Kin recognition and inbreeding reluctance in Penelope R. Whitehorn, Matthew C. Tinsley, Dave Goulson

To cite this version:

Penelope R. Whitehorn, Matthew C. Tinsley, Dave Goulson. Kin recognition and inbreeding reluctance in bumblebees. Apidologie, Springer Verlag, 2009, 40 (6), ￿10.1051/apido/2009050￿. ￿hal-00892042￿

HAL Id: hal-00892042 https://hal.archives-ouvertes.fr/hal-00892042 Submitted on 1 Jan 2009

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie 40 (2009) 627–633 Available online at: c INRA/DIB-AGIB/EDP Sciences, 2009 www.apidologie.org DOI: 10.1051/apido/2009050 Original article

Kin recognition and inbreeding reluctance in bumblebees*

Penelope R. Whitehorn, Matthew C. Tinsley,DaveGoulson

School of Biological and Environmental Sciences, University of Stirling, Stirling, FK9 4LA, UK

Received 12 January 2009 – Revised 24 March 2009 – Accepted 27 March 2009

Abstract – Inbreeding frequently has a costly impact on fitness, thus selection has favoured the evolution of kin recognition and inbreeding avoidance behaviour in many species. As haplodiploid , bumblebees are susceptible to additional costs of inbreeding due to their single-locus complementary sex determination (sl-CSD) system, which means that incest can result in the production of costly diploid males. Here we test whether Bombus terrestris reproductives are able to discriminate between kin and non-kin and whether their willingness to mate is adjusted accordingly. We found that B. terrestris reproductives took significantly longer to mate with siblings compared to non-relatives. This indicates that this species exhibits kin recognition and uses this information to determine mating behaviour.

Bombus terrestris / mating / inbreeding avoidance /

1. INTRODUCTION Todrank and Heth, 2003). Incest avoidance via kin discrimination has been reported for sev- In species that suffer from inbreeding de- eral species, including halictine bees pression, mechanisms to avoid mating with (Smith and Ayasse, 1987), the field cricket close relatives are expected to be selected Gryllus bimaculatus (Simmons, 1989), the for (Pusey and Wolf, 1996). Kin recogni- ant Iridomyrmex humilis (Keller and Passera, tion is one such mechanism and close rela- 1993) the termite Zootemopsis nevadensis tives can be identified using either environ- (Shellman-Reeve, 2001) and the cockroach mental (extrinsic) or genetic (intrinsic) clues Blattella germanica (Lihoreau et al., 2007). (Holmes and Sherman, 1983). Extrinsic kin recognition is often context based: individ- It might be expected that bumblebees have uals learn environmental cues, such as the evolved methods of kin recognition as they scent of their nest environment, then identify are particularly susceptible to costs of inbreed- kin as those possessing the same environmen- ing due to their single-locus complementary tal cues (Holmes and Sherman, 1982). Intrin- sex determination (sl-CSD) system (Zayed and sic kin recognition is independent of learning Packer, 2005). The sex-determining locus is and is mediated by recognition alleles: indi- polyallelic; individuals that are heterozygous viduals bearing the same alleles consider one develop into diploid females, whereas hemizy- another as kin (Keller and Ross, 1998). In- gotes become haploid males. However, if indi- creasingly, the definition of kin recognition viduals are homozygous at the sex locus they is restricted only to intrinsic mechanisms, al- develop as diploid males. This occurs rarely in though extrinsic mechanisms, such as nest- large outbreeding populations because many mate recognition, can also lead to clear kin CSD alleles can be maintained by negative fre- discrimination (Barnard and Aldhous, 1991; quency dependant selection (Duchateau et al., 1994). However, genetic drift in small popula- Corresponding author: P.R. Whitehorn, tions is expected to increase diploid male pro- [email protected] duction (DMP) by reducing CSD allelic rich- * Manuscript editor: Jean-Noël Tasei ness (Cook and Crozier, 1995).

Article published by EDP Sciences 628 P.R. Whitehorn et al.

