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Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China
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HARALG-901; No. of Pages 9 Harmful Algae xxx (2013) xxx–xxx
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Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China Wei Han a,b, Li-Ping Chen a,b, Jian-Heng Zhang a,b, Xiao-Ling Tian a,b, Liang Hua a,b, Qing He a,b, Yuan-Zi Huo a,b, Ke-Feng Yu a,b, Ding-Ji Shi a,c, Jia-Hai Ma a, Pei-Min He a,b,* a b c
College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China Institutes for Marine Science Research, Shanghai Ocean University, Shanghai 201306, China Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 February 2013 Received in revised form 23 May 2013 Accepted 23 May 2013
In this paper, species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area, Jiangsu Province of China were investigated during 2010–2011. Based on the sequences analysis of nuclear-encoded ITS (including 5.8S rDNA regions) and 5S rDNA spacer regions, dominant species of both attached and free-floating Ulva samples were identified as Ulva compressa, Ulva linza, Ulva prolifera and Ulva flexuosa. Phylogenetic tree based on sequences of ITS and 5S rDNA spacer regions for attached and free-floating Ulva species was constructed, respectively. Species compositions of the Ulva population attached on aquaculture rafts varied with seasons, and U. prolifera was only found on aquaculture rafts in March 2011 during the 2010– 2011 Porphyra yezoensis cultivation season, which had the same sequences of ITS and 5S rDNA spacer regions as that of the dominant species bloomed in the Yellow Sea of China in 2008. Dominant species of the free-floating Ulva population at the early stage of the green tide were U. compressa, U. flexuosa, and U. linza. Free-floating U. prolifera appeared in the middle of May, 2011. ITS sequence similarity rates of U. compressa and U. flexuosa between the attached and free-floating species were 100%. And ITS and 5S rDNA spacer sequences of the attached and the free-floating U. prolifera population also showed no differences. Further study showed that there were two types of free-floating U. prolifera population (Type 5S-A and Type 5S-B) based on 5S rDNA spacer sequences. The present study would provide some useful information for clarifying the outbreak mechanism of green tides occurred in the Yellow Sea, China. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Attached population Free-floating population Green tides ITS sequences Molecular identification Porphyra aquaculture raft Ulva prolifera 5S rDNA spacer regions
1. Introduction The ‘‘green tide’’ was an abnormal phenomenon which was aroused by vast accumulations of floating large, filamentous green algae in the shallow and muddy intertidal zone of the sea (Schories and Reise, 1993). Eutrophication was generally considered to be responsible for the high growth rate and huge biomass accumulation of the harmful green algae (Fletcher, 1996; Morand and Briand, 1996; Schories et al., 2000; Largo et al., 2004; Wang et al., 2008). Green tides occurred in Europe, North America, South America, Asia and Australia and became a troublesome problem worldwide (Morand and Briand, 1996; Herna´ndez et al., 1997; Blomster et al., 2002; Hiraoka et al., 2004; Merceron et al., 2007). In China, the Yellow Sea was continuously attacked by green tides
* Corresponding author at: College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China. Tel.: +86 021 61900467/15692165272; fax: +86 021 61900467. E-mail address: [email protected] (P.-M. He).
from 2007 to 2012, and a great economic losses to marine industries (e.g., fishery aquaculture and tourism) were caused (Sun et al., 2008; Wang et al., 2008; Ye et al., 2008; Liu et al., 2009; Hu et al., 2010; Pang et al., 2010; Zhang et al., 2010, 2012, 2013; Qiao et al., 2011; Luo et al., 2012). In 2011, the green tide extended nearly 19 thousand km2 and covered about 500 km2 of the sea surface in the Yellow Sea (Zhang et al., 2012, 2013). Green tides have drawn a lot of attention from governments and researchers since 2007 in China, however, the causes of green tides have still remained unknown so far. The geographical origin and propagule source of the green tide algae have been considered to be the two key points to clarify the outbreak mechanism of green tides (Lotze et al., 1999; Phillips, 2006; Zhang et al., 2010). So far, there have been three main hypotheses about the origin of green tides in the Yellow Sea as follows: 1. From the eutrophic river estuary and its adjacent sea area. Waters of the Changjiang River estuary have abundant nutrient and favorable temperature and salinity which benefit the growth of Ulva species. Xu et al. (2009) inferred that a large number of Ulva individuals drifted toward the south of the Yellow Sea with
1568-9883/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hal.2013.05.018
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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32.80° 33.00°
N
N Rudong
32.60°
32.00°
S4 Sun Island S3 S2 S1
Yangtze River Esturary
31.00° 120.00°
121.00°
122.00°
E 123.00°
32.40°
32.20° 121.00°
Rudong
121.20°
121.40°
121.60° E 121.80°
Fig. 1. Sampling sites of attached Ulva species on Porphyra aquaculture rafts.
the Changjiang Diluted Water. At first these Ulva individuals gathered together, and then they proliferated at a high rate (Xu et al., 2009). The Ulva population maintained a high growth rate due to favorable conditions of the South Yellow Sea (Ding et al., 2009). 2. From aquaculture ponds in Jiangsu Province, China. Molecular analysis indicated that the algae collected from landbased animal aquaculture ponds mostly resembled the dominating species of bloom-forming algae in 2008 based on ITS sequences and the chloroplast-encoded rbcL gene (Pang et al., 2010). This hypothesis was also in accord with the analysis results of MODIS (moderate-resolution imaging spectroradiometer) on the drift pathway of green tide alga (Qiao et al., 2011). However, Liu et al. (2011) directly denied that the green algal species in shrimp ponds of the Jiangsu coast was the same as the green-tide species in the Yellow Sea using interval amplified polymorphism markers (ISSR). 3. From Porphyra yezoensis aquaculture rafts in Jiangsu Province, China. Many researchers (Liu et al., 2009, 2010; Hu et al., 2010; Keesing et al., 2011) assumed that there was a close relationship between the expansion of P. yezoensis aquaculture in China and the green tides blooming in the Yellow Sea. The coast of Rudong, Jiangsu Province in China is very likely to be the earliest green tide algae floating area (Zhang et al., 2013). Liu et al. (2009, 2010) proposed that the free-floating green tides alga was from Porphyra aquaculture rafts, which was supported by the rapid expansion of Porphyra aquaculture area and the drift pathway of Ulva prolifera during the bloom. However, samples collected from Porphyra aquaculture rafts and the green tides caused species showed certain interspecies divergence based on ITS and rbcL gene sequences analysis (Pang et al., 2010). Though limited by the sampling intervals and survey scope, it is necessary to make further research on the relationship between attached green alga Ulva on P. yezoensis aquaculture rafts and free-floating green tide caused algae. Internal transcribed spacer (ITS) was widely applied in the identification of Ulva species (Tan et al., 1999; Hayden et al., 2003; Coat et al., 1998; Malta et al., 2009), but it could hardly identify the LPP group (Ulva linza-procera-prolifera) (Tian et al.,
2011). Species in the LPP group can be distinguished from each other with 5S rDNA spacer sequences analysis (Shimada et al., 2008; Zhang et al., 2011). In the present study, both ITS and 5S rDNA spacer sequence analysis were used to investigate species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area to clarify the relationship between the green alga attached on Porphyra aquaculture rafts and free-floating green tide algae. 2. Materials and methods 2.1. Collection of attached Ulva samples from Porphyra aquaculture rafts In China, P. yezoensis is cultivated from October to the next April, so the aquaculture rafts are set up in September and removed in the next April. There were four attached Ulva sample collection stations in the Rudong coast, Jiangsu Province, China (see Fig. 1 and Table 1). There were three sites (three Porphyra aquaculture rafts) with 1 km distance between two rafts) for sample collection in each station. One raft included the mooring rope, bamboo and net (Fig. 2). A total of 72 samples were collected in November 2010 and January, March, April, November and December 2011. In the meantime, the sea surface temperature (SST) was measured in each sample collection site every month. 2.2. Collection of free-floating Ulva samples in the Rudong coast By boat monitoring, the earliest free-floasting green algae were found around the Sun Island on April 20, 2011. From April 20 to June 12, sample collections of free-floating green tide algae were carried out in Rudong coastal area, and the information of collection sites was listed in Table 2. Meanwhile, morphology characteristics of the free floating Ulva population were observed and the SST of the survey area was measured.
