Insect. Soc. DOI 10.1007/s00040-015-0428-0

Insectes Sociaux

RESEARCH ARTICLE

Mating frequency and maternity of males in Melipona mondury (Hymenoptera: Apidae) M. V. C. Viana1 • C. A. L. de Carvalho2 • H. A. C. Sousa1 • A. K. Francisco1 • A. M. Waldschmidt1

Received: 29 December 2014 / Revised: 19 July 2015 / Accepted: 23 July 2015 Ó International Union for the Study of Social Insects (IUSSI) 2015

Abstract In eusocial bees, multiple mating can increase colony fitness, but decreases relatedness between female offspring and causes a conflict between workers for male parentage. In this work, the paternity and maternity of males in Melipona mondury were evaluated using microsatellite loci. Contrary to expectation, 78 % of M. mondury queens had multiple mates (2–7 mates). The genetic relatedness between sister workers ranged from 0.53 to 0.90 in polyandrous colonies. The production of males by workers ranged from 0 to 100 %, with a mean value of 50 %, following the common pattern reported for stingless bees, mainly Melipona species. The high worker–worker relatedness in polyandrous colonies favored worker reproduction. These results corroborate inclusive fitness theory and can support future conservation genetics studies of stingless bees. Keywords Polyandry  Maternity  Microsatellites  Stingless bee

Introduction Eusocial behavior, as observed in bees, imposes a trade-off at a colony level once the reduced effective population size inherent to this life mode reduces the genetic variability

& M. V. C. Viana [email protected] 1

Departamento de Cieˆncias Biolo´gicas, Universidade Estadual do Sudoeste da Bahia, Av. Jose´ Moreira Sobrinho s/n, Jequie´, Bahia Zip Code 45206-190, Brazil

2

Nu´cleo de Estudo dos Insetos (Insecta), Universidade Federal do Recoˆncavo da Bahia, Rua Rui Barbosa, 710, Cruz das Almas, Bahia Zip Code 44380-000, Brazil

(Graur 1985). On the other hand, multiple mating can maximize colony adaptive value by increasing genetic variability (Baer and Schmid-Hempel 1999; Palmer and Oldroyd 2000) and effective population size (Borges et al. 2010) and by decreasing the frequency of diploid males (Tarpy and Page 2001). Most reports show that queens of Bombus species and stingless bees mate once or a few times (Imperatriz-Fonseca et al. 1998; Paxton et al. 1999; Palmer et al. 2002; Owen and Whidden 2013; Jaffe´ et al. 2014), while polyandry is common within Apis (Palmer and Oldroyd 2000; Kraus et al. 2005). Theoretical predictions state that in the cases of monogyny and polyandry, the workers are, on average, more related to their brothers (r = 0.25) than to their nephews (r = 0.125). This triggers a social conflict among workers that is controlled by collective policing, i.e., strategies to suppress the laying of workers, such as aggression or egg destruction so the proportion of offspring from the queen bee is increased (Ratnieks 1988; Ratnieks et al. 2006). When monogyny and monandry are present, the relatedness between a worker and its brother (r = 0.25) is lower than that compared to the nephews (r = 0.375) and favors worker reproduction (Ratnieks 1988). The stingless bee Melipona mondury Smith, 1863 is found in the Atlantic Forest of Brazil, ranging from states of Bahia to Santa Catarina (Melo 2003). Its habitat has been reduced and fragmented (Fundac¸a˜o SOS Mata Atlaˆntica and Instituto Nacional de Pesquisas Espaciais 2010), leading to population decreases and low genetic variation (Tavares et al. 2007). In the state of Bahia, this species inhabits humid, low forest regions (de Sousa et al. 2012), being poorly known by beekeepers. Little information is available about its biological and genetic aspects. Therefore, the goal of this study was to test theoretical predictions of intracolony conflicts and to support conservation studies by

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evaluating the frequency of queen matings, intracolonial genetic diversity, and maternity of males in M. mondury.

