西北太平洋海岸帶大彈涂魚復(fù)合體的隱存種與進(jìn)化歷史

陳 卉Gianluca Polgar殷 維傅萃長

(1. 復(fù)旦大學(xué)生物多樣性科學(xué)研究所, 生物多樣性與生態(tài)工程教育部重點(diǎn)實(shí)驗(yàn)室, 上海 200433;

2. Biology Programme, Universiti Brunei Darussalam, Bandar Seri Begawan, Gadong, Negara Brunei Darussalam)

西北太平洋海岸帶大彈涂魚復(fù)合體的隱存種與進(jìn)化歷史

陳 卉1Gianluca Polgar2殷 維1傅萃長1

(1. 復(fù)旦大學(xué)生物多樣性科學(xué)研究所, 生物多樣性與生態(tài)工程教育部重點(diǎn)實(shí)驗(yàn)室, 上海 200433;

2. Biology Programme, Universiti Brunei Darussalam, Bandar Seri Begawan, Gadong, Negara Brunei Darussalam)

大彈涂魚Boleophthalmus pectinirostris間斷分布于西太平洋海岸帶東亞與馬來西亞馬六甲海峽, 但馬來西亞種群的分類地位尚存爭議。研究使用線粒體ND5基因序列(718 bp)與核位點(diǎn)Rag1基因序列(1395 bp)對西北太平洋海岸帶11個地點(diǎn)的45尾大彈涂魚屬魚類進(jìn)行系統(tǒng)發(fā)育關(guān)系重建, 結(jié)果表明大彈涂魚包括東亞與馬來西亞兩個單系群, 兩者形成姊妹群關(guān)系。GMYC分析、*BEAST物種樹支持大彈涂魚東亞譜系和馬來西亞譜系是不同種。分子測定年齡分析表明大彈涂魚東亞譜系與馬來西亞譜系之間的分化時間為2.73百萬年。因此, 西北太平洋海岸帶大彈涂魚是復(fù)合體, 包括兩個物種: 東亞種群是大彈涂魚 Boleophthalmus pectinirostris sensu stricto, 而馬來西亞種群是隱存種Boleophthalmus sp.。大彈涂魚與隱存種之間的物種分化可能是晚上新世冰期海平面下降產(chǎn)生的地理隔離以及間冰期洋流對基因交流的阻礙兩方面相互作用的結(jié)果。

蝦虎魚科; 大彈涂魚; 系統(tǒng)發(fā)育; 物種界定; 西北太平洋

大彈涂魚屬(Boleophthalmus Valenciénnes, 1837)魚類是一群生活在太平洋和印度洋沿岸潮間帶淤泥質(zhì)潮灘上的小型魚類, 隸屬于鱸形目 Perciformes、蝦虎魚亞目Gobioidei、蝦虎魚科Gobiidae、背眼蝦虎魚亞科Oxudercinae[1]。該屬包括5種魚類: 北澳洲大彈涂魚Boleophthalmus birdsong Murdy, 1989、薄氏大彈涂魚B. boddarti(Pallas, 1770)、綠斑大彈涂魚B. caeruleomaculatus McCulloch & Waite, 1918、杜氏大彈涂魚B. dussumieri Valenciennes, 1837和大彈涂魚B. pectinirostris(Linnaeus, 1758)。依據(jù)Fish Base[2]與the Catalog of Fishes[3]數(shù)據(jù)庫的記錄, 分布于西北太平洋的大彈涂魚屬魚類包括兩種: 薄氏大彈涂魚與大彈涂魚。其中, 薄氏大彈涂魚主要分布于南中國海北部灣以南, 而大彈涂魚則間斷分布于東亞(南中國海北部灣以北)與馬來西亞的馬六甲海峽[1,4—9]。大彈涂魚東亞種群的體長一般不超過135 mm[4], 而馬來西亞種群的體長可達(dá)175 mm[9]。 Murdy[1]對大彈涂魚屬魚類進(jìn)行分類校訂后認(rèn)為大彈涂魚馬來西亞種群可能是一個隱存種(Cryptic species)。分子證據(jù)已在魚類分類與系統(tǒng)學(xué)研究中廣泛運(yùn)用[10,11], 其中線粒體ND5與核位點(diǎn)Rag1基因是蝦虎魚科魚類分類與系統(tǒng)學(xué)研究中最常用的分子標(biāo)記之一[12,13]。因此, 本研究選擇ND5與Rag1基因作為分子標(biāo)記, 使用分子證據(jù)探討大彈涂魚馬來西亞種群的物種狀態(tài)。

