BiologicalJoumal ofthe Linnean Sock?& (2000), 69: 483-502. With 2 figures
doi:10.1006/bij1.1999.0371, available online at http://www.idealibrary.com on
lBEal@
Microsatellite DNA analysis reveals low diploid
male production in a comrnunal bee with
inbreeding
R. J. PAXTON*, P. A. THOREN AND N. GYLLENSTRAND
Department o f Genetics, Uppsala Universip, Box 7003, $75007 Uppsala, Sweden
J. TENGO
Ecological Research Station o f Uppsala Universip, Olands Skogsty 6280, S-38693
FdGestaden, Sweden
Received 25 September 1998; acceptedfor publication 5 3u4 I999
The mechanism of sex determination assumed widespread in parthenogenetically arrhenotokous Hymenoptera is that of single locus complementary sex determination (CSD).
Functionally sterile diploid males are produced under CSD and generate a genetic load, the
cost of which increases with inbreeding. We quantlfy diploid male production (DMP,
proportion of diploid individuals that are male) using a morphological criterion (adult fresh
weight) and genetical (microsatellite DNA) markers in a communal, sexually size-dimorphic
bee, Andma scotica, which inbreeds. Male genotypes suggesteda DMP of0.003. The inbreeding
coefficient,A was sipficantly positive (+0.165), equivalent to 44% of matings being among
full sibs (predicted DMP of 0.1 1). We hypothesize three non-mutually exclusive explanations
to account for the large difference between the low observed (in males) and high expected
(derived fromffor females) DMP: (i) multilocus CSD, (ii) ‘sex allele signalliig’ tied to mate
selection, and (i) sperm selection within mated females. The costs of inbreeding through
DMP are apparently low in A. scotica.
0 2000 The Linnean Society of London
ADDITIONAL KEYWORDS:-complementary sex determination - morphology - heterozygosity - sex locus - Hymenoptera - Andrenidae - Andrena scotica.
CONTENTS
Introduction . . . . . . . . . . .
Material and methods . . . . . .
The study organism, Andrena scotica .
Collection of biological material . .
Microsatellite analysis . . . . .
Results . . . . . . . . . . . .
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* Corresponding author. Present address: Zoologisches Institut der Univemitat Tubingen, Auf der
Morgenstelle 28, D-72076 Tubingen, Germany. E-mail: [email protected]
0024-4066/00/040483+20 $35.0010
483
0 2000 The Linnean Society of London
484
R. J. PAXTON ETAL.
Adult morphology, sex ratio at emergence and intranidal mating
Estimation of diploid male production from morphological data
Genetic analysis of females . . . . . . . . . . . .
Estimation of diploid male production from genetic data . . .
Discussion . . . . . . . . . . . . . . . . . . .
Mating structure . . . . . . . . . . . . . . . .
Mechanism of sex determination . . . . . . . . . .
Functional significance of male size variation . . . . . .
Acknowledgements . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . .
Appendix . . . . . . . . . . . . . . . . . . . .
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INTRODUCTION
The basic mode of reproduction in most species of Hymenoptera is thought to
be parthenogenetic arrhenotoky (Crozier & Pamilo, 1996); fertilized eggs develop
into diploid females whilst unfertilized eggs develop into haploid males. Sex determination in such haplodiploids at the genic level is less clearly understood. Of
the competing models, genic balance, genomic imprinting and complementary sex
determination (CSD)have received most attention. There is little evidence supporting
the former and, though recent data suggest genomic imprinting in one chalcid wasp
(Dobson & Tanouye, 1998), CSD is considered the most plausible and widespread
mechanism of sex determination in Hymenoptera (Crozier, 1971, 1977; Cook &
Crozier, 1995).Other models of sex determination have not received much theoretical
or empirical investigation (Beukeboom, 1995; Cook & Crozier, 1995; Crozier &
Pamilo, 1996).
Within CSD, individuals heterozygous at one or more sex loci develop into
females. Haploid (hemizygous) individuals and those homozygous at the sex locus
or loci develop into males. Distinguishing between multilocus and single locus modes
of CSD is difficult for two reasons. Firstly, confirmation of multilocus CSD requires
controlled inbreeding across several generations and, secondly, a multilocus system
easily collapses into a single locus system without judicious technical care to avoid
loss of allelic diversity at several sex loci when founding an experimental population
(Crozier, 1971, 1977; Cook, 1993a).Inbreeding and pedigree analyses support single
locus CSD, albeit merely for a few species of Hymenoptera (reviewed in Cook,
1993b; Crozier & Pamilo, 1996). However, most parthenogenetically arrhenotokous
Hymenoptera are believed to possess single locus CSD (Cook & Crozier, 1995).
For species with CSD, diploid males are occasionally produced. These are
individuals who are the product of a fertilized egg but homozygous at the sex locus/
loci. Though they can have high viability (Camargo, 1982; Duchateau, Hoshiba &
Velthuis, 1994; Duchateau & Marien, 1995), diploid males rarely reproduce, and
would anyhow theoretically give rise to triploid offspring (e.g. Naito & Suzuki, 1991).
Thus diploid males are generally considered to have low to zero fitness (Cook,
1993b; Ross et al., 1993). Their presence rules out genic balance theories of sex
determination and supports, though not exclusively, those based on CSD (Crozier
& Pamilo, 1996). Diploid males have been detected in many aculeate Hymenoptera
(ants, bees and wasps), suggesting they have CSD; indeed, CSD is considered
ancestral within the Aculeata (Cook, 199313; Cook & Crozier, 1995).
Because diploid males generally have very low fitness, CSD imposes a genetic
DIPLOID COMMUNAL BEE MALES
485
load which is particularly strong under inbreeding. Crozier (1971, 1977) proposed
multilocus CSD for hymenopterans with regular, though not exclusive, inbreeding.
He argued that diploid male production (DMP) would remain low under multilocus
CSD as long as occasional outcrossing occurred to restore heterozygosity. However,
inbreeding Aculeata, such as socially parasitic Epimyrmu ants and other ants with
cycles of inbreeding within the nest, are thought to have a mechanism of sex
determination other than CSD (Buschinger, 1989; Cook, 1993b; Cook & Crozier,
1995). The only regularly inbreeding aculeate hymenopteran whose mechanism of
sex determination has been studied intensively is Gonimus nephanfidis. It has neither
single nor multilocus CSD, presumably because of the penalty of diploid male
production under inbreeding (Cook, 1993a). However, to what extent G. nephuntidis
and Epimyrma are exclusive inbreeders is not entirely clear.
Solitary Hymenoptera with CSD are thought to have evolved mating systems
which reduce inbreeding and the costs of DMP (e.g. Bracon hebetor, Antolin & Strand,
1992; Ode, Antolin & Strand, 1995). For social Hymenoptera with CSD, DMP is
likely to impose high fitness costs for individual colonies and their queens because
it results in the potential loss of workers (females with reduced fertility) which are
important for colony survival and reproduction. The mating system of several
eusocial bees has been suggested to have evolved as a means of reducing inbreeding
(Plowright & Pallett, 1979; Foster, 1992) and the costs of DMP (Page, 1980; Crozier
& Page, 1985). There are, however, a number of fully eusocial ants which produce
many diploid males (e.g. Sohopsis invicta: Ross et al., 1999; Formicu spp.: Pamilo et
al., 1994)
In this paper, we study the production of diploid males using a morphological
criterion and genetic (microsatelliteDNA) markers in a European bee, Andrenu scoticu
Perkins 1916 ( =Andrenu jucobi Perkins 1921) (Hymenoptera: Andrenidae). It is an
aculeate species which exhibits a relatively simple social organization and which has
regular, though not exclusive, inbreeding (Paxton & Tengo, 1996; Paxton et ul.,
1996b). We use the very low frequency of diploid males to shed light on A. scoticds
mechanism of sex determination and mating system.
