西北太平洋海岸带大弹涂鱼复合体的隐存种与进化历史

陈 卉Gianluca Polgar殷 维傅萃长

(1. 复旦大学生物多样性科学研究所, 生物多样性与生态工程教育部重点实验室, 上海 200433;

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

西北太平洋海岸带大弹涂鱼复合体的隐存种与进化历史

陈 卉1Gianluca Polgar2殷 维1傅萃长1

(1. 复旦大学生物多样性科学研究所, 生物多样性与生态工程教育部重点实验室, 上海 200433;

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

大弹涂鱼Boleophthalmus pectinirostris间断分布于西太平洋海岸带东亚与马来西亚马六甲海峡, 但马来西亚种群的分类地位尚存争议。研究使用线粒体ND5基因序列(718 bp)与核位点Rag1基因序列(1395 bp)对西北太平洋海岸带11个地点的45尾大弹涂鱼属鱼类进行系统发育关系重建, 结果表明大弹涂鱼包括东亚与马来西亚两个单系群, 两者形成姊妹群关系。GMYC分析、*BEAST物种树支持大弹涂鱼东亚谱系和马来西亚谱系是不同种。分子测定年龄分析表明大弹涂鱼东亚谱系与马来西亚谱系之间的分化时间为2.73百万年。因此, 西北太平洋海岸带大弹涂鱼是复合体, 包括两个物种: 东亚种群是大弹涂鱼 Boleophthalmus pectinirostris sensu stricto, 而马来西亚种群是隐存种Boleophthalmus sp.。大弹涂鱼与隐存种之间的物种分化可能是晚上新世冰期海平面下降产生的地理隔离以及间冰期洋流对基因交流的阻碍两方面相互作用的结果。

虾虎鱼科; 大弹涂鱼; 系统发育; 物种界定; 西北太平洋

大弹涂鱼属(Boleophthalmus Valenciénnes, 1837)鱼类是一群生活在太平洋和印度洋沿岸潮间带淤泥质潮滩上的小型鱼类, 隶属于鲈形目 Perciformes、虾虎鱼亚目Gobioidei、虾虎鱼科Gobiidae、背眼虾虎鱼亚科Oxudercinae[1]。该属包括5种鱼类: 北澳洲大弹涂鱼Boleophthalmus birdsong Murdy, 1989、薄氏大弹涂鱼B. boddarti(Pallas, 1770)、绿斑大弹涂鱼B. caeruleomaculatus McCulloch & Waite, 1918、杜氏大弹涂鱼B. dussumieri Valenciennes, 1837和大弹涂鱼B. pectinirostris(Linnaeus, 1758)。依据Fish Base[2]与the Catalog of Fishes[3]数据库的记录, 分布于西北太平洋的大弹涂鱼属鱼类包括两种: 薄氏大弹涂鱼与大弹涂鱼。其中, 薄氏大弹涂鱼主要分布于南中国海北部湾以南, 而大弹涂鱼则间断分布于东亚(南中国海北部湾以北)与马来西亚的马六甲海峡[1,4—9]。大弹涂鱼东亚种群的体长一般不超过135 mm[4], 而马来西亚种群的体长可达175 mm[9]。 Murdy[1]对大弹涂鱼属鱼类进行分类校订后认为大弹涂鱼马来西亚种群可能是一个隐存种(Cryptic species)。分子证据已在鱼类分类与系统学研究中广泛运用[10,11], 其中线粒体ND5与核位点Rag1基因是虾虎鱼科鱼类分类与系统学研究中最常用的分子标记之一[12,13]。因此, 本研究选择ND5与Rag1基因作为分子标记, 使用分子证据探讨大弹涂鱼马来西亚种群的物种状态。

