Ulva Linnaeus, which is a large genus within Ulvaceae J.V. Lamouroux ex Dumortier, includes about 100 species worldwide (Guiry and Guiry 2014). This genus is found in all aquatic habitats from freshwater to seawater environments (Loughnane et al. 2008). In the past, the taxonomy of Ulva had been based mainly on morphology (Bliding 1963, 1968, Koeman and van den Hoek 1981, Hoeksema and van den Hoek 1983). However, this makes it difficult to identify Ulva species, because of the simple thallus structure (Heesch et al. 2009). Recently, molecular analysis, which is an useful tool for differentiating taxa, has been adopted in the identification, taxonomy and phylogeny of species within this genus (Malta et al. 1999, Tan et al. 1999, Hayden et al. 2003, Loughnane et al. 2008, Hofmann et al. 2010). Many synonyms reported in Ulva have been the result of previous molecular analyses (Hayden et al. 2003, Brodie et al. 2007, Sfriso 2010, Alongi et al. 2014, Guiry and Guiry 2014).

A total of 15 species are currently recorded in marine algal flora of Korea (Lee and Kang 1986, 2002, Lee 2008, Bae 2010, Kim et al. 2013). During survey of indigenous species, two marine ulvalean species (Chlorophyta) were collected from the southern and eastern coasts of Korea. These two Korean entities were identified based on morphological and molecular analyses and are newly recorded in marine algal flora of Korea in the present study.

Samples for the present study were collected from Busan and Pohang, Korea, in 2014. All specimens were preserved in 5-10% formalin seawater, and pressed on herbarium sheets. A portion of the material was dried and preserved in silica gel for molecular analysis. Species identification was based on thallus morphology following the criteria of Bliding (1963, 1968) and Koeman and van den Hoek (1981). Sections of the thallus were mounted in 20% corn syrup for permanent preparation.

Total genomic DNA was extracted from silica-gel-preserved sample using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Before extraction, dried material was crushed with liquid nitrogen using a mortar and pestle. Concentrations of extracted DNA were assessed by using gel electrophoresis on a 1% agarose gel. Extracted DNA was used for amplification of the internal transcribed spacer (ITS) regions using published primers (Blomster et al. 1998). PCR amplifications were performed in a TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio Inc., Otsu, Japan) with an initial denaturation step at 94℃ for 5 min followed by 35 cycles at 94℃ for 1 min, 56℃ for 1 min, and 72℃ for 2 min and a final extension at 72℃ for 7 min. The reaction volume was 20 μL, consisting of 20 ng of genomic DNA, 2 μL of 10x PCR buffer, 2 μL of 200 μM dNTP, 1 μL of each forward and reverse primer, and 0.5 units of Taq polymerase (TaKaRa Bio Inc.). Amplifications were examined using gel electrophoresis in a 1% agarose gel and amplified ITS region products were purified using a QIAquick Gel Extraction Kit (Qiagen). The PCR products were moved to Macrogen Sequencing Service for sequencing (Macrogen, Seoul, Korea). The PCR primers were also used for sequencing.

Sequences for the ITS region were aligned using BioEdit (Hall 1999). Phylogenetic analyses were performed using the neighbor-joining (NJ) and maximum-likelihood (ML) methods. Bootstrap values were calculated with 1,000 replications. ITS sequences of other species (excluding U. flexuosa and U. rigida collected from Korea during the present study) were obtained from GenBank. Umbraulva japonica was used as an outgroup.

Korean name: Yeon-gal-pa-rae nom. nov. (신칭: 연갈파래)

Specimens examined: NIBRAL0000143269, PKNU0000134878 (Cheongsapo: 17 May 2014).

Habitat: Epilithic near the lower intertidal.

