Estuarine, Coastal and Shelf Science 163 (2015) 96e102

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Estuarine, Coastal and Shelf Science

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Genetic analyses of floating Ulva prolifera in the Yellow Sea suggest a unique ecotype

* * Jin Zhao a, Peng Jiang a, , Song Qin b, , Xiaojie Liu a, c, Zhengyi Liu d, Hanzhi Lin a, Fuchao Li a, Huaxin Chen a, Chunhui Wu a, c a Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China b Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China c University of Chinese Academy of Sciences, Beijing 100049, China d College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China article info abstract

Article history: Large-scale green tides caused by the green algae Ulva prolifera have occurred in the Yellow Sea every Accepted 5 May 2015 summer since 2007. The genetic variation and relationships at the intra-species level among floating and Available online 14 May 2015 attached samples of U. prolifera collected from 2007 to 2011 were analysed using ISSR (inter-simple sequence repeat) markers. The results showed that all of the floating samples collected from the Yellow Keywords: Sea during the past five years formed a single genetic entity that was different from the attached samples Yellow Sea in intertidal zone. A SCAR (sequence characterized amplified region) marker highly specific to the green tide floating samples of U. prolifera was identified and it showed that the same population dominated in the Ulva prolifera ecotype blooms from 2007 to 2013. In combination with the morphology and physiological features, the genetic genetic variation analysis results suggested that a unique ecotype of U. prolifera was responsible for the largest green tides in the world. These findings indicated that attached U. prolifera along the coast does not seem to be the originator of the green tide in the Yellow Sea and gene flow between attached and floating populations does not readily take place. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Ulva prolifera (¼Enteromorpha prolifera) has been confirmed as the only dominant species in the Yellow Sea green tides by both Green tides have become a globally severe problem due to the morphology and molecular analysis (Jiang et al., 2008; Ye et al., increased eutrophication of coastal waters (Charlier et al., 2007). In 2011; Zhao et al., 2013). Detailed studies tracing the blooms addition to various ecological impacts on indigenous biodiversity discovered the coexistence of Ulva compressa, Ulva flexuosa and (Berger et al., 2003) and biogeochemical cycles (Sandjensen and Ulva linza-procera-prolifera (LPP) complex in the floating algal Borum, 1991), these algal blooms affect aquaculture and tourism assemblage, but U. prolifera gradually dominated during the drifting (Sun et al., 2008). Most green tides involve Ulva (Blomster et al., period (Tian et al., 2011). 2002; Shimada et al., 2003)orEnteromorpha, which is a synonym Since quite a few attached Ulva prolifera populations distributed of Ulva (Hayden et al., 2003). China experienced a small-scale widely along the coast of China, and some even have high biomass (82 km2) green tide in the Yellow Sea caused by Ulva in 2007 near the bloom area (Zhang et al., 2011), comparative genetic analysis from May to July. A green algal bloom on a much larger scale at the intra-species level between populations has been a key to figure (3489 km2) occurred again in 2008 (Keesing et al., 2011). In the out the origin of the floating algae. Preliminary study of the samples following years, the Yellow Sea underwent consecutive green tide collected from 2007 to 2009 using ISSR (inter-simple sequence repeat) blooms, which have been documented as the largest green tides markers revealed that the floating samples exhibited a close genetic ever to occur worldwide (Liu et al., 2013a). relationship and that they were different from all of the U. prolifera samples attached to hard substrates in coastal areas (Zhao et al., 2011). Phylogenetic analysis of the 5S rDNA spacer region also indicated that the floating U. prolifera in the Yellow Sea from 2008 to 2009 formed a * Corresponding authors. E-mail addresses: [email protected] (P. Jiang), [email protected] (S. Qin). monophyletic clade (Lin et al., 2011; Zhang et al., 2011). http://dx.doi.org/10.1016/j.ecss.2015.05.027 0272-7714/© 2015 Elsevier Ltd. All rights reserved. J. Zhao et al. / Estuarine, Coastal and Shelf Science 163 (2015) 96e102 97

