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 Ulvaceae 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.