To estimate the mating frequency of queen bees and maternity of males, we sampled the queen, 10 worker pupae, and about 10 males from each of 10 colonies (Table 1) from the meliponary on Ouro Fino farm in Jaguaquara (13°430 29.8200 S, 39°470 24.7200 W, 292 m), state of Bahia, within the natural range of M. mondury. The colonies of this meliponary were removed from dead or chopped trees and from fire risk areas in a 15-km transect and kept in boxes for over 20 years, without artificial selection. Additional 28 colonies from the same meliponary were sampled, and a total of 38 colonies were used to estimate allele frequencies, observed heterozygosity (Ho), and unbiased expected heterozygosity (He) at the population level, using one worker per colony. Total DNA was extracted according to Waldschmidt et al. (1997). After testing the polymorphism of 49 microsatellite loci in polyacrylamide gels, the loci Mmo15, Mmo21 (Lopes and da Silva 2010), Mru14 (Lopes et al. 2009), Mbi232, Mbi233, and Mbi254 (Peters et al. 1998) were amplified as described by Schuelke (2000). The amplicons were marked with VIC fluorescence and genotyped separately in automatic sequencer ABI 3500 (Applied Biosystems) and analyzed using the software GeneMapper v5.0. The paternal genotypes were obtained by subtraction, based on genotypes of queen bees and workers. The effective mating frequency, related to the proportion of offspring from each male parent, was calculated as me = 1/(Ry2i ), where yi is the proportion of individuals of patriline i in a colony (Starr 1984). The relatedness coefficient among workers was

estimated using the software KINGROUP v2.09 (Konovalov et al. 2004), based on the algorithm proposed by Queller and Goodnight (1989). The likelihood of non-detection of two male parents with the same genotype was calculated at population level as dp = P(Rq2i ), where qi is the frequency of allele i in a locus. The sum for each locus is multiplied among loci (Boomsma and Ratnieks 1996). The former likelihood on colony level (dc) was calculated by multiplying across loci the allele frequencies of the male parent alleles (Palmer et al. 2002), and summing the calculated value of each male parent in the same colony. When the queen and male parent alleles could not be discriminated, we summed the frequencies of the detected alleles. For aforementioned, colony-level and population-level non-detection likelihoods, we used the population allele frequencies based on the 38 colonies sampled from meliponary. A male produced by a worker was detected based on inheritance from its grandfather of one allele at an informative locus (Foster et al. 2000). Due to sample size effects and inheritance pattern, the number of detected males produced by workers in each colony (Nwm) was corrected and used to estimate its percentage in the colony. The probability that a male inherited an allele from its grandfather is 0.5, so the proportion of males that can be detected using the formula Pj = (1 - 0.5i), where i is the number of informative loci (Palmer et al. 2002). The proportion of males produced by workers that can be detected in a given sample number (Na) was estimated as Na = (PjNj) where Nj is the number of male samples in a colony. The percentage of male offspring from workers is estimated by dividing the observed number of males produced by workers by Na and then multiplying it by 100 (Foster et al. 2001). A Spearman’s Rank Correlation Coefficient was calculated to test the correlation between me and percentage of males produced by workers.

Table 1 Number of sampled queens and workers, number of sampled males (Nj), patrilines (Patril.), number of informative loci (Iloc), effective mating (me), relatedness coefficient (R),

number of detected worker produced males (Nwm), and estimated proportion of worker’s offspring (Pwm) in each colony of Melipona mondury

Methods

Colony

Queen

Worker

Nj

Patril.

Iloc

me

R

Nwm

Pwm (%)

c09

1

10

10

7

4

6.25

0.53 ± 0.22

10

100.00

c11

1

10

–

–

0

–

0.63 ± 0.21

–

–

c13

1

10

10

4

2

3.57

0.89 ± 0.07

8

100.00

c16

1

10

10

1

3

1.00

0.75 ± 0.16

0

0.00

c24

1

10

10

2

2

1.72

0.90 ± 0.07

4

53.33

c24f2

1

10

6

2

2

1.72

0.81 ± 0.18

2

44.44

c25

1

10

4

2

4

1.92

0.57 ± 0.31

2

53.33

c26

1

10

10

2

2

1.92

0.55 ± 0.30

0

0.00

c29

1

10

10

5

3

3.85

0.75 ± 0.13

9

100.00

c33

1

10

10

1

0

1.00

0.98 ± 0.03

–

Mean

1

10

2.55 ± 1.71

0.74 ± 0.16

123

2.89 ± 2.03

– 56.38 ± 39.09

Mating frequency and maternity of males in Melipona mondury (Hymenoptera: Apidae)

