Journal of Heredity 2004:95(1)
© 2004 The American Genetic Association 95:46-52
Genetic Structure of Asian Populations of Bombus ignitus (Hymenoptera: Apidae)
From the Laboratory of Coevolution, Shanghai Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 20032, P. R. China (Shao, Mao, and Fu), Laboratory of Molecular Biology of Domestic Animals, and Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, P. R. China (Shao and Zhang), Laboratory for Conservation and Utilization of Bioresources, Yunnan University, Kunming 650091, China (Zhang), Laboratory of Insect Technology, Department of Bioresource Sciences, Faculty of Agriculture, Tamagawa University, Machida, Tokyo 194-8610, Japan (Ono), Shanghai Key Laboratory of Protected Horticultural Techniques, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd., Shanghai 201106, China (Wang), and Department of Animal Biology, University of Pavia, Piazza Botta 9, 27100 Pavia, Italy (Bonizzoni). We are indebted to many friends and colleagues: Myenong-Lyeol Lee (Korea), W.-J. Yu, and M.-F. Yang for sample collection; Y.-G. Yao, J.-F. Pang, H.-R. Luo, K.-X. Qu, D. Pang, G.-S. Wu, Y.-X. Luan, J.-G. Xiangyu, J. Luo, and X.-L. Song for technical and theoretical help; and A. Widmer (Switzerland) and R. Malacrida (Italy) for manuscript revisions. This work was supported by the Chinese Academy of Sciences (KSCX2-1-05), Program for Key International S&T Cooperation Project of China (2001CB711103), and Natural Science Foundation of China (NSFC).
Address correspondence to Ya-Ping Zhang, Laboratory for Conservation and Utilization of Bioresources, Yunnan University, Kunming 650091, China, or e-mail: zhangyp{at}public.km.yn.cn.
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Abstract |
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The genetic structure of seven mainland and island Asian populations of Bombus ignitus was investigated using nine microsatellite markers and the sequences of part of the mitochondrial cytochrome b (cytb) gene. While microsatellite markers showed high genetic variability, no sequence variation was found in the cytb gene fragment analyzed. The number of microsatellite alleles ranged from 9 to 24. Gene diversities per locus per population ranged from 0.378 to 0.992. Analysis of molecular variance (AMOVA) and most pairwise FST values showed significant genetic differentiation between mainland and island populations. Cytb sequences data and microsatellite bottleneck tests indicated that almost all populations were subjected to recent bottlenecks. Our results suggest that B. ignitus populations diverged due to recent bottlenecks and geographic isolation.
Bumblebees are widely used as tomato pollinators in greenhouses because they are more efficient than honeybees in low-light conditions. After the refinement of bee-rearing techniques (Eijnde et al. 1991; Pouvreau and Marileau 1980), many companies could provide colonies at any developmental stage. However, there were only a few species merchandized, most commonly Bombus terrestris. As it was inevitable that bees were transported between different regions (Ortiz-Sanchez 1992), thus promoting gene exchange, it is urgent to characterize the genetic structure of different geographic populations to avoid the spreading of some dangerous parasites and phenomena analogous to the hybridized European honeybee problem from the African bee invasion (Goka et al. 2001; Visscher 2001).
Bombus ignitus was mainly distributed in China, Japan, and Korea (Williams 1996) and was used in China and Japan as a commercially available pollinator. Many merchandisers were trying to export it to all over the world because of its biology and efficiency for pollinating, similar to B. terrestris (Asada and Ono 2000; Bilinski 1999). Two main European bumblebee species, B. terrestris and Bombus pascuorum, were studied for geographic genetic variation and both of them showed that insular populations differentiated from continental ones (Estoup et al. 1996; Pirounakis et al. 1998; Widmer et al. 1998, 1999). However, the population structure of B. ignitus has been rarely explored.
