Chinese Journal of Oceanology and Limnology Vol. 31 No. 6, P. 1210-1215, 2013 http://dx.doi.org/10.1007/s00343-013-3045-7

Molecular diversity of Enteromorpha from the coast of Yantai: a dual-marker assessment*

LIU Haiyan (刘海燕)1, 2 , LIU Zhengyi (刘正一) 3 , WANG Yinchu (王寅初) 1, 2 , ZHAO Yushan (赵玉山) 4 , QIN Song (秦松) 1, 2, ** 1 University of Chinese Academy of Sciences, Beijing 100049, China 2 Yantai Institute of Coastal Zone, Chinese Academy of Sciences, Yantai 264003, China 3 Key Lab of Marine Biology in Jiangsu, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China 4 Shandong Oriental Ocean Sci- Tech Co. Ltd., Yantai 264003, China

Received Feb. 4, 2013; accepted in principle Apr. 20, 2013; accepted for publication Aug. 29, 2013 © Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2013

Abst r act We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS (internal transcribed spacer) and 5S rDNA NTS (non-transcribed spacer) sequences. The ITS sequences showed slight nucleotide divergences between Enteromorpha linza and Enteromorpha prolifera . In contrast, multiple highly variable regions were found in the ITS region of Enteromorpha fl exuosa . In general, there were more variable sites in the NTS region than in the ITS region in the three species. The variations in 5S rDNA NTS sequences indicated that the molecular diversity of Enteromorpha from the coast of Yantai is very high. However, a phylogenetic tree constructed using 5S rDNA NTS sequence data indicated that genetic differences were not directly related to geographical distribution.

Keyword : Enteromorpha; internal transcribed spacer (ITS); 5SrDNA non-transcribed spacer (NTS); molecular diversity

1 INTRODUCTION Enteromorpha species (Tanner, 1986; Tan et al., 1999). Thus, the identifi cation of E nteromorpha spp. Enteromorpha is a green algal genus widespread from morphological characters has resulted in in coastal areas. Species in this genus can attach to inaccurate estimates of biodiversity in some cases. reefs or artifi cial substrata, or they can be free fl oating Classifi cations based on molecular data have given (van den Hoek et al., 1995; Ding and Luan, 2009). more accurate biodiversity estimates. A few They have ecological functions and economic value molecular sequences have been shown to be useful to as materials for food and medicine (Rouxel et al., assess molecular diversity in the genus Enteromorpha 2001). There are more than 617 Enteromorpha (Hayden and Waaland, 2002; Hayden et al., 2003). species listed in Algaebase, of which only 24 are These include the sequences of two nuclear-encoded fl agged as taxonomically accepted (Guiry and Guiry, internal transcribed spacer (ITS) regions of ribosomal 2012). Of the 25 species known in China (Dong, cistrons, ITS1 and ITS2 (Coat et al., 1998; O’Kelly et 1963; Zeng and Zhang, 1984; Zhang et al., 2005), al., 2010) and rbcL (Zechman, 2003; Loughnane et only fi ve are common in coastal areas: Enteromorpha al., 2008). Although these molecular markers are prolifera , Enteromorpha linza , Enteromorpha compressa , Enteromorpha intestinalis , and Enteromorpha clathrata. The taxonomic separation of species is based on their morphological features; * Supported by the National Key Technology Research and Development Program (No. 2008BA49B01) and the Key Innovation Program of Chinese however, taxonomists have observed vast Academy of Sciences (No. KZCX2-YW-209) morphological variability within and among ** Corresponding author: [email protected] No.6 LIU et al.: Molecular diversity of Enteromorpha of Yantai 1211

Table 1 Details of collection dates and sites for the three Enteromorpha species used in this study

