Cryptic species in the Pyropia yezoensis complex (Bangiales, Rhodophyta): Sympatric occurrence of two cryptic species even on same rocks Kyosuke Niwa, 1 * Norio Kikuchi, 2 Mi Sook Hwang, 4 Han-Gu Choi 5 and Yusho Aruga 3 1 Fisheries Technology Institute, Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Akashi, 2 Coastal Branch of Natural History Museum and Institute, Chiba, Katsuura, 3 Nishikamata 2-4-21, Tokyo, Japan, 4 Seaweed Research Center, NFRDI, Mokpo and 5 Division of Life Sciences, Korea Polar Research Institute, Incheon, Korea SUMMARY In a previous study on wild populations of Pyropia, the occurrence of two possible new species (Pyropia sp. 2 and Pyropia sp. 3) which are closely related to the two commercially important Pyropia species, P. yezoensis and P. tenera, was confirmed as the result of molecular phylogenetic analyses. To characterize the morphologi- cal features of the two wild Pyropia species, we col- lected Pyropia blades in a natural population in which Pyropia sp. 3 was known to occur, and carried out molecular identification before detailed morphological observations. Through the molecular identification we found, unexpectedly, that Pyropia sp. 2 blades grew sympatrically in the same site. Therefore, after molecu- lar identification, we examined in detail the external morphology and anatomy of the two wild Pyropia species using more than 10 blades each. As a result, it is concluded that all of the blades of the two species are morphologically identical to P. yezoensis, but dis- tinct from P. tenera. It is therefore considered that both of the two wild Pyropia species are cryptic species within the P. yezoensis complex. Furthermore, this study revealed that the two cryptic species grew sympatrically, even on the same rocks within the natural habitat. Key words: cryptic species, polymerase chain reaction- restriction fragment length polymorphism, Pyropia yezoensis, speciation. INTRODUCTION The red algal order Bangiales was comprised of only two genera, blade-forming Porphyra and filamentous Bangia until three additional filamentous genera were proposed (Müller et al. 2005; Nelson et al. 2005). In Porphyra, 264 species have been reported from cold temperate to tropical waters (Guiry & Guiry 2012). Recently, Sutherland et al. (2011) proposed that Porphyra and Bangia are revised as eight and seven genera (including the abovementioned three genera) respectively on the basis of molecular phylogenetic analysis. Their study demonstrated that there is a large genetic diversity in the Bangiales despite the simple morphological features, and the two commercially important species, Porphyra yezoensis and Porphyra tenera, were transferred to the genus Pyropia, the genus that contains the largest number of species among the blade-forming genera. On the other hand, recent molecular studies have revealed that cryptic diversity was found not only among genera of Bangiales but also in the closely related Porphyra/Pyropia species (Brodie et al. 2007; Lindstrom 2008; Niwa et al. 2009). Consequently, some new species have been described from each complex com- prising several morphologically similar but genetically distinct taxa (Neefus et al. 2002; Lindstrom & Fredericq 2003; Brodie et al. 2007). In wild Pyropia populations, the presence of two possible new species Pyropia sp. 2 and Pyropia sp. 3 which are closely related to P. yezoensis and P. tenera have been confirmed by molecular phylogenetic analysis of the plastid ribulose- 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene and sequence divergences of plastid RubisCO spacer and nuclear internal transcribed spacer 1 (ITS-1) rDNA regions (Niwa et al. 2009; Niwa & Kobiyama 2009). In particular, the sequence divergence of ITS-1 strongly supported that the sample (P7) of Pyropia sp. 2 and the samples (P10, TG-1) of Pyropia sp. 3 are considered as separate species, and that these two entities are clearly different species from P. yezoensis and P. tenera (see Table 1 in Niwa et al. 2009). Further- more, polymerase chain reaction-restriction fragment *To whom correspondence should be addressed. Email: kyousuke_niwa@pref.hyogo.lg.jp Communicating Editor: M. Kamiya. Received 17 December 2012; accepted 8 July 2013. doi: 10.1111/pre.12035 Phycological Research 2014; 62: 36–43 © 2013 Japanese Society of Phycology