Behav Ecol Sociobiol (2002) 52:143–150 DOI 10.1007/s00265-002-0498-7
O R I G I N A L A RT I C L E
David R. Tarpy · Robert E. Page Jr.
Sex determination and the evolution of polyandry in honey bees (Apis mellifera)
Received: 8 December 2001 / Revised: 5 April 2002 / Accepted: 10 April 2002 / Published online: 5 June 2002 © Springer-Verlag 2002
Abstract Many hypotheses attempt to explain why queens of social insects mate multiply. We tested the sex locus hypothesis for the evolution of polyandry in honey bees (Apis mellifera). A queen may produce infertile, diploid males that reduce the viability of worker brood and, presumably, adversely affect colony fitness. Polyandry reduces the variance in diploid male production within a colony and may increase queen fitness if there are non-linear costs associated with brood viability, specifically if the relationship between brood viability and colony fitness is concave. We instrumentally inseminated queens with three of their own brothers to vary brood viability from 50% to 100% among colonies. We measured the colonies during three stages of their development: (1) colony initiation and growth, (2) winter survival, and (3) spring reproduction. We found significant relationships between brood viability and most colony measures during the growth phase of colonies, but the data were too variable to distinguish significant non-linear effects. However, there was a significant step function of brood viability on winter survival, such that all colonies above 72% brood viability survived the winter but only 37.5% of the colonies below 72% viability survived. We discuss the significance of this and other “genetic diversity” hypotheses for the evolution of polyandry. Keywords Mating systems · Polyandry · Social insects · Sex determination
Communicated by L Sundström D.R. Tarpy (✉) · R.E. Page Jr. Entomology Department, University of California, Davis, CA 95616, USA Present address: D.R. Tarpy, Department of Neurobiology and Behavior, Seeley G. Mudd Hall, Cornell University, Ithaca, NY 14853, USA email:
[email protected] Tel.: +1-607-2544377, Fax: +1-607-2544308
Introduction Kin selection (Hamilton 1964) is the leading explanation for the evolution of insect social behavior. Individuals forfeit their own reproductive success in exchange for inclusive fitness benefits derived by raising close relatives (particularly female sexuals). Multiple mating by queens, or polyandry, occurs in several social insect taxa (Page 1986), and creates a difficulty for kin selection (reviewed by Bourke and Franks 1995; Crozier and Pamilo 1996). As mating number increases, the average genetic relatedness among sister nestmates declines by the function 0.25+0.5/me, where me is the effective mating number of the queen mother. Thus polyandry reduces the relatedness among workers within a colony and diminishes their potential inclusive fitness. The inconsistency between kin selection and polyandry has been a focus of sociobiology beginning with Hamilton (1964) and continuing to the present day (Bourke and Franks 1995; Crozier and Pamilo 1996; Schmid-Hempel 1998; Keller and Reeve 1999). Extreme levels of polyandry are reported in honey bees (Apis spp.), where queens mate with 8– 27 males on average depending on the species in question (reviewed by Palmer and Oldroyd 2000). Many hypotheses have been proposed to explain the potential benefit gained from polyandry (Arnqvist and Nilsson 2000; Jennions and Petrie 2000), but several reviews have concluded that the “genetic diversity” hypotheses are the most plausible explanations for polyandry in honey bees and other social Hymenoptera (Crozier and Page 1985; Keller and Reeve 1994; Oldroyd et al. 1998; Cole and Wiernasz 1999; Palmer and Oldroyd 2000). The genetic diversity hypotheses propose that multiple mating is adaptive because it increases the genotypic variation within a colony. As mating number increases for a queen, the number of subfamilies (female offspring that share the same father) similarly increases. Therefore, genes for different traits become varied among colony members, enabling them to better adapt to different ecological conditions. Although this basic principle is