Table 1 Ulva sampling sites collected from Porphyra aquaculture rafts in Rudong coast, Jiangsu Province. Collection date
Sample No.
GPS
2010.11.19 2011.1.18 2011.3.16 2011.4.16 2011.11.24 2011.12.16
RD1048 RD1053 RD1106 RD1118 RD1145 RD1146
Site Site Site Site
Thallus type Attached 1 2 3 4
N N N N
328270 45.3600 328280 49.6400 328290 59.7800 328310 36.7000
E E E E
1218190 02.4400 1218190 57.1800 1218210 19.2000 1218220 37.1700
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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Fig. 2. (A–D) Growth of ulvoid algae attached on Porphyra aquaculture rafts along the coasts of Rudong, Jiangsu Province: (A) bamboo; (B and D) ropes; (C) nets.
2.3. DNA extraction and PCR amplification
2.4. Phylogenetic analysis
Algal samples collected in the field were cleaned in situ and put in boxes with ice immediately and then transported back to the laboratory within 24 h. The samples were classified roughly according to their morphotypes, and 3–4 morphotypes were identified. Then three single thalli (three reduplicates) in each morphotype were picked up and cleaned several times with sterile seawater. Total DNA was extracted from each Ulva blade using Tiangen DNAsecure Plant Kits (Zhang et al., 2011). The primers for ITS sequences and 5S rDNA spacer sequences PCR amplification were designed according to Coat et al. (1998) and Shimada et al. (2008), respectively. And the PCR amplification program followed the description of Tian et al. (2011). PCR-amplified products of the ITS were sent to Sangon Biotech (Shanghai, China) Co., Ltd. for sequencing. If the samples belonged to the LPP group, 5S rDNA spacer sequences PCR amplification was carried out and then detected by agarose gel electrophoresis, and the shortest DNA fragments of PCR products were sent to Sangon Biotech (Shanghai, China) Co., Ltd for sequencing (Tian et al., 2011).
Sequences of ITS and 5S rDNA regions were aligned using Clustal X (Thompson et al., 1997). Neighbor-joining (NJ) analyses of the aligned sequences were performed with Mega 4.0 (Tamura et al., 2007), and the reliability of each branch was tested by 1000 bootstrap replications. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980).
Table 2 Free-floating Ulva samples collected around Sun Island in Rudong coast at the early stage of the green tide, 2011. Collection date
Sample No.
GPS
Thallus type
4.20 4.23 5.6 5.14 5.24 6.12
RD1120 RD1131 RD1136 RD1137 RD1139 RD1140
N 328310 04.1000 E 1218250 11.9900
Free-floating
3. Results 3.1. Species composition of attached Ulva population on Porphyra aquaculture rafts The ITS and 5S rDNA spacer regions sequences of 70 attached Ulva samples were analyzed. Dominant species compositions of the Ulva population and the SST value of every month were shown in Table 3, and the phylogenetic tree was shown in Fig. 3. With ITS sequences analyses, 13 samples were identified as Ulva compressa and their ITS sequences showed no difference. Thirty-six samples were identified as the LPP group, in which 19 samples were identified as U. prolifera and 17 samples were identified as U. linza with 5S rDNA spacer regions analyses. The similarity rates of ITS sequences between samples from Porphyra aquaculture rafts and reports of U. compressa were almost 100%, and the same thing happened to the LPP group. Twenty-one samples were identified as Ulva flexuosa and the genetic distance ranged from 0.103 to 0.108, and the similarity rate of the ITS sequences between them and U. flexuosa (AB097646.1; HM031176.1), U. flexuosa (HM481178.1) reported by Blomster et al. (2002) was 99.7% and 96.7%, respectively. It indicated that the dominant species attached on
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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Table 3 Species succession of the attached Ulva population on Porphyra aquaculture rafts in Rudong coast, Jiangsu Province. Collection date
2010.11.19
2011.1.18
2011.3.16
2011.4.20
2011.11.24
2011.12.16
SST/8C Composition species
18.0 U. flexuosa
10.0 U. flexuosa U. linza
7.5 U. flexuosa U. linza U. compressa U. prolifera (A)
13.5 U. flexuosa U. linza U. compressa
13.7 U. flexuosa
4.5 U. flexuosa U. linza U. compressa U. prolifera (A, B)
U. prolifera (A, B)
RD111 8c RD104 8b 87
RD114 6d U.flexuosa AB09 764 6.1
U. flexuosa
99 RD105 3c RD105 3b RD110 6c 65
95 RD104 8a U. flexou sa HM031176 .1 RD110 6a RD114 5 RD114 6a
98
U. prolifera FJ026732 .1
82
41 U.linza AJ00020 3.1
LPP
RD114 6b RD110 6d 65 91
68
RD105 3a RD111 8a U.flexuosa HM481178 .1 U.compressa EU93398 1.1 RD110 6b
99 RD114 6c 70 RD111 8b
U. compressa
U.compressa HM03 1178.1 Percursaria percursa AY260 570 .1 Mono stroma grevillei GU06 2560.1
0.05
Fig. 3. NJ tree constructed from the analysis based on ITS sequences of collected samples along Jiangsu coasts and those downloaded from GenBank. Bootstrap values (1000 replicates) (a, b, c, d indicated different composition species, the same as follows).