However, after successive blooms of Ulva prolifera in the Yellow volume of 25 ml containing 10.5 ml of ddH2O, 12.5 mlof2 Taq Sea, gene exchange between the floating and attached populations Master Mix (Sinobio, Beijing, China), 1 ml of primer (10 mM, Sangon, may occur to a certain extent and the genetic composition of the Shanghai, China) and 1 ml of template DNA (approximately 30 ng/ dominant population of the Yellow Sea green tides might change. ml). Amplification was conducted by a thermocycler (Biometra, Different genotypes of floating U. prolifera have been proposed Germany) using a program described by Zhao et al. (2011). The ISSR based on the results of phylogenetic analyses using the 5S rDNA primers used in this study and their annealing temperatures are spacer region as a genetic marker (Huo et al., 2013; Liu et al., listed in Table 2. The ISSR reactions were replicated twice for each 2013b). But the difference could result from an inhomogeneity of frond. the 5S rDNA spacer region, which has been previously revealed in other organisms (Sajdak et al., 1998; Baum et al., 2001). Thus, genome wide and specific molecular markers are more appropriate 2.4. Visualization and scoring of the ISSR fragments for analysis of genetic variation in floating seaweed. In this study, we applied ISSR markers to investigate the genetic The PCR products were separated on a 2% agarose gel stained diversity of the floating and attached population samples of Ulva with ethidium bromide in 1 TAE buffer and then visualized and prolifera in the Yellow Sea from 2007 to 2011. Compared to the scored under ultraviolet light (Bio-Rad, USA). D2000 plus DNA single molecular marker with potential heterogeneous multicopies marker (Transgene, Beijing, China) was loaded onto both sides of at limited locus in the genome, such as the 5S rDNA spacer region, the gel as the fragment-sized standard. The gel images were pro- the ISSR markers that generate the fingerprint of the whole genome cessed for scoring using BandScan version 5.0 (Drets, 1978). The use could provide more sufficient resolution to reveal the genetic var- of digital imaging to identify and score bands helped establish a iations among populations and have been widely used to analyze consistent scoring protocol and eliminate bias during the scoring the intra-species genetic diversity for seaweeds (Wang et al., 2008; process (Wolfe and Liston, 1998). Polymorphic fragments of each Mostafa et al., 2011). We further developed a SCAR (sequence locus were scored in a binary manner as present (1) or absent (0). fi fi characterized amplified region) marker that is highly specific to the Only those bands that were clearly identi ed in both ampli ed floating population using the ISSR fingerprints. SCAR markers are a replicates were recorded. type of genetic marker that is used to identify a specific population from other populations (Zijlstra, 2000). To find out if the genetic 2.5. Data analysis makeup of the floating U. prolifera in lower case unchanged across several years, we examined samples collected through 2007 to Assuming HardyeWeinberg equilibrium, the Nei's unbiased 2013. genetic distances (D) between samples were determined. A matrix of pairwise distances between all of the individual fronds was 2. Materials and methods calculated using