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異齒亞綱貝類DNA條形碼與系統發生學研究

DNA Barcoding and Molecular Phylogeny of Heterodonta

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【作者】 于貞貞

【導師】 李琪

【作者基本信息】 中國海洋大學, 水產養殖, 2014, 博士

【摘要】 異齒亞綱(Heterodonta)隸屬于雙殼綱軟體動物門,種類繁多,現存約2600種,分布于簾蛤目(Veneroida)和海螂目(Myoida)兩大目,是雙殼貝類中種類最為豐富、分布最為廣泛的一個亞綱。異齒亞綱貝類外部形變化大,貝殼形式多種多樣,具有極其豐富的生物多樣性。和其他軟體動物門類一樣,異齒亞綱貝類傳統分類學建立的重要依據是貝殼的表觀特征(大小、質地、殼型、鉸合部、鉸合齒、內外韌帶、殼表二級結構等)、成體軟體部的組織和器官的解剖結構(閉殼肌的數目和形狀、鰓的結構、外套竇、外套線、接合線、水管觸手的形態與分布等)以及生態學特征(食性、棲息環境等)。然而,這些分類特征不但多樣性程度高而且可塑性極大,因為受到外部環境、種間平行進化與種內生態型繁雜等因素的影響,某些分類群種內差異性較大而種間差異性較小甚至存在相互重疊的現象,而有些物種的不同地理群體之間卻因長期隔離具有較大的差異性。這都使得利用傳統的形態學方法對異齒亞綱貝類進行物種鑒定和系統發育分析變得極其困難,從而導致異齒亞綱貝類的分類處于相對混亂狀態,特別是種以上水平。因此,形態學方法亟需一種效率更高、通用性更強、更加經濟便捷的方法來進行分類和系統發育學研究以輔助其發展。利用DNA序列進行物種多樣性分析成為一種有效的研究途徑。最新提出的DNA條形碼(DNAbarcoding)技術,作為一種用于物種鑒定的新興技術正引起廣泛的關注。它利用一段短的線粒體DNA序列作為物種快速鑒定的標記,并希望以此建立DNA序列和生物物種之間一一對應的關系。DNA序列不僅能用來有效地鑒定物種,而且還能推斷物種間的系統發生關系,這就是所謂的分子系統學。分子系統學的發展有助于我們更深刻地理解各物種間的演化關系。DNA條形碼技術能彌補傳統形態學鑒定的缺陷,已成功應用于動物、植物及微生物界,極大地推動了生物多樣性研究的發展。但它的推行也引起了許多的爭議。本研究我們首先驗證了DNA條形碼技術及其分析方法的可行性,然后利用DNA條形碼技術和相關標記對異齒亞綱貝類進行了全面的物種鑒定和系統發育研究。具體研究結果如下:1.五種DNA條形碼分析方法鑒定櫻蛤總科貝類的比較研究對采自中國沿海的櫻蛤總科16個種68個個體的線粒體COI和16S rRNA基因的部分序列進行了測序,其它10個物種的相應序列從GenBank中獲得。以現行的形態學分類系統為基準,以鳥蛤總科4個物種為外類群,評估了五種DNA條形碼方法(傳統的距離法,ABGD法,單系法,GMYC法和CAOS法)在科、屬、種不同分類水平上鑒定櫻蛤總科26個形態學物種的有效性。研究結果顯示:(1)對于COI和16S rDNA基因,特征分析法(CAOS)均可以正確鑒定所有研究的櫻蛤總科的種類,并且可以有效區分一些屬。所以CAOS特征法的適用性最高,特別是在較高的分類階元。(2)對于距離法而言,它們能有效的區分大部分種類,新提出的ABGD法的鑒定效率等于或者略高于傳統距離法,并沒有顯現出較大的優勢。基于COI基因我們在種的水平上發現了寬度為1%的條形碼間隙(barcoding gap),但是種以上分類階元的遺傳距離出現明顯的重疊區,所以屬及以上分類階元的界限無法界定。(3)對于傳統的聚類法(單系法)而言,基于COI和16S基因的貝葉斯樹顯示所有物種都以較高的支持度形成相互獨立的單系群,但是僅有一個屬顯示為單系發生,也就是說單系法能夠成功地鑒定出櫻蛤總科所有的26個形態學物種,但是同樣不適用于種上階元的鑒定。