Open Access

Genetic structure of Mount Huang honey bee (Apis cerana) populations: evidence from microsatellite polymorphism

  • Fang Liu1,
  • Tengfei Shi1,
  • Sisi Huang1,
  • Linsheng Yu1, 3Email author and
  • Shoudong Bi2
Contributed equally
Hereditas2016153:8

DOI: 10.1186/s41065-016-0010-4

Received: 4 February 2016

Accepted: 13 June 2016

Published: 6 July 2016

Abstract

Background

The Mount Huang eastern honey bees (Apis cerana) are an endemic population, which is well adapted to the local agricultural and ecological environment. In this study, the genetic structure of seven eastern honey bees (A. cerana) populations from Mount Huang in China were analyzed by SSR (simple sequence repeat) markers.

Results

The results revealed that 16 pairs of primers used amplified a total of 143 alleles. The number of alleles per locus ranged from 6 to 13, with a mean value of 8.94 alleles per locus. Observed and expected heterozygosities showed mean values of 0.446 and 0.831 respectively. UPGMA cluster analysis grouped seven eastern honey bees in three groups.

Conclusion

The results obtained show a high genetic diversity in the honey bee populations studied in Mount Huang, and high differentiation among all the populations, suggesting that scarce exchange of honey bee species happened in Mount Huang. Our study demonstrated that the Mount Huang honey bee populations still have a natural genome worth being protected for conservation.

Keywords

Apis cerana Honey bee Microsatellite Mount huang SSRs

Background

Mount Huang lies in the southern end of the Anhui province of China (Wannan), between 117°02′–118°55′ east longitude and 29°24′–30°24′ north latitude. It has a typical humid subtropical monsoon climate, luxuriantly diverse flora and fauna, including honey bees. The Mount Huang eastern honey bees (A. cerana) are an endemic population, which is well adapted to the local agricultural and ecological environment. They have a particularly strong foraging instinct and resistance to parasitic mites as well as American foulbrood. This endemic population was listed in the first directory of potential breeding stock for conservation in 2001. The A. cerana conservation document Wannan (Wan D/XM01-19-87) was published according to ecological standards in Anhui province. However, since then, a massive replacement of the endemic honey bees has occurred, especially after the importation of Apis mellifera [1]. In some regions, this has resulted in almost complete replacement of endemic A. cerana populations with imported stocks.

Microsatellites are suitable nuclear markers used in population genetic studies in various taxonomic groups [2, 3]. This is due to their mass polymorphic loci, codominance, abundance throughout the genome, and relative ease of scoring [4]. The genetic structure of eastern honey bee populations from many regions of China have been analyzed using microsatellite markers. These regions mostly include areas in the middle east (Fujian, Jiangsu and Zhejiang provinces) and the south west (Tibei, Yunnan province and Hainan province). Zhu et al. [5] used five microsatellite loci to analyze the genetic structure of six A. cerana populations from Fujian province, their results showed that the mean expected heterozygosities (He) of populations from most of regions in Fujian was 0.5171 . In another study, a significant difference in genetic structures was found among honey bees from six regions in Wuyi Mountain located in Fujian province, the mean He of the six different region population was range from 0.4525–0.5613 [6]. Using six microsatellite loci, Ji et al. [7] found higher population heterozygosity and abundant genetic diversity in Apis cerana as well as in Apis mellifera (western European honey bee) populations in Jiangsu province, they found the mean He of A. cerana population in this province was 0.4424. Cao et al. [8] analyzed the genetic structure of more than ten A. cerana populations from different regions in Zhejiang province, their results showed that the genetic diversity of A. cerana in Zhejiang was relatively low, with the mean He was 0.3179. Guo et al. [9] used 8 microsatellite markers found the A. cerana populations in Qinling-Data mountain areas has rich genetic polymorphism, with the mean He, number of alleles, polymorphism information content (PIC) were 0.6877, 7.71, 3.7488 respectively. Ji et al. [10] found significant differentiation among 20 A. cerana populations from geographically different regions in China using 21 microsatellite loci. 502 alleles were found, and the average value of He of 21 microsatellites was 0.8689. Additionally, the mean He of the populations from Wuding (Yunnan province), Hainan, Bomi Tibet was 0.695, 0.6641 and 0.562 respectively, revealing their relatively high genetic diversity. Genetic diversity of A. cerana populations from Lushui in Yunnan province was evaluated with 21 microsatellite markers. 121 alleles were found, and the mean number of effective alleles was 5.76, the average He and PIC of all loci were 0.6752 and 0.6192, respectively, these results indicated the high level of genetic diversity in Lushui [11]. Six A. cerana populations distributed in different geographic areas in the south west Yunnan province were also surveyed using 21 microsatellite loci. All populations showed high levels of heterozygosity except the population from Diqing, the average He of the total populations was 0.739, and a significant genetic differentiation was found among all six populations [12]. Taken together, the genetic variation of A. cerana populations is overall higher in the southwest of China than in the middle east of China.

