張 順, 林施泉, 孟凡旭, 吳 敏, 許學(xué)偉, 王春生,*

1 國(guó)家海洋局第二海洋研究所, 杭州 310012 2 浙江大學(xué)生命科學(xué)學(xué)院, 杭州 310058

太平洋克拉里昂-克利伯頓斷裂帶嘴刺目線蟲多樣性

張 順1,2, 林施泉1, 孟凡旭1, 吳 敏2, 許學(xué)偉1, 王春生1,*

1 國(guó)家海洋局第二海洋研究所, 杭州 310012 2 浙江大學(xué)生命科學(xué)學(xué)院, 杭州 310058

從太平洋深?？死锇?克利伯頓斷裂帶(Clarion-Clipperton fracture zone,簡(jiǎn)稱CC區(qū))4個(gè)站位采集的深海沉積物樣品中檢出26條嘴刺目(Enoplida)線蟲個(gè)體。綜合應(yīng)用形態(tài)學(xué)和分子生物學(xué)方法,共鑒定嘴刺目線蟲6科8屬,其中尖口線蟲科(Oxystominidae)個(gè)體數(shù)量最多,占總數(shù)的57.7%,其次為前感線蟲科(Anticomidae,19.2%)、光皮線蟲科(Phanodermatidae, 7.7%)、鉤線蟲科(Oncholaimidae, 7.7%)、烙線蟲科(Ironidae, 3.8%)和矛線蟲科(Enchelidiidae, 3.8%)。科、屬組成與相鄰站點(diǎn)同期采樣所獲的線蟲近似,而豐度組成比例有所差異。分子生物學(xué)方法獲得了線蟲rRNA基因序列16條,經(jīng)GenBank數(shù)據(jù)庫(kù)比對(duì),其與已有的序列相似性范圍為94%—99%,以此為依據(jù)可確定到科的水平和大部分屬的水平(84.6%)。DNA條形碼比對(duì)結(jié)果和形態(tài)學(xué)鑒定結(jié)果有較高一致性,表明分子條形碼技術(shù)可作為深海線蟲鑒定的有效手段。系統(tǒng)發(fā)育分析結(jié)果顯示,基于18S和28S rRNA基因序列,采用不同方法構(gòu)建系統(tǒng)發(fā)育樹,其分支結(jié)構(gòu)基本一致；鉤線蟲科和矛線蟲科聚類在一起,光皮線蟲科和前感線蟲科聚類在一起,顯示出彼此間較近的遺傳關(guān)系。

嘴刺目線蟲；多樣性；系統(tǒng)發(fā)育；克拉里昂-克利伯頓斷裂帶

自由生活海洋線蟲屬于線蟲動(dòng)物門,記錄種約4000種,但尚有大量種類未被鑒定,估計(jì)全球線蟲物種數(shù)量超過2萬(wàn)種[1]。海洋線蟲是最豐富的后生動(dòng)物類群,分布廣、數(shù)量多。在多數(shù)生境中海洋線蟲豐度可占后生動(dòng)物數(shù)量的70%—90%；在某些生境中,如有機(jī)質(zhì)豐富的沉積物中,可達(dá)90%以上[2]。在底棲食物鏈中,海洋線蟲是較高級(jí)生物的食物來源,同時(shí)也影響低級(jí)生物群落組成,在食物鏈中具有承上啟下作用[3]。

早期Filipjev以側(cè)器作為主要性狀,其它性狀為補(bǔ)充,建立了線蟲分類系統(tǒng)。首次提出包括當(dāng)時(shí)已知全部種的海洋線蟲分類系統(tǒng),將線蟲劃分為1個(gè)綱11個(gè)目,并首次對(duì)線蟲起源進(jìn)行了討論,提出線蟲共同祖先是嘴刺目海洋線蟲的觀點(diǎn)[4]。依據(jù)WORMS[5]最新數(shù)據(jù),嘴刺目線蟲包含14科,20屬,1742種。嘴刺目是體長(zhǎng)較長(zhǎng)的海洋線蟲類群,某些種類可達(dá)數(shù)毫米。研究表明,嘴刺目線蟲是活躍的捕食者(在形態(tài)學(xué)分類單元上,有多個(gè)科的線蟲具有排列復(fù)雜的齒),在小型底棲動(dòng)物群落內(nèi)具有重要的生態(tài)功能。然而,相對(duì)于陸生和寄生線蟲,海洋嘴刺目線蟲進(jìn)化關(guān)系鮮有研究[1,6-7]。