Number of samples Species Collection site or area Ecological type Collection date S125 E . prolifera No.1 Bathing Beach, Yantai Attached 18-Apr-09 S389 No.1 Bathing Beach, Yantai Attached 21-May-10 S395 No.1 Bathing Beach, Yantai Attached 29-May-10 S390 E . fl exu o s a Yangmadao, Yantai Attached 22-May-10 S391 Yangmadao, Yantai Attached 22-May-10 S392 Yangmadao, Yantai Attached 22-May-10 S398 E . linza Yangmadao,Yantai Attached 22-May-10 S106 Yangmadao, Yantai Attached 18-Apr-09 S107 Yangmadao, Yantai Attached 18-Apr-09 generally not developed using type specimens, such markers allow the precise identifi cation and assessment of diversity in this genus (Coat et al., 1998; Hiraoka et al., 2003). Several studies on major Enteromorpha species and populations have been conducted in temperate and boreal waters in many parts of China, especially in Qingdao, south of the Yellow Sea (Jiang et al., 2008; Zhang et al., 2008; Shimada et al., 2010). This is because large-scale green tides consisting of Enteromorpha species have occurred in this area (Wang et al., 2010; Pang et al., 2010; Duan et al., 2012). However, molecular screening of Enteromorpha species in other geographical regions, e.g., Yantai, which is located in the north of the Yellow Sea, has indicated that their diversity is often underestimated. Fig.1 Sampling location on Yantai coast Understanding the molecular diversity of the Site 1: No.1 Bathing Beach; Site 2: Yangmadao along the coast of Yantai will be useful to study the biodiversity of Enteromorpha and to manage this resource. In addition, molecular data has not been used 2.2 DNA extraction to distinguish between E . prolifera and E . linza in Each thallus was washed three to four times with Yantai. In this study, we analyzed ITS nrDNA sterilized seawater. Algal tissues were ground in (Blomster et al., 1998) and 5SrDNA non-transcribed liquid nitrogen with a pestle and mortar for 2–3 min, spacer (NTS) sequences to investigate the molecular and the fi ne powder was transferred to a micro-tube. diversity of Enteromorpha collected from the coast of Total genomic DNA was extracted by the modifi ed Yantai. CTAB method (Doyle and Doyle, 1990). Briefl y, 2 MATERIAL AND METHOD each sample was mixed with DNA extraction solution and then digested with Protease K, added 2.1 Sample collection to a fi nal concentration of 100 μg/mL. The mixed liquid was incubated at 55°C for 2 h with shaking Nine Enteromorpha samples were collected in every 10 min, and then cooled to room temperature. early summer of 2009 and 2010 from two sites from An equal volume of 5 mol/L potassium acetate was the coast of Yantai (Table 1, Fig.1). Living specimens added and mixed before extraction with phenol: and herbarium specimens were deposited in our chloroform: isoamyl alcohol (25:24:1). The extract laboratory. Epiphytic algae were removed and all was fi rst precipitated with ethanol at -20°C for 12 h, algae samples were examined under a microscope to and then centrifuged and washed with 70% ethanol. identify the species. Dried DNA was dissolved in Tris-EDTA buffer (pH 1212 CHIN. J. OCEANOL. LIMNOL., 31(6), 2013 Vol.31

Table 2 Details of ITS and 5SrDNA NTS sequences obtained from GenBank

GenBank accession number Taxon Origin of sequenced material and reference ITS 5SrDNA NTS E . linza Carraroe Co. Galway, Ireland (Shimada et al., 2008) AB298633 AB298669 Zhanqiao, Qingdao (Duan et al., 2011) HM584729 HM584771 E . prolifera Mumomi River, Fukuoka, 2.7 km upstream, Japan (Shimada et al., 2008) AB298312 AB298661 Xiaomaidao, Qingdao (Duan et al., 2011) HM584741 HM584779 E . fl exuosa Shizuoka, Shimoda, Japan (Shimada et al., 2008) AB097646 Ulvaria obscura Padilla Bay, WA, USA (Hayden et al., 2003) AY260571 Umbraulva olivascens Portaferry, Strangford Lough, Northern Ireland (Hayden et al., 2003) AY260564

=8.0) and stored for use as templates for PCR amplifi cation.