the Jaccard similarity coefficient, which empha- sizes shared traits among fronds and ignores the absence of shared 2.1. Sample collection traits. Clustering with unweighted pair-group mean analysis (UPGMA) was performed based on the Jaccard similarity coefficient From 2007 to 2011, nine attached population samples from using MAGA 4 (Tamura et al., 2007) and NTSYS-pc, version 2.1 substrates in littoral zone and 14 floating samples of Ulva prolifera (Rohlf, 2000). were collected at different sites in the Yellow Sea and East China Sea, and in 2012 and 2013, an additional 10 floating algae samples fi were collected (Fig. 1 and Table 1). The species identi cation of the 2.6. SCAR marker development samples was based on both morphological characteristics and an analysis of the ribosomal DNA internal transcribed spacers (ITS). Based on the ISSR profiles of the samples collected from 2007 to The algal thalli were rinsed carefully in sterilized seawater to 2009, those bands occurring solely in the floating samples with remove debris and epiphytes and then cultured in Von Stosch's high intensity, clear separation and a molecular weight between Enriched Medium (VSE medium). 200 bp to 2000 bp were selected. The selected bands were purified (Gel Midi Purification Kit, TianGen, Beijing, China), ligated into a 2.2. DNA extraction pMD18-T vector (Takara, Dalian, China), and transformed into Top- 10 Escherichia coli competent cells. The positive clones were sent to For the samples collected from 2007 to 2011, four fragments of a business enterprise (Co. Majorbio, Shanghai China) for separated fronds from each collection site were selected randomly sequencing. SCAR primers consisting of more than 18 bases were for DNA extraction; for the samples collected from 2012 to 2013, designed from the sequences of the cloned ISSR fragments using one fragment was selected to represent a sample. The genomic DNA Primer 5.0 (Clarke and Gorley, 2001) and were synthesized by from each tubular thallus (approximately 5 cm) was separated Sangon (Shanghai, China). using a modified CTAB method (Zhao et al., 2010). The quality and concentration of the obtained DNA were tested by comparison with the lDNA/EcoRIþ Hind III markers (Fermentas, Shanghai, China) 2.7. SCAR amplification on a 0.8% agarose gel. The SCAR amplifications were performed in 10 ml reaction 2.3. ISSR amplification mixtures containing 4.2 ml of water, 5 mlof2 Taq Master Mix (SinoBio, Beijing, China), 0.2 ml of each primer (10 mM), and 0.4 mlof Six out of the 50 ISSR primers made available by the University template DNA (approximately 30 ng/ml). The PCR conditions were of British Columbia Biotechnology Laboratory were selected to 94 C for 10 min followed by 35 cycles of 94 C for 45 s, annealing amplify the profiles of Ulva prolifera fronds on the basis that they temperature for 45 s, and 72 C for 1 min and a final elongation at generated clear, reproducible bands ranging from 200 bp to 72 C for 10 min. The SCAR amplifications were analysed by the 2000 bp. The samples collected from 2007 to 2011 were subjected electrophoresis of 3 ml of the PCR products on a 2% (w/v) agarose gel to ISSR analysis. The ISSR-PCR reactions were performed in a stained with ethidium bromide. 98 J. Zhao et al. / Estuarine, Coastal and Shelf Science 163 (2015) 96e102