西北太平洋的一个独特地形特点是具有南中国海、东中国海、黄海和日本海等一系列相互连接的边缘海[14]。上新世和更新世的冰期及间冰期旋回导致的海平面涨落对边缘海的面积和结构造成了剧烈影响[14]。基于分子证据的系统发育研究揭示在晚上新世和更新世冰期阶段海平面下降导致连接边缘海的海峡形成陆桥成为地理障碍, 导致近海鱼类谱系分化和物种形成[12,15]; 间冰期阶段海平面上升, 边缘海重新连接形成复杂的水文情况[16], 中国海沿岸流、黑潮(Kuroshio Current)、南中国海暖流亦能成为自然屏障阻碍种群间的基因流, 促进鱼类谱系分化与物种形成[17,18]。因此, 本研究假设冰期阶段海平面下降马六甲海峡成为陆桥以及间冰期阶段复杂的洋流系统可能在西北太平洋海岸带大弹涂鱼属鱼类的遗传分化过程中扮演了重要角色。本研究目的是通过沿西北太平洋海岸带进行广泛取样, 基于线粒体与核基因证据来揭示西北太平洋海岸带大弹涂鱼东亚种群与马来西亚种群的间断分布格局的进化历史。

1 材料与方法

1.1 取样和分子标记

西北太平洋地区大弹涂鱼属鱼类标本包括来自我国广西党江、广东湛江、福建泉州、浙江温州、上海崇明、韩国顺天(Suncheon)和日本六角川(Rokkaku River)7个地点的大弹涂鱼东亚种群19尾(图1), 来自马来西亚龟咯岛(Pulau Kukup, PK)和丹戎比艾(Tanjung Piai, TP)2个地点的大弹涂鱼马来西亚种群11尾, 来自马来西亚龟咯岛、丹戎比艾、凯莉岛(Carey Island)和双溪槟榔(Sungai Pinang)4个地点的薄氏大弹涂鱼15尾(图1)。外类群选择拉氏狼牙虾虎鱼Odontamblyopus lacepedii 2尾、里贝卡狼牙虾虎鱼 Odontamblyopus rebecca、弹涂鱼Periophthalmus modestus 和 大 鳍 弹 涂 鱼Periophthalmus magnuspinnatus各1尾(表1)。采集标本的肌肉用 95%乙醇固定保存。选择线粒体NADH 脱氢酶亚基5基因(ND5)与核位点重组激活基因1(Rag1)作为分子标记。

图1 西北太平洋海岸带大弹涂鱼属鱼类的采样地点Fig. 1 Sample sites of Boleophthalmus fishes along the northwestern Pacific coast

1.2 DNA提取、PCR扩增与产物测序

采用高盐法从鱼体肌肉组织中提取全基因组DNA。扩增ND5基因的引物为L12321(5′-GGTCTT AGGAACCAAAAACTCTTGGTGCAA-3′)与H13396 (5′-CCTATTTTTCGGATGTCTTG-3′)[19]; PCR反应条件为: 94℃预变性5min; 94℃变性35s, 55℃退火35s, 72℃延伸40s; 循环35次; 72℃终末延伸8min。扩增Rag1基因片段的方案为巢式PCR。第一轮使用的引物为 RAG1F1(5′-CTGAGCTGCAGTCAG TACCATAAGATGT-3′)和RAG1R1(5′-CTGAGTCC TTGTGAGCTTCCATRAAYTT-3′)[20], 第二轮使用本研究设计的引物 GOBRAG1F1(5′-GCCAGATCTT CCAGCCTCT-3′)和XRAG1R(5′-TACTTGGADGTG TAGAGCC-3′); PCR反应条件为: 94℃预变性5min; 94℃变性35s, 55℃退火40s, 72℃延伸40s; 循环35次; 72℃终末延伸8min。PCR产物经2.0%琼脂糖电泳凝胶纯化, 使用第二轮扩增引物在 ABI 3730 DNA测序仪中进行测序。

表1 种名、采样地点、个体编号、单倍型及GenBank 登录号Tab. 1 Species, sampling localities, codes, haplotypes and GenBank accession numbers

续表

1.3 单倍型网络图与系统发育关系重建

使用CLUSTAL X version 1.83 软件[21], 采用默认参数对 ND5和 Rag1基因序列进行对位。使用DnaSP version 4.20软件选择ND5基因的单倍型[22]。使用Phase version 2.1软件[23,24], 设置后验概率阈值为 0.6, 选择 Rag1基因的单倍型。一些个体的Rag1基因具有两个单倍型(表 1)。使用 Network version 4.6软件[25]构建Median-Joining单倍型网络图, 并采用最大简约(Maximum parsimony)过程[26]确定单倍型之间的连接。