Mitochondrial DNA (mtDNA) has been widely used in vertebrates and invertebrates as a molecular genetic marker because of its high mutation rate, haploid maternal inheritance, and its abundance in cells, more than 10,000 copies (Brown et al. 1979; Gillham 1994; Moritz et al. 1987). After the honeybee mtDNA genome was completely sequenced (Crozier and Crozier 1993), mtDNA sequences were used to study the evolutionary relationships among many Hymenoptera taxa (Estoup et al. 1996; Koulianos 1999; Koulianos and Schmid-Hempel 2000; Widmer et al. 1998, 1999). Estoup et al. (1996) found little difference within and between continent and island B. terrestris populations based on mitochondrial COII gene sequences, while Widmer et al. (1998) found more in the cytb gene. Widmer and Schmid-Hempel (1999) and Pirounakis et al. (1998) also found many variations in the cytb sequence in B. pascuorum.
Microsatellites are popular genetic markers because of their high polymorphism (Paxton et al. 2001; Queller et al. 1993; Widmer et al. 1998, 1999), and are used for studying bumblebee population structure and paternal identity (Estoup et al. 1995a–c, 1996; Paxton et al. 2001; Widmer et al. 1998, 1999).
In the present study we analyzed the variation in nine microsatellite loci and part of the cytb gene sequence to study the genetic structure of different populations of B. ignitus from China, South Korea, and Japan. Our aim was to determine whether there was genetic differentiation between different local populations.
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Materials and Methods |
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Materials
Queen and worker bees were collected from three localities in mainland China (Beijing, Zhejiang, and Guizhou), three localities in mainland South Korea (W
nju, Muju, and Jeongseon), and three localities in Japan (Nagano, Niigata, and Yamanashi) between 1999 and 2001 (Figure 1). Wnju, Muju, and Jeongseon bees were regarded as one population because of the small sample size. All bees were kept in pure ethanol at room temperature. Individuals were taken more than 500 m apart to decrease the possibility of sampling individuals from the same colony (Estoup et al. 1996).
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DNA Extraction
DNA was isolated from about 50 mg of tissue from the head, thorax, or legs using standard phenol/chloroform methods (Garnery et al. 1991; Koulianos 1999).
Microsatellite Analysis
A total of 124 individuals were analyzed at nine microsatellite loci (B10, B11, B124, B96, B100, B126, B119, B118, and B132), previously described in Estoup et al. (1995b, 1996). Polymerase chain reactions (PCRs) were set up with 5–10 ng total DNA, 0.4 pM for each pair of primers, 0.2 mM for each dNTP (including 1/100 florescent dye-labeled dNTP), 0.4 µg/µl BSA, 1x PCR buffer, 0.5–1.0 mM MgCl2, 0.5 U Taq polymerase to a final 10 µl volume. PCR was performed at 96°C for 5 min, 35 cycles of 96°C for 50 s, 53–58°C (referring primers as described in Estoup et al. 1995b, 1996) for 40 s, 72°C for 40 s, followed by a 10 min extension at 72°C. The PCR products were scanned with internal standard Mark GS350 [ROX] using an ABI 377 sequencer (Perkin-Elmer). The results were read with Gene Analysis 3.1 and further analyzed with Genotyper 2.0, then checked by eye.
PCR Amplification and Sequencing
Sixteen individuals selected from all the populations (Figure 1) were amplified and sequenced for part of the cytb gene by using the CB1 and CB2 primers of Simon et al. (1994). These primers locate to 11,400–11,425 bp and 11,859–11,884 bp in the honeybee mtDNA sequence (Crozier and Crozier 1993). Sequencing was performed using a Bigdye Terminator cycle sequencing kit (PE Biosystems) on an ABI 377 sequencer. Each individual was sequenced in both directions. Sequences were submitted to GenBank with the accession number AF498398.
