摘要
如何准确地界定种和鉴定种对生物学家们来讲仍然是个难题。尽管已经提出许多界定种的方法,尤其是基于DNA的方法,但在实际应用中仍存在很大困难。近年来提出的DNA条形码技术由于能快速鉴别大量样品而迅速成为生物多样性研究的热点,但是其作为种界定工具的有效性却备受质疑。相反,基于多种不同来源标记的整合性种界定方法正逐渐成为种界定的有力工具。海洋双壳贝类虽然是海洋中一个多样性极丰富的门类,但由于多数种类研究不足以及研究手段的限制,其多样性很可能被低估。本论文中,我们验证了DNA条形码技术在两种双壳贝类(贻贝科和牡蛎科)种类鉴定中的应用,以及整合性分类方法在海洋隐存种(栉江珧)发现中的应用,为研究海洋物种分类、多样性保护和演化等问题提供借鉴。
1.基于线粒体COI的贻贝科DNA条形码的研究
本研究首次验证了DNA条形码技术在贻贝科中的应用性。通过线粒体COI(mtCOI)和16S rDNA片段对贻贝科11种52个样品进行DNA条形码分析。通过构建mtCOI和16S rDNA K2P邻接树,发现基本上所有种均可形成各自独立的单系。种内和种间K2P(Kimura’s2-parameter)遗传距离计算结果显示,除个别种和个体外,其种间距离一般高达20%以上,远远大于其种内遗传差异。同时在紫贻贝、凸壳肌蛤和曲线索贻贝中检测到线粒体异质性现象。结果说明,基于mtCOI的DNA条形码技术在贻贝科物种的鉴定中具有可行性;然而由于线粒体双单亲遗传模式和线粒体异质性现象的存在,应用DNA条形码进行贻贝科种类鉴定时要分外谨慎。
2.基于系统发生和距离法的牡蛎科DNA条形码研究
牡蛎科牡蛎是一类形态极多变而且分布较广泛的生物类群,其分类和发生关系长期以来一直困扰着分类学家和系统发生学家。本研究首次利用公共数据库的DNA序列(mtCOI和16S rDNA),通过基于距离法的DNA条形码技术和系统发生方法,对牡蛎科种类进行了鉴定分析。首先利用系统发生分析重构牡蛎科两种线粒体基因的系统发生关系,其次计算各种类种内和种间K2P遗传分化距离。通过系统发生关系的重建,发现所有种类(包括近缘种在内)在两种基因发生树上均可以形成分化明显的支系。16SrDNA种内变异与种间分化有所重叠。但是mtCOI基因的种内平均种内距离仅为0-1.4%(最大2.2%),平均种间距离则为2.6-32.2%(最小为2.2%),表明虽然距离差距较小,但是条形码间隙(barcoding gap)仍然是存在的。分析结果证实了DNA条形码和系统发生方法在牡蛎种类鉴定中的有效性。
3.基于多重标记分析的栉江珧隐存多样性和杂交研究
越来越多的研究通过结合多种数据类型(例如,mtDNA、核基因和形态)的分析方法在海洋中发现了隐存种的存在。对这些隐存种的阐释增加了我们对海洋物种多样性和演化历史的认识。栉江珧(Atrina Pectinata)具有多变的外部形态,因此被认为可能存在隐存种。本研究利用mtCOI、核ITS(nrITS)基因以及形态特征标记对采自中国沿海的16个及日本沿海的1个栉江珧群体进行分析,探讨隐存种的存在以及隐存种之间是否曾经发生过杂交。DNA条形码基因mtCOI系统发生分析结果发现了6个分化明显的线粒体支系,支系内变异仅为0.4-0.8%,而支系间分化则达4.3-22.0%,说明可能为6个隐存种。通过nrITS系统发生关系重建发现了5个分化显著的支系,支系间遗传距离为3.7-30.3%。这5个核基因演化支系分别与线粒体支系1-4和(5+6)基本相对应,证明了5个独特的演化支系的存在。同时发现单个个体中存在分化明显的多个nrITS序列,清楚地表明这些演化支系间在不远的过去曾经发生过杂交,这也是造成核基因和线粒体基因分析结果不完全一致的一个原因。形态上发现5种形态类型,虽然中间形态的存在模糊了形态类型间的清晰界限,但是这5个形态类型基本上与5个演化支系一一相对应。本研究结果证明了应用多种标记综合分析方法在隐存多样性及演化历史研究中的重要性。
Accurate species delimitation and identification is a challenging problem for biologists. Adozen operational methods, mostly based on DNA sequences, have been proposed, but it isstill problematic when delimiting species. DNA barcoding has become a well-funded, globalenterprise since its proposition as a rapid and practical molecular method to identify anddiscovery species, although the latter utility has been repeatedly criticized. On the contrary, anintegrative approach using multiple lines of evidence to delimit species is increasinglypopularized. As a big, diverse group in marine realm, bivalve biodiversity has probably beenunderestimated because of a lack of scrutiny of most taxa as well as the absence of powerfultools. In the present disertation, we assessed the utility of DNA barcoding techniques in theidentification of specimens from Mytilidae and Ostreidae, and we also explored the crypticdiversity in the pen shell Atrina pectinata in combination of different sources of data tomethodologically aid studies of taxonomy, biodiversity conservation and evolution of marinetaxa.