aquaculture rafts included U. compressa, U. prolifera, U. linza, and U. flexuosa. 3.2. Seasonal variations of species compositions of the attached Ulva population on Porphyra aquaculture rafts Based on ITS and 5S rDNA spacer regions sequence analyses for 70 samples collected from November 2010 to April 2011, as well as November and December of 2011, dominant Ulva species compositions on Porphyra aquaculture rafts in the Rudong coast varied with seasons. U. flexuosa was found during the whole Porphyra aquaculture seasons from November 2010 to April 2011. U. linza appeared on aquaculture rafts later than U. flexuosa and covered from January to April, 2011. U. compressa was collected on aquaculture rafts in March and April 2011. U. prolifera was checked out in March 2011 with low biomass. However, in November 2011, there was huge biomass of attached U. prolifera on Porphyra aquaculutre rafts according to the analysis of ITS and 5S spacer region sequence. Though the similarity rates of ITS sequences were all 100% in the U. prolifera population, they can be separated into
5S-A and 5S-B two groups based on 5S rDNA spacer regions. Sequence divergence between the Type 5S-A and Type 5S-B was 1.9%, due to a 53-bp-sequence deletion and several bases substitution in 5S-A (Fig. 4). U. prolifera was collected in March 2011 belonged to the 5S-A group. The two type sequences were also found in the U. prolifera samples from the big scale green tides at Qingdao coast (Chen, unpublished). And then in December 2011, U. prolifera (including Type 5S-A and Type 5S-B) was still the dominant species and U. compressa, U. linza and U. flexuosa also appeared on Porphyra aquaculutre rafts (Table 3). 3.3. Dominant species composition of the free-floating Ulva population at the early stage of the green tide A total of 38 Ulva individuals collected at the early stage of green tides were analyzed with ITS sequence, and the LPP group was later identified with the 5S rDNA sequences analysis. They were identified to be U. linza (4 samples), U. flexuosa (8 samples), U. compressa (16 samples) and U. prolifera (10 samples), respectively (Table 4).
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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Fig. 4. Two sequence-types (5S-A and 5S-B) of 5S rDNA spacer regions in free-floating U. prolifera samples.
Table 4 Species succession of the free-floating Ulva population in Rudong coast in 2011. Collection date
2011.4.20
2011.4.23
2011.5.6
2011.5.14
2011.5.24
2011.6.12
SST/8C Composition species
13.5 U. compressa U. flexuosa
14.0 U. compressa U. flexuosa U. linza
16.0 U. compressa U. flexuosa U. linza
17.5 U. compressa U. flexuosa
19.3 U. compressa
22.4
U. prolifera (A, B)
3.4. Dominant species succession in the free-floating Ulva population at the early stage of the green tide In 2011, the earliest sporadic floating Ulva population was found around the Sun Island in Rudong coast on April 20, and the SST was 13.5 8C. With ITS and 5S rDNA spacer sequences analysis, these earliest floating Ulva species were identified as U. flexuosa and U. compressa. On May 14 of 2011, the U. Prolifera population presented on the sea surface and the SST was 17.5 8C. And further molecular identification and quantity analysis showed that the ratio of the free-floating U. prolifera population to the whole freefloating green tide algae population was about 3 to10. Then the U. prolifera population reached up to 99% of the whole green tide population on June 12. The results indicated that there was a species succession in the free-floating Ulva population at the early stage of the green tide, and U. compressa, U. flexuosa and U. linza were also important components in the whole free-floating Ulva population before the green tide broke out. 3.5. Comparison between the attached and free-floating Ulva species For comparing the free-floating and attached Ulva population in the early stage of the green tide in April 2011, the phylogenetic tree was constructed based on ITS sequences, and Percursaria percursa and Monostroma grevillei were used as out groups. The NJ tree was shown in Fig. 5. The attached and free-floating U. compressa and U. flexuosa populations gathered together and their similarity rates were 100%. And the unrooted phylogenetic NJ tree for the LPP group was generated with nucleotide sequences of 5S rDNA spacer regions, and U. linza and U. prolifera were identified (Fig. 6).
U. linza U. prolifera (A, B)
U. prolifera (A, B)
4. Discussion 4.1. Seasonal variations of dominant species of the attached Ulva population on Porphyra aquaculture rafts Temperature and irradiance are the most important environmental factors for green macroalgae growth (Nelson et al., 2008). Different Ulva species had different adaptabilities to temperature and irradiance (Taylor et al., 2001; Kim et al., 2011). The seasonal variation of dominant species was due to their different ecological adaptabilities. According to our studies, there was a seasonal variation of Ulva dominant species attached on Porphyra aquaculture rafts at Rudong coast from November 2010 to April 2011. U. flexuosa has tolerance for wide ranges of temperature and irradiance, so it can grow on the Porphyra aquaculture rafts during the whole Porphyra cultivation seasons. The most rapid growth of U. compressa and U. linza is both at 15 8C (Wang et al., 2010b; Taylor et al., 2001). And U. linza also tends to grow well at low irradiance and reduce its growth rate at high irradiance (Taylor et al., 2001). Due to their adaption to the low temperature and/or low irradiance, U. compressa and U. linza were dominant species during March-April and low temperature seasons (from November to next April), respectively. While, U. prolifera shows a broader tolerance of temperature which grows well at both low temperature (5 8C) and high temperature (25 8C), and its specific growth rate (SGR) increases with temperature within 5–25 8C (Fu et al., 2008). However, we found U. prolifera attached on Porphyra aquaculture rafts in Rudong coast only in March, 2011, and until now there is no data showing U. prolifera appeared on Porphyra aquaculture rafts in 2008 and 2009 (Pang et al., 2010; Zhang et al., 2011; Tian et al., 2011). In the
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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200 8-F FJ026732 .1 61 RD114 0
LPP
RD113 7b
49
RD113 9b 96 U.linza AJ00 0203.1 RD113 1c 91
RD113 9a 84
RD113 6c U.flexuosa HM481178.1 U.flexousa HM0311 76.1
59 72
RD112 0b U.flexuosa AB097646 .1
99
U. flexuosa
RD113 7a 96
RD113 1a RD113 6b U.compressa EU933981 .1 RD113 6a
96 U.compressa HM03 117 8.1 RD112 0a
U. compressa
86 RD113 1b RD113 7c RD113 9c Percursaria percursa AY26057 0.1 Mono stroma grevillei GU062560 .1
0.05 Fig. 5. NJ tree constructed from the analysis based on ITS sequences of unattached Ulva samples at the early stage of green tide in Jiangsu coast and attached Ulva samples on the aquaculture raft in April 2011 and those downloaded from GenBank. Bootstrap values (1000 replicates).
