全 文 ：第 32卷 第 6期 生 态 科 学 32(6): 703-710
2013年 11月 Ecological Science Nov. 2013
收稿日期：2012-07-04收稿，2012-12-10接受
基金项目：国家海洋局项目 (201205010), (MATHAB00000000)
作者简介：高嵩（1988—），男，硕士，海藻养殖技术及种苗工程。
∗通讯作者，马家海，Email: jhma@shou.edu.cn

高嵩，马家海，Shimada Satoshi，陈斌斌，雎敏，蔡永超，曾宴平. 2010年中国黄海绿潮藻优势种分子生物学鉴定及形态特征研
究[J]. 生态科学, 2013, 32(6): 703-710.
GAO Song, MA Jia-hai, Shimada Satoshi, CHEN Bin-bin, JU Min, CHAI Yong-chao, Zeng Yan-ping. Molecular identification of dominant
green tide algae in Yellow Sea China 2010, and their morphological characteristics[J]. Ecological Science, 2013, 32(6): 703-710.

2010 年中国黄海绿潮藻优势种分子生物学鉴定及形
态特征研究
高嵩 1，马家海 1 *，Shimada Satoshi 2， 陈斌斌 1，雎敏 1，蔡永超 1，曾宴平 1
1. 上海海海洋大学水产与生命学院，201306，上海
2. Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo 112-8610, Japan

【摘要】近五年来，在中国南黄海海域连续爆发了大规模的绿潮灾害。该论文主要针对采集自 2010年绿潮中绿潮优势种藻体，
结合分子生物学方法和主要形态特征进行研究。对样品 ITS序列分析的结果表明，绿潮优势种为 Ulva linza-procera-prolifera类
群（LPP complex）。5S rDNA间隔区序列分析结果显示，样品中 LPP类群的种类主要为 Ulva prolifera，说明绿潮中最为主要
的种类为 Ulva prolifera。此外，在 2010年绿潮的整个过程中，漂浮 Ulva compressa一直存在，而且部分基本形态特征发生了
改变。在绿潮中，Ulva compressa可以生长为气囊状而漂浮于海面，成为其他藻体的附着基质，从而有利于绿潮的迁移和扩大。
尽管发生了形态变化，绿潮的主要种类是 LPP和 Ulva compressa，其中管状中空分枝繁多的 Ulva prolifera是最为主要的。
关键词： 绿潮藻；形态特征；序列分析；气囊状；LPP；Ulva compressa
doi:10.3969/j.issn. 1008-8873.2013.06.005 中图分类号：Q173 文献标识码：A 文章编号：1008-8873(2013)06-703-08
Molecular identification of dominant green tide algae in Yellow Sea China 2010,
and their morphological characteristics
GAO Song1, MA Jia-hai1*, Shimada Satoshi2, CHEN Bin-bin1, JU Min1, CHAI Yong-chao1, Zeng Yan-ping1
1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
2. Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo 112-8610, Japan

Abstract: During the last five years, massive green tide disasters have broken out annually in southern Yellow Sea. This study aimed
mainly at the dominant species collected from 2010 green tide, using molecular methods and morphological characteristics in
combination. The result of ITS sequencing analysis showed that the dominant species was clustered into LPP (Ulva
linza-procera-prolifera ) clade. Further study of LPP clade by using 5S rDNA spacer region showed that the most dominant species was
Ulva prolifera. Besides, Ulva compressa existed during the entire 2010 green tide, experiencing some changes in basic morphology. It
transformed into a bubble-like structure, which enabled it to float on the surface of the water. This kind of thalli can be attached by other
thalli, thus making it conducive to the migration and expansion of green tide. As some morphological characteristics had changed,
species belonging to LPP complex and Ulva compressa were major species in green tide. Of these species, the tubular and intensively
branched thalli of Ulva prolifera were the most dominant.
Key words: Green tide algae; Morphology; Phylogeny; LPP; Ulva compressa.
生 态 科 学 Ecological Science 32卷 704
Introduction

