Chinese Journal of Oceanology and Limnology Vol. 28 No. 4, P. 762-768, 2010 DOI: 10.1007/s00343-010-9069-3 Ulva and Enteromorpha (Ulvaceae, Chlorophyta) from two sides of the Yellow Sea: analysis of nuclear rDNA ITS and plastid rbcL sequence data* WANG Jinfeng (王金锋)†,††,**, LI Nan (李楠)††,†††,**, JIANG Peng (姜鹏)†,***, BOO Sung Min††††, LEE Wook Jae†††††, CUI Yulin (崔玉琳)†,††, LIN Hanzhi (林瀚智)†,††, ZHAO Jin (赵瑾)†,††, LIU Zhengyi (刘正一)††††††, QIN Song (秦松)†,*** † Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China †† Graduate University of Chinese Academy of Sciences, Beijing 100049, China ††† Yantai Institute of Coastal Zone Research for Sustainable Development, Chinese Academy of Sciences, Yantai 264003, China †††† Department of Biology, Chungnam National University, Daejeon 305764, Korea ††††† Jeju Biodiversity Research Institute, Hidi, Jeju 690701, Korea †††††† College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China Received Apr. 13, 2009; revision accepted Jun. 30, 2009 © Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2010 Abstract Ulvacean green seaweeds are common worldwide; they formed massive green tides in the Yellow Sea in recent years, which caused marine ecological problems as well as a social issue. We investigated two major genera of the Ulvaceae, Ulva and Enteromorpha, and collected the plastid rbcL and nuclear ITS sequences of specimens of the genera in two sides of the Yellow Sea and analyzed them. Phylogenetic trees of rbcL data show the occurrence of five species of Enteromorpha (E. compressa, E. flexuosa, E. intestinalis, E. linza and E. prolifera) and three species of Ulva (U. pertusa, U. rigida and U. ohnoi). However, we found U. ohnoi, which is known as a subtropical to tropical species, at two sites on Jeju Island, Korea. Four ribotypes in partial sequences of 5.8S rDNA and ITS2 from E. compressa were also found. Ribotype network analysis revealed that the common ribotype, occurring in China, Korea and Europe, is connected with ribotypes from Europe and China/Japan. Although samples of the same species were collected from both sides of the Yellow Sea, intraspecific genetic polymorphism of each species was low among samples collected worldwide. Keyword: ITS; phylogeography; rbcL; Ulvaceae; warm current; Yellow Sea 1 INTRODUCTION Ulva and Enteromorpha, two major genera in the Ulvaceae, are well known for their many species, global distributions and morphological variation (van den Hoek et al., 1995). More than 140 Ulva and 135 Enteromorpha species are distributed globally in coastal areas, and a few species occur in fresh water (Guiry et al., 2002; Hayden et al., 2003). These two genera are also known for their frequent blooms, which occur worldwide. A huge biomass of unattached fronds, called “green-tide”, originates from fragmentation or reproduction over a short time (Fletcher, 1990; Schories et al., 1993; Valiela et al., 1997; Raffaelli et al., 1998; Malta et al., 1999; Bäck et al., 2000; Blomster et al., 2002). Massive E. prolifera blooms appeared in the Yellow Sea during summer of 2008, and this caused social problems as well as marine ecological issues (Sun et al., 2008). Using molecular analysis, our previous studies implied that the blooms only consisted of this single species (Jiang et al., 2008; Wang et al., 2010). However, worldwide, green-tide forming algae are never limited to one species (Fletcher, 1990; Hiraoka et al., 2003; Sun et al.,  Supported by the National Key Technology Research and Development Program (No. 2008BAC49B01), National High Technology Research and Development Program of China (863 Program) (No. 2009AA10Z106) and the Knowledge Innovation Project of the Chinese Academy of Sciences (No. KZCX2-YW-209) ** Contributed equally as the first author  Corresponding authors: jiangpeng@qdio.ac.cn; sqin@ms.qdio.ac.cn No.4 2008). Investigations on the distribution of Ulvaceae in the Yellow Sea are helpful for recognizing blooms and for preventing blooms from