李興安 牛慶生 薛運波

（吉林省養(yǎng)蜂科學(xué)研究所，吉林132108）

西方蜜蜂的線粒體DNA細(xì)胞色素氧化酶亞基-Ⅰ～細(xì)胞色素氧化酶亞基-ⅠⅠ富含A+T非編碼區(qū)單倍型類群（續(xù)）

李興安 牛慶生 薛運波

（吉林省養(yǎng)蜂科學(xué)研究所，吉林132108）

續(xù)《中國蜂業(yè)》2016年第10期

因此，根據(jù)A.m.的COI-COII NC單倍型類群分析的原始數(shù)據(jù)，A.m.非洲世系為DraI RFLP譜圖表征的3個COI-COII NC單倍型類群（表1）。

在A.m.進化過程中，阿爾卑斯山以北直至烏拉爾山的寬闊地域及其比鄰的突尼斯、摩洛哥、撒哈拉沙漠近地中海地帶等地是A.m.的一個自然分布區(qū)[5，8]。昆蟲形態(tài)變異分析（和昆蟲系統(tǒng)地理學(xué)分析），將分布于那里的歐洲黑蜂等A.m.地理亞種（及其相關(guān)生態(tài)類型）概括為A.m.西部歐洲世系[25]。基于DraI RFLP分析，本文將A.m.西部歐洲世系樣本的90種DraI RFLP譜圖歸納為1種COI-COII NC單倍型類群，即P、Q元件組成類群[19，21-24，26，27，30，32-35]。這個單倍型類群具有1個P元件和5個不同串聯(lián)重復(fù)頻率Q元件，至少包括PQ、PQQ、PQQQ、PQQQQ以及PQQQQQ 5種mtDNA單倍型類型[19，22-24，26，27，30，32-34，36]。因此，根據(jù)A.m.的COI-COII NC單倍型類群分析原始數(shù)據(jù)，A.m.西部歐洲世系為RFLP譜圖表征的1個COI-COII NC單倍型類群（表1）。

在A.m.進化過程中，阿爾卑斯山南麓、地中海東北部沿岸以及喀爾巴阡山之間的廣袤地域是A.m.的另外一個自然分布區(qū)[5，8]。昆蟲形態(tài)變異分析（和昆蟲系統(tǒng)地理學(xué)分析），將分布于那里的意大利蜂、卡呢鄂拉蜂等地理亞種（及其相關(guān)生態(tài)類型）概括為A.m.東部歐洲世系[25]?；赟NP分析，本文將A.m.東部歐洲世系樣本的25種SNP譜圖歸納為1種COI-COII NC單倍型類群,即Q元件組成類群[23，24，37-39]。這個單倍型類群僅為Q元件1種mtDNA單倍型類型。因此，根據(jù)A.m.的COICOII NC單倍型類群分析的原始數(shù)據(jù)，A.m.東部歐洲世系為SNP譜圖表征的1個COI-COII NC單倍型類群（表1）。

西部亞洲或（和）中部亞洲是A.m.的第四個自然分布區(qū)[5，8]。昆蟲形態(tài)變異分析（和昆蟲系統(tǒng)地理學(xué)分析），將分布于那里的高加索蜂等A.m.地理亞種（及其相關(guān)生態(tài)類型）概括為A.m.亞洲世系[25]。基于DraI RFLP分析，本文將A.m.亞洲世系樣本的29種DraI RFLP譜圖歸納為3種COI-COII NC單倍型類群，即P0、Q元件組成類群，P0'、Q元件組成類群以及P1'、Q元件組成類群[22，35，40，41]。其中，P0、Q元件組成類群具有1個P0元件和3個不同串聯(lián)重復(fù)頻率Q元件，至少包括P0Q、P0QQ以及P0QQQ 3種mtDNA單倍型類型；P0'、Q元件組成類群具有1個P0'元件和1個Q元件，僅為P0'Q 1種mtDNA單倍型類型[22]；P1'、Q元件組成類群具有1個P1'元件和1個Q元件，僅為P1'Q 1種mtDNA單倍型類型[42]。因此，根據(jù)A.m.的COI-COII NC單倍型類群分析的數(shù)據(jù)，A.m.亞洲世系為DraI RFLP譜圖表征的3個COI-COII NC單倍型類群（表1）。

3 總結(jié)與展望

通過上述分析，本文依據(jù)A.m.地理亞種的COICOII NC多態(tài)性規(guī)律，將A.m.歸納為8個COI-NCCOII單倍型類群。A.m.地理亞種的COI-COII NC單倍型類群為西方蜜蜂分類學(xué)研究提供了新思路。

