黄海绿潮藻分子鉴定与类群演替研究
|
摘要
2007-2011年，我国黄海海域连续五年暴发绿潮，特别是2008年青岛海域暴发了世界上最大规模的绿潮，甚至危及到奥帆赛的顺利举行，从而引起了有关政府机构和相关学者的高度重视和关注。形成绿潮的藻类主要为绿藻门石莼属（Ulva）海藻。绿潮藻的分类鉴定、遗传溯源，绿潮的暴发机制、预测预警、防控治理和综合利用等问题都是研究的重点和热点。本论文首先对黄海绿潮藻主要种类进行形态特征和分子检测初步研究，并对江苏沿海岸基绿潮藻种类做了调查和研究，接着于2009-2011年连续三年对我国南黄海海域早期漂浮绿潮藻进行种类构成与演替分析，同时对2008-2011年连续四年暴发的黄海绿潮优势种浒苔（Ulva prolifera）进行种群演替分析。最后利用GenBank中已有的石莼属种类DNA序列和本实验室采集的全国沿海石莼属种类探讨了ITS和rbcL基因序列作为石莼属绿潮藻基因条形码的可行性。主要研究结果如下：
对分子鉴定后进行纯种培养的4种黄海绿潮藻进行了形态学特征研究。结果表明，U. prolifera、Ulva compressa和Ulva linza的外部形态特征和解剖学特征均与《中国经济海藻志》（曾呈奎，1962）一致，前两种均具有管状结构，U. linza均具有缘管结构。Ulva sp.外观形态学特征和解剖学特征与U.linza无显著差异。在室内培养条件，前面3种能根据形态学分类特征区分开来，而无法鉴定出U. linza和Ulva sp.，因此仍需借助分子手段。
从不同野外生境条件下的绿潮藻样品中，挑选了15个形态特征非常相似的样品进行了分子鉴定。形态鉴定结果表明，所有样品均具有缘管结构，通过ITS全长序列可将15个样品划分为2类，其中12个样品属于LPP类群，3个样品属于Ulva sp.类群。5S rDNA间隔区序列进一步将LPP类群的12个样品为U.prolifera和U.linza两大类群。
2009年和2010年对江苏沿海岸基采集的绿潮藻进行了种类构成和分子系统分析（其中2009年35个样品、2010年36个样品）。2年的ITS序列和5S rDNA间隔区序列分析结果相似，江苏沿海岸基绿潮藻种类主要为U.linza、U.prolifera、Ulvasp.、U.compressa和Blidingia sp.5个类群。其中Ulva sp.、U.compressa、Blidingiasp.的ITS序列相应地与海区漂浮的绿潮藻Ulva sp.、U.compressa和Blidingia sp.的ITS序列完全一样。从地区来看，如东、大丰、射阳沿海岸基几乎都分布有海区早期漂浮绿潮藻所有种类；从生境来看，紫菜筏架、沿岸堤坝分布有海区早期漂浮绿潮藻的所有种类，而盐度较低的养殖池塘、入海河道主要为U.prolifera。ITS序列结合5S rDNA间隔区序列分析发现采自养殖池塘、入海河道的U.prolifera类群的ITS序列（同HM031181）与2008年黄海绿潮藻优势种（RD802）相差2bp，5S rDNA间隔区序列具有与RD802相同的三种亚型，其中Ⅱ型占绝对优势，未见Ⅰ型；漂浮绿潮藻U.prolifera类群（ITS序列同2008年漂浮优势种）5S rDNA间隔区序列同样存在这三种亚型，但2008年漂浮优势种的5S rDNA间隔区序列还具有其独特的Ⅰ型。2009年的调查结果表明，连云港沿海岸基主要为U.linza，且其ITS序列和5S rDNA间隔区序列与如东、大丰、射阳的U.linza及海区漂浮的U.linza序列差异较大，所以漂浮绿潮藻来源于连云港沿海岸基的可能较小。2010年调查中，作者增加了养殖池塘和入海河道样本量，在养殖池塘样本中作者检测到了一些ITS同RD802、5S rDNA间隔区序列属于Ⅱ型的U.prolifera类群，此类群藻体可能是ITS同RD802、5S rDNA间隔区序列属于Ⅰ型的U.prolifera类群（RD802）随海水进入养殖池塘后与养殖池塘的主要优势U.prolifera类群（ITS同HM031181、5S rDNA间隔区序列属于Ⅱ型）杂交产生的后代。
接着作者对2009-2011年南黄海早期漂浮绿潮藻进行分子系统分析和类群演替研究。结果表明，3年的早期漂浮绿潮藻种类组成一致，均为Ulva sp.、U.compressa、U.linza、U.prolifera和Blidingia sp.。从连续3年的监测结果来看，每年均是4月中下旬在如东海区首先出现大规模漂浮绿潮藻，分子研究结果表明，最先出现的漂浮绿潮藻并非2008年黄海漂浮绿潮藻优势种U.prolifera，而是Ulvasp.和U.compressa，每年的5月中旬开始出现漂浮绿潮藻优势种U.prolifera，随后漂浮绿潮藻优势种U.prolifera生物量迅速增加，至6月初漂浮绿潮藻优势种U.prolifera已经占到了90%以上，随后藻团继续向北漂移。但在大丰海区、射阳海区看不到这种类群演替现象。另外，2009年早期漂浮绿潮藻ITS序列同RD802的样品，5S rDNA间隔区序列只发现一种（Ⅰ型）；2010年早期漂浮绿潮藻ITS序列同RD802的样品存在四种亚型：Ⅰ型（同2008年黄海漂浮优势种）、Ⅱ型（同养殖池塘优势种）、Ⅲ型、Ⅳ型，Ⅲ型、Ⅳ型样品极少仅在大丰早期漂浮样品中出现，且采集回来的样品中混有空心莲子草，说明Ⅲ型、Ⅳ型样品可能是通过入海河道漂过来的。2011年早期漂浮绿潮藻ITS序列同RD802的样品5S rDNA间隔区序列同时存在Ⅰ型和Ⅱ型两种亚型，且Ⅱ型较前一年出现频率增大。为了弄清黄海绿潮大规模暴发时，漂浮优势种ITS序列同RD802，其5S rDNA间隔区序列是否也存在Ⅰ型和Ⅱ型两种亚型以及这两种亚型是如何变化的，作者对采自2008-2011年黄海海域暴发绿潮时的藻样进行ITS序列和5S rDNA间隔区序列分析，结果表明，这些样品ITS序列全部相同且同RD802，2008年样品的5S rDNA间隔区序列全部Ⅰ型，2009年以后同时存在Ⅰ型和Ⅱ型两种亚型，且5S rDNA间隔区序列Ⅱ型的比例由2009年的1:10.25增加到2010年的1:2.33，至2011年已经超过Ⅰ型成为优势亚型，比例已经高达4.625:1。
最后作者对GenBank中下载的石莼属9个物种和石莼科盘苔的rbcL和ITS序列以及本实验全国沿海绿潮藻预调查采集的我国沿海地区常见绿藻种类进行了绿潮藻DNA条形码技术的初步研究。根据最大种内遗传距离小于最小种间遗传距离的严格DNA条形码判断标准，rbcL和ITS序列虽都不适合单独作为绿潮藻的DNA条形码，但分别能区分10个物种中的7个物种和8个物种。rbcL的最小种间遗传距离变异程度低于ITS的，说明前者比后者更为保守，更适合更高级分类阶元的分类。ITS在石莼属绿潮藻中能够较好地区分其他种类，却不能很好区分LPP复合体，而本论文的前两章研究结果证实5S rDNA间隔区序列能够有效地区分LPP复合体。故作者提出ITS序列和5S rDNA间隔区序列组合使用可以作为绿潮藻的DNA条形码。
A serious outbreak of green tide continuously struck the Yellow Sea of China from2007to2011. More seriously, the largest outbreak of green tide in the sea area ofQingdao in2008hampered the regatta sailing during the Beijing Olympic Games. Thusgovernment and researchers had paid much attention to green tide. Ulva is the maincomponents of green tide algae. The research hotpots include taxonomic identificationand genetic origin of the green tide algae, and outbreak mechanism, forecast and earlywarming, prevention and control management and comprehensive utilization of greentide. This paper dealt with the molecular identification and population succession ofgreen tide algae in the Yellow Sea. In the second chapter, the morphological featuresand molecular identification of the main pure culture green tide algae were preliminarilyinvestigated. The molecular identification and species composition of shore-based greentide algae were studied in the next chapter. The fourth chapter focused on the speciescomposition and their succession of early free-floating green tide algae in the southernYellow Sea from2009to2011, and on the4-year (from2008to2011) variation of thedominant outbreak population U. prolifera in this Sea. In the final chapter, thefeasibility of ITS sequences and rbcL sequences as gene barcode of Ulva green tidealgae was explored.
The morphological characteristics of4molecularly identified and subsequentlypurely cultured species of green tide algae originally collected from the Yellow Seawere investigated. The external morphological and anatomical features of U. prolifera,U.compressa and U.linza were in accordance with those described in CommercialMarine Algae of China (Zeng,1962), the two former with tubular structure and U.linza with distromatic blades. Ulva sp. didn t differ with U.linza in the externalmorphological and anatomical features. In conclusion, in the culture condition inlaboratory, the three species, U. prolifera, U.compressa and U.linza, could be identifiedfrom each other in term of taxonomic features, but couldn t be done for U.linza andUlva sp., so the molecular method is necessary. The15samples with similarmorphological features sorted from green tide algae collected in different habitats. Allthe15samples possessing distromatic blades were classified into two groups, with12samples for LPP group and3samples for Ulva sp. group by the phylogenetic tree of ITSDNA sequences.12samples of LPP group were further classified into two groups, i.e.,U.prolifera and U.linza.