forming. Despite a relatively large number of studies on the taxonomy of Ulva and Enteromorpha in the Yellow Sea (i.e. east coast of China and west coast of Korea) (Dong, 1963; Tseng et al., 1963; Kang, 1966; Lee et al., 1981; Lee et al., 1986; Yoon et al., 1991; Koh et al., 1992; Cho et al., 1996; 1998), there have been very few molecular analyses focused on the phylogeographic structure and genetic variation of the species. The aim of this study was to investigate different species of Ulva and Enteromorpha using plastid rbcL sequence data from specimens of the two genera collected from both sides of the Yellow Sea, and to make reference to occurrences of these genera in different parts of the world. Genetic variation of representative species of the family Ulvaceae in the Yellow Sea were also investigated by analyzing the nuclear rDNA ITS. 2 MATERIALS AND METHODS 2.1 Collection of samples Ulva 763 WANG et al.: Ulvaceae species form the Yellow Sea and Enteromorpha specimens were collected from both sides of the Yellow Sea. They included 18 specimens from 12 sites on the east coast of China, and six specimens from four sites on the west coast of Korea (Table 1, Fig.1). The general morphological taxonomy of Ulva and Enteromorpha specimens was based on the descriptions and references in Algaebase (www.algaebase.org). Living specimens or herbariums were deposited in our lab. 2.2 DNA extraction After transfer to the lab, the fronds were washed 3–4 times with sterilized seawater. The algal tissue was milled with a mortar in liquid nitrogen for 2–3 min. Total DNA was extracted from the fresh material according to the method described in Doyle et al. (1990). DNA quality was examined on 1% TAE agarose gels stained with ethidium bromide. 2.3 PCR amplification, purification and sequencing PCR amplification of the ribosomal DNA containing internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS2) sequence was performed using primers (Table 2) and the thermal cycling profile from Leskinen et al. (1997). The cycle was 6 min initial Table 1 Enteromorpha and Ulva specimens collected for the study Sample Species E. compressa Collection location Collection date S007 P01 (36°15′ N, 120°40′ E) 27 Jun. 2007 S032 E. flexuosa P01 (36°15′ N, 120°40′ E) 3 Aug. 2007 S034 E. compressa P02 (35°23′ N, 119°33′ E) 4 Aug. 2007 S035 E. linza P02 (35°23′ N, 119°33′ E) 4 Aug. 2007 S036 E. compressa P03 (34°05′ N, 120°20′ E) 5 Aug. 2007 S046 E. linza P04 (36°05′ N, 120°28′ E) 8 Aug. 2007 S047 E. compressa P04 (36°05′ N, 120°28′ E) 8 Aug. 2007 S048 E. prolifera P05 (35°57′ N, 120°46′ E) 9 Aug. 2007 S049 E. linza P06 (35°55′ N, 120°09′ E) 9 Aug. 2007 S050 U. rigida P06 (35°55′ N, 120°09′ E) 9 Aug. 2007 S052 E. compressa P07 (35°58′ N, 120°18′ E) 9 Aug. 2007 S053 E. compressa P07 (35°58′ N, 120°18′ E) 9 Aug. 2007 S055 U. pertusa P08 (35°38′ N, 119°53′ E) 9 Aug. 2007 S057 U. pertusa P09 (35°38′ N, 119°48′ E) 9 Aug. 2007 S059 E. intestinalis P10 (35°35′ N, 119°45′ E) 9 Aug. 2007 S068 U. pertusa P11 (36°03′ N, 120°18′ E) 11 Aug. 2007 S078 U. pertusa P12 (36°03′ N, 120°20′ E) 12 Aug. 2007 S090 E. compressa P12 (36°03′ N, 120°20′ E) 14 Aug. 2007 S471 E. compress P13 (36°19′ N, 126°30′ E) 21 Sept. 2008 S472 U. pertusa P13 (36°19′ N, 126°30′ E) 21 Sept. 2008 S473 U. ohnoi P14 (33°30′ N, 126°28′ E) 26 Sept. 2008 S474 U. ohnoi P15 (33°18′ N, 126°50′ E) 25 Sept. 2008 S476 E. flexuosa P16 (35°37′ N, 126°28′ E) 22 Sept. 2008 S477 E. flexuosa P16 (35°37′ N, 126°28′ E) 22 Sept. 2008 764 CHIN. J. OCEANOL. LIMNOL., 28(4), 2010 Fig.1 Sample collection sites denaturing at 94°C, followed by 35 cycles at 94°C for 1 min 10 s, 54°C for 50 s and 72°C for 1 min 30 s, and the final step was 72°C for 10 min. The rbcL gene was amplified using the primers (Table 2) and reaction profile (94°C for 3 min, followed by 35 cycles of 1 min at 94°C, 2 min at 45°C, and 3 min at 65°C) based on the method by Manhart (1994). Amplifications were checked on 1% TAE agarose gels after staining with ethidium bromide. PCR primers were synthesized by Shanghai Sangon Corp. The final fagments for sequencing were purified with a Bioteke PCR Purification Kit (Bioteke Corp., Beijing, China) according to the manufacturer’s instructions. After purification, the amplified products were sequenced by the National Human Genome Center at Shanghai. 