Insectes soc. 45 (1998) 277 – 287 0020-1812/98/030277-11 $ 1.50+0.20/0 © Birkhäuser Verlag, Basel, 1998
Insectes Sociaux
Research article
Estimating the contribution of laying workers to population fitness in African honeybees (Apis mellifera) with molecular markers R.F.A. Moritz 1, *, M. Beye 1 and H.R. Hepburn 2 1
2
Institut für Zoologie, Martin Luther Universität Halle, Kröllwitzer Strasse 44, D-06099 Halle, Germany, e-mail:
[email protected] Department of Entomology and Zoology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa, e-mail:
[email protected]
Key words: Worker reproduction, mitochondrial DNA, nuclear DNA, genetic diversity, Apis mellifera, hybrid zone. Summary Two subspecies of honeybees, Apis mellifera capensis and A. m. scutellata provide an ideal model to test for the significance of reproductive workers in natural populations of honeybees. Laying workers of A. m. capensis parthenogenetically produce female offspring (thelytoky) whereas workers of the other subspecies produce male offspring (arrhenotoky). By using a two allele marker system in both the mitochondrial (mt) and in the nuclear (nuc) DNA, a deterministic population genetical model shows that through the differences in laying worker reproduction alone, clines of the mt and the nuc marker should be shifted. The stronger the impact of laying workers the further should the capensis mt type introgress into the scutellata population. The theoretical model is supported by empirical data from the hybrid zone between the two subspecies. The nuc hybrid zone begins 200 km south of the mt hybrid zone indicating a significant impact of the laying workers on colony reproduction.
Introduction Reproductive hierarchies are typical for insect societies. In the socially most advanced species, a queen dominates her daughter workers which help to raise the queen’s offspring. Reproductive division of labour is particularly strict in honeybee colonies, Apis mellifera. The queen is the only reproducing female and the workers actively eliminate the few eggs which are occasionally laid by other workers, a process which has been coined “policing” (Ratnieks and Visscher, 1989). Queens, however, are not immortal. After loss of the queen, the honeybee colony usually rears an emergency queen from the young female larvae in the colony. In some * Author for correspondence.
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cases however emergency rearing may fail and the colonies become hopelessly queenless, a situation which typically occurs in the case of queen loss after swarming. There is no young brood in these colonies to rear emergency queens and eventually laying workers will develop. The same is seen in cases of failed supersedure (Allsopp and Hepburn, 1997). Laying workers are supposed to represent the last chance of a colony to produce sexual reproductives (drones) before it finally completely dies. Yet in the Cape honeybee, Apis mellifera capensis, this is not so. Here laying workers can produce female offspring (Onions, 1912; Anderson, 1963) which eventually can develop into a new queen (Hepburn and Crewe, 1991). Since the queen is mated with several males, the genotypic composition can change dramatically during take-over by laying worker “pseudoqueens” (Moritz et al., 1996) and many subfamilies in the colony may disappear. Nevertheless, the colony persists at the same nest site although the genotypic composition completely changes. Interestingly, A. m. scutellata, the adjacent honeybee race, does not show this trait of worker thelytoky and laying workers produce male offspring. Here the colony prevails for a few weeks, produces drones until the workers disappear, and finally dies. Since both subspecies form a hybrid zone (Crewe et al., 1993) it may seem surprising that thelytoky does not readily spread throughout the scutellata populations because in theory a capensis colony need never die. However, the situation is not trivial. It could be shown that stable equilibria between both laying worker strategies are unlikely (Moritz, 1986). Greef (1996a) found a wider parameter space for the fixation of the thelytokous rather than the arrhenotokous worker reproductive strategy. The critical question however is, if laying