形態(tài)學(xué)鑒定準(zhǔn)確度受到多種因素影響,例如,不同生境中的同種線蟲可表現(xiàn)出不同的外形特征,僅通過形態(tài)學(xué)特征將線蟲鑒定到種水平難度大,線蟲鑒定缺乏快速準(zhǔn)確的技術(shù)方法。盡管線蟲分類研究歷史已有上百年,然而依然缺乏用于高級(jí)分類單元(如科以上分類單元)系統(tǒng)發(fā)育重建的同源形態(tài)性狀的客觀標(biāo)準(zhǔn)。

DNA條形碼(又稱分子條形碼)技術(shù)是通過對(duì)某一類DNA序列進(jìn)行分析達(dá)到區(qū)分物種目的的技術(shù)。DNA條形碼在運(yùn)用于海洋線蟲研究中,有兩類分子標(biāo)記顯示出較高的價(jià)值,分別是核糖體小亞基序列(SSU rDNA)和核糖體大亞基序列(LSU rDNA),其中核糖體小亞基序列在多數(shù)線蟲系統(tǒng)發(fā)育分析中具有普遍適用性。其半保守序列可用來研究科以上分類單元的系統(tǒng)發(fā)育關(guān)系,其可變區(qū)序列能在科或?qū)俚乃缴蠈⒕€蟲區(qū)分,甚至在種水平上區(qū)分不同的種類[8]。同時(shí)由于其序列包含較多的系統(tǒng)發(fā)育信息、較少的多態(tài)性,因此在揭示不同分類單元遺傳關(guān)系時(shí)也非常有效。核糖體大亞基序列作為分子標(biāo)記用于線蟲鑒定的歷史只有20多年,主要集中于D2或D3延伸片段[9],此分子標(biāo)記具有顯著的種內(nèi)多態(tài)性,在區(qū)分某些類群的不確定種時(shí)具有較高價(jià)值,為系統(tǒng)發(fā)育分析提供遺傳信息。其他分子標(biāo)記,如轉(zhuǎn)錄間隔區(qū)(ITS)和線粒體基因(COI),也曾應(yīng)用于線蟲分類研究,但其具有較高的種內(nèi)變異性、大量插入,使比對(duì)工作較為困難[10],序列極難擴(kuò)增。以往研究表明,形態(tài)學(xué)分類和DNA分子條形碼鑒定結(jié)果之間存在較高的一致性[11-12]。

克拉里昂-克利伯頓斷裂帶富含多金屬結(jié)核,具有重要經(jīng)濟(jì)價(jià)值。國(guó)際海底管理局承擔(dān)了保護(hù)海洋環(huán)境免受破壞和過度開發(fā)的使命。為了順利完成這一任務(wù),需要對(duì)海洋生物進(jìn)行研究。線蟲作為海洋底棲生物的主要類群,其群落結(jié)構(gòu)變化可反映環(huán)境變化。嘴刺目線蟲是深海線蟲最重要的類群之一,形態(tài)學(xué)資料較為齊全[1],已發(fā)布的核酸序列較多。然而以往關(guān)于嘴刺目線蟲的研究主要集中于近海潮間帶海灘[13],深海(如CC區(qū))線蟲研究報(bào)道鮮見,研究方法主要采用形態(tài)學(xué)分類法,分子技術(shù)應(yīng)用少[14-15]。本研究結(jié)合形態(tài)學(xué)分類和分子生物學(xué)方法,研究CC區(qū)嘴刺目線蟲的多樣性,以期能初步探知該區(qū)嘴刺目線蟲類群組成,并對(duì)其遺傳關(guān)系進(jìn)行探討。

