青島近海路氏雙髻鯊群體遺傳學(xué)分析

宋 娜王航寧劉淑德涂 忠高天翔

(1. 中國(guó)海洋大學(xué)水產(chǎn)學(xué)院, 青島 266003; 2. 乳山市水產(chǎn)技術(shù)推廣站, 乳山 264500; 3. 山東省水生生物資源養(yǎng)護(hù)管理中心, 煙臺(tái) 264003)

青島近海路氏雙髻鯊群體遺傳學(xué)分析

宋 娜1王航寧2劉淑德3涂 忠3高天翔1

(1. 中國(guó)海洋大學(xué)水產(chǎn)學(xué)院, 青島 266003; 2. 乳山市水產(chǎn)技術(shù)推廣站, 乳山 264500; 3. 山東省水生生物資源養(yǎng)護(hù)管理中心, 煙臺(tái) 264003)

研究采集了青島近海23尾路氏雙髻鯊(Sphyrna lewini), 通過(guò)線粒體DNA控制區(qū)片段對(duì)其遺傳多樣性進(jìn)行分析。研究結(jié)果顯示: 在23個(gè)個(gè)體的控制區(qū)序列上存在13個(gè)變異位點(diǎn), 未檢測(cè)到插入/缺失位點(diǎn); 檢測(cè)到7個(gè)單倍型, 其中3個(gè)為個(gè)體共享單倍型(Hap1、Hap3和Hap5), 4個(gè)為個(gè)體獨(dú)有單倍型; 青島近海路氏雙髻鯊呈現(xiàn)中等水平的單倍型多樣度和較低的核苷酸多樣度; 與已報(bào)道的日照、霞浦群體間的遺傳分化指數(shù)Fst值分別為–0.0571和–0.0328, 表明青島群體與其他兩個(gè)群體間不存在顯著差異。以Sphyrna zygaena為外群構(gòu)建NJ系統(tǒng)樹(shù)顯示本研究中7個(gè)單倍型共分成兩支, 分別與來(lái)自太平洋、印度洋的單倍型類群聚類。中國(guó)近海的路氏雙髻鯊作為一個(gè)具有較低遺傳多樣性的瀕危物種, 其資源保護(hù)更應(yīng)該引起足夠的重視。

路氏雙髻鯊; 遺傳多樣性; 瀕危物種; 資源保護(hù)

路氏雙髻鯊(學(xué)名: Sphyrna lewini; 英文名: the scalloped hammerhead shark)是雙髻鯊屬(Sphyrna)中一種全球性熱帶和暖溫帶水域分布的鯊魚(yú)[1,2],分類上隸屬于真鯊目(Carcharhiniformes)、雙髻鯊科(Sphyrnidae)。雙髻鯊因具有特殊橫向延伸的頭部而得名, 路氏雙髻鯊是其中體型較大的種類, 其生性兇猛, 喜食小型魚(yú)類, 繁殖方式為胎生, 一次可產(chǎn)下15至31尾幼鯊。與其他類型海洋魚(yú)類相比較,鯊魚(yú)因生長(zhǎng)緩慢、性成熟時(shí)間長(zhǎng)等特點(diǎn)限制了其自我補(bǔ)充能力[3—5], 這使得鯊魚(yú)極易受到過(guò)度捕撈的影響。鯊魚(yú)具有較高的經(jīng)濟(jì)價(jià)值和藥用價(jià)值, 所以一直以來(lái)都是商業(yè)和休閑漁業(yè)的重要捕撈對(duì)象。據(jù)報(bào)道近年來(lái)鯊魚(yú)的資源都已經(jīng)遭到不同程度的破壞[6,7], 路氏雙髻鯊資源量也大大減少[8], 已被世界自然保護(hù)聯(lián)盟(IUCN)列為瀕危物種, 2013年被收錄至《瀕危野生動(dòng)植物種國(guó)際貿(mào)易公約(CITES)》的附錄Ⅱ中。