Fig. 1. Sampling areas in the Yellow Sea and East China Sea. Floating samples collected (open circles), littoral attached samples collected (filled circles) and both floating and littoral attached samples collected (half-filled circles).

Table 1 3. Results Details of the specimens used in this study.

Sample Collection site Collection date Sample state 3.1. Species identification

S096 Yumingzui, Huangdao, Qingdao 29 Jul 2007 Floating S196 Sculpture Park, Qingdao 15 Jun 2008 Floating The ITS sequences of the 33 samples used in this study have S266 Lianyungang 8 Jul 2008 Floating been submitted to GenBank, and their accession numbers were S349 Sculpture Park, Qingdao 16 Jul 2008 Floating KP966511 e KP966543. A phylogenetic analysis showed that 31 out S380 Donghai Restaurant, Qingdao 25 Jul 2008 Floating of the 33 samples were clustered into the LPP clade. The two 09DH Donghai Restaurant, Qingdao 18 Jul 2009 Floating fl 10YY No.1 bathing beach, Qingdao 23 Jul 2010 Floating remaining samples were two oating samples (QD156 and S792) U146 Zhujialin, Lvsi, Qidong 2 Jul 2011 Floating that clustered into the Ulva compressa clade, which was excluded U149 Laobagang, Nantong 2 Jul 2011 Floating from the intra-specific genetic analysis (Fig. S1). The morphology of U152 Sheyang, Yancheng 3 Jul 2011 Floating all 31 LPP samples exhibited strong characteristics of branched and U156 Haitou, Ganyu, Lianyungang 4 Jul 2011 Floating tubular thalli with monostromatic walls, as described by Wang U158 Guanxi, Lianyungang 4 Jul 2011 Floating U162 Rizhao 4 Jul 2011 Floating et al. (2010). Combining the results of molecular and morpholog- N161 No.1 bathing beach, Qingdao 7 Jul 2011 Floating ical analyses, the 31 samples collected in this study were identified QD075 Donghai Restaurant, Qingdao 17 Jun 2012 Floating as Ulva prolifera. Only the 31 samples of U. prolifera were considered QD076 No.1 bathing beach, Qingdao 17 Jun 2012 Floating in a further study of the intra-specific genetic diversity. QD156 Liuqinghe, Qingdao 1 Jul 2012 Floating S792 Rudong, Nantong 29 Apr 2013 Floating S791 Rudong, Nantong 7 May 2013 Floating 3.2. ISSR amplification S793 Xuejiadao, Qingdao 7 Jun 2013 Floating S795 No.3 bathing beach, Rizhao 7 Jun 2013 Floating S796 No.1 bathing beach, Qingdao 8 Jun 2013 Floating A total of 104 high-intensity and well-separated bands were S989 Xuejiadao, Qingdao 6 Jul 2013 Floating recorded from the profiles generated by the six ISSR primers S986 Haiyang, Yantai 31 Jul 2013 Floating (Table 2). All of these ISSR fragments were polymorphic among the S565 Gangshan Port, Rizhao 26 Mar 2009 Attached 92 Ulva prolifera individual fronds collected from 2007 to 2011 S605 Da'ao Village, Fuqing 7 Apr 2009 Attached (Figs. S2 and S3). The fingerprints were digitized and subjected to S703 Fenghua, Ningbo 27 Mar 2009 Attached S744 Dafeng, Yancheng 1 Apr 2009 Attached further phylogenetic analysis. S746 Dafeng, Yancheng 1 Apr 2009 Attached U148 Rudong, Nantong 2 Jul 2011 Attached U150 New Beilingzha 2 Jul 2011 Attached 3.3. Phylogenetic analysis U159 Guanxi, Lianyuangang 4 Jul 2011 Attached U161 Beach, Guanxi, Lianyungang 4 Jul 2011 Attached The phylogenetic tree of UPGMA based on the Jaccard distance fl S096, S196, S266, S349, S380, 09DH, S565, S605, S703, S744 and S746 were used in a between Ulva prolifera fronds revealed that all of the oating preliminary study (Zhao et al., 2011) and were considered together with the newly samples were grouped into one cluster and were significantly collected samples in the present study. separated from the attached samples (Fig. 2). The cophenetic J. Zhao et al. / Estuarine, Coastal and Shelf Science 163 (2015) 96e102 99

Table 2 Primers for ISSR amplification.

Primer name Sequence (50/30) Annealing temperature (C) Number of polymorphic bands

816 (CA)8T 46.9 15 818 (CA)8G 49.5 20 826 (AC)8C 49.6 16 855 (AC)8YT 48.8 16 857 (AC)8YG 51.2 21 890 HVH(TG)7 47.0 16

correlation indicated a very good fit of the cluster analysis to the 3.4. Results of the amplification of the SCAR marker data (r ¼ 0.97). To better understand the pattern of variation among the sam- A specific band (928 bp) of the floating samples was selected ples, a UPGMA dendrogram was also produced using Nei's unbiased based on fingerprints amplified by ISSR primer 855. Its sequence has genetic distances (D) among the samples (Table S1). Similar to the been submitted to GenBank, and the accession number is KP966544. dendrogram obtained based on the distance between fronds, the No significant homologous sequence was found in the NCBI data- Ulva prolifera samples were grouped into two distinct clusters base. Based on the complete sequencing data, a pair of SCAR primers (Fig. 3). The floating samples formed one major cluster, whereas the was designed. The sequences of the primers were YSF-F (ACACC- nine attached samples formed another cluster. TACAAACCCTAACC) and YSF-R (TTGTCGCCCAACCACTTC), and the