next Porphyra auqaculure season (October 2011–April 2012), we found that the U. prolifera population (which bloomed with large scale in Qingdao coast in 2008) almost spread on the whole Porphyra aquaculture rafts with huge biomass. It seemed that U. prolifera were gradually adapting to the local environment in 2010. But it was perplex that there was no the U. prolifera population spreading on Porphyra aquaculture rafts in April 2011. 4.2. Variation of dominant species compositions of the free-floating Ulva population at the early stage of the green tide Different adaptive abilities to irradiance and temperature resulted in the species succession of the free-floating population during the early stage of the green tide. The earliest sporadic freefloating Ulva species were U. compressa and U. flexuosa which collected in Rudong coast on April 20, 2011, and the local seawater temperature was about 13.5 8C. The free-floating U. flexuosa disappeared when the SST was 18 8C above, so did the freefloating U. compressa when the SST increased to 20 8C (Table 4). Though the optimum temperature for the growth of U. linza was 15 8C (Taylor et al., 2001), the free-floating U. linza were still found at the end of May 2011 when the SST was around 20 8C. U. linza disappeared in the middle of July 2011 probably due to its low irradiance tolerance. For U. prolifera could survive when the temperature increased up to 30 8C and be exposed to extremely high irradiance levels (Wang et al., 2007), Free-floating U. prolifera samples were collected from 14 May to 12 June when the SST was
higher than 17 8C (Table 4) at Rudong Coast, as well as during the summer time in Qingdao coast. The optimum seasons for the growth of U. prolifera ranged from mid of May and to end of June (Liu et al., 2009). In conclusion, there was a species succession for the free-floating Ulva population at the early stage of the green tide, they appeared in turn: U. compressa, U. flexuosa, U. linza and U. prolifera. Similar species succession at the early stage of the green tide algae was also found in 2009 and 2010 (Tian et al., 2011). Freefloating U. compressa, U. flexuosa and the LPP group first appeared in Rudong coastal area in April 2009, and U. prolifera which had the same ITS sequences and 5S rDNA spacer region as that of the dominant species bloomed in 2008 became the most dominant species gradually (Tian et al., 2011). After the middle of May, U. compressa, U. flexuosa and U. linza grew at low rates in higher temperature and irradiance conditions. U. prolifera proliferated at a high rate with the SST increasing (Fu et al., 2008). According to our ship-monitoring, the earlier freefloating U. prolifera patches were firstly found around Sun Island, Rudong coast. For the influence of northwards current, they moved to the north. In July, when the SST was up to 30 8C, the free-floating U. prolifera population almost disappeared in Rudong coast. 4.3. The origin of the dominant species of the large scale green tide in the Yellow Sea of China The Green tide occurred every year in the Yellow Sea of China since 2007. However, no convincing biological evidence of the algal
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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RD1137a RD1139a
5S-A
59 RD1106a RD1145a 71
RD1140a RD1146a
100
U.prolifera HM031149 .1 U.prolifera HM58478 6.1 RD1137b 60
5S-B
RD1139b 94 RD1140b RD1145b 98 U.linza HM031140 .1 RD1106b
47 RD1139 62 RD1139c
U.linza AB298 685 .1
U. linza
RD1146b RD1136 62
RD1131 RD1053
0.005 Fig. 6. NJ tree constructed from the analysis based on 5S rDNA spacer sequences of attached samples in March, 2011 and free-floating Ulva samples at the early stage of green tide in Jiangsu coast and those downloaded from GenBank. Bootstrap values (1000 replicates).
source is available so far. Accurate localization of the original algal source is the first step in understanding this large-scale green tide and finding possible solutions to the problems. Based on morphological characteristics and molecular phylogenetic analysis, the dominant species of the green tide algae was identified as U. prolifera (Sun et al., 2008; Ding et al., 2009; Zhao et al., 2011, 2012; Wang et al., 2010a). However, instead of U. prolifera, U. flexuosa and U. compressa were firstly found on the sea surface in the earlier stage for green tides bloomed in 2009 and 2010 based on the results of both field investigation and laboratory identification (Tian et al., 2011). In this paper, the similar species succession was also found in Rudong coast in 2011. Sequence identities of U. flexuosa and U. compressa between attached and free-floating population were 100% indicating that the early freefloating green tide algae might originate from the Porphyra aquaculture raft. According to satellite remote sensing observation, it was confirmed that a large scale of green tide algae originated from Subei inshore and then floated to Qingdao sea area (Qiao et al., 2011). Therefore, attached green algae on Porphyra aquaculture rafts along Sunbei seashore were considered as the source of the green tide algae (Liu et al., 2009, 2010; Hu et al., 2010; Keesing et al., 2011). In 2010, the nori cultivation area in Rudong coast was up to 84.67 km2 (Zhang et al., 2012, 2013), and the biomass of ulvoid algae attached on Porphyra rafts in Rudong coast was investigated in April 2010. The average biomass on the mooring
ropes, bamboos and nets was 238 g, 183 g and 24 g per net frame, respectively, and the total biomass of ulvoid algae attached on Porphyra rafts was about 3951 t (FW) in Rudong coast (Zhang et al., 2012). Though the attached green tide algae biomass was huge on the Porphyra aquaculture rafts, the molecular evidence was negative. The population of U. prolifera whose ITS and 5S rDNA sequence were the same as the U. prolifera population bloomed in Qingdao coast in 2008 was not found on Porphyra aquaculture rafts in Rudong coast before 2011 (Pang et al., 2010; Zhang et al., 2011; Tian et al., 2011). Only in March 2011, we found U. prolifera attached on Porphyra aquaculture rafts, which had the same ITS and 5S rDNA spacer region sequences as that of the green tide algae U. prolifera bloomed in the Yellow Sea in 2008. Though in November and December 2011, the attached U. prolifera developed as the dominant species population on Porphyra aquaculture rafts, it is difficult to conclude that green algae on Porphyra aquaculture rafts along Rudong coast is the source of the green tide algae. In addition, reproductive cells of U. prolifera were not found, while reproductive cells of U. linsa, U. flexuosa and U. compressa were found in Rudong coast in 2010 (data is unpublished). Further study is needed to clarify the relationship between the attached Ulva species and the green tide caused species. It indicated that the free-floating population of U. prolifera bloomed in the Yellow Sea with large scale in 2008 adapted to the local ecological environment and gradually became one of the dominant species on Porphyra aquaculture rafts in 2011, which
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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might develop a new source for green tides blooming in Yellow Sea. It seemed that the dominant population of the free-floating U. prolifera have settled down at Porphyra aquaculture rafts, and gradually developed different population types of 5S rDNA spacer region sequences, such as 5S-A and 5S-B. It indicated that we further need to monitor the population succession change for the free-floating population types in U. prolifera carefully, and tracking the origin of the green tide for U. prolifera population bloomed with large scale in Qingdao in 2008 would be very difficult. Acknowledgements This study was supported by the National Ocean Public Welfare Scientific Research Project (201205010 and 201105023), National Science & Technology Supporting Program (2012BAC07B03), Key Laboratory of Integrated Marine Monitoring and Applied Technologies for Harmful Algal Blooms (MATHAB20100202, MATHAB200907), Doctoral Fund of Shanghai Ocean University (A-2400-10-0130) and National Funds for Distinguished Young Scientists of China (SSC10001), Shanghai Universities First-class Disciplines Project, Discipline name: Marine Science. [SS]
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HARALG-901; No. of Pages 9 Harmful Algae xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Harmful Algae journal homepage: www.elsevier.com/locate/hal
Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China Wei Han a,b, Li-Ping Chen a,b, Jian-Heng Zhang a,b, Xiao-Ling Tian a,b, Liang Hua a,b, Qing He a,b, Yuan-Zi Huo a,b, Ke-Feng Yu a,b, Ding-Ji Shi a,c, Jia-Hai Ma a, Pei-Min He a,b,* a b c