MATERIAL AND METHODS
irhe s t u 4 oqpnism, Andrena scotica
Andrena scotica is a univoltine, fossorially nesting bee, common in North and Central
Europe (Westrich, 1989). The study population, at site Tornbottens Stugby, is
located on the island of Oland, SE Sweden (16" 34' E, 56" 29' N). A previous study
into the social and genetic organization of A. scoticu at the same site (Paxton et ul.,
199613) supports the notion that females are facultatively communal, a less complex
form of social organization (Wilson, 197 1). Michener (1974) has defined communal
species as those in which (i) females share a common nest in which they provision
their offspring yet in which (ii) there is no overlap of generations amongst nestmate
females and (iii) no reproductive division of labour amongst nestmate females. At
the field site, over 500 A. scotica females may have shared a common nest entrance
(in 1993: median=92 females, range 1-594 females, N=37 nests), there was no
overlap of generations of nestmate females, and there was no support for a
486
R.J. PAXTON E T A .
reproductive division of labour amongst nestmates (Paxton et al., 199613); each female
is thought to provision her own brood ( = offspring) cells with self-collected pollen
and nectar in a subterranean, self-constructed cell, attached by a gallery to the
communal nest entrance.
Each A. scotica brood cell is ‘mass provisioned’ with resources (principally pollen
and nectar) by a mother, who then lays a single egg upon the provision mass and
seals the cell. An offspring, female or male, consumes its provisions during summer,
completes development, and then overwinters as adults within its natal brood cell.
Offspring first emerge above ground through their mother’s gallery and nest entrance,
which happens in the following May at our study site (Paxton, Tengo & Hedstrom,
1996). The species shows sexual size dimorphism. Females are generally larger than
males (Paxton & Tengo, 1996), a situation that is common to many aculeates (e.g.
Helms, 1994).
For the majority of bees, including Andrena, females are thought to be receptive
only at or soon after emergence, to mate just once, and to lose receptivity soon
thereafter (Eickwort & Ginsberg, 1980). Exceptions to this female mating system
occur among bees, in particular multiple mating (Page & Metcalf, 1982) and
prolonged receptivity (e.g. Danforth, 1991). For A. scotica, mating appears to occur
only at or soon after emergence, though female mating frequency is not known. A
quirk of A. scotica’s mating behaviour is that mating often occurs before bees first
emerge above ground in spring, and thus within the natal nest (Paxton & Tengo,
1996), and generates a degree of inbreeding (Paxton et aL, 1996b). As well as mating
intranidally, A. scotica males that have already emerged from their natal nest scour
vegetation at between 1 4 m above the ground in the vicinity of nesting sites,
attracting and searching for receptive females (Tengo, 1979).
Collection of biological material
All nest entrances at our field site were permanently and uniquely marked with
metal and plastic tags in 1993 during the flight season of A. scotica (May and June).
Also in 1993, 160 brood cell-provisioningfemales were collected from the entrances
of 8 nests as each female returned to its nest carrying pollen provisions. They were
stored individually at - 80°C for genetic analysis.
In 1994, nylon netting ‘emergence traps’ (for details see Paxton & Tengo, 1996)
were secured over 17 nest entrances (females had been sampled from three of these
nests in 1993) on 29 April, prior to adult emergence. Traps were left in situ until 30
June, after the period of emergence of A. scotica. Adults, the offspring of mothers
that had used these nest entrances the previous year for nesting, emerged from
overwintering in their natal cells and crawled upwards from their natal nest entrances
into the traps. The traps were examined twice or more per day throughout the
period of their use. All adults that emerged from the 17 nest entrances into traps
were counted. They were either released directly or, for those emerging on 57 of
the 63 days of trap use, were first weighed (fresh weight) to +O.l mg to give an
estimate of size at emergence. In other bees, fresh weight has been considered an
accurate measure of size (Rust, 1991), and fresh weight is very closely correlated
with head width in A. scotica (Paxton & Tengo, 1996). The 1994 emergence data
are used to generate the size distributions of females and males as well as the
numerical and investment sex ratios at emergence.
DIPLOID COMMUNAL BEE MALES
487
During the 1995 and 1996 fight seasons, emergence traps were placed over a
smaller number of nest entrances than in 1994, and for a slightly shorter duration
within each season. All adults emerging into traps were weighed. In 1995, a random
sample of 55 females and 103 males that emerged into these traps from 15 nests
was collected and stored at -80°C for genetic analysis. An additional 47 males
that appeared to be much larger than usual and 39 normal-sized males were also
collected fiom emergence traps during all three years of trap use and were weighed
and then stored individually at -80°C for genetic analysis.
To determine whether females had mated intranidally in their natal nest before
their first emergence above ground in spring, some of those emerging in 1994 and
1995 were collected from traps, dissected under insect saline (0.9% NaCl), and their
spermathecae examined using phase contrast microscopy ( x 400 magnification) for
the presence of spermatozoa. The contents of the crop and rectum of each dissected
individual were also examined for pollen or lack of meconium respectively, signs
that the individual had probably been trapped not on its first emergence.
Mimsatellik?anabsis
Each individual was examined at three polymorphic microsatellite DNA loci, AJ07, AJ-25 and AJ-26, previously developed for this species (Paxton et al., 1996b).
PCRs and resolution and visualization of alleles followed Paxton et al. (1996b) except
that PCR products for samples other than 1993females were labelled by incorporation
of CL 33P-dATPinto reactions and were visualized by exposure to Kodak Biomax
film for 24-4-8h. One individual of known genotype was run for every 10 test
individuals for every locus to facilitate scoring of alleles across gels. The DNA extract
of one individual, a female collected in 1993, did not ampl@ at the three loci
employed here nor at four other A. scotica-microsatelltie loci, suggesting its DNA
had become degraded or was lost.
The independence of loci was tested by genotypic (for female data) and gametic
(for male data) linkage disequilibrium for pairs of loci. To determine whether females
and males sampled in different years could have been drawn from a common
population, the significance of variation amongst years and sexes in allele frequencies
was evaluated by exact tests (Raymond & Rousset, 1995a), with the null hypothesis
of identical allelic distribution across samples. For females (diploid individuals), these
values may be inflated due to positive inbreeding coefficients (see below); alleles
drawn from one individual are not a random sample. Thus genotypic differentiation
between females sampled in 1993 ahd 1995 was also calculated.
For females, expected, Hap,(Levene, 1949; Nei, 1978) and observed, Hobs,single
locus heterozygosities were calculated. Mating structure was measured as a departure
Theoretically,
from random mating with the inbreedingcoefficient,$ (Hap-Hds)/Hmp
f ranges from negative values (minimum - 1: negative assortative mating) through
0 (no deviation from random mating) to
1 (complete inbreeding or selfing).
Significance of departure of genotypic proportions from Hardy-Weinberg (HW)
equilibrium was evaluated using the score (qtest with the null hypothesis of
HW proportions and the alternative hypothesis of heterozygote deficit (Rousset &
Raymond, 1995). Where the alternative hypothesis of heterozygote deficit can be
stated in advance, the score test is more powerful than one employing the null
hypothesis of any departure from HW proportions. Previous genetic and behavioural
+
488
R.J. PAXTON ETAL.
analyses of A. scotica (Paxton & Tengo, 1996; Paxton et al., 1996b) have suggested
heterozygote deficit through inbreeding.
Population genetic parameters were estimated and their statistical significance
was evaluated using GENEPOP (Version 3.lb: Raymond & Rousset, 1995b). It
determines the significance of the estimates of parameters using exact tests, these
being suited to the analysis of microsatellite data where there may be many alleles,
each at low frequency. Statistical tests follow Zar (1984) and were performed either
by hand or with the statistical packages STATVIEW 4.5 and SPSS 4.0 for the
Macintosh. Where many painvise comparisons were made, we applied a sequential
Bonferroni correction (Rice, 1989) to control the probability of a Type I statistical
error.