Data Analyses
The cytb sequences were edited by Editseq and aligned by Segman in the DNAStar package. Unless specified, microsatellite data were gathered using Arlequin 2.0 (Schneider et al. 2000). Pairwise linkage disequilibrium and the Hardy-Weinberg equilibrium (HWE) were checked to analyze the association between the microsatellite loci and population states (Guo and Thompson 1992; Lewontin and Kojima 1960; Nei 1978; Slatkin and Excoffier 1996). The pairwise FST and RST could be used as short-term genetic distances between populations, the former fitting for the infinite alleles model (IAM) and the latter for single stepwise mutation model (SMM; Balloux and Lugon-Moulin 2002; Schneider et al. 2000; Slatkin 1995). We tested the relationship between local populations by calculating pairwise FST because B. ignitus mainly performed IAM based on previous studies (discussed in the results of the bottleneck test). To estimate the degree of population differentiation, an analysis of molecular variance (AMOVA) was performed (Excoffier et al. 1992; Schneider et al. 2000; Slatkin 1991; Weir 1996; Weir and Cockerham 1984; Wright 1931). For this analysis, the populations analyzed were divided into two groups: a group of island populations and a group of mainland populations. Because there was no definite boundary separating individuals and the population definition might be not appropriate, the distribution of the assignment criterion for individuals of a given population was used to confirm the probability that an individual belonged to the population with the software GeneClass (Cornuet et al. 1999). This analysis was done with the Bayesian likelihood method, with individuals assigned to the population in which the likelihood of their genotype was highest, which was better on the basis of Cornuet's description (Cornuet et al. 1999).
When a population was subjected to a bottleneck or expansion event, the microsatellite observed heterozygosity would be larger or smaller than the expected heterozygosity (Cornuet and Luikart 1996). The bottleneck test was performed with Bottleneck version 1.2.02 (Cornuet and Luikart 1996). The significance of the test was assessed with the Sign and Wilcoxon tests. The first test suffers from low statistical power; the second test provides relatively high power and can be used with as few as four polymorphic loci and any number of individuals (Cornuet and Luikart 1996). The test was run with the IAM mutation model because it was the fittest model for this species based on present results (outlined in the results section).
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Results |
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mtDNA Variation
A segment of 450 bp from the cytb gene was sequenced from 16 individuals chosen from the eight bee populations. We did not observe any variation in this region in B. ignitus. However, extensive variation has been observed between bumblebee species (Figure 2).
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Gene Diversity, HWE, and Linkage Disequilibrium
Table 1 shows the polymorphism results at nine microsatellite loci in the eight populations of B. ignitus. The number of microsatellite alleles ranged from 9 to 24. Gene diversities per locus per population ranged from 0.378 to 0.992. Departure from HWE was found in 29 of 72 cases, most commonly in the B96 and B119 loci (Table 1). The reason for the deviations from HWE might be high selective stress (bottleneck; discussed later) and small sample size. No departure was found when the exact test was performed in each population. Linkage disequilibrium was found in 35 of 252 cases among the analyzed loci and populations, but no common pair of loci deviated from equilibrium in all the populations (data not showed).
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Population Differentiation
Population differentiation was tested first by FST distance. In addition to the fact that all the mainland-island comparisons were significant, FST comparisons were also significant between the two mainland populations (Zhejiang and Beijing) and between the island populations (Nagano and Niigata, and Nagano and Yamanashi) (Table 2). Gene diversity per population per locus was similar for island and mainland populations, which is in contrast with the results of Estoup et al. (1996).
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Analysis of molecular variance is a method of estimating population differentiation directly from molecular data and testing hypotheses about differentiation (Excoffier et al. 1992). The result of the AMOVA test showed that the island group was differentiated from the mainland group (P <.05, data not shown).
An individual assignment test showed 64.52% of those individuals were correctly identified, and only 5 of the 44 wrongly assigned individuals were interchanged between the mainland and island populations. Thus samples from those localities were, to some extent, typical (data not shown).
Bottleneck Test
The bottleneck test with the IAM model showed deviations from mutation equilibrium for three populations (Beijing, Zhejiang, and Nagano) with the Sign test, and only two populations (Guizhou and South Korea) showed no deviation with the Wilcoxon test (Table 3). All the deviations were due to excess observed heterozygosity.