2.4 Data analysis Sequences were aligned with published data (Table 3) using ClustalX (Larkin et al., 2007) and edited by BioEdit (Hall, 1999). A sequence from Monostroma nitidum (AF387110) was selected as the outgroup on the basis of its taxonomy and morphology (Bliding, 1968; van den Hoek et al., 1995). Maximum parsimony (MP) analyses of the rbcL data were conducted using the PAUP* 4.0b10 program (Swofford, 2002). All heuristic searches were Vol.28 performed using 1 000 replicates and the following procedure: employ the random addition of taxa, retain only the best tree, hold ten trees at each step, use tree bisection-reconnection (TBR) branch swapping, collapse to zero-length branches, and use MULTREES. Bootstrap support values were calculated using 1 000 replicates with the following options selected: heuristic search; TBR branch swapping; collapse of zero-length branches; and random-sequence-addition with one replicate. Maximum likelihood (ML) phylogenetic analyses were conducted using the RAxML program (Stamatakis, 2006) with the GTR + Γ + I model. We used 300 independent tree inferences using the -# option. Bootstrap values were calculated using 1 000 replicates and the same substitution model. Bayesian analyses were conducted with MrBayes v.3.1.2 (Ronquist et al., 2003) using the Metropoliscoupled Markov chain Monte Carlo (MC3) method with the GTR + Γ + I model for both the combined and individual data sets. For each matrix, six million generations of two independent runs were performed with four chains, and trees were sampled every 100 generations. The burn-in period was identified graphically by tracking the likelihoods at each generation to determine whether they reached a plateau. The 72802 trees sampled at stationarity were used to infer the Bayesian posterior probability. Majority-rule consensus trees were calculated using PAUP*. Reported ITS sequences of E. compressa and E. flexuosa in GenBank were downloaded. Along with the sequence data from this study, ribotype network analyses of the ITS sequences were performed using TCS 1.21 (Clement et al., 2000). Each line between two connecting ribotypes corresponded to one base substitution. 3 RESULTS 3.1 Analysis of rbcL sequences The chloroplast-encoded rbcL gene was sequenced for 16 specimens of Enteromorpha and 8 sides of the Yellow Sea. Phylogenetic trees of the Table 2 PCR primers used in the study Primers 1 FW RV1 RBCL24F RBCL1383R2 2 1 2 Leskinen et al. (1997) Manhart (1994) Sequence Target Direction 5′-TCGTAACAAGGTTTCCGTAGG-3′ ITS Forward 5′-TTCCTTCCGCTTATTGATATGC-3′ ITS Reverse 5′-TAAAGCAGGTGCAGGATTTAAAGC-3′ rbcL Forward 5′-TATCAAATTCAAATTTAATTTCTTTCCAAAC-3′ rbcL Reverse No.4 765 WANG et al.: Ulvaceae species form the Yellow Sea Table 3 Published sequences of Ulva and Enteromorpha used in the study Species E. compressa Source and/or reference Date Ythan Estuary, Aberdeenshire, Scotland (Tan et al., 1999) Ythan Estuary, Aberdeenshire, Scotland (Tan et al., 1999) ITS AF013981 AF013982 Portaferry, Strangford Lough, N. Ireland (Blomster et al., 1998) 18 Apr. 1996 AF035345 Ballyhenry Is., Strangford Lough, N. Ireland (Blomster et al., 1998) 11 Dec. 1995 AF035346 Portaferry, Strangford Lough, N. Ireland (Blomster et al., 1998) 12 Apr. 1996 AF035350 Portaferry, Strangford Lough, N. Ireland (Blomster et al., 1998) 12 Apr. 1996 AF035351 Greatman’s Bay, Co. Galway, Ireland (Blomster et al., 1998) 21 Mar. 1997 AF035352 8 May 2005 AB280824 13 May 1997 AB097641 Aichi, Mikawa Bay, Nizaki, Japan Teguma, Nagasaki Prefecture, Japan (Shimada et al., 2003) rbcL Wembury, Devon, England (Blomster et al., 2000) AF201763 Laganside, Belfast