workers play any significant role at all for reproduction under natural and not only theoretical conditions. Laying workers occur in all honeybees after loss of the queen. On the one hand, Ratnieks (1990) argued that the risk of loosing a queen is extremely low. On the other hand, the losses of queens on mating flights have been found to exceed 20% in western Europe (Tiesler, 1972). It seems to be a tedious task to reliably determine parameters such as queen survival in the field. One might easily be trapped by artefacts e.g., due to artificially high colony densities, and queen loss through orientation errors as they may occur on beekeeper managed mating stations. It would therefore be desirable to measure the impact of laying workers in an empirical study of a natural population. The hybrid zone between A. m. capensis and A. m. scutellata in South Africa serves as a prime natural experimental setting to evaluate the reproductive significance of laying workers. Capensis laying workers produce female offspring and hence they pass on both mitochondrial (mt) and nuclear (nuc) genes to further generations. Scutellata workers, however, produce only males which cannot contribute to the mt gene pool. Since honeybees have a limited rate of dispersal during reproductive swarming and mating, the stepping stone model of Kimura and Weiss (1964) may be a helpful tool to understand the genetic demography in the hybridzone. Given suitable genetic markers were found typical for A. m. suctellata and A. m. capensis, the stepping stone model predicts a monotonic cline correlated with geographic distance. The cline for nuc markers, however, should be different to mt markers because the capensis mitochondrial genome is propagated by both queenright and queenless colonies whereas the scutellata mtDNA is exclusively propagated by queenright colonies. Therefore, even given everything else being equal and
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under neutrality of the markers, one would expect mt genes of A. m. capensis to introgress further into adjacent scutellata populations than nuc genes. This shift should be particularly expressed in the male sex. This becomes clear if we imagine the case were laying workers contribute very strongly to the production of sexuals. Males are then primarily produced by scutellata type laying workers whereas queens are of the capensis type. Resulting workers will all have the capensis mitotype but show scutellata markers at considerable frequencies through the male contribution. Materials and methods DNA sampling The theory predicts that there should be a shift between the clines of mt DNA and nuc DNA, the latter particularly strong between male nuc markers and female mt markers. In order to estimate nuc DNA allele frequencies in males we used an indirect method. We sampled 20 workers of each colony to determine the queens’ genotypes and that of the fathering males. Since a queen mates with many males it is possible to determine maternal alleles with single locus DNA fingerprinting. Worker samples of 89 colonies collected in 1993 and 1995 including the putative hybrid zone between both subspecies in South Africa were studied (Fig. 1). Ethanol-preserved worker samples were DNA extracted with routine techniques as described by Moritz et al. (1994).
Fig. 1. Map of southern Africa showing the sample locations
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Mitochondrial DNA analysis Mitochondrial variability was analyzed using a variable DNA repeat region in the COI-COII region (Garnery et al., 1992) which had been shown to reveal a Dra I restriction fragment pattern which is monomorphic in capensis (Moritz et al., 1994). It consists of a 54 bp fragment P0 and a 196 bp sequence Q. Depending on the subspecies, the Q fragment can occur in various repeats making it an extremely useful tool for studies at the subspecies level (Garnery et al., 1993). The fragment was amplified with the following DNA-primer pair: E2: 5′-GGCAGAATAAGTGCATTG-3′; H2: 5′-CAATATCATTGATGACC-3′ The polymerase chain reaction was done with 5 ml 1/5 diluted sample DNA and 25 ml PCR reaction buffer including 60 mM dNTP, 1 U Taq polymerase, and 1 mM of each primer. After denaturation at 92°C, thermocycle