基于环境DNA宏条形码技术的秦淮河生物多样性研究

秦淮河是南京的母亲河,其生物多样性受城市化进程影响面临严重威胁,而物种资源调研是生物多样性保护的基础。环境DNA宏条形码技术较形态学监测是一种简单高效、灵敏度高的新型监测技术。为探究秦淮河浮游生物、底栖动物及鱼类的生物多样性,于2019年7月,采用环境DNA宏条形码技术对其进行了探究,并分析了秦淮河上下游间的差异及环境因子对其群落结构的影响。结果表明:秦淮河共监测到浮游动物13属22种407个操作分类单元(Operational Taxonomic Units, OTUs),浮游植物85属60种4445个OTUs,底栖动物16属17种212个OTUs,鱼类53属44种1663个OTUs。其中浮游动物以游泳轮虫目(Ploima)和双甲目(Diplostraca)为主,共占浮游动物63.37%,浮游植物以隐藻门(Cryptomonas)和褐藻门(Ochrophyta)为主,共占浮游植物88.11%,底栖动物中节肢动物门(Arthropoda)占比最高,达91.67%,鱼类中鲤形目(Cypriniformes)占比最高,达69.99%。与秦淮河历史形态学监测数据相比,环境DNA宏条形码技术在...     

瓯江口夏、秋季浮游动物种类组成及其多样性

依据2007年6和9月瓯江12水域两个航次的浮游动物调查资料,研究瓯江口浮游动物种类组成和多样性特征,并探索水团等因素对浮游动物优势种及物种多样性分布的影响.结果表明:调查水域共鉴定出浮游动物76种(不含18种浮游幼体和仔鱼),隶属于7门8纲20目41科,其中桡足类是最主要的优势类群;瓯江口水域浮游动物主要由亚热带种构成;就盐度适应而言,浮游动物种类数以外海种居多,而近海种在浮游动物总丰度中占有明显优势;秋季浮游动物种类数显著高于夏季;秋季的Shannon-Wiener多样性指数(2.89)明显高于夏季(2.23),这与秋季物种数较高有关;夏季水团交汇处的大门岛邻近水域多样性指数最高,秋季该水域多样性指数呈现从河口到外海逐渐增加的趋势.     

哥斯达黎加外海夏季表层浮游动物种类组成及分布

根据2009年7—8月在东热带太平洋哥斯达黎加外海(4°30’—10°24’N、91°20’—100°00’W)进行茎柔鱼探捕调查期间采集的表层浮游动物资料,对其种类组成、生物量及其分布进行了分析。结果表明,调查海域,鉴定出腔肠动物8目10科17属23种;甲壳动物8目30科43属74种;毛颚动物1目1科1属4种;尾索动物3目3科5属5种;环节动物为1目3科5属6种;软体动物1目1科3属3种;其他浮游动物有头足类的幼体、鱼卵和仔稚鱼以及浮游幼虫。调查海域总生物量和丰度的平均值分别为(124.78±176.83)mg/m3和(848±1219)个/m3,生物量较大的类群主要是甲壳动物的桡足类和毛颚动物的箭虫类其中桡足类的丰度(727个/m3)为最高,其次为箭虫类(373个/m3);出现频率较高的为桡足类、箭虫类、长尾类、头足类和鱼类仔稚鱼。     

中山市淡水浮游动物区系调查

2005年6～7月和12月对中山市主要淡水水体的浮游动物区系进行了调查。结果表明,中山市主要淡水水体浮游动物由38目92科129属317种组成。区系特点以原生动物最丰富,有30目82科102属263种,占浮游动物物种总数的83%;其次为轮虫,有4目4科19属37种组成,占12%;枝角类和桡足类物种数最少。各样点浮游动物种类在11～168种之间,种类最少的出现在洪奇沥,最多的出现在逍遥谷。研究结果表明,中山市淡水浮游动物资源比较丰富。各区系浮游动物的分布与水体的营养状态密切相关,污染严重的水体,种类数较少,清洁水体,种类数较多。     

