone is accounted for by two factors. First, D.
chrysippus is a species of high vagility (Smith & Owen, 1997). Numerous anecdotal
observations and evidence from seasonal changes in morph frequency support the
interpretation that immigration and emi%grationare responsible for gene flow, at
least at Nairobi and Dar es Salaam and probably elsewhere. Migration over long
distances can account for- the observed wide dispersal of genes on the isolation-bydistance model of Wright (1969). Second, the hybrid zone involves not the usual
two but possibly 4-5 races in varied proportions, which have hybridized in different
parts of East Africa, probably at many different times and with diverse consequences.
We are unable at present to describe the distributions of the proposed races in more
than rather general terms as there are few data from large areas of the continent
but further research to rectify this problem is in hand. Migratory movements,
seasonal changes and introgression present further problems.
D.chrysippus, which is the only African member of its genus, probably dispersed
from south-east Asia, across the Indian sub-continent and into Africa, at some time
during the Pliocene or Pleistocene. It may have arrived by two separate routes,
chrysippus coming overland via Arabia and liboria by island hopping across the Indian
Ocean. O n this interpretation, the hypothetical karyotype with sex-linked B/C genes
is the primitive one. Isolation of the African populations from Asia probably occurred
during one or more of the long dry periods, coincident with Northern Hemisphere
glaciations, when the Sahara Desert was often much more extensive than now, both
within and beyond Africa (Williams & Faure, 1980). The wide desert belt across
the Middle East and into India would effectively isolate the African and Asian
populations, at least in the north-east of the African continent. The climatic
vicissitudes within the continent also created refugia, isolated by lowland and
montane forest during pluvials and by sand desert during glacials. Therefore we
envisage that subspeciation would follow a pattern familiar in African birds (Moreau,
1966) and similar to that proposed for several groups of forest animals and plants
in the Neotropical Region (Brown et al., 1974; Whitmore & Prance, 1987).
We suggest that five races of D.chvsippus should be designated in Africa, the first
two of which have a wide distribution outside the continent: orange chrysippus, liboria
( zorientis), brown chrysz$pus, dorippus and alcippus. The remaining named phenotypes
such as transien.s, alcippoides (= weak alcippus of Owen & Chanter, 1968)) albinus,
semialbinus (= weak albinus of Owen & Chanter) and brown dorippus (Smith, 197513,
1980) are hybrid forms scarcely, if at all, extending beyond the hybrid zone. Some
of the hybrid forms, far from being disadvantaged, are common, exhibit positive
heterosis (Smith, 1980) or have acquired heterozygote advantage (Smith, 1981): this
suggests that the polymorphisms, though allopatric in origin, have subsequently
undergone sympatric evolutionary adjustment, perhaps as an adaptation to the
remarkably high frequency of mimia in the area which may threaten the safety of
the models (Owen, 1971; Smith et al., 1993).
Further research is needed more precisely to define the boundaries of the hybrid
zone and the ranges of the races around it. It is also recognized that the five races
are divided between two semispecies (incipient biological species) since, despite
widespread hybridization, two hypothetical karyotypes are substantially isolated by
both pre-zygotic (Smith, 1984) and post-zygotic mechanisms (Smith et al., in prep.).
76
D. A. S. SMITH E7'11f..
It is premature to name the proposed semispecies which are as yet morphologically
ill-defined. To tcst our hypothesis, both cytological examination and mtDNA analysis
of African populations inside and beyond the hybrid zone are highly desirable.
ACKNOWIJDCEMENTS
We are pleased to acknowlcdgc a grant from the Royal Society to thc thrcc scnior
authors to carry out field work in Africa. DASS also thanks the administrators of
the Bonhote Bequest, Idinncan Society of London for a grant towards this work. Dr
Ikrek Whiteley drew the fiLgures.
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