Results The number of alleles detected for loci Mmo15, Mmo21, Mru14, Mbi232, Mbi233, and Mbi254 were equal to 2, 2, 4, 3, 9, and 10, respectively. Expected heterozygosity values ranged from 0.298 to 0.860 (Table 2). At the population level, null alleles were detected at loci Mmo15, Mbi233, and Mbi254 with a frequency of 0.12, 0.04, and 0.05, respectively. At colony level, null alleles were detected in locus Mru14, in three colonies, based on allele inheritance pattern (see below). The probability of non-detection of two parents with the same genotype at population level was dp \ 0.002 and at colony level was dc \ 0,026 for all colonies. Worker pupae not sharing alleles with the queen, but sharing one allele with other workers, were detected in locus Mbi233 of colony c11. This was caused probably by queen supersedure, since the queen bee was young (small body size and pale abdominal coloration) and hence from the same generation as the sampled worker pupae. Null alleles were found in locus Mru14 in colonies c24-F2, c25, and c26, as the incompatible genotypes were homozygous, and the alleles were inherited as expected in the remaining loci. Except for the colony with overlapping generations (c11), only two or three alleles were identified in a single locus. Queens of nine colonies mated from one to seven times, with effective mate frequency ranging from 1 to 6.25, taking into account the hypothesis of null alleles in colonies c24F2, c25, and c26. No informative loci were obtained in colony c33, so it was considered to be monandrous. The mean relatedness values between sister workers ranged from 0.53 to 0.90 in polyandrous colonies, and from 0.75 to 0.98 in monandrous colonies (Table 1). The workers of M. mondury are capable of reproduction. The variation of worker’s offspring proportion ranged from 0 to 100 % within colonies (Table 1). Worker reproduction was identified in both polyandrous and monandrous colonies. Diploid males were absent and the maternity of all individuals could be assigned to workers or the queen. No Table 2 Number alleles per locus, observed heterozygosity (Ho), and unbiased expected heterozygosity (He) of the 38 meliponary colonies Locus

Alelles

Ho

He

Mmo15

2

0.333

0.501

Mmo21

2

0.359

0.298

Mru14

4

0.462

0.543

Mbi232

3

0.313

0.537

Mbi233

9

0.711

0.838

Mbi254

10

0.838

0.860

Mean

5

0.502

0.596

SE

3.57

0.090

0.088

males were found in colony c11 and no informative loci were observed for the colony c33. Even with the estimation of null alleles, paternal alleles (present in workers, but not in the queen) could be identified in locus Mru14 and were used to determine males produced by worker in colonies c25 and c24f2. Spearman’s Rank Correlation Coefficient between me and percentage of male offspring from workers was r = 0.851 (p \ 0.01).