海洋魚(yú)類在其地理分布范圍內(nèi)通常呈現(xiàn)出較弱的遺傳分化, 這主要是由于地理群體間缺少交流障礙以及其較強(qiáng)的擴(kuò)散能力所致[9—11], 對(duì)鯊魚(yú)、金槍魚(yú)等具有較強(qiáng)運(yùn)動(dòng)能力的海洋魚(yú)類來(lái)說(shuō)更是如此。但近年來(lái)大量的群體遺傳學(xué)研究發(fā)現(xiàn)如棲息地的不連續(xù)分布、海洋魚(yú)類的底棲生活習(xí)性等都可以成為阻礙海洋魚(yú)類基因交流的障礙[12,13]。鯊魚(yú)繁殖方式一般為產(chǎn)沉性卵或者胎生, 其生活史特征中缺乏能夠促進(jìn)基因交流的卵和仔魚(yú)的浮游期,且鯊魚(yú)會(huì)在固定的近海水域進(jìn)行產(chǎn)卵[14,15], 存在戀出生地(Philopatry)行為, 這些生活史特性可能會(huì)阻礙鯊魚(yú)群體間的基因交流, 從而使不同群體間產(chǎn)生遺傳分化[16,17]。路氏雙髻鯊為胎生魚(yú)類, 因此同樣缺乏卵和仔魚(yú)的浮游期, 其季節(jié)性聚集行為也暗示其可能存在戀出生地行為[18—21], 暗示其種群間可能存在基因交流的障礙。

到目前為止, 線粒體DNA控制區(qū)因其具有較高的種內(nèi)多態(tài)性成為研究群體水平遺傳多樣性的理想標(biāo)記, 黑梢真鯊Carcharhinus limbatus[17,22]等多種鯊魚(yú)線粒體DNA的研究都顯示其種群內(nèi)存在顯著的遺傳結(jié)構(gòu)。國(guó)外學(xué)者對(duì)路氏雙髻鯊已有較多的研究[18,23,24], 群體遺傳結(jié)構(gòu)也有研究報(bào)道[25,26]。例如, Nance等[25]采用微衛(wèi)星標(biāo)記以及線粒體DNA測(cè)序兩種方法分析了東太平洋6個(gè)地點(diǎn)的路氏雙髻鯊樣品, 結(jié)果顯示其種群內(nèi)不存在顯著的遺傳結(jié)構(gòu)。Duncan等[26]分析了來(lái)自太平洋、印度洋和大西洋的路氏雙髻鯊樣品, 結(jié)果顯示同海區(qū)沿岸分布的群體之間有很強(qiáng)的基因交流, 但是三個(gè)大洋間群體遺傳差異顯著。國(guó)內(nèi)關(guān)于路氏雙髻鯊的研究較少[27,28], 尚未見(jiàn)關(guān)于青島近海路氏雙髻鯊群體遺傳學(xué)的研究報(bào)道。本研究采用線粒體DNA控制區(qū)部分序列測(cè)定對(duì)青島近海的23尾路氏雙髻鯊進(jìn)行分析, 闡明青島近海路氏雙髻鯊群體的遺傳多樣性現(xiàn)狀, 以期為我國(guó)近海路氏雙髻鯊的種群分布和基因交流現(xiàn)狀提供證據(jù)。

1 材料與方法

1.1 樣品采集

于2012年9月在山東青島沿海采集到23尾路氏雙髻鯊樣品(體長(zhǎng)59.0—69.0 cm; 體重977.3—1669.1 g), 經(jīng)形態(tài)鑒定后, 將樣品冷凍保存至中國(guó)海洋大學(xué)漁業(yè)生態(tài)學(xué)實(shí)驗(yàn)室, 并取背部肌肉于95%的酒精中保存。