而新提出的GMYC法鑒定效率十分不理想,基于COI基因,多增加了34.6%的物種;基于16S rRNA基因,多鑒定了約58.8%的物種。所以基于樹形的DNA條形碼方法僅適用于研究種及以下水平的親緣關系。因此我們推斷利用特征法DNA條形碼技術鑒定物種的鑒定效率最好、適用性最廣,但在初期鑒定物種階段我們也推薦使用ABGD法和單系法。同時,本研究還發現COI比相對保守的16S rRNA基因更適合DNA條形碼標記,特別是對于基于樹形的條形碼方法。2.基于距離法的異齒亞綱近緣種DNA條形碼研究本節研究了異齒亞綱67個屬263個近緣種共1042個個體的標準DNA條形碼序列——COI基因序列。利用K2P遺傳距離模型計算所得序列種內和屬內種間兩兩間的遺傳距離,并分析了其遺傳距離相對分布頻率。種內遺傳距離和種間遺傳距離存在著顯著的重疊,條形碼間隙(barcoding gap)在異齒亞綱近緣種間并不存在。我們分析了傳統距離法,即10倍法則和3%閾值標準鑒定物種的效率。結果顯示,10倍法則無法鑒定55.59%的物種,而3%閾值標準無法鑒定42.58%的物種。另外,我們檢測了新提出的基于遺傳距離的DNA條形碼分析方法——條形碼間隙自動檢索法鑒定物種的效率。該方法較之于傳統的遺傳距離方法顯現出較明顯的優勢。結果顯示,它雖然能將這1042條序列劃分為264個群組,在這264個群組的組成中有207個假定種的組成與NCBI分類系統相吻合。也就是說條形碼間隙自動檢索法能有效地區分78.70%的物種。與傳統的距離DNA條形碼方法相比,條形碼間隙自動檢索法有以下幾個方面的優點:(1)不依賴于根據經驗設定的單一的閾值標準,鑒定標準更加客觀,而且針對于同一個數據集可以使用不同的鑒定標準;(2)適用條件更加寬泛,即使種內和種間遺傳距離存在重疊也不影響它的有效性;(3)操作更加簡便,省時、省力,更適用于大規模的物種初期鑒定工作。3.基于COI和16S rRNA基因的異齒亞綱系統發生學關系研究研究利用線粒體基因COI和16S rRNA進行貝葉斯和最大似然法分析呈現了異齒亞綱的系統發生關系。我們將獲得的拓撲結構與傳統劃分的目、超科、科和屬以及已發表過的分子學研究一起對比分析。結果顯示在系統發生樹上簾蛤目和海螂目并沒有像期待的那樣形成兩個獨立進化的分支。在總科的水平上,袖扣蛤總科、飾貝總科、同心蛤總科、鳥蛤總科、心蛤總科、竹蟶總科和厚殼蛤總科為單系;滿月蛤總科、簾蛤總科、櫻蛤總科、蛤蜊總科、蜆總科、海螂總科、海筍總科為并系或者復系。厚科蛤、星形蛤科和心蛤科顯示了較近的親緣關系,作為異齒亞綱內其它科的姐妹群形成了獨立的分支。本研究中索足蛤科位于系統發育樹的較基部的位置,遠離了滿月蛤總科。支持了Williams et al.(2004)將無齒蛤科移除滿月蛤總科的觀點。鳥蛤總科和櫻蛤總科、簾蛤總科和蛤蜊總科、竹蟶總科和袖扣蛤總科分別為姐妹群關系。櫻蛤總科縊蟶屬與竹蟶總科刀蟶屬顯示了較近的親緣關系,具體分類地位需要進一步研究。簾蛤科與蛤蜊總科、北極蛤科、同心蛤總科和猿頭蛤總科聚合成高支持度的分支。本研究有力說明了目前異齒亞綱的分類非常混亂,需要利用形態學、古生物學和分子數據進行校正。4.利用三種不同DNA條形碼分析方法探究縊蟶屬系統發生學關系本研究分析了采自中國沿海的櫻蛤總科的16個物種以及竹蟶總科的4個物種的線粒體COI基因和16S rRNA基因序列,對縊蟶屬系統發生關系和分類地位進行了研究。距離法、單系法和特征法條形碼分析方法在本研究中得到成功的應用。研究結果表明,縊蟶屬與櫻蛤總科其他屬的遺傳距離遠遠大于其與竹蟶總科各屬的遺傳距離,與刀蟶屬的遺傳距離最小,序列的差異度最低、相似性最高。以鳥蛤總科為外群,利用COI和16SrRNA整合序列構建的貝葉斯系統發生樹顯示縊蟶屬作為刀蟶屬的姐妹群與莢蟶屬緊緊地聚為一支,說明縊蟶屬、刀蟶屬和莢蟶屬具有最近的共同祖先。特征法分析顯示縊蟶屬與參考系中刀蟶科特征堿基的匹配度最高,共同特征堿基數遠遠多于三個。綜合形態學數據,本研究表明縊蟶屬應劃歸竹蟶總科的刀蟶科。