Shore-based investigation along the coastline of Jiangsu Province in2009and in2010showed that filamentous Ulvaceae algae were prevalent in various environmentsof this Province including floating rafts for Porphyra cultivation, landbased culturingponds, estuaries, embankments, intertidal stones, wharfs, and hard muddy coasts. Thenuclear encoded internal transcribed spacer (ITS) and the associated5.8S rDNA regionsas well as5S rDNA spacer sequences were sequenced and analyzed for the green algaecollected from the coastline of the Jiangsu Province in2009（22samples）and in2010(36samples). The phylogenetic analysis of ITS sequence and5S rDNA spacersequences revealed that the samples of2009fell into five distinct clades: the U.prolifera clade, the U. compressa clade, the Ulva sp. clade, the U.linza clade and theBlidingia sp. Clade. The similar pattern for the samples collected in2010was revealedtoo. The sequences of Ulva sp., U. compressa, and Blidingia sp. were completely inagree with those floating in the sea area, respectively. Regionally, the all early floatinggreen tide algae of sea area almost occurred in the seashore of Rudong, Dafeng, andSheyang. All the species of early floating green tide algae were distributed in laver raftand coastal levee, however, U. prolifera was predominant in the culture pond and tidalriver with low salinity. Further analysis with combination of ITS DNA sequence and5SrDNA spacer sequence found that the ITS sequence of U.prolifera sampled from culture pond and tidal river, identical with HM031181, differed in2bp with those of thedominant green tide algae of Yellow Sea in2008(numbered as RD802, the same in thenext text), the5S rDNA spacer sequence had3hypotypes, of which hypotypeⅡpredominated. The same3hypotypes of5S rDNA spacer sequence occurred forfree-floating U. prolifera, with the same ITS DNA sequence as RD802, but hypotypeⅠwas only found in RD802.
The results from samples of2009showed that U. linza was the main component ofgreen tide algae in base shore of Lianyungang, and its ITS DNA sequence and5S rDNAspacer sequence differed largely with those collected from base shore of Rudong,Dafeng and Sheyang, and those of free-floating U. linza. It was suggested that thefree-floating green tide algae would not originated from base shore of Lianyungangcoastline in terms of those above differences. More samples from culture pond and tidalriver were collected in the investigation journey of2010. The U. prolifera group withITS sequence identical to RD802and hypotypesⅡof5S rDNA spacer sequence. This U.prolifera group might be the hybrid progeny of U. prolifera proup (RD802), with ITSsequence identical to RD802and hypotype of5S rDNA spacer sequence, and U.prolifera group prevailed in culture pond (with ITS sequence identical to HM031181and hypotypeⅡof5S rDNA spacer sequence). It need more evidence to support theabove inference.
From2009to2011, we investigated the green tide algae species composition andcommunity succession in the early stage of green tide in the southern Yellow Sea bymolecular phylogenic analysis. The results showed that the green tide algae species inthe early stage were Ulva sp., U.compressa, U.linza, U.prolifera and Blidingia sp., andthe three-year composition of green tide algae were identical. As the monitoring resultof three years, large-scale green tide algae first occurred in Rudong sea in mid-to-lateApril of every year, the dominant bloom forming alga were Ulva sp. and U.compressaother than U. prolifera, the dominant free-floating green tide algae U. prolifera emergedin mid May every year, and subsequently its biomass mushroomed accounting for90% of the whole biomass of green tide algae until June, and then the floating algae groupcontinued to drift to the north. However, the sea area of Dafeng and Sheyang hardlywitnessed the community succession pattern mentioned above. Only one hypotype of5S rDNA spacer sequence (hypotypeⅠ) was found from the samples of early floatinggreen tide algae collected in2009with ITS sequence identical to RD802, however,4hypotypes were found from the samples in2010with the same ITS sequence as RD802,which were hypotypeⅠidentical to RD802, hypotypeⅡ to the dominant species foundin culture pond, hypotype Ⅲ and Ⅳ. Hypotype Ⅲ and Ⅳ were hardly found in theearly floating samples collected from Dafeng sea area, which were mixed with Alligatoralternanthera, indicating samples of hypotype Ⅲ and Ⅳ were brought to there throughtidal river. There appeared two hypotypeⅠand Ⅱ of5S rDNA spacer sequence amongthe samples of early floating green tide algae collected in2011with ITS sequenceidentical to RD802, and the occurrence frequency of the latter hypotype increasedcompared with in2010. The analysis on ITS sequence and5S rDNA spacer sequence ofalgae collected from2008to2011, leading to green tide in theYellow Sea, showed thatthe proportion of hypotypeⅡ to hypotypeⅠincreased largely year by year, with zero ofthe proportion in2008,0.098in2009,0.429in2010, and4.625in2011. So, thehypotypeⅡsuperseded hypotyeⅠto become the dominant hypotype in2011.Using the rbcL sequences and ITS sequences of10species (9species from Ulva andthe other one was Blidingia minima), part of which were download from GenBank andthe other obtained from the common green algae collected during pre investigation ofour laboratory along coastline of China，I explored the DNA barcoding of green tidealgae. Although rbcL and ITS seperately was not fit to be used as DNA barcoding interm of the DNA barcoding judgement standard of the maximum intra-specific geneticdistance being less than the minimum inter-specific genetic distance, they coulddiscriminate7and8species of the10selected species. The variation of minimuminter-specific genetic distance of rbcL sequence was lower than that of ITS, indicatingthat the former was more conservative that the latter and thus more useful for idenfication at the higher taxonomic level. ITS sequence can very good distinguish theother species of green tide algae except LPP complex. Because the arcticle researchresults confirmed the5S rDNA spacer sequence could effectively distinguish LPPcomplex. So author put forward ITS sequence combined with5S rDNA spacer sequencecould be as green tide algae DNA barcode.
对分子鉴定后进行纯种培养的4种黄海绿潮藻进行了形态学特征研究。结果表明，U. prolifera、Ulva compressa和Ulva linza的外部形态特征和解剖学特征均与《中国经济海藻志》（曾呈奎，1962）一致，前两种均具有管状结构，U. linza均具有缘管结构。Ulva sp.外观形态学特征和解剖学特征与U.linza无显著差异。在室内培养条件，前面3种能根据形态学分类特征区分开来，而无法鉴定出U. linza和Ulva sp.，因此仍需借助分子手段。
从不同野外生境条件下的绿潮藻样品中，挑选了15个形态特征非常相似的样品进行了分子鉴定。形态鉴定结果表明，所有样品均具有缘管结构，通过ITS全长序列可将15个样品划分为2类，其中12个样品属于LPP类群，3个样品属于Ulva sp.类群。5S rDNA间隔区序列进一步将LPP类群的12个样品为U.prolifera和U.linza两大类群。
2009年和2010年对江苏沿海岸基采集的绿潮藻进行了种类构成和分子系统分析（其中2009年35个样品、2010年36个样品）。2年的ITS序列和5S rDNA间隔区序列分析结果相似，江苏沿海岸基绿潮藻种类主要为U.linza、U.prolifera、Ulvasp.、U.compressa和Blidingia sp.5个类群。其中Ulva sp.、U.compressa、Blidingiasp.的ITS序列相应地与海区漂浮的绿潮藻Ulva sp.、U.compressa和Blidingia sp.的ITS序列完全一样。从地区来看，如东、大丰、射阳沿海岸基几乎都分布有海区早期漂浮绿潮藻所有种类；从生境来看，紫菜筏架、沿岸堤坝分布有海区早期漂浮绿潮藻的所有种类，而盐度较低的养殖池塘、入海河道主要为U.prolifera。ITS序列结合5S rDNA间隔区序列分析发现采自养殖池塘、入海河道的U.prolifera类群的ITS序列（同HM031181）与2008年黄海绿潮藻优势种（RD802）相差2bp，5S rDNA间隔区序列具有与RD802相同的三种亚型，其中Ⅱ型占绝对优势，未见Ⅰ型；漂浮绿潮藻U.prolifera类群（ITS序列同2008年漂浮优势种）5S rDNA间隔区序列同样存在这三种亚型，但2008年漂浮优势种的5S rDNA间隔区序列还具有其独特的Ⅰ型。2009年的调查结果表明，连云港沿海岸基主要为U.linza，且其ITS序列和5S rDNA间隔区序列与如东、大丰、射阳的U.linza及海区漂浮的U.linza序列差异较大，所以漂浮绿潮藻来源于连云港沿海岸基的可能较小。2010年调查中，作者增加了养殖池塘和入海河道样本量，在养殖池塘样本中作者检测到了一些ITS同RD802、5S rDNA间隔区序列属于Ⅱ型的U.prolifera类群，此类群藻体可能是ITS同RD802、5S rDNA间隔区序列属于Ⅰ型的U.prolifera类群（RD802）随海水进入养殖池塘后与养殖池塘的主要优势U.prolifera类群（ITS同HM031181、5S rDNA间隔区序列属于Ⅱ型）杂交产生的后代。
接着作者对2009-2011年南黄海早期漂浮绿潮藻进行分子系统分析和类群演替研究。结果表明，3年的早期漂浮绿潮藻种类组成一致，均为Ulva sp.、U.compressa、U.linza、U.prolifera和Blidingia sp.。从连续3年的监测结果来看，每年均是4月中下旬在如东海区首先出现大规模漂浮绿潮藻，分子研究结果表明，最先出现的漂浮绿潮藻并非2008年黄海漂浮绿潮藻优势种U.prolifera，而是Ulvasp.和U.compressa，每年的5月中旬开始出现漂浮绿潮藻优势种U.prolifera，随后漂浮绿潮藻优势种U.prolifera生物量迅速增加，至6月初漂浮绿潮藻优势种U.prolifera已经占到了90%以上，随后藻团继续向北漂移。但在大丰海区、射阳海区看不到这种类群演替现象。另外，2009年早期漂浮绿潮藻ITS序列同RD802的样品，5S rDNA间隔区序列只发现一种（Ⅰ型）；2010年早期漂浮绿潮藻ITS序列同RD802的样品存在四种亚型：Ⅰ型（同2008年黄海漂浮优势种）、Ⅱ型（同养殖池塘优势种）、Ⅲ型、Ⅳ型，Ⅲ型、Ⅳ型样品极少仅在大丰早期漂浮样品中出现，且采集回来的样品中混有空心莲子草，说明Ⅲ型、Ⅳ型样品可能是通过入海河道漂过来的。2011年早期漂浮绿潮藻ITS序列同RD802的样品5S rDNA间隔区序列同时存在Ⅰ型和Ⅱ型两种亚型，且Ⅱ型较前一年出现频率增大。为了弄清黄海绿潮大规模暴发时，漂浮优势种ITS序列同RD802，其5S rDNA间隔区序列是否也存在Ⅰ型和Ⅱ型两种亚型以及这两种亚型是如何变化的，作者对采自2008-2011年黄海海域暴发绿潮时的藻样进行ITS序列和5S rDNA间隔区序列分析，结果表明，这些样品ITS序列全部相同且同RD802，2008年样品的5S rDNA间隔区序列全部Ⅰ型，2009年以后同时存在Ⅰ型和Ⅱ型两种亚型，且5S rDNA间隔区序列Ⅱ型的比例由2009年的1:10.25增加到2010年的1:2.33，至2011年已经超过Ⅰ型成为优势亚型，比例已经高达4.625:1。
最后作者对GenBank中下载的石莼属9个物种和石莼科盘苔的rbcL和ITS序列以及本实验全国沿海绿潮藻预调查采集的我国沿海地区常见绿藻种类进行了绿潮藻DNA条形码技术的初步研究。根据最大种内遗传距离小于最小种间遗传距离的严格DNA条形码判断标准，rbcL和ITS序列虽都不适合单独作为绿潮藻的DNA条形码，但分别能区分10个物种中的7个物种和8个物种。rbcL的最小种间遗传距离变异程度低于ITS的，说明前者比后者更为保守，更适合更高级分类阶元的分类。ITS在石莼属绿潮藻中能够较好地区分其他种类，却不能很好区分LPP复合体，而本论文的前两章研究结果证实5S rDNA间隔区序列能够有效地区分LPP复合体。故作者提出ITS序列和5S rDNA间隔区序列组合使用可以作为绿潮藻的DNA条形码。