Lough, N. Ireland (Blomster et al., 2000) AF202466 Portaferry, Strangford Lough, N. Ireland (Tan et al., 1999) AJ234302 Skagerrak, Sweden (Leskinen et al., 2004) AJ550764 Westcapelle, Netherlands (Leskinen et al., 2004) AJ550765 Cashel Bay, Co. Galway, Ireland (Loughnane et al., 2008) 5 Apr. 2003 AY255859 EU484397 E. flexuosa Marlborough Sounds, Picton, New Zealand 8 Apr. 2005 EF110051 E. intestinalis Sheep Bay, Valdez-Cordova, Alaska, USA (Hayden et al., 2004) 8 Jul. 1997 AY422552 E. linza Yoshino River, Tokushima Prefecture, Japan (Shimada et al., 2003) 12 Mar. 2000 AB097620 DQ813497 No data E. procera Osaka Bay, Japan (Hayden et al., 2004) E. prolifera Blakely Is., WA, USA (Hayden et al., 2002) AF499670 No data AY422554 U. rigida U. ohnoi U. pertusa Monostroma nitidum 26 Apr. 2000 AY422562 Pelluco Beach, SE of Pucrto Montt, Chile (Hayden et al., 2004) 17 Oct. 2000 AY422564 Kilmore Quay, Co. Wexford, Ireland (Loughnane et al., 2008) 24 Sep. 2002 EU484417 Naha, Okinawa Prefecture, Japan (Hiraoka et al., 2003) 10 Dec. 1998 AB116035 Yura, Hyogo Prefecture, Japan (Hayden et al., 2004) 21 Mar. 2000 AY422549 Sept. 2006 EF372236 No data rbcL data showed the occurrence of five species of Enteromorpha (E. compressa, E. flexuosa, E. intestinalis, E. linza and E. prolifera) and three species of Ulva (U. pertusa, U. rigida, and U. ohnoi). According to the RAxML tree of rbcL AF387110 (Fig.2), the S007, S034, S036, S047, S052, S053 and S090 specimens from the west coast and the S471 specimen from east coast of the Yellow Sea fell into the largest E. compressa clade. S055, S057, S068, S078 and S472 formed the U. pertusa clade; 766 CHIN. J. OCEANOL. LIMNOL., 28(4), 2010 S059 belonged to the E. intestinalis clade; S473 and S474 from Jeju Island resembled the U. ohnoi clade; S050 fell into the U. rigida clade; S035, S046 and S049 formed the E. linza and E. procera (this name is currently regarded as a synonym of E. linza Linnaeus, www.algaebase.org) clade; S048 belonged to the E. prolifera clade; S032, S476 and S477 resembled the E. flexuosa clade. U. ohnoi from the north (S473) and south coast (S474) of Jeju Island, Korea, fell into one group with a rather distinct frond size (Fig.3). The species had a strong sister relationship to U. rigida. As a sister species, E. flexuosa and E. prolifera formed a cluster with E. linza, E. compressa and E. intestinalis. Bootstrap support under ML criteria was strong for all groups. Vol.28 3.2 Analysis of ITS sequences A ribotype network analysis of E. compressa partial sequences of the 5.8S ribosomal RNA gene and internal transcribed spacer 2, including data of specimens from both sides of the Yellow Sea, and reported data of Japanese and European specimens, were performed to show the phylogeographic structure, distribution and genetic variation of Ulvaceae species from the Yellow Sea. (Fig.4). In this network, four ribotypes of E. compressa were found, and the groups differed by 0–2 nucleotide substitutions. There are three variable sites in the 229 bp alignment. A group consisting of 13 specimens from China, Korea and Europe appeared to be basal. A group comprising samples from China and Japan was connected with the basal group and was related to two European lineages from Scotland and Northern Ireland (two European groups). Fig.4 Ribotype network of ITS sequences from E. compressa. Each line between two connecting ribotypes corresponds to one base substitution Fig.2 RAxML tree from rbcL data of Ulva and Enteromorpha The bootstrap values shown above the branches are from 1 000 bootstrap resamplings with RAxML and MP and Bayesian inference When the sequences from E. flexuosa were analyzed, the specimens fell into two ribotypes. One ribotype was formed by the specimens from China, Japan and Korea, and the others formed a Europe ribotype. These two ribotypes differed by only one nucleotide substitution (not shown). 