RESULTS
Adult morpholoQ, sex ratio at e m q e n c e and intranzdal mating
Andrena scotica showed clear sexual size dimorphism in 1994.Males were significantly
smaller than females (Mann-Whitney test: r = 5 1.123, P<O.O01), weighing c. 40%
of a female at emergence (mean weight +SE, females: 72.1 kO.2 mg, N=3898;
males: 29.3 k0.2 mg, JV= 1142). Many mass provisioning bees are sexually size
dimorphic, females being larger than males as a consequence of the greater food
masses with which cells destined to contain fertilized eggs (daughters)are provisioned
by their mothers (Klostermeyer, Mech & Rasmussen, 1973;Danforth, 1990).Though
provision mass size is not known for A. scotica, unfertilized eggs of this species are
undoubtedly laid most often in brood cells with small provision masses and fertilized
eggs in cells containing large provision masses.
For adults emerging into traps in 1994, the numerical sex ratio (M/F=0.295,
N= 8970 adults) was significantly female biased (xZ1= 1434.79, P<O.OOOl), assuming
no differential mortality of the sexes. Adults collected from emergence traps in 1995
and 1996, though fewer in number and possibly partially incomplete, similarly
showed significantly female biased numerical sex ratios (1995: M/F = 0.4 12, N=
767 adults, xZ1=69.38, P<O.0001; 1996: M/F=0.219, J\r=278 adults, x2,=63.49,
P<O.OOO 1).
Using mean adult fresh weight at emergence as a measure of cost, the cost ratio
(M/FJ was 0.406 for A. scoticu in 1994. The expected investment ratio under the
assumption of equal investment in the two sexes is given by the reciprocal of the
cost ratio (Boomsma, 1989). The observed numerical sex ratio in 1994 was also
significantly female biased relative to that expected under equality of investment
(x2,= 4205.66, KO.0001; observed ‘fresh weight’ investment ratio in 1994: M / F =
0.1 19). The emergence sex ratio was remarkably consistent across all nests in 1994
(sex ratio data for nest S7 were presented in Paxton & Tengo, 1996); it did not vary
with the number of adults emerging from each nest (Spearman rank correlation of
nests from which at least one individual of each sex emerged, nests weighted equally,
r,=0.103, N==16, NS).
Female and male fresh weights at emergence had a similar absolute range
(minimum and maximum recorded fresh weights for females: 38.7-106.8 mg; males:
[email protected]), but female fresh weights were less variable than those of males
DIPLOID COMMUNAL BEE MALES
489
1000
A
800
m
al
*
$600
d
Q-l
0
8
P
E 400
n'
200
0
I"
I "
~
~
~
~
~
~
~
Weight in mg
Figure 1. The fresh weight distributions at emergence for (A) females (N = 3898) and (B) males (N=
1142) of Andrena scoticu from 17 nests in 1994; weights are grouped into 5 mg classes and show the
class mid-point. The arrow shows the threshold weight (45 mg) above which all males are considered
diploids.
(coefficient of variation (SD/mean as a "/o), female V= 13.1%; male V=26.3%;
variance ratio test F,141, = 3.141, P<O.OOl). Female weight distribution at emergence was unimodal (Fig. 1A). However, it was slightly negatively skewed (GI=
-0.1 1 1 , p<0.05), statistically significantly different from a normal distribution
~
R.J. PAXTON ETAL.
490
(Kolmogorov-Smirnov D-max = -0.024, P<0.05). In contrast, male weight distribution at emergence appeared bimodal (Fig. lB), and was highly positively skewed
(G, = 1.740, PcO.00 1) and significantly different from normal (KolmogorovSmirnov D-max= $0.096, P<O.OOl).
Of more than 600 females emerging into traps in 1994 and who were dissected,
75% contained spermatozoa in the spermatheca whereas 25% did not (data presented
in Paxton & Tengo, 1996), suggesting that many females had mated within their
natal nest before first emergence. The frequency of pre-emergence intranidal mating
was independent of the number of adults emerging from nests (Paxton & Tengo,
1996). Of the 55 A. scotica females who were collected at first emergence in
1995, 25 (circa 45%) contained spermatozoa in their spermathecae and 30 had a
spermatheca devoid of spermatozoa. Crops of all dissected females were without
trace of pollen and their recta were full of meconium, suggesting that all had freshly
emerged from natal nest entrances into emergence nets.
+
Estimation
of dipLoid maLe productionj o m morphological data
If mothers lay fertilized eggs in cells containing large, and unfertilized eggs in
cells containing small provision masses, as female and male weight distributions
suggest, the positive skew of the male weight distribution may have been caused by
diploid males. That is, offspring of fertilized eggs may have been laid in cells
containing large provision masses (destined for female offspring) yet they were
homozygous at the sex locus or loci. Under this hypothesis, males above a given
threshold weight are considered to be diploid, those under the threshold haploid.
Determination of this hypothetical threshold weight is not straighfonvard. Examination of the female weight distribution by eye (Fig. 1A) suggests that males
above circa 40 mg could have been putative diploid males. However, knowledge is
lacking on whether A. scotica shows sex differential conversion of provision mass into
wet weight at emergence; for other aculeates, males are less efficient converters of
provisions to adult body mass (Boomsma, 1989;Danforth, 1990;Visscher & Danforth,
1993; Helms, 1994; Boomsma, Keller & Nielsen, 1995).Also, there may be a range
of intermediate provision mass weights upon which A. scotica regularly lays both
fertilized and unfertilized eggs, possibly a function of the mother’s own absolute
size. Bimodality of the male weight distribution (Fig. 1B) suggests a threshold of
around 50 mg. Deviation of the putative haploid male weight distribution from
normality following removal of all males above various weights was statistically
evaluated with the Kolmogorov-Smirnov test and by examination of skewness.
There was lack of statistical significance at a threshold of between 40 and 50mg
(results not shown). However, this analysis assumes that haploid male fresh weight
at emergence follows a normal distribution, which may not hold.
If all males above a threshold of 45 mg were diploids, then 3.94% of all males
that emerged in 1994 and were weighed were putatively diploid (proportion of
males as diploids =0.039). Put an alternative way, 1.1 Yo of all diploids that emerged
and were weighed were putative diploid male (diploid male production, DMP=
0.0 1 1).
Using a threshold male weight of 45 mg, there was much variation in the number
of putative diploid males that emerged among the 17 nests. However, putative DMP
was not significantly related to the number of females that emerged from nests
DIPLOID COMMUNAL BEE MALES
491
TMLE1. Expected and observed heterozygosities (Hapand Hob$respectively), inbreeding coefficients v;'
+SE in parentheses), and single and multilocus probabilities (f SE in parentheses) of heterozygote
deficit relative to Hardy-Weinberg expectations based on the score (v) test at three microsatellite loci
for a total of 214 Andrena scohu females (159 females from 8 nests in 1993 and 55 females from 9 nests
in 1995)
Locus
Number
of alleles
H"p
Hob$
8
0.566
0.602
0.774
0.647
0.472
0.495
0.654
0.540
x (fSE)
Score (v) test,
Exact P ( f SE)
~~~~~
AJ-07
AJ-25
AJ-26
ALL LOCI
4
9
.
0.000 ( f O.Oo0)
o.Oo0 (~0.000)
0.003 ( f0.000)
O.Oo0 (fO.000)
+0.166 (kO.091)
f0.177 (kO.080)
f0.155 (f0.076)
+0.165 ( f 0.064)
H,: unbiased estimate (LRvene, 1949; Nei, 1978).Standard error offwas obtained by jackknifing over n e a .
(ANOVA of linear regression for the 16 nests from which at least one female or
large male emerged Fj, j 4 = 0.504, NS). Males above the hypothetical threshold
weight emerged across the entire period of emergence, and did not differ in their
timing of emergence from males below the threshold weight (ANOVA Fz,1140=
0.628, NS; mean date of emergence for both groups= 1st June). For either group
of males above and below the threshold weight, there was no relationship of weight
at emergence upon date of emergence (ANOVA of linear regressions, males>45 mg:
FI, 43 = 0.012, NS; males <45 mg: Fi, 1095 = 2.423, NS).
Genetic anabsis offmales
All three microsatellite loci had several alleles, many at intermediate frequency
(Appendix). No significant linkage disequilibrium (genotypic linkage for females,
gametic linkage for males) was detected for females or males collected in different
years (-0.05 for all pairwise comparisons).