A serious outbreak of green tide continuously struck the Yellow Sea of China from2007to2011. More seriously, the largest outbreak of green tide in the sea area ofQingdao in2008hampered the regatta sailing during the Beijing Olympic Games. Thusgovernment and researchers had paid much attention to green tide. Ulva is the maincomponents of green tide algae. The research hotpots include taxonomic identificationand genetic origin of the green tide algae, and outbreak mechanism, forecast and earlywarming, prevention and control management and comprehensive utilization of greentide. This paper dealt with the molecular identification and population succession ofgreen tide algae in the Yellow Sea. In the second chapter, the morphological featuresand molecular identification of the main pure culture green tide algae were preliminarilyinvestigated. The molecular identification and species composition of shore-based greentide algae were studied in the next chapter. The fourth chapter focused on the speciescomposition and their succession of early free-floating green tide algae in the southernYellow Sea from2009to2011, and on the4-year (from2008to2011) variation of thedominant outbreak population U. prolifera in this Sea. In the final chapter, thefeasibility of ITS sequences and rbcL sequences as gene barcode of Ulva green tidealgae was explored.
The morphological characteristics of4molecularly identified and subsequentlypurely cultured species of green tide algae originally collected from the Yellow Seawere investigated. The external morphological and anatomical features of U. prolifera,U.compressa and U.linza were in accordance with those described in CommercialMarine Algae of China (Zeng,1962), the two former with tubular structure and U.linza with distromatic blades. Ulva sp. didn t differ with U.linza in the externalmorphological and anatomical features. In conclusion, in the culture condition inlaboratory, the three species, U. prolifera, U.compressa and U.linza, could be identifiedfrom each other in term of taxonomic features, but couldn t be done for U.linza andUlva sp., so the molecular method is necessary. The15samples with similarmorphological features sorted from green tide algae collected in different habitats. Allthe15samples possessing distromatic blades were classified into two groups, with12samples for LPP group and3samples for Ulva sp. group by the phylogenetic tree of ITSDNA sequences.12samples of LPP group were further classified into two groups, i.e.,U.prolifera and U.linza.
Shore-based investigation along the coastline of Jiangsu Province in2009and in2010showed that filamentous Ulvaceae algae were prevalent in various environmentsof this Province including floating rafts for Porphyra cultivation, landbased culturingponds, estuaries, embankments, intertidal stones, wharfs, and hard muddy coasts. Thenuclear encoded internal transcribed spacer (ITS) and the associated5.8S rDNA regionsas well as5S rDNA spacer sequences were sequenced and analyzed for the green algaecollected from the coastline of the Jiangsu Province in2009（22samples）and in2010(36samples). The phylogenetic analysis of ITS sequence and5S rDNA spacersequences revealed that the samples of2009fell into five distinct clades: the U.prolifera clade, the U. compressa clade, the Ulva sp. clade, the U.linza clade and theBlidingia sp. Clade. The similar pattern for the samples collected in2010was revealedtoo. The sequences of Ulva sp., U. compressa, and Blidingia sp. were completely inagree with those floating in the sea area, respectively. Regionally, the all early floatinggreen tide algae of sea area almost occurred in the seashore of Rudong, Dafeng, andSheyang. All the species of early floating green tide algae were distributed in laver raftand coastal levee, however, U. prolifera was predominant in the culture pond and tidalriver with low salinity. Further analysis with combination of ITS DNA sequence and5SrDNA spacer sequence found that the ITS sequence of U.prolifera sampled from culture pond and tidal river, identical with HM031181, differed in2bp with those of thedominant green tide algae of Yellow Sea in2008(numbered as RD802, the same in thenext text), the5S rDNA spacer sequence had3hypotypes, of which hypotypeⅡpredominated. The same3hypotypes of5S rDNA spacer sequence occurred forfree-floating U. prolifera, with the same ITS DNA sequence as RD802, but hypotypeⅠwas only found in RD802.
The results from samples of2009showed that U. linza was the main component ofgreen tide algae in base shore of Lianyungang, and its ITS DNA sequence and5S rDNAspacer sequence differed largely with those collected from base shore of Rudong,Dafeng and Sheyang, and those of free-floating U. linza. It was suggested that thefree-floating green tide algae would not originated from base shore of Lianyungangcoastline in terms of those above differences. More samples from culture pond and tidalriver were collected in the investigation journey of2010. The U. prolifera group withITS sequence identical to RD802and hypotypesⅡof5S rDNA spacer sequence. This U.prolifera group might be the hybrid progeny of U. prolifera proup (RD802), with ITSsequence identical to RD802and hypotype of5S rDNA spacer sequence, and U.prolifera group prevailed in culture pond (with ITS sequence identical to HM031181and hypotypeⅡof5S rDNA spacer sequence). It need more evidence to support theabove inference.
From2009to2011, we investigated the green tide algae species composition andcommunity succession in the early stage of green tide in the southern Yellow Sea bymolecular phylogenic analysis. The results showed that the green tide algae species inthe early stage were Ulva sp., U.compressa, U.linza, U.prolifera and Blidingia sp., andthe three-year composition of green tide algae were identical. As the monitoring resultof three years, large-scale green tide algae first occurred in Rudong sea in mid-to-lateApril of every year, the dominant bloom forming alga were Ulva sp. and U.compressaother than U. prolifera, the dominant free-floating green tide algae U. prolifera emergedin mid May every year, and subsequently its biomass mushroomed accounting for90% of the whole biomass of green tide algae until June, and then the floating algae groupcontinued to drift to the north. However, the sea area of Dafeng and Sheyang hardlywitnessed the community succession pattern mentioned above. Only one hypotype of5S rDNA spacer sequence (hypotypeⅠ) was found from the samples of early floatinggreen tide algae collected in2009with ITS sequence identical to RD802, however,4hypotypes were found from the samples in2010with the same ITS sequence as RD802,which were hypotypeⅠidentical to RD802, hypotypeⅡ to the dominant species foundin culture pond, hypotype Ⅲ and Ⅳ. Hypotype Ⅲ and Ⅳ were hardly found in theearly floating samples collected from Dafeng sea area, which were mixed with Alligatoralternanthera, indicating samples of hypotype Ⅲ and Ⅳ were brought to there throughtidal river. There appeared two hypotypeⅠand Ⅱ of5S rDNA spacer sequence amongthe samples of early floating green tide algae collected in2011with ITS sequenceidentical to RD802, and the occurrence frequency of the latter hypotype increasedcompared with in2010. The analysis on ITS sequence and5S rDNA spacer sequence ofalgae collected from2008to2011, leading to green tide in theYellow Sea, showed thatthe proportion of hypotypeⅡ to hypotypeⅠincreased largely year by year, with zero ofthe proportion in2008,0.098in2009,0.429in2010, and4.625in2011. So, thehypotypeⅡsuperseded hypotyeⅠto become the dominant hypotype in2011.Using the rbcL sequences and ITS sequences of10species (9species from Ulva andthe other one was Blidingia minima), part of which were download from GenBank andthe other obtained from the common green algae collected during pre investigation ofour laboratory along coastline of China，I explored the DNA barcoding of green tidealgae. Although rbcL and ITS seperately was not fit to be used as DNA barcoding interm of the DNA barcoding judgement standard of the maximum intra-specific geneticdistance being less than the minimum inter-specific genetic distance, they coulddiscriminate7and8species of the10selected species. The variation of minimuminter-specific genetic distance of rbcL sequence was lower than that of ITS, indicatingthat the former was more conservative that the latter and thus more useful for idenfication at the higher taxonomic level. ITS sequence can very good distinguish theother species of green tide algae except LPP complex. Because the arcticle researchresults confirmed the5S rDNA spacer sequence could effectively distinguish LPPcomplex. So author put forward ITS sequence combined with5S rDNA spacer sequencecould be as green tide algae DNA barcode.