1.2 DNA提取

采用傳統(tǒng)酚-氯仿方法提取路氏雙髻鯊基因組DNA, 用超純水將乙醇沉淀后的DNA溶解, –20℃保存?zhèn)溆谩?/p>

1.3 PCR擴(kuò)增與產(chǎn)物測(cè)序

采用引物Pro-L和SLcr-H[25]擴(kuò)增控制區(qū)第一高變區(qū)。PCR反應(yīng)體系為25 μL (基因組DNA約為10 ng、正反向引物各200 nmol/L、Taq DNA聚合酶1.25單位、200 μmol/L的每種dNTP、50 mmol/L KCl、1.5 mmol/L MgCl2和10 mmol/L Tris)。PCR反應(yīng)均在TaKaRa熱循環(huán)儀上完成, 反應(yīng)循環(huán)參數(shù)參照宋娜等[28]。設(shè)陰性對(duì)照來(lái)檢測(cè)PCR是否存在污染。取2 μL PCR產(chǎn)物用1.5%的瓊脂糖凝膠進(jìn)行檢測(cè), 回收純化的目的片段送上海桑尼生物科技有限公司進(jìn)行雙向測(cè)序。

1.4 數(shù)據(jù)分析

從GenBank下載了日照和霞浦兩個(gè)群體的序列(序列號(hào)為KU942394-KU942398)以及Ducan等報(bào)道的25個(gè)單倍型(序列號(hào)為DQ438148-DQ43817)用于比較分析。將本研究測(cè)得序列用Lasergene軟件包中Seqman軟件進(jìn)行比對(duì), 對(duì)測(cè)序膠圖進(jìn)行核對(duì)并輔以人工校正; 與GenBank中的序列比對(duì)后截取同源序列進(jìn)行分析; 序列的堿基組成、變異位點(diǎn)、轉(zhuǎn)換/顛換值以及群體遺傳分化指數(shù)Fst、確切P檢驗(yàn)采用Arlequin3.11軟件包進(jìn)行分析; 采用jModeltest軟件計(jì)算反映突變速率在不同位點(diǎn)間異質(zhì)性的gamma分布形狀參數(shù)。基于AICc準(zhǔn)則, 篩選出適合序列分析的模型為HKY+I+G (G=0.7010)[29], 基于HKY+ I+G模型, 以Sphyrna zygaena (序列號(hào)GU385314)為外群, 使用PAUP 4.0軟件構(gòu)建單倍型的鄰接樹(shù)(Neighbor joining, NJ)[30], 采用1000次非參數(shù)自展分析進(jìn)行重復(fù)檢驗(yàn)計(jì)算系統(tǒng)樹(shù)各分支的置信度[31]。采用Arlequin 3.11軟件來(lái)進(jìn)行中性假說(shuō)檢驗(yàn)以及核苷酸不配對(duì)分布分析[32,33]。

2 結(jié)果

將獲得的序列經(jīng)過(guò)人工比對(duì)校正, 得到長(zhǎng)度681 bp的控制區(qū)序列, 與GenBank中的序列進(jìn)行比對(duì)后截取548 bp同源片段序列進(jìn)行分析。在本研究中, 青島群體的序列堿基含量分別為C: 23.14%、T: 37.08%、A: 31.17%、G: 8.60%, A+T堿基含量(68.3%)明顯大于C+G含量(31.7%)。青島群體的控制區(qū)序列上共存在13處核苷酸替換(12個(gè)轉(zhuǎn)換和1個(gè)顛換), 未檢測(cè)到插入/缺失位點(diǎn)(圖 1), 共定義了7個(gè)單倍型, 其中3個(gè)單倍型為個(gè)體共享單倍型(Hap1、Hap3和Hap5), 其余4個(gè)單倍型為個(gè)體獨(dú)有單倍型(表 1)。與日照和霞浦兩個(gè)群體的序列比對(duì)后, 共獲得8個(gè)單倍型, 其中4個(gè)單倍型為群體共享單倍型(Hap1、Hap2、Hap4和Hap5), 其余4個(gè)單倍型為群體獨(dú)有單倍型。在本研究中, 青島群體的單倍型多樣度(Haplotype diversity)、核苷酸多樣度(Nucleotide diversity)、兩兩序列比較的平均堿基差異數(shù)(The mean number of pairwise nucleotide dif-ferences)分別為0.6957±0.0880、0.0034±0.0023和1.8658±1.1080。

