Insect. Soc. 55 (2008) 43 – 50 0020-1812/08/010043-8 DOI 10.1007/s00040-007-0976-z
Birkhuser Verlag, Basel, 2008

Insectes Sociaux

Research article

Diploid male production in a rare and locally distributed bumblebee, Bombus florilegus (Hymenoptera, Apidae) J. Takahashi 1 , 2 , T. Ayabe2,3, M. Mitsuhata 2 , I. Shimizu 1 and M. Ono 2 1 2 3

Center for Ecological Research, Kyoto University, Otsu, Shiga, 520-2113 Japan, email: [email protected] Laboratory of Entomology, Graduate School of Agriculture, Tamagawa University, Machida, Tokyo, 194-8610 Japan Department of Oncology and Pharmacodynamics, Meji Pharmaceutical University, 2-522-1 Noshio, Tokyo 204-8588, Japan

Received 19 July 2007; revised 13 October 2007; accepted 15 October 2007. Published Online First 23 January 2008

Abstract. The European bumblebee B. terrestris was recently introduced in Japan for agricultural purposes and has now become naturalized. The naturalization of this exotic species may have great detrimental effects on closely related native Japanese bumblebees. The Japanese bumblebee Bombus florilegus is a rare and locally distributed species found in the Nemuro Peninsula of Hokkaido, Japan. In order to assess its population genetics, we estimated the genetic structure of B. floriACHTUNGRElegus in 16 breeding colonies (queen, workers, and males) and 20 foraging queens by analyzing microsatellite DNA markers. Of the 36 queens analyzed by genotyping and dissection, 32 had been inseminated by a male. The remaining 4 had not been inseminated at all. Of the 4 nonmated queens, one was triploid. Diploid males were found in 4 breeding colonies. Based on the microsatellite data, it appears that B. florilegus has low reproductive success. Since matched mating and nonmating within local populations are high, the extinction risk is correspondingly higher. Our results suggest that conservation of the Japanese B. florilegus is required in order to protect it from both habitat destruction and the naturalization of alien species. Keywords: Bombus florilegus, diploid males, triploid females, sex determination, microsatellites.

Introduction One of the most serious threats to the continued survival of many organisms is the widespread destruction of natural ecosystems resulting from human economic activities. Rare species characterized by small population sizes are at a considerably greater risk than widespread

species with large populations. The International Union for Conservation of Nature and Natural Resources (IUCN) has established a 5-point classification system to categorize the degree of threat to a species (Frankham et al., 2002), and the habitat size for all species is listed as one of the most important factors. Previous case studies have demonstrated that the risk of extinction is high if the habitat size is small (Frankham et al., 2002; Keller and Wallker, 2002). Thus, the risk of extinction is greater in a regionally distributed species than in a widely distributed species. In addition, the importance of maintaining genetic diversity is a crucial factor in the long-term conservation of threatened species with local populations. The loss of genetic diversity decreases the capacity of natural populations to adapt to environmental change and to natural enemies such as parasites and pathogens. Moreover, species with a local population structure may accumulate harmful genetic mutations through the loss of genetic diversity within the population. Recent reviews have emphasized the importance of genetic factors in the conservation of both invertebrates and vertebrates (Frankham et al., 2002; Keller and Wallker, 2002; Spielman et al., 2004). For example, reduced heterozygosity in small isolated populations of butterflies has been demonstrated to increase extinction risk (Saccheri et al., 1998; Schmitt and Hewitt, 2004). In the absence of frequent immigration, species with local populations, such as those persisting in fragmented habitats, have been observed to lose an inclusive fitness through inbreeding (Ross et al., 1993; Yamauchi et al., 2002). The depressive impact of inbreeding may reduce both individual and population fitness through the expression of harmful recessive genes. Locally distributed social insects with a small habitat size are particularly susceptible to the loss of genetic diversity because most of