西北太平洋的一個獨(dú)特地形特點(diǎn)是具有南中國海、東中國海、黃海和日本海等一系列相互連接的邊緣海[14]。上新世和更新世的冰期及間冰期旋回導(dǎo)致的海平面漲落對邊緣海的面積和結(jié)構(gòu)造成了劇烈影響[14]。基于分子證據(jù)的系統(tǒng)發(fā)育研究揭示在晚上新世和更新世冰期階段海平面下降導(dǎo)致連接邊緣海的海峽形成陸橋成為地理障礙, 導(dǎo)致近海魚類譜系分化和物種形成[12,15]; 間冰期階段海平面上升, 邊緣海重新連接形成復(fù)雜的水文情況[16], 中國海沿岸流、黑潮(Kuroshio Current)、南中國海暖流亦能成為自然屏障阻礙種群間的基因流, 促進(jìn)魚類譜系分化與物種形成[17,18]。因此, 本研究假設(shè)冰期階段海平面下降馬六甲海峽成為陸橋以及間冰期階段復(fù)雜的洋流系統(tǒng)可能在西北太平洋海岸帶大彈涂魚屬魚類的遺傳分化過程中扮演了重要角色。本研究目的是通過沿西北太平洋海岸帶進(jìn)行廣泛取樣, 基于線粒體與核基因證據(jù)來揭示西北太平洋海岸帶大彈涂魚東亞種群與馬來西亞種群的間斷分布格局的進(jìn)化歷史。

1 材料與方法

1.1 取樣和分子標(biāo)記

西北太平洋地區(qū)大彈涂魚屬魚類標(biāo)本包括來自我國廣西黨江、廣東湛江、福建泉州、浙江溫州、上海崇明、韓國順天(Suncheon)和日本六角川(Rokkaku River)7個地點(diǎn)的大彈涂魚東亞種群19尾(圖1), 來自馬來西亞龜咯島(Pulau Kukup, PK)和丹戎比艾(Tanjung Piai, TP)2個地點(diǎn)的大彈涂魚馬來西亞種群11尾, 來自馬來西亞龜咯島、丹戎比艾、凱莉島(Carey Island)和雙溪檳榔(Sungai Pinang)4個地點(diǎn)的薄氏大彈涂魚15尾(圖1)。外類群選擇拉氏狼牙蝦虎魚Odontamblyopus lacepedii 2尾、里貝卡狼牙蝦虎魚 Odontamblyopus rebecca、彈涂魚Periophthalmus modestus 和 大 鰭 彈 涂 魚Periophthalmus magnuspinnatus各1尾(表1)。采集標(biāo)本的肌肉用 95%乙醇固定保存。選擇線粒體NADH 脫氫酶亞基5基因(ND5)與核位點(diǎn)重組激活基因1(Rag1)作為分子標(biāo)記。

圖1 西北太平洋海岸帶大彈涂魚屬魚類的采樣地點(diǎn)Fig. 1 Sample sites of Boleophthalmus fishes along the northwestern Pacific coast

1.2 DNA提取、PCR擴(kuò)增與產(chǎn)物測序

采用高鹽法從魚體肌肉組織中提取全基因組DNA。擴(kuò)增ND5基因的引物為L12321(5′-GGTCTT AGGAACCAAAAACTCTTGGTGCAA-3′)與H13396 (5′-CCTATTTTTCGGATGTCTTG-3′)[19]; PCR反應(yīng)條件為: 94℃預(yù)變性5min; 94℃變性35s, 55℃退火35s, 72℃延伸40s; 循環(huán)35次; 72℃終末延伸8min。擴(kuò)增Rag1基因片段的方案為巢式PCR。第一輪使用的引物為 RAG1F1(5′-CTGAGCTGCAGTCAG TACCATAAGATGT-3′)和RAG1R1(5′-CTGAGTCC TTGTGAGCTTCCATRAAYTT-3′)[20], 第二輪使用本研究設(shè)計的引物 GOBRAG1F1(5′-GCCAGATCTT CCAGCCTCT-3′)和XRAG1R(5′-TACTTGGADGTG TAGAGCC-3′); PCR反應(yīng)條件為: 94℃預(yù)變性5min; 94℃變性35s, 55℃退火40s, 72℃延伸40s; 循環(huán)35次; 72℃終末延伸8min。PCR產(chǎn)物經(jīng)2.0%瓊脂糖電泳凝膠純化, 使用第二輪擴(kuò)增引物在 ABI 3730 DNA測序儀中進(jìn)行測序。