使用 MrBayes version 3.2软件[27]与 RAxML version 7.2.6 软件[28], 基于密码子分区构建贝叶斯(Bayesian)和最大似然(Maximum likelihood)树。使用jModeltest version 0.1.1软件[29]选择各分区的最适碱基替换模型。贝叶斯分析基于马可夫蒙特卡罗(Markov chain Monte Carlo)进行1.2×107代运算, 每1000代对系统树进行1次抽样。抛弃前3000棵抽样树, 构建 50%的多数原则一致树, 并计算每个节点的后验概率值(Bayesian Posterior Probability, BPP)。最大似然分析使用快速登山算法(Rapid-hillclimbing algorithm), 在 GTRGAMMA 模型下进行100次重复找出分值最高的最大似然树(Best-scoring ML tree)。再进行1000次自展(Bootstrap)分析, 从而估计节点的自展支持度(Bootstrap support, BS)。

1.4 物种界定与基因流

基于ND5 + Rag1联合基因序列, 使用General Mixed Yule-Coalescent(GMYC)方法确定进化显著性单元(Evolutionary Significant Units, ESU)。GMYC方法结合种间分化的Yule过程和种内分化的溯祖过程, 通过确定两个过程之间的转换点, 为物种界定提供依据[30]。首先, 使用BEAST version 1.7.2软件[31]构建超度量树(Ultrametric tree), 种群动态模型设置为稳定大小模型(Constant-size model), 突变率设置为平均值为 1的对数正态分布(Lognormal distribution)。运行 4个重复, 每个重复运行 5×107代, 每1000代取样一次, burnin为1/10。接着, 4个重复的结果用 BEAST软件包中的 LogCombiner version 1.7.2合并, 利用Tracer verson 1.5软件[32]查看有效取样大小(Effective Sampling Size, ESS)值(ESS大于200), 在BEAST软件包中的 TreeAnnotator version 1.7.2中生成最大谱系置信树(Maximum Clade Credibility Tree)。最后, 在R环境[33]中使用SPLITS软件包[34]进行单阈值(Single-threshold)的 GMYC方法分析。

使用 IMa2软件[35,36]在隔离迁移(Isolation with migration, IM)模型下估算物种间的基因流。在Hasegawa-Kishino-Yano(HKY)进化模型下运行MCMC链5×107代, 舍弃前5×106代之后保证ESS值大于200, 参数间的自相关(Parameter autocorrelations)小于 0.05。运算过程中未指定具体的突变率 u时, IMa2可估计种群突变率(Population mutation rates, θ)和每突变迁移率(Migration rates per mutation, M)。而有效种群大小(Effective population size)N = θ/4u, 每世代迁移率(Migration rates per generation)m = Mu, 故而每世代基因迁移有效数目(Effective numbers of gene migrants per generation) 2Nm = 2×θ/4u×Mu = θM/2。

图2 基于ND5基因(a)与Rag1基因(b)构建的贝叶斯50%多数原则一致树Fig. 2 The Bayesian 50% major rule consensus tree based on ND5 (a) gene and Rag1 (b) gene

1.5 遗传距离、物种树与分化时间

使用MEGA version 5.05 软件[37]计算物种间的成对 Kimura双参数(K2P)平均遗传距离。使用*BEAST version 1.7.2软件[31,38]构建物种树(Species tree)。分子钟采用对数正态分布的松散钟, 先验树(Tree prior)设置为Yule过程。共运行了4个重复, 每个重复运行5×107代, 每1000代取样一次, burnin为1/10。使用LogCombiner软件合并4重复的结果, 利用Tracer软件检验其ESS值。在TreeAnnotator软件中生成最大谱系置信树。由于缺乏虾虎鱼科鱼类化石, 本研究使用 Mukai, et al.[39]估算的吻虾虎鱼属鱼类ND5基因的突变率, 每百万年每谱系每位点(1.95 ± 0.17)%估算分化时间。