引文
[1]Blomster J, Back S, Fewer DP etc. Novel morphology in. Enteromorpha (Ulvophyceae)forming green tides. American Journal of Botany,2002,89:1756～1763
[2]Anderson DM, Glibert PM, Burkholder JM. Harmful algal blooms and eutrophication:nutrient sources, composition and consequences. Estuaries,2002,25:704-726
[3]Nelson TA, Lee DJ, Smith BC. Are “green tide” harmful algal blooms? Toxic properties of watersoluble extracts from two bloom forming macroalgae, Ulva fenestrata and Ulvaria obscura(Ulvophyceae). Journal of Phycology,2003a,39:874～879
[4]Anderson RJ, Monteiro PMS, Levitt G.J. The effect of localised eutrophication on competitionbetween Ulva lactuca (Ulvaceae, Chlorophyta) and a commercial resource of Gracilaria verrucosa(Gracilariaceae, Rhodophyta). Hydrobiologia,1996,326:291～296
[5]Hiraoka M, Shimada S, Uenosono M etc. Anew green tide forming alga, Ulva ohnoi Hiraoka etShimada sp. nov.(Ulvales, Ulvophyceae) from Japan. Phycology Reseach,2003,51:17～29
[6]Largo DB, Sembrano J, Hiraoka M etc. Taxonomic and ecological profile of green tides speciesof Ulva (Ulvales, Chlorophyta) in central Philippines. Hydrobiologia,2004,5(12):247～253
[7]Charlier RH, Morand P, Finkl CW etc. Green tides on the Brittany coasts. EnvironmentalResearch, engineering and Management,2007,3:52～59
[8]Nelson TA, Haberlin K, Nelson AV etc. Ecological and physiological controls of speciescomposition in green macroalgal blooms. Ecology,2008,89:1287～1298
[9]Sun S, Wang F, Li C, etc. Emerging challenges: Massive green algae blooms in the YellowSea. Nature Precedings,2008: hdl:10101/npre.12008.12266.10101
[10]Yabe T, IshiiY, AmanoY etc. Green tide formed byfree floating Ulva spp. atYatsu tidal flat,Japan. Limnology,2009,10:239～245
[11]曾呈奎,张德瑞,张峻甫.中国经济海藻志.北京:科学出版社,1962:43～50
[12]Tseng CK. Common sea weeds of China. Beijing, China: Science Press.1983.
[13]唐启升,张晓雯,叶乃好等.绿潮研究现状与问题.中国科学基金,2010,(1):5～9
[14]李大秋,贺双颜,杨倩等.青岛海域浒苔来源与外海分布特征研究.环境保护,2008,16:45～46
[15]董海.青岛发布绿潮Ⅳ级警报.现代渔业信息,2009,24(7):29
[16]王建伟,阎斌伦,林阿朋等.浒苔（Enteromorpha prolifera）生长及孢子释放的生态因子研究.海洋通报,2007,26(2):60～65
[17]田千桃.绿潮藻的生长、营养盐吸收及其对微藻的克生效应研究[硕士研究生学位论文].上海:上海海洋大学,2010
[18]王阳阳,霍元子,曹佳春等.低温、低光照对扁浒苔生长的影响.中国水产科学,2010,17(3):593～599
[19]Vermaat JE, Sand-Jensen K. Survival, metabolism and growth of Ulva lactuca under winterconditions: a laboratory study of bottlenecks in the life cycle. Marine Biology,1987,95:55～61
[20]Back S, Lehvo A, Blomster J. Mass occurrence of unattached Enteromorpha intestinalis onthe Finnish Baltic Sea coast. Annales Botanici Fennici,2000,37:155～161
[21]Taylor R, Fletcher RL, Raven JA. Preliminarystudies on the growth of selected “green tide”algae in laboratoryculture: effects of irradiance, temperature, salinityand nutrients on growthrate. Botanica Marina,2001,44:327～336
[22]郭赣林,董双林,董云伟.温度及其波动对孔石莼生长及光合作用的影响.中国海洋大学学报,2006,36:941～945
[23]Levavassur G, Edwards GE, Osmond CB etc. Inorganic carbon limitation of photosynthesisin Ulva rotundata (ChloroPhyta). Journal of Phycology,1991,27:667～672
[24]刘静雯,董双林.温度和盐度对几种大型海藻生长率和NH4-N吸收的影响.海洋学报,2001,23:109～116
[25]Lartigue J, Neill A, Hayden BL etc.The impact of salinity fluctuations on net oxygenproduction and inorganic nitrogen uptake by Ulva lactuca (Chlorophyceae).Aquatic Botany，2003，75:339～350
[26]Dan A, Hiraoka M, Ohno M etc. Observations on the effect of salinity and photon fluencerate on the induction of sporulation and rhizoid formation in the green alga Enteromorphaprolifera (Müller) J. Agardh (Chlorophyta, Ulvales). Fishery Science,2002,68:1182～1188
[27]李德,周亮,林东年.生态因子对缘管浒苔生长和孢子附着的影响.现代渔业信息,2009,24(5):22～24
[28]Chan KY, Wong PK, Ng SL. Growth of Enteromorpha linza in sewage effluent and sewageeffluent seawater mixtures. Hydrobiologia,1982,97:9-13
[29]Beer S, Eshel A. Photosynthesis of Ulva SPP. Effects of desiccation when exposed to air.Joumal of Experimental Marine Biology and Ecology,1983,70:91～97
[30]Gao K, Ji Y, Aruga Y. Relationshipo f CO2concentrations to photosynthesis of intertidalmacroalgae during emersion. Hydrobiologia,1999,398:355～359
[31]Raffaelli DG, Raven J, Poole L. Ecological impact of macroaglal blooms. OceanographyMarine Biology Annuual Review,1998,36:97～125
[32]Hiraoka M, Dan A, Shimada S etc. Different life histories of Enteromorpha prolifera (Ulvales,Chlorophyta) from four rivers on Shikoku Island, Japan. Phycologia,2003,42(3):275～284
[33]王晓坤,马家海,叶道才等.浒苔(Enteromorpha prolifera)生活史的初步研究.海洋通报,2007,26(5):112～116
[34]林阿朋,王建伟,阎斌伦,等.浒苔Enteromorpha prolifera组织培养初步研究.生态科学,2006,25(4):320～324
[35]Lee TF, Wichroski M. Polar regeneration in Enteromorpha prolifera. Phycologia,1996,32:2
[36]孙修涛,王翔宇,王文俊,等.绿潮中浒苔的抗逆能力和药物灭杀效果初探.海洋水产研究,2008,29(5):130～136
[37]Reed RH, Russell G. Salinity fluctuations and their influence on”bottle brush”morphogenesis in Enteromorpha intestinalis (L.) Link. European Journal of Phycology,1978,13:149～153
[38]Valiela I, McClelland J, Hauxwell Jl etc. Macroalgal blooms in shallow estuaries: controlsand ecophysiological and ecosystem consequences. Limnology and Oceanography,1997,42:1105～1118
[39]叶乃好,张晓雯,毛玉泽等.黄海绿潮浒苔（Enteromorpha prolifera）生活史的初步研究.中国水产科学,2008,15(5):853～559
[40]Lotze HZ, Schram W, Schories D etc. Control of macroalgal blooms at early developmentalstages: Pilayella littoralis versus Enteromorpha spp. Oecologia.1999,119:46～54
[41]Lotze HK, Worm B, Sommer U. Propagule banks, herbivory and nutrient supply controlpopulation development and dominance patterns in macroalgal blooms. Oikos.2000,85:46～58
[42]Worm B, Heike K, Sommer U. Algal propagules banks modify competition, consumer andresource control on Baltic rocky shores. Oecologia,2001,128:281～293
[43]Camey LT, Edwards MS. Cryptic process in the sea: a review of delayed development in themicroscopic life stages of marine macroalgae. Algae.2006,21:161～168
[44]Dan A, Ohno M, Matuoka M. Cultivation of the green alga Enteromorpha prolifera usingchopped tissue for artificial seeding. Suisan Zoshoku.1997,45:5～8
[45]Kamermans P, Malta EJ, Verschuure JM etc. Role of cold resistance and burial for wintersurvival and spring initiation of an Ulva spp.(Chlorophyta) bloom in a eutrophic lagoon(Veerse Meer lagoon, The Netherlands). Marine Biology (Berlin).1998,131:45～51
[46]Zhang XW, Wang HX, Mao YZ etc. Somatic cells serve as potential propagule bank ofEnteromorpha prolifera forming a green tide in the Yellow Sea, China. Journal of ApplliedPhycology,2010,22:173～180
[47]Matsukawa Y, Umebayashi O. Ulva pertusa biomass growth rate and its limiting factors inan intertidal flat. Bull Tokai Reg Fish Res Lab,1988,126:25～35
[48]梁宗英,林祥志,马牧,张静,闫晓波,刘涛.浒苔漂流聚集绿潮现象的初步分析.中国海洋大学学报,2008,38(4):601～604
[49]Foster GL. Indications regarding the source of combined nitrogen for Ulva laetuca. Annalsof the Missouri Botanical Garden,1914, I:229～235
[50]Villares R, Carballeira A. Nutrient Limitation in Macroalgae (Ulva and Enteromorpha)from the Rias Baixas (NW Spain). Marine Ecology,2004,25(3):225～243
[51]Naldi M, Viarolinaldi P. Nature uptake and storage in the seaweed Ulva rigida C．Agardh inrelation to nitrate availability and thallus nitrate content in a eutrophic coastal lagoon (SaccadiGoro, Po River Delta, Italy). Joumal of Experimental Marine Biology and Ecology,2002,269:65～83
[52]Pedersen MF. Transient ammonium uptake in the macroalga Ulva lactuca (Chlorophyta):nature, regulation, and the consequences for choice of measuring technique. Journal ofPhycology,1994,30:980～986