表1 種名、采樣地點(diǎn)、個體編號、單倍型及GenBank 登錄號Tab. 1 Species, sampling localities, codes, haplotypes and GenBank accession numbers

續(xù)表

1.3 單倍型網(wǎng)絡(luò)圖與系統(tǒng)發(fā)育關(guān)系重建

使用CLUSTAL X version 1.83 軟件[21], 采用默認(rèn)參數(shù)對 ND5和 Rag1基因序列進(jìn)行對位。使用DnaSP version 4.20軟件選擇ND5基因的單倍型[22]。使用Phase version 2.1軟件[23,24], 設(shè)置后驗(yàn)概率閾值為 0.6, 選擇 Rag1基因的單倍型。一些個體的Rag1基因具有兩個單倍型(表 1)。使用 Network version 4.6軟件[25]構(gòu)建Median-Joining單倍型網(wǎng)絡(luò)圖, 并采用最大簡約(Maximum parsimony)過程[26]確定單倍型之間的連接。

使用 MrBayes version 3.2軟件[27]與 RAxML version 7.2.6 軟件[28], 基于密碼子分區(qū)構(gòu)建貝葉斯(Bayesian)和最大似然(Maximum likelihood)樹。使用jModeltest version 0.1.1軟件[29]選擇各分區(qū)的最適堿基替換模型。貝葉斯分析基于馬可夫蒙特卡羅(Markov chain Monte Carlo)進(jìn)行1.2×107代運(yùn)算, 每1000代對系統(tǒng)樹進(jìn)行1次抽樣。拋棄前3000棵抽樣樹, 構(gòu)建 50%的多數(shù)原則一致樹, 并計算每個節(jié)點(diǎn)的后驗(yàn)概率值(Bayesian Posterior Probability, BPP)。最大似然分析使用快速登山算法(Rapid-hillclimbing algorithm), 在 GTRGAMMA 模型下進(jìn)行100次重復(fù)找出分值最高的最大似然樹(Best-scoring ML tree)。再進(jìn)行1000次自展(Bootstrap)分析, 從而估計節(jié)點(diǎn)的自展支持度(Bootstrap support, BS)。

1.4 物種界定與基因流

基于ND5 + Rag1聯(lián)合基因序列, 使用General Mixed Yule-Coalescent(GMYC)方法確定進(jìn)化顯著性單元(Evolutionary Significant Units, ESU)。GMYC方法結(jié)合種間分化的Yule過程和種內(nèi)分化的溯祖過程, 通過確定兩個過程之間的轉(zhuǎn)換點(diǎn), 為物種界定提供依據(jù)[30]。首先, 使用BEAST version 1.7.2軟件[31]構(gòu)建超度量樹(Ultrametric tree), 種群動態(tài)模型設(shè)置為穩(wěn)定大小模型(Constant-size model), 突變率設(shè)置為平均值為 1的對數(shù)正態(tài)分布(Lognormal distribution)。運(yùn)行 4個重復(fù), 每個重復(fù)運(yùn)行 5×107代, 每1000代取樣一次, burnin為1/10。接著, 4個重復(fù)的結(jié)果用 BEAST軟件包中的 LogCombiner version 1.7.2合并, 利用Tracer verson 1.5軟件[32]查看有效取樣大小(Effective Sampling Size, ESS)值(ESS大于200), 在BEAST軟件包中的 TreeAnnotator version 1.7.2中生成最大譜系置信樹(Maximum Clade Credibility Tree)。最后, 在R環(huán)境[33]中使用SPLITS軟件包[34]進(jìn)行單閾值(Single-threshold)的 GMYC方法分析。

使用 IMa2軟件[35,36]在隔離遷移(Isolation with migration, IM)模型下估算物種間的基因流。在Hasegawa-Kishino-Yano(HKY)進(jìn)化模型下運(yùn)行MCMC鏈5×107代, 舍棄前5×106代之后保證ESS值大于200, 參數(shù)間的自相關(guān)(Parameter autocorrelations)小于 0.05。運(yùn)算過程中未指定具體的突變率 u時, IMa2可估計種群突變率(Population mutation rates, θ)和每突變遷移率(Migration rates per mutation, M)。而有效種群大小(Effective population size)N = θ/4u, 每世代遷移率(Migration rates per generation)m = Mu, 故而每世代基因遷移有效數(shù)目(Effective numbers of gene migrants per generation) 2Nm = 2×θ/4u×Mu = θM/2。