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the same among the hypotheses, each attributes the adaptive advantage of genetic variation to one of the following selective forces: 1. Genetic diversity increases the behavioral diversity of the worker task force (Oster and Wilson 1978; Fuchs and Schade 1994; Oldroyd et al. 1994; Fuchs and Moritz 1999), possibly to allow a colony to exploit different foraging environments more efficiently (Oldroyd et al. 1992; Lobo and Kerr 1993). 2. Genetic diversity provides a buffer against fluctuations in the environment (Crozier and Page 1985; Page et al. 1995). 3. Genetic diversity reduces the prevalence of parasites and pathogens among colony members (Hamilton 1987; Sherman et al. 1988; Shykoff and SchmidHempel 1991a, b; Schmid-Hempel 1995, 1998; Baer and Schmid-Hempel 1999). 4. Genetic diversity reduces the optimal sex ratio of the workers to that of the queen (Moritz 1985; Queller 1993; Ratnieks and Boomsma 1995). 5. Genetic diversity reduces the variation in diploid male production as a consequence of the single-locus sex determination system (Page 1980; Crozier and Page 1984; Ratnieks 1990; Pamilo et al. 1994; Crozier and Pamilo 1996). The last of these hypotheses has a simple, well-known genetic mechanism that underlies the potentially-adaptive effect of polyandry. The mechanism involves the sex determination system of most social ants, bees, and wasps, where the sex of an individual is governed by a single gene with numerous alleles (Cook 1993). Because of the haplodiploid genetic structure of the order Hymenoptera, females have two copies of the sex gene while normal males have only one. Fertilized eggs that possess two different alleles at the sex locus are heterozygous and develop into females. In honey bees, fertilized eggs that are homozygous at the sex locus develop into sterile males, but are not raised to maturity (Woyke 1963, 1965). A high proportion of these “diploid drones” within a colony results in low worker brood viability, which presumably has a negative impact on colony fitness (Page 1980). Previous theoretical work has demonstrated a direct relationship between queen mating number and colony brood viability, such that the variance in diploid male production decreases as effective mating number increases (Page 1980; Page and Marks 1982; Crozier and Page 1985; Pamilo et al. 1994; Crozier and Pamilo 1996; Palmer and Oldroyd 2000). Colony brood viability converges upon a population average proportion as mating number increases, the value of which depends on the number and frequency of sex alleles in the population. Similar to many risk sensitivity models (e.g., Smallwood 1996), these studies have shown that polyandrous queens only gain an adaptive advantage there are diminishing fitness returns associated with increasing brood viability, i.e. there is a concave fitness relationship around the average viability of the population (Page 1986).
Woyke (1980, 1981) tested the effect of brood viability on several colony variables. He demonstrated that colonies in the 50% brood viability group had significantly smaller worker populations and honey yields than did colonies in the 75% or 100% brood viability groups, suggesting a concave effect of brood viability on colony fitness (Woyke 1980). Other factors, however, suggest convex relationships, such as brood area (Woyke 1980). In a repeated study, the relationships between brood viability and these same colony variables appear to be linear (Woyke 1981). Regardless, these measures are fitness correlates in a managed apicultural setting, and it is yet unclear how brood viability impacts fitness components. It would be helpful, therefore, to vary brood viability on a more continuous scale (rather than in groups) and to test the hypothesis in a context that more closely resembles the annual life cycle of a colony to determine direct fitness effects. New honey bee colonies are initiated by swarms in the spring that grow and develop through the summer (Seeley 1985; Winston 1987). Nectar collected during the foraging season is stored as honey for the workers to consume during the winter. Surviving colonies then increase their worker populations, produce reproductive males (drones), and, if possible, produce reproductive females through colony fission, or swarming. The present study simultaneously established colonies with varying brood viability and measured them during three important phases of their life history: (1) colony initiation and growth, (2) winter survival, and (3) spring reproduction. The impact that brood viability has on these three stages determines the fitness effects of diploid male production and the potential for polyandry to evolve.