Caused by accumulations of green macro-algae,
green tides usually occur in eutrophic marine
environments, which can substantially alter marine
community structures and functions, and lead to
economic losses in marine industries[1]. This harmful
ecological phenomenon happens worldwide [2-5].
From year 2007 to 2010, massive green tide
disasters broke out annually in southern Yellow Sea.
The green tide that broke out in the coastal region of
Qingdao China between May and July 2008 covered
an area of approximately 13000 km2-30000km2,
occupying about a third of the area that was planned to
be used for the sailing competition. Nearly 1 million
fresh weight tons of algae were removed by more than
16,000 people, using more than 1,600 fishing and
transportation vessels [6, 7].
Morphological characteristics for classification of
blooming causing Ulva algae are limited, and these
characteristics can be changed greatly due to the
influence of environmental conditions and their own
growth characteristics[2, 4, 8], resulting in vague species
boundaries in this genus. Thus morphological
identification of conducive species is difficult.
Therefore, molecular methods are important to
identify the green tide algae. Based on ITS rDNA
and/or rbcL analysis, Zhang et al. [9] and Pang et al. [10]
suggested that the bloom-forming alga was U.
prolifera; Leliaert et al. [11] believed the dominating
species belonged to LPP (Ulva linza-procera-prolifera)
complex; Nonetheless, ITS and rbcL gene are not able
to distinguish species in LPP clade. 5S rDNA spacer
region was proved to be useful for interspecies
phylogenetic analysis of Phaeophyceae [12, 13] and was
used by Shimada et al. [14] for phylogeography of Ulva
in Japan. However, researches using 5S rDNA spacer
region as a molecular marker on green tide algae in
China are rare.
The aims of this study are: firstly, to identify the
algal bloom in Yellow Sea 2010, combined with
several green tide algae samples from 2008 and 2009,
by analyzing ITS and 5S rDNA spacer region; and
secondly, to assess the main morphological
characteristics of the algae.

1 Material and methods
1.1 Sample collection and processing
Samples were mainly collected from the green
tide in 2010. Twelve samples were collected along the
drifting path of the green tide at five different sites:
Rudong, Dafeng, Sheyang, Rizhao and Qingdao (Tab.
1). In addition, cultured samples (LYGf2a, LYGf2b
LYGf3, RDa, RDb) collected from Lianyungang and
Rudong in 2008 and 2009 were also used in the study.
All the samples were refrigerated immediately after
collecting and taken to laboratory within 24 hours.
Single thallus of the dominant species were selected to
be cultured under the following conditions:
temperature 15-20 ℃, salinity 26.9, illumination
100-160 µmol·m-2·s-1, and illumination period:
12L:12D, PES nutrient concentration: 2%.

1.2 Morphological observation
Identification was based on morphology of the
thalli following five criteria [15-17]. Characteristic
criteria were: (1) macro-morphology including branch
situation; (2) cell shape and arrangement; (3) cross
section view; (4) chloroplast position in the cell;
(5)number of pyrenoid per cell.

1.3 DNA extraction, amplification of ITS and 5S
regions, and sequencing
All the samples were washed clean by distilled
water. For the axis, branches were removed as much
as possible. Total DNA was extracted with e.Z.N.A.
Plant DNA mini kit (Omega Bio-tek, Norcross,
Norcross, Georgia, U.S.) according to the specification
of the manufacturer. Polymerase chain reaction (PCR)
amplification of ITS (including ITS1, 5.8S and ITS2)
was taken using primers (forward: 5’-
GTAACAAGGTCTCCGTAGGTG-3’ and reverse: 5’
- AGTTCAGCGGGTAGCCTCAC-3’ ) and thermal
cycling profile (94 ℃ for 3 min, followed by 35 cycles
of 30 s at 94 ℃, 30 s at 60 ℃, 45 s at 72 ℃, and