Inbreeding coefficients,_f;for the female genotypic data were positive for all three
loci and for both years of collection (1993: +0.16, +0.19, +0.13; 1995: +0.17,
0.14, 0.24 for AJ-07, AJ-25 and AJ-26 respectively; data for 1993were presented
in Paxton et al., 1996b). There were no statistical differences between years of
collection (1993 and 1995) in genotypic proportions (-0.25 for all loci). When the
data from the two years were combined, all three loci obviously also showed positive
inbreeding coefficients (Table 1). Analysis of departure of genotypic proportions
from Hardy-Weinberg equilibrium by the score (U)test revealed significant heterozygote deficit at all loci (Table 1).
Assuming inbreeding to cause the heterozygote deficit, the degree of sib mating
can be estimated. Across loci and years, there was a mean f of 0.165 (Table 1).
At equilibrium, the frequency of full-sib mating, a, for females of haplodiploid
species can be given under simplifylng assumptions by equation 4a of Pamilo (1985),
rearranged as:
+
+
+
a=4f/(3f+ 1)
(and see Li, 1955; Pamilo & Varvio-Aho, 1979; Laidlaw & Page, 1986). For A.
scoctca, the observed f can then be predicted to arise where 01 =0.44, that is, when
circa 44% of matings are between f d sibs.
R. J. PAXTON ETAL.
492
50
40
(0
Z 30
E
Icl
h
n
g
20
z
10
0
Figure 2. Numbers of haploid and diploid Andma scotica males, as determined by zygosity of individuals
at three highly variable microsatellite loci (homozygous or hernizygous: ; heterozygous: m), with
respect to their fresh weight at emergence. Males were collected from 1994 to 1996, but do not represent
a random sample of all males that emerged across these years. The arrow shows the threshold weight
(45mg) above which all males were considered diploids based on morphological criteria.
Estimation of diploud male production j b m genetic data
All 142 males whose fresh weights were less than 45 mg showed a haploid pattern
(homozygousor hemizygous) at all three loci (Fig. 2). For males heavier than 45 mg,
9 of 47 were heterozygous at one locus apiece (Fig. 2). Heterozygous male fresh
weight ranged between 48.0 mg and 73.5 mg.
Using the observed number of heterozygous males to calculate DMP, there was
no relationship between DMP and the number of females emerging from nests in
1994 (ANOVA of linear regression Fl, = 0.0 10, NS).
Allele frequencies (Appendix) did not vary among years or between females and
males (D0.05 for all loci; for diploid individuals, positive inbreeding coefficients
lead to slightly inflated probabilities of allelic differentiation, yet still these are not
significant). Using observed heterozygosities as opposed to HcXp
allows calculation of
the probability that a randomly chosen diploid individual is heterozygous at one or
more neutral loci (Phet):
where i = the ith locus and L = the number of unlinked loci.
Table 1 gives values of HobSfor 214 females. However, approximately 2.5 males
heavier than 45 mg would be expected to occur in a sample of 2 14 females (see Fig.
1). Conservatively assuming all males above 45 mg to be diploid and homozygous
at all microsatellite loci gives revised (minimal)estimates of Hob$
for randomly chosen
diploids of 0.467, 0.490 and 0.647 (loci AJ-07, AJ-25 and AJ-26 respectively), a
DIPLOID COMMUNAL BEE MALES
493
(maximal)estimate forfof +0.174 and a Phct of 0.904 for our three loci. However,
diploid males are unlikely be a random sample of all diploid individuals.
Assumption of singk? locus complementary sex determination
Under CSD, diploid males are likely to come from the most inbred portion of the
population and therefore Phetbased on the Hqb3
of randomly chosen diploid individuals
will be an overestimate of the probability that a diploid male was heterozygous at
one or more loci. We use Wright’s method of path coefficients (Iaidlaw & Page,
1986) to derive an expectedffor diploid males that are produced by inbreeding.
Thereby, we obtain estimates of Hobs( =Hup(l-f)) and Phet for diploid males, and
their expected and total observed numbers.
Assuming single locus CSD, a single generation of brother-fd sister mating
(equivalent to a ‘maternal mother-daughter’ mating for diplodiploids) will lead to,
on average, a DMP of 0.25 (Cook & Crozier, 1995), likely accounting for most
diploid males. With this level of inbreeding, f at generation t can be given as:
J;=0.25 (1+2f;-,
+Ap2)
(Laidlaw & Page, 1986).
If diploid males are functionally sterile andfmeasured in females (mothers) is
+ 0.165 (Table l), then expected f for diploid males brought about through close
inbreeding will be + 0.374.
Allele and genotype frequencies were identical across loci, years, and sexes. Hence
we use H+ from Table 1 and anfof + 0.374 to generate estimates of Hobsfor diploid
males: 0.354, 0.377 and 0.485 (loci AJ-07, AJ-25 and AJ-26 respectively). Slight
error may be introduced into these estimates because males may also be generated
through both lesser and greater degrees of inbreeding, leading to under- and
overestimates of HobJfor diploid males respectively. However, our estimates of Hqb5
should be close approximations.
If all 47 analysed males heavier than 45 mg had been diploid males then, using
our estimates of
we would have expected to have detected 37 diploid males as
heterozygotes at one or more loci (upper and lower limits, based on fk 2 SE for
females across loci and years: 30-39 diploid males as heterozygotes). Only 9 were
actually detected, suggesting that most males heavier than 45mg were in fact
haploid.
Under the above assumptions and estimates of Hd5,Phet for diploid males d be
0.793. The total number of diploid males (&) is:
Nd = N h / p h e t
where & is the observed number of males heterozygous at one or more loci.
Hence we estimate that the total number of diploid males in the group of 47
whose fresh weights were greater than 45 mg, N,, =9/0.793 = 11.4. Given the high
probability of detecting diploid males as heterozygous at one or more loci (phet=
0.793), we assume that all 142 analysed males lighter than 45mg were indeed
haploid.
Notwithstanding our data indicating that diploid males were heavier than haploid
males, our calculations suggest that weight alone cannot be used to determine the
ploidy of a male. Only circa 24% of all males heavier than 45 mg were putative
diploid males, the rest being putative haploid males. For the 1994 emergence data
set, there were 3898 females, 1097 males lighter than 45 mg in fresh weight and 45
494
R. J. PAXTON ETAL.
males heavier than 45 mg. DMP in 1994 can be estimated at 10.9/(3898
0.003.
+ 10.9)=
Assumption of multilocus complmentay sex determination
Under multilocus CSD, a single generation of full sib inbreeding will theoretically
lead to a lower DMP (for 2 sex loci, expected DMP = 0.063; for 3 sex loci, expected
DMP= 0.016) than that expected assuming single locus CSD. Diploid males are
only likely to arise following inbreeding across a greater number of generations
compared to expectations assuming single locus CSD. But, conversely,such extremely
inbred matings are also likely to be rare events becauseffor randomly selected
diploids was maximally estimated at 0.174. Under the hypothesis of multilocus
CSD, diploid males are thus likely to be very rare, as appeared to be the case.
Hence the expectedfof diploid males may be higher under multilocus CSD than
the +0.374 expected assuming single locus CSD. In agreement with the hypothesis
of multilocus CSD, we note that the inbreeding coefficient for the nine males
heterozygous at microsatellite loci was 0.485, undoubtedly an underestimate as
some diploid males will have gone undetected because they were homozygous at
all loci. The actual number of diploids in our sample of 47 males heavier than
45mg is unlikely to have been much higher under multilocus CSD than that
predicted under single locus CSD (c. 1 1 males). This is because, for a fixed f for
randomly chosen diploids, as the degree of inbreeding of some matings increases,
so must the frequency of such inbred matings decrease. Multilocus CSD could
explain the high f and low frequency of diploid males despite the estimated 44%
full sib mating.
+
+
DISCUSSION
Mating structure
The genetic analysis of A. scotica females revealed considerable heterozygote deficit
relative to Hardy-Weinberg expectations. Heterozygote deficit can arise through a
number of causes, in particular through (i) null alleles, (ii) selection, (iii) population
subdivision (the Wahlund effect) or (iv) inbreeding (e.g. Rousset & Raymond, 1995).