Bumblebee diploid males yield no ge- given a choice queens of Bombus frigidus and netic return for the resources invested in Bombus bifarius preferentially mated with un- them. Bombus terrestris diploid males have related males (Foster, 1992). Males of these smaller testes and fewer spermatozoa than two species exhibit similar pre-mating be- haploid males, and hence suffer reduced fertil- haviour known as ‘patrolling’, where males ity (Duchateau and Marien, 1995). Queens that mark prominent objects with a pheromone and do mate with diploid males may produce a vi- visit them sequentially to encounter potential able colony containing triploid offspring, but mates attracted by the scent (Alford, 1975; the triploid queens are infertile (Ayabe et al., Williams, 1991). In such a situation it is un- 2004). Additionally, as diploid males are pro- likely that reproductives will encounter both duced from the first brood, the majority are siblings and non-siblings at the same time and on the wing too early in the season to en- so choice experiments such as Foster’s (1992) counter virgin queens. The social nature of perhaps do not represent the natural situation. bumblebees predisposes them to further costs Here we take an alternative approach to inves- of DMP: diploid males are produced instead of tigate kin recognition in B. terrestris, another 50% of the female workforce and do not con- species in which males exhibit patrolling be- tribute to colony productivity. This slows the haviour in the wild. rate of colony growth in under B. terrestris is an annual, primitively eu- laboratory conditions (Plowright and Pallett, social species. Under natural con- 1979) and significantly reduces survival of ditions, queens emerge from hibernation in B. terrestris colonies in the field (Whitehorn spring and individually found colonies. Once et al., 2009). the first batch of offspring has been produced, Diploid males occur in rare and localised they take over the tasks of foraging, brood care bumblebee species in the wild. In the Japanese and nest maintenance. Towards the end of the bumblebee Bombus florilegus 28% of sam- colony cycle, usually in the late summer, sex- pled colonies contained diploid males; simi- uals (young queens and males) are produced larly, in the UK, 5% of Bombus muscorum and leave the nest to find mates. The young males were found to be diploid. In both cases queens mate only once and then enter hiberna- this is thought to result from low genetic diver- tion; the old queen, the workers and the males sity, small population size and fragmentation then die. The following spring the queens that (Takahashi et al., 2008; Darvill et al., 2006). have survived hibernation give rise to the next Recent modelling has demonstrated that DMP generation (Alford, 1975). can initiate a rapid extinction vortex (Zayed We present B. terrestris reproductives with and Packer, 2005), which has implications for either siblings or non-siblings and measure the persistence of small genetically impover- their propensity to mate. This may be a more ished populations of bumblebees. In contrast, realistic measure of inbreeding avoidance as in large populations the risk of matings be- a delayed propensity to mate in natural situa- tween bees with identical sex determination tions will reduce the chance of successful cop- locus genotype is low, so long as siblings ulation. do not mate. However, bumblebee nests often produce large numbers of queens and males si- multaneously, so encounters between siblings 2. MATERIALS AND METHODS are likely and inbreeding avoidance behaviour is therefore beneficial. 2.1. Experimental protocol The mating behaviour of bumblebees has Eight laboratory colonies of B. terrestris, pur- been well studied in the laboratory (Djegham chased from Koppert Biological Systems (The et al., 1994; Tasei et al., 1998;Sauterand Netherlands) in February 2008, provided young Brown, 2001; Baer, 2003). By comparison, lit- queens and males. The colonies were checked each tle is known about inbreeding avoidance be- day and new sexuals that had emerged were re- haviours. One study suggested that at least moved and housed in single sex sibling groups. The two bumblebee species recognise kin; when sexuals were mated when between two and ten days Kin recognition and inbreeding reluctance in bumblebees 629