1 研究方法

1.1 樣品采集分離和鑒定

采用箱式(9 cm ? 61 cm,BC)和多管(MC)取樣的方式從太平洋CC區(qū)4個(gè)站位采集沉積物樣品(表1),立即固定于DESS溶液(20%的二甲基亞砜,0.2 M乙二胺四乙酸二鈉,加入氯化鈉使溶液飽和,pH 8.0)[16]。

表1 采樣記錄表

BC: 箱式取樣器Box-Corer；MC: 多管取樣器Multi-Corer

沉積物先用孔徑為38 μm的濾網(wǎng)過濾,再用LudoxTM(密度為1.18 g/mL)重懸浮[17]。在解剖顯微鏡下,將線蟲(嘴刺目)樣本轉(zhuǎn)移到載玻片上,制備線蟲臨時(shí)玻片。在顯微鏡(Leica DM5500)下用微分干涉和相差模式對(duì)線蟲形態(tài)進(jìn)行觀察、拍照和測(cè)量。形態(tài)學(xué)鑒定主要依據(jù)Gerlach、Rieman和Lorenzen的方法[18-20]。顯微觀察后,為了防止線蟲基因組降解,立即提取線蟲基因組[21]。

1.2 DNA提取、PCR擴(kuò)增、鑒定

線蟲從玻片分離,用無(wú)菌水清洗3次,清除線蟲表面的化學(xué)試劑和其它雜質(zhì)。依據(jù)線蟲的大小,將線蟲切成幾段并轉(zhuǎn)移至0.5 mL的PCR管中,PCR中含有20 μL線蟲裂解液(WLB)(50 mmol/L KCl,1% Gelatin, 10 mmol/L Tris-Cl pH 8.2, 2.5 mmol/L MgCl2, 0.45%(v/v)Tween 20, 60 μg/mL蛋白酶K)。線蟲樣品在-70℃凍融15 min,65 ℃消化1 h以上去除蛋白質(zhì),95 ℃處理15 min使蛋白酶K失活。冷卻至室溫后,于13000 r/min離心1 min。上清液即可作為PCR模板,用于兩種片段(片段長(zhǎng)度分別為～990 bp的18S rRNA gene和～260 bp的28S rRNA gene D3區(qū))擴(kuò)增。18S rRNA gene用988F(5′-CTCAAAGATTAAGCCATGC- 3′)和1912R(5′-TTTACGGTCAGAA CTAGGG- 3′)引物對(duì)擴(kuò)增[22]。28S rRNA基因D3區(qū)使用引物對(duì)D3a(5′-GACCCGT CTTGAAACACGGA- 3′)、D3b(5′-TCGGAAGGAACCAGCTACTA- 3′)擴(kuò)增。50 μL PCR體系包括ddH2O 34.5 μL、10×Ex buffer 5 μL、引物各2 μL(10 μM)、dNTP(每種2.5 mmol/L)4 μL、Ex Taq(Takara, 5 U/μL)0.5 μL、模板DNA 2 μL。PCR反應(yīng)條件為：98℃變性10 s,退火30 s,72℃延伸1 min,循環(huán)40次,72 ℃延伸10 min。引物對(duì)988F、1912R的退火溫度為49 ℃,引物對(duì)D3a、D3b的退火溫度為54 ℃。

PCR產(chǎn)物用2%的瓊脂糖電泳檢測(cè)、純化后,連接到pMD19-T,有插入片段的單克隆送鉑尚生物技術(shù)(上海)有限公司測(cè)序。

1.3 數(shù)據(jù)分析

從GenBank網(wǎng)絡(luò)數(shù)據(jù)庫(kù)下載相關(guān)序列,使用MEGA5軟件包內(nèi)置的Clustal W[23]對(duì)序列進(jìn)行比對(duì),然后使用鄰接法、最大似然法和最大簡(jiǎn)約法構(gòu)建系統(tǒng)發(fā)育樹。其中鄰接法為雙木村雙參數(shù)模型,最大似然法(ML)采用田村3參數(shù)模型,其他參數(shù)為：G+I。最大簡(jiǎn)約法(MP)、最大似然法(ML)和鄰接樹(NJ)自舉值：MP和NJ為1000,ML為100。