Null alleles, those non-scorable due to lack of a PCR amplification product, have
been detected in other studies employing microsatellite loci, where they have led to
apparent heterozygote deficit (Callen et al., 1993; Paetkau & Strobeck, 1995;
Pemberton et al., 1995; Neumann & Wetton, 1996). 'Null allele homozygotes',
individuals that cannot be scored at a locus due to lack of any amplification product,
should then exist, their number being a function of how many individuals are
analysed and the frequency of the putative null allele (Chakraborty et al., 1992;
BrooMield, 1996).
For our data set of 2 14 females, we estimate that-at least 2.7 null allele homozygotes
should have occured, and we would have expected (D0.93) at least one null allele
homozygote, if the observed heterozygote deficit was accounted for entirely through
null alleles. This conservatively assumes DNA degredation led to the one individual
whose DNA was not amplifiable at any locus. Our analysis does not allow categorical
rejection of null alleles explaining the heterozygote deficit in the present data set
DIPLOID COMMUNAL BEE MALES
495
due to a small sample size. However, we have not detected null allele homozygotes
in 180 additional A. scotica females with the same loci, though one would have been
expected (BO.99). Furthermore, none of the putative haploid males has shown a
null allele at any locus. Thus null alleles probably do not account for the heterozygote
deficit observed in A. scatica.
Microsatellite loci are considered to be neutral genetic markers (Queller, Strassmann & Hughes, 1993). Further, selection would need to be of similar magnitude
on three unlinked loci to account for the similar absolute heterozygote deficit
observed in them. Thus selection is unlikely to account for the observed heterozygote
deficit. There is no evidence of subdivision of the population of A. scoha at the
study site, which comprises a single aggregation of communal nests across an area
of 7.5.m x 330.0 m and in which females are no more closely related to nestmates
than to females from distant nests (Paxton et al., 1996b). Inbreeding is then the most
likely cause of the heterozygote deficit in A. scotica.
Female biased sex ratios are predicted under local mate competition and inbreeding
(Hamilton, 1967; Charnov, 1982; Herre, l985), and were observed in A. scotica. The
observed sex ratio in 1994 (M/F= 0.1 1-0.30) would be predicted where competition
occurred for mates Aongst the offspring of 1-2 mothers (Charnov, 1982: 68-88).
Though natural nests contained up to hundreds of provisioning females, a likely
explanation is that the species has a mating system incorporating local mate
competition plus inbreeding within subsections of the communal nest.
Approximately 44% of full sib mating was predicted by the genetic analysis of
females. This high degree of inbreeding is all the more noteworthy given the low
relatedness of female nestmates (Paxton et al., 1996b). The frequency of preemergence intranidal mating varied between years (7545%) but was higher than
the frequency of fdl sib mating. Some, but not all, of the pre-emergence mating
was most likely a result of full sib mating. However, a more detailed analysis of the
relationship between intranidal mating, sib mating and f is hampered by our lack
of knowledge of the synchrony of emergence of kin from subsections of the communal
nest, female mating frequency, nest philopatry, and duration of nest use.
Mechanism
of sex determination
The identification of diploid A. kohca males (heterozygous at one or more loci)
suggests that they have a CSD-based mechanism of sex determination, though the
evidence is not unequivocal. Cytological analysis of heterozygous males is required
to confirm that they are diploid, or demonstration is needed that their alleles are
inherited biparentally (Cook, 1993b). Given several independent lines of evidence
for inbreeding in A. scotica, the suggested presence of CSD is surprising as it
theoretically leads to a genetic load through diploid male production. Predictions
of DMP in A. scotica can be made under varying assumptions. These can be compared
with the DMP observed from the genetic analysis of the zygosity of males, thereby
testing assumptions underlying methods to identlfy diploid males, and also shedding
light on the species’ mechanism of sex determination.
Where male size is used to identlfy putative diploid males and a threshold weight
of 45 mg is used to define male ploidy, DMP is c. 0.01 1. This is lower than that
reported for other bees (e.g. Kukuk & May, 1990; Packer & Owen, 1990). Yet it is
still far higher than that observed through the genetic analysis of the zygosity of A.
496
R. J. PAXTON E'TAL.
scotica males, where DMP is c. 0.003. Moreover, if all heavy males were assumed to
be diploid, they would have an inbreeding coefficient of 0.904, equivalent to 97 Yo
full sib mating, and inconsistent with the genetic and observational data derived
from females. Size is therefore a poor predictor of the ploidy of an A. scotica male.
This is surprising because A. scotica practises mass provisioning of brood cells and
has large sexual size dimorphism. Though the size of diploid males often differs
from that of haploid males in other mass provisioning bees (Packer & Owen, 1990;
Mueller, Eickwort & Aquadro, 1994),our data caution against the use of morphology
alone to define male ploidy in mass provisioning, sexually size-dimorphic Aculeata
(see also Chapman & Stewart, 1996).
Under simplifylng assumptions, the reciprocal of DMP gives an estimate of the
effective number of alleles segregating within a population at a putative single sex
locus (Adams et al., 1977; Owen & Packer, 1994). Assuming panmixia and single
locus CSD, 360 sex alleles would need to exist to account for the extremely low
level of observed DMP in A. scotica. Lack of panmixia would increase the predicted
number of sex alleles (Yokoyama & Nei, 1979). Observations of DMP in other
aculeates suggest they have up to tens of sex alleles (Kerr, 1987; Kukuk & May,
1990; Packer & Owen, 1990; Ross et al., 1993; Duchateau et al., 1994; reviewed in
Cook & Crozier, 1995), far fewer than the number in A. scotica suggested by us. It
is unlikely that selection could be strong enough to maintain such allelic diversity
in a finite population as that predicted in A. scotica under single locus CSD (see
Yokoyama & Nei, 1979).
Our genetic and behavioural data suggest that A. scotica does not have a panmictic
mating structure, but rather that c. 44% of female matings are between full sibs.
Full sib mating will, on average, lead to a DMP of 0.25 under single locus CSD
(Cook & Crozier, 1995). With the degree of inbreeding seen in A. scotica, and
assuming single locus CSD, DMP should be c. 0.110, independent of mating
frequency and the number of sex alleles. Even though the frequency of full sib
mating, a,may be inaccurate, the DMP expected in A. scotica under single locus
CSD is almost two orders of magnitude higher than the observed DMP of 0.003.
The data are inconsistent with single locus CSD in A. scotica.
Differential mortality of diploid males may be one reason for the low DMP in A.
scotica. However, our limited excavation of brood cells suggests that mortality from
egg to adult is generally low (Paxton et al., 1996a).Diploid males in other aculeates
also have high viability (Camargo, 1982; Ross et al., 1993; Duchateau et al., 1994;
Pamilo et al., 1994; Duchateau & Marien, 1995), though those of one Hymenoptera
Parasitica (Bracon hebetor) have very low viability (Whiting, 1943).
The large discrepancy between the observed and the expected DMP can be
accounted for by three other non-exclusive explanations. First, the species may have
multilocus CSD, or another mechanism permitting occasional DMP. For multilocus
CSD, the relationship between the effective number of alleles per locus (k), DMP,
and the effective number of sex loci (n) is:
+
k= 1/("JDMP)
(Cook & Crozier, 1995).
To account for the given DMP in A. scotica, between 3 and 4 sex loci would have
to be invoked. This low number of loci is consistent with the view that selection is
unlikely to be strong enough to maintain heterozygosity at many loci unless they
have pleiotropic effects (Crozier, 1971; 1977; Bull, 1981). Reviewing earlier studies,
Moritz (1986) suggested that a two locus mode of CSD exists in other bees, and
DIPLOID COMMUNAL BEE MALES
497
Chapman & Stewart (1996) present data on low DMP in an inbreeding aculeate
wasp that are more consistent with a two or three locus mode of CSD. If multilocus
CSD exists in A. scotica, the few diploid (heterozygous) males detected might then
have been very highly inbred individuals, as also suggested by their h i g h j Practical
difficulties of controlling matings preclude inbreeding or pedigree studies on multilocus CSD in A. scotica.