6期 高嵩，等. 2010年中国黄海绿潮藻优势种分子生物学鉴定及形态特征研究 705

Tab. 1 Specimens of Ulva used in this study, including GenBank accession numbers used for ITS and 5S spacer analyses.
Accession No. Species Samples Date Collection sites Geodetic coordinates
ITS 5S spacer
U. linza LYGf2a 8 Jul. 2008 Lianyungang, Jiangsu n/a JN712227 JN712244
U. prolifera LYGf2b 8 Jul.2008 Lianyungang, Jiangsu n/a JN712228 JN712245
U. prolifera LYGf3 8 Jul. 2008 Lianyungang, Jiangsu n/a JN712229 n/a
U. compressa RDa 9 May. 2009 Rudong, Jiangsu
N 32°30.657
E 121°34.141
JN712230 n/a
U. compressa RDb 9 May. 2009 Rudong, Jiangsu
N 32°30.582
E 121°26.007
JN712231 n/a
U. compressa RDc 28 May. 2010 Rudong, Jiangsu
N 32°30.231
E 121°38.336
JN712232 n/a
U. compressa RDd 28 May. 2010 Rudong, Jiangsu
N 32°30.450
E 121°44.907
JN712233 n/a
U. prolifera RDe 28 May. 2010 Rudong, Jiangsu
N 32°30.240
E 120°42.786
JN712234 JN712246
U. prolifera DFa 29 May. 2010 Dafeng, Jiangsu
N 33º18.581
E 121º07.928
JN712235 JN712247
U. prolifera DFb 29 May. 2010 Dafeng, Jiangsu
N 33º18.581
E 121º07.928
JN712236 JN712248
U. prolifera SYa 12 Jun. 2010 Sheyang, Jiangsu
N 33°50.100
E 120°48.680
JN712237 JN712249
U. prolifera SYb 12 Jun. 2010 Sheyang, Jiangsu
N 33°50.165
E 120°42.450
JN712238 JN712250
U. prolifera RZa 2 Jul. 2010 Rizhao, Shandong
N 35°20.253
E 119°30.662
JN712239 JN712252
U. compressa RZb 2 Jul. 2010 Rizhao, Shandong
N 35°20.253
E 119°30.662
JN712240 n/a
U. prolifera RZc 2 Jul. 2010 Rizhao, Shandong
N 35°20.253
E 119°30.662
JN712241 JN712251
U. prolifera QDa 4 Jul. 2010 Qingdao, Shandong
N 36°03.704,
E 120°19.005
JN712242 JN712253
U. prolifera QDb 4 Jul. 2010 Qingdao, Shandong
N 36°03.704
E 120°19.005
JN712243 JN712244

finally 10 min at 72 ℃). 5S regions were obtained as
described [26]. Amplification products were detected
under UV light after 1.0% agarose gel electrophoresis
and stained with ethidium bromide. The synthesis of
primers, purification and sequencing of PCR products
were carried out by Shanghai Sangon Biologic
Engineering Technology & Service Co., Ltd.

1.4 Phylogenetic analysis
Sequences were edited and aligned using
BioEdit[30] and ClustalX (ver1.83) [31] respectively. The
obtained sequences were searched using National
Center for Biotechnology Information (NCBI) basic
local alignment search tool (BLAST), and downloaded
from GenBank. The construction of neighbor-joining
tree was preformed with the Kimura two-parameter
model using MEGA 4.1 refer to Tamura et al. [32].
Blidingia sp. served as out-group, all bootstrap values
were based on 1000 re-samplings.

2 Results

2.1 Phylogenetic analysis
The neighbor-joining tree based on ITS analysis
is presented in Fig 2. Seventeen samples were
clustered into two large clades. The majority (twelve
samples) clustered into LPP clade and was supported
by 100% bootstrap value, indicating that the dominant
species in 2010 belonged to LPP complex. The
remaining five samples belonged to U. compressa
生 态 科 学 Ecological Science 32卷 706
clade which was supported by 100% bootstrap value.
This result showed that U. compressa existed in green
tide both in 2009 and 2010.
Two amplification products of 5S rDNA spacer
were not used in this study, which were much longer
than the target fragments. The unrooted phylogenetic
tree obtained with N-J analysis of 5S rDNA spacer
region using the reliable data is shown in Fig. 3.
Thirteen samples of the LPP complex were divided
into two groups: The U. linza group including only
one sample (LYGf2a), and the U. prolifera group
composed of the remaining samples. These two groups
were clearly separated supported by 100% bootstrap
values. The result indicated that U. linza was present
in green tide, but the dominant species was U.
prolifera.