old in mesh-sided flight cages (70 cm × 70 cm × 3. RESULTS 70 cm) between 1st April and 16th April 2008. The matings took place in the laboratory, adjacent to The mating behaviour of 173 young queens large windows, between 1000 h and 1500 h so there from eight colonies was recorded; 70 with were considerable quantities of natural light. non-relatives, 103 with siblings. A mean of Young queens from each colony were either of- 10.8 minutes (± 0.94) passed before a sib- fered their brothers as mates or unrelated males ling mating occurred, compared to a mean from one other randomly chosen colony. Bees were of only 4.5 minutes (± 1.15) for a mating mated in groups (n = 15 to 60), always in a 1:2 ra- between non-relatives. Pooling the data in tio of young queens to males. Only sibling groups this way revealed a highly significant differ- were used, i.e. all males in the mating cage at any ence between sibling and non-sibling matings one time were brothers, and all queens were sisters. (F1,171 = 22.21, P < 0.001). Bees originated Mating pairs were removed from the flight cage dur- from eight maternal colonies; for offspring ing copulation. The mated queens then went into a from seven of these, sibling matings were no- separate study that we have published elsewhere, tably delayed relative to unrelated matings, in which required that we performed twice as many one case time to mate with siblings and non- sibling matings as non-sibling matings. The will- relatives was similar (see Fig. 1). Two-sample ingness of queens to mate with their brothers com- t-tests showed that these differences were sig- pared to non-relatives was investigated by measur- ing the time between the release of bees into the nificant for three out of the eight colonies flight cage and a copulation. All mating sessions (P ranged from 0.018 to 0.024). were terminated after one hour. The proportions of Maternal colony, the number of bees in the mated and unmated queens were recorded for each mating cage and the interaction between the mating batch where more than one mating occurred. maternal colony and the identity of the mate did not significantly influence time to mate (F7,152 = 0.70, P = 0.70; F5,152 = 0.73, P = 0.60; F7,152 = 0.97, P = 0.452 respec- tively). 2.2. Statistical analyses A mean proportion of 0.59 (± 0.06) queens mated within non sibling batches, compared to a mean proportion of 0.43 (± 0.05) queens within sibling batches. This difference was not Data were analysed in Minitab 15 (Minitab significant (F1,22 = 3.46, P = 0.076). The Inc., State College, PA, USA) with a General Lin- proportion of mated queens was influenced by ear Model. The response variable, time to mate, maternal colony (F7,23 = 4.87, P = 0.002) but was box-cox transformed to fulfil the assump- not by the number of bees in the mating cage tions of normality. Mate identity (sibling vs. non- (F6,16 = 0.94, P = 0.497). sibling), maternal colony, number of individuals in the cage and their interactions were included in the model. The model was sequentially simpli- 4. DISCUSSION fied by the step-wise removal of non-significant terms. A further General Linear Model was used For the first time, this study has demon- to analyse an additional response variable, propor- strated that B. terrestris reproductives are less tion of queens mated within a batch. Mate identity, maternal colony and number of individuals in the willing to mate with their siblings than with cage were included in the model, which was again unrelated individuals. Successful copulations sequentially simplified. For bees originating from between siblings took more than twice as long each of the eight colonies, individual t-tests were to initiate than matings between non-relatives. carried out to determine the significance of differ- This suggests that B. terrestris has the abil- ences between the time to mate for sib and non-sib ity to recognise kin and modulates its mating matings. Tests did not assume equal variance and behaviour accordingly. Additionally, a greater were uncorrected for multiple comparisons. Means proportion of queens mated with non-relatives, are recorded ± their standard errors throughout. compared to siblings, but this difference was 630 P.R. Whitehorn et al.