However, information about genetic variation of A. cerana in Mount Huang of Anhui province, in south east China, is presently not available. Therefore, the objectives of this study were to examine the genetic structure of Mount Huang honey bee populations using microsatellite analyses. Our findings provide important information that can be used for effective management and conservation of A. cerana in Wannan.

Methods

Samples

Adult workers from 190 colonies were collected from seven locations covering the main bee keeping areas in Huangshan [13], south of Anhui province in China (Wannan). Over 25 colonies were obtained from Jing County (JC), Huangshan District (HSD), Qimen County (QMC), and Yi County (YC), whereas 30 colonies were collected from Jixi County (JXC), She County (SC) and Huizhou District (HZD). The distances between the locations are more than 10 km. Ten bees from each colony were subjected to genetic analysis. Honey bee samples were preserved in 95 % ethanol and kept at −20 °C until needed.

DNA extraction and microsatellites amplification

Total DNA was extracted from the thorax of each bee, following the method of Smith and Hagen [14], and the concentration was then estimated using ultraviolet spectrophotometer MV-2201. DNA was stored at −20 °C until needed. The primer sequences of these loci were as previously described by Solignac et al. [15]. The annealing temperature and the concentration of MgCl2 are listed in Table 1. The PCR conditions were as follow: one cycle at 95 °C for 5 min; 35 cycles of 95 °C for 45 s, 50–60 °C for 45 s, 72 °C for 60 s; and one cycle at 72 °C for 8 min. The PCR products were electrophoretically fractionated in 6% denaturating polyacrylamide gels and visualized by silver staining. The size of amplified microsatellites was measured using EDA20 Kodak imaging system (Eastman Kodak, USA).
Table 1

Location of 16 microsatellite loci in chromosome or linkage group and PCR conditions

Locus

Chromosome or linkage group

GenBank Accession number

Mg 2+ (mmol/L)

Annealing temperature (°C)

AP243

ChrLG1

AJ509466

2.2

57.5

A043

ChrLG1

AJ509256

1.5

55

AP274

ChrLG3

AJ509486

2.0

55.0

AT105

ChrLG5

AJ509553

2.2

56.5

A113

ChrLG6

AJ509290

2.0

58.2

A107

ChrLG7

AJ509287

2.0

57.0

A024d

ChrLG7

AJ509241

2.0

57.0

A014

ChrLG8

AJ509239

2.0

55.6

A088

ChrLG8

AJ509283

1.6

58.0

A007

ChrLG8

AJ509236

2.0

55

AP033

ChrLG10

AJ509319

2.2

56.5

AP297

ChrLG12

AJ509499

1.0

54

A029

ChrLG12

AJ509245

1.2

58

A035

ChrLG14

AJ509251

2.0

53.4

A028

ChrLG14

AJ509244

2.0

55.0

AG005C

ChrLG16

AJ509723

2.0

55.0

Statistical analyses

The number of alleles, allele frequencies, observed and expected heterozygosity (Ho and He) of each microsatellite locus were computed with Microsatellite-Toolkit software [16]. Genetic and genotypic differentiation tests were conducted among the 7 populations using GENEPOP 4.1. The exact test for Hardy-Weinberg equilibrium (HWE), genetic structure and gene flow (Nm) were calculated with GENEPOP 4.1 [17]. F-statistics between populations were calculated using the software FSTAT (v2.9.3) [18]. The significance level of multiple comparisons was calculated using the sequential Bonferroni procedure [19].