College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China Institutes for Marine Science Research, Shanghai Ocean University, Shanghai 201306, China Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 February 2013 Received in revised form 23 May 2013 Accepted 23 May 2013
In this paper, species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area, Jiangsu Province of China were investigated during 2010–2011. Based on the sequences analysis of nuclear-encoded ITS (including 5.8S rDNA regions) and 5S rDNA spacer regions, dominant species of both attached and free-floating Ulva samples were identified as Ulva compressa, Ulva linza, Ulva prolifera and Ulva flexuosa. Phylogenetic tree based on sequences of ITS and 5S rDNA spacer regions for attached and free-floating Ulva species was constructed, respectively. Species compositions of the Ulva population attached on aquaculture rafts varied with seasons, and U. prolifera was only found on aquaculture rafts in March 2011 during the 2010– 2011 Porphyra yezoensis cultivation season, which had the same sequences of ITS and 5S rDNA spacer regions as that of the dominant species bloomed in the Yellow Sea of China in 2008. Dominant species of the free-floating Ulva population at the early stage of the green tide were U. compressa, U. flexuosa, and U. linza. Free-floating U. prolifera appeared in the middle of May, 2011. ITS sequence similarity rates of U. compressa and U. flexuosa between the attached and free-floating species were 100%. And ITS and 5S rDNA spacer sequences of the attached and the free-floating U. prolifera population also showed no differences. Further study showed that there were two types of free-floating U. prolifera population (Type 5S-A and Type 5S-B) based on 5S rDNA spacer sequences. The present study would provide some useful information for clarifying the outbreak mechanism of green tides occurred in the Yellow Sea, China. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Attached population Free-floating population Green tides ITS sequences Molecular identification Porphyra aquaculture raft Ulva prolifera 5S rDNA spacer regions
1. Introduction The ‘‘green tide’’ was an abnormal phenomenon which was aroused by vast accumulations of floating large, filamentous green algae in the shallow and muddy intertidal zone of the sea (Schories and Reise, 1993). Eutrophication was generally considered to be responsible for the high growth rate and huge biomass accumulation of the harmful green algae (Fletcher, 1996; Morand and Briand, 1996; Schories et al., 2000; Largo et al., 2004; Wang et al., 2008). Green tides occurred in Europe, North America, South America, Asia and Australia and became a troublesome problem worldwide (Morand and Briand, 1996; Herna´ndez et al., 1997; Blomster et al., 2002; Hiraoka et al., 2004; Merceron et al., 2007). In China, the Yellow Sea was continuously attacked by green tides
* Corresponding author at: College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China. Tel.: +86 021 61900467/15692165272; fax: +86 021 61900467. E-mail address: [email protected] (P.-M. He).
from 2007 to 2012, and a great economic losses to marine industries (e.g., fishery aquaculture and tourism) were caused (Sun et al., 2008; Wang et al., 2008; Ye et al., 2008; Liu et al., 2009; Hu et al., 2010; Pang et al., 2010; Zhang et al., 2010, 2012, 2013; Qiao et al., 2011; Luo et al., 2012). In 2011, the green tide extended nearly 19 thousand km2 and covered about 500 km2 of the sea surface in the Yellow Sea (Zhang et al., 2012, 2013). Green tides have drawn a lot of attention from governments and researchers since 2007 in China, however, the causes of green tides have still remained unknown so far. The geographical origin and propagule source of the green tide algae have been considered to be the two key points to clarify the outbreak mechanism of green tides (Lotze et al., 1999; Phillips, 2006; Zhang et al., 2010). So far, there have been three main hypotheses about the origin of green tides in the Yellow Sea as follows: 1. From the eutrophic river estuary and its adjacent sea area. Waters of the Changjiang River estuary have abundant nutrient and favorable temperature and salinity which benefit the growth of Ulva species. Xu et al. (2009) inferred that a large number of Ulva individuals drifted toward the south of the Yellow Sea with
1568-9883/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hal.2013.05.018
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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32.80° 33.00°
N
N Rudong
32.60°
32.00°
S4 Sun Island S3 S2 S1
Yangtze River Esturary
31.00° 120.00°
121.00°
122.00°
E 123.00°
32.40°
32.20° 121.00°
Rudong
121.20°
121.40°
121.60° E 121.80°
Fig. 1. Sampling sites of attached Ulva species on Porphyra aquaculture rafts.
the Changjiang Diluted Water. At first these Ulva individuals gathered together, and then they proliferated at a high rate (Xu et al., 2009). The Ulva population maintained a high growth rate due to favorable conditions of the South Yellow Sea (Ding et al., 2009). 2. From aquaculture ponds in Jiangsu Province, China. Molecular analysis indicated that the algae collected from landbased animal aquaculture ponds mostly resembled the dominating species of bloom-forming algae in 2008 based on ITS sequences and the chloroplast-encoded rbcL gene (Pang et al., 2010). This hypothesis was also in accord with the analysis results of MODIS (moderate-resolution imaging spectroradiometer) on the drift pathway of green tide alga (Qiao et al., 2011). However, Liu et al. (2011) directly denied that the green algal species in shrimp ponds of the Jiangsu coast was the same as the green-tide species in the Yellow Sea using interval amplified polymorphism markers (ISSR). 3. From Porphyra yezoensis aquaculture rafts in Jiangsu Province, China. Many researchers (Liu et al., 2009, 2010; Hu et al., 2010; Keesing et al., 2011) assumed that there was a close relationship between the expansion of P. yezoensis aquaculture in China and the green tides blooming in the Yellow Sea. The coast of Rudong, Jiangsu Province in China is very likely to be the earliest green tide algae floating area (Zhang et al., 2013). Liu et al. (2009, 2010) proposed that the free-floating green tides alga was from Porphyra aquaculture rafts, which was supported by the rapid expansion of Porphyra aquaculture area and the drift pathway of Ulva prolifera during the bloom. However, samples collected from Porphyra aquaculture rafts and the green tides caused species showed certain interspecies divergence based on ITS and rbcL gene sequences analysis (Pang et al., 2010). Though limited by the sampling intervals and survey scope, it is necessary to make further research on the relationship between attached green alga Ulva on P. yezoensis aquaculture rafts and free-floating green tide caused algae. Internal transcribed spacer (ITS) was widely applied in the identification of Ulva species (Tan et al., 1999; Hayden et al., 2003; Coat et al., 1998; Malta et al., 2009), but it could hardly identify the LPP group (Ulva linza-procera-prolifera) (Tian et al.,
2011). Species in the LPP group can be distinguished from each other with 5S rDNA spacer sequences analysis (Shimada et al., 2008; Zhang et al., 2011). In the present study, both ITS and 5S rDNA spacer sequence analysis were used to investigate species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area to clarify the relationship between the green alga attached on Porphyra aquaculture rafts and free-floating green tide algae. 2. Materials and methods 2.1. Collection of attached Ulva samples from Porphyra aquaculture rafts In China, P. yezoensis is cultivated from October to the next April, so the aquaculture rafts are set up in September and removed in the next April. There were four attached Ulva sample collection stations in the Rudong coast, Jiangsu Province, China (see Fig. 1 and Table 1). There were three sites (three Porphyra aquaculture rafts) with 1 km distance between two rafts) for sample collection in each station. One raft included the mooring rope, bamboo and net (Fig. 2). A total of 72 samples were collected in November 2010 and January, March, April, November and December 2011. In the meantime, the sea surface temperature (SST) was measured in each sample collection site every month. 2.2. Collection of free-floating Ulva samples in the Rudong coast By boat monitoring, the earliest free-floasting green algae were found around the Sun Island on April 20, 2011. From April 20 to June 12, sample collections of free-floating green tide algae were carried out in Rudong coastal area, and the information of collection sites was listed in Table 2. Meanwhile, morphology characteristics of the free floating Ulva population were observed and the SST of the survey area was measured.