圖1 刺嘴目線蟲類群組成Fig.1 Community composition of Enoplid

2 研究結(jié)果

2.1 形態(tài)學(xué)鑒定

從CC區(qū)4個(gè)站位分離獲得26個(gè)嘴刺目線蟲個(gè)體(圖1),分屬6個(gè)科(表2和表3),線蟲中文學(xué)名依據(jù)文獻(xiàn)[24]。其中,尖口線蟲科線蟲15條(占線蟲總數(shù)的57.7%),包括Oxystominasp.1條、Halalaimussp.13條及無(wú)法鑒定到屬的線蟲1條；前感線蟲科線蟲5條(19.2%),均為Anticomasp.；光皮線蟲科線蟲2條(7.7%),Phanodermopsissp.和Crenopharynxsp.各1條；鉤線蟲科線蟲2條(7.7%),均為Viscosiasp.；烙線蟲科線蟲1條(3.4%),為Syringolaimussp.；矛線蟲科線蟲1條(3.4%),為Bathyeurystominasp.。

箱式采樣站位(BC站)和多管采樣站位(MC站)均為2個(gè)采樣點(diǎn)。BC站和MC站共有的嘴刺目線蟲包括劍口線蟲科、前感線蟲科、鉤線蟲科和光皮線蟲科。BC站樣品中,前感線蟲科為最主要類群；MC站樣品中,劍口線蟲科為主要類群。烙線蟲科和矛線蟲科僅在MC站樣品中發(fā)現(xiàn),比例為10%。

表2 鑒定線蟲基因片段數(shù)量

本次研究的烙線蟲科未獲得核酸序列

形態(tài)學(xué)測(cè)量值上,嘴刺目線蟲體長(zhǎng)一般在1 mm以上,其中最長(zhǎng)線蟲Viscosiasp.體長(zhǎng)約1.8 mm,最短的線蟲Oxystominid長(zhǎng)度僅為0.2 mm。在尾長(zhǎng)方面,Crenopharynxsp.最長(zhǎng),Thalassomonhysterasp.最短(表3)。

表3 嘴刺目線蟲形態(tài)測(cè)量值/μm

M：雄性 male；F：雌性 Female；J：幼體 Juvenile；de Man比值,a=全長(zhǎng)/最大體寬;b=全長(zhǎng)/食道長(zhǎng);c=全長(zhǎng)/尾長(zhǎng)；如有多條線蟲,則a、b、c取平均值；- NA：數(shù)據(jù)無(wú)法獲得 not available

2.2 18S和28SrRNA基因序列

采用分子生物學(xué)方法,共獲得16條DNA序列(18S rRNA基因序列和28S rRNA基因序列各8條,表2)。序列已經(jīng)提交到GenBank,基因登陸號(hào)為KR0811950- KR0811957和KR0811958- KR0811965。18S rRNA基因序列片段長(zhǎng)度約為991 bp,其可變區(qū)比例低于保守區(qū),具有較高的保守性。28S rRNA基因序列片段長(zhǎng)度為306 bp,可變性區(qū)比例高于18S rRNA基因(表4)。序列與數(shù)據(jù)庫(kù)比對(duì)后均可確定到科的水平和84.6%屬的水平。尚有少量18S和28S rRNA基因序列在數(shù)據(jù)庫(kù)中無(wú)法搜索到相近的線蟲核酸序列,表明部分種類的線蟲核酸序列尚未納入核酸數(shù)據(jù)庫(kù)中,線蟲分子數(shù)據(jù)尚需補(bǔ)充。

表4 18S和28S rRNA基因的堿基組成

堿基長(zhǎng)度包括空位,可變區(qū)包括缺失片段; 堿基頻率A, 腺嘌呤 Adenine; T, 胸腺嘧啶 Thymine; C 胞嘧啶 Cytosine; G 鳥嘌呤 Guanine