Discussion The number of identified alleles in Mbi loci was higher in M. mondury than in Melipona bicolor (Peters et al. 1998). Our results suggest that when the primer annealing sites are conserved (Francini et al. 2010; Viana et al. 2011), allele diversity depends on factors that contribute to the evolutionary dynamics of microsatellites, such as repeat number, type of the nucleotide motif, perfection of repeat structure, and recombination (Bhargava and Fuentes 2010). Also, the allele diversity was higher than those reported by other studies of stingless bees using microsatellites loci (Tavares et al. 2007; Lopes et al. 2010; Koser et al. 2014). A population study would help to understand the difference in genetic structure in the State of Bahia and the other regions in the country. The sampling of pupae to estimate the frequency of matings assured the analysis of individuals born in the sampled colony (Palmer et al. 2002). Sampling of queens was essential to deduce the parental genotypes. Without this information, the number of estimated matings would be equal to 1, except in colonies c24-F2 and c25 (two matings). Furthermore, it would be impossible to identify the overlapped generations in c11 and the mean relatedness value within colonies of 0.74 ± 0.16 could have been interpreted as a confirmation of a putative monandry. The procedure herein presented is able to allow the detection of polyandry in stingless bees in further studies, challenging the overall view that these insects are mainly monandrous. Seven out of nine colonies exhibited polyandry, a described but not common result for stingless bees (Paxton et al. 1999; Palmer et al. 2002; Jaffe´ et al. 2014). One subspecies of bumblebee, which are mostly monandrous, also showed some degree of polyandry (Owen and Whidden 2013). Multiple mating might have evolved in eusocial insects after the workers have lost their abilities to sexual reproduction and the eusocial behavior became irreversible (Hamilton 1964). The occurrence of worker reproduction in polyandrous colonies could be explained by the detected relatedness values among workers, from 0.53 to 0.98 (Table 1). In many cases, reproduction of workers is favored because the mean relatedness values between workers and nephews (r [ 0.25) are higher than these values between workers and siblings

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(r = 0.25) (Ratnieks 1988). If the high relatedness values between workers were due to genetic relationship between parents, we could expect the detection of diploid males (Tarpy and Page 2001). In this study, the non-detection could be the result of sampling effects (Toth et al. 2004), killing of diploid males (Francini et al. 2012), or queen and her mate not sharing a csd allele. The variation in male production per worker among colonies might be related to sampling effects (Toth et al. 2004) and sampling period (Moo-Valle et al. 2001; Alves et al. 2009). In stingless bees, the proportion of males produced by workers was estimated through microsatellites (0 to 100 % in eight species) (To´th et al. 2002), direct observation (94.5 % in M. favosa) (Sommeijer et al. 1999), and by isozymes (38 % in M. subnitida) (Contel and Kerr 1976). Thus, the occurrence of reproduction by stingless bees workers should not be regarded as an aberration but a reproductive option (Beekman and Oldroyd 2008). Behavioral studies need to be performed to help us understand the reproductive strategies of workers in this and other stingless bee species.

Conclusion Our study corroborates theoretical predictions about worker reproduction and relatedness by showing that in polyandrous colonies, relatedness favors production of sons and tolerance of nephews. Future studies of population genetics and conservation of this species will be supported by the knowledge of parameters involved in reproduction aspects, as population effective size. Acknowledgments The authors acknowledge Dr. Iracilda Sampaio for opening the doors of the Laborato´rio de Filogenoˆmica of the Universidade Federal do Para´. This research was supported by funding from Programa Nacional de Po´s Doutorado/Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (PNPD/CAPES), Fundac¸a˜o de Amparo a` Pesquisa do Estado da Bahia (FAPESB), Universidade Estadual do Sudoeste da Bahia (UESB) and Universidade Federal do Recoˆncavo da Bahia (UFRB).

References Alves DA, Imperatriz-Fonseca VL, Santos-Filho PS (2009) Production of workers, queens and males in Plebeia remota colonies (Hymenoptera, Apidae, Meliponini), a stingless bee with reproductive diapause. Genet Mol Res 8:672–683. doi:10.4238/vol82kerr030 Baer B, Schmid-Hempel P (1999) Experimental variation in polyandry affects parasite loads and fitness in a bumble-bee. Nature 397:151–154 Beekman M, Oldroyd BP (2008) When workers disunite: intraspecific parasitism by eusocial bees. Annu Rev Entomol 53:19–37. doi:10. 1146/annurev.ento.53.103106.093515 Bhargava A, Fuentes FF (2010) Mutational dynamics of microsatellites. Mol Biotechnol 44:250–266. doi:10.1007/s12033-009-9230-4