4 DISCUSSION Fig.3 Morphology and habitat of Ulva ohnoi collected from the north (a, S473) and south (b, S474) of Jeju Island, Korea. Scale bars=5 cm (a) and 20 cm (b) The present study demonstrates the distribution of Ulva and Enteromorpha on both sides of the Yellow Sea using molecular analysis. Mass specimens of E. flexuosa, E. compressa and U. pertusa were found, while E. intestinalis, E. linza, E. prolifera and U. rigida were only found in specimens from the west coast of the Yellow Sea. The results strongly support traditional morphological classification. At two sites No.4 WANG et al.: Ulvaceae species form the Yellow Sea on Jeju Island, Korea, U. ohnoi is known as a subtropical to tropical species. However, the morphology and habitat of U. ohnoi showed some differences between thalli collected from opposite sides of Jeju Island. The length of attached thalli from the northern area was several centimeters, and the length of free-floating thalli from the southern area was more than 1 m; in Japan, thalli are 20–50 cm (Hiraoka et al., 2003). Water characteristics and the warm currents around Jeju may influence this variation. The Tsushima Warm Current flows into the Japan Sea and the Yellow Sea Warm Current flows into the Yellow Sea from south of Jeju Island (Ichikawa et al., 2002). Temperature, salinity and nutrition in sea water south of the island are higher than those in the north, especially in autumn when the currents are warm (Lie et al., 2000). As a result, U. ohnoi has a large frond size in the south of Jeju Island. This species of green-tideforming alga was previously described from only southern and western areas of Japan, which are close to the Yellow Sea (Hiraoka et al., 2003). Our study reported the occurrence of U. ohnoi outside of Japan. In addition, the rbcL sequence (AY422562) of putative material of E. procera from Japan is identical to that of the S046 specimen from the west coast of the Yellow Sea and the S046 specimens of E. linza in the present study and from published data (AB097620). Because there are no reports on the species in Japan (Yoshida, 1998), the published sequence of E. procera from Japan should be reexamined. Phylogenetic analyses of plastid rbcL gene sequences divided specimens from both sides of the Yellow Sea into eight groups. Unlike the PPL complex (E. prolifera, E. procera and E. linza) that was formed by analyzing nuclear rDNA ITS sequences in a study by Shimada et al. (2008), in our study, as sister species, E. flexuosa and E. prolifera formed a single cluster. This result was similar to that of Hiraoka et al. (2003), which also revealed a close phylogenetic relationship between E. flexuosa and E. prolifera. Based on their tubular, monostromatic blades, Enteromorpha are typically separated from Ulva by morphological taxonomy (Link, 1820); however, the genera Ulva and Enteromorpha could not be separated from each other by analysis of the rbcL sequence data. This is consistent with the studies by Tan et al. (1999) and Hayden et al. (2003), in which they suggested Ulva and Enteromorpha were not distinct genera. 767 On the basis of partial sequences of 5.8S rDNA and the ITS2 of E. compressa, ribotype network analysis revealed that the common ribotype occurring in China, Korea and Europe, is connected with ribotypes from Europe and China/Japan. However, these four groups differed from each other by 0–2 nucleotide substitutions, and analyses of the E. flexuosa ITS sequence data revealed a similar result. Although samples of the same species were collected from both sides of the Yellow Sea, intraspecific genetic polymorphism of each species was low among samples collected worldwide. 5 ACKNOWLEDGEMENTS We are grateful to K M LEE and G H BOO of Chungnam National University, Korea for their assistance with collecting samples and providing experimental support. References Bäck S, Lehvo A, Blomster J. 2000. Mass occurrence of unattached Enteromorpha intestinalis on the Finnish Baltic Sea coast. Ann. Bot. 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