2.2 Morphological characteristics
Morphologies of all samples were mainly divided
into four types, and were summarized in Tab. 2 and
shown in Fig. 3.
The dominant free-floating thalli have intensive
branches, with second, third or even more degree
branches in most cases. Generally, diameters of the
branches are shorter than those of the axis. Primary
branches were counted, percentage of which of some
samples could reach to 33.3%, indicating the
considerable large number of false branches.
As for the bubble-like thalli, there was no stringent
classification criterion in traditional morphological
research. Although this kind of thalli took up a
significant part of the green tide, little research has
been carried out.

3 Discussion

Green tides caused by Ulva are common in many
parts of the world. Those occurred in the southern
Yellow Sea in recent years were rare for the scale and
the impact they had. But the number of the species in
the green tides remains difficult to conclude currently.
One of the dominant thalli has some typical
morphological characteristics: tubular and intensively
branched [11, 21], which is relatively similar to U.
prolifera but with a much higher branch frequency. In
this study, this type of thalli has been shown to be U.
prolifera. Among the primary branches, false branches
accounted for about 1/3. Secondary false branches
caused by attachment of other thalli to primary
branches were observed. Large numbers of false
branches are responsible for the intensive branches
and serious interference in identification of green tide
algae. Sample LYGf2a was the offspring of thalli with
this morphology and was identified as U. linza. This
result indicated that although U. linza was not the
dominant species, it could probably attach on other
thalli in the form of false branch. Parts of those false
branches grew from spores which were attached to
thalli; parts of them grew from unreleased spores
directly which were still in sporangium [22]. Those true
or false branches interweaved with each other,
together with the tubular structure the algae possessed,
enabling the pervasive algae to float and expand over
the sea surface.

U. compressa was present in green tide. This
study examined the morphological characteristics of
bubble-like free-floating U. compressa in the Chinese
green tide for the first time. According to the results, it
is easy to get a conclusion that even though the thalli
were free-floating and could be transformed into an
abnormal structure, round cornered cells with
marginal distributed chloroplast is an unchangeable
characteristic of mature U. compressa. This
morphological characteristic is similar to the attached
U. compressa reported in previous studies [17, 23].
Branching used to be a primary morphological
characteristic of U. compressa in traditional taxonomy,
but in the study, some samples with few branches or
none were identified as U. compressa, suggesting that
branching is not effective in identification of
free-floating U. compressa. These five samples were
collected from Rudong, Jiangsu (the starting period)
and Rizhao, Shandong (the late period) respectively,
suggesting that U. compressa probably existed in the
whole drifting process of green tide from south to north.
6期 高嵩，等. 2010年中国黄海绿潮藻优势种分子生物学鉴定及形态特征研究 707


Fig.1 N-J phylogenetic tree based on ITS+5.8S rDNA sequence analysis. Blidingia sp. served as outgroups. Numerals at internal
nodes are bootstrap values for 1,000 replicates greater than 50%
生 态 科 学 Ecological Science 32卷 708

Fig. 2 Unrooted N-J phylogenetic tree based on 5S rDNA spacer region analysis of LPP clade. Numerals at internal nodes are
bootstrap values for 1,000 replicates greater than 50%.

Tab. 2 Main morphological characteristics of samples

Type Macro-morphology Shapes and arrangements of cells
Cross section view

Chloroplast
position in
surface view
Number of
pyrenoids
per cell
1 U. prolifera
(LYGf2b, RDe, DFa,
SYa, SYb, RZc, QDa,
QDb)
Intensively
branched, with
multi-degree
branches
Rectangle to polygonal, irregular
arrangement in mature parts,
longitudinal arrangement in
younger parts and branches
Tubular, cells located in
the central part
Fully
filled/partially
filled in cells

1-2
2 U. prolifera
(LYGf3,RZa,DFb)
Normally
branched, with
obvious axis
Rectangle to polygonal,
longitudinal arrangement in some
part
Tubular, cells located in
the central part
Fully filled