西太平洋浮游动物种类多样性

西太平洋区是全球海洋生物种源中心,许多类群的最高物种多样性都出现于该区域,因此,在该区开展种类多样性的研究不仅重要和必要,而且具有在跨国尺度上进行综合管理和相互合作的迫切性。本文在西太平洋的浮游动物样品鉴定分类、编目、文献资料整理和分析的基础上,记录和编入西太平洋10个浮游动物类群2,658种(含亚种),隶属于206科675属,其中水螅水母类99科251属697种,栉水母类12科22属59种,浮游软体动物14科35属86种,介形类8科89属416种,桡足类51科156属908种,糠虾类4科58属202种,磷虾类2科8属56种,十足类8科22属105种,毛颚类5科8属48种,被囊类5科26属81种。     

DNA条形码及其在海洋浮游动物生态学研究中的应用

浮游动物的准确鉴定是浮游动物生态学研究的基础.传统的基于形态特征的鉴定不仅费时费力,而且部分类群特别是浮游幼体由于形态差异细微,鉴定存在困难,导致物种多样性被低估.DNA条形码(DNA barcodes)技术为浮游动物物种鉴定提供了一个有力工具,已迅速应用于海洋浮游动物生态学研究.本文介绍了DNA条形码的基本概念、优势及局限性,总结了该技术(主要是基于线粒体细胞色素C氧化酶第一亚基(mtCOI)基因序列片段的DNA条形码)在海洋浮游动物物种快速鉴定、隐种发现、营养关系研究、生物入侵种监测、群落历史演变反演、种群遗传学以及生物地理学中的成功应用.随着DNA条形码数据库信息量覆盖率的不断提高和新一代测序技术的快速发展,DNA条形码将提供除了种类鉴定外更加丰富的信息,从而帮助人们更好地理解海洋浮游动物的多样性及其在生态系统中的功能,推动海洋浮游动物生态学的发展.     

DNA宏条形码技术在海洋浮游动物多样性和生态学研究中的应用

浮游动物是海洋生态系统的关键类群,其覆盖门类广泛,多样性高。传统形态鉴定技术需要检测人员具备专业的形态鉴定知识,且费时费力。宏条形码技术无需分离生物个体,而是提取拖网采集到的浮游动物混合样本的总DNA,或者水体中的环境DNA （eDNA）,依托高通量测序平台测序,能够实现对大规模样本快速、准确、经济的分析,在海洋浮游动物生态学研究中得到越来越广泛的应用。分析了DNA宏条形码技术常用的核糖体和线粒体分子标记,在浮游动物多样性和数量研究中的可靠性和不足,并给出在海洋浮游动物群落监测,食物关系分析及生物入侵早期预警等研究中的应用。未来,开发多基因片段组合条形码,发展完备的参考数据库及实现准确的量化研究是DNA宏条形码技术发展的重要方向。     

基于宏基因组的保安湖浮游动物多样性研究

研究于2019年春、夏、秋、冬四季对保安湖进行了水样采集, 基于宏基因组测序, 在优化物种鉴定和丰度计算方法的基础上, 考察了保安湖浮游动物的多样性、群落结构及其影响因素。共鉴定到浮游动物OTU 374种, 其中原生动物282个; 枝角类45个; 桡足类26个; 轮虫21个。从季节来看, 夏、秋季保安湖的浮游动物多样性高; 从湖区来看, 肖四海和主湖区浮游动物多样性高。季节因素对保安湖浮游动物群落结构的影响高于湖区影响。保安湖营养状态为中营养型, 水体温度、叶绿素a是影响保安湖浮游动物群落结构的主要环境因子, 不同类群与环境因子相关性不同, 总体可分为5类。其中原生动物优势类群为混合营养的纤毛虫和丝足虫, 同硝氮、化学需氧量、温度有明显的相关性, 而枝角类和桡足类同环境因子的关系较为相似, 与溶氧、叶绿素a、正磷酸盐存在明显相关。研究利用宏基因组方法对保安湖浮游动物多样性开展了研究, 为从浮游动物这一角度来理解保安湖这一江湖阻隔型湖泊的生物多样性的变化提供了支撑。     