Morphology: Thalli 15-25 cm high (Fig. 1a and 1b), erect, membranous, distromatic, usually unbranched or little branched conical to ligulate shape, light to dark green in color, soft in texture, attached by a small holdfast (<5 mm) on rocks near the lower intertidal; frond ribbon-shaped, with a spirally twisted basal portion, usually undulate at the margin, 40-60 μm thick in the upper portion (Fig. 1d), 80-120 μm thick in the basal portion (Fig. 1f); cells usually arranged in pairs, rectangular to polygonal near the middle to upper portion (Fig. 1c), oval to rectangular with round corners near the basal portion in the surface view (Fig. 1e), transformed into rhizoidal cells near the base, 15-25 μm × 10-15 μm, with a length to width ratio of 1.6-1.8 in the transverse section; chloroplasts cap-like, parietal, with 1 (-2) pyrenoids (5-6 μm).

Korean name: Gyeong-gal-pa-rae nom. nov. (신칭: 경갈파래).

Specimens examined: NIBRAL0000143268, PKNU 0000134875, PKNU 0000134879 (Duhodong: 3 July 2014).

Habitat: Epilithic near the subtidal.

Morphology: Thalli 20-30 cm high (Fig. 2a), membranous, distromatic, irregular in shape, light to dark green in color, stiff in texture, attached by small holdfast on rocks near subtidal; frond irregular in shape, usually undulate and dentate at the margin (Fig. 2d), with small macroscopic denticulations along the margin (Fig. 2b), 40-60 μm thick (Fig. 2f); denticulations usually branched (Fig. 2c); cells usually arranged in pairs, rectangular to polygonal with round corners in the surface view (Fig. 2e), 5-15 μm × 5-10 μm, with length to width ratio of 1.2-2.0 in the transverse section; chloroplasts cap-like, parietal, with (1-) 2-3 pyrenoids (2-3 μm).

Ulva flexuosa, which occurs in various localities including China and Japan (Guiry and Guiry 2014), was originally described from Duino, Adriatic Sea (Womersley 1984). This species is very similar to U. linza and U. lactuca in habit. However, U. flexuosa is distinguished from the two species by thalli with a spirally twisted basal portion, undulate margin and ribbon-shaped frond. Specimens collected from Cheongsapo, Busan, during the present study share these features as described above (Fig. 1a and 1b).

Ulva rigida resembles U. laetevirens Areschoug in having macroscopic denticulations along the thallus margin (Sfriso 2010). However, the denticulations in U. rigida are very numerous and branched, while those in U. laetevirens are occasionally isolated and rarely branched (Sfriso 2010). This type of denticulation is also found in Korean specimens collected during the present study.

Many studies have used the ITS region to analyze the phylogenetic relationships among Ulva species (Malta et al. 1999, Hayden et al. 2003, Hayden and Waaland 2004, Hofmann et al. 2010, O’Kelly et al. 2010). In phylogenetic tree obtained from the present study based on the molecular data (Figs. 3 and 4), the former Korean entity forms the same clade with U. californica as a subclade of the group containing U. flexuosa from Greece (near type locality) and China and U. californica from USA (type locality) and Japan with relatively high bootstrap value of 92% in NJ tree and 86% in ML tree. In this group supported by the undulate margin of thalli, genetic distance between the Korean entity and U. flexuosa ranged from 1.1% to 1.3%, while that between U. californica and U. flexuosa was 0.8 - 1.3%. According to Mareš et al. (2011), the genetic distance for ITS sequences among U. flexuosa subspecies range from 3.8% to 5.9%. Based on this report, the Korean entity and U. californica are categorized into a specific variation within U. flexuosa. The present analyses, which show interspecific genetic distance of 2.1-13.3% in ulvalean species, also support strongly the intraspecific variation between those species. Accordingly, Ulva californica Wille in Collins et al. (1899), which is currently accepted (Guiry and Guiry 2014), is synonymized with U. flexuosa, here.

The second Korean entity nests in the same clade with U. armoricana and U. scandinavica. Recently, however, U. armoricana and U. scandinavica were reduced to synonymy with U. rigida based on molecular studies (Brodie et al. 2007). Although this Korean entity forms a sister clade to other U. rigida from Spain (type locality) and Scotland in NJ tree, genetic distance between those species is 0.5% within intraspecific variation range.

Based on these morphology and molecular data, our Korean entities are identified as U. flexuosa and U. rigida, respectively. This is the first record of these two ulvalean species in Korea.