Fig. 2. Dendrogram of the UPGMA cluster analysis constructed using the Jaccard distance calculated for 23 samples (92 individuals) of U. prolifera based on ISSR fragments. Samples for the Yellow Sea floating algae are shown surrounded by a red oval. All of the floating individuals were grouped together and formed a clade separating from the littoral attached samples. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 100 J. Zhao et al. / Estuarine, Coastal and Shelf Science 163 (2015) 96e102

and far-distance drifting of them, than the attached thalli (Kong et al., 2011). And the morphological characteristics displayed continuously through several generations cultured in the labora- tory (Hiraoka et al., 2011). What is more, living conditions, such as the temperature range (Huo et al., 2013), irradiance (Kim et al., 2011), desiccation (Gao et al., 2011) and salinity (Wei et al., 2010), are obviously different between the littoral attached and the floating populations in the Yellow Sea. Compared to the attached populations, the floating population was better adapted to the habitat on the sea surface (Hiraoka et al., 2011). Combining the facts that the dominant floating population in the Yellow Sea was distinct in genetics, morphology, physiology and living conditions, it was reasonable to conclude that the Yellow Sea green tide was dominated by a unique ecotype of Ulva prolifera,a genetically distinct population adapted to the drifting life style on sea surface. Many ecotypes have been reported within several HAB (harmful algae bloom)-caused microalgae species based on genetic analysis (Fredrickson et al., 2011; Piccini et al., 2011). The intra- specific variations, such as morphological, genetic characters, maximum growth, photosynthesis rates, and tolerance of adversity (Thessen et al., 2009), were possibly important factors for their competitive success (Hallegraeff et al., 2012). The definition of this Fig. 3. Dendrogram based on Nei's unbiased genetic distance by the method of floating U. prolifera as a unique ecotype is critical to the further UPGMA. Samples for the Yellow Sea floating algae are shown surrounded by a red oval. investigation and monitor of the Yellow Sea green tide. All of the floating samples were grouped together and formed a clade separated from First, the detection of a different ecotype raises a question as to the littoral attached samples. There was no significant relationship between the ge- fl the origin of the green tide. There are two different meanings netic distances and the sampling time of the oating samples. (For interpretation of fl the references to colour in this figure legend, the reader is referred to the web version involved: the annual seed bank for the oating biomass and the of this article). initial biological origin of the floating algae since 2007. Upon the annual seed bank, Liu et al. (2013b) proposed that propagules in intertidal seawater and sediments constituted an over-winter annealing temperature was 50.5 C. The expected size of the SCAR “seed stock”, and proliferated attached to Porphyra rafts in the product obtained for the Yellow Sea floating Ulva prolifera was Subei Shoal as a “nursery” (Liu et al., 2010; Zhang et al., 2015). 830 bp. However, to confirm this hypothesis, it would have been necessary We used the developed SCAR marker to test the 33 algal samples to obtain molecular data, by use of SCAR marker specific to the collected from 2007 to 2013. The PCR results showed that the target floating ecotype, on algae attached to the rafts, which we did not band was amplified for 21 out of the 22 floating Ulva prolifera do. We are uncertain about the initial origin of the green tide. In samples, with the exception of S793. In contrast, none of the nine the early years of green tide in the Yellow Sea, Ulva prolifera were samples of attached U. prolifera showed the specific band (Fig. 4). rarely found on the rafts during the Porphyra aquaculture season (Shen et al., 2012). It is only since 2011 that the quantity of 4. Discussion U. prolifera attached to the Porphyra aquaculture rafts began to increase significantly (Han et al., 2013). These results suggest that The results revealed that the floating Ulva prolifera samples the Porphyra aquaculture rafts may not be the initial origin of the collected between 2007 and 2013 were genetically similar (except floating U. prolifera in 2007. Our present results indicate that the for one sample in 2013) and that the attached U. prolifera were attached populations along the coast of the Yellow Sea could not genetically different from the floating U. prolifera, corroborating be the source, neither. It may be a unique ecotype of U. prolifera previous results (Zhao et al., 2011). may have invaded into the Subei Shoal and initially caused the Besides the genetic traits, the floating population was also green tides in 2007. different from the attached populations on morphology, physiology Second, after successive blooms, genetic variation kept between and living environments. Most remarkably, the bloom-forming the floating and the attached populations in the coastal area sug- thalli exhibited denser branches, which facilitated the long-time gested the limited gene flow between them. That could result from