A second explanation for the simultaneous occurrence of inbreeding, CSD and
low DMP in A. scotica is the avoidance of ‘matched matings’, in which either or
both sexes refrain from mating with an individual carrying the same sex allele. In
other aculeates, individuals may be able to recognize conspecifics and avoid mating
with kin (Smith, 1983; Smith & Ayasse, 1987; Foster, 1992; Keller & Passera, 1993),
likely as a means of inbreeding avoidance (Pusey & Wolf, 1996). Avoidance of
matched matings, namely specific sex allele recognition, has been suggested to exist
in Hymenoptera with CSD and low DMP (Duchateau et al., 1994; Butcher et al.,
1997). Yet, to date, there is little empirical support for it in any species (Ratnieks,
1991; Cook & Crozier, 1995). In A. scotica, the subterranean mating behaviour and
difficulties in breeding it in the laboratory make the testing of matched mating
avoidance problematic. However, the existence of at least a few diploid males
suggests that avoidance of matched matings in A. scotica is not totally effective, if it
occurs at all.
Thirdly, and more speculatively, of all the spermatozoa received by a female
during mating, one with a sex d e l e different to those of her or her eggs may be
preferentially used for egg fertilization. Such sperm selection, whether active by the
female or passive, could also account for low DMP in A. scotica. Testing this hypothesis
would require comparison by genetic markers linked to the sex locus of a mother,
her spermathecal contents, and her diploid offspring.
Functional signijcance
of male size variation
There was much variation in A. scotica male size at emergence. Brood cell provision
mass undoubtedly provides a proximate explanation for this variation in adult size.
DMP accounts for why a very small proportion of males were much larger in size
than others. Is there a functional explanation for the c. 76% of heavy males (>45 mg)
that were putative haploids?
When mating in the vicinity of the natal site is a regular feature of the mating
system, hymenopteran males have morphological adaptations, such as large overall
size and enlarged head and mandibles (so-calledmacrocephalic males), to monopolize
matings at the natal site (Hamilton, 1979; Kinomura & Yamauchi, 1987; Heinze
& Holldobler, 1993; Heinze et al., 1993; Cook et ul., 1997). A mixed mating system,
in which females mate both intranidally and at some distance from their nest, can
generate opposing selection pressures on male morphology. For communal Perdita
bees with mixed mating systems, males display great size variation (Z? texana: Danforth
& Neff, 1992) or even discrete dimorphism (Rportalis: Danforth, 1991);in the latter
case, large males monopolize intranidal matings whilst small males obtain matings
at flowers (Danforth, 1991). Similar bimodality in male size occurs in Formica ants
and has also been interpreted as an adaptation to alternative male mating tactics
(Fortelius et al., 1987).
As well as mating intranidally, A. scoticu males regularly attract and search for
498
R. J. PAXTON E T A .
females above ground at vegetation away from nests (Tengo, 1979), where matings
have occasionally been seen (JT, pers. obs.). Excavation of nests (Paxton et al.,
1996a) suggests that males do not remain with their natal nest after egress from
their natal cells. The wide variation in A. scotica male size might then reflect the
species’ mixed mating system. If so, large males would be expected to remain within
their natal nest for longer than small males. However there was no size-related
difference in the timing of emergence of males, suggesting male size variation may
be of little or no functional significance with respect to male intranidal versus
extranidal mating.
Alternatively the wide variation in male size, and in particular the presence of
very large males, may have a non-functionalexplanation. A female may underestimate
the amount of provisions within a brood cell and lay an unfertilized egg onto a large
provision mass. Furthermore, the spermatozoa released by a female at oviposition may
fail to fertilize the egg, giving rise to a haploid son within a brood cell containing
sufficient provision mass for a diploid daughter. These and other ‘errors’ have been
suggested to explain why bees produce sons when daughters are otherwise expected
(Raw & O’Toole, 1979; Knerer, 1980; McCorquodale & Owen, 1994). Approximately 3% of putative haploid A. scotica males weighed more than 45 mg, two
standard deviations above the mean, as might possibly be expected, and therefore
not demanding any functional explanation.
Our data suggest that, despite a moderate degree of inbreeding, the costs of
inbreeding through DMP may be very low in A. scotica. More speculatively, a high
frequency of pre-emergence intranidal mating beyond that arising from inbreeding
may provide a selective advantage to communal living in A. scotica; by nesting
communally, a mother may provide her sons with access to additional intranidal
mates and her daughters with a choice of mates in a predator-safe location. Other
communal bees that have been examined in detail also mate intranidally (Danforth,
1991; Danforth & Neff, 1992; Paxton et al., 1999), or possess macrocephalic males
(Kukuk & Schwarz, 1988), suggestive of intranidal mating. Given the practical
difficulties in detecting intranidal mating, it may be more widespread than currently
recognized, and could provide a selective benefit to communal nesting in addition
to those of improved nest defence (Lin & Michener, 1972; Abrams & Eickwort,
1981) and economy of nest construction (Michener, 1974; Eickwort, 1981).
ACKNOWLEDGEMENTS
We are grateful to Arnaud Estoup for advice, help and assistance during all stages
of the development of microsatellites and Pekka Pamilo for a stimulating and
rewarding environment in which to work. For discussion and many helpful and
constructive comments on the manuscript we thank Pia Gertsch, Katja Hogendoorn,
Remko Leijs, Pekka Pamilo, Perttu Seppa and an anonymous referee. Susanne
Gustafsson assisted greatly in the laboratory, as did Helle Sonne in the collection
and weighing of bees. Financial support was provided by the European Union, the
Wenner-Gren Foundation, the DFG PJP) and the NFR (JT).
REFERENCES
AbramsJ, Eickwort GC. 1981. Nest switching and guarding by the communal sweat bee Agapostemon
virescens (Hymenoptera: Halictidae). Insectes Sociuw 2 8 105-1 16.
DIPLOID COMMUNAL BEE MALES
499
Adams J, Rothman ED, Kerr WE, Paulino ZL. 1977. Estimation of the number of sex alleles
and queen matings from diploid male frequencies in a population of Apir mllfera. Genetics 86:
583-596.
Antolin MF, Strand MR. 1992. Mating system of Bracon hebetor (Hymenoptera:Braconidae). Ecological
Entomology 17: 1-7
Beukeboom LW. 1995. Sex determination in Hymenoptera: a need for genetic and molecular studies.
BWESS~S
17: 813-817.
BoomsmaJJ. 1989. Sex-investment ratios in ants: has female bias been systematically overestimated.
American Naturalist 133: 517-532.
Boomsma JJ, Keller L, Nielsen MG. 1995. A comparative analysk of sex ratio investment
parameters in ants. Functional Ecology 9: 743-753.
BrookfieldJFY. 1996. A simple new method for estimating null allele frequency from heterozygote
deficiency. MoLular Ecology 5: 453455.
Bullg. 1981. Coevolution of haplodiploidy and sex determination in the Hymenoptera. Evolution 35:
568-580.
Buschinger A. 1989. Evolution, speciation, and inbreeding in the parasitic ant genus Epimyna
(Hymenoptera, Formicidae).Journal of Evolutiomzy Biology 2: 265-283.
Butcher R, Driessen G, Whitfield W, Hubbard S. 1997. Venturia canescens Gravenhorst: a model
for the investigation of evolutionary selection of mating strategies under complimentary sex
determination.In: Sixth Congress ofthe European Socie93r Evolutionazy Biology. Amhem, The Netherlands,
P1.
Callen DF, Thompson AD, Shen Y, Phillips H, Richards RI, Mulley JC, Sutherland GR.
1993. Incidence and origin of 'null' alleles in the (AC)n microsatellite markers. A ~ n ~ k m J o u r n aof l
Human Genetus 52: 922-927.
C-go
CA de. 1982. Longevity of diploid males, haploid males, and workers of the social bee
Melipona quadn@s&, Hymenoptera, Apidae. Journal ofthe K&as Entomological S O & ~5 5 8-12.