2.3 系統(tǒng)發(fā)育分析

采用NJ、MP或ML法進(jìn)行基于18S和28S rRNA基因序列的系統(tǒng)發(fā)育分析結(jié)果顯示,不同方法構(gòu)建的系統(tǒng)發(fā)育樹分支結(jié)構(gòu)基本一致(圖2)。28S rRNA基因序列的系統(tǒng)發(fā)育樹中,鉤線蟲科和矛線蟲科聚類在一起,光皮線蟲科和前感線蟲科聚類在一起,分別顯示出其彼此間較近的遺傳關(guān)系。

2.4 形態(tài)學(xué)與分子數(shù)據(jù)比較分析

本研究獲得的分子序列與GenBank數(shù)據(jù)庫(kù)中的序列相似性為94%—99%(表5),分子條形碼比對(duì)結(jié)果和形態(tài)學(xué)鑒定結(jié)果有較高的一致性。在科水平上,所有DNA條形碼比對(duì)結(jié)果和形態(tài)學(xué)鑒定結(jié)果一致；在屬水平上,87.5%的線蟲DNA條形碼比對(duì)結(jié)果與形態(tài)學(xué)鑒定結(jié)果一致。

圖2 鄰接法構(gòu)建的系統(tǒng)發(fā)育樹(a, 18S rRNA gene；b, 28S rRNA gene)Fig.2 The phylogenetic tree (a, 18S rRNA gene; b, 28S rRNA gene)圓心表明3種系統(tǒng)發(fā)育樹(NJ、MP、ML)均一致。括號(hào)中為本次研究所用線蟲的編號(hào)或NCBI的登陸號(hào)

線蟲編號(hào)ID形態(tài)學(xué)鑒定Morphologicalidentification18SrRNA基因相似性Similarityof18SrRNAgene28SrRNA基因相似性Similarityof28SrRNAgene科Family屬Genus屬Genus相似性/%Similarity屬Genus相似性/%SimilarityBC05-6OxystominidaeHalalaimusHalalaimus99BC05-7OxystominidaeOxystominaOxystomina99BC05-22PhanodermatidaePhanodermopsPhanodermatid99MC12-3PhanodermatidaeCrenopharynxPhanoderma99Phanoderma94MC12-5EnchelidiidaeBathyeurystominaBathyeurystomina99Bathyeurystomina99MC12-20OxystominidaeHalalaimusHalalaimus98MC12-1OxystominidaeHalalaimusHalalaimus98Halalaimus98MC12-12OxystominidaeHalalaimusHalalaimus99MC12-2OncholaimidaeViscosiaViscosia98MC12-7OxystominidaeHalalaimusHalalaimus99MC12-8OxystominidaeHalalaimusHalalaimus99MC12-9AnticomidaeAnticomaAnticomid99BC06-1OncholaimidaeViscosiaViscosia98MC12-10Ironidae(1)Syringolaimus

(1) 未獲得核酸序列

3 討論

本次研究圍繞CC區(qū)嘴刺目線蟲開展,科水平上,尖口線蟲科數(shù)量最多,烙線蟲科數(shù)量最少,屬水平上,Halalaimus是主要的屬,這與Lambshead等人研究結(jié)論一致[14]。此外,本次研究發(fā)現(xiàn)光皮線蟲科線蟲2條,占線蟲總數(shù)的6.9%,Phanodermopsissp.和Crenopharynxsp.各1條,這在以往CC區(qū)線蟲研究文獻(xiàn)中鮮見[14-15]。上述結(jié)果表明,CC區(qū)尚有部分線蟲種類未被發(fā)現(xiàn),該區(qū)嘴刺目線蟲多樣性需進(jìn)一步研究。