1-2
3 U. linza
(LYGf2a)
No branch
Round to polygonal, irregular
arrangement
Tubular, cells located in
the central part
Fully filled 1, rarely2
4 U. compressa
(RDa,RDb,RDc,RDd,R
Zb)
Bubble-liked, with
few branches or
none
Oval to polygonal, irregular
arrangement
Tubular, cells located in
the central part, some
have thicker cell wall
on the inner side
Hood-shaped,
distributes at
the margin of
the cell
1-2, rarely 3
6期 高嵩，等. 2010年中国黄海绿潮藻优势种分子生物学鉴定及形态特征研究 709

Fig. 3 Morphological characteristics of samples. 1-5 Main morphological characteristics of intensively branched U. prolifera. 1 Wet
habit thallus, 2 Cross section view, 3 Surface view of cell, showing the arrangement, shape, chroloplast and pyrenoids. 4 True branch. 5
False branch. 6-10 Main morphological characteristics of normally branched U. prolifera. 6 Wet habit thallus, 7 Cross section view, 8
Surface view of cell, showing the arrangement, shape, chloroplast and pyrenoids, 9-10 True branch. 11-13 U. linza. 11 Wet habit thallus,
12 Cross section view, 13 Surface view of cell, showing the arrangement, shape, chloroplast and pyrenoids. 14-16 Bubble-like U.
compressa. 14 Wet habit thallus, 15 Cross section view. 16 Surface view of cell, showing the arrangement, shape, chloroplast and
pyrenoids


The bubble-like structure enabled thalli to float on the
surface of water to get more irradiation for growth and
can be attached by other thalli. This is conducive to
the migration and expansion of green tide.
Traditional identification of green macro-algae is
mostly based on classification criterion of attached
species, yet green tide algae are free-floating ones.
This particular growth pattern is the reason for great
changes of morphological characteristics, bringing
difficulties to study. Moreover, these changes are an
internal reason for the explosion of a green tide.
This study revealed that the dominant species in the
green tide were the ones belonging to the LPP
complex (including U. prolifera and U. linza) and U.
compressa. Of these, the tubular and intensively
branched thalli of U. prolifera were the most dominant
species of algal bloom in Yellow Sea. We hope that
this study can be of use in researching taxonomy of
green tide algae, as well as finding the reason and
tracing the source of green tides.
生 态 科 学 Ecological Science 32卷 710
References