圖2 基于ND5基因(a)與Rag1基因(b)構(gòu)建的貝葉斯50%多數(shù)原則一致樹Fig. 2 The Bayesian 50% major rule consensus tree based on ND5 (a) gene and Rag1 (b) gene

1.5 遺傳距離、物種樹與分化時間

使用MEGA version 5.05 軟件[37]計算物種間的成對 Kimura雙參數(shù)(K2P)平均遺傳距離。使用*BEAST version 1.7.2軟件[31,38]構(gòu)建物種樹(Species tree)。分子鐘采用對數(shù)正態(tài)分布的松散鐘, 先驗(yàn)樹(Tree prior)設(shè)置為Yule過程。共運(yùn)行了4個重復(fù), 每個重復(fù)運(yùn)行5×107代, 每1000代取樣一次, burnin為1/10。使用LogCombiner軟件合并4重復(fù)的結(jié)果, 利用Tracer軟件檢驗(yàn)其ESS值。在TreeAnnotator軟件中生成最大譜系置信樹。由于缺乏蝦虎魚科魚類化石, 本研究使用 Mukai, et al.[39]估算的吻蝦虎魚屬魚類ND5基因的突變率, 每百萬年每譜系每位點(diǎn)(1.95 ± 0.17)%估算分化時間。

圖3 基于ND5和Rag1基因聯(lián)合數(shù)據(jù)構(gòu)建的貝葉斯一致樹Fig. 3 The Bayesian consensus tree based on combined data of ND5 and Rag1 genes

2 結(jié)果

2.1 序列特征

ND5基因部分序列長度718 bp, 可變位點(diǎn)117個、簡約信息位點(diǎn)109個, Rag1基因部分序列長度1395 bp, 可變位點(diǎn)51個、簡約信息位點(diǎn)40個。取樣的45尾大彈涂魚屬魚類包括23個ND5基因單倍型和31個Rag1基因單倍型。GenBank登錄號信息見表1。

2.2 系統(tǒng)發(fā)育關(guān)系與單倍型網(wǎng)絡(luò)圖

圖4 西北太平洋大彈涂魚屬魚類單倍型的Median-Joining網(wǎng)絡(luò)圖Fig. 4 Haplotype Median-Joining network of Boleophthalmus fishes in the northwestern Pacific

ND5、Rag1單基因貝葉斯分析(圖 2)以及聯(lián)合基因的貝葉斯和最大似然法分析(圖 3)得到了一致的拓?fù)浣Y(jié)構(gòu)。這些分析顯示大彈涂魚與薄氏大彈涂魚是單系群(后驗(yàn)概率 BPP = 84%—100%, 自展支持度BS = 100%); 大彈涂魚東亞與馬來西亞種群分別是單系群并形成姊妹群關(guān)系(BPP = 97%—100%;自展支持度 BS =100%), 以下稱為東亞譜系與馬來西亞譜系(圖 2、3)。單倍型網(wǎng)絡(luò)圖進(jìn)一步顯示大彈涂魚東亞譜系、馬來西亞譜系與薄氏大彈涂魚之間具有明顯的遺傳分化(圖4)。ND5基因單倍型網(wǎng)絡(luò)圖顯示大彈涂魚東亞譜系與馬來西亞譜系之間具有64步突變, 兩譜系與薄氏大彈涂魚之間具有73步突變,而譜系內(nèi)相互連接單倍型之間不超過 5步突變(圖4a)。Rag1基因單倍型網(wǎng)絡(luò)圖顯示大彈涂魚東亞譜系與馬來西亞譜系之間具有16步突變, 兩譜系與薄氏大彈涂魚之間具有24步突變, 而譜系內(nèi)相互連接單倍型之間不超過2步突變(圖4b)。

圖5 基于ND5和Rag1基因聯(lián)合數(shù)據(jù)在BEAST中構(gòu)建的超度量樹(a)、時間和譜系間的關(guān)系(b)以及似然值和時間的變化曲線(c)Fig. 5 The ultrametric tree implemented with BEAST (a), relationship between time and lineage (b), and relationship between time and likelihood value (c) based on combined data of ND5 and Rag1 genes