本研究中,所獲得的線蟲兩種rRNA基因序列(18S rRNA和28S rRNA基因)與GenBank數(shù)據(jù)庫(kù)中的序列相似性為94%—99%(表5),分子條形碼比對(duì)結(jié)果和形態(tài)學(xué)鑒定結(jié)果具有較高一致性,表明分子條形碼技術(shù)是海洋自由生活線蟲快速鑒定的有效手段。18S rRNA基因由于保守性較高,常用于高級(jí)分類單元的系統(tǒng)分析；28S rRNA基因往往表現(xiàn)出較高變異性、難以確定不同類群同源性及高級(jí)分類單元的遺傳關(guān)系,研究者通常采用28S rRNA基因序列研究線蟲屬或更低分類水平之間的遺傳關(guān)系[11]。形態(tài)學(xué)方法由于步驟繁瑣、鑒定耗時(shí)長(zhǎng),影響線蟲基因組完整性,從而降低PCR擴(kuò)增效率[21]。同時(shí),在PCR擴(kuò)增過程中,容易擴(kuò)增獲得真菌或細(xì)菌序列。此外, DNA制備技術(shù)對(duì)線蟲序列獲取效率具有影響,例如堿裂解法制備的基因組容易擴(kuò)增獲得真菌序列,這在研究線蟲與微生物之間關(guān)系方面具有一定價(jià)值。

DNA條形碼技術(shù)是通過使用DNA基因片段作為條形碼來對(duì)物種進(jìn)行快速、準(zhǔn)確識(shí)別的技術(shù)。它主要具有兩個(gè)作用：1、利用已知種類對(duì)未知種類輔助鑒定；2、辨別往期研究種類之間的不同。對(duì)于研究充分、采樣全面、基因和形態(tài)廣泛研究的類群,DNA條形碼顯示出較高的價(jià)值,特別是在已知種類對(duì)未知種類的輔助鑒定方面。本次研究中,經(jīng)形態(tài)鑒定,MC12- 1號(hào)線蟲屬于Halalaimus屬,但與其他Halalaimus屬的線蟲存在顯著差異；系統(tǒng)發(fā)育分析上,MC12- 1號(hào)線蟲與其他Halalaimus屬線蟲歸為同一類群,但形成不同分支,表現(xiàn)出一定的遺傳分化。這顯示出DNA條形碼在形態(tài)學(xué)鑒定結(jié)果的補(bǔ)充上具有一定價(jià)值。

DNA條形碼應(yīng)用于海洋線蟲鑒定中需要以下幾步：(1)創(chuàng)建海洋線蟲綜合條形碼數(shù)據(jù)庫(kù)；(2)需要成熟方法,新的類群能比對(duì)和匹配到數(shù)據(jù)庫(kù)中已有數(shù)據(jù),或能進(jìn)行有效補(bǔ)充；(3)條形碼需要與分類學(xué)數(shù)據(jù)結(jié)合,應(yīng)用于生物種類確定。截至2015年7月,GenBank數(shù)據(jù)庫(kù)有104個(gè)屬、150個(gè)種共計(jì)600多條海洋線蟲條形碼序列[25]。其中41種海洋線蟲序列屬于嘴刺綱,109種屬于色矛綱。嘴刺綱和色矛綱中,分子序列(SSU/LSU/COI)最多的是鉤線蟲科和色矛線蟲科,它們包含的種均不超過20個(gè)；而Oncholaimus屬僅包含了30種已經(jīng)描述的種。與現(xiàn)有線蟲多樣性相比,具有分子數(shù)據(jù)的海洋線蟲較少。為促進(jìn)DNA條形碼準(zhǔn)確性,需增加不同種類海洋線蟲的分子序列數(shù)據(jù)。

