Aquatic Botany 93 (2010) 25–31 Contents lists available at ScienceDirect Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot Molecular analysis of green-tide-forming macroalgae in the Yellow Sea Jinfeng Wang a,b , Peng Jiang a,∗ , Yulin Cui a,b , Nan Li b,c , Mingqing Wang d , Hanzhi Lin a,b , Peimin He e , Song Qin a,∗ a Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, China b Graduate University of Chinese Academy of Sciences, Beijing 100049, China c Yantai Institute of Coastal Zone Research for Sustainable Development, Chinese Academy of Sciences, Yantai 264003, China d College of Marine Life Science, Ocean University of China, Qingdao 266003, China e College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 200090, China article info Article history: Received 5 September 2009 Received in revised form 25 February 2010 Accepted 3 March 2010 Available online 9 March 2010 Keywords: The Yellow Sea Free-floating Enteromorpha ITS rbcL psbA abstract In the summer of 2008, free-floating green algae bloomed in the Yellow Sea. Samples were collected in a wide area (119 ◦ 32 ′ –122 ◦ 00 ′ E, 32 ◦ 25 ′ –36 ◦ 49 ′ N). We calculated the sequence divergences of nuclear ITS, chloroplast rbcL, and psbA data of free-floating samples collected from the Yellow Sea and Ulvaceae from Europe and Japan. In the ITS sequence, 19 out of the 21 Yellow Sea samples of 2008 were iden- tical to those of a sample taken at Qingdao in 2007. A low divergence (0.2%) was found in remaining two samples. Similar evidence was shown by pairwise distances of rbcL and psbA gene sequence data, implying the uniformity of the Yellow Sea blooms in 2007 and 2008. The ITS sequence of the Yellow Sea samples differed 8.1–10.8% from free-floating Enteromorpha or Ulva reported worldwide. ITS-based molecular phylogenetic results and rbcL sequence data grouped the free-floating alga in the Yellow Sea into one clade with Enteromorpha procera, Enteromorpha linza and Enteromorpha prolifera. Further- more, both morphological characteristics and ribotype network of the ITS sequences imply that the blooming algae in 2007 and 2008 were E. prolifera. The haplotypes of the Yellow Sea free-floating E. prolifera are closely related to those from the Japanese coast but less to European and American algae. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Enteromorpha or Ulva can multiply strongly from fragmentation or reproduction, forming a “green-tide” and resulting in ecological and social problems (Fletcher et al., 1990; Valiela et al., 1997; Bäck et al., 2000; Blomster et al., 2002). Enteromorpha species have been distinguished morphologically from Ulva sp. for having tubular and monostromatic blades (Link, 1820). Infrageneric classification and phylogeographic studies of Enteromorpha are difficult to perform due to the species’ exten- sive distribution (Van den Hoek et al., 1995), tiny intraspecific differences (Bliding, 1963), a high variability in morphology and cytology resulting from the change of developmental stages and environmental conditions (De Silva and Burrows, 1973; Mshigeni and Kajumulo, 1979; Tanner, 1986), and the free-floating nature (Malta et al., 1999; Blomster et al., 2002). In recent years, molec- ular analyses have been conducted to address these difficulties. Along with the sequences in the GenBank, nuclear rDNA ITS and ∗ Corresponding authors. Tel.: +86 532 82898500; fax: +86 532 82880645. E-mail addresses: jiangpeng@qdio.ac.cn (P. Jiang), sqin@qdio.ac.cn (S. Qin). chloroplast rbcL have been frequently cited to identify species of Ulvaceae (Malta et al., 1999; Hayden et al., 2003; Shimada et al., 2003), and are often used as genetic markers in phylogeographic studies on Ulva and Enteromorpha (Blomster et al., 1999; Hayden and Waaland, 2002; Leskinen et al., 2004; Shimada et al., 2008). In the summer of 2008, Enteromorpha sp. bloomed in Qingdao City off the coast of the Yellow Sea, causing concern among scien- tists and coastal managers (Sun et al., 2008; Leliaert et al., 2009). Studies were carried out on the morphological characteristics, life history, physiological parameters, and reasons for the blooming of the green alga (Fu et al., 2008; Ding et al., 2009). Using ITS sequences, Jiang et al. (2008) demonstrated that 10 free-floating Enteromorpha sp. samples from Qingdao coast in 2007 were one species. The attached Ulvaceae species in this area did not consti- tute the biogeographic cradle of the blooms. Subsequently, Leliaert et al. (2009) reported on the 2008 bloom event based on ITS and rbcL gene sequences. However, the green-tide forming samples from the Qingdao coast in these two studies were obtained only within a span of a year and from a restricted area. In the present study, nuclear rDNA ITS, chloroplast rbcL, and psbA sequences of free-floating samples from a much larger area of the Yellow Sea are reported and discussed for species identification, genetic diversity, and phylogeographical structure. 0304-3770/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2010.03.001