123

Boomsma JJ, Ratnieks FLW (1996) Paternity in Eusocial Hymenoptera. Philos Trans R Soc B Biol Sci 351:947–975. doi:10.1098/ rstb.1996.0087 Borges AA, de Oliveira Campos LA, Saloma˜o TMF, Tavares MG (2010) Genetic variability in five populations of Partamona helleri (Hymenoptera, Apidae) from Minas Gerais State, Brazil. Genet Mol Biol 33:781–4. doi:10.1590/S1415-47572010005000087 Contel EPB, Kerr WE (1976) Origin of males in Melipona subnitida estimated from data of an isozymic polymorphic system. Genetica 46:271–277. doi:10.1007/BF00055470 de Sousa HAC, Viana MVC, Oliveira RM De, et al (2012) Distribution of Melipona mondury Smith 1863 (Hymenoptera: Apidae, Meliponini) from state of Bahia. Magistra 24:99–104 Foster KR, Ratnieks FLW, Gyllenstrand N, Thoren PA (2001) Colony kin structure and male production in Dolichovespula wasps. Mol Ecol 10:1003–1010. doi:10.1046/j.1365-294X.2001.01228.x Foster KR, Ratnieks FLW, Raybould AF (2000) Do hornets have zombie workers? Mol Ecol 9:735–742. doi:10.1046/j.1365-294x. 2000.00920.x Francini IB, Nunes-Silva CG, Carvalho-Zilse GA (2012) Diploid Male Production of Two Amazonian Melipona Bees (Hymenoptera: Apidae). Psyche A J Entomol 2012:1–7. doi:10.1155/2012/ 484618 Francini IB, Sousa ACB, Sforc¸a DA et al (2010) Isolation and characterization of microsatellite loci in the stingless bee Melipona interrupta manaosensis (Apidae: Meliponini). Conserv Genet Resour 2:27–30. doi:10.1007/s12686-009-9130-8 Fundac¸a˜o SOS Mata Atlaˆntica, Instituto Nacional de Pesquisas Espaciais (2010) Atlas dos Remanescentes Florestais da Mata Atlaˆntica. Perı´odo 2008-2010. http://www.inpe.br/noticias/arquivos/pdf/atlasrelatoriofinal.pdf. Accessed 16 July 2015 Graur D (1985) Gene diversity in Hymenoptera. Evolution 39:190–199 Hamilton WD (1964) The genetical evolution of social behaviour. II. J Theor Biol 7:17–52. doi:10.1016/0022-5193(64)90039-6 Imperatriz-Fonseca VL, Matos ET, Ferreira F, Velthuis HHW (1998) A case of multiple mating in stingless bees (Meliponinae). Insectes Soc 45:231–233. doi:10.1007/s000400050083 Jaffe´ R, Pioker-Hara FC, dos Santos CF et al (2014) Monogamy in large bee societies: A stingless paradox. Naturwissenschaften 101:261–264. doi:10.1007/s00114-014-1149-3 Konovalov DA, Manning C, Henshaw MT (2004) kingroup: a program for pedigree relationship reconstruction and kin group assignments using genetic markers. Mol Ecol Notes 4:779–782. doi:10. 1111/j.1471-8286.2004.00796.x Koser JR, Francisco FDO, Moretto G (2014) Genetic Variability of Stingless Bees Melipona mondury Smith and Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae) from a Meliponary. Sociobiology 61:313–317. doi:10.13102/sociobiology.v61i3. 313-317 Kraus FB, Neumann P, Moritz RFA (2005) Genetic variance of mating frequency in the honeybee (Apis mellifera L.). Insectes Soc 52:1–5. doi:10.1007/s00040-004-0766-9 Lopes D, da Silva F (2010) A scientific note on the characterization of microsatellite loci for Melipona mondury (Hymenoptera: Apidae). Apidologie 41:138–140. doi:10.1016/j.ymthe.2005.01.005 Lopes DM, da Silva FO, Fernandes Saloma˜o TM et al (2009) Microsatellite loci for the stingless bee Melipona rufiventris (Hymenoptera: Apidae). Mol Ecol Resour 9:923–5. doi:10.1111/ j.1755-0998.2008.02486.x Lopes DM, de Oliveira Campos LA, Saloma˜o TMF, Tavares MG (2010) Comparative study on the use of specific and heterologous microsatellite primers in the stingless bees Melipona rufiventris and M. mondury (Hymenoptera, Apidae). Genet Mol Biol 33:390–393. doi:10.1590/S1415-47572010005000017 Melo GAR (2003) Notas sobre meliponı´neos neotropicais, com a descric¸a˜o de treˆs novas espe´cies (Hymenoptera, Apidae). In:

Mating frequency and maternity of males in Melipona mondury (Hymenoptera: Apidae) Melo GAR, Alves-dos-Santos I (eds) Apoidea Neotropica: Homenagem aos 90 Anos de Jesus Santiago Moure. UNESC, Criciu´ma, pp 85–91 Moo-Valle H, Quezada-Eua´n JJG, Wenseleers T (2001) The effect of food reserves on the production of sexual offspring in the stingless bee Melipona beecheii (Apidae, Meliponini). Insectes Soc 48:398–403. doi:10.1007/PL00001797 Owen RE, Whidden TL (2013) Monandry and polyandry in three species of North American bumble bees (Bombus) determined using microsatellite DNA markers. Can J Zool 91:523–528. doi:10.1139/cjz-2012-0288 Palmer KA, Oldroyd BP (2000) Evolution of multiple mating in the genus Apis. Apidologie 31:235–248 Palmer KA, Oldroyd BP, Quezada-Euan JJG et al (2002) Paternity frequency and maternity of males in some stingless bee species. Mol Ecol 11:2107–2113. doi:10.1046/j.1365-294X.2002.01589.x Paxton RJ, Weißschuh N, Engels W et al (1999) Not Only Single Mating in Stingless Bees. Naturwissenschaften 86:143–146. doi:10.1007/s001140050588 Peters JM, Queller DC, Fonseca VLI, Strassmann JE (1998) Microsatellite loci for stingless bees. Mol Ecol 7:784–787 Queller DC, Goodnight KF (1989) Estimating Relatedness Using Genetic Markers. Evolution (N Y) 43:258. doi:10.2307/2409206 Ratnieks FLW (1988) Reproductive harmony via mutual policing by workers in eusocial Hymenoptera. Am Nat 132:217–236. doi:10. 2307/2461867 Ratnieks FLW, Foster KR, Wenseleers T (2006) Conflict resolution in insect societies. Annu Rev Entomol 51:581–608. doi:10.1146/ annurev.ento.51.110104.151003 Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18:233–4. doi:10.1038/72708

Sommeijer MJ, Chinh TX, Meeuwsen FJAJ (1999) Behavioural data on the production of males by workers in the stingless bee Melipona favosa (Apidae, Meliponinae). Insectes Soc 46:92–93. doi:10.1007/s000400050118 Starr CRK (1984) Sociality in the Aculeate Hymenoptera. In: Smith RL (ed) Sperm Competition and the Evolution of Animal Mating Systems. Academic Press, New York, pp 427–464 Tarpy DR, Page R (2001) The curious promiscuity of queen honey bees (Apis mellifera): evolutionary and behavioral mechanisms. Ann Zool Fennici 38:255–265 Tavares MG, Dias LA dos S, Borges AA, et al (2007) Genetic divergence between populations of the stingless bee uruc¸u amarela (Melipona rufiventris group, Hymenoptera, Meliponini): is there a new Melipona species in the Brazilian state of Minas Gerais? Genet Mol Biol 30:667–675. doi: 10.1590/S141547572007000400027 Toth E, Queller DC, Dollin A, Strassmann JE (2004) Conflict over male parentage in stingless bees. Insectes Soc 51:1–11. doi:10. 1007/s00040-003-0707-z To´th E, Strassmann JE, Nogueira-Neto P et al (2002) Male production in stingless bees: Variable outcomes of queen-worker conflict. Mol Ecol 11:2661–2667. doi:10.1046/j.1365-294X.2002.01625.x Viana MV, Miranda EA, de Francisco AK et al (2011) Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona. Genet Mol Res 10:3942–3947. doi:10.4238/2011.November.22.11 Waldschmidt AM, Saloma˜o TMF, Barros EG de, de Campos LAO (1997) Extraction of genomic DNA from Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae). Brazilian J Genet. doi:10. 1590/S0100-84551997000300011

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