采用分子生物學(xué)方法對(duì)CC區(qū)嘴刺目線蟲研究鮮有報(bào)道。依據(jù)近期嘴刺目線蟲分子系統(tǒng)發(fā)育分析[1,26],嘴刺目線蟲主要分為5大分支：(1)：桿咽科(Rhabdolaimidae)；(2)：無(wú)咽科(Alaimidae)和烙線蟲科；(3)：似三孔線蟲科(Tripyloididae)和長(zhǎng)尾線蟲科(Trefusiidae)；(4)：尖口線蟲科、鉤線蟲科和矛線蟲科；(5)：腹口線蟲科(Thoracostomopsidae)、嘴刺線蟲科(Enoplidae)、光皮線蟲科、狹線蟲科(Leptosomatidae)、烙口線蟲科、裸口線蟲科(Anoplostomatidae)和前感線蟲科。本研究結(jié)果顯示,采用18S和28S rRNA基因分別構(gòu)建系統(tǒng)發(fā)育樹,運(yùn)用不同建樹方法,獲得的系統(tǒng)發(fā)育樹拓?fù)浣Y(jié)構(gòu)基本一致。遺傳分析結(jié)果顯示,鉤線蟲科和矛線蟲科,遺傳關(guān)系較近(NJ、ML和MP法分別為：100%、100%和99%),這和形態(tài)學(xué)分類一致,它們都?xì)w為鉤線蟲超科(Oncholaimoidea),與往期結(jié)果一致,鉤線蟲超科為單系群；尖口線蟲科所包含的屬,在本次系統(tǒng)發(fā)育樹中的拓?fù)浣Y(jié)構(gòu)不穩(wěn)定,表明尖口線蟲科可能為并系群,其中屬之間關(guān)系需要進(jìn)一步研究；光皮線蟲科和前感線蟲科的遺傳關(guān)系較近(NJ、ML和MP法分別為：99%、98%和98%),可歸為嘴刺線蟲超科(Enoploidea),關(guān)于其是否為單系群或并系群,以往爭(zhēng)議多,本次研究支持其為單系群。為了確定線蟲的遺傳關(guān)系,后期的研究可以使用合并的序列(18S和28S rRNA基因)進(jìn)行系統(tǒng)發(fā)育分析或開展多基因綜合分析,并參考口器等形態(tài)學(xué)特征。

分子條形碼技術(shù)缺點(diǎn)是缺乏通用引物,導(dǎo)致核酸序列擴(kuò)增效率不高。因此,未來線蟲分子鑒定一方面需要設(shè)計(jì)或?qū)ふ腋线m的引物、新的基因位點(diǎn)和采用宏基因組分析等新技術(shù)；另一方面需要改進(jìn)形態(tài)學(xué)分析方法,如視頻采集與編輯方法(VCE)[27]。未來對(duì)深海嘴刺目線蟲的研究不僅要有形態(tài)分類和種群遺傳關(guān)系研究,還需要結(jié)合形態(tài)可視化技術(shù),如串型玻片掃描電鏡、三維重建[28]、共聚焦成像和肌肉組織功能分析[29],增進(jìn)對(duì)線蟲形態(tài)學(xué)進(jìn)化和功能多樣性的了解。綜合運(yùn)用形態(tài)學(xué)和分子生物學(xué)的方法將有助于探知生物類群的同形種(Cryptic species),提升對(duì)海洋線蟲生物多樣性的認(rèn)識(shí),完善線蟲的分類體系。

4 結(jié)論

CC區(qū)嘴刺目線蟲科水平上,尖口線蟲科數(shù)量最多,烙線蟲科數(shù)量最少；屬水平上,Halalaimus屬是優(yōu)勢(shì)屬。本次研究發(fā)現(xiàn)的光皮線蟲科線蟲在以往CC區(qū)線蟲研究文獻(xiàn)中鮮見。這表明,CC區(qū)尚有部分線蟲種類未被發(fā)現(xiàn),該區(qū)嘴刺目線蟲多樣性需進(jìn)一步系統(tǒng)研究。所獲得的分子序列與數(shù)據(jù)庫(kù)中的序列比對(duì)結(jié)果顯示,DNA條形碼比對(duì)結(jié)果和形態(tài)學(xué)鑒定結(jié)果有較高一致性,表明DNA條形碼技術(shù)可作為深海線蟲鑒定的有效手段。基于18S和28S rRNA基因序列,采用不同方法構(gòu)建系統(tǒng)發(fā)育樹,其分支結(jié)構(gòu)基本一致；鉤線蟲科和矛線蟲科聚類在一起,光皮線蟲科和前感線蟲科聚類在一起,顯示出彼此間較近的遺傳關(guān)系。

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Nematode (Enoplida) diversity in sediment samples collected from the Clarion-Clipperton Fracture Zone