圖 1 路氏雙髻鯊青島群體控制區(qū)序列變異位點(diǎn)Fig. 1 Variable sites of S. lewini from Qingdao based on mitochondrial control region sequences

青島群體與日照、霞浦群體間的遺傳分化指數(shù)Fst值分別為–0.0571和–0.0328(表 2), 兩兩群體之間的遺傳差異較小且統(tǒng)計(jì)檢驗(yàn)結(jié)果均不顯著, 表明青島群體與其他兩個(gè)群體間不存在顯著差異。確切P檢驗(yàn)的結(jié)果也均不顯著(表 2), 表明青島群體與其他兩個(gè)群體存在隨機(jī)交配現(xiàn)象。

以Sphyrna zygaena為外群, 構(gòu)建了青島、日照、霞浦3個(gè)群體8個(gè)單倍型和Ducan等25個(gè)單倍型的NJ系統(tǒng)樹(shù)。結(jié)果顯示存在4個(gè)顯著分化的單倍型類群, 其中來(lái)自大西洋的隱存種(DQ438148)形成單獨(dú)一支, 中國(guó)近海路氏雙髻鯊8個(gè)單倍型分別與來(lái)自太平洋、印度洋的單倍型類群聚類(Clade A、Clade C)聚為兩支, 而在單倍型分支B (Clade B, 大西洋、印度-西太平洋)中未檢測(cè)到中國(guó)近海的單倍型分布(圖 2)。

在本研究中, 路氏雙髻鯊的核苷酸不配對(duì)分布圖呈多峰類型(圖 3a), 而個(gè)體數(shù)較多的單倍型分支A的核苷酸不配對(duì)分布呈明顯的單峰狀態(tài)(圖 3b),暗示其可能經(jīng)歷過(guò)近期的群體擴(kuò)張事件。分支A的Tajima’D檢驗(yàn)結(jié)果并未顯著偏離中性(Tajima’s D=–1.302, P=0.095), 但Fu’Fs檢驗(yàn)結(jié)果卻是顯著的(Fs=–6.857, P=0.001), 這可能是由于Fu’Fs檢驗(yàn)對(duì)近期群體擴(kuò)張更為敏感所致。對(duì)觀測(cè)值和模擬值的擬合度進(jìn)行檢驗(yàn)結(jié)果顯示, 觀測(cè)值與期望值非常吻合(Harpending’s指數(shù)=0.01, P＞0.05), 沒(méi)有顯著偏離擴(kuò)張模型[34]。基于單倍型類群A的τ值2.387和0.8%/ Myr的分歧速率計(jì)算顯示路氏雙髻鯊群體擴(kuò)張事件大約發(fā)生在540kyr前。

表 1 路氏雙髻鯊3個(gè)群體的單倍型頻率分布表Tab. 1 Haplotype distribution of three S. lewini populations

表 2 路氏雙髻鯊兩兩群體間的遺傳分化指數(shù)Fst(對(duì)角線下)和確切P檢驗(yàn)(對(duì)角線上)Tab. 2 Pairwise Fst(below diagonal) and the significant nondifferentiation exact P test value (above diagonal) among three populations of S. lewini

圖 2 基于路氏雙髻鯊線粒體控制區(qū)單倍型構(gòu)建的鄰接關(guān)系樹(shù)Fig. 2 Nj tree for S. lewini control region haplotypes以Sphyrna zygaena為外群構(gòu)建系統(tǒng)樹(shù)，分支上的數(shù)字表示支持率, 支持率＜60%的未列出Sphyrna zygaena was used to root the tree as an outgroup. Bootstrap with ＜60% were unshown