虾稻综合种养系统浮游动物群落结构及其与环境因子的关系

为探究虾稻综合种养(integrated crayfish-rice co-culture,ICRC)系统浮游动物的群落结构特征及其主要影响因素,对潜江市典型ICRC稻田渔沟和田面水体的浮游动物进行了季度研究,并分析了浮游动物和浮游植物、水体理化因子之间的相关性。结果显示:(1)共采集浮游动物60种,分别包含原生动物、轮虫、枝角类和桡足类12、28、10、10种(属),其中,优势类群为剑水蚤目无节幼体、桡足幼体以及蚤状溞(Daphnia pulex);Shannon-Wiener指数、Margalef指数和Pielou指数的年均值分别为1. 48、3. 42和0. 55;(2)浮游动物密度和生物量的变化范围分别为7. 65～1309. 50 ind./L、0. 08～50. 04 mg/L,年均值分别为360. 0 ind./L、17. 45 mg/L,桡足类和枝角类在密度和生物量上均占有绝对优势;(3)浮游动物物种数、多样性指数(田面除外)、密度和生物量均表现出明显的季节差异,其中密度和生物量在春季显著最高,相关性分析表明浮游动物物种数和资源量主要受总磷、浮游植物密度与生物量的影响;(4)浮游动物的物种数、多样性、密度和生物量在渔沟和田面两种生境间无明显差异。本研究表明ICRC系统中有丰富的大型浮游动物资源未得到利用,同时鉴于浮游动物可作为小龙虾早期生活史阶段的食物来源,需进一步研究秋冬季浮游动物变化,以及如何培育和合理利用此阶段的浮游动物以促进幼虾生长、提高存活率。     

中国北方常见芫菁分子物种界定(鞘翅目: 芫菁科)

【目的】应用线粒体COI和核CAD基因片段探讨自动条形码间隔探索(automatic barcode gapdiscovery, ABGD)、广义混合Yule溯祖模型(generalized mixed Yule coalescent, GMYC)、贝叶斯泊松树进程(Bayesian Poisson tree processes, bPTP)和贝叶斯系统发育和系统地理分析(Bayesianphylogenetics and phylogeography, BPP) 4种分析方法在芫菁科(Meloidae)昆虫分子物种界定中的适用性。【方法】分别基于COI, CAD和COI+CAD串联序列数据集,应用ABGD, GMYC, bPTP和BPP 4种方法对中国北方芫菁科常见的6属(沟芫菁属Hycleus、斑芫菁属Mylabris、豆芫菁属Epicauta、绿芫菁属Lytta、星芫菁属Megatrachelus和短翅芫菁属Meloe)18个形态种进行分子物种界定,并与形态学鉴定结果进行比较。【结果】利用COI+CAD串联序列数据集所得物种界定结果与形态鉴定结果一致;COI数据集使用ABGD和GMYC方法的界定结果与形态鉴定结果一致,而bPTP划分的物种数较形态鉴定结果多;基于CAD序列在3种单基因物种界定方法的结果中,除GMYC与形态划分一致外,其余均显示部分结果与形态划分不同。【结论】在芫菁科分子物种界定中,多基因联合序列、多种界定方法分析所得结果优于单一基因片段和界定方法的分析结果。本研究的结果为芫菁科昆虫的分子物种界定和整合分类提供了数据支持和参考。     

A comparative analysis of metabarcoding and morphology‐based identification of benthic communities across different regional seas

In a world of declining biodiversity, monitoring is becoming crucial. Molecular methods, such as metabarcoding, have the potential to rapidly expand our knowledge of biodiversity, supporting assessment, management, and conservation. In the marine environment, where hard substrata are more difficult to access than soft bottoms for quantitative ecological studies, Artificial Substrate Units (ASUs) allow for standardized sampling. We deployed ASUs within five regional seas (Baltic Sea, Northeast Atlantic Ocean, Mediterranean Sea, Black Sea, and Red Sea) for 12–26 months to measure the diversity and community composition of macroinvertebrates. We identified invertebrates using a traditional approach based on morphological characters, and by metabarcoding of the mitochondrial cytochrome oxidase I (COI) gene. We compared community composition and diversity metrics obtained using the two methods. Diversity was significantly correlated between data types. Metabarcoding of ASUs allowed for robust comparisons of community composition and diversity, but not all groups were successfully sequenced. All locations were significantly different in taxonomic composition as measured with both kinds of data. We recovered previously known regional biogeographical patterns in both datasets (e.g., low species diversity in the Black and Baltic Seas, affinity between the Bay of Biscay and the Mediterranean). We conclude that the two approaches provide complementary information and that metabarcoding shows great promise for marine monitoring. However, until its pitfalls are addressed, the use of metabarcoding in monitoring of rocky benthic assemblages should be used in addition to classical approaches rather than instead of them.     