在同領(lǐng)域的相關(guān)研究中，相關(guān)文獻既缺乏A.m.非洲世系、A.m.西部歐洲世系以及A.m.東部歐洲世系的SNP分析數(shù)據(jù)，又缺乏A.m.亞洲世系的DraI RFLP分析數(shù)據(jù)（表1）。其原因可能是低通量分析技術(shù)限制了相關(guān)研究在研究期間獲得大量COI-COII NC數(shù)據(jù)，即低分辨率瓊脂糖（和聚丙烯酰胺）凝膠電泳技術(shù)和低通量第一代DNA序列測定技術(shù)不適合進行大量A.m.地理亞種樣品的COI-COII NC多態(tài)性分析。然而，在一些其它生物樣品的mtDNA單倍型分析中，高分辨率毛細(xì)管DNA電泳技術(shù)和高通量第二代DNA序列測定技術(shù)，能夠克服上述研究的技術(shù)瓶頸[9]。鑒于高分辨率、高通量技術(shù)具有方法學(xué)優(yōu)勢，A.m.的mtDNA多態(tài)性分析最終將在分子水平建立A.m.地理亞種的COI-COII NC單倍型數(shù)據(jù)庫，COI-COII NC單倍型數(shù)據(jù)庫將更透徹地反映A.m.的COI-NC-COII多態(tài)性規(guī)律。

表1 A.m.的COI-COII NC單倍型類群分析統(tǒng)計表

表1 A.m.的COI-COII NC單倍型類群分析統(tǒng)計表(續(xù))

表1 A.m.的COI-COII NC單倍型類群分析統(tǒng)計表(續(xù))

在同領(lǐng)域的相關(guān)研究中，除了美洲A.m.的COICOII NC多態(tài)性分析之外，較少文獻系統(tǒng)地報道其它非自然分布區(qū)A.m.的研究進展。美洲A.m.包括17世紀(jì)初期引入的歐洲黑蜂等A.m.西部歐洲世系后代，19世紀(jì)引入的意大利蜂、卡呢鄂拉蜂等A.m.東部歐洲世系后代和高加索蜂等A.m.亞洲世系后代，以及20世紀(jì)中期引入的非洲蜂等A.m.非洲世系后代。本文將美洲A.m.非洲世系的20種DraI RFLP譜圖歸納為P0、Q元件組成類群的3種mtDNA單倍型類型（即P0Q、P0QQ和P0QQQ），將美洲A.m.西部歐洲世系的7種DraI RFLP譜圖歸納為P、Q元件組成類群的3種mtDNA單倍型類型（即PQ、PQQ和PQQQ），美洲A.m.東部歐洲世系的2種SNP譜圖歸納為Q元件組成類群的1種mtDNA單倍型類型（即Q），將美洲A.m.亞洲世系的4種DraI RFLP譜圖歸納為P0、Q元件組成類群的3種mtDNA單倍型類型（即P0Q、P0QQ和P0QQQ）[29]。美洲A. m.的COI-COII NC多態(tài)性分析將為其它非自然分布區(qū)A.m.的同類型研究提供可靠的文獻依據(jù)。

在同領(lǐng)域的相關(guān)研究中，除了東方蜜蜂、沙巴蜂、綠努蜂以及蘇拉威西蜂等A.m.近緣物種的COI-COII NC多態(tài)性分析外，較少文獻報道大蜜蜂等蜜蜂屬其它物種的同類型研究。盡管如此，這些A.m.近緣物種的COI-COII NC多態(tài)性分析還是揭示了這樣一個事實：相對于A.m.，它們具有短的COI-COII NC，其長度范圍是30～100堿基。這或許為我們透露出這樣一點信息：蜜蜂屬生物的COI-NC-COII多態(tài)性與蜜蜂屬生物的物種多樣性之間是否存在某種關(guān)系。而且，鑒于A.m.的COI-COII NC多態(tài)性分析已積累了豐富的知識和成熟的技術(shù)，這些蜜蜂屬其它物種的同類型研究有了可借鑒的知識儲備和技術(shù)手段。因此，在蜜蜂屬范圍內(nèi)，有必要系統(tǒng)研究每個物種的COI-COII NC單倍型類群。

[1]Williams DL.A veterinary approach to the European honey bee (Apis mellifera L.)[J].Veterinary journal,2000,160(1)∶61-73.

[2]Kremen C,Williams NM,Thorp RW.Crop pollination from native bees at risk from agricultural intensification[J].Proceedings of the National Academy of Sciences of the United States of America, 2002,99(26)∶16812-16816.