图3 基于ND5和Rag1基因联合数据构建的贝叶斯一致树Fig. 3 The Bayesian consensus tree based on combined data of ND5 and Rag1 genes

2 结果

2.1 序列特征

ND5基因部分序列长度718 bp, 可变位点117个、简约信息位点109个, Rag1基因部分序列长度1395 bp, 可变位点51个、简约信息位点40个。取样的45尾大弹涂鱼属鱼类包括23个ND5基因单倍型和31个Rag1基因单倍型。GenBank登录号信息见表1。

2.2 系统发育关系与单倍型网络图

图4 西北太平洋大弹涂鱼属鱼类单倍型的Median-Joining网络图Fig. 4 Haplotype Median-Joining network of Boleophthalmus fishes in the northwestern Pacific

ND5、Rag1单基因贝叶斯分析(图 2)以及联合基因的贝叶斯和最大似然法分析(图 3)得到了一致的拓扑结构。这些分析显示大弹涂鱼与薄氏大弹涂鱼是单系群(后验概率 BPP = 84%—100%, 自展支持度BS = 100%); 大弹涂鱼东亚与马来西亚种群分别是单系群并形成姊妹群关系(BPP = 97%—100%;自展支持度 BS =100%), 以下称为东亚谱系与马来西亚谱系(图 2、3)。单倍型网络图进一步显示大弹涂鱼东亚谱系、马来西亚谱系与薄氏大弹涂鱼之间具有明显的遗传分化(图4)。ND5基因单倍型网络图显示大弹涂鱼东亚谱系与马来西亚谱系之间具有64步突变, 两谱系与薄氏大弹涂鱼之间具有73步突变,而谱系内相互连接单倍型之间不超过 5步突变(图4a)。Rag1基因单倍型网络图显示大弹涂鱼东亚谱系与马来西亚谱系之间具有16步突变, 两谱系与薄氏大弹涂鱼之间具有24步突变, 而谱系内相互连接单倍型之间不超过2步突变(图4b)。

图5 基于ND5和Rag1基因联合数据在BEAST中构建的超度量树(a)、时间和谱系间的关系(b)以及似然值和时间的变化曲线(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、物种树与基因流

基于ND5 + Rag1基因联合数据的GMYC分析(图 5)显示西北太平洋大弹涂属鱼类包括三个进化显著性单元, 分别对应于大弹涂鱼东亚谱系、马来西亚谱系与薄氏大弹涂鱼。种间或谱系间属于种间分化之Yule过程, 种内或谱系内属于种内分化的溯祖过程, 大弹涂鱼东亚谱系、马来西亚谱系与薄氏大弹涂鱼应界定为三个物种(图5)。*BEAST物种树(图6)进一步支持基于GMYC分析的物种划分并具有强的统计支持(BPP = 100%)。隔离迁移模型估计亦显示大弹涂鱼东亚谱系、马来西亚谱系与薄氏大弹涂鱼之间基因流非常小, 每世代基因迁移有效数目接近于零(图7)。

2.4 遗传距离和分化时间

基于ND5基因序列计算的大弹涂鱼东亚谱系、马来西亚谱系与薄氏大弹涂鱼种间或谱系间 K2P遗传距离为10.41%—11.87%, 种内或谱系内K2P遗传距离为0.32%—0.57%(表2)。基于Rag1基因序列计算的大弹涂鱼东亚谱系、马来西亚谱系与薄氏大弹涂鱼种间或谱系间K2P遗传距离为1.50%— 1.80%, 种内或谱系内K2P遗传距离为0.18%— 0.30%。分子测定年龄结果表明大弹涂鱼东亚谱系与马来西亚谱系之间的分化时间为 2.73百万年, 两谱系与薄氏大弹涂鱼之间的分化时间为3.865百万年(图6)。

图6 基于ND5和Rag1基因联合数据的*BEAST物种树与时间树Fig. 6 *BEAST species tree and time tree based on combined data of ND5 and Rag1 genes