The number and frequency of alleles were used to obtain the Da genetic distance between each population. Population tree based on Da values was constructed using the neighbor-joining (NJ) algorithms and unweighted pair group method with arithmetic mean (UPGMA) using PHYLIP software package (version 3.69) [20].

Results

The locations where honey bee samples were collected were mentioned in Yu et al. [13]. The number and size of alleles, the value of Ho and He are listed in Additional file 1: Table S1. A total of 143 alleles were detected at 16 microsatellite loci. The overall parameters per population are shown in Additional file 1: Table S1. The gene diversity (the mean He) varied between 0.597 ± 0.109 in the HSD population and 0.71 ± 0.056 in the SC population, and mean observed heterozygosities between 0.398 ± 0.186 in HSD population and 0.495 ± 0.235 in JC population. The observed heterozygosity (Ho) was relatively high in all populations except that of JC at loci A035, and HSD population at loci A107. The mean number of alleles for each population ranged from 2.936 ± 0.853 in HSD to 6.438 ± 1.632 in SC population.

We detected significant deviations (P < 0.05) from Hardy-Weinberg equilibrium in 86 out of 112 population-locus combination and all deviations were in favor of homozygotes (Table 2). Both genic and genotypic differentiation tests showed highly significant (P < 0.0001) differentiation among all populations. Multilocus FIS values varied between 0.181 (A029) and 0.618 (A088), while the FIT values varied from 0.314 (A028) to 0.701 (AP297). A high heterozygote deficiency within populations and the total population were revealed by positive FIS and FIT (Table 3). The value of FST for the total populations was 0.171, all loci contributed significantly to the differentiation (P < 0.001).
Table 2

The P -value of every population and every locus in Hardy-Weinberg test

locus

P value

JC

HSD

JXC

YC

QMC

SC

HZD

AP243

0.4447

0.2713

0

0.0087

0.2283

0.0084

0.0034

A043

0.0001

0.0046

0

0

0.3076

0

0.0008

AP274

0.0003

0.0995

0.001

0.0058

0.2845

0

0.0017

AT105

0.003

0.3695

0.0002

0.071

0.0716

0

0.0813

A113

1

0

0

0.0023

0.0725

0

0.0025

A107

0.1234

0

0.1747

0.322

0.0402

0

0.7541

A024d

0.0001

0

0.0019

0.0027

0.0106

0

0.0009

A014

0.0001

0.0092

0

0.0117

0

0.0004

0

A088

0.0115

0.0001

0

0

0.0001

0

0

AP033

1

0.0025

0.0029

0.0034

0.0057

0.007

0.2327

AP297

0.0052

0.7253

0

0

0.0018

0.0001

0

A029

0.0243

0.9913

0.0195

0.8768

0.9816

0.1932

0

A007

0.0148

0.0001

0.0033

0.001

0.0027

0

0

A035

0

0.9728

0

0.101

0.0111

0

0.0026

A028

0.3279

0.0358

0.309

0.0004

0

0

0.0008

AG005C

0.0009

0

0.0014

0.0023

0.2447

0

0.0012

Table 3

F. coefficients of total of populations including 7 A. cerana in Mount Huangshan of Wannan

Locus

F IT

F ST

F IS

AP243

0.361***

0.108***

0.283***

A043

0.631***

0.203***

0.541***

AP274

0.453***

0.131***

0.372***

AT105

0.390***

0.134***

0.295***

A113

0.361***

0.126***

0.268***

A107

0.360***

0.198***

0.200***

A024d

0.527***

0.110***

0.470***

A014

0.568***

0.170***

0.479***

A088

0.681***

0.166***

0.618***

AP033

0.349***

0.181***

0.208***

AP297

0.701***

0.268***

0.589***

A029

0.404***

0.272***

0.181***

A007

0.444***

0.077***

0.398***

A035

0.604***

0.277***

0.458***

A028

0.314***

0.116***

0.225***

AG005C

0.481***

0.150***

0.390***

Mean ± SD

0.47 ± 0.032***

0.171 ± 0.016***

0.369 ± 0.035***

Note: Mean estimates from jack-knife over loci, standard deviations (SD) are given in parentheses; ***p < 0.001