conditions were 45 s at 92°C, 45 s at 47°C, 120 s at 70°C for 40 cycles with a final step of 10 min at 70°C. The PCR products were digested with Dra I and restriction patterns were visualized with ethidium bromide after polyacrylamide gel (8%) electrophoresis (150 V for 1.5 h) to identify the haplotype according to the procedure of Garnery et al., (1992). From each colony the haplotype of two workers was determined to identify the haplotype of the mother queen. In addition to the samples already published by Moritz et al. (1994) 64 colonies at 14 locations were screened in the putative hybrid zone. Nuclear DNA analysis Estoup et al. (1995) used single locus DNA fingerprinting for discrimination of several honeybee subspecies. However, we were unable to use this tool for our study of A. m. scutellata and A. m. capensis because intrapopulation variability of the loci A8 and A88 was equally large as between population variation (data not shown). Although Estoup et al. (1995) did discriminate the two subspecies from other honeybee races on the basis of 13 microsatellite loci, overlap in allele frequencies between both subspecies rendered single locus DNA fingerprinting unsuitable for this study. We therefore searched for alternative nuclear markers with a better discriminative power. The variability at the Z-locus which is closely linked to the highly variable sex locus in honeybees (Beye et al., 1994, 1996) proved to have sufficient discriminatory power. A primer pair+ Z1 (5′-AGCCGACTAATATAATTTC-3′) and Z2 (5′-GGAAAGAGGGTTATTATAC-3′) amplified a variable region which yielded DNA fragments ranging from 340 bp to 880 bp. PCR conditions were as follows: 1 ml of a 1/5 dilution of each DNA sample was used in the PCR cocktail which consisted of 9 ml Taq polymerase reaction buffer including 1.5 mM MgCl2 , 0.2 units Taq polymerase, 60 mM dNTP, and 0.4 mM of each primer. The reaction mix was topped with a droplet of mineral oil. After 180 s of DNA denaturation at 94°C the polymerase reaction cycle was repeated 35 times following a thermal regime of 60 s at 94°C, 60 s at 49°C, and 60 s at 70°C . The PCR product was separated in an 8% acrylamide gel (250 V for 1.5 h), stained with ethidium bromide and photographed over a UV light screen.
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Results Mitochondrial markers As reported previously (Moritz et al., 1994) we identified haplotypes with either one, two or three Q repeats in the PCR product. The QQQ type was less frequent than the other two types in the sample. The QQ type was more abundant in areas with the capensis phenotype (sensu Hepburn and Crewe, 1991) whereas the Q type was more frequent in scutellata habitats (Table 1). Particularly north of 30° latitude do we observe a high variability of mitotypes (Fig. 1). South of this line the QQ type is highly abundant with frequencies ranging from 0.89–1.0. The restriction pattern
Table 1. Three different haplotypes characterized by different numbers of the Q-repeat sequence in the COI-COII region of the mtDNA were detected in the transect sampling region from Cape Town to Hoedspruit (Q, QQ, QQQ). The haplotype frequencies were determined at 32 sample locations in South Africa
Location
#
km
n
Q
QQ
QQQ
Cape Town Heidelberg Knysna Addo Ft. Beaufort Stutterheim Cradock Tarkastad Citrusdal Sutherland Queenstown Beaufort West Port St. Johns Hofmeyr Sterkstroom Molteno Dordrecht Victoria West Steynsburg Jamestown Burgersdorp Aliwal North Britstown Zastron Smithfield Ixopo Durban Warrenton Nigel Piet Retief Warmbath Hoedspruit
1* 2* 3* 4 5 6 7 8 9* 10* 11 12* 13 14 15 16 17 18* 19 20 21 22 23* 24 25 26* 27* 28* 29* 30* 31* 32*
0 0 0 50 145 160 205 220 160 180 235 190 240 265 275 290 295 300 307 325 335 375 390 420 425 440 455 550 600 650 690 780
3 5 7 9 3 6 6 6 3 2 9 2 3 4 6 4 6 1 6 3 4 6 4 4 6 3 3 4 3 8 4 4
0 0 0.14 0 0 0 0 0 0 0 0 0 0 0 0.20 0 0 0 0 0 0 0 0 0 0 0.33 0.33 0.5 0.67 0.38 0.25 0.75
1 1 0.86 0.89 1 1 1 1 1 1 0.89 1 1 0.75 0.80 1 1 1 0.83 1 1 0.80 1 1 0.67 0.67 0 0.5 0.33 0.5 0.5 0.25
0 0 0 0.11 0 0 0 0 0 0 0.11 0 0 0.25 0 0 0 0 0.17 0 0 0.20 0 0 0.33 0 0.67 0 0 0.13 0.25 0
n = number of colonies tested. * includes data from Moritz et al., 1994. km = distance north of 34° latitude.