Table 1 Ulva sampling sites collected from Porphyra aquaculture rafts in Rudong coast, Jiangsu Province. Collection date
Sample No.
GPS
2010.11.19 2011.1.18 2011.3.16 2011.4.16 2011.11.24 2011.12.16
RD1048 RD1053 RD1106 RD1118 RD1145 RD1146
Site Site Site Site
Thallus type Attached 1 2 3 4
N N N N
328270 45.3600 328280 49.6400 328290 59.7800 328310 36.7000
E E E E
1218190 02.4400 1218190 57.1800 1218210 19.2000 1218220 37.1700
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Fig. 2. (A–D) Growth of ulvoid algae attached on Porphyra aquaculture rafts along the coasts of Rudong, Jiangsu Province: (A) bamboo; (B and D) ropes; (C) nets.
2.3. DNA extraction and PCR amplification
2.4. Phylogenetic analysis
Algal samples collected in the field were cleaned in situ and put in boxes with ice immediately and then transported back to the laboratory within 24 h. The samples were classified roughly according to their morphotypes, and 3–4 morphotypes were identified. Then three single thalli (three reduplicates) in each morphotype were picked up and cleaned several times with sterile seawater. Total DNA was extracted from each Ulva blade using Tiangen DNAsecure Plant Kits (Zhang et al., 2011). The primers for ITS sequences and 5S rDNA spacer sequences PCR amplification were designed according to Coat et al. (1998) and Shimada et al. (2008), respectively. And the PCR amplification program followed the description of Tian et al. (2011). PCR-amplified products of the ITS were sent to Sangon Biotech (Shanghai, China) Co., Ltd. for sequencing. If the samples belonged to the LPP group, 5S rDNA spacer sequences PCR amplification was carried out and then detected by agarose gel electrophoresis, and the shortest DNA fragments of PCR products were sent to Sangon Biotech (Shanghai, China) Co., Ltd for sequencing (Tian et al., 2011).
Sequences of ITS and 5S rDNA regions were aligned using Clustal X (Thompson et al., 1997). Neighbor-joining (NJ) analyses of the aligned sequences were performed with Mega 4.0 (Tamura et al., 2007), and the reliability of each branch was tested by 1000 bootstrap replications. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980).
Table 2 Free-floating Ulva samples collected around Sun Island in Rudong coast at the early stage of the green tide, 2011. Collection date
Sample No.
GPS
Thallus type
4.20 4.23 5.6 5.14 5.24 6.12
RD1120 RD1131 RD1136 RD1137 RD1139 RD1140
N 328310 04.1000 E 1218250 11.9900
Free-floating
3. Results 3.1. Species composition of attached Ulva population on Porphyra aquaculture rafts The ITS and 5S rDNA spacer regions sequences of 70 attached Ulva samples were analyzed. Dominant species compositions of the Ulva population and the SST value of every month were shown in Table 3, and the phylogenetic tree was shown in Fig. 3. With ITS sequences analyses, 13 samples were identified as Ulva compressa and their ITS sequences showed no difference. Thirty-six samples were identified as the LPP group, in which 19 samples were identified as U. prolifera and 17 samples were identified as U. linza with 5S rDNA spacer regions analyses. The similarity rates of ITS sequences between samples from Porphyra aquaculture rafts and reports of U. compressa were almost 100%, and the same thing happened to the LPP group. Twenty-one samples were identified as Ulva flexuosa and the genetic distance ranged from 0.103 to 0.108, and the similarity rate of the ITS sequences between them and U. flexuosa (AB097646.1; HM031176.1), U. flexuosa (HM481178.1) reported by Blomster et al. (2002) was 99.7% and 96.7%, respectively. It indicated that the dominant species attached on
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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Table 3 Species succession of the attached Ulva population on Porphyra aquaculture rafts in Rudong coast, Jiangsu Province. Collection date
2010.11.19
2011.1.18
2011.3.16
2011.4.20
2011.11.24
2011.12.16
SST/8C Composition species
18.0 U. flexuosa
10.0 U. flexuosa U. linza
7.5 U. flexuosa U. linza U. compressa U. prolifera (A)
13.5 U. flexuosa U. linza U. compressa
13.7 U. flexuosa
4.5 U. flexuosa U. linza U. compressa U. prolifera (A, B)
U. prolifera (A, B)
RD111 8c RD104 8b 87
RD114 6d U.flexuosa AB09 764 6.1
U. flexuosa
99 RD105 3c RD105 3b RD110 6c 65
95 RD104 8a U. flexou sa HM031176 .1 RD110 6a RD114 5 RD114 6a
98
U. prolifera FJ026732 .1
82
41 U.linza AJ00020 3.1
LPP
RD114 6b RD110 6d 65 91
68
RD105 3a RD111 8a U.flexuosa HM481178 .1 U.compressa EU93398 1.1 RD110 6b
99 RD114 6c 70 RD111 8b
U. compressa
U.compressa HM03 1178.1 Percursaria percursa AY260 570 .1 Mono stroma grevillei GU06 2560.1
0.05
Fig. 3. NJ tree constructed from the analysis based on ITS sequences of collected samples along Jiangsu coasts and those downloaded from GenBank. Bootstrap values (1000 replicates) (a, b, c, d indicated different composition species, the same as follows).