Genetic distances between pairs of Mount Huang A. cerana, in the present study, were 0.2104-0.5027 (Table 4, lower triangle). The pairwise Nm value (Table 4, upper triangle) between populations varied between 0.6049 (SC and JXC) and 0.8345 (YC and HSD). The neighbor-joining tree (Fig. 1) was constructed according to the Da genetic distance between each pair of populations. The SC population formed a group separating from other populations. JXC and HZD formed a cluster, JC, HSD, YC and QMC grouped together.
Table 4

Nei’s genetic distance, Da (lower triangle) and the gene flow, Nm (up triangle) between populations

Population

JX

HSQ

JXX

YX

QMX

SX

HZQ

JX

–––––

0.7812

0.7326

0.8103

0.7714

0.6623

0.7503

HSQ

0.2469

–––––

0.6594

0.8345

0.6827

0.6053

0.6148

JXX

0.3112

0.4164

–––––

0.7201

0.6678

0.6049

0.7016

YX

0.2104

0.1809

0.3284

–––––

0.8050

0.6749

0.7239

QMX

0.2595

0.3817

0.4038

0.2169

–––––

0.6335

0.6568

SX

0.4120

0.5020

0.5027

0.3932

0.4565

–––––

0.6456

HZQ

0.2872

0.4864

0.3544

0.3231

0.4204

0.4375

–––––

The line means blank. There is no genetic distance between the same populations

https://static-content.springer.com/image/art%3A10.1186%2Fs41065-016-0010-4/MediaObjects/41065_2016_10_Fig1_HTML.gif
Fig. 1

Dendogram of phylogenetic relationships among 7 Apis cerana populations based on Nei’s genetic distance using neighbor-joining method

Discussion

Population differentiation was detected in Mount Huangshan A. cerana through analysis of microsatellite polymorphism. There are limited population genetic studies of Mount Huangshan A. cerana, using microsatellite analysis. A previous study by Yu et al. [13], using morphometric analysis reported similar finding. Therefore, our study adds to the knowledge of the genetic changes that may be occurring in this important endemic honey bee population. This knowledge can be used for genetic management and conservation efforts, for the preservation of this population, which was first listed in the directory of potential breeding stock for conservation in 2001. The pure A. cerana population has several desirable advantages biological features, including disease resistance and the ability to get rid of bee mites from colonies. To date, other studies of A. cerana by microsatellite loci have focused on the eastern and southern populations of China. According to those reports, the expected heterozygosity levels were highest among honey bee populations in the south west region, which ranged between 0.5625 and 0.738 [1012], and were lowest among honey bees in the middle east reported as 0.3179-0.5613 [58]. In the present study, the microsatellite mean expected heterozygosity values for ranged between 0.597 and 0.781, which is similar to those of honey bees in Yunnan. This similarity may be attributable to similar geographic environments in Yunnan and Mount Huang, which are both mountainous with high altitude.

The microsatellite data also showed that, the Mount Huang populations seem to be more variable compared to those in the eastern Chinese region. Average allele numbers and heterozygosities were higher in Mount Huang populations than in Fujian [5, 6], Jiangsu [7], Zhejiang [8]. This difference may, in part, be attributable to differences in the evaluated population sizes.

Significant deviations from Hardy-Weinberg equilibrium were observed at loci A024d, A014, A088, and A007 in all investigated populations even with correction by sequential Bonferroni method. Such deviation may be due to miscoring of alleles resulting from the existence of shatter bands [21]. However, in this case it is unlikely this is the cause, because the band intensity among a group of stutter bands was very carefully examined in order to obtain consistent allele designation. Due to the use of heterospecific primers in this species, the other factor may be a result of the presence of null alleles at these microsatellite loci [22]. It will be premature to conclude which of these factors contribute heterozygote deficiencies of A. cerana in Mount Huang until these bees are examined using microsatellite primers developed from A. cerana rather than those from A. mellifera [23].