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Table 2. Drone allele frequencies at the Z-locus at 19 sample locations in the hybrid zone between A. m. capensis and A. m. scutellata. Site numbering as in Table 1
Site Allele
3
4
5
6
7
8
11
14
15
16
17
18
19
20
21
22
23
24
25
340 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 840 860 880
– – – 0.11 – 0.11 0.22 0.11 0.11 0.17 0.17 – – – – – – – – –
– – – 0.11 – – – 0.06 0.11 – 0.28 – – – – 0.10 – – – 0.28
– – – – – – 0.24 – – 0.05 0.14 0.14 – 0.38 – – – – 0.05 –
0.08 – 0.16 – 0.08 – 0.16 0.08 0.08 0.08 – 0.08 – – 0.16 – – – – –
– – 0.20 0.10 – 0.20 0.10 – 0.30 0.10 – – – – – – – – – –
– – – 0.17 – 0.17 0.08 0.17 – 0.17 0.13 – 0.04 – 0.04 – 0.04 0.04 – –
– – – – – 0.06 0.16 0.19 0.09 0.34 0.03 0.03 0.03 0.06 – – – – – –
– – – – – – 0.21 0.17 – 0.08 0.17 0.17 0.13 – – – – 0.08 – –
– – 0.06 0.09 0.03 0.06 0.09 0.06 0.06 0.22 0.09 0.06 0.13 – 0.03 – – – – –
– – – – – – – 0.25 0.25 0.25 0.25 – – – – – – – – –
– 0.03 0.03 0.07 0.07 – 0.20 0.13 0.07 0.30 0.07 – 0.03 – – – – – – –
– – – – – – 0.20 0.05 0.15 0.20 – 0.25 0.10 – – – 0.05 – – –
– – – – – – 0.32 – 0.23 – 0.14 – 0.14 0.09 – – 0.05 0.05 – –
– – – – – – 0.25 0.06 0.06 0.25 0.19 – 0.13 – 0.06 – – – – –
– – – – – – 0.20 0.10 0.60 – – – 0.10 – – – – – – –
– – – 0.21 0.03 – 0.21 – 0.32 0.06 0.06 – 0.06 – – – 0.06 – – –
– – – – – 0.08 0.17 0.42 0.08 – 0.08 – 0.17 – – – – – – –
– – – – – 0.04 0.21 – – 0.08 0.13 0.25 0.29 – – – – – – –
– – – – 0.19 0.13 – 0.44 – – 0.06 – 0.19 – – – – – – –
n
18
18
22
12
10
24
32
24
32
8
30
20
22
16
10
34
12
24
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n = number of paternal chromosomes sampled.