Figure 1. The mean time for reproductives from 8 colonies to copulate with siblings (light grey bars) and non-siblings (dark grey bars). Bars represent the least squares means and their standard errors as predicted by a GLM. The GLM demonstrated that significantly more time elapsed before a queen mated with a sibling compared to a non-relative. Asterisks mark individual within-colony differences that were significant with 2-sample t-tests. The x-axis shows colony ID and sample size in parentheses. not significant. Variation in the reluctance to timately the result of female choice for two mate existed between maternal colonies but main reasons. Firstly, the queen controls the the trend for delayed sib-mating was evident onset of copulation as she must move her sting in experiments on bees from seven of the for the male to be able to insert his genitalia; eight maternal colonies tested. Among-family queens are very choosy, often rejecting many variation is common in bumblebees and has males in the laboratory (Djegham et al., 1994; been found in a number of different fitness Duvoisin et al., 1999)aswellasinthefield traits (Gerloff et al., 2003;Gerloff and Schmid- (Kindl et al., 1999). Secondly, because bum- Hempel, 2005). blebee queens mate only once (Estoup et al., A successful copulation is the result of 1995; Schmid-Hempel and Schmid-Hempel, a number of interacting factors, which can 2000) and males are capable of mating many include male choice, female choice, male times (Tasei et al., 1998) selection acts more courtship behaviour, female response to this strongly on females to choose a mate that will courtship and female reproductive status maximise her fitness. This suggests that the ff (Halliday, 1983). Several precopulatory be- di erent propensities to mate observed in our haviours occur in bumblebees. Conditions in experiment were a result of variations in fe- the laboratory are too artificial for males to male behaviour and B. terrestris queens have set up nuptial routes and exhibit their pa- the ability to recognise siblings. trolling behaviour, but other behaviours that B. terrestris colonies are almost invariably occur once potential mates have encountered headed by a single, singly mated queen, en- each other can be observed. Males approach suring high relatedness of all colony members. females, inspect them with their antennae and Queens may recognise siblings either because then attempt to copulate. Females respond to they are close kin or because they are nest- males in three ways, either by remaining im- mates; the former suggests they use intrinsic mobile, flying away, or exhibiting threat be- genetic cues, whereas the latter suggests ex- haviour by raising their middle legs (Djegham trinsic environmental cues are employed to de- et al., 1994). However, it is not known how dif- termine mating relunctance. Because bumble- ferences in these behaviours influence copula- bees are social it is possible that kin are tion success (Sauter and Brown, 2001). recognised extrinsically through prior associa- Despite the uncertainty surrounding the role tion as has been found in other species (for ex- of precopulatory behaviours, it is likely that ample, Frommen et al., 2007). Alternatively, a successful copulation in bumblebees is ul- an intrinsic mechanism may have developed. Kin recognition and inbreeding reluctance in bumblebees 631