Chakraborty R, De Andrade M, Diager SP, Budowle B. 1992. Apparent heterozygote deficiencies
observed in DNA typing data and their implications in forensic applications.Annuls of Human Genetics
5 6 45-57.
Chapman TW,Stewart SC. 1996. Extremely high levels of inbreeding in a natural population of
the free-living wasp Ancirtrocacs antilope (Hymenoptera: Vespidae: Eumenidae). Heredity 7 6 65-69.
Charnov EL. 1982. 7 7 ~
t?~ozyofsex allocation. Princeton, New Jersey: Princeton University Press.
Cook JM. 1993a. Experimental tests of sex determination in Goniozus nephantidir (Hymenoptera:
Bethylidae). HerediQ 71: 13G-137.
CookJM. 1993b. Sex determination in the Hymenoptera: a review of models and evidence. H m d @
71: 421435.
CookJM, Compton SG, Herre EA, West SA. 1997. Alternative mating tactics and extreme male
dimorphism in fig wasps. Proceedings ofthe Royal Sock9 ofLondon B 264 747-754.
CookJM, Crozier RH, 1995. Sex determination and population biology in the Hymenoptera. T r d s
in Ecologv and Evolution 1 0 281-286.
Crozier RH. 1971. Heterozygosity and sex determination in haplo-diploidy. Ammian Naturalist 105:
39-12.
Crozier RH. 1977. Evolutionary genetics of the Hymenoptera. Annual Review ofEntomoloQ 22: 263-288.
Crozier RH, Page RE. 1985. On being the right size: male contributions and multiple mating in
the social Hymenoptera. Behauioral Ecology and SoGiobiology 1 8 105-1 15.
Crozier RH, Parnilo P. 1996. Evolution ofsocial insect colonies. Sex allocation and kin selection. Oxford,
UK: Oxford University Press.
Danforth BN. 1990. Provisioning behavior and the estimation of investment ratios in a solitary bee,
Calliopsis (Hypoomarrotma) persimilis (Cockerell) (Hymenoptera: Andrenidae). Behavioral Ecology and
So~iobioloD27: 159-168.
Danforth BN. 1991. The morphology and behavior of dimorphic males in Perdituportulir (Hymenoptera:
Andrenidae). Behavioral Ecology and Sociobiology 2 9 235-247.
Danforth BN, NeffJL. 1992. Male polymorphism and polyethism in Pmlita texana (Hymenoptera:
Andrenidae). Annals ofthe Entomological Sock9 of Amen'ca 85: 616-626.
Dobson SL, Tanouye MA. 1998. Evidence for a genomic imprinting sex determination mechanism
in Nasonia uitripennir (Hymenoptera; Chalcidoidea). Gmtics 149: 233-242.
Duchateau MJ, Hoshiba H, Velthuis HHW. 1994. Diploid males in the bumble bee Bornbzu
terrestris. Entomologica Experimentalis et Applicata 71: 263-269.
500
R.J. PAXTON ETAL.
Duchateau MJ, Marien J. 1995. Sexual biology of haploid and diploid males in the bumble bee
B m b u s t t m s t k . Inrecta Sociaux 42: 255-266.
Eickwort GC. 1981. Presocial insects. In: Hermann HE, ed. Social insects. New York, USA Academic
Press, 199-279.
Eickwort GC, Ginsberg HS. 1980. Foraging and mating in Apoidea. Annual Re& ofEntomologv
25: 421-446.
Fortelius W, Pamilo P, Rosengren R, Sundstrom L. 1987. Male size dimorphism and alternative
reproductive tactics in Fonnica exsecta ants (Hymenoptera, Formicidae). Annuli <oobgica Fennici 24:
45-54.
Foster RL. 1992. Nestmate recognition as an inbreeding avoidance mechanism in bumble bees
(Hymenoptera: Apidae).Journal ofthe Kansas Entomological Sockp 65: 238-243.
Hamilton WD. 1967. Extraordinary sex ratios. Science 156 477488.
Hamilton WD. 1979. Wingless and fighting males in fig wasps and other insects. In: Blum MS, Blum
NA, eds. Sexual selection and reproductive competition in inseck New York Academic Press, 167-220.
Heinze J, Holldobler B. 1993. Fighting for a harem of queens: physiology of reproduction in
Cardiocondyla male ants. Aoceediigs ofthe National Academy o f Sciences 9 0 8412-84 14.
Heinze J, Hiihnholz K, Schilder K, Hiilldobler B. 1993. Behavior of ergatoid males in the ant,
Cardwcondyla. Inrectes SoCiaux 40: 273-282.
Helms KR. 1994. Sexual size dimorphism and sex ratios in bees and wasps. American Naturalist 143:
4 18-434.
Herre EA. 1985. Sex ratio adjustment in fig wasps. Science 228 896-898.
Keller L, Passera L. 1993. Incest avoidance, fluctuating asymmetry and the consequences of
inbreeding in Iridomymx humilis, an ant with multiple queen colonies. Behavioral Ecology and Sociobiobgy
33: 191-199.
Kerr WE. 1987. Sex determination in bees. XXl. Number of XO heteroalleles in a natural population
of Mel$ona com@ress+es fm&latu (Apidae).Inrectes Sociaux 3 4 274-279.
Kinomura K, Yamauchi K. 1987. Fighting and mating behaviors of dimorphic males in the ant
Cardiocondyla wughtoni. J o u m l o f EthobD 5: 75-8 1.
Klostermeyer EC, Mech SJ, Rasmussen WB. 1973. Sex and weight of Megachile rotundatu
(Hymenoptera: Megachilidae) progeny associated with provision weights. Journal of the Kansas
Entomological Sociep 46: 536-548.
Knerer G. 1980. Evolution of halictine castes. N a t u k s e n s c h a f i 67: 133-1 35.
Kukuk PF, May B. 1990. Diploid males in a primitively eusocial bee, Lariogbssum (Diali.tuS) zephyncm
(Hymenoptera, Halictidae). Evolution 44: 1522-1528.
Kukuk PF, Schwarz MP. 1988. Macrocephalic male bees as functional reproductives and probable
guards. Pan-P@c Entomobgist 6 4 131-137.
Laidlaw HH, Page RE. 1986. Mating designs. In: Rinderer TE, ed. Beegenetics and breediig. Orlando,
Florida: Academic Press, 323-344.
Levene H. 1949. On a matching problem arising in genetics. Annals ofMaffiematica1 Statistics 2 0 91-94.
Li CC. 1955. Population genetis. Chicago, USA University of Chicago Press.
Lin N, Michener CD. 1972. Evolution of sociality in social insects. Quartmb Rariav OfBiobgy 47:
131-159.
McCorquodale DB, Owen RE. 1994. Laying sequence, diploid males, and nest usurpation in the
leafcutter bee, Megachile mtundata (Hymenoptera: Megachilidae).Journal ofInrct Behauior 7: 73 1-738.
Michener CD. 1974. Ihe social behavior o f the bees. A comparative study. Cambridge, Massachusetts:
Belknap Press of Harvard University Press.
Moritz RFA. 1986. Sex determination in bees other than Apis. In: Rinderer TE, ed. Bee genetics and
breeding. Orlando, Florida: Academic Press, 121-1 45.
Mueller UG, Eickwort GC, Aquadro CF. 1994. DNA fingerprinting analysis of parent-offspring
conflict in a bee. Aoceedings ofthe National Academy OfSCiences 91: 5143-5147.
Naito T, Suzuki H. 1991. Sex determination in the sawfly, Athalia msae @cornis (Hymenoptera):
occurrence of triploid males. Journal ofHeredip 82: 101-104.
Nei M. 1978. Estimation of average heterozygosity and genetic distance from a small number of
individuals. Genetis 8 9 583-590.
Neumann K, Wetton JH. 1996. Highly polymorphic microsatellites in the house sparrow (Paser
domesticus). Molecular Ecobgv 5: 307-309.