Site Allele
3
4
5
6
7
8
11
14
15
16
17
18
19
20
21
22
23
24
25
340 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 840 860 880
– – – 0.25 – – – 0.50 0.25 – – – – – – – – – – –
– – – 0.17 – – – – 0.17 – 0.33 – – – – – – – – 0.33
– – – – – – 0.33 – – – 0.17 0.17 – 0.33 – – – – – –
– – 0.13 – 0.25 – 0.13 – – 0.13 – 0.13 – – 0.25 – – – – –
– – 0.25 – – 0.25 – – – 0.5 – – – – – – – – – –
– – – 0.10 – 0.10 – 0.40 – 0.20 – – – – 0.10 – – 0.10 – –
– – – – – – 0.20 0.20 0.10 0.50 – – – – – – – – – –
– – – – – – 0.17 0.17 – – 0.50 0.17 – – – – – – – –
– – 0.08 0.08 – – – 0.16 0.08 0.16 0.16 0.08 0.08 – 0.08 – – – – –
– – – – – – – 0.33 0.17 0.17 0.33 – – – – – – – – –
– – 0.10 0.10 – – 0.30 – 0.10 0.30 0.10 – – – – – – – – –
– – 0.25 – – – – – 0.25 – – 0.50 – – – – – – – –
– – – – – – 0.33 – 0.50 – – – – 0.17 – – – – – –
– – – – – – 0.33 – – 0.17 0.17 – 0.17 – 0.17 – – – – –
– – – – – – 0.33 0.17 0.50 – – – – – – – – – – –
– – – 0.16 – – 0.25 – 0.42 – 0.08 – – – – – 0.08 – – –
– – – – – 0.50 – – – – – – 0.50 – – – – – – –
– – – – – – 0.25 – – – 0.25 0.25 0.25 – – – – – – –
– – – – 0.17 0.33 – 0.50 – – – – – – – – – – – –
n
4
6
6
8
4
10
10
6
12
6
10
4
6
6
6
12
2
4
6
Contribution of laying workers to population fitness in African honeybees
Table 3. Queen allele frequencies at the Z-locus at 19 sample locations in the hybrid zone between A. m. capensis and A. m. scutellata. Site numbering as in Table 1
n = number of maternal chromosomes sampled.
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of the PCR product with Dra I showed that it was identical to the haplotype supposed be typical for A. m. capensis (Moritz et al., 1994). The Q type which is atypical for capensis is missing in all but two sampled colonies south of 30° latitude, one of which is in Knysna and the other in Sterkstroom. Nuclear markers Twenty different alleles were identified at the Z-locus (Table 2). Since several workers were screened from each colony, it was possible to deduce the genotype of the queen from the segregation pattern. Only the two alleles which were alternatively present in all workers could represent the queen’s genotype (Table 3). The other alleles were of male origin and were used to determine allele frequencies in the male population. Most allele frequencies did not reveal any particular pattern related to geographic distribution of the two subspecies under study. Allele Z-700 however followed a distinct cline along the chosen transect. It was absent in all colonies sampled south of 32° latitude and increased to a frequency of up to 0.29 near 30° latitude in the male population (Fig. 2). The Z-700 allele which is lacking in the capensis samples (locations 3–7) was rare in the queen population. Only four heterozygous queens were identified which each carried one copy of this allele (locations 15, 20, 23, 24). This was significantly lower than in the male chromosome sample in the hybrid zone (locations 14–25; c 2 = 4.08, p < 0.05).
Fig. 2. Frequency of the Z-marker alleles (left y-axis, circles) and the mitochondrial Q-haplotype (right y-axis, squares) plotted against the distance from the 34th° latitude (in km). The putative clines are estimated from a linear regression including the data exceeding 160 km and 370 km for nuc- and mt-markers respectively
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Discussion The stepping stone model predicts that the mtDNA and nucDNA clines should be shifted in the hybrid zone between A. m. scutellata and A. m. capensis if laying workers have a significant impact on reproduction at the population level. Although we only make qualitative predictions, the data nevertheless seems to support that laying workers contribute substantially to the population fitness. The shift between the clines for mt and nuc DNA markers can be explained alone on the basis of neutral selection and symmetrical migration. This does not exclude that selection or asymmetrical migration as suggested by Hepburn et al. (1993) further exaggerates the phenomena. The nucDNA hybrid zone begins about 200 km further south than does the zone of increased Q-haplotype frequencies. Interestingly the molecular marker allele Z-700 which is atypical for capensis rises in frequency in the drone population at the same sample sites where the hybrid zone has been detected by morphometric means (Crewe et al., 1993). The typical capensis mitochondrial haplotype (QQ) extends well into this area. The atypical Q-haplotype increases its frequency only north of 30° latitude. Moreover, the frequency of the scutellata type Z-700 allele remains significantly lower in the queen population than in the drone population throughout the hybrid zone. This was predicted if laying workers contribute to the gene pool by parthenogenetically producing both queens and male sexual reproductives. Worker produced males are expected to resemble