基于环境DNA宏条形码技术的辽东湾典型围海养殖池塘内水母多样性研究

研究使用环境DNA宏条形码技术(eDNA metabarcoding)检测辽东湾东北部河口区围海养殖池塘水母种类多样性,探索适用于水母种类物种鉴定和监测的新方法。利用环境DNA宏条形码技术,分别基于18S rDNA和COI宏条形码检测了辽东湾东北部河口区围海养殖池塘水母种类多样性,通过水样采集、过滤、eDNA提取、遗传标记扩增、测序与生物信息分析的环境DNA宏条形码标准化分析流程,从围海养殖池塘7个采样点中获得可检测的采样点数据。结果显示,基于18S rDNA宏条形码检测出8种水母种类,其中钵水母纲大型水母2种、水螅水母总纲小型水母6种;基于COI宏条形码技术共检测出19种水母种类,其中钵水母纲大型水母5种、水螅水母总纲小型水母14种;两种DNA条形码标记都显示养殖种类海蜇(Rhopilema esculentum)为优势种。研究结果表明,环境DNA宏条形码技术作为一种新兴的生物多样性监测手段可用于快速检测水母种类多样性,在水母类物种鉴定、监测及早期预警中有较大的应用潜能。     

Assessing morphological and DNA-based diet analysis techniques in a generalist predator, the arrow squid Nototodarus gouldi

Establishing the diets of marine generalist consumers is difficult, with most studies limited to the use of morphological methods for prey identification. Such analyses rely on the preservation of diagnostic hard parts, which can limit taxonomic resolution and introduce biases. DNA-based analyses provide a method to assess the diets of marine species, potentially overcoming many of the limitations introduced by other techniques. This study compared the effectiveness of morphological and DNA-based analysis for determining the diet of a free-ranging generalist predator, the arrow squid (Nototodarus gouldi). A combined approach was more effective than using either of the methods in isolation. Nineteen unique prey taxa were identified, of which six were found by both methods, 10 were only detected using DNA and three were only identified using morphological methods. Morphological techniques only found 50% of the total number of identifiable prey taxa, whereas DNA-based techniques found 84%. This study highlights the benefits of using a combination of techniques to detect and identify prey of generalist marine consumers.     

Metabarcoding Is Powerful yet Still Blind: A Comparative Analysis of Morphological and Molecular Surveys of Seagrass Communities

In the context of the sixth wave of extinction, reliable surveys of biodiversity are increasingly needed to infer the cause and consequences of species and community declines, identify early warning indicators of tipping points, and provide reliable impact assessments before engaging in activities with potential environmental hazards. DNA metabarcoding has emerged as having potential to provide speedy assessment of community structure from environmental samples. Here we tested the reliability of metabarcoding by comparing morphological and molecular inventories of invertebrate communities associated with seagrasses through estimates of alpha and beta diversity, as well as the identification of the most abundant taxa. Sediment samples were collected from six Zostera marina seagrass meadows across Brittany, France. Metabarcoding surveys were performed using both mitochondrial (Cytochrome Oxidase I) and nuclear (small subunit 18S ribosomal RNA) markers, and compared to morphological inventories compiled by a long-term benthic monitoring network. A sampling strategy was defined to enhance performance and accuracy of results by preventing the dominance of larger animals, boosting statistical support through replicates, and using two genes to compensate for taxonomic biases. Molecular barcodes proved powerful by revealing a remarkable level of diversity that vastly exceeded the morphological survey, while both surveys identified congruent differentiation of the meadows. However, despite the addition of individual barcodes of common species into taxonomic reference databases, the retrieval of only 36% of these species suggest that the remaining were either not present in the molecular samples or not detected by the molecular screening. This finding exemplifies the necessity of comprehensive and well-curated taxonomic reference libraries and multi-gene surveys. Overall, results offer methodological guidelines and support for metabarcoding as a powerful and repeatable method of characterizing communities, while also presenting suggestions for improvement, including implementation of pilot studies prior to performing full “blind” metabarcoding assessments to optimize sampling and amplification protocols.     