[3]Le Conte Y and Navajas M.Climate change∶impact on honeybee populations and diseases[J].Rev.sci.tech.Off.int.Epiz,2008, 27(2)∶499-510.

[4]Rogers SR,Tarpy DR,Burrack HJ.Bee species diversity enhances productivity and stability in a perennial crop[J].PLoS One, 2014,9(5)∶e97307.

[5]Whitfield C W,Behura S K,Berlocher S H,et al.Thrice out of Africa∶Ancient and recent expansions of the honey bee,Apis mellifera[J].Science,2006,314∶642-645.

[6]Chen C,Liu Z,Pan Q,et al.Genomic Analyses Reveal Demographic History and Temperate Adaptation of the Newly Discovered Honey Bee Subspecies Apis mellifera sinisxinyuan n.ssp[J].Mol Biol Evol,2016,33(5)∶1337～1348.

[7]Ruttner F,Tassencourt L,Louveaux J.Biometrical statistical analysis of the geographic variability of Apis mellifera L.[J].Apidologie,1978,9∶363-381.

[8]Han F,Wallberg A,Webster MT.From where did the Western honeybee(Apis mellifera L.)originate[J]Ecology and Evolution, 2012,2(8)∶1949-1957.

[9]Meixner MD,Pinto MA,Bouga M,et al.Standard methods for characterising subspecies and ecotypes[J].Journal of Apicultural Research,2013,52(4)∶1-25.

[10]Corlett RT.Flower visitors and pollination in the Oriental(Indomalayan)Region[J].B Biological reviews of the Cambridge Philosophical Society,2004,79(3)∶497-532.

[11]Bouga M,Alaux C,Bienkowska M,et al.A review of methods for discrimination of honey bee populations as applied to European beekeeping[J].Journal of Apicultural Research,2011,50(1)∶51-84.

[12]De la R′ua P,Jaff′e R,Dall'Olio R,et al.Biodiversity,conser-vation and current threats to European honeybees[J].Apidologie, 2009,40∶263-284.

[13]vanEngelsdorp D,Meixner MD.A historical review of managed honey bee populations in Europe and the United States and factors that may affect them[J].Journal of Invertebrate Pathology,2010, 103∶580-595.

[14]Roxane M.Magnus,Amber D.Tripodi,Allen L.Szalanski.Mitochondrial DNA Diversity of Honey Bees(Apis mellifera L.)from Unmanaged Colonies and Swarms in the United States[J].Biochemical Genetics,2014,52(5-6)∶245-257.

[15]Cornuet J-M,Garnery L,Solignac M.Putative origin and function of the intergenic region between COI and COII of Apis mellifera L.Mitochondrial DNA[J].Genetics,1991,1128∶393-403.

[16]Crozier RH,Crozier YC,Mackinlay AG.The COI-COII region of honeybee mitochondrial DNA∶Evidence for variation in insect mitochondrial evolutionary rates[J].Molecular Biology and Evolution,1989∶6(4)∶399-411.

[17]Davolos D1,Maclean N.Mitochondrial COI-NC-COII sequences in talitrid amphipods(Crustacea)[J].Heredity(Edinb), 2005,94(1)∶81-86.

[18]Cann RL,Wilson AC.Length mutations in human mitochondrial DNA[J].Genetics,1983,104∶699-711.

[19]Rortais A,Arnoid G,Alburaki M,et al.Review of the DraI test for the conservation of the black honeybee(Apis mellifera mellifera) [J].Conservation Genet Resour,2011,3∶383-391.

[20]Crozier RH,Crozier YC.The mitochondrial genome of the honeybee Apis mellifera L.∶complete sequence and genome organization[J].Genetics,1993,133∶97-117.

[21]Franck P,Garnery L,Loiseau A,et al.Genetic diversity of the honeybee in Africa∶microsatellite and mitochondrial data[J]. Heredity,2001,420-430.

[22]Franck P,Garnery L,Solignac M,et al.Molecular confirmation of a fourth lineage in honeybees from the Near East[J].Apidologie, 2000,31∶167-180.

[23]Franck P,Garnery L,Celebrano G,et al.Hybrid origins of honeybees from Italy(Apis mellifera ligustica)and Sicily(Apis mellifera sicula)[J].Molecular Ecology,2000,9∶907-921.

[24]Nedic N,Stanisavljvic L,Mladenovic M,et al.Molecular characterization of the honeybee Apis mellifera carnica in Serbia[J]. Archives of Biological Science Belgrade,2009,61(4)∶587-598.

