利用PCR-RFLP技术鉴定传粉榕小蜂隐种混合样品的物种组成

Identifying cryptic species in pollinating-fig wasps by PCR-RFLP on mtDNA COI gene

隐种(cryptic species)是指形态上几乎完全相同但遗传组成存在显著分化的物种。在榕树-榕小蜂一对一共生系统中,传粉榕小蜂隐种的发现对协同进化、物种共存等重要的生态和进化理论提出了严峻的挑战。因为很难从形态上直接区分隐种,所以,相关研究中的一个迫切需要解决的问题就是如何快速而准确地鉴定隐种。本文采用PCR-RFLP方法分析了mtDNA COI基因片段,对木瓜榕(Ficus auriculata)和鸡嗉果榕(F.semicordata)的传粉榕小蜂隐种进行了区分。结果表明为木瓜榕传粉的大果榕小蜂(Ceratosolen emarginatus)存在两个隐种(A和B),分别包含1个XhoI和1个BssSI酶切位点。将两个隐种的样品按不同比例混合,提取基因组DNA,PCR扩增mtDNA COI片段,经XhoI和BssSI分别酶切,均能通过酶切图谱准确检测出混合样品的隐种组成。鸡嗉果榕小蜂(C.gravelyi)两个隐种的mtDNA COI基因序列也存在较大差别,分别包含1个BmrI和1个AvaI酶切位点,隐种混合样品经BmrI和AvaI分别酶切的结果也能准确鉴定混合样品的物种组成。我们的结果表明基于PCR和DNA酶切技术能快速而准确地区分传粉榕小蜂的隐种。

Cryptic species are morphologically undetected yet genetically divergent species.The presence of cryptic pollinating-fig wasp species has potentially important implications for understanding fig/fig-wasp mutualisms and the coexistence of cryptic species.It is essential to identify cryptic species quickly and accurately using molecular markers since the species are morphologically undistinguishable.We used PCR-RFLP to analyze the mtDNA COI gene in cryptic species of Ceratosolen emarginatus and C.gravelyi.The PCR-RFLP was useful for determining the composition of cryptic pollinating-fig wasp species.Ceratosolen emarginatus included two cryptic species（A and B） with an XhoI and a BssSI digesting site in the COI gene,respectively.Ceratosolen gravelyi included two cryptic species with a BmrI and an AvaI digesting site,respectively.Genomic DNA samples were extracted from a mixture of cryptic species A and B within C.emarginatus,and XhoI and BssSI endonucleases were used to digest the amplified COI fragments.XhoI-specific fragments were found when cryptic species A was present,and BssSI-specific fragments were found when cryptic species B was present.The digestion of COI gene with BmrI and AvaI in two cryptic species of C.gravelyi showed similar results.Taken together,PCR-RFLP,based on PCR and DNA restriction enzyme digestion,can quickly and accurately identify cryptic pollinating-fig wasp species.