图7 基于IM模型估算的大弹涂鱼东亚谱系、马来西亚谱系和薄氏大弹涂鱼之间的基因流Fig. 7 Gene flows among East Asia lineage and Malaysia lineage of Boleophthalmus pectinirostris and B. boddarti estimated by the IM model

3 讨论

表2 大弹涂鱼属鱼类种内和种间的Kimura 双参数(K2P)遗传距离Tab. 2 Intra-specific and inter-specific Kimura 2-parameter genetic distance (K2P) of Boleophthalmus fishes

3.1 物种界定与隐存种

近年来, 分子证据揭示虾虎鱼科鱼类中发现隐存种的现象十分普遍[12,39—43]。本研究基于线粒体与核基因分子标记的系统发育重建显示大弹涂鱼东亚种群和马来西亚种群均是单系群, 并形成姊妹群关系。物种的谱系概念认为物种可看作是系统发育分析中一个单系群所代表的一组群体[44,45]。根据这个标准, 大弹涂鱼东亚谱系和马来西亚谱系能被界定为2个种。GMYC分析、*BEAST物种树重建以及基因流估算亦表明大弹涂鱼东亚谱系和马来西亚谱系是不同种。从遗传距离的角度看, 线粒体基因的种间分化水平一般比种内分化水平大10倍[46,47]。大弹涂鱼东亚谱系和马来西亚谱系ND5基因谱系间分化是谱系内分化的18—19倍, 因而也支持两物种的界定。基于大弹涂鱼模式产地在我国[1,4], 因此,可推断大弹涂鱼东亚谱系是大弹涂鱼(Boleophthalmus pectinirostris sensu stricto), 大弹涂鱼马来西亚谱系是隐存种(Boleophthalmus sp.)。这个发现扩展了对西太平洋大弹涂鱼属鱼类的认识, 并把大弹涂鱼的分布范围限定在东亚(南中国海北部湾以北), 而隐存种(Boleophthalmus sp.)目前局限于马六甲海峡。

3.2 进化历史

晚上新世和更新世最大冰期的海平面比现今海平面低120—140 m[48], 当时黄、渤海整个区域及南中国和海东中国海的部分区域都露出了水面[14]。先前的研究表明晚上新世海平面最低时期台湾海峡、对马海峡露出水面造成的东中国海与南中国海之间、东中国海与日本海之间的隔离导致了西北太平洋近岸狼牙虾虎鱼属(Odontamblyopus)鱼类与鲻属(Mugil)鱼类的物种形成[12,18]。本研究发现大弹涂鱼(东亚谱系)和隐存种(马来西亚谱系)的分化时间估计为2.73百万年。因此, 晚上新世海平面最低时期马六甲海峡露出水面造成的南中国海与印度洋之间的隔离可能导致大弹涂鱼和隐存种分化为姊妹群。

另一方面, 南中国海及其邻近海域复杂的水文特征对物种的扩散和分布也有重大的影响[49,50]。大弹涂鱼生活史中适合进行长距离迁徙的只有其幼鱼的浮游阶段, 该阶段持续约35d[51]。大弹涂鱼在每年的 4—9月间进行繁殖, 而该时期南中国海暖流沿越南中部–海南岛–广东这一路线向西北方向流动, 同时在南中国海暖流的南方, 南中国海环流则在夏季季风的影响下向东南方向流动[52]。这两个方向相反的洋流只可能将大弹涂鱼与隐存种的浮游幼体向相反的方向输送, 使它们之间的基因交流受到强烈的隔离, 从而进一步促进物种形成。最近的一些研究亦推断间冰期洋流作用引起的生物地理障碍是导致西北太平洋海洋生物物种形成的原因之一[18, 53—57]。

综上所述, 西北太平洋海岸带大弹涂鱼是复合体, 包括两个物种: 东亚种群是大弹涂鱼(Boleophthalmus pectinirostris sensu stricto), 而马来西亚种群是隐存种(Boleophthalmus sp.)。大弹涂鱼与隐存种之间的物种分化可能是晚上新世冰期海平面下降产生的地理隔离以及间冰期洋流对基因交流的阻碍两方面相互作用的结果。为了揭示这两个种分化的机制, 还需要进一步研究。

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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