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the colony members are sterile workers. Reproductive individuals emerge only during the reproductive season; consequently, the total number of parents in a population of social insects is relatively small compared to that in a population of nonsocial insects (Chapman and Bourke, 2001; Packer and Owen, 2001). The social Hymenoptera, such as ants and some species of bees and wasps, are particularly vulnerable to the loss of genetic diversity through inbreeding because of the haplo-diploid system of sex determination (Pamilo and Crozier, 1997; Chapman and Bourke, 2001). Population fitness, however, will not be reduced by inbreeding in the haplo-diploid social Hymenoptera because haploid males allow a more rapid purging of deleterious recessive alleles (Sorati et al., 1996). A decrease in genetic diversity by complementary sex determination (CSD) in social Hymenoptera will have important consequences for the fitness of a population. In the single-locus (sl) CSD model, sex is determined by the complementary action of codominant alleles at a sex-determining autosomal locus (Crozier, 1971; Bull, 1981). In this model, males develop when the sex loci are either hemizygous (haploid) or homozygous (diploid) and females develop when the loci are heterozygous (Whiting, 1939). Therefore, the production of sterile diploid males is expected to occur by the matched mating of parents homozygous at the sex locus. In social Hymenoptera, diploid male production has been demonstrated to increase colony mortality and decrease colony growth rates (Plowright and Pallett, 1979; Ross and Fletcher, 1986). Therefore, the degree of genetic diversity and the frequency of diploid males are important parameters in hymenopteran conservation genetics since high frequencies of homozygotes at any loci may indicate genetic impoverishment resulting from matched mating, small habitat size, population fragmentation, loss of genetic diversity, or a combination of these factors (Packer and Owen, 2001). Bumblebees are social Hymenoptera in which annual colonies are typically founded by a single queen. The European and North American species of Bombus generally mate once or twice (Estoup et al., 1995; Schmid-Hempel and Schmid-Hempel, 2000; Sauter et al., 2001; Paxton et al., 2001; Payne et al., 2003). Bumblebees are highly susceptible to inbreeding and matched mating (Chapman and Bourke, 2001) because most species are generally monogynous and monoandrous under the conditions of random mating. Recent studies on numerous bumblebee populations in Europe have revealed a reduction in population size due to agricultural intensification and the destruction of the natural habitats of bumblebees (Goulson, 2003). Of the 25 native species that have been recorded in the UK, three have already become extinct and several remain only in small, isolated populations (Goulson, 2003). Bumblebees play an important role as pollinators of entomophilous plants such as wild flowers and crops; therefore, a reduction in the number of pollinators may have serious consequences for both agricultural plants

Diploid male production in Bombus florilegus

and wild flowers. Many studies on bumblebee population genetics have focused on common and widespread species like B. terrestris (Estoup et al., 1996; Widmer et al., 1998), B. pascuorum (Pirounakis et al., 1998; Widmer and Schmid-Hempel, 1999), and B. ignitus (Shao et al., 2004). These species essentially maintain high genetic diversity and exhibit high levels of genetic differentiation among isolated populations. In contrast, little is known about the population genetic structures of rare and threatened species that live in fragmented habitats. To date, such data exist for only two species, namely B. muscorum and B. sylvarum, in Europe (Darvill et al., 2006; Ellis et al., 2006). Isolated populations of B. muscorum have been demonstrated to be genetically differentiated, and no evidence has been found for inbreeding and bottlenecks caused by geographical isolation (Darvill et al., 2006). B. sylvarum exhibits genetic differences among the fragmented populations, and the low genetic variation of this species is affected by drift (Ellis et al., 2006). In 1992, the European bumblebee B. terrestris was introduced in Japan as a pollinator of crops grown in greenhouses. Some of the queens escaped from these greenhouses had the species has now become naturalized (Washitani, 1998). In Hokkaido and Honshu islands, the naturalization of this species is having a negative impact on native bumblebee species (Washitani, 1998). Due to its larger colony size and greater adaptability to the environment, the European B. terrestris tends to possess greater fecundity than the native Japanese bumblebees (Ito, 1998). B. terrestris males, being vigorous reproducers, copulate with females of the native Japanese species. Hybrid female bees are unable to establish colonies since they can only lay unfertilized eggs (Ito, 1998). Further, it is suggested that immigrant exotic bumblebees compete with native species for resources such as food and nesting places. Consequently, the effective size of native bumblebee populations may be decreased due to the naturalization of B. terrestris. In Japan, 14 species of bumblebee from the genera Bombus and Psithyrus are distributed from Kyushu to Hokkaido. One rare bumblebee, B. florilegus, is distributed in a limited area (approximately 150 km2) in the Nemuro Peninsula of Hokkaido, Japan (Fig. 1). In 2001, B. florilegus was designated as a rare species in the Red Data Book of Hokkaido. This species is predicted to have suffered a decline in population size because of habitat development and competition with naturalized B. terrestris (Nakatani, 1999). The population genetics and reproductive structures of Japanese bumblebees are, however, almost completely unknown. The aim of this study was to determine the genetic diversity and reproductive structure of the rare and locally distributed bumblebee, B. florilegus, using DNA-microsatellite genotyping, data that can then be used to preserve the species.