【Abstract】 DNA barcoding is attracting the wide attention as a new method to indentify species. Itcan propose a solution to the limitations of the traditional morphological identification andpromote the development of the biodiversity studies. DNA barcoding has been usedsuccessfully in many animal groups since it was proposed. But the practice of barcoding hasalso received much criticism. DNA sequences enable not only the identification of specieseffectively, but also the inference of phylogenetic relationships, i.e., molecular systematics.The development of molecular systematics can help us understand the evolutionaryrelationships among species profoundly. The Heterodonta is the commercially importantgroup in bivalve molluscs and many species of the Heterodonta have been cultured andexploited, but few studies focus on the biodiversity and phylogenetics of Heterodonta. Thisstudy firstly assesses the utility of various DNA barcoding methods using the superfamilyTellinoidea, then compares the effectiveness of the new proposed Automatic Barcode GapDiscovery (ABGD) to the traditional distance-based DNA barcoding method in discriminatingthe closely related species of Heterodonta, and finally applies the DNA barcoding to andSinonovacula and the whole Heterodonta to present their phylogenetic relationships usingmultiple molecular markers. The main results are listed as follows:1. Utility of DNA barcoding for Tellinoidea: a comparison of distance, coalescent andcharacter-based methods on two different gene regionsWe newly sequenced the COI and16S rRNA fragmentes of68individuals of16tellinoidean species, and downloaded65sequences of other10tellinoideans from GenBank.The character-based method (CAOS) showed fairly high applicability in all studied cases, butthe tree-based methods were found efficient just on the species-level identification. For distance-based methods, ABGD gained a success ratio equal or greater than the classical one.For coalescent-based methods, the monophyly-based method successfully delimited all of thetellinoidean species, but the GMYC model clearly inflated the number of species units by34.6%for COI gene and by58.8%for16S gene. Thus we conclude that CAOS performs best,but we also suggest the use of the ABGD method and the monophyly-based method forprimary partitions. Additionally, we reveal that COI gene may be more suitable as a standardbarcode marker than16S gene, particularly for tree-based methods.2. DNA barcoding of the Heterodonta closely related speciesWe analyzed the sequences of1042individuals belonging to263closely reated species of67genera of Heterodonta to assess the effectiveness of two distance-based DNA barcodingmethods. The K2P pairwise genetic distances between sequences were calculated and therelative frequency of intraspecifc and interspecific distances was counted. Results showed thatthere was an obvious overlap between intraspecific and interspecific divergences, thus nobarcoding gap exited among the genetic distances of closely related species of Heterodonta.The10×rule threshold resulted in lumping about55.57%of distinct species, while the“3%” standard criterion splitted42.58%species. Then we evaluated the usefulness of theAutomatic Barcode Gap Discovery method (ABGD). We could found significant superiorityof ABGD over the traditional distance-based method in identifying the sisiter taxon. Althoughit assigned the1042individuals to264groups,207of the groups coincided with the currentNCBI taxonomy. In other words, ABGD could successfully distinguish78.70%of theanalyzed species. Comparing with the traditional method, ABGD has three benefits:(1) itdoes not rely on an empirical value to distinguish speices, and it allows multiple criterions inone dataset;(2) this method proposes a standard definition of the barcode gap and it can beused even when the two distributions overlap;(3) it is simpler and easier for users to handle,and is excellent in terms of computation time. 3. Molecular phylogeny of the HeterodontaIn our study, we use two mitochondrial (COI and16S) genes to reconstruct thephylogenetic relationships of the Heterodonta. All of the sequences from GenBank togetherwith the sequences we obtained were analysed. The results showed that the two classes:Veneroida and Myoida do not form two major clades with high support values as expected, oncontrary, they mixed together. In our trees, at the superfamily level, Galeommatoidea,Dreissenoidea, Glossoidea, Cardioidea, Carditoidea, Solenoidea and Crassatelloidea displayas monophyletic taxon, while the monophyly of Lucinoidea, Veneroidea, Tellinoidea,Mactroidea, Corbiculoidea, Myoidea and Pholadoidea were not supported.Crassatellidae/Astartidae/Carditidae formed a clade that is sister to all other heterodontbivalves. The results supported the conclusion of Williams et al., that move Thyasiridae outof Lucinoidea. The present molecular analyses also indicate a possible sister relationship withCardioidea/Tellinoidea, Veneridae/Mactroidea, and Solenoidea/Galeommatoidea. Thephylogenetic results of our study powerfully challenge to the current classification systemsbased on morphology.4. New insight into phylogeny of Sinonovacula (Bivalvia: Solecurtidae) revealed bycomprehensive DNA barcoding analyses on two mitochondrial genesThe present study was undertaken to clarify the genetic relationships of Sinonovaculathrough comprehensive DNA barcoding analyses on COI and16S rRNA genes. For bothgenes, the K2P distances between species of Sinonovacula and species of other generabelonging to Tellinoidea were much bigger than those between Sinonovacula and genera ofSolenoidea. Bayesian tree of combined data demonstrated that Sinonovacula and Cultellusformed a monophylic clade with well supports. An extremely high matching rate of CAsbetween Sinonovacula and the reference family Cultellidae was found. Collaborating withmorphological evidences, we suggest that the former Solecurtidae genus Sinonovacula shouldbe placed in Cultellidae, as a sister taxa to Cultellus.

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