ZHANG Shun1,2, LIN Shiquan1, MENG Fanxu1, WU Min2, XU Xuewei1, WANG Chunsheng1,*

1SecondInstituteofOceanography,Hangzhou310012,China2Collegeoflifesciences,ZhejiangUniversity,Hangzhou310058,China

Marine free-living nematodes (Phylum Nematoda) are widespread and abundant in marine sediments, often representing 70%—90% of the benthic metazoans. However, marine nematode taxonomy is severely underdeveloped, and about only 4,000 species of free-living marine nematodes have been described. Nematode identification by using traditional morphological methods is time consuming and expensive. Some marine nematodes are small, displaying similar morphological characters and are difficult to identify by traditional methods. Molecular technology, or “barcoding,” offers the potential of a fast and objective way of species identification. The small and large subunit ribosomal DNA (SSU rDNA and LSU rDNA) gene-based phylogenetic analysis is a powerful tool for clarifying evolutionary relationships among nematode taxa. The order Enoplida is one of the most important groups of marine nematodes. Many enoplids are possibly active predators, and play important ecological roles in marine environments. Here we reported the isolation of 26 nematodes, belonging to Enoplida, from sediment samples collected at four sites in the Clarion-Clipperton Fracture Zone of the Pacific Ocean. The specimens were preserved in DESS solution (20% dimethyl sulphoxide, 0.25 mol/L disodium EDTA pH 8.0, saturated with NaCl) immediately after collection. Each sediment sample was rinsed through a 38-μm sieve using filtered seawater, and extracted using the Ludox flotation method. Nematodes were placed on temporary slides and observed using a Leica DM5500 microscope. After image capturing, each specimen was washed, cut into several pieces, transferred into micro-centrifuge tubes, and digested with Proteinase K. A series of frozen specimens was subsequently thawed and subjected to PCR amplification of the 18S rRNA gene and D3 expansion segments of the 28S rRNA gene. Sequences were analyzed and compared with published data from GenBank by means of a BLAST search. Phylogenetic trees were constructed using the neighbor-joining, maximum likelihood, and maximum parsimony methods with the MEGA5 program package, after multiple alignment of the data by CLUSTAL W. Based on morphological and molecular analyses, these 26 nematodes were classified into six families and eight genera, among which Oxystominidae was the most abundant family, accounting for 57.7%. The other families included Anticomidae (19.2%), Phanodermatidae (7.7%), Oncholaimidae (7.7%), Ironidae (3.85%), and Enchelidiidae (3.85%). At the family and genus level, the community composition at adjacent sites during the same period showed similar results, but the abundance was different. Sixteen sequences of rRNA gene were obtained in the present study and their similarity to the sequences in GenBank ranged from 94 to 99%. According to the results from BLAST, all sequences could be identified at the level of family, while 84.6% of the 16 sequences could be identified at the level of genus. The results from morphological and molecular analysis showed high consistency, which suggested that molecular barcoding is an efficient method to identify deep-sea nematodes. Phylogenetic trees constructed from the sequences of 18S and 28S rRNA gene showed similar topological structures; the species of Oncholaimidae and Enchelidiidae were clustered into one group, whereas those of Phanodermatidae and Anticomidae were clustered into another group, indicating their close genetic relationships.

enoplida; diversity; phylogeny; Clarion-Clipperton Fracture Zone

國(guó)際海域資源調(diào)查與開發(fā)“十二五”課題(DY125- 14-E-02; DY125- 22-QY- 29)

2015- 09- 18;

日期:2016- 07- 13

10.5846/stxb201509181920

*通訊作者Corresponding author.E-mail: wang-sio@163.com

張順, 林施泉, 孟凡旭, 吳敏, 許學(xué)偉, 王春生.太平洋克拉里昂-克利伯頓斷裂帶嘴刺目線蟲多樣性.生態(tài)學(xué)報(bào),2017,37(5):1630- 1638.

Zhang S, Lin S Q, Meng F X, Wu M, Xu X W, Wang C S.Nematode (Enoplida) diversity in sediment samples collected from the Clarion-Clipperton Fracture Zone.Acta Ecologica Sinica,2017,37(5):1630- 1638.