Coupling between taxonomic and functional diversity in protistan coastal communities

The study of protistan functional diversity is crucial to understand the dynamics of oceanic ecological processes. We combined the metabarcoding data of various coastal ecosystems and a newly developed trait-based approach to study the link between taxonomic and functional diversity across marine protistan communities of different size-classes. Environmental DNA was extracted and the V4 18S rDNA genomic region was amplified and sequenced. In parallel, we tried to annotate the operational taxonomic units (OTUs) from our metabarcoding dataset to 30 biological traits using published and accessible information on protists. We then developed a method to study trait correlations across protists (i.e. trade-offs) in order to build the best functional groups. Based on the annotated OTUs and our functional groups, we demonstrated that the functional diversity of marine protist communities varied in parallel with their taxonomic diversity. The coupling between functional and taxonomic diversity was conserved across different protist size classes. However, the smallest size-fraction was characterized by wider taxonomic and functional groups diversity, corroborating the idea that nanoplankton and picoplankton are part of a more stable ecological background on which larger protists and metazoans might develop.     

Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island,France)

Diatoms are excellent ecological indicators of water quality because they are broadly distributed, they show high species diversity and they respond rapidly to human pressures. In Europe, the Water Framework Directive (WFD) gives the legal basis for the use of this indicator for water quality assessment and its management. Several quality indices, like the Specific Polluosensitivity Index (SPI), were developed to assess the ecological quality status of rivers based on diatom communities. It is based on morphological identifications and count of diatom species present in natural biofilms using a microscope. This methodology requires high taxonomic skills and several hours of analysis per sample as 400 individuals must be identified to species level. Since several years, a molecular approach based on DNA metabarcoding combined to High-Throughput Sequencing (HTS) is developed to characterize species assemblages in environmental samples which is potentially faster and cheaper. The ability of this approach to provide reliable diatom inventories has been demonstrated and its application to water quality assessment is currently being improved. Despite optimization of the DNA metabarcoding process with diatoms, few studies had yet extended it at the scale of a freshwater monitoring network and evaluated the reliability of its quality assessment compared to the classical morphological approach.In the present study we applied DNA metabarcoding to the river monitoring network of the tropical Island Mayotte. This island is a French département since 2011 and the WFD has to be applied. This offered the opportunity to scale up the comparison of molecular and morphological approaches and their ability to produce comparable community inventories and water quality assessments. Benthic diatoms were sampled following WFD standards in 45 river sites in 2014 and 2015 (80 samples). All samples were submitted in parallel to the molecular and the morphological approaches. DNA metabarcoding was carried out using Genelute DNA extraction method, rbcL DNA barcode and PGM sequencing, while microscopic counts were carried out for the classical methodology. Diatom community structures in terms of molecular (OTUs) and of morphological (species) were significantly correlated. However, only 13% of the species was shared by both approaches, with qualitative and quantitative variation due to i) the incompleteness of the reference library (82% of morphological species are not represented in the database), ii) limits in taxonomic knowledge and iii) biases in the estimation of relative abundances linked to diatom cell biovolume. However, ecological quality status assessed with the molecular and morphological SPI values were congruent, and little affected by sequencing depth. DNA metabarcoding of diatom communities allowed a reliable estimation of the quality status for most of the rivers at the scale of the full biomonitoring network of Mayotte Island.     

DNA from soil mirrors plant taxonomic and growth form diversity

Ecosystems across the globe are threatened by climate change and human activities. New rapid survey approaches for monitoring biodiversity would greatly advance assessment and understanding of these threats. Taking advantage of next-generation DNA sequencing, we tested an approach we call metabarcoding: high-throughput and simultaneous taxa identification based on a very short (usually <100 base pairs) but informative DNA fragment. Short DNA fragments allow the use of degraded DNA from environmental samples. All analyses included amplification using plant-specific versatile primers, sequencing and estimation of taxonomic diversity. We tested in three steps whether degraded DNA from dead material in soil has the potential of efficiently assessing biodiversity in different biomes. First, soil DNA from eight boreal plant communities located in two different vegetation types (meadow and heath) was amplified. Plant diversity detected from boreal soil was highly consistent with plant taxonomic and growth form diversity estimated from conventional above-ground surveys. Second, we assessed DNA persistence using samples from formerly cultivated soils in temperate environments. We found that the number of crop DNA sequences retrieved strongly varied with years since last cultivation, and crop sequences were absent from nearby, uncultivated plots. Third, we assessed the universal applicability of DNA metabarcoding using soil samples from tropical environments: a large proportion of species and families from the study site were efficiently recovered. The results open unprecedented opportunities for large-scale DNA-based biodiversity studies across a range of taxonomic groups using standardized metabarcoding approaches.     