2.3 PCR amplifi cation, purifi cation, and sequencing

The PCR amplifi cation of ribosomal DNA containing ITS and 5SrDNA NTS sequences was performed with following primers: ITS (IF: 5'-TTTGTACACACCGCCCG-3'; IR: 5'- TCCTCCGCTTATTGATATGC-3'), and NTS (FS: 5'-GGTTGGGCAGGATTAGTA-3', RS: 5'- Fig.2 Phylogenetic tree based on ITS sequences constructed AGGCTTAAGTTGCGAGTT-3') and the thermal by neighbor joining (NJ) method cycling profi le described by Leskinen and Pamilo (1997) and Yotsukura et al. (2002). Amplicons were checked on 1% TAE agarose gels after staining with joining (NJ)-based phylogenetic analyses were GeneFinder DNA dye (Beijing Bio-V Gentech Co. performed with MEGA 4.0 (Tamura et al., 2007), and Ltd., Beijing, China). The PCR primers were NJ trees were constructed using the Kimura two- synthesized by the Shanghai Sangon Biological parameter model, considering gaps, transitions, and Engineering Technology and Service Co. Ltd., transversions. A bootstrap analysis with 1 000 Shanghai, China. The fi nal fragments were purifi ed replicates was applied to assess clade stability. using a Bioteke PCR Purifi cation Kit (Bioteke Corp., Beijing, China) according to the manufacturer’s 3 RESULT instructions. Purifi ed DNA fragments were introduced into the 3.1 Analysis of ITS sequences pMD19-T vector (TaKaRa, Dalian, China) and Alignments of 522 bp (n =16) were produced for transformed into Escherichia coli strain TG1. the ITS sequences, and the samples identifi ed as E . Recombinant clones were screened and sequenced as linza and E . prolifera showed only minor sequence described by Wei (1999). divergence (0–3 bp). There were only very slight 2.4 Data analysis divergences in the ITS sequence among E . linza and E . prolif e ra samples from Yantai and Qingdao in Plasmids were sequenced by the Shanghai Sangon China, Japan, and Ireland. However, the ITS sequence Biological Engineering Technology and Service Co. of E . fl exuosa showed greater divergence. The NJ tree Ltd. The resulting sequences were used for homology constructed using ITS sequence data was shown in searches with the Basic Local Alignment Search Tool Fig.2. In the phylogenetic analysis, the nine (BLAST) at NCBI. The sequences from each strain Enteromorpha samples were clearly separated into and reference sequences from GenBank (Table 2) two groups based on ITS sequences. The larger group were aligned using Clustal X (Thompson et al., 1997), contained six samples and was designated as the E . and then adjusted by BioEdit (Hall, 1999). Neighbor linza -procera - prolifera (LPP) group (Shimada et al., No.6 LIU et al.: Molecular diversity of Enteromorpha of Yantai 1213

Table 3 Sequence divergence of 5SrDNA NTS sequences among Enteromorpha samples from the Yantai coast

el1 el2 elS106 elS107 elS398 ep1 ep2 epS125 epS389 eoS395 efS390 efS391 efS392 el1 el2 0.036 elS106 0.031 0.005 elS107 0.031 0.005 0.000 S398 0.005 0.031 0.026 0.026 ep1 0.126 0.144 0.138 0.138 0.132 ep2 0.144 0.163 0.156 0.156 0.150 0.020 epS125 0.144 0.163 0.156 0.156 0.150 0.020 0.000 epS389 0.150 0.169 0.163 0.163 0.156 0.026 0.026 0.026 epS395 0.150 0.169 0.163 0.163 0.156 0.026 0.026 0.026 0.000 efS390 0.634 0.648 0.636 0.636 0.634 0.651 0.690 0.690 0.690 0.690 efS391 0.634 0.648 0.636 0.636 0.634 0.651 0.690 0.690 0.690 0.690 0.000 efS392 0.635 0.649 0.638 0.638 0.635 0.653 0.692 0.692 0.692 0.692 0.036 0.036

el1: Enteromorpha linza AB298669; el2: Enteromorpha linza HM584771; ep1: Enteromorpha prolifera AB298661; ep2: Enteromorpha prolifera HM584779; el: Enteromorpha linza ; ep: Enteromorpha prolifera ; ef: Enteromorpha fl exuosa

