Examination of species delimitation of ambiguous DNA-based Ulva (Ulvophyceae, Chlorophyta) clades by culturing and hybridisation MASANORI HIRAOKA 1 *, KENSUKE ICHIHARA 2 ,WENRONG ZHU 3 ,SATOSHI SHIMADA 4 ,NAOHIRO OKA 5 ,JIANJUN CUI 3 , SHUNTARO TSUBAKI 6 AND PEIMIN HE 7 1 Usa Marine Biological Institute, Kochi University, Inoshiri 194, Usa, Tosa, Kochi 781-1164, Japan 2 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan 3 Graduate School of Kuroshio Science, Kochi University, 2-5-1 Akebono, Kochi 780-8520, Japan 4 Division of the Natural/Applied Sciences, Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka 2-1-1, Bunkyo-ku, Tokyo 112-8610, Japan 5 Department of Bioscience and Bioindustry, Tokushima University, 2-1 Minami-josanjima, Tokushima, 770-8506, Japan 6 Department of Chemical Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, 2-12-1, Ookayama, Meguro, Tokyo 152-8550, Japan 7 College of Marine Ecology and Environment, Shanghai Ocean University, Shanghai 201306, China ABSTRACT: Phylogenetic clades based on DNA sequence data are heavily used to delimit species in the current taxonomy of Ulva. However, because hybridisation within clades and among other clades for other species has been seldom tested, it remains unclear if molecular clades agree with species boundaries based on the biological species concept. An ITS-based Ulva clade including many specimens collected worldwide was provisionally named ‘U. flexuosa’; its species boundary is ambiguous, and in the literature this group has been variously merged with the closely related U. californica clade. In the present study, we clarified the species boundary of this clade and its taxonomic status. Namely reproductive relationships among strains in this and closely related clades were examined by culturing and hybridisation. The culture experiment demonstrated that the ITS-based clade includes a sexual variant and an obligate asexual variant. The sexual strains having 0%–0.4% divergence in ITS2 sequence successfully crossed with each other. In combinations among the closely related clades including true U. flexuosa with . 1.2% distance in the sequence, no hybridisation occurred. With U. californica strains having a 1.2% sequence divergence, the provisional ‘U. flexuosa’ strains had few hybrid sporophytes, and these produced zoids that failed to develop normally. Based on these observations of prezygotic and postzygotic isolation, we conclude that the ITS-based clade delimits a different species from U. flexuosa and U. californica. Taxonomically, both the sexual and the asexual variants are assigned to U. mediterranea. KEY WORDS: Asexual, Life history, Prezygotic isolation, Postzygotic isolation, Sexual, Species boundary, Taxonomy, Ulva mediterranea INTRODUCTION Species of the genus Ulva Linnaeus are ubiquitous and abundant in the seashore littoral zone as well as brackish or freshwater areas, particularly in temperate areas and increasingly all over the world. Some Ulva species cause massive macroalgal blooms called green tides, which produce significant impacts on the local environment and economy (Liu et al. 2013), and others are utilised as an important food item (Ohno 1993). Because the genus Ulva includes various species and varieties with different ecophysiological charac- teristics, species identification is essential for furthering ecological and environmental studies. In the current Ulva taxonomy, a combination of classical morphological meth- ods and molecular phylogenetic analyses has been applied for species identification. Recently, reports on Ulva species identification using this combined approach have greatly increased (e.g. Kraft et al. 2010; O’Kelly et al. 2010; Mareˇ s et al. 2011; Wolf et al. 2012; Guidone et al. 2013; Kirkendale et al. 2013; Kazi et al. 2016). In these studies, the ITS region of nuclear ribosomal DNA is the most common molecular marker, and it is often used with other markers, such as the plastid rbcL and tufA genes, to create phylogenetic trees with better topological support. However, it remains unclear if the phylogenetic clades based on these molecular markers carefully reflect species boundaries because the relationships of the phylogenetic clades have been seldom examined in the context of the biological species concept. Therefore, each study has arbitrarily assessed whether a range of sequence variation in a certain clade is within or outside of a single species boundary. This has caused uncertainties in the taxonomic circumscription of some Ulva clades. A typical example is a clade generally indicated as ‘U. flexuosa Wulfen’, which includes various specimens collected worldwide, mainly from eastern Asia. This ITS-based clade, originally formed by four Japanese and Greek specimens (GenBank accession number AB097644-7), was reported with the provisional name ‘U. flexuosa’ by Shimada et al. (2003) because their sequences were closely related to the sequence of a Swedish sample identified as U. flexuosa (AJ234306) by Tan et al. (1999). Since then, many more specimens belonging to this clade have been sequenced from * Corresponding author (mhiraoka@kochi-u.ac.jp). DOI: 10.2216/16-109.1 Ó 2017 International Phycological Society Phycologia Volume 56 (5), 517–532 Published 16 May 2017 517