Metabarcoding improves detection of eukaryotes from early biofouling communities: implications for pest monitoring and pathway management

In this experimental study the patterns in early marine biofouling communities and possible implications for surveillance and environmental management were explored using metabarcoding, viz. 18S ribosomal RNA gene barcoding in combination with high-throughput sequencing. The community structure of eukaryotic assemblages and the patterns of initial succession were assessed from settlement plates deployed in a busy port for one, five and 15 days. The metabarcoding results were verified with traditional morphological identification of taxa from selected experimental plates. Metabarcoding analysis identified > 400 taxa at a comparatively low taxonomic level and morphological analysis resulted in the detection of 25 taxa at varying levels of resolution. Despite the differences in resolution, data from both methods were consistent at high taxonomic levels and similar patterns in community shifts were observed. A high percentage of sequences belonging to genera known to contain non-indigenous species (NIS) were detected after exposure for only one day.     

Environmental DNA metabarcoding studies are critically affected by substrate selection

Effective biomonitoring is critical for driving management outcomes that ensure long‐term sustainability of the marine environment. In recent years, environmental DNA (eDNA), coupled with metabarcoding methodologies, has emerged as a promising tool for generating biotic surveys of marine ecosystems, including those under anthropogenic pressure. However, more empirical data are needed on how to best implement eDNA field sampling approaches to maximize their utility for each specific application. The effect of the substrate chosen for eDNA sampling on the diversity of marine taxa detected by DNA metabarcoding has not yet been systematically analysed, despite aquatic systems being those most commonly targeted for eDNA studies. We investigated the effect of four commonly used eDNA substrates to explore taxonomic diversity: (a) surface water, (b) marine sediment, (c) settlement plates and (d) planktonic tows. With a focus on coastal ports, 332 eDNA samples from Australia (Indian and Southern oceans) and Kazakhstan (Caspian Sea) were collected and analysed by multi‐assay DNA metabarcoding. Across study locations, between 30% and 52% of eukaryotic families detected were unique to a particular substrate and <6% of families were found in all four substrates. Taxonomic composition varied significantly depending on the substrate sampled implying that the suitability (and bias) of an eDNA substrate will depend on the focal taxa. These findings demonstrate that single substrate eDNA metabarcoding likely underestimates the total eukaryotic diversity. Future eDNA experimental design should consider incorporating multiple substrates or select substrate(s) best suited to the specific detection of target taxa.     

Identification of larval fish in mangrove areas of Peninsular Malaysia using morphology and DNA barcoding methods

The identification of larval fish has been an important morphological issue in marine biology due to the dramatic transformations that most species undergo from early larval stages to adulthood. Insufficient morphological diagnostic characters in larval fishes made it easy to misidentify them and a difficult process to key to genus and species level. The experiment aims to find out, by applying DNA barcoding, how consistent the morphological identifications can be among larval fish. Larval fish were mainly collected using plankton nets around mangrove areas in Pendas (Johor), Setiu (Terengganu), Pekan (Pahang) and Matang (Perak) Malaysia between April 2015 and October 2015. A total of 354 samples were morphologically identified, mostly to the family level and a few to the genus level. Larval fish ranged from 1.5 mm to 31 mm of total length, with the most abundant individuals being <3 mm. Among them, a total of 177 individuals were selected for DNA barcoding analyses. Molecular works involved polymerase chain reaction (PCR) and sequencing of mitochondrial Cytochrome c Oxidase I (COI) gene fragment (655 base pairs) methods. DNA barcoding enabled all samples to be identified down to species level. The overall genetic identities ranged from 91% to 100%. Morphological identification classified the specimens into 19 families and 11 genera while DNA barcoding identified them into 19 families 33 genera and 40 species. A comparison between the two methods showed a mismatched identification of 42.6% where the accuracy percentage for morphological identification was moderate for the family level (67.8%) but was low for genus level identification (30%). The DNA barcoding method also managed to successfully identify 86.4% of the samples up to their species level where morphological method has failed to do so. The most misidentified families in the study were Blenniidae, Sparidae, Apogonidae Ambassidae and Monachantidae while almost all samples from the family Gobiidae and Engraulidae were correctly identified to family level because of their distinct morphology. In conclusion, taxonomic studies of larval fish should continue using combination of both morphology and DNA barcoding methods. Morphological identification should be more conservative i.e., when in doubt, it is better to key only to family and not to the genus and species level. DNA barcoding is a better method for deeper taxonomic levels identification with the existence of robust sequence reference libraries and should be able to validate the accuracy of traditional larval fish identification.