2.3 GMYC分析、物種樹與基因流

基于ND5 + Rag1基因聯(lián)合數(shù)據(jù)的GMYC分析(圖 5)顯示西北太平洋大彈涂屬魚類包括三個進(jìn)化顯著性單元, 分別對應(yīng)于大彈涂魚東亞譜系、馬來西亞譜系與薄氏大彈涂魚。種間或譜系間屬于種間分化之Yule過程, 種內(nèi)或譜系內(nèi)屬于種內(nèi)分化的溯祖過程, 大彈涂魚東亞譜系、馬來西亞譜系與薄氏大彈涂魚應(yīng)界定為三個物種(圖5)。*BEAST物種樹(圖6)進(jìn)一步支持基于GMYC分析的物種劃分并具有強(qiáng)的統(tǒng)計支持(BPP = 100%)。隔離遷移模型估計亦顯示大彈涂魚東亞譜系、馬來西亞譜系與薄氏大彈涂魚之間基因流非常小, 每世代基因遷移有效數(shù)目接近于零(圖7)。

2.4 遺傳距離和分化時間

基于ND5基因序列計算的大彈涂魚東亞譜系、馬來西亞譜系與薄氏大彈涂魚種間或譜系間 K2P遺傳距離為10.41%—11.87%, 種內(nèi)或譜系內(nèi)K2P遺傳距離為0.32%—0.57%(表2)?；赗ag1基因序列計算的大彈涂魚東亞譜系、馬來西亞譜系與薄氏大彈涂魚種間或譜系間K2P遺傳距離為1.50%— 1.80%, 種內(nèi)或譜系內(nèi)K2P遺傳距離為0.18%— 0.30%。分子測定年齡結(jié)果表明大彈涂魚東亞譜系與馬來西亞譜系之間的分化時間為 2.73百萬年, 兩譜系與薄氏大彈涂魚之間的分化時間為3.865百萬年(圖6)。

圖6 基于ND5和Rag1基因聯(lián)合數(shù)據(jù)的*BEAST物種樹與時間樹Fig. 6 *BEAST species tree and time tree based on combined data of ND5 and Rag1 genes

圖7 基于IM模型估算的大彈涂魚東亞譜系、馬來西亞譜系和薄氏大彈涂魚之間的基因流Fig. 7 Gene flows among East Asia lineage and Malaysia lineage of Boleophthalmus pectinirostris and B. boddarti estimated by the IM model

3 討論

表2 大彈涂魚屬魚類種內(nèi)和種間的Kimura 雙參數(shù)(K2P)遺傳距離Tab. 2 Intra-specific and inter-specific Kimura 2-parameter genetic distance (K2P) of Boleophthalmus fishes

3.1 物種界定與隱存種

近年來, 分子證據(jù)揭示蝦虎魚科魚類中發(fā)現(xiàn)隱存種的現(xiàn)象十分普遍[12,39—43]。本研究基于線粒體與核基因分子標(biāo)記的系統(tǒng)發(fā)育重建顯示大彈涂魚東亞種群和馬來西亞種群均是單系群, 并形成姊妹群關(guān)系。物種的譜系概念認(rèn)為物種可看作是系統(tǒng)發(fā)育分析中一個單系群所代表的一組群體[44,45]。根據(jù)這個標(biāo)準(zhǔn), 大彈涂魚東亞譜系和馬來西亞譜系能被界定為2個種。GMYC分析、*BEAST物種樹重建以及基因流估算亦表明大彈涂魚東亞譜系和馬來西亞譜系是不同種。從遺傳距離的角度看, 線粒體基因的種間分化水平一般比種內(nèi)分化水平大10倍[46,47]。大彈涂魚東亞譜系和馬來西亞譜系ND5基因譜系間分化是譜系內(nèi)分化的18—19倍, 因而也支持兩物種的界定?；诖髲椡眶~模式產(chǎn)地在我國[1,4], 因此,可推斷大彈涂魚東亞譜系是大彈涂魚(Boleophthalmus pectinirostris sensu stricto), 大彈涂魚馬來西亞譜系是隱存種(Boleophthalmus sp.)。這個發(fā)現(xiàn)擴(kuò)展了對西太平洋大彈涂魚屬魚類的認(rèn)識, 并把大彈涂魚的分布范圍限定在東亞(南中國海北部灣以北), 而隱存種(Boleophthalmus sp.)目前局限于馬六甲海峽。