aquaculture rafts included U. compressa, U. prolifera, U. linza, and U. flexuosa. 3.2. Seasonal variations of species compositions of the attached Ulva population on Porphyra aquaculture rafts Based on ITS and 5S rDNA spacer regions sequence analyses for 70 samples collected from November 2010 to April 2011, as well as November and December of 2011, dominant Ulva species compositions on Porphyra aquaculture rafts in the Rudong coast varied with seasons. U. flexuosa was found during the whole Porphyra aquaculture seasons from November 2010 to April 2011. U. linza appeared on aquaculture rafts later than U. flexuosa and covered from January to April, 2011. U. compressa was collected on aquaculture rafts in March and April 2011. U. prolifera was checked out in March 2011 with low biomass. However, in November 2011, there was huge biomass of attached U. prolifera on Porphyra aquaculutre rafts according to the analysis of ITS and 5S spacer region sequence. Though the similarity rates of ITS sequences were all 100% in the U. prolifera population, they can be separated into
5S-A and 5S-B two groups based on 5S rDNA spacer regions. Sequence divergence between the Type 5S-A and Type 5S-B was 1.9%, due to a 53-bp-sequence deletion and several bases substitution in 5S-A (Fig. 4). U. prolifera was collected in March 2011 belonged to the 5S-A group. The two type sequences were also found in the U. prolifera samples from the big scale green tides at Qingdao coast (Chen, unpublished). And then in December 2011, U. prolifera (including Type 5S-A and Type 5S-B) was still the dominant species and U. compressa, U. linza and U. flexuosa also appeared on Porphyra aquaculutre rafts (Table 3). 3.3. Dominant species composition of the free-floating Ulva population at the early stage of the green tide A total of 38 Ulva individuals collected at the early stage of green tides were analyzed with ITS sequence, and the LPP group was later identified with the 5S rDNA sequences analysis. They were identified to be U. linza (4 samples), U. flexuosa (8 samples), U. compressa (16 samples) and U. prolifera (10 samples), respectively (Table 4).
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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Fig. 4. Two sequence-types (5S-A and 5S-B) of 5S rDNA spacer regions in free-floating U. prolifera samples.
Table 4 Species succession of the free-floating Ulva population in Rudong coast in 2011. Collection date
2011.4.20
2011.4.23
2011.5.6
2011.5.14
2011.5.24
2011.6.12
SST/8C Composition species
13.5 U. compressa U. flexuosa
14.0 U. compressa U. flexuosa U. linza
16.0 U. compressa U. flexuosa U. linza
17.5 U. compressa U. flexuosa
19.3 U. compressa
22.4
U. prolifera (A, B)
3.4. Dominant species succession in the free-floating Ulva population at the early stage of the green tide In 2011, the earliest sporadic floating Ulva population was found around the Sun Island in Rudong coast on April 20, and the SST was 13.5 8C. With ITS and 5S rDNA spacer sequences analysis, these earliest floating Ulva species were identified as U. flexuosa and U. compressa. On May 14 of 2011, the U. Prolifera population presented on the sea surface and the SST was 17.5 8C. And further molecular identification and quantity analysis showed that the ratio of the free-floating U. prolifera population to the whole freefloating green tide algae population was about 3 to10. Then the U. prolifera population reached up to 99% of the whole green tide population on June 12. The results indicated that there was a species succession in the free-floating Ulva population at the early stage of the green tide, and U. compressa, U. flexuosa and U. linza were also important components in the whole free-floating Ulva population before the green tide broke out. 3.5. Comparison between the attached and free-floating Ulva species For comparing the free-floating and attached Ulva population in the early stage of the green tide in April 2011, the phylogenetic tree was constructed based on ITS sequences, and Percursaria percursa and Monostroma grevillei were used as out groups. The NJ tree was shown in Fig. 5. The attached and free-floating U. compressa and U. flexuosa populations gathered together and their similarity rates were 100%. And the unrooted phylogenetic NJ tree for the LPP group was generated with nucleotide sequences of 5S rDNA spacer regions, and U. linza and U. prolifera were identified (Fig. 6).
U. linza U. prolifera (A, B)
U. prolifera (A, B)
4. Discussion 4.1. Seasonal variations of dominant species of the attached Ulva population on Porphyra aquaculture rafts Temperature and irradiance are the most important environmental factors for green macroalgae growth (Nelson et al., 2008). Different Ulva species had different adaptabilities to temperature and irradiance (Taylor et al., 2001; Kim et al., 2011). The seasonal variation of dominant species was due to their different ecological adaptabilities. According to our studies, there was a seasonal variation of Ulva dominant species attached on Porphyra aquaculture rafts at Rudong coast from November 2010 to April 2011. U. flexuosa has tolerance for wide ranges of temperature and irradiance, so it can grow on the Porphyra aquaculture rafts during the whole Porphyra cultivation seasons. The most rapid growth of U. compressa and U. linza is both at 15 8C (Wang et al., 2010b; Taylor et al., 2001). And U. linza also tends to grow well at low irradiance and reduce its growth rate at high irradiance (Taylor et al., 2001). Due to their adaption to the low temperature and/or low irradiance, U. compressa and U. linza were dominant species during March-April and low temperature seasons (from November to next April), respectively. While, U. prolifera shows a broader tolerance of temperature which grows well at both low temperature (5 8C) and high temperature (25 8C), and its specific growth rate (SGR) increases with temperature within 5–25 8C (Fu et al., 2008). However, we found U. prolifera attached on Porphyra aquaculture rafts in Rudong coast only in March, 2011, and until now there is no data showing U. prolifera appeared on Porphyra aquaculture rafts in 2008 and 2009 (Pang et al., 2010; Zhang et al., 2011; Tian et al., 2011). In the
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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200 8-F FJ026732 .1 61 RD114 0
LPP
RD113 7b
49
RD113 9b 96 U.linza AJ00 0203.1 RD113 1c 91
RD113 9a 84
RD113 6c U.flexuosa HM481178.1 U.flexousa HM0311 76.1
59 72
RD112 0b U.flexuosa AB097646 .1
99
U. flexuosa
RD113 7a 96
RD113 1a RD113 6b U.compressa EU933981 .1 RD113 6a
96 U.compressa HM03 117 8.1 RD112 0a
U. compressa
86 RD113 1b RD113 7c RD113 9c Percursaria percursa AY26057 0.1 Mono stroma grevillei GU062560 .1
0.05 Fig. 5. NJ tree constructed from the analysis based on ITS sequences of unattached Ulva samples at the early stage of green tide in Jiangsu coast and attached Ulva samples on the aquaculture raft in April 2011 and those downloaded from GenBank. Bootstrap values (1000 replicates).