divergences in the NTS sequence among E . linza , E . prolifera , and E . fl exuosa . However, smaller intraspecifi c divergence was found in different areas. The results indicated that the molecular diversity of Enteromorpha was higher for 5S rDNA sequences than for ITS sequences, and the divergence of sample S125 in its 5SrDNA NTS sequence corresponded with its taxonomy in Enteromorpha . 4 DISCUSSION Fig.3 Unrooted tree based on 5SrDNA NTS sequences constructed by neighbor-joining (NJ) method In this study, we determined the sequences of the ITS and the NTS regions to evaluate the molecular diversity of Enteromorpha samples collected from the 2008). Samples S390, S391, and S392 were well Yantai coast in early summer. Previous studies supported (BP=100/100 ITS) as a monophyletic revealed complex intraspecifi c and interspecifi c group; this group included E . fl exuosa from China variations in Enteromorpha , based on ITS sequence and Japan. However, because of the low variations analyses (Shimada et al., 2008; Hayden et al., 2003; among ITS sequences, the group E . linza and the Pang et al., 2010). In this study, the ITS sequence data group E . prolifera were not clearly separated. suggested that there were complex interspecifi c 3.2 Analysis of 5SrDNA sequences variations between E . linza and E . prolifera , consistent with the results of previous studies (Shimada et al., Alignments of 323 bp (n =13) were produced for 2008; Duan et al., 2012). One of our samples (S125) the NTS marker. The unrooted phylogenetic tree and some reported previously (Shimada et al., 2008) obtained from NJ analysis of the NTS sequence data were clustered into the E . linza group based on their is shown in Fig.3. The 13 samples were divided into ITS sequences. This fi nding indicated that there was a three groups: E . linza , E . prolifera, and E . fl exuosa . very low level of molecular diversity in the ITS region Cluster analysis showed no direct relationship between E . linza and E . prolifera. Thus, the ITS between genetic differences and geographical sequence was not suitable for assessing the molecular distance. The intraspecifi c nucleotide diversities of diversity of E . linza and E . prolifera . It seemed Enteromorpha were 0–3.6%, while the interspecifi c unlikely that further sampling would reveal a higher ones were 12.6%–69.3% (Table 3). There were large degree of variation between E . linza and E . prolifera . 1214 CHIN. J. OCEANOL. LIMNOL., 31(6), 2013 Vol.31

More research is required to clarify the taxonomic NTS sequences than among ITS sequences in relevance of these species. Enteromorpha species. The sequences were clearly Studies on the molecular diversity of eukaryotic clustered by species and not by geographic location. organisms such as Enteromorpha have often used the In fact, the 5S rDNA NTS sequences are conserved ribosomal DNA ITS sequence (Heesch et al., 2009; within a species, even among members of that species Kraft et al., 2010). However, in eukaryotes, the non- in different parts of the world. Among the nine transcribed spacer region in tandem-repeated 5S collected samples, the principle component rDNAs (namely, the 5SrDNA NTS region) shows a contributed the most variability within E . linza and E . higher degree of variability (Soltis and Soltis, 1999). prolifera clusters. However, sequence comparison Thus, it is often more suitable for phylogenetic showed that there was no relationship between analysis. Compared with 5SrDNA NTS sequences, 5SrDNA sequence divergence and geographical ITS sequences are relatively conservative, and despite distribution of Enteromorpha . This may be related to the differences in ITS sequences among samples, the biological behavior of Enteromorpha . Since it is these differences were not suffi cient to clearly an opportunistic seaweed, human activities such as separate the samples. Therefore, ITS sequences alone fi shing and marine culture would affect its distribution should not be used to evaluate the molecular diversity (Valiela et al., 1997). On the other hand, this study of Enteromorpha species. In fact, Enteromorpha was conducted on a small geographical scale. samples with the same or slightly different ITS Although the geographical region was limited to sequences may not have the same origin (Leskinen Yantai, we observed high levels of molecular diversity and Pamilo, 1997). among members of the widespread taxon In this study, we report for the fi rst time the Enteromorpha . Similar studies using 5S NTS 5SrDNA NTS sequences of E . fl exuosa . The 5SrDNA molecular markers to analyze Enteromorpha NTS sequence exhibited wide divergence among the distributed in other locations will provide information three species. Hence, this NTS sequence would be about the genetic diversity Enteromorpha that cannot suffi cient to clarify the phylogenetic relationships be inferred from morphological analyses alone. among Enteromorpha specimens. 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