3 討論

鯊魚(yú)通常處于食物鏈的頂端, 因此在生態(tài)系統(tǒng)中具有十分重要的作用。路氏雙髻鯊是為數(shù)不多的近海常見(jiàn)且數(shù)量較多的鯊魚(yú)之一, 是近海鯊魚(yú)捕撈業(yè)的重要漁獲對(duì)象, 因此承受了巨大的捕撈壓力,同時(shí)路氏雙髻鯊的集群行為使其更易受到商業(yè)捕撈的影響。根據(jù)NMFS (National Marine Fisheries Service)[35]報(bào)道, 有些海域的路氏雙髻鯊資源已經(jīng)處于過(guò)度捕撈狀態(tài)。本研究中青島近海路氏雙髻鯊呈現(xiàn)中等水平的單倍型多樣度和較低的核苷酸多樣度, 與日照、霞浦兩群體的遺傳多樣性水平(h=0.51±0.01—0.60±0.13; π=0.0024±0.0017—0.0046±0.0031)相當(dāng)[28], 與太平洋東海岸與西海岸的路氏雙髻鯊群體遺傳多樣性指數(shù)也相近[26]。較低的遺傳多樣性在其他一些受威脅的鯊魚(yú)中也有報(bào)道, 例如Mustelus schmitti[36]、Negaprion brevirostris和N. acutidens[37]等。

圖 3 路氏雙髻鯊線粒體控制區(qū)核苷酸不配對(duì)分布圖Fig. 3 The mismatch distributions for the control region haplotypes in S. lewinia. 所有樣品; b. 分支A (柱形為觀測(cè)值; 曲線為群體擴(kuò)張模型下的預(yù)期分布)a. all samples; b. Clade A (Bars: observed pairwise differences; solid line: the expected mismatch distributions under the sudden expansion model)

群體間遺傳分化指數(shù)計(jì)算結(jié)果表明中國(guó)近海路氏雙髻鯊3個(gè)群體間不存在顯著的遺傳分化, 確切P檢驗(yàn)的結(jié)果暗示群體間可能存在頻繁的基因交流, 雖然路氏雙髻鯊大洋間存在顯著的遺傳差異,但是沿海岸線分布的群體間并不缺乏基因交流[26]。路氏雙髻鯊的系統(tǒng)地理格局類型與許多全球性分布的軟骨魚(yú)類相似, 如Carcharhinus limbutus[17,18]、Lagenorhynchus obscurus[38]等。基因交流可以維持物種的遺傳多樣性[39], 但是本研究結(jié)果暗示路氏雙髻鯊較低的遺傳多樣性可能并不是有限的基因交流導(dǎo)致的, 這一結(jié)果提示其資源狀況可能并不樂(lè)觀。為保持物種對(duì)環(huán)境的適應(yīng)能力, 其有效群體大小以及群體遺傳多樣性都應(yīng)該維持在某一水平[39]。對(duì)路氏雙髻鯊的歷史動(dòng)態(tài)研究結(jié)果表明其有效種群與過(guò)去相比明顯減小[24], 過(guò)度捕撈可能降低了有效種群大小[40], 使種群更易發(fā)生遺傳漂變進(jìn)而降低遺傳多樣性[41]。

NJ樹(shù)中中國(guó)近海的8個(gè)單倍型分別與來(lái)自太平洋、印度洋單倍型聚類, 且不與地理位置相對(duì)應(yīng),而大西洋和印度-西太平洋的單倍型單獨(dú)構(gòu)成分支。Ducan等[26]認(rèn)為巴拿馬海峽的封閉導(dǎo)致了大西洋群體的隔離, 而印度-西太平洋的單倍型是大西洋群體從西向東擴(kuò)散的結(jié)果, 夏威夷或者南非的群島可能是路氏雙髻鯊進(jìn)入東太平洋的基石。復(fù)雜的動(dòng)態(tài)歷史導(dǎo)致了路氏雙髻鯊單倍型類群的分化。印度-太平洋與大西洋間歷史上的隔離是許多海洋生物存在類似系統(tǒng)地理格局的主要因素, 如Thunnus obesus[42]、Lagenorhynchus obscurus[38]等。