Methods Experimental subjects and colony establishment The experimental queens were derived from the same mother and father so that they were related by G=0.75 (Crozier and Pamilo 1996). We raised experimental drones from the same colony by placing frames of male-sized brood cells within the hive. The queens were, therefore, super sisters to each other and sisters of the experimental drones (see also Woyke 1980, 1981). We instrumentally inseminated each queen with the semen of three brothers. We collected approximately 1 µl of semen successively from each of the three males in an insemination syringe (Laidlaw 1977). To mix the semen, we injected it into a 1.5 ml Ependorf tube, repeatedly expelled and recollected it with the syringe, and spun it for 30 s at 14,000 g using a microcentrifuge (Moritz 1984). We only used queens in the experiment that laid fertilized eggs within their respective ‘mating nuclei’ prior to the experiment. We initiated colonies to mimic the founding of a swarm during the spring. Each hive consisted of a single Langstroth chamber containing two frames of drawn comb, one frame of honey, and two frames containing wax foundation. Wax foundation is given to hived bees to provide the substrate for constructing new combs. Each ‘foundation frame’ contained only half a frame of foundation so that colonies would construct new comb using their own resources. We weighed each empty hive unit (including their frames) to the nearest 0.114 kg (0.25 lb) on a platform scale.
145 We randomly introduced commercially-purchased 0.9 kg (2 lb) packages of bees (approximately 7,000 workers) into each hive. During the worker introductions, we also placed a queen cage– each containing a single experimental queen– into each hive following standard introduction methods (Laidlaw and Page 1997). We inspected the colonies 1 week later to determine if the queens were accepted by the workers and laying eggs. 3. Brood viability measurements We measured the viability of worker brood in each colony 13 days after the queens began to lay eggs. We chose this time because the first fertilized eggs that were laid by the introduced queens had reached the capped, pupal stage. Diploid drones derived from those first egg cohorts had been killed after hatching, leaving unoccupied cells among those patches of brood. Any worker eggs subsequently laid within those empty cells would have been in the larval stage and, therefore, unsealed. Counting the number of sealed cells versus empty cells or those with larvae within contiguous areas of brood provides an estimate of the brood viability within a particular colony (Mackensen 1951). We performed counts by randomly sampling three to five regions of the comb containing 100 brood cells. We conducted second measurements of brood viability well after the colonies had been established using a different method. We placed sections of clear plastic over contiguous areas of eggs within each colony and held them in place on the top bars of the frames with thumb tacks. We marked brood cells that contained eggs with a blue pen on the plastic sheets. Nine days later, we placed the plastic sheets over the same patches of brood, and we re-marked uncapped cells with a black pen over top of the blue mark. We calculated the ratio of blue to black marks for each colony to estimate the proportion of diploid drones produced by the queen. In several instances, entire sections of eggs were consumed by the workers between the two measurement periods. For example, one colony with approximately 100% brood viability had its entire patch of eggs removed and replaced with nectar. Therefore, we used the highest brood viability measure obtained from the two methods in the analyses. In no case was the second measurement significantly higher than the first. There was a continuous distribution of brood viability among the colonies, probably a result of variation in contributions of sperm for the three males used to inseminate each queen. The viability scores in the experimental colonies ranged from 46% to 95%, slightly lower than the idealized 50–100% range, most likely because of factors other than the sex locus (Woyke 1962b, 1976; Fukuda and Sakagami 1968). We expected this distribution, and it provided a more continuous scale for the experimental objective. Colony evaluations We inspected the colonies every week to ensure the presence of the experimental queens and to add additional frames. We added a foundation frame to a colony when the comb on an outermost frame was under construction. We added foundation frames to the colonies as needed until a maximum of 10 frames occupied a colony, slightly less than the total surface area of a median-sized feral nest studied by Seeley and Morse (1978). We measured the colonies every 3 weeks after they were established. We conducted the evaluations using a hand-held metal grid with 2.54-cm squares to estimate the surface area of several factors on each frame in each colony. 1. Worker population was measured by estimating the surface coverage of adult bees, taking into account the relative density of the workers. The number of workers was estimated by approximating one worker per square centimeter. 2. Total brood area comprised the total amount of comb in the brood nest. It was impractical to measure the “effective” brood area– the actual amount of brood comb that contained developing workers– because of the inconsistency of queen oviposi-
4. 5.