3.2 進(jìn)化歷史

晚上新世和更新世最大冰期的海平面比現(xiàn)今海平面低120—140 m[48], 當(dāng)時黃、渤海整個區(qū)域及南中國和海東中國海的部分區(qū)域都露出了水面[14]。先前的研究表明晚上新世海平面最低時期臺灣海峽、對馬海峽露出水面造成的東中國海與南中國海之間、東中國海與日本海之間的隔離導(dǎo)致了西北太平洋近岸狼牙蝦虎魚屬(Odontamblyopus)魚類與鯔屬(Mugil)魚類的物種形成[12,18]。本研究發(fā)現(xiàn)大彈涂魚(東亞譜系)和隱存種(馬來西亞譜系)的分化時間估計為2.73百萬年。因此, 晚上新世海平面最低時期馬六甲海峽露出水面造成的南中國海與印度洋之間的隔離可能導(dǎo)致大彈涂魚和隱存種分化為姊妹群。

另一方面, 南中國海及其鄰近海域復(fù)雜的水文特征對物種的擴(kuò)散和分布也有重大的影響[49,50]。大彈涂魚生活史中適合進(jìn)行長距離遷徙的只有其幼魚的浮游階段, 該階段持續(xù)約35d[51]。大彈涂魚在每年的 4—9月間進(jìn)行繁殖, 而該時期南中國海暖流沿越南中部–海南島–廣東這一路線向西北方向流動, 同時在南中國海暖流的南方, 南中國海環(huán)流則在夏季季風(fēng)的影響下向東南方向流動[52]。這兩個方向相反的洋流只可能將大彈涂魚與隱存種的浮游幼體向相反的方向輸送, 使它們之間的基因交流受到強(qiáng)烈的隔離, 從而進(jìn)一步促進(jìn)物種形成。最近的一些研究亦推斷間冰期洋流作用引起的生物地理障礙是導(dǎo)致西北太平洋海洋生物物種形成的原因之一[18, 53—57]。

綜上所述, 西北太平洋海岸帶大彈涂魚是復(fù)合體, 包括兩個物種: 東亞種群是大彈涂魚(Boleophthalmus pectinirostris sensu stricto), 而馬來西亞種群是隱存種(Boleophthalmus sp.)。大彈涂魚與隱存種之間的物種分化可能是晚上新世冰期海平面下降產(chǎn)生的地理隔離以及間冰期洋流對基因交流的阻礙兩方面相互作用的結(jié)果。為了揭示這兩個種分化的機(jī)制, 還需要進(jìn)一步研究。

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CRYPTIC SPECIES AND EVOLUTIONARY HISTORY OF BOLEOPHTHALMUS PECTINIROSTRIS COMPLEX ALONG THE NORTHWESTERN PACIFIC COAST

CHEN Hui1, POLGAR Gianluca2, YIN Wei1and FU Cui-Zhang1
(1. Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, Fudan University, Shanghai 200433, China; 2. Biology Programme, Universiti Brunei Darussalam, Bandar Seri Begawan, Gadong, Negara Brunei Darussalam)

The species range of Boleophthalmus pectinirostris sensu lato includes two disjunctive areas, i.e., East Asia and Strait of Malacca in Malaysia along the northwestern Pacific coast. However, the species status of Malaysian populations remains disputed. Mitochondrial ND5 gene (718 bp) and nuclear Rag1 gene (1395 bp) were used to reconstruct phylogenetic relationships among Boleophthalmus pectinirostris fishes by sampling 45 specimens from 11 locations in the northwestern Pacific. The results showed that Boleophthalmus pectinirostris fishes could be divided into two major monophyletic groups, i.e., East Asian lineage and Malaysian lineage, and which together formed the sister-group relationship. Species delineation using the analyses of GMYC and *Beast species tree supports that East Asian lineage and Malasian lineage of Boleophthalmus pectinirostris sensu lato should be placed into two different species. Molecular dating revealed that the divergence time between East Asian lineage and Malaysian lineage of Boleophthalmus pectinirostris sensu lato was 2.73 Ma. We concluded that Boleophthalmus pectinirostris sensu lato was a complex, including two species. The East Asian populations is Boleophthalmus pectinirostris sensu stricto, and the Malaysian populations is a cryptic species (Boleophthalmus sp.). Our findings suggested that species split between Boleophthalmus pectinirostris sensu stricto and Boleophthalmus sp. was attributed to geographical isolation during lowing sea levels of ice ages and the barrier of gene flow induced by ocean currents during interglacial period in the late Pliocene.

Gobiidae; Boleophthalmus; Phylogeny; Species delineation; Northwestern Pacific

10.7541/2014.10

Q111

A

1000-3207(2014)01-0075-12