[53]Bjoernsaeter BR, Wheeler PA. Effeet of nitrogen and phosphorus supply on growth andtissue composition of Ulva fenestrata and Enteromorpha intestinalis (Ulvales, Chlorophyta).Joumal of Phycology,1990,26:603～611
[54]Duke CS, Litaker W, Ramus J. Effects of temperature, nitrogen supply and tissue nitrogenon ammonium uptake rates of the chlorophyte sea weeds Ulva curvata and Codiumdecorticatum. Joumal of Phycology,1989,25:113～120
[55]李瑞香,吴晓文,韦钦胜,王宗灵,李艳,孙萍.不同营养盐条件下浒苔的生长.海洋科学进展,2009,2:211～216
[56]Lapointe BE, Littler MM, Littler DS. Nutrient availability to marine macroalgae insilieiclastic versus carbonate-rich coastal waters. Estuaries and Coasts,1992,15:75～82
[57]Morand P, Merceron M. Macroalgal Population and sustainability. Journal of CoastalResearch,2005,21:1009～1020
[58]Liu DY, Keesing JK, Xing QG etc. World's largest macroalgal bloom caused by expansionof seaweed aquaculture in China. Marine Pollution Bulletin,2009,58:888～895.
[59]Wang BD. Cultural eutrophication in the Changjiang(Yangtze River) plume: History andperspective. Estuarine, Coastal and Shelf Science,2003,68:60～65
[60]徐姗楠,何培民.我国赤潮频发现象分析与海藻栽培生物修复作用.水产学报,2006,30(4):554～561
[61]Levitus S, Antonov JI, Boyer TP etc. Warming of the World Ocean. Science,2000,287:2224～2229
[62]Jiao NZ, Zhang Y, Zeng YH etc. Ecological anomalies in the East China Sea: Impacts of theThree Gorges Dam? Water reseach,2007,41:1287～1293
[63]Sfriso A, Pavoni B, Marcomini A etc. Macroalgae, nutrient cycles and pollutants in theLagoon of Venice. Estuaries and Coasts,1992,15:517～528
[64]Cohen RA, Fong P. Using opportunistic green macroalgae as indicators of nitrogen supplyand sources to estuaries. Ecological Applications,2006,16:1405～1420
[65]de Paula Silva PH, McBride S, de Nys R etc.2008. Integrating filamentous green tidealgae into tropical pond based aquaculture. Aquaculture,284:74～80
[66]NelsonTA, NelsonAV, Tjoelker M. Seasonal and spatial patterns of “green tides”(Ulvoidalgal blooms) and related water qualityparameters in the coastal waters of Washington State,USA. Botanica Marina,2003b,46:263～275
[67]Dahlgren S, Kautsky L. Can different vegetative states in shallow coastal bays of the BalticSea be linked to internal nutrient levels and external nutrient load? Hydrobiologia,2004,514:249～258
[68]Jones M, Pinn E. The impact of a macroalgal mat on benthic biodiversity in Poole Harbour.Marine Pollution Bulletin,2006,53:63～71
[69]Lopes RJ, Pardal MA, Marques JC. Impact of macroalgal blooms and wader predatidalmacroinvertebrates: experimental evidence from the Mondego estuary (Portugal). Journal ofExperimental Marine Biology and Ecology,2000,249:165～179
[70]Berezina NA, Tsiplenkina IG, Pankova ES etc. Dynamics of invertebrate communities instonylittoral of the Neva Estuary(Baltic Sea) under macroalgal blooms and bioinvasions.Transactions of Water Bulliten,2007,1:65～76
[71]Berezina NA. Spatial distribution of macrofauna in a littoral zone with drifting macroalgaein the Neva estuary. Estonian Journal of Ecology,2008,57:198～213
[72]Atkins RP, Deeley DM, McAlpine KW. Managing the aquatic environment. NutrientCycling in Agroecosystems,1993,36:171～175
[73]Rogers RJ, Dake N, Dussart GBJ. Ulva Problem in Kent:1973-2003. Marine PollutionBulletin,2004,49:145～146
[74]Gao S, Chen X,Yi Q etc. Astrategyfor the proliferation of Ulva prolifera, main causativespecies of green tides, with formation of sporangia by fragmentation. PLoS One,2010,5(1):e8571
[75]Linnaeus C. Species Plantarum. Impensis GC Nauk,1800
[76]Silva PC. A review of nomenclatural conservation in the algae from the point of view of thetype method. University of California Press:1952
[77]Bonneau ER. Polymorphic behavior of Ulva Lactuca (Chlorophyta) in axenic eulture.1.Occurrence of Enteromorpha-like plants in haPloid clones. Journal of Phycology,1977,13:133～140
[78]Provasoli L, Pintner IJ. Bacteria induced polymorphism in an axenic laboratory strain of UlvaLactuca (Chlorophyceae). Journal of Phycology,1980,16:196～201
[79]Blomster J, Maggs CA, Stanhope MJ. Molecular and morphological analysis ofEnteromorpha intestinalis and E.compressa (Chlorophyta) in the British Isles. Journal ofphycology,1998,34:319～340
[80]Leskinen E, Alstrom-RapaPort C, Pamilo P. Phylogeographical structure, distribution andgenetic variation of the green algae Ulva intestinalis and U.compressa (Chlorophyta) in theBaltic Sea area. Molecular Ecology,2004,13:2257～2265
[81]Malta EJ, Draisma SGA, Kamermans P. Free-floating Ulva in the southwest Netherlands：species or morphotypes? a morphological，molecular and ecological comparison. EuropeanJournal of Phycology,1999,34:443～454
[82]Tan IH, Blomster J, Hansen G etc. Molecular phylogenetic evidence for a reversiblemorphogenetic switch controlling the gross morphology of two common genera of greenseaweeds, Ulva and Enteromorpha. Molecular Biology and Evolution,1999,16:1011～1018
[83]Shimada S, Hiraoka M, Nabata S etc. Molecular phylogenetic analyses of the Japanese Ulvaand Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva.Phycological Research,2003,51:99～108
[84]Hayden HS, Blomster J, Maggs CA etc. Linnaeus was right all along: Ulva andEnteromorpha are not distinct genera. European Journal of Phycology,2003,38:277～294
[85]Hayden HS, Waaland JR. Phylogenetic systematics of the Ulvaceae (Ulvales, Ulvophyceae)using chloroplast and nuclear DNA sequences. Journal of Phycology,2002,38:1200～121
[86]Shimada S,Yokoyama N, AraiA etc. Phylogeographyof the genus Ulva (Ulvophyceae,Chlorophyta), with special reference to the Japanese freshwater and brackish taxa. Journal ofAppllied Phycology,2008,20:979～989
[87]Coat G, Dion P, Noailles MC etc. Ulva armoricana (UIvales, Chlorophyta) from the coastsof Brittany (France). Ⅱ. Nuclear rDNA ITS sequence analysis. European Journal ofPhycology,1998,33:81～86
[88]Leliaert F, Zhang X, Ye N etc. Identity of the Qingdao algal bloom. Phyeological Research,2009,57:147～151
[89]张晓雯,毛玉泽,庄志猛等.黄海绿潮浒苔的形态学观察及分子鉴定.中国水产科学,2008,15:522～529
[90]Pang SJ, Liu F, Shan TF etc.Tracking the algal origin of the Ulva bloom in the Yellow Seaby a combination of molecular, morphological and physiological analyses. MarineEnvironmental Research,2010,69(4):207～215
[91]OTT F D．Synthetic media and techniques for the axenic cultivation of marine algae andflagellate．Va J Sci，1965，16:205～218
[92]丁兰平,栾日孝.浒苔（Enteromorpha prolifera）的分类鉴定、生境习性及分布.海洋与湖沼,2009,40(1):68～71
[93]汪文俊,王飞久,陈松林等.浒苔ITS区的扩增和分析.海洋水产研究,2008,29(5):124～129
[94]Leskinen E, Pamilo P. Evolution of the ITS sequences of ribosomal DNA in Enteromorpha(Chlorophyceae). Hereditas,1997,126:17～23
[95]Thompson JD, Higgins DG, GibsonTJ. CLUSTALW: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting, position specific gappenalties and weight matrix choice. Nucleic Acids Research,1994,22:4673～4680
[96]Saitou N, Nei M. The neighbor joining method:A new method for reconstructingphylogenetic trees. Molecular Biology and Evolution,1978,4:406～425
[97]Zhang XW, Xu D, Mao YZ etc. Settlement of vegetative of Ulva prolifera confirmed as an425important seed sourse for succession of a large-scale green tide bloom. Limnology andoceanography,2011,56(1):233～242
[98]Mares J, Leskinen E, Sitkowska M etc. True identity of the European reshwater Ulva(Chlorophyta) revealed by a combined molecular and morpholofical approach. Journal ofPhycology,2011,47:1～15.