許多沿大陸架海域分布的鯊魚(yú)移動(dòng)能力較弱,通常只在沿海區(qū)域活動(dòng), 并不向大洋擴(kuò)散[43,44]。Kohler和Turner[45]對(duì)路氏雙髻鯊進(jìn)行標(biāo)志放流研究, 結(jié)果顯示標(biāo)記點(diǎn)與回捕點(diǎn)的距離一般不超過(guò)100 km,且很少在開(kāi)闊海域捕到路氏雙髻鯊, 但是有一個(gè)個(gè)體移動(dòng)距離甚至超過(guò)了1600 km。因此這種鯊魚(yú)一般不進(jìn)行長(zhǎng)距離擴(kuò)散, 但偶然也有跨大洋運(yùn)動(dòng)的行為[41]。因此盡管不能確定路氏雙髻鯊是否存在戀出生地行為, 但是考慮到路氏雙髻鯊沿岸分布以及很少長(zhǎng)距離擴(kuò)散的特性, 如果某一海域的路氏雙髻鯊資源嚴(yán)重破壞, 將很難從其他海域進(jìn)行補(bǔ)充, 因此禁止過(guò)度捕撈以及對(duì)路氏雙髻鯊的保護(hù)是十分重要的。作為一個(gè)遺傳多樣性較低的瀕危物種, 路氏雙髻鯊資源保護(hù)管理更應(yīng)該引起足夠的重視。

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GENETIC STUDY ON SPHYRNA LEWINI FROM QINGDAO COASTAL WATERS

SONG Na1, WANG Hang-Ning2, LIU Shu-De3, TU Zhong3and GAO Tian-Xiang1
(1. Fisheries College, Ocean University of China, Qingdao 266003, China; 2. Rushan Fishery Technology Popularization Center, Rushan 264500, China; 3. Shandong Hydrobios Resources Conservation and Management Center, Yantai 264003, China)

The scalloped hammerhead shark, Sphyrna lewini, is a small to medium-sized shark that circumglobally distributes along continental margins and oceanic islands in tropical waters. It has been listed as one globally endangered species by IUCN because of continuous resource recession by habitat degradation and influence of human activities. The present study analyzed mitochondrial DNA sequences of a total of 23 specimens collected from Qingdao coastal waters, and found 13 variable sites in the 548 bp fragments without indel/deletion. These polymorphic sites defined 7 haplotypes, 3 of them (Hap1, Hap3 and Hap5) were different individuals and the other 4 haplotypes were unique for one individual. Moderate haplotype diversity and low nucleotide diversity were detected for Qd population. There was no genetic differentiation between Qd and other reported two populations, and Fstwere –0.0571 and –0.0328, respectively. Haplotypes from Chinese coastal waters were clustered into two clades with haplotypes from the Pacific and Indian Ocean by the topology of the NJ tree rooted with the out-group S. zygaena. More attentions should be paid to resource protection for Sphyrna lewini with, and the endangered species (Sphyrna lewini) from Chinese coastal waters should be considered one population, which need greater resource protection because of low genetic diversity.

Sphyrna lewini; Genetic diversity; Endangered species; Resource protection

Q346+.5

A

1000-3207(2017)03-0603-06

10.7541/2017.77

2016-06-22;

2016-11-07

海洋公益性行業(yè)科研專項(xiàng)(201305043和201405010); 國(guó)家科技基礎(chǔ)條件平臺(tái)-水產(chǎn)種質(zhì)資源平臺(tái)項(xiàng)目資助 [Supported by the Public Science and Technology Research Funds Projects of Ocean (201305043 and 201405010); National Infrastructure of Fishery Germplasm Resources]

宋娜(1983—), 女, 山東乳山人; 博士; 主要從事漁業(yè)生態(tài)學(xué)研究。E-mail: songna624@163.com

高天翔(1962—), 男, 遼寧遼陽(yáng)人; 博士; 主要從事漁業(yè)生態(tài)學(xué)研究。E-mail: gaotianxiang0611@163.com