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DNA extraction from individuals and spermathecae DNA was extracted from individuals using the Walsh et al. method (1991). Template DNA was extracted from the individuals by boiling macerated tissue in 400 ml 5 % Chelex (Bio-Rad) resin at 95 8C for 10 min. Sperm, present in the spermathecae of the queens, was collected and processed as described by Peters et al. (1995). The spermathecae were stored in 1 phosphate-buffered saline solution, and the sperm clumps were dissected from the membranes using insect pins. Genomic DNA was extracted from the sperm by using a DNeasy Tissue Kit (Qiagen).

Figure 1. Distribution area (shaded area) of Bombus florilegus in Hokkaido, Japan.

Materials and methods Sample collection From 1998 to 2004, B. florilegus individuals were collected in May and June from eastern Hokkaido, Japan (Fig. 2). A total of 36 samples were obtained from a 60 km2 area. The 16 queens collected in 1998 and 2004 were used for genetic analysis. The 20 queens collected in 1999, 2000, and 2001 were colonized by standard methods as described by Ayabe et al. (2004). For DNA analysis, queens and their offspring were collected from each of the breeding colonies over a period of approximately 1 month and preserved in 99 % ethanol prior to storage at –20 8C.

Microsatellite genotyping Microsatellite DNA analysis was conducted using 13 microsatellite primers from B. terrestris (Estoup et al., 1995, 1996). The genotypes of 36 queens, the sperm in their spermathecae, workers (n = 90), and males (n = 32) were examined using DNA microsatellites. All polymerase chain reactions (PCR) were performed in a total volume of 15 ml reaction mixture containing 1.0 ml (approximately 20 ng) template DNA, 0.2 mm primer, 1.2 ml dNTP mix (250 mm), 1.5 ml 10 reaction buffer, 1.5 mm MgCl2 and 0.05 units Taq DNA polymerase (Takara). All PCR reactions were performed under the following conditions. After denaturation at 948 C for 3 min, the samples were subjected to 30 cycles at 94 8C for 30 s, 50 – 58 8C for 30 s (Estoup et al., 1995, 1996), and 72 8C for 30 s. The forward primer of each marker was 5’-end-labelled with fluorescent phosphoramidite (NED, 6-FAM, VIC, and PET). The PCR products were visualized with an ABI 3100 Genetic Analyzer using 500 LIZTM, an internal size standard. The fragments were then analyzed using ABI GeneScan software (version 3.7) and ABI Genotyper DNA fragment analysis software (version 3.7).

Diploid male detection Individuals were sexed based on their morphology prior to DNA extraction. Diploid males were identified by heterozygosity at any locus (B10, B11, B121, and B126). Males were sampled during the early stages (first brood) of the colony when workers were produced; only then can F, the proportion of diploids that are males, can be estimated with a high degree of confidence. Single locus estimates of F were made using the following equation (Owen and Packer, 1994): ^ ¼ B=ðA þ BÞ F

(1)

where A is the total number of heterozygous females and B is the total number of heterozygous males per locus per sample. The frequency of inbreeding q was calculated using the following equation (Owen and Packer, 1994): ^ ¼ 2F=1
s, q

(2)

where s is the selection coefficient against diploid males. This value is assumed to be zero but is unknown. The effective number of alleles at the sex-determining locus K, by assuming sl-CSD, was estimated as follows (Adams et al., 1977): ^ ^ ¼ 2=q, K

(3)

These equations provide minimum estimates of q and K, i.e. where s is zero (Zayed and Packer, 2001). Figure 2. Bombus florilegus queen in spring.

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J. Takahashi et al.