Ode PJ, Antolin MF, Strand MR. 1995. Blood-mate avoidance in the parasitic wasp Bracon hebetor
Say. Animal Behaviour 49: 1239-1248.
DIPLOID COMMUNAL BEE MALES
501
Owen RE, Packer L. 1994. Estimation of the proportion of diploid males in populations of
Hymenoptera. Heredip 7 2 2 19-227.
Packer L, Owen RE. 1990. Allozyme variation, linkage disequilibrium and diploid male production
in a primitively social bee Auglochlorella stria& (Hymenoptera; Halictidae). Heredip 65: 241-248.
Paetkau D, Strobeck C. 1995. The molecular basis and evolutionary history of a microsatellite null
allele in bears. Moleculur Ecology 4: 519-520.
Page RE. 1980. The evolution of multiple mating behavior by honey bee queens (Apis mellfera L.).
GW&S 96: 263-273.
Page RE, Metcalf RA. 1982. Multiple mating, sperm utilization and social evolution. American
N&dd I19: 263-28 1 .
Pamilo P. 1985. Effect of inbreeding on genetic relatedness. Hereditas 103 195-200.
Pamilo P, Gertsch P, Thorkn PA, Seppi P. 1997. Molecular population genetics of social insects.
Annual Reviau of&stematics and E c o l o ~28: 1-25.
Pamilo P, Sundstrijm L, Fortelius W, Rosengren R. 1994. Diploid males and colony-level
selection in F m u a ants. Ethology Ecology and Evolution 6: 221-235.
Pamilo P, Varvio-Ah0 S. 1979. Genetic structure of nests in the ant Formica sanguinea. Behavioral
E c ~ l and
o ~ SociobioloQ 6 91-98.
Paxton RJ, Tengo J. 1996. Intranidal mating, emergence, and sex ratio in a communal bee Andrena
jacobi Perkins 1921 (Hymenoptera: Andrenidae).Journal ofZnsect Behavior 9 4 2 1 4 0 .
Paxton RJ, Tengo J, Hedstrijm L. 1996a. Fly parasites and other associates of a communal bee,
Andma sco&a (Hymenoptera: Apoidea), on Oland, SE Sweden. Entomologisk Tuiskn$117: 165-178.
Paxton RJ, Thokn PA, Tengo J, Estoup A, Pamilo P. 199613. Mating structure and nestmate
relatedness in a communal bee, Andrma jacobi (Hymenoptera: Andrenidae), using microsatellites.
M~leculurE c o l o ~5 5 1 1-5 19.
Paxton RJ, Giovanetti MyAndrietti F, Scarnod E, Scanni B. 1999. Mating in a primitively
social bee, A n d m agilissima (Hymenoptera: Andrenidae). Ethology, Ecology and Evolution 11: 371-382.
PembertonJM, Slate J, Bancroft DR, Barrett JA. 1995. Nonamp-g
alleles at microsatellite
loci: a caution for parentage and population studies. Molenrlar E c o h ~4: 24S252.
Plowright RC, Pallett MJ. 1979.Worker-male conflict and inbreeding in bumble bees (Hymenoptera:
Apidae). C d i m Entomologist 111: 289-294.
Pusey A, Wolf M. 1996.Inbreeding avoidance in animals. T i in Ecology and Evolution 11: 201-206.
Queller DC, StrassmannJE, Hughes CR. 1993. Microsatellites and kinship. T i in E c o l o ~and
Evolution 8: 285-288.
Ratnieks FLW. 1991. The evolution of genetic odor-cue diversity in social Hymenoptera. American
Muralist 137: 202-226.
Raw A, O’Toole C. 1979. Errors in the sex of eggs laid by the solitary bee Osmia rufa (Megachilidae).
Behmi1ur70 16€&171.
Raymond MyRousset F. 1995% An exact test for population differentiation.Evolution 49: 1280-1283.
Raymond MyRousset F. 1995b. GENEPOP (V. 1.2): population genetics software for exact tests
and ecumenicism. Joumal ofHeredip 86: 248-249.
Rice WR. 1989. Analyzing tables of statistical tests. Evolution 43: 223-225.
Ross KG, Vargo EL, Keller L, Trager JC. 1993. Effects of a founder event on variation in the
genetic sex-determination system of the fire ant Sohopsis inuictu. Genehs 135 843-854.
Rousset F, Raymond M. 1995.Testing heterozygote excess and deficiency. GenetiGs 140: 1413-1419.
Rust RW. 1991. Size-weight relationships in Osmia lignaria Pr0p;nqua Cresson (Hymenoptera: Megachilidae).Journal ofthe Kansas Entomological So&p 62: 174-178.
Smith BH. 1983. Recognition of female kin by male bees through olfactory signals. hceedings ofthe
N i n a 1 Academy ofsciences 8 0 45514553.
Smith BH, Ayasse M. 1987. Kin-based male mating preferences in two species of halictine bees.
Behaviwal EGOhgy and Sociobiology 2 0 3 13-3 18.
Tengo J. 1979. Odour-released behaviour in A n d m male bees (Apoidea, Hymenoptera).
7:
1548.
Visscher PK, D d o r t h BN. 1993.Biology of Ca1l;OpJ;Fpugionis (Hymenoptera: Andrenidae): nesting,
foraging, and investment sex ratio. Annals of the Entomological Sociep ofAmeriGa 8 6 822-832.
Westrich P. 1989. Die WUienm Badm Wiirttembergs. Stuttgart: Verlag Eugen Ulmer.
Whiting PW. 1943. Multiple alleles in complementary sex determination of Habmbracon. Genetics 28:
365-382.
Wilson EO. 1971. 7he insect societies. Cambridge, Massachusetts: Belknap Press of Harvard University
Press.
zoon
R. J. PAXTON ETAL.
502
Yokoyaxna S, Nei M. 1979. Population dynamics of sex-determining alleles in honey bees and selfincompatibility alleles in plants. Genetics 91: 609-626.
Zar JH. 1984. Biostatktical ana$szi (2nd ed.). Englewood Cliffs, New Jersey: Prentice-Hall.
APPENDIX
Allele frequcncies at three microsatellite loci for Andma scotica females and males collected across 4
years at Tornbottens Stugby. Alleles are designated by a lower case letter, with putative number of
dinucleotide repeat motifs per allele in parentheses. For males, No. alleles counted assumes all nonheterozygotes were haploid (hemizygous).
locus AJ-07
Sample
females 1993
females 1995
males 1994
males 1995
malrs 1996
bees
No.
alleles
a
(22)
b
(23)
(24)
d
(25)
k
(32)
(33)
e
(34)
f
(35)
h
(36)
159
55
70
113
6
318
110
79
113
6
0.082
0.145
0.152
0.150
0
0.654
0.555
0.570
0.549
0.833
0
0
0.025
0
0
0.145
0.164
0.089
0.124
0.167
0.006
0
0
0
0
0.006
0
0
0
0
0.082
0.118
0.165
0.124
0
0.013
0.018
0
0.044
0
0.013
0
0
0.009
0
C
No.
C
g
IOCUSAJ-25
No.
bees
No.
alleles
(12)
d
(21)
(22)
b
(23)
females 1993
females 1995
males 1994
males 1995
males 1996
159
55
70
113
6
318
110
79
113
6
0.208
0.209
0.228
0.22 1
0.333
0.129
0.100
0.127
0.053
0
0.075
0.145
0.089
0.159
0
0.588
0.545
0.557
0.566
0.667
Sample
No.
bees
No.
a
(15)
g
alleles
(20)
b
(21)
(22)
h
(23)
d
(25)
e
(26)
f
(27)
(28)
159
55
70
113
6
318
110
79
113
6
0.352
0.382
0.380
0.310
0.500
0.016
0
0.038
0
0
0.129
0.100
0.127
0.142
0
0.236
0.236
0.291
0.301
0.167
0.003
0
0
0.018
0
0.085
0.091
0.063
0.088
0.167
0.135
0.127
0.051
0.097
0
0.041
0.064
0.051
0.044
0.167
0.003
0
0
0
0
Sample
females 1993
females 1995
males 1994
males 1995
males 1996
a
C
1