Considering the FST estimates, all 16 microsatellite loci contributed mostly to differentiate the populations (Table 3), corresponding to the low Nm value (Nm < 1) between populations, which indicated. The FST value of AP033 loci here is similar to Ji et al. [7]. The bee samples we collected in present study included wild honey bees and domestic honey bees, which also may contribute to the large differentiation among honey bees from different locations. A total of seven Apis cerana populations in Huangshan were isolated by high mountains [24], it is unlikely gene flow for these honey bees.

Large genetic distances were observed among all populations, especially those between A. cerana from SC population and other populations (d = 0.3932-0.5027). The seven A. cerana populations in this study could then be genetically divided into three different groups, which included one group with JC, HSD, YC, and QMC populations, another with JXC and HZD populations, while the SC population was a separate group (Fig. 1). YC was firstly linked to HSD, which was also indicated by the relative high Nm values (0.7812) between them. While SC formed one group alone, it is located between Mount Huang and Mount Tianmu, this may have been an important barrier to gene flow for SC A. cerana and other populations.

Conclusion

Microsatellite data obtained in this study demonstrated that the Mount Huanghoney bee populations still have a natural genome worth being protected for conservation. However, further screening of A. cerana on a larger geographic scale should be carried out to fully elucidate the patterns of conspecific and intraspecific population structure in Wannan A. cerana.

Declarations

Acknowledgements

This work was supported by grants earmarked for Modern Agro-industry Technology Research System (CARS-45-KXJ9), and National Natural Science Foundation of China (No, 31272511; No. 31302039). We are grateful to Chengwu Wu, the director of Bureau of Animal husbandry of Huangshan in Anhui Province, and the Five Bee Home Limited Company for their help in bee-collecting. And we thank Ting Ji and Ling Yin from Yangzhou University for their kindly help in the research.

Authors’ contributions

FL and LSY designed research; FL and STF performed research; FL, SSH and SDB analyzed data and wrote the paper. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Honey bee Research Institute, College of Animal Science & Technology, Anhui Agricultural University
(2)
College of Forest and Garden, Anhui Agricultural University
(3)
Apiculture Research Institute, College of Animal Science & Technology, Anhui Agricultural University