[99]Kraft LGK, Kraft GT, Waller RF. Investications into southen Australian Ulva(Chlorophyta)taxonomy and molecular phylogeny indicate both cosmopolitanism and cryptic species. Journalof Phycology,2010(46):1257～1277
[100]田晓玲,霍元子,陈丽平等.江苏如东近海绿潮藻分子监测与类群演替分析.科学通报,2011,56(4-5):309～317
[101]徐兆礼,叶属峰,徐韧.2008年中国浒苔灾害成因条件和过程推测.水产学报,2009,33(3):430～437
[102]Keesing JK, Liu DY, Fearns P etc. Inter-and intra-annual patterns of Ulva prolifera green tidesin the Yellow Sea. Marine Pollution Bulletin,2011(62):1169～1182
[103]Hiraoka M, Ichihara K, Zhu WR etc. Culture and hybridization experiments on an Ulva cladeincluding the Qingdao strain blooming in the Yellow Sea. PLoS One,2011(6):1～6
[104]Fletcher RT. The occurrence of green-tide. In: Schramm, W., Nienhuis, P.H.(Eds.), MarineBenthic Vegetation: Recent Changes and the Effects of Eutrophication. Springer Verlag, Berlin,1996,7～43
[105]Ye NH, Zhuang ZM, Jin XS etc. China is on the tracking Enteromorpha spp. forming greentide. Nature Proceedings,2008, hdl:10101/npre.2008.2352.1
[106]Liu DY, Keesing JK, Dong ZJ etc. Recurrence of the world′s largest green-tide in2009inYellow Sea, China: Porphyra yezoensis aquaculture rafts confirmed as nursery for macroalgalblooms. Marine Pollution Bulletin,2010,60(9):1423～1432
[107]应成琦,张婷,李信书等.我国近海浒苔漂浮种类ITS与18S rDNA序列相似性分析.水产学报,2009,33(2):215～219
[108]刘峰.黄海绿潮的成因以及绿潮浒苔的生理生态学和分子系统学研究[博士学位论文].青岛:中国科学院海洋研究所
[109]王建伟,林阿朋,李艳燕等.浒苔(Enteromorpha prolifera)藻体发育的显微观察.生态科学,2006,25(5):400～404
[110]陈群芳.漂浮绿潮藻浒苔繁殖特性与藻体气囊形成研究[硕士学位论文].上海：上海海洋大学
[111]劳方业,符成,陈仲华等.斑茅杂交后代的分子鉴定.甘蔗糖业,2006(1):6～11
[112]闫化学,于杰. DNA条形码技术在植物中的研究现状.植物学报,2010,45(1):102～108
[113]Schindel DE, Miller SE. DNA barcoding a useful tool for taxonomists. Nature,2005,435:17
[114]Ebach MC, Holdrege C. DNA barcoding is no substitute for taxonomy. Nature,2005,434:697–697
[115]Agnarsson I, Kuntner M. Taxonomy in a changing world: seeking solutions for a science incrisis. Systematic Biology,2007,56:531～539
[116]Kress WJ, Wurdack KJ, Zimmer EA etc. Use of DNA barcodes to identify flowering plants.Proc Natl Acad Sci USA,2005,102:8369～8374
[117]Taberlet P, Coissac E, Pompanon F etc. Power and limitations of the chloroplast trnL (UAA)intron for plant DNA barcoding. Nucleic Acids Research,2007,35: e14
[118]Meyer CP, Paulay G. DNA barcoding: error rates based on comprehensive sampling. PLoSBiology,2005,3: e422
[119]Kimura M. A simple method for estimating evolutionary rate of base substitutions throughcomparative studies of nucleotide sequences. Journal of Molecular Evolution,1980,16:111～120
[120]Newmaster SG, Fazekas AJ, Ragupathy S. DNA barcoding in land plants: evaluation of rbcL ina multigene tiered approach. Canadian Journal of Botany,2006,84,335～341
[121]Kress WJ, Erickson DL. A two-locus global DNA barcode for land plants: the coding rbcL genecomplements the non-coding trnH-psbA spacer region. PLoS One,2007,2(6): e508
[122]Sass C, Little DP, Stevenson DW etc. DNA barcoding in the cycadales: testing the potential ofproposed barcoding markers for species identification of cycads. PloS One,2007,2: e1154
[123]Lazaro EM, Sluys R, Pala M etc. Molecular barcoding and phylogeography of sexual andasexual freshwater planarians of the genus Dugesia in the westem Mediterranean(Platyhelminthes, Tricladida, Dugesiidae). Molecular phylogeneties and Evolution,2009,52:835～845
[2]Anderson DM, Glibert PM, Burkholder JM. Harmful algal blooms and eutrophication:nutrient sources, composition and consequences. Estuaries,2002,25:704-726
[3]Nelson TA, Lee DJ, Smith BC. Are “green tide” harmful algal blooms? Toxic properties of watersoluble extracts from two bloom forming macroalgae, Ulva fenestrata and Ulvaria obscura(Ulvophyceae). Journal of Phycology,2003a,39:874～879
[4]Anderson RJ, Monteiro PMS, Levitt G.J. The effect of localised eutrophication on competitionbetween Ulva lactuca (Ulvaceae, Chlorophyta) and a commercial resource of Gracilaria verrucosa(Gracilariaceae, Rhodophyta). Hydrobiologia,1996,326:291～296
[5]Hiraoka M, Shimada S, Uenosono M etc. Anew green tide forming alga, Ulva ohnoi Hiraoka etShimada sp. nov.(Ulvales, Ulvophyceae) from Japan. Phycology Reseach,2003,51:17～29
[6]Largo DB, Sembrano J, Hiraoka M etc. Taxonomic and ecological profile of green tides speciesof Ulva (Ulvales, Chlorophyta) in central Philippines. Hydrobiologia,2004,5(12):247～253
[7]Charlier RH, Morand P, Finkl CW etc. Green tides on the Brittany coasts. EnvironmentalResearch, engineering and Management,2007,3:52～59
[8]Nelson TA, Haberlin K, Nelson AV etc. Ecological and physiological controls of speciescomposition in green macroalgal blooms. Ecology,2008,89:1287～1298
[9]Sun S, Wang F, Li C, etc. Emerging challenges: Massive green algae blooms in the YellowSea. Nature Precedings,2008: hdl:10101/npre.12008.12266.10101
[10]Yabe T, IshiiY, AmanoY etc. Green tide formed byfree floating Ulva spp. atYatsu tidal flat,Japan. Limnology,2009,10:239～245
[11]曾呈奎,张德瑞,张峻甫.中国经济海藻志.北京:科学出版社,1962:43～50
[12]Tseng CK. Common sea weeds of China. Beijing, China: Science Press.1983.