Fig. 4. Amplification of the specific SCAR marker. a) Floating and littoral attached samples collected from 2007 to 2011. b) Floating samples collected from 2012 to 2013. S096 was used as a positive control. All of the samples were confirmed to be U. prolifera, except two (QD156 and S792), which were U. compressa. J. Zhao et al. / Estuarine, Coastal and Shelf Science 163 (2015) 96e102 101 the asexual reproduction, which was reported to be the dominant processes occurring during the early northward floating period. Limnol. Oce- e proliferation style for the bloom-forming Ulva in eutrophic condi- anogr. 58, 2206 2218. Jiang, P., Wang, J.F., Cui, Y.L., Li, Y.X., Lin, H.Z., Qin, S., 2008. Molecular phylogenetic tions (Kamermans et al., 1998; Blomster et al., 2002). And the analysis of attached Ulvaceae species and free-floating Enteromorpha from oceanographic factors, such as eddies (Lin et al., 2011) and current Qingdao coasts in 2007. Chin. J. Oceanol. Limnol. 26, 276e279. variations, might provide the geographical barrier for the floating Kamermans, P., Malta, E.J., Verschuure, J.M., Lentz, L.F., Schrijvers, L., 1998. Role of cold resistance and burial for winter survival and spring initiation of an Ulva ecotype, which need to be taken into account. spp. (Chlorophyta) bloom in a eutrophic lagoon (Veerse Meer lagoon, The Third, aside from some necessary environmental conditions, Netherlands). Mar. Biol. 131, 45e51. such as eutrophication, the biological property of this floating Keesing, J.K., Liu, D.Y., Fearns, P., Garcia, R., 2011. Inter- and intra-annual patterns of Ulva prolifera green tides in the Yellow Sea during 2007-2009, their origin and ecotype may also offer essential contributions to the Yellow Sea relationship to the expansion of coastal seaweed aquaculture in China. Mar. green tide and should thus receive greater attention with respect to Pollut. Bull. 62, 1169e1182. understanding the mechanism underlying the formation of the Kim, J.H., Kang, E.J., Park, M.G., Lee, B.G., Kim, K.Y., 2011. Effects of temperature and irradiance on photosynthesis and growth of a green-tide-forming species (Ulva bloom. linza) in the Yellow Sea. J. Appl. Phycol. 23, 421e432. In addition, the SCAR marker developed in this study was Kong, F., Mao, Y., Cui, F., Zhang, X., Gao, Z., 2011. Morphology and molecular iden- proven to be highly specific to the floating ecotype, which is an tification of Ulva forming green tides in Qingdao, China. J. Ocean Univ. China 10, e effective tool to examine the genetic similarity of the bloom- 73 79. Lin, H.Z., Jiang, P., Zhang, J.X., Wang, J.F., Qin, S., Sun, S., 2011. Genetic and marine forming algae in the coming years, to monitor the settlement of cyclonic eddy analyses on the largest macroalgal bloom in the world. Environ. the floating ecotype in the coastal area and to assess the gene ex- Sci. Technol. 45, 5996e6002. change between the floating and the attached populations. Liu, D.Y., Keesing, J.K., Dong, Z.J., Zhen, Y., Di, B.P., Shi, Y.J., Fearns, P., Shi, P., 2010. Recurrence of the world's largest green-tide in 2009 in Yellow Sea, China: Porphyra yezoensis aquaculture rafts confirmed as nursery for macroalgal Acknowledgements blooms. Mar. Pollut. Bull. 60, 1423e1432. Liu, D.Y., Keesing, J.K., He, P.M., Wang, Z.L., Shi, Y.J., Wang, Y.J., 2013a. The world's largest macroalgal bloom in the Yellow Sea, China: formation and implications. The authors are grateful to Wei Wei, Jianheng Zhang and Estuar. Coast. Shelf Sci. 129, 2e10. Wenting Xu for their help with sample collection. We also thank Liu, F., Pang, S.J., Chopin, T., Gao, S.Q., Shan, T.F., Zhao, X.B., Li, J., 2013b. 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