References

  1. Yu LS, Zou YD, Bi SD S. Relation between population dynamics and distribution of honey bees and ecological environment. Chin J Appl Ecol. 2006;17:1465–8. In Chinese.Google Scholar
  2. Bowcock AM, Linares RA, Tomforhde J, Minch E, Kidd JR, Sforza CLL. High resolution of human evolutionary trees with polymorphic microsatellites. Nature. 1994;368:455–7.View ArticlePubMedGoogle Scholar
  3. Taylor AC, Sherwin WB, Wayne RK. Genetic variation of microsatellite loci in a bottlenecked species: the northern hairy-nosed wombat Lasiorhinus krefftii. Mol Ecol. 1994;3:277–99.View ArticlePubMedGoogle Scholar
  4. Bodur ÇM, Kence KA. Genetic structure of honeybee, Apis mellifera L. (Hymenoptera:Apidae) populations of Turkey inferred from microsatellite analysis. J Apicult Res. 2007;46:50–6.View ArticleGoogle Scholar
  5. Zhu XJ, Zhou BF, Wu XD, Xu JX, Xia XC. Genetic diversity of Apis cerana cerana in Fujian based on microsatellite markers. J Fujian Agric Forestry Univ (National Science Edition). 2011;40(4):407–11. In Chinese.Google Scholar
  6. Zhu XJ, Xu XJ, Zhou MQ, Wu XD, Zhou BF. Genetic analysis of Apis cerana cerana in Wuyi mountain nature reserve based on microsatellite DNA. Fujian J Agric Sci. 2011;26:935–40 (In Chinese).Google Scholar
  7. Ji T, Chen J, Yin L, Bao WB, Chen GH. Study on microsatellite genetic polymorphism of wild and domestic Apis cerana cerana populations from Jiangsu province. China Anim Husbandry Vet Med. 2007;34:39–41 (In Chinese).Google Scholar
  8. Su XL, Cao L, Zhao DX, Hua Q, Hu FL. Genetic Diversity of Microsatellite DNA for Apis cerana cerana in Zhejiang. Apicult Chin. 2013;64:10–1 (In Chinese).Google Scholar
  9. Guo HP, Zhou SJ, Xu XJ, Xu JX, Yu YL, Yang KJ, et al. Population genetic analysis of Apis cerana cerana from the Qinling-Daba mountain areas based on microsatellite DNA. Acta Entomol Sin. 2016;59:337–45 (In Chinese).Google Scholar
  10. Ji T, Yin L, Chen G. Genetic diversity and population structure of Chinese honeybees (Apis cerana) under microsatellite markers. Afr J Biotechnol. 2011;10:1712–20.Google Scholar
  11. Lin D, Yin L, Jing T, Liu M, Chen HG. Study on genetic diversity of Apis cerana cerana populations from Lushui by microsatellite markers. Apiculture of China. 2009;60:9–11 (In Chinese).Google Scholar
  12. Yin L, Ji T, Chen GH. Study on genetic diversity and genetic differentiation of six geographic populations of Apis cerana (Hymenoptera: Apidae) in Yunnan by microsatellite markers. Southwest China J Agric Sci. 2011;24:772–9 (In Chinese).Google Scholar
  13. Yu LS, Liu F, Huang SS, Bi SD, Zong C, Wang TS. Morphmetric analysis of Apis cerana populations in Huangshan. China J Apic Sci. 2013;57:117–24.Google Scholar
  14. Smith DR, Hagen RH. The biogeography of Apis cerana as revealed by mitochondrial DNA sequence data. J Kansas EntomolSoc. 1996;69:294–310.Google Scholar
  15. Solignac M, Vautrin D, Baudry E, Mougel F, Loiseau A, Cornuet JM. A microsatellite-based linkage Map of the honeybee, Apis mellifera L. Genetics. 2004;167(1):253–62.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Nei M. Analysis of gene diversity in subdivided populations. P Natl Acad Sci USA. 1973;70:3321–3.View ArticleGoogle Scholar
  17. Rousset F. GENEPOP’ 007: a complete reimplementation of the GENEPOP software for windows and linux. Mol Ecol Resour. 2008;8:103–6.View ArticlePubMedGoogle Scholar
  18. Goudet J. FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3). Available from http://www2.unil.ch/popgen/softwares/fstat.htm. 2001. Updated from Goudet (1995).
  19. Rice WS. Analyzing tables of statistical tests. Evolution. 1989;43:223–5.View ArticleGoogle Scholar
  20. Nei MF, Tajima TY. Accuracy of estimated phylogenetic trees from molecular data. J Mol Evol. 1983;19:153–70.View ArticlePubMedGoogle Scholar
  21. O’Reilly P, Wright JM. The evolving technology of DNA fingerprinting and its application to fisheries and aquaculture. J Fish Biol. 1995;47:29–55.View ArticleGoogle Scholar
  22. Callen DF, Thompson AD, Shen Y, Phillips HA, Richards RI, Mulley JC, et al. Incidence and origin of ‘null’ alleles in the (AC) n microsatellite markers. Am J Hum Genet. 1993;52:922–7.PubMedPubMed CentralGoogle Scholar
  23. Sittipraneed S, Laoaroon S, Klinbunga S, Wongsiri S. Genetic differentiation of the honey bee (Apis cerana) in Thailand: evidence from microsatellite polymorphism. J Apicult Res. 2001;40:9–16.View ArticleGoogle Scholar
  24. Jing CR. Mount Huangshan. Beijing: Science Press; 1994.Google Scholar

Copyright

© The Author(s) 2016

Advertisement