Diploid male production in Bombus florilegus

Table 1. Genetic variation in the microsatellite marker loci studied, where NA is the number of alleles detected in the 90 study females and HO the observed heterozygosity at each locus. Locus

No. of females analyzed

NA

B10

90

B11 B96

Allele frequencies

HO

a

b

c

d

4

0.58

0.29

0.26

0.13

90

4

0.56

0.33

0.22

0.11

90

1

1.00

B100

90

2

0.73

0.27

B101

90

2

0.89

0.11

B116

90

1

1.00

0.00

B118

90

1

1.00

0.00

B119

90

1

1.00

B121

90

4

0.57

0.22

B124

90

2

0.61

0.31

B126

90

4

0.49

0.25

B131

90

2

0.72

0.28

0.39

B132

90

2

0.70

0.30

0.27

0.69 0.65 0.00 0.53 0.27

0.00 0.18

0.03

0.63

0.17

0.09

0.64

0.54

Colony genetic structure Genetic structure of B. florilegus We estimated the genetic relatedness among workers (n = 14 colonies), and between queens (n = 32) and their mate (n = 32), inbreeding coefficient (F), and allele frequencies using the Relatedness 4.2 and Kinship 1.3 computer programs (Goodnight and Queller 1994; 1999).

Results Allelic variation at the microsatellite loci of B. florilegus By using B. florilegus as the template DNA, the target regions were successfully amplified with 13 primer pairs (Table 1). The genotypes of all females, sperm, and males were polymorphic at 4 loci. At 9 of the 13 loci, 1 or 2 allelic numbers were observed in 36 queens and their offspring (n = 122). The B10, B11, B121, and B126 microsatellite loci in all individuals each exhibited 4 alleles and the mean observed heterozygosity (HO) for these loci ranged from 0.63 to 0.69 (Table 1). Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium tests were conducted for the 4 variable loci using the genepop program (Raymond & Rousset, 1995; web version 3.4). For this population, the allele distributions for the 4 variable loci (B10, B11, B121, and B126) exhibited statistically significant deviation from HWE (P < 0.05). None of the 4 loci pairs in this population exhibited a significant deviation from random association. No significant linkage disequilibrium was observed between the polymorphic loci.

Of the 36 queens collected, 16 succeeded in establishing a colony within the laboratory. Generally, in male Japanese bumblebees begin to emerge in the middle of August. All colonies were collected for analysis before August. Thus, we analyzed the males and females that emerged in the laboratory before the reproductive stage. Of the 16 colonies examined, the queens from 14 had been inseminated by a male. Queens of the remaining 2 colonies had not been inseminated at all (Table 2). Workers emerged from the 14 mated colonies. The number of workers ranged from 2 to 24 (mean = 6.4). Four mated and 2 nonmated queen colonies produced males during the first broodemergence stage. The mating frequencies of a queen were estimated to be 1 based on the genotypes of the sperm in the spermathecae and the female offspring. The genotypes of males were heterozygous at any loci, indicating that these males were diploid. The estimated heterozygosity for the proportion of diploids that were males, F, was high across all loci (Table 2). Diploid males were only detected in 4 mated queen colonies (28 %). Approximately half of these colony members were male. The number of males ranged from 3 to 9 in 4 colonies. The sex ratio of queen-laid offspring was compared with an even sex ratio using Fishers exact probability test. In diploid male-producing colonies, the sex ratio between females and males was approximately 1:1, with no significant difference from an even sex ratio (all test, P > 0.91). The frequencies of matched mating q in the population ranged from 0.182 to 0.302 and averaged 0.255 (Table 2). The number of effective alleles at the sex-determining locus K ranged from 6 to 11 and averaged 8 alleles in this population. No female offspring were born in the 2 nonmated queen colonies; the number of males born in these colonies was 8

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Table 2. The data of offspring emerged colonies in B. florilegus. Proportion of diploid males (F), the matched mating (q), and effective number of alleles at the sex-determining locus (K). Locus

Probability of diploid males

F

q

K

Range

0.28 – 0.50

0.091 – 0.151

0.182 – 0.302

6.625 – 11.0

Mean  SD

0.414  0.106

0.128  0.029

0.255  0.058

8.2  2.07

Table 3. Paternity frequency and genetic relatedness among individuals of 14 B. florilegus colonies. Colony no.

