Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal waters via use of quantitative PCR assays Bum Soo Park a , Pengbin Wang a , Jin Ho Kim a , Joo-Hwan Kim a , Christopher J. Gobler b , Myung-Soo Han a,c, * a Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea b School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA c Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, South Korea 1. Introduction The ichthyotoxic unarmored dinoflagellate Cochlodinium poly- krikoides a taxa of harmful algal blooms (HAB) responsible for substantial mortality to both wild and farmed fish (Kudela and Gobler, 2012). The presence of C. polykrikoides has been reported in tropical, subtropical and temperate waters, such as British Columbia, Canada (Whyte et al., 2001), the US east coast (Gobler et al., 2008), Mexico in the eastern Pacific, the coastal waters of Costa Rica (Vargas-Montero et al., 2004, 2006), Japan (Yuki and Yoshimatsu, 1989), China (Qi et al., 1993), and Korea (Kim, 1998a; Cho and Costas, 2004). In recent decades, harmful algal blooms caused by C. polykrikoides Margalef have exhibited an apparent increase in harmful impacts worldwide (Kudela and Gobler, 2012). At the same time, the economic damage to fisheries and aquaculture due to massive mortality has also increased sharply, particularly in Korea (Kim et al., 2001, 2007). In 1995, a particularly severe and widespread C. polykrikoides bloom persisted for nearly eight weeks along the entire south coast of Korea, ultimately resulting in economic losses of up to 95 million US dollars (Kim, 1998b). Since then, harmful algal blooms of this species have been an annual feature along southern Korean coastal waters. Therefore, intensive investigation, including studies of growth characteristics and vertical migration, have been conducted to obtain a better understanding of the factors influencing the formation of C. polykrikoides blooms (Park et al., 2001; Kim et al., 2004; Jeong et al., 2004). In addition, various studies have been undertaken to clarify the mechanisms of C. polykrikoides bloom formation, such as overwintering strategies, hyaline cysts (temporary cyst), and resting cysts (Matsuoka and Fukuyo, 2000; Kim et al., 2002; Tang and Gobler, 2012), and the current-driven movement of C. polykrikoides blooms in the Andaman Sea, the East China Sea, and the East Sea/the Sea of Japan have been evaluated using ocean- color satellite imagery (Azanza and Baula, 2005; Miyahara et al., Harmful Algae 37 (2014) 133–141 A R T I C L E I N F O Article history: Received 9 September 2013 Received in revised form 30 April 2014 Accepted 30 April 2014 Keywords: Cochlodinium polykrikoides Ribotype Quantitative real-time PCR Field application A B S T R A C T While the toxic dinoflagellate Cochlodinium polykrikoides is known to form blooms that are maintained for extended periods, the genetic differentiation of these blooms are currently unknown. To assess this, we developed a real-time PCR assay to quantify C. polykrikoides at the intra-specific level, and applied this assay to field samples collected in Korean coastal waters from summer through fall. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides: Philippines, East Asian, and American/Malaysian. Significant linear relationships (r 2  0.995) were established between C t and the log of the copy number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms in Korean coastal waters were found to be comprised of Philippines and East Asian ribotypes but not the American/Malaysian ribotype. The Philippines ribotype was found to be highly abundant during summer bloom initiation and peak, whereas the East Asian ribotype became the dominant ribotype in the fall. As such, this newly developed qPCR assay can be used to quantify the cryptic ecological succession of sub-populations of C. polykrikoides during blooms that light microscopy and previously developed qPCR assays cannot resolve. ß 2014 Published by Elsevier B.V. * Corresponding author at: Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea. Tel.: +82 2 2220 0956; fax: +82 2 2296 1741. E-mail address: hanms@hanyang.ac.kr (M.-S. Han). Contents lists available at ScienceDirect Harmful Algae jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/hal http://dx.doi.org/10.1016/j.hal.2014.04.019 1568-9883/ß 2014 Published by Elsevier B.V.