[1] Nelson T A, Haberlin K, Nelson A V, Ribarich H,
Hotchkiss R, Van Alstybe K L, Buckingham L, Simunds
D J, Fredrickson K. Ecological and physiological
controls of species composition in green macroalgal
blooms[J]. Ecol, 2008, 89(5): 1287-1298.
[2] Blomster J, Bäck S, Fewer D P, Kiirikki M, Lehvo A,
Maggs C A, Stanhope M J. Novel morphology in
Enteromorpha (Ulvophyceae) forming green tide[J]. Am
J Bot, 2002, 89(11): 1756-1763.
[3] Hiraoka M, Shimada S, Uenosono M, Masuda M. A new
green-tide-forming alga, Ulva ohnoi Hiraoka et Shimada
sp. Nov. (Ulvales, Ulvophyceae) from Japan[J]. Phycol
Res, 2004, 52(1): 17-29.
[4] Malta E J, Draisma S GA, Kamermans P. Free-floating
Ulva (Ulvaceae, Chlorophyta) in the southwest
Netherlands: species or morphotypes? A morphological,
molecular and ecological comparison[J]. Eur J Phycol,
1999, 34(9): 443-454.
[5] Valiela I., Mcclelland J., Hauxwell J., Behr P.J., Hersh D.,
Foreman K., Macroalgal blooms in shallow estuaries:
controls and ecophysiological and ecosystem
consequences[J]. Limnol Oceanogr, 1997, 42(5):
1105-1118.
[6] Leliaert F, Malta E J, Engelen A H, Mineur F, Clerck O
D. Qingdao algal bloom culprit identified[J]. Mar Pollut
Bull, 2008, 56(9): 1515-1516.
[7] Sun S, Wang F, Li C, Qin S, Zhou M, Ding L, Pang S,
Duan D, Wang G, Yin B, Yu R, Jiang P, Liu Z, Zhang G,
Fei X, Zhou M. Emerging challenges: massive green
algal blooms in the Yellow Sea[J]. Nature Proceedings,
2008. http://hdl.handle.net/10101/npre.2008.2266.1
[8] Kim K Y, Ahn Y S, Lee I K. Growth and Morphology of
Enteromorpha linza (L.) J. Ag. and E. prolefera
(MüllerO J. Ag. (Ulvales, Chlorophyceae)[J]. The
Korean Journal of Phycology, 1991, 6(1): 31-45.
[9] Zhang X, Mao Y, Zhuang Z, Liu S, Wang Q, Ye N.
Morphological characteristics and molecular
phylogenetic analysis of green tide Enteromorpha sp.
occurred in the Yellow Sea[J]. Journal of Fishery
Sciences of China, 2008, 15(5): 822-829.
[10] Pang S, Liu F, Shan T, Xu N, Zhang Z, Gao S, Chopin T,
Sun S. Tracking the algal origin of the Ulva bloom in the
Yellow sea by a combination of molecular morphological
and physiological analyses[J]. Mar Environ Res, 2009,
69(4): 207-215.
[11] Leliaert F, Zhang X, Ye N, Malta E J, Engelen A H,
Mineur F, Verbruggen H, Clerck O D. Identity of the
Qingdao algal bloom[J]. Phycol Res, 2009, 57(2):
147-151.
[12] Yotsukura N, Kawai T, Motomura T, Ichimura T.
Tandem 5S ribosomal RNA genes and spacer region
sequences of three Japanese Laminaria species[J]. J Appl
Phycol, 2002, 14: 233-239 .
[13] Yotsukura N, Kawai T, Kawashima S, Ebata H, Ichimura
T. Nucleotide sequence diversity of the 5S rDNA spacer
in the simple blade kelp genera Laminaria, Cymathaere
and Kjellmaniella (Laminariales, Phaeophyceae) from
northern Japan[J]. Phycol Res, 2006, 54(4): 269–279.
[14] Shimada S, Yokoyama N, Arai S, Hiraoka M.
Phylogeography of the genus Ulva (Ulvophyceae,
Chlorophyta), with special reference to the Japanese
freshwater and brackish taxa[J]. J Appl Phycol, 2008, 20:
979–989.
[15] Yoshida T. Marine Algae of Japan[M]. Tokyo: Uchida
Rokakuko Publishing Co, 1998. 33-36.
[16] Zeng C. Seaweeds in Yellow Sea and Bohai Sea of
China[M]. Beijing: Science Press, 2009. 387-392.
[17] Zeng C, Zhang D, Zhang J, Xia E, Xia B, Dong M, Yang
Z. Chinese Economic Seaweeds[M]. Beijing: Science
Press, 1962. 36-50.
[18] Hall T A. BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows
95/98/NT[J]. Nucleic Acids Symp, 1999, 41: 95-98.
[19] Thompson J D, Higgins D G, Gibson T J. CLUSTAL W:
improving the sensitivity of progressive multiple
sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix
choice[J]. Nucleic Acids Res, 1994, 22: 4673-4680.
[20] Tamura K, Dudley J, Nei M, Kumar S. MEGA4:
Molecular Evolutionary Genetics Analysis (MEGA)
Software Version 4.0[J]. Mol Biol Evol, 2007, 24(8):
1596-1599.
[21] Wang J, Jiang P, Cui Y, Li N, Wang M, Lin H, He P, Qin
S. Molecular analysis of green-tide-forming macroalgae
in the Yellow Sea[J]. Aquat Bot, 2010, 93(1): 25-31.
[22] Wang X, Ma J, Ye D, Chen X. Preliminary Study on the
Life History of Enteromorpha prolifera[J]. Mar Sci Bull,
2007, 26(25): 112-116.
[23] Blomster J, Maggs C A, Stanhope M J, Molecular and
morphological analysis of Enteromorpha intestinalis and
E. compressa (Chlorophyta) in the british Isles[J]. J
Phycol, 1998, 34: 319-340.