Number of workers and males

Number of mating frequency

Regression relatiedness worker to worker

queen to mated male

9801

Workers (2) and diploid males (3)

1

0.75

0.15

9902

Workers (10) and diploid males (9)

1

0.77

0.22

0003

Workers (4) and diploid males (3)

1

0.79

0.13

0004

Workers (5) and diploid males (4)

1

0.74

0.08

9802

Workers (7)

1

0.74

0.01

9804

Workers (8)

1

0.75

0.00

9901

Workers (10)

1

0.73

0.00

9903

Workers (6)

1

0.75

0.09

9905

Workers (5)

1

0.83

0.11

0002

Workers (7)

1

0.76

0.00

0101

Workers (8)

1

0.74

0.00

0102

Workers (6)

1

0.80

0.05

0301

Workers (7)

1

0.77

0.00

0401

Workers (5)

1

0.76

0.00

1

0.76  0.03

0.06  0.07

Mean  SD

and 12. Based on spermathecae dissection and PCR analysis, it was determined that 18 of the 20 non-nesting queens examined had been inseminated by a single male, while the remaining 2 had not been inseminated at all. Of the 2 nonmated queens, one was triploid at 2 loci (B121 and B126). Worker-to-worker relatedness was 0.76  0.03 across 14 colonies with the inbreeding coefficient not significantly different from zero (F = 0.01  0.01). This result strongly suggests that all of the colonies were monogynous and monoandrous under random mating (Table 3). The relatedness of a queen and its mated male was 0.05  0.07 (mean  SD) across 32 pairs. In the 4 diploid male colonies, it was 0.15  0.06 (mean  SD). The highly relatedness of pairs showed a strong possibility of matched mating (Table 3).

Discussion We found that B. florilegus queens typically mate with only 1 male. These findings are consistent with the observations of other Bombus species in which monogyny is the norm (Estoup et al. , 1995). The European and North American species of Bombus generally mate

only once (Estoup et al. , 1995 ; Schmid-Hempel and Schmid-Hempel, 2000 ; Sauter et al. , 2001; Paxton et al. , 2001; Payne et al. , 2003). Similarly, B. florilegus does not differ from the majority of Bombus species with respect to the number of fathering males per colony. The mating structure of B. florilegus is typically monoandrous, which reduces the amount of genetic diversity present in each colony relative to that of polyandrous species and thus increases the susceptibility to matched mating (Chapman and Bourke, 2001). The frequency of nonmated queens in this population as determined by spermathecae dissection was 11 %. This study is the first to demonstrate such a high frequency of nonmated queens in bumblebee colonies. Two colonies (colony codes 0103 and 0104) produced only haploid males. The high frequency of nonmated queens is an important problem faced by species with small local populations. The colonies founded by these queens will produce only haploid males, and these colonies will be unable to survive to the reproductive stage due to the absence of worker individuals. Therefore, the occurrence of nonmated queens is predicted to be one of the causes of some colonies having a small population size. Consequently, in order to preserve the Japanese population of

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B. florilegus, it is necessary to maintain a large number of successful colonies. Diploid male production is disadvantageous in that these males are practically sterile (Agoze et al., 1994). Queens copulating with diploid males produce female offspring that are sterile triploids (Hung et al., 1974; Stouthamer et al., 1992; Ayabe et al., 2004). The production of diploid males, therefore, exacts a fitness cost at the colony level by reducing genetic diversity at the sex locus (Cook and Crozier, 1995). Further, matched mating is not expected to occur at a high frequency in normal populations (Kukuk and May, 1990; Packer and Owen, 1991). We found diploid males in 28 % of the B. florilegus colonies examined in this study. The frequency of these diploid male colonies was higher than that of other bumblebees, namely, B. terrestris (Duchateau et al., 1994) and B. sylvarum (Ellis et al., 2006). The causes of such high levels of diploid males appear to be related not only to monoandry, but also to the low genetic diversity present in local populations. Further, the level of genetic variation in B. florilegus is considerably lower than that in other bumblebees, including the rare and declining species B. muscorum (Darvill et al., 2006) and B. sylvarum (Ellis et al., 2006) and the common and widespread species such as B. terrestris (Estoup et al., 1996; Widmer et al., 1998), B. pascuorum (Pirounakis et al., 1998; Widmer and Schmid-Hempel, 1999), and B. ignitus (Shao et al., 2004). This result suggests that low genetic diversity in local populations can cause matched matings in B. florilegus. We first found 2.7 % triploid females among the natural population of the bumblebees examined. We do not, however, have any data on the sexual viability of diploid B. florilegus males. Ayabe et al. (2004) reported the successful production of diploid males and sterile triploid females in B. terrestris by artificial inbreeding. Although we found diploid males and triploid queens in the present study, triploid females are nonmated queens in natural populations. Our findings suggest that diploid males of B. florilegus copulate with females. The frequency of diploid male colonies in B. florilegus was higher than that in other eusocial Hymenoptera (Ross and Fletcher, 1986; Kerr, 1987; Ross et al., 1993; Duchateau et al., 1994; Foster et al., 2000; Yamauchi et al., 2002; Tsuchida et al., 2004; Ellis et al., 2006). Our results indicated that the inbreeding coefficient estimated for this population does not significantly differ from zero. The genetic diversity (number of alleles or observed heterozygosity) of B. florilegus, however, was low across all loci when compared with that of other monoandrous Bombus species (Estoup et al., 1996; Pirounakis et al., 1998; Widmer et al., 1998; Widmer and Schmid-Hempel, 1999; Darvill et al., 2006; Ellis et al., 2006). Similarly, the effective number of alleles at the sex-determining locus K in B. florilegus population was lower (mean = 8) than that in other Hymenoptera (the honeybee Apis mellifera: 19, Adams et al., 1977; the fire ant Solenopsis invicta: 15, Ross and Fletcher, 1986; Ross et al., 1993; the stingless