the scutellata type whereas worker produced queens should be of the capensis type. We interpret these findings as supportive evidence that laying workers in the honeybee contribute substantially to the overall population fitness. Alternative explanations not invoking laying worker reproduction seem to be less suited to explain the introgression of a mitochondrial type into a nuclear hybrid zone. Drones and queens of both subspecies readily interbreed and there are no indications for any assortative matings which might also result in a similar introgression pattern. A potential selective advantage of the one over the other racial type in specific environments is unlikely since this would require a fitness advantage associated with the mitotype. Although, mitochondria – genotype incompatibilities have been claimed to be significant for honeybee fitness (Harrison and Hall, 1993) these effects were weak and observed for distantly related honeybee subspecies from Africa and Europe. Although we cannot rule out these alternative mechanisms in principle, they clearly require complex additional assumptions which would render the data interpretation less parsimonious. The phenomenon seems to be primarily dependent on worker reproduction in queenless colonies. The honeybee colony has a well developed system of worker policing which efficiently prevents worker reproduction in queenright colonies (Ratnieks and Visscher, 1989). Ratnieks (1988) noted that workers should evolve policing under conditions of queen matings with more than 2 males, because they gain more from the queen’s sons rather than sons of worker sisters. On the other hand Greeff (1996b) showed that policing of thelytokously produced worker eggs in A. m. capensis has no advantage. Laying workers produce mainly worker offspring and only few larvae will be reared to queens. The relatedness to the virgin queen, however, is equal to the queenright condition for all workers (but for the one laying the royal egg) rendering policing nonadaptive. Therefore the reproductive
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potential of thelytokous and arrhenotokous laying workers in queenright colonies may be very different indeed. Whereas the male producing workers primarily reproduce in queenless colonies due to policing, the female producing workers may not be policed. Indeed reproductive workers lay substantial numbers of eggs in queenright colonies (Hillesheim et al., 1989) whereas this only occurs in extremely low frequencies in the other subspecies with arrhenotokous laying workers (Oldroyd et al., 1994). If the effect of laying thelytokous workers in queenright colonies in the hybrid zone is strong (i.e., resulting in worker produced queens), the capensis haplotype should introgress even further into adjacent scutellata populations than predicted under the assumption of neutrality. The impact of worker reproduction in queenright colonies on the gene pool composition in A. m. capensis is unexplored. It will be extremely difficult to discriminate between queen and worker produced brood in queenright colonies because laying capensis workers produce clonal offspring with so far undetected genetic recombination (Moritz and Haberl, 1994). Detection of worker produced females in natural queenright colonies will therefore be infeasible even with advanced molecular genetic tools. Dividing the colony into a queenright and a queenless compartment with queen excluders is regular beekeeping practice and does allow for the observation of laying workers. However, the biological significance of such observations is unclear since queen excluder screens are clearly not forming a particularly natural condition in honeybee colonies. Irrespective of the significance of worker reproduction under queenright conditions in A. m. capensis, our analysis supports the concept that laying workers contribute substantially to the gene pool in honeybee populations. In spite of the strict reproductive hierarchy in the sophisticated honeybee society with the presence of an “infertile” caste, workers seem to be able to directly contribute to the population fitness through the production of sexual reproductives. Most likely queenless colonies will provide most to this worker contribution but also in queenright colonies worker reproduction is not zero. Worker reproduction which is not restricted to more primitively social insects. Moreover, it is not just an “odd” phenomenon in highly eusocial insects but actually a substantial contribution which is measurable at the population level.
Acknowledgements We wish to thank Gregor Slosarek, and Rosemarie Hoffmann for technical assistance. We are grateful to Marius Felder for providing the Z-marker sequence. Financial support was granted by the Deutsche Forschungsgemeinschaft.
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