[13]唐启升,张晓雯,叶乃好等.绿潮研究现状与问题.中国科学基金,2010,(1):5～9
[14]李大秋,贺双颜,杨倩等.青岛海域浒苔来源与外海分布特征研究.环境保护,2008,16:45～46
[15]董海.青岛发布绿潮Ⅳ级警报.现代渔业信息,2009,24(7):29
[16]王建伟,阎斌伦,林阿朋等.浒苔（Enteromorpha prolifera）生长及孢子释放的生态因子研究.海洋通报,2007,26(2):60～65
[17]田千桃.绿潮藻的生长、营养盐吸收及其对微藻的克生效应研究[硕士研究生学位论文].上海:上海海洋大学,2010
[18]王阳阳,霍元子,曹佳春等.低温、低光照对扁浒苔生长的影响.中国水产科学,2010,17(3):593～599
[19]Vermaat JE, Sand-Jensen K. Survival, metabolism and growth of Ulva lactuca under winterconditions: a laboratory study of bottlenecks in the life cycle. Marine Biology,1987,95:55～61
[20]Back S, Lehvo A, Blomster J. Mass occurrence of unattached Enteromorpha intestinalis onthe Finnish Baltic Sea coast. Annales Botanici Fennici,2000,37:155～161
[21]Taylor R, Fletcher RL, Raven JA. Preliminarystudies on the growth of selected “green tide”algae in laboratoryculture: effects of irradiance, temperature, salinityand nutrients on growthrate. Botanica Marina,2001,44:327～336
[22]郭赣林,董双林,董云伟.温度及其波动对孔石莼生长及光合作用的影响.中国海洋大学学报,2006,36:941～945
[23]Levavassur G, Edwards GE, Osmond CB etc. Inorganic carbon limitation of photosynthesisin Ulva rotundata (ChloroPhyta). Journal of Phycology,1991,27:667～672
[24]刘静雯,董双林.温度和盐度对几种大型海藻生长率和NH4-N吸收的影响.海洋学报,2001,23:109～116
[25]Lartigue J, Neill A, Hayden BL etc.The impact of salinity fluctuations on net oxygenproduction and inorganic nitrogen uptake by Ulva lactuca (Chlorophyceae).Aquatic Botany，2003，75:339～350
[26]Dan A, Hiraoka M, Ohno M etc. Observations on the effect of salinity and photon fluencerate on the induction of sporulation and rhizoid formation in the green alga Enteromorphaprolifera (Müller) J. Agardh (Chlorophyta, Ulvales). Fishery Science,2002,68:1182～1188
[27]李德,周亮,林东年.生态因子对缘管浒苔生长和孢子附着的影响.现代渔业信息,2009,24(5):22～24
[28]Chan KY, Wong PK, Ng SL. Growth of Enteromorpha linza in sewage effluent and sewageeffluent seawater mixtures. Hydrobiologia,1982,97:9-13
[29]Beer S, Eshel A. Photosynthesis of Ulva SPP. Effects of desiccation when exposed to air.Joumal of Experimental Marine Biology and Ecology,1983,70:91～97
[30]Gao K, Ji Y, Aruga Y. Relationshipo f CO2concentrations to photosynthesis of intertidalmacroalgae during emersion. Hydrobiologia,1999,398:355～359
[31]Raffaelli DG, Raven J, Poole L. Ecological impact of macroaglal blooms. OceanographyMarine Biology Annuual Review,1998,36:97～125
[32]Hiraoka M, Dan A, Shimada S etc. Different life histories of Enteromorpha prolifera (Ulvales,Chlorophyta) from four rivers on Shikoku Island, Japan. Phycologia,2003,42(3):275～284
[33]王晓坤,马家海,叶道才等.浒苔(Enteromorpha prolifera)生活史的初步研究.海洋通报,2007,26(5):112～116
[34]林阿朋,王建伟,阎斌伦,等.浒苔Enteromorpha prolifera组织培养初步研究.生态科学,2006,25(4):320～324
[35]Lee TF, Wichroski M. Polar regeneration in Enteromorpha prolifera. Phycologia,1996,32:2
[36]孙修涛,王翔宇,王文俊,等.绿潮中浒苔的抗逆能力和药物灭杀效果初探.海洋水产研究,2008,29(5):130～136
[37]Reed RH, Russell G. Salinity fluctuations and their influence on”bottle brush”morphogenesis in Enteromorpha intestinalis (L.) Link. European Journal of Phycology,1978,13:149～153
[38]Valiela I, McClelland J, Hauxwell Jl etc. Macroalgal blooms in shallow estuaries: controlsand ecophysiological and ecosystem consequences. Limnology and Oceanography,1997,42:1105～1118
[39]叶乃好,张晓雯,毛玉泽等.黄海绿潮浒苔（Enteromorpha prolifera）生活史的初步研究.中国水产科学,2008,15(5):853～559
[40]Lotze HZ, Schram W, Schories D etc. Control of macroalgal blooms at early developmentalstages: Pilayella littoralis versus Enteromorpha spp. Oecologia.1999,119:46～54
[41]Lotze HK, Worm B, Sommer U. Propagule banks, herbivory and nutrient supply controlpopulation development and dominance patterns in macroalgal blooms. Oikos.2000,85:46～58
[42]Worm B, Heike K, Sommer U. Algal propagules banks modify competition, consumer andresource control on Baltic rocky shores. Oecologia,2001,128:281～293
[43]Camey LT, Edwards MS. Cryptic process in the sea: a review of delayed development in themicroscopic life stages of marine macroalgae. Algae.2006,21:161～168
[44]Dan A, Ohno M, Matuoka M. Cultivation of the green alga Enteromorpha prolifera usingchopped tissue for artificial seeding. Suisan Zoshoku.1997,45:5～8
[45]Kamermans P, Malta EJ, Verschuure JM etc. Role of cold resistance and burial for wintersurvival and spring initiation of an Ulva spp.(Chlorophyta) bloom in a eutrophic lagoon(Veerse Meer lagoon, The Netherlands). Marine Biology (Berlin).1998,131:45～51
[46]Zhang XW, Wang HX, Mao YZ etc. Somatic cells serve as potential propagule bank ofEnteromorpha prolifera forming a green tide in the Yellow Sea, China. Journal of ApplliedPhycology,2010,22:173～180
[47]Matsukawa Y, Umebayashi O. Ulva pertusa biomass growth rate and its limiting factors inan intertidal flat. Bull Tokai Reg Fish Res Lab,1988,126:25～35
[48]梁宗英,林祥志,马牧,张静,闫晓波,刘涛.浒苔漂流聚集绿潮现象的初步分析.中国海洋大学学报,2008,38(4):601～604
[49]Foster GL. Indications regarding the source of combined nitrogen for Ulva laetuca. Annalsof the Missouri Botanical Garden,1914, I:229～235
[50]Villares R, Carballeira A. Nutrient Limitation in Macroalgae (Ulva and Enteromorpha)from the Rias Baixas (NW Spain). Marine Ecology,2004,25(3):225～243
[51]Naldi M, Viarolinaldi P. Nature uptake and storage in the seaweed Ulva rigida C．Agardh inrelation to nitrate availability and thallus nitrate content in a eutrophic coastal lagoon (SaccadiGoro, Po River Delta, Italy). Joumal of Experimental Marine Biology and Ecology,2002,269:65～83
[52]Pedersen MF. Transient ammonium uptake in the macroalga Ulva lactuca (Chlorophyta):nature, regulation, and the consequences for choice of measuring technique. Journal ofPhycology,1994,30:980～986
[53]Bjoernsaeter BR, Wheeler PA. Effeet of nitrogen and phosphorus supply on growth andtissue composition of Ulva fenestrata and Enteromorpha intestinalis (Ulvales, Chlorophyta).Joumal of Phycology,1990,26:603～611
[54]Duke CS, Litaker W, Ramus J. Effects of temperature, nitrogen supply and tissue nitrogenon ammonium uptake rates of the chlorophyte sea weeds Ulva curvata and Codiumdecorticatum. Joumal of Phycology,1989,25:113～120
[55]李瑞香,吴晓文,韦钦胜,王宗灵,李艳,孙萍.不同营养盐条件下浒苔的生长.海洋科学进展,2009,2:211～216
[56]Lapointe BE, Littler MM, Littler DS. Nutrient availability to marine macroalgae insilieiclastic versus carbonate-rich coastal waters. Estuaries and Coasts,1992,15:75～82
[57]Morand P, Merceron M. Macroalgal Population and sustainability. Journal of CoastalResearch,2005,21:1009～1020
[58]Liu DY, Keesing JK, Xing QG etc. World's largest macroalgal bloom caused by expansionof seaweed aquaculture in China. Marine Pollution Bulletin,2009,58:888～895.