Diploid male production in Bombus florilegus

bee Melipona compressipe fasciculata: 20, Kerr, 1987; the halictine bee Lasioglossum zephyrum: between 10 and 25, Kukuk and May, 1990; the parasitic wasp Diadromus pulchellus: 15, Periquet et al., 1993; B. terrestris: 24, Duchateau et al., 1994; the paper wasp Polistes chinensis antennalis: 33, Tsuchida et al., 2002). We found the estimated number of alleles at the sex locus to be 6 to 11 (mean = 8) in the B. florilegus population. This number, however, may overestimate the number actually occurring in natural populations. The frequency of matched mating of queens in this population was high; consequently, triploid females and diploid males were borne by the sterile offspring carrying the sl-CSD allele. Based on the results of the microsatellite marker analysis, we demonstrated that B. florilegus in Hokkaido has fewer alleles than other bumblebees (Estoup et al., 1996; Pirounakis et al., 1998; Widmer et al., 1998; Widmer and Schmid-Hempel, 1999; Darvill et al., 2006; Ellis et al., 2006). The effective population size is determined by the number of successfully reproducing colonies in a population rather than by the number of individuals. It was shown recently that the population of B. florilegus has declined, primarily due to land-development activities such as the development of farmlands, residential areas, and roads (Nakatani, 1999). In addition, the European bumblebee B. terrestris was first introduced in Japan for agricultural purposes in 1992 and naturalized colonies of B. terrestris were found to be established in some areas a few years later (Washitani, 1998). The naturalization of the exotic species B. terrestris suggests that the greatest detrimental effects will be exerted on closely related species such as native Japanese bumblebees (Dafni and Shmida, 1996; Thomson, 1997; Washitani, 1998; Hingston and McQuillan, 1999; Goka et al., 2001). Based on the past examples of B. terrestris naturalization, the presence of this exotic species is predicted to have several deleterious effects on native B. florilegus populations, including competition for nest sites and food sources, hybridization, and transmittance of parasites and pathogens. Therefore, in addition to stemming the loss of habitat, the conservation of B. florilegus will require protection from the harmful influence of the exotic B. terrestris. Our results showed that B. florilegus foundress queens are generally monogynous and monoandrous under random-mating conditions. However, 28 % of the colonies included diploid males due to the small population size. Additionally, the results obtained from the spermathecae dissection and PCR of extracted DNA indicated that 11 % of the queens did not copulate at all. This species is in danger of extinction in Japan due to its matched mating or nonmating caused by the small population size. Recently, many colonies of the European bumblebee B. terrestris were introduced in Hokkaido and are used annually as a biological source of pollination in greenhouses. Exotic queens have subsequently escaped from these colonies and have now become naturalized in Hokkaido (Washitani, 1998). There is a possibility that B.

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terrestris is an aggressive competitor for nesting site, habitat, and food resources and possesses the ability to hybridize with native Japanese bumblebees, including B. florilegus (Ito, 1998; Washitani, 1998; Nakatani, 1999; Matsumura et al., 2004). Since local populations of B. florilegus with low reproductive success may be particularly susceptible to the naturalization of B. terrestris, the conservation of B. florilegus will thus require measures that not only protect it from habitat destruction but also from the impact of competition with exotic species.

Acknowledgements We sincerely thank the laboratory members of Hokkaido University and Kyoto University for kindly helping in sample collection for this works. This study was supported by JSPS research fellowships for J.T. and Grant-in-Aid for Scientific Research (B) of JSPS (17380038) for M.O.

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