1. A test of the utility of DNA barcoding in the bivalve family Mytilidae
This study firstly tested whether the standard DNA barcoding method is suitable for speciesidentification of the bivalve family Mytilidae. Here,52samples belonging to eleven speciesof Mytilidae were analyzed using mitochondrial COI (mtCOI) gene as well as mitochondrial16S rDNA. Neighbor-joining trees of mtCOI and16S rDNA sequences using Kimura’s2-parameter (K2P) substitution model showed that most of the species were reciprocallymonophyletic with no exceptions. Pairwise K2P genetic distances among all species andwithin species were calculated in MEGA. And the results indicated that the genetic distancesbetween species showed up to>20%, much greater than distances within species when exceptional species and individuals were excluded. Mitochondrial DNA heteroplasmy wasdetected in Mytilus galloprovincialis, Musculus senhousia and Hormomya mutalilis. Ourresults suggested that DNA barcoding is a promising tool for species identification ofMytilidae, but the interpretation should be cautious in light of the doubly uniparentalinheritance of mtDNA (DUI) and mtDNA heteroplasmy.
2. Identifying the true oysters (Ostreidae) with mitochondrial phylogeny anddistance-based DNA barcoding
Oysters (family Ostreidae), with high levels of phenotypic plasticity and wide geographicdistribution, are a challenging group for taxonomists and phylogenetics. Based on theGenBank database, the present study used two mitochondrial fragments, mtCOI and16SrDNA, to assess whether oyster species could be identified by phylogeny and distance-basedDNA barcoding techniques. Relationships among species were estimated by the phylogeneticanalyses of both genes, and then pairwise K2P inter-and intraspecific genetic divergenceswere assessed. Species forming well differentiated clades in the molecular phylogenies wereidentical for both genes even when the closely related species were included. Intraspecificvariability of16S rDNA overlapped with interspecific divergence. However, average intra-and interspecific genetic divergences for mtCOI were0–1.4%(maximum2.2%) and2.6–32.2%(minimum2.2%), respectively, indicating the existence of a barcoding gap. Theseresults confirm the efficacy of species identification in oysters via DNA barcodes andphylogenetic analysis.
3. Cryptic diversity and hybridization revealed by multiple sources of data in Atrinapectinata
Cryptic species have been increasingly revealed in the marine realm through an analyticalapproach incorporating multiple lines of evidence (e.g., mtDNA, nuclear genes andmorphology). Illustrations of cryptic taxa improve our understanding of species diversity andevolutionary histories within marine animals. The pen shell Atrina pectinata is known toexhibit extensive morphological variations that may harbour cryptic diversity. In this study,we investigated16A. pectinata populations along the coast of China and one from Japan to explore possible cryptic diversity and hybridization using a combination of mitochondrial(mtCOI) and nuclear (ribosomal internal transcribed spacer, nrITS) genes as well asmorphology. Phylogenetic analyses of mtCOI ‘DNA barcoding gene’ sequences resolved sixdivergent lineages with intralineage divergences between0.4%and0.8%. Interlineagesequence differences ranged from4.3%to22.0%, suggesting that six candidate crypticspecies are present. The nrITS gene revealed five deep lineages with Kimura2-parameterdistances of3.7–30.3%. The five nuclear lineages generally corresponded to mtCOI lineages1–4and (5+6), suggestive of five distinct evolutionary lineages. Multiple nrITS sequences ofsignificant variance were found within an individual, clearly implying recent hybridizationevents between among the evolutionary lineages, which contributed to cytonucleardiscordance. Morphologically, five morphotypes matched the five genetic lineages, althoughthe intermediates may well blur the boundaries of different morphotypes. This studydemonstrates the importance of combining multiple lines of evidence to explore speciescryptic diversity and past evolutionary histories.
引文
[1]‘Species are groups of actually or potentially interbreeding natural populations, which arereproductively isolated from other such groups.’(Mayr1942)
[2]‘…a lineage of an ancestor-descendent populations which maintains its identity from other suchlineages and which has its own evolutionary tendencies and historical fate.’(Wiley1981)
[3]‘A phylogenetic species is an irreducible (basal) cluster of organisms, diagnosably distinct from othersuch clusters, and with which there is a parental pattern of ancestry and descent.’(Cracraft1989)
[4]‘… a geographically constrained groups of individuals with some unique apomorphous character, is theunit of evolutionary significance.’(Rosen1978)
[5]‘…equates species with separately evolving metapopulation lineages…’(de Queiro2007)
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