INTRODUCTION

The red algal genus Chondrus (Gigartinales, Gigartinaceae) was typified with Chondrus crispus Stackhouse, which is distributed across the North Atlantic coast (Brodie et al. 1991;Hommersand et al. 1993, 1994). This genus is primarily characterized by lacking an envelope separating carposporangia from gametophytic tissues (Mikami 1965;Hommersand et al. 1993), and currently includes 17 species worldwide (Hommersand et al. 1993;Brodie et al. 1997;Guiry and Guiry 2020). These species are mainly distinguished by thallus shape and position of reproductive structures (Taylor and Chen 1994;Matsumoto and Shimada 2013).

Of these, Chondrus verrucosus Mikami (1965) was originally described from Inubosaki, Chiba Prefecture, Japan, based on Chondrus ocellatus Holmes f. canaliculatusOkamura (1932) with linear to cuneate, canaliculated fronds. This species seems to be delimited from other Chondrus species by strongly canaliculated fronds, verruciform cysto-carps, and reproductive structures limited to distal portions of the thallus (Mikami 1965). According to Matsumoto and Shimada (2013), C. retortus is very similar to Chondrus verrucosus in morphology. In the present study, a species similar to Chondrus verrucosus and C. retortus was collected from the southern coast of Korea, and is newly recorded in Korean marine algal flora based on morphological and molecular analysis.

MATERIALS AND METHODS

Specimens for this study were collected from Honghyeon- ri, Namhae located in the southern coast of Korea. Morphological data were obtained from fresh, liquid-preserved and herbarium specimens. Liquid-preserved material was stored in a 10% solution of Formalin/seawater. Blades dissected from the cleared materials were hand sectioned, transferred to a slide with distilled water, and mounted in pure glycerin. Measurements are given as width and length. For permanent slides, the glycerin was exchanged with 10-20% corn syrup.

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. Extracted DNA was used for amplification of ribulose-1, 5-bisphosphate carboxylase large subunit (rbcL) regions. For rbcL, the gene was amplified in three overlapping parts with the primer pairs FrbcL start (5ʹ-TGTGTTGTCGACATGTCTAACTCTGTAGAAG- 3ʹ) - R753 (5ʹ-GCTCTTTCATACATATCTTCC- 3ʹ), F492 (5ʹ-CGTATGGATAAATTTGGTCG- 3ʹ) - R1150 (5ʹ-GCATTTGTCCGCAGTGAATACC- 3ʹ), and F993 (5ʹ-GGTACTGTTGTAGGTAAATTAGAAGG- 3ʹ) - RrbcS (5ʹ-TGTGTTGCGGCCGCCCTTGTGTT AGTCTCAC-3ʹ) (Freshwater and Rueness 1994). PCR amplifications were performed in a TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio Inc., Otsu, Japan). PCR was performed with an initial denaturation step at 94°C for 4 min, followed by 35 cycles of 1 min at 94°C, 1 min at 50°C, and 2 min at 72°C, with a final 7-min extension at 72°C. The PCR products were moved to Macrogen Sequencing Service for sequencing (Macrogen, Seoul, Korea). Sequences for the rbcL region were aligned using BioEdit (Hall 1999). Phylogenetic analyses were performed using neighbor joining and maximum- likelihood methods. Bootstrap values were calculated with 1,000 replications. RbcL sequences of other species were obtained from GenBank. Gelidium corneum (Hudson) J.V. Lamouroux was used as an outgroup.

RESULTS AND DISCUSSION

Chondrus retortus K. Matsumoto & S. Shimada 2013

Korean name: Ae-gi-jin-du-bal-sa-chon nom. nov.

(신칭: 애기진두발사촌)

Type locality. Enoshima, Kanagawa Prefecture, Japan.

Specimens examined: NIBRRD0000004364-NIBRRD 0000004366 (Honghyeon-ri, Namhae: 25.x.2018).

Habitat: Epilithic near upper to lower intertidal.

Morphology: Thalli up to 5-10 cm high, compressed, thin, somewhat canaliculated, crispy, yellowish to dark brown in color, cartilaginous in texture, attached to substrate by small discoid holdfast; main axes issuing numerous branches and proliferations on margin and surface; proliferations lanceolate, pinnately arranged, more or less constricted at base, 5-10 mm in length, 1-2 mm in width; cortex 5-8 cell layer thick, with 2-3 cell layer thick in outer cortex; inner cortical cells ellipsoid to round in shape; medullary cells densely arranged; tetrasporangial sori round to polygonal, scattered on both sides of branches; tetrasporangia round to ellipsoidal, developed from shallow medullary cells, 10- 30 μm in diam., divided tetrahedrally. Sexual plants were not collected during the present study.

According to the original description (Matsumoto and Shimada 2013), C. retortus appears to be characterized by short, canaliculated and crisped fronds, carposporangia developing among roundish medullary and tetrasporangial development from shallow medullary. The vegetative and tetrasporangial features are found in the Korean alga collected from Namhae (Fig. 1).

Chondrus retortus is readily distinguished from the similar species, C. verrucosus, by the cell shape of the medullary layer around young carposporangia (Matsumoto and Shimada 2013). In the former, the cells are roundish, while the latter has a linear form (Matsumoto and Shimada 2013). In addition, both species are also distinguishable from each other in some vegetative features including thallus size in the mature stage. C. retortus has thinner, somewhat canaliculate and relatively small mature fronds rather than thicker, comparatively regularly canaliculate and typical large mature fronds (Matsumoto and Shimada 2013).

The position of tetrasporangial sori is an important diagnostic feature among the Gigartinaceae, though it is paraphyletic with respect to some genera (Mikami 1965;Kim 1976;Hommersand et al. 1999;Matsumoto and Shimada 2013). In Chondrus, the type species C. crispus has tetrasporangial sori filling the whole medulla, whereas in some species such as C. elatus, C. ocellatus, C. verrucosus and C. yendoi, they are developed from shallow medullary cells (Mikami 1965;Brodie et al. 1991;Fredericq et al. 1992;Matsumoto and Shimada 2013). Chondrus retortus also shows the same developmental features of tetrasporangial sori as C. verrucosus and C. elatus of the sister clade.

Although female plants were not collected during the present study, our Korean specimens can be identified as C. retortus based on the vegetative features. Their identity is supported by molecular data.

In general, the value of interspecific divergence in the Gigartinales varies from 2.8 to 16.5% (Hommersand et al. 1994;Kato et al. 2009). In the phylogenetic tree based on rbcL sequence, the Korean alga nests in the same clade as Chondrus retortus with a genetic distance of 0.0-0.1% (Fig. 2). Based on these morphological and molecular data, this species is identified as Chondrus retortus. This is the first record of C. retortus in Korean marine algal flora.