[59]Wang BD. Cultural eutrophication in the Changjiang(Yangtze River) plume: History andperspective. Estuarine, Coastal and Shelf Science,2003,68:60～65
[60]徐姗楠,何培民.我国赤潮频发现象分析与海藻栽培生物修复作用.水产学报,2006,30(4):554～561
[61]Levitus S, Antonov JI, Boyer TP etc. Warming of the World Ocean. Science,2000,287:2224～2229
[62]Jiao NZ, Zhang Y, Zeng YH etc. Ecological anomalies in the East China Sea: Impacts of theThree Gorges Dam? Water reseach,2007,41:1287～1293
[63]Sfriso A, Pavoni B, Marcomini A etc. Macroalgae, nutrient cycles and pollutants in theLagoon of Venice. Estuaries and Coasts,1992,15:517～528
[64]Cohen RA, Fong P. Using opportunistic green macroalgae as indicators of nitrogen supplyand sources to estuaries. Ecological Applications,2006,16:1405～1420
[65]de Paula Silva PH, McBride S, de Nys R etc.2008. Integrating filamentous green tidealgae into tropical pond based aquaculture. Aquaculture,284:74～80
[66]NelsonTA, NelsonAV, Tjoelker M. Seasonal and spatial patterns of “green tides”(Ulvoidalgal blooms) and related water qualityparameters in the coastal waters of Washington State,USA. Botanica Marina,2003b,46:263～275
[67]Dahlgren S, Kautsky L. Can different vegetative states in shallow coastal bays of the BalticSea be linked to internal nutrient levels and external nutrient load? Hydrobiologia,2004,514:249～258
[68]Jones M, Pinn E. The impact of a macroalgal mat on benthic biodiversity in Poole Harbour.Marine Pollution Bulletin,2006,53:63～71
[69]Lopes RJ, Pardal MA, Marques JC. Impact of macroalgal blooms and wader predatidalmacroinvertebrates: experimental evidence from the Mondego estuary (Portugal). Journal ofExperimental Marine Biology and Ecology,2000,249:165～179
[70]Berezina NA, Tsiplenkina IG, Pankova ES etc. Dynamics of invertebrate communities instonylittoral of the Neva Estuary(Baltic Sea) under macroalgal blooms and bioinvasions.Transactions of Water Bulliten,2007,1:65～76
[71]Berezina NA. Spatial distribution of macrofauna in a littoral zone with drifting macroalgaein the Neva estuary. Estonian Journal of Ecology,2008,57:198～213
[72]Atkins RP, Deeley DM, McAlpine KW. Managing the aquatic environment. NutrientCycling in Agroecosystems,1993,36:171～175
[73]Rogers RJ, Dake N, Dussart GBJ. Ulva Problem in Kent:1973-2003. Marine PollutionBulletin,2004,49:145～146
[74]Gao S, Chen X,Yi Q etc. Astrategyfor the proliferation of Ulva prolifera, main causativespecies of green tides, with formation of sporangia by fragmentation. PLoS One,2010,5(1):e8571
[75]Linnaeus C. Species Plantarum. Impensis GC Nauk,1800
[76]Silva PC. A review of nomenclatural conservation in the algae from the point of view of thetype method. University of California Press:1952
[77]Bonneau ER. Polymorphic behavior of Ulva Lactuca (Chlorophyta) in axenic eulture.1.Occurrence of Enteromorpha-like plants in haPloid clones. Journal of Phycology,1977,13:133～140
[78]Provasoli L, Pintner IJ. Bacteria induced polymorphism in an axenic laboratory strain of UlvaLactuca (Chlorophyceae). Journal of Phycology,1980,16:196～201
[79]Blomster J, Maggs CA, Stanhope MJ. Molecular and morphological analysis ofEnteromorpha intestinalis and E.compressa (Chlorophyta) in the British Isles. Journal ofphycology,1998,34:319～340
[80]Leskinen E, Alstrom-RapaPort C, Pamilo P. Phylogeographical structure, distribution andgenetic variation of the green algae Ulva intestinalis and U.compressa (Chlorophyta) in theBaltic Sea area. Molecular Ecology,2004,13:2257～2265
[81]Malta EJ, Draisma SGA, Kamermans P. Free-floating Ulva in the southwest Netherlands：species or morphotypes? a morphological，molecular and ecological comparison. EuropeanJournal of Phycology,1999,34:443～454
[82]Tan IH, Blomster J, Hansen G etc. Molecular phylogenetic evidence for a reversiblemorphogenetic switch controlling the gross morphology of two common genera of greenseaweeds, Ulva and Enteromorpha. Molecular Biology and Evolution,1999,16:1011～1018
[83]Shimada S, Hiraoka M, Nabata S etc. Molecular phylogenetic analyses of the Japanese Ulvaand Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva.Phycological Research,2003,51:99～108
[84]Hayden HS, Blomster J, Maggs CA etc. Linnaeus was right all along: Ulva andEnteromorpha are not distinct genera. European Journal of Phycology,2003,38:277～294
[85]Hayden HS, Waaland JR. Phylogenetic systematics of the Ulvaceae (Ulvales, Ulvophyceae)using chloroplast and nuclear DNA sequences. Journal of Phycology,2002,38:1200～121
[86]Shimada S,Yokoyama N, AraiA etc. Phylogeographyof the genus Ulva (Ulvophyceae,Chlorophyta), with special reference to the Japanese freshwater and brackish taxa. Journal ofAppllied Phycology,2008,20:979～989
[87]Coat G, Dion P, Noailles MC etc. Ulva armoricana (UIvales, Chlorophyta) from the coastsof Brittany (France). Ⅱ. Nuclear rDNA ITS sequence analysis. European Journal ofPhycology,1998,33:81～86
[88]Leliaert F, Zhang X, Ye N etc. Identity of the Qingdao algal bloom. Phyeological Research,2009,57:147～151
[89]张晓雯,毛玉泽,庄志猛等.黄海绿潮浒苔的形态学观察及分子鉴定.中国水产科学,2008,15:522～529
[90]Pang SJ, Liu F, Shan TF etc.Tracking the algal origin of the Ulva bloom in the Yellow Seaby a combination of molecular, morphological and physiological analyses. MarineEnvironmental Research,2010,69(4):207～215
[91]OTT F D．Synthetic media and techniques for the axenic cultivation of marine algae andflagellate．Va J Sci，1965，16:205～218
[92]丁兰平,栾日孝.浒苔（Enteromorpha prolifera）的分类鉴定、生境习性及分布.海洋与湖沼,2009,40(1):68～71
[93]汪文俊,王飞久,陈松林等.浒苔ITS区的扩增和分析.海洋水产研究,2008,29(5):124～129
[94]Leskinen E, Pamilo P. Evolution of the ITS sequences of ribosomal DNA in Enteromorpha(Chlorophyceae). Hereditas,1997,126:17～23
[95]Thompson JD, Higgins DG, GibsonTJ. CLUSTALW: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting, position specific gappenalties and weight matrix choice. Nucleic Acids Research,1994,22:4673～4680
[96]Saitou N, Nei M. The neighbor joining method:A new method for reconstructingphylogenetic trees. Molecular Biology and Evolution,1978,4:406～425
[97]Zhang XW, Xu D, Mao YZ etc. Settlement of vegetative of Ulva prolifera confirmed as an425important seed sourse for succession of a large-scale green tide bloom. Limnology andoceanography,2011,56(1):233～242
[98]Mares J, Leskinen E, Sitkowska M etc. True identity of the European reshwater Ulva(Chlorophyta) revealed by a combined molecular and morpholofical approach. Journal ofPhycology,2011,47:1～15.
[99]Kraft LGK, Kraft GT, Waller RF. Investications into southen Australian Ulva(Chlorophyta)taxonomy and molecular phylogeny indicate both cosmopolitanism and cryptic species. Journalof Phycology,2010(46):1257～1277
[100]田晓玲,霍元子,陈丽平等.江苏如东近海绿潮藻分子监测与类群演替分析.科学通报,2011,56(4-5):309～317
[101]徐兆礼,叶属峰,徐韧.2008年中国浒苔灾害成因条件和过程推测.水产学报,2009,33(3):430～437
[102]Keesing JK, Liu DY, Fearns P etc. Inter-and intra-annual patterns of Ulva prolifera green tidesin the Yellow Sea. Marine Pollution Bulletin,2011(62):1169～1182
[103]Hiraoka M, Ichihara K, Zhu WR etc. Culture and hybridization experiments on an Ulva cladeincluding the Qingdao strain blooming in the Yellow Sea. PLoS One,2011(6):1～6
[104]Fletcher RT. The occurrence of green-tide. In: Schramm, W., Nienhuis, P.H.(Eds.), MarineBenthic Vegetation: Recent Changes and the Effects of Eutrophication. Springer Verlag, Berlin,1996,7～43
[105]Ye NH, Zhuang ZM, Jin XS etc. China is on the tracking Enteromorpha spp. forming greentide. Nature Proceedings,2008, hdl:10101/npre.2008.2352.1
[106]Liu DY, Keesing JK, Dong ZJ etc. Recurrence of the world′s largest green-tide in2009inYellow Sea, China: Porphyra yezoensis aquaculture rafts confirmed as nursery for macroalgalblooms. Marine Pollution Bulletin,2010,60(9):1423～1432
[107]应成琦,张婷,李信书等.我国近海浒苔漂浮种类ITS与18S rDNA序列相似性分析.水产学报,2009,33(2):215～219
[108]刘峰.黄海绿潮的成因以及绿潮浒苔的生理生态学和分子系统学研究[博士学位论文].青岛:中国科学院海洋研究所
[109]王建伟,林阿朋,李艳燕等.浒苔(Enteromorpha prolifera)藻体发育的显微观察.生态科学,2006,25(5):400～404
[110]陈群芳.漂浮绿潮藻浒苔繁殖特性与藻体气囊形成研究[硕士学位论文].上海：上海海洋大学
[111]劳方业,符成,陈仲华等.斑茅杂交后代的分子鉴定.甘蔗糖业,2006(1):6～11
[112]闫化学,于杰. DNA条形码技术在植物中的研究现状.植物学报,2010,45(1):102～108
[113]Schindel DE, Miller SE. DNA barcoding a useful tool for taxonomists. Nature,2005,435:17
[114]Ebach MC, Holdrege C. DNA barcoding is no substitute for taxonomy. Nature,2005,434:697–697
[115]Agnarsson I, Kuntner M. Taxonomy in a changing world: seeking solutions for a science incrisis. Systematic Biology,2007,56:531～539
[116]Kress WJ, Wurdack KJ, Zimmer EA etc. Use of DNA barcodes to identify flowering plants.Proc Natl Acad Sci USA,2005,102:8369～8374
[117]Taberlet P, Coissac E, Pompanon F etc. Power and limitations of the chloroplast trnL (UAA)intron for plant DNA barcoding. Nucleic Acids Research,2007,35: e14
[118]Meyer CP, Paulay G. DNA barcoding: error rates based on comprehensive sampling. PLoSBiology,2005,3: e422
[119]Kimura M. A simple method for estimating evolutionary rate of base substitutions throughcomparative studies of nucleotide sequences. Journal of Molecular Evolution,1980,16:111～120
[120]Newmaster SG, Fazekas AJ, Ragupathy S. DNA barcoding in land plants: evaluation of rbcL ina multigene tiered approach. Canadian Journal of Botany,2006,84,335～341
[121]Kress WJ, Erickson DL. A two-locus global DNA barcode for land plants: the coding rbcL genecomplements the non-coding trnH-psbA spacer region. PLoS One,2007,2(6): e508
[122]Sass C, Little DP, Stevenson DW etc. DNA barcoding in the cycadales: testing the potential ofproposed barcoding markers for species identification of cycads. PloS One,2007,2: e1154
[123]Lazaro EM, Sluys R, Pala M etc. Molecular barcoding and phylogeography of sexual andasexual freshwater planarians of the genus Dugesia in the westem Mediterranean(Platyhelminthes, Tricladida, Dugesiidae). Molecular phylogeneties and Evolution,2009,52:835～845