[25]Arias MC,Sheppard WS.Molecular phylogenetics of honey bee subspecies(Apis mellifera L.)inferred from Mitochondrial DNA sequence[J].Molecular Phylogenetics and Evolution,1996,5∶3557-3566.

[26]Garnery L,Franck P,Baudry E,et al.Genetic diversity of the west European honey bee(Apis mellifera mellifera and Apis mellifera iberica)II.Microsatellite loci[J].Molecular Biology and Evolution,1998,30,S49-S74.

[27]Franck P,Garnery L,Solignac M,et al.The origin of west European subspecies of honeybees(Apis mellifera L.)∶New insight from microsatellite and mitochondrial data[J].Evolution,1998,52 (4)∶1119-1134.

[28]Eelarua P,Galian J,Serrano J,et al.Genetic structure and distinctness of Apis mellifera L.population from the Canary Islands[J]. Molecular Ecology,2001,10∶1733-1742.

[29]Collet T,Ferreira KM,Arias MC,et al.Genetic structure of Africanized honeybee populations(Apis mellifera L.)from Brazil and Uruguay viewed through mitochondrial DNA COI-COII patterns [J].Heredity(Edinb),2006,97(5)∶329-335.

[30]Miguel I,Iriondo M,Garnery L,et al.Gene flow within the M evolutionary lineage of Apis mellifera L.∶role of the Pyrenees,isolation by distance and post-glacial recolonization routes in the Western Europe[J].Apidologie,2007,38∶141-155.

[31]El-Niweiri MAA,Moritz RFA.Mitohondrial discrimination of honeybees(Apis mellifera L.)of Sudan[J].Apidologie,2008,39∶566-573.

[32]Garnery L,Franck P,Baudry E,et al.Genetic diversity of the west European honeybee(Apis mellifera mellifera and Apis mellifera iberica).I.Mitochondrial DNA[J].Molecular Biology and Evolution,1998,30(Suppl.1)∶S31-S47.

[33]De la Rua P,Jimenez Y,Galian J,et al.Evaluation of the biodiversity of honey bee(Apis mellifera L.)populations from Eastern Spain[J].Journal of Apicultural Research,2004,43∶162-166.

[34]Jensen AB,Palmer KA,Boomsma JJ,et al.Varing degrees of Apis mellifera ligustica introgression in protected populations of the black honeybee,Apis mellifera mellifera,in northwest Europe[J]. Molecular Biology and Evolution,2005,14∶93-106.

[35]Alburaki M,Bertrand B,Legout H,et al.A fifth major genetic group among honeybees revealed in Syria[J].BMC Genetics,2013, 14∶117.

[36]Garnery L,Mosshine EH,Cornuet J-M.Mitochondrial DNA variation in Moroccan and Spanish honey bee populations[J]. Molecular ecology,1995,4∶465-471.

[37]Kandemir L,Kence M,Sheppard WS,et al.Mitochondrial DNA variation in honeybee(Apis mellifera L.)populations from Turkey[J].Journal of Apicultural Research,2006,45∶33-38.

[38]Solorzano CD,Szalanski AL,Kence M,et al.Phylogeography and population genetics of honeybees(Apis mellifera L.)from Turkey based on COI-COII sequence data[J].Sociobiology,2009, 53(2)∶237-246.

[39]Ozdil F,Yildiz MA,Hall HG.Molecular characterization of Turkish honeybee populations(Apis mellifera L.)inferred from mitochondrial DNA RFLP and sequence results[J].Apidologie,2009, 40（5）：570-576.

[40]Garnery L,Solignac M,Celebrano G,et al.A simple test using restricted PCR-amplified mitochondrial DNA to study the genetic structure of Apis mellifera L.[J].Experimentia,1993,49∶1016-1021.

[41]Alburaki M,Moulin S,Legout H,et al.Mitochondrial structure of Eastern honeybee populations from Syria,Lebanon and Iraq[J]. Apidologie,2011,42∶628-641.

[42]Palmer MR,Smith DR,Kaftanoglu O.Turkish honeybees∶Genetic Variation and Evidence for a fourth lineage of Apis mellifera L.mtDNA[J].The Journal of Heredity,2009,91(1)∶42-46.

吉林省科技發(fā)展計劃項目（20130101094JC；20140101132JC）、國家蜂產(chǎn)業(yè)技術(shù)體系（CARS-45-KXJ2；CA2S-45-SYZ4）

李興安（1965-），男，研究員，E-mail∶Lxingan@sina.com