next Porphyra auqaculure season (October 2011–April 2012), we found that the U. prolifera population (which bloomed with large scale in Qingdao coast in 2008) almost spread on the whole Porphyra aquaculture rafts with huge biomass. It seemed that U. prolifera were gradually adapting to the local environment in 2010. But it was perplex that there was no the U. prolifera population spreading on Porphyra aquaculture rafts in April 2011. 4.2. Variation of dominant species compositions of the free-floating Ulva population at the early stage of the green tide Different adaptive abilities to irradiance and temperature resulted in the species succession of the free-floating population during the early stage of the green tide. The earliest sporadic freefloating Ulva species were U. compressa and U. flexuosa which collected in Rudong coast on April 20, 2011, and the local seawater temperature was about 13.5 8C. The free-floating U. flexuosa disappeared when the SST was 18 8C above, so did the freefloating U. compressa when the SST increased to 20 8C (Table 4). Though the optimum temperature for the growth of U. linza was 15 8C (Taylor et al., 2001), the free-floating U. linza were still found at the end of May 2011 when the SST was around 20 8C. U. linza disappeared in the middle of July 2011 probably due to its low irradiance tolerance. For U. prolifera could survive when the temperature increased up to 30 8C and be exposed to extremely high irradiance levels (Wang et al., 2007), Free-floating U. prolifera samples were collected from 14 May to 12 June when the SST was
higher than 17 8C (Table 4) at Rudong Coast, as well as during the summer time in Qingdao coast. The optimum seasons for the growth of U. prolifera ranged from mid of May and to end of June (Liu et al., 2009). In conclusion, there was a species succession for the free-floating Ulva population at the early stage of the green tide, they appeared in turn: U. compressa, U. flexuosa, U. linza and U. prolifera. Similar species succession at the early stage of the green tide algae was also found in 2009 and 2010 (Tian et al., 2011). Freefloating U. compressa, U. flexuosa and the LPP group first appeared in Rudong coastal area in April 2009, and U. prolifera which had the same ITS sequences and 5S rDNA spacer region as that of the dominant species bloomed in 2008 became the most dominant species gradually (Tian et al., 2011). After the middle of May, U. compressa, U. flexuosa and U. linza grew at low rates in higher temperature and irradiance conditions. U. prolifera proliferated at a high rate with the SST increasing (Fu et al., 2008). According to our ship-monitoring, the earlier freefloating U. prolifera patches were firstly found around Sun Island, Rudong coast. For the influence of northwards current, they moved to the north. In July, when the SST was up to 30 8C, the free-floating U. prolifera population almost disappeared in Rudong coast. 4.3. The origin of the dominant species of the large scale green tide in the Yellow Sea of China The Green tide occurred every year in the Yellow Sea of China since 2007. However, no convincing biological evidence of the algal
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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RD1137a RD1139a
5S-A
59 RD1106a RD1145a 71
RD1140a RD1146a
100
U.prolifera HM031149 .1 U.prolifera HM58478 6.1 RD1137b 60
5S-B
RD1139b 94 RD1140b RD1145b 98 U.linza HM031140 .1 RD1106b
47 RD1139 62 RD1139c
U.linza AB298 685 .1
U. linza
RD1146b RD1136 62
RD1131 RD1053
0.005 Fig. 6. NJ tree constructed from the analysis based on 5S rDNA spacer sequences of attached samples in March, 2011 and free-floating Ulva samples at the early stage of green tide in Jiangsu coast and those downloaded from GenBank. Bootstrap values (1000 replicates).
source is available so far. Accurate localization of the original algal source is the first step in understanding this large-scale green tide and finding possible solutions to the problems. Based on morphological characteristics and molecular phylogenetic analysis, the dominant species of the green tide algae was identified as U. prolifera (Sun et al., 2008; Ding et al., 2009; Zhao et al., 2011, 2012; Wang et al., 2010a). However, instead of U. prolifera, U. flexuosa and U. compressa were firstly found on the sea surface in the earlier stage for green tides bloomed in 2009 and 2010 based on the results of both field investigation and laboratory identification (Tian et al., 2011). In this paper, the similar species succession was also found in Rudong coast in 2011. Sequence identities of U. flexuosa and U. compressa between attached and free-floating population were 100% indicating that the early freefloating green tide algae might originate from the Porphyra aquaculture raft. According to satellite remote sensing observation, it was confirmed that a large scale of green tide algae originated from Subei inshore and then floated to Qingdao sea area (Qiao et al., 2011). Therefore, attached green algae on Porphyra aquaculture rafts along Sunbei seashore were considered as the source of the green tide algae (Liu et al., 2009, 2010; Hu et al., 2010; Keesing et al., 2011). In 2010, the nori cultivation area in Rudong coast was up to 84.67 km2 (Zhang et al., 2012, 2013), and the biomass of ulvoid algae attached on Porphyra rafts in Rudong coast was investigated in April 2010. The average biomass on the mooring
ropes, bamboos and nets was 238 g, 183 g and 24 g per net frame, respectively, and the total biomass of ulvoid algae attached on Porphyra rafts was about 3951 t (FW) in Rudong coast (Zhang et al., 2012). Though the attached green tide algae biomass was huge on the Porphyra aquaculture rafts, the molecular evidence was negative. The population of U. prolifera whose ITS and 5S rDNA sequence were the same as the U. prolifera population bloomed in Qingdao coast in 2008 was not found on Porphyra aquaculture rafts in Rudong coast before 2011 (Pang et al., 2010; Zhang et al., 2011; Tian et al., 2011). Only in March 2011, we found U. prolifera attached on Porphyra aquaculture rafts, which had the same ITS and 5S rDNA spacer region sequences as that of the green tide algae U. prolifera bloomed in the Yellow Sea in 2008. Though in November and December 2011, the attached U. prolifera developed as the dominant species population on Porphyra aquaculture rafts, it is difficult to conclude that green algae on Porphyra aquaculture rafts along Rudong coast is the source of the green tide algae. In addition, reproductive cells of U. prolifera were not found, while reproductive cells of U. linsa, U. flexuosa and U. compressa were found in Rudong coast in 2010 (data is unpublished). Further study is needed to clarify the relationship between the attached Ulva species and the green tide caused species. It indicated that the free-floating population of U. prolifera bloomed in the Yellow Sea with large scale in 2008 adapted to the local ecological environment and gradually became one of the dominant species on Porphyra aquaculture rafts in 2011, which
Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018
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might develop a new source for green tides blooming in Yellow Sea. It seemed that the dominant population of the free-floating U. prolifera have settled down at Porphyra aquaculture rafts, and gradually developed different population types of 5S rDNA spacer region sequences, such as 5S-A and 5S-B. It indicated that we further need to monitor the population succession change for the free-floating population types in U. prolifera carefully, and tracking the origin of the green tide for U. prolifera population bloomed with large scale in Qingdao in 2008 would be very difficult. Acknowledgements This study was supported by the National Ocean Public Welfare Scientific Research Project (201205010 and 201105023), National Science & Technology Supporting Program (2012BAC07B03), Key Laboratory of Integrated Marine Monitoring and Applied Technologies for Harmful Algal Blooms (MATHAB20100202, MATHAB200907), Doctoral Fund of Shanghai Ocean University (A-2400-10-0130) and National Funds for Distinguished Young Scientists of China (SSC10001), Shanghai Universities First-class Disciplines Project, Discipline name: Marine Science. [SS]
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Please cite this article in press as: Han, W., et al., Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China. Harmful Algae (2013), http://dx.doi.org/10.1016/j.hal.2013.05.018