One such mechanism is known as phenotypic sex pheromone, which is more similar within matching in which an individual recognises than among families (Herzner et al., 2006). kin by assessing the similarities and differ- In some social Hymenoptera, this indication ences between its own phenotype and that has been shown to be mediated through the of unfamiliar conspecifics (Blaustein, 1983). chemical composition of cuticular hydrocar- This latter mechanism is thought to occur in bon recognition pheromones, for example in the field cricket Gryllus bimaculatus where the the bee Lasioglossum zephyrum (Smith and females use their own cuticular compounds as Wenzel, 1988), the wasp, Polistes fuscatus a phenotypic template (Simmons, 1989). (Gamboa et al., 1996)andthefireantSolenop- It is beyond the scope of this paper to at- sis invicta (Keller and Ross, 1998). tempt to distinguish which method of dis- The delayed propensity for B. terrestris crimination B. terrestris may be employing. to mate with siblings, demonstrated in this Nevertheless, whatever the underlying cogni- study, is likely to have been selected for as tive mechanisms, the behaviour observed in an inbreeding avoidance mechanism. This in this study represents a clear example of kin turn decreases the production of costly diploid discrimination (Tang-Martinez, 2001). These males. However, in small fragmented popu- findings augment those of Foster (1992), who lations, mate choice is substantially reduced found that queens of B. frigidus and B. bifar- and sibling matings and diploid male produc- ius preferentially mated with unrelated males. tion may become inevitable. This endorses In Foster’s experiment the queens were given the importance of habitat, and hence popula- the choice between mating with a nestmate tion connectivity when considering bumblebee or a non-nestmate, which might suggest that conservation. queens compare males to see which are most different to themselves. However, in our exper- iment B. terrestris queens only ever encoun- ACKNOWLEDGEMENTS tered one type of male and still appeared to discriminate between kin and non-kin. We thank Steph O’Connor, Christiane Nitsch Such kin recognition and avoidance be- and Calum Brown for help with the laboratory haviour is expected to have been strongly se- work. We are grateful to James Weir for technical support. PRW was funded by a NERC studentship. lected for in bumblebees to avoid the costs of diploid male production that result from a mating between siblings. This is in accor- Reconnaissance de la parentèle et mécanismes dance with the model of genetic complemen- pour éviter la consanguinité chez les bourdons. tarity, which assumes that females do not al- Bombus terrestris / accouplement / consanguinité ways choose a male with intrinsically superior / haplodiploidie / reconnaissance de parentèle genes. They may instead choose males with whom they have a higher genetic compatibil- ity, i.e. the viability of offspring depends on Zusammenfassung – Verwandtschaftser- kennung und Inzuchtvermeidung bei Hum- the interaction between the male and female meln. Bei vielen Tierarten führt die Paarung genotypes (Tregenza and Wedell, 2002). Many mit nahen Verwandten bei den Nachkommen studies of genetic complementarity have fo- zu einer geringerer Fitness, ein Phänomen, das cused on polyandrous species where there is als Inzuchtdepression bekannt ist. Solche Arten the potential for postcopulatory female choice sollten daher in der Lage sein, ihre Verwandten zu erkennen und eine Paarung mit ihnen zu (Birkhead and Pizzari, 2002; Colegrave et al., vermeiden. Das Ziel dieses Experimentes war es zu 2002). However, the majority of bumblebee prüfen, ob Hummeln aus der Art Bombus terrestris species are monoandrous (Estoup et al., 1995; ihre Verwandten erkennen können, da Hummeln Schmid-Hempel and Schmid-Hempel, 2000) allgemein als besonders anfällig gegenüber In- ff and in such species there must be some pre- zuchte ekte gelten. Dies vor allem deshalb, da aufgrund der genetischen Strukturen im Hummel- copulatory indication of a male’s relatedness. volk die Paarung zwischen verwandten Individuen In the solitary wasp Philanthus triangulum this zu diploiden Männchen führen kann. Diploide indication is through variation in the male’s Männchen sind steril und werden auf Kosten der 632 P.R. Whitehorn et al.