6.
7.
tion patterns within and among colonies. Although a “corrected” brood area could be calculated by multiplying the total brood area by a colony’s brood viability, it would not be justified because queens may revisit brood patches and oviposit in empty cells made available from consumed diploid males (Woyke 1963; unpublished data). Therefore, this measure signifies ‘overall brood nest size’ rather than ‘amount of brood’. Stored honey was determined by measuring the total surface area of both capped and uncapped honey and nectar. This measure did not take into account any differences in comb thickness, therefore greater honey stores as a result of deeper cells were not represented by this variable (but see colony biomass). The surface area of stored pollen was also measured, often by summing partial square grids because pollen cells were patchily distributed. Empty space, the amount of comb outside the brood nest that was not used for honey or pollen storage, was also calculated for each colony. Unconstructed wax foundation was not included in this measure. The total amount of wax comb was determined for each colony by adding the surface areas of brood, honey, pollen, and empty comb. These factors totaled 1,720 cm2 for both sides of a full frame, so differences in total comb area were only observed among colonies after new wax substrate was constructed on the foundation frames. Drone brood area was determined by counting the number of square grids of male brood cells that contained larvae or pupae. The outside frames only contained half sheets of wax foundation with worker-sized cells. Colonies typically reared the majority of their drones on the bottom half of the outside frames, therefore most of the drone comb was newly-constructed by the colony rather than existing previously.
We measured the variables on each frame and entered the data into a Tandy 102 laptop computer, which totaled each measurement for each colony. We weighed each colony to the nearest 0.114 kg using a platform scale during the early evening on the days of evaluation when foraging activity had significantly diminished. We calculated the change in colony biomass by taking the measured weight and subtracting the initial empty hive weight plus the combined weights of any introduced foundation frames (0.193 kg each). Finally, we obtained worker dry weight measurements 3 times, twice during the summer of 1998 and once in the spring of 1999. We placed 8– 25 newly-emerged workers into zip-lock plastic bags and frozen at –20°C several hours later. We dried the workers in an oven and weighed them on a Mettler H20 scale to the nearest 0.1 mg. We conducted a total of six evaluations during the course of the active season in 1998, the last of which occurred 18 weeks after colony initiation. We provided each colony with standard disease treatments, including two Apistan strips to control the parasitic mite Varroa destructor and an antibiotic grease patty (containing Terramycin, sugar, and vegetable shortening) to control American foulbrood (Penabacillus larvae), European foulbrood (Melissococcus pluton), and tracheal mites (Acarapis woodi). All colonies remained at the field site, without manipulation, after we performed the final evaluations of the summer. We inspected the colonies periodically throughout the winter months to verify colony survival. We resumed the colony measurements the following spring, which were conducted every 3 weeks until mid-July.
Results Colony initiation, growth, and development “Tokens” of fitness are factors that correlate with colony fitness but are not direct measures of reproduction (Page et al. 1995). For honey bee colonies, these factors include worker population, brood area, stored honey and
146 Table 1 Pairwise correlations for end-summer colony fitness tokens in the honey bee (Apis mellifera). Significance level is subject to a type I error correction (see text) Workers
Brood
Honey
Pollen
Empty
Comb
Biomass
Bee weight
1
0.888 ****
0.804 ****
0.433 *
–0.776 ****
0.762 ****
0.865 ****
0.369 *
Workers
1
0.574 ***
0.394 *
–0.605 ***
0.710 ***
0.653 ****
0.372 *
Brood
1
0.455 **
–0.854 ****
0.762 ***
0.9679 ****
0.321 n.s.
Honey
1
–0.594 ***
0.391 *
0.459 **
0.278 n.s.
Pollen
1
–0.442 *
–0.836** **
–0.032 n.s.
Empty
1
0.785 ****
0.290 n.s.
Comb
1
0.323 n.s.
Biomass
1
Bee weight
*P