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Three models of evolution have been proposed for microsatellites: SMM, IAM, and TPM (Cornuet and Luikart 1996; Di Rienzo et al. 1994; Kimura and Crow 1964; Kimura and Otha 1978). Estoup (1995a,c) found that, for honeybees and bumblebees, microsatellites did not fit the SMM, but did fit the IAM. Most recent studies have shown that dinucleotides evolved in several steps (Huang et al. 2002). Although there was no definite model for B. ignitus, the nine microsatellite loci we studied were dinucleotide repeats. Thus we followed the IAM. Under this model, excess heterozygosity was found for most populations, which may be a response to recent bottlenecks. The small sample size of Guizhou and South Korea possibly contributed to their results of no significance.
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Discussion |
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Genetic Variation
The analysis of nine microsatellite markers and part of the cytb sequences of 124 individuals from seven local populations of B. ignitus showed that while microsatellite markers were polymorphic, no variation was found in the cytb sequences. Previous studies of B. pascuorum showed six haplotypes in the same part of the cytb sequence (Figure 2), which suggests that genetic diversity of mtDNA in B. pascuorum is at least several times higher than that in B. ignitus (Pirounakis et al. 1998). Another kind of bumblebee, B. terrestris, also showed high diversity in this part of cytb (Widmer et al. 1998). A high level of cytb sequence variation among different bumblebee species has also been observed (Figure 2).
There were three possible explanations for the lack of cytb sequence variation in B. ignitus. First, B. ignitus diverged later than the other two bee species. Second, the B. ignitus cytb sequence evolved slower than that of the other two bee species. Third, B. ignitus populations experienced a recent bottleneck. Our previous phylogenetic analysis made us reject the first explanation because B. ignitus diverged earlier than B. terrestris (Shao et al. 2002). The similar branch length of B. ignitus and B. terrestris (Shao et al. 2002) and the results of the relative rate test did not support that the B. ignitus cytb gene evolved slower. However, the third explanation was supported by the results of the bottleneck test, which found that most populations were subject to bottlenecks. Further studies are required to verify our explanation.
Population Differentiation
Genetic variability of B. ignitus was high for microsatellites. Most of the variation was present within populations rather than among populations, as was previously found in B. pascuorum (Widmer and Schmid-Hempel 1999). Most pairwise FST comparisons were not significant between pairs of mainland populations or between pairs of island populations, which might result from long-time gene pool homogenizing. Although no migration of B. ignitus has so far been reported, bumblebees are capable of flying long distances, and migration of B. terrestris, B. lucorum, and B. lapidarius have been observed (Mikkola 1978; Philip 1957). Moreover, a small number of migrants can be enough to homogenize different populations (Hartl and Clark 1989; Ibrahim et al. 1996).
Most island populations displayed significant genetic differentiation from mainland populations. Geographic isolation might be the cause of this genetic differentiation. An analogous situation of geographic isolation was found in two other bee species, B. terrestris and B. pascuorum (Estoup et al. 1996; Pirounakis et al. 1998; Widmer and Schmid-Hempel 1999; Widmer et al. 1998).
Another reason for the homogenization within island or mainland populations and differentiation between populations may be recent bottlenecks. Indeed, the lack of mtDNA diversity and the results of the bottleneck test on microsatellite data support the theory that those populations were subjected to recent bottlenecks.
Potential Influence of Human Activity
As B. ignitus is now used for pollination in China and Japan and may be commercially transported all over the world, one may have concern about the ecological effect. Our survey indicates that there is little differentiation in continental populations, so there is no need to worry about the impact of B. ignitus within isolated fields. However, the Japanese bees showed remarkable differentiation from continental populations, so there is a need to avoid homogenizing both of the separated gene pools when using this bumblebee as pollinator.
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Footnotes |
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Corresponding Editor: R. C. Woodruff
Received October 4, 2002
Accepted August 15, 2003
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