fleißigen Arbeiterinnen produziert, wodurch das Darvill B., Ellis J.S., Lye G.C., Goulson D. (2006) Hummelvolk geschwächt wird. Daher sollte die Population structure and inbreeding in a rare natürliche Selektion zu Verwandtschaftserkennung and declining bumblebee, Bombus muscorum und Inzuchtvermeidung führen, um die Kosten für (Hymenoptera: ), Mol. Ecol. 15, 601–611. die Produktion diploider Männchen zu umgehen. Djegham Y., Verhaeghe J.C., Rasmont P. (1994) Das Paarungsexperiment wurde in einem großen Copulation of Bombus terrestris L. (Hymenoptera, Flugkäfig (70 cm × 70 cm × 70 cm) aus Gaze Apidae) in captivity, J. Apicult. Res. 33, 15–20. durchgeführt und den jungen Königinnen wurden Duchateau M.J., Hoshiba H., Velthuis H.H.W. (1994) entweder ihre Brüder oder unverwandte Männchen Diploid males in the bumble bee Bombus ter- als Paarungspartner angeboten. Die Bereitschaft restris, sex determination, sex alleles and viability, der Königinnen sich mit ihren Brüdern bzw. den Entomol. Exp. Appl. 71, 263–269. unverwandten Männchen zu paaren wurde ermit- Duchateau M.J., Marien J. (1995) Sexual biology of telt, indem die Zeitspanne zwischen dem Freilassen haploid and diploid males in the bumble bee der Paarungspartner (Königin und Männchen) und Bombus terrestris, Insectes Soc. 42, 255–266. der erfolgreichen Kopulation gemessen wurde. Durchschnittlich 10,8 Minuten (± 0,94) vergingen, Duvoisin N., Baer B., Schmid-Hempel P. (1999) bis eine Verwandtenpaarung stattfand, während Sperm transfer and male competition in a bumble- im Durchschnitt lediglich 4,5 Minuten (± 1,15) bee, Anim. Behav. 58, 743–749. für eine Paarung zwischen nicht verwandten Estoup A., Scholl A., Pouvreau A., Solignac M. Partnern benötigt wurden. Diese Ergebnisse lassen (1995) Monoandry and polyandry in bumble vermuten, dass B. terrestris die Fähigkeit zur Ver- bees (Hymenoptera – Bombinae) as evidenced by wandtschaftserkennung besitzt und entsprechend highly variable microsatellites, Mol. Ecol. 4, 89– dem Verwandtschaftsgrad das Paarungsverhal- 93. ten ändert. Weitere Untersuchungen sollten die Foster R.L. (1992) Nestmate recognition as an in- Mechanismen der Verwandtschaftserkennung breeding avoidance mechanism in bumble bees aufklären. (Hymenoptera, Apidae), J. Kansas Entomol. Soc. 65, 238–243. Bombus terrestris / Paarung / Vermeidung von Frommen J.G., Luz C., Bakker T.C.M. (2007) Kin dis- Inzucht / Haplodiploidie crimination in sticklebacks is mediated by social learning rather than innate recognition, Ethology 113, 276–282. Gamboa G.J., Grudzien T.A., Espelie K.E., Bura REFERENCES E.A. (1996) Kin recognition pheromones in social wasps: Combining chemical and behavioural evi- Alford D.V. (1975) Bumblebees Davis-Poynter, dence, Anim. Behav. 51, 625–629. London. Gerloff C.U., Ottmer B.K., Schmid-Hempel P. (2003) ff Ayabe T., Hoshiba H., Ono M. (2004) Cytological ev- E ects of inbreeding on immune response and idence for triploid males and females in the bum- body size in a social insect, Bombus terrestris, blebee, Bombus terrestris, Chromosome Res. 12, Funct. Ecol. 17, 582–589. 215–223. Gerloff C.U., Schmid-Hempel P. (2005) Inbreeding de- pression and family variation in a social insect, Baer B. (2003) Bumblebees as model organisms to Bombus terrestris (Hymenoptera: Apidae), Oikos study male sexual selection in social insects, 111, 67–80. Behav. Ecol. Sociobiol. 54, 521–533. Halliday T.R. (1983) The study of mate choice, Barnard C.J., Aldhous P. (1991) Kinship, kin discrim- in: Bateson P. (Ed.), Mate choice, Cambridge ination and mate choice, in: Hepper P.G. (Ed.), University Press, Cambridge, pp. 3–32. Kin recognition, Cambridge University Press, Cambridge, pp. 125–146. Herzner G., Schmitt T., Heckel F. Schreier P., Strohm E. (2006) Brothers smell similar: Variation in the Birkhead T.R., Pizzari T. (2002) Postcopulatory sexual sex pheromone of male European Beewolves selection, Nat. Rev. Genet. 3, 262–273. Philanthus triangulum F. (Hymenoptera: Blaustein A.R. (1983) Kin Recognition Mechanisms Crabronidae) and its implications for inbreeding - Phenotypic Matching Or Recognition Alleles, avoidance, Biol. J. Linnean Soc. 89, 433–442. Am. Nat. 121, 749–754. Holmes W.G., Sherman P.W. (1982) The ontogeny of Colegrave N., Kotiaho J.S., Tomkins J.L. (2002) Mate kin recognition in two species of ground squirrels, choice or polyandry: reconciling genetic compati- Am. Zool. 22, 491–517. bility and good genes sexual selection, Evol. Ecol. Holmes W.G., Sherman P.W. (1983) Kin recognition in Res. 4, 911–917. , Am. Sci. 71, 46–55. Cook J.M., Crozier R.H. (1995) Sex determination and Keller L., Passera L. (1993) Incest avoidance, fluctuat- population biology in the Hymenoptera, Trends ing asymmetry, and the consequences of inbreed- Ecol. Evol. 10, 281–286. ing in Iridomyrmex humilis, an ant with multiple Kin recognition and inbreeding reluctance in bumblebees 633

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