INTRODUCTION

Derbesia Solier, which belongs to Bryopsidales J.H. Schaffner (Lam and Zechmann 2006; Verbruggen et al. 2009), is a siphonous green algal genus with a thallus composed of erect and prostrate filaments and multiflagellate zoospores. This genus was established based on D. marina (Lyngbye) Solier (1846), which was originally described from Quivig, Faeröes, Denmark (Lyngbye 1819). At that time, only the general features of filaments and sporangia with gross morphology were given to the type species (Lyngbye 1819). However, many studies on life history, with great interest, increased our knowledge of Derbesia (Kornmann 1938; Feldmann 1950; Ziegler and Kingsbury 1964; Page 1970; MacRaild and Womersley 1974; Mayhoub 1976; Kobara and Chihara 1981, 1984). It was first clarified by Kornmann (1938) that Derbesia marina and Halicystis ovalis (Lyngbye) Areschoug are sporophytic and gametophytic phases of the one species. This suggested that Derbesia shows an alternation of heteromorphic generations with a Halicystis phase, which had been regarded as an independent genus characterized as having a coenocytic thallus consisting of an erect vesicular part and a rhizoidal part, although some authors (Kornmann 1966, 1970; Sears and Wilce 1970) have shown that direct reproduction of the sporophyte of D. marina can occur, as in Pedobesia established based on D. clavaeformis from Australia (MacRaild and Womersley 1974; Silva et al. 1996).

Derbesia is distinguished from the most similar genus Pedobesia MacRaild & Womersley in having basal septum at the portion of branching and heteromorphic life history with a gametophytic Halicystis stage and in lacking calcium carbonate deposited inside the cell wall (MacRaild and Womersley 1974; Woolcott et al. 2000, Table 2). This genus is globally distributed from the tropics to Arctic waters, and includes about 20 species worldwide (Guiry and Guiry 2018). Of these, three species have been recorded in Korea (Lee and Kang 1986, 2002; Kim et al. 2013). In the course of a floristic study of Korean marine algae, two siphonous green algae were collected from the eastern coast of Korea. This study reports unrecorded two Derbesia species based on morphological and molecular analyses.

MATERIALS AND METHODS

Samples for the present study were collected from the eastern coast of Korea. Some 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. Sections of the thallus were mounted in 20% corn syrup for permanent slides.

Total genomic DNA was extracted from a silica-gel-preserved sample using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturerʼs protocol. Before extraction, dried material was crushed with liquid nitrogen using a mortar and pestle. Concentrations of extracted DNA were assessed using gel electrophoresis on a 1% agarose gel. Extracted DNA was used for amplification of rbcL regions using following primers (forward: 5ʹ-TCCAA AAACTGAAACTAAAGCAGG-3ʹ, reverse: 5ʹ-AATTTCT TTCCAAACTTCACAAGC-3ʹ) (Lam and Zechman 2006).

PCR amplifications were performed in a TaKaRa PCR Thermal Cycler Dice with an initial denaturation step at 94°C for 5 min followed by 35 cycles at 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min and a final extension at 72°C 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 each of forward and reverse primer, and 0.5 units of Taq polymerase (Takara Korea, Korea). Amplifications were examined using gel electrophoresis in a 1% agarose gel and amplified rbcL region products were purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). 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 the maximum-likelihood (ML), neighbor-joining (NJ) and unweighted pair group method using arithmetic averages (UPGMA) methods. Bootstrap values were calculated with 1,000 replications. RbcL sequences of other species were obtained from GenBank. Bryopsis pennata J.V. Lamouroux was used as an outgroup.

RESULTS AND DISCUSSION

Derbesia minimaWeber-van Bosse 1913: 95.

Type locality: Kalimantan Selatan, Selat Makassar, Lumu- Lumu, Borneo bank, Indonesia (Huisman 2015).

Korean name: Jag-eun-eong-kin-sil nom. nov. (신칭: 작은 엉킨실).

Specimens examined: NIBRCL0000112655, MGARB01 2361, MGARB012362, MGARB012363, MGARB012364 (Gisung-ri: 5.vi.2016).

Habitat: Epiphytic near the lower intertidal.

Morphology: Sporophytic thalli up to 1 cm high, usually epiphytic on other algae, tufted, light green in color (Fig. 1A, B), with erect and prostrate systems; many erect filaments arising from a prostrate system with rhizoidal base, siphonous, cylindrical, 30-60 μm diam., subdichotomously branched several times (Fig. 1C); filamentous lateral branches with a basal septum (Fig. 1C); chloroplasts numerous, subspherical to lenticular, relatively small, measuring 3-10 μm long and 2-5 μm wide, each with a pyrenoid (Fig. 1D). Sporangia and gametophytes were not observed during the present study.

Derbesia indica (R. Patel & Isabella) P.C. Silva 1996: 810.

Type locality: Mithapur near Port Okha, Gujarat, India (Silva et al. 1996).

Korean name: Na-gog-eong-kin-sil nom. nov. (신칭: 나곡 엉킨실).

Specimens examined: NIBRCL0000112654, MGARB01 2365, MGARB012366, MGARB012367, MGARB012368 (Nagok-ri: 24.vii.2015).

Habitat: Epilithic or epiphytic near the intertidal.

Morphology: Sporophytic thalli up to 2 cm high, epilithic or epiphytic on other algae, tufted, light green in color (Fig. 2A, B), composed of erect and prostrate systems; many erect filaments arising from rhizoidal base of a prostrate system, siphonous, cylindrical, 50-80 μm diam., sparsely subdichotomously branched (Fig. 2C); filamentous lateral branches with a basal septum (Fig. 2C); chloroplasts numerous, spherical, measuring 5-15 μm diam., each with a pyrenoid (Fig. 2D). Sporangia and gametophytes were not observed during the present study.

Derbesia minima, which occurs in various localities including Japan, Australia, New Zealand and Micronesia (Guiry and Guiry 2018), was originally described from Indonesia (Huisman 2015). This species is characterized by the combined features of relatively small tufted sporophytic thalli arising from an irregularly lobed rhizoidal base, cylindrical erect branches with transverse and basal septum, ovoid or pyriform sporangia with a short pedicel and double basal septa, and each subspherical chloroplast with one pyrenoid (Huisman 2015; the present study). Of these features, the basal septum at point of branching is one of the important generic features of Derbesia (MacRaild and Womersley 1974; Woolcott et al. 2000). According to Huisman (2015), apart from this basal septum, transverse septa in vegetative siphons are also occasionally observed in this species from Australia. D. minima is distinguished from similar species with a small thallus (lower than 5 mm in height), such as D. attenuate E.Y. Dawson, D. furcate R.W. Ricker and D. padinae Trono, by the occasional presence of the septum (Dawson 1954; Trono 1971; Ricker 1987; Huisman 2015). Derbesia minima appears to be distinct in having a transverse septum, which is not a typical feature of the genus. These transverse septa were not found in Korean specimens. The Korean specimens also differ from the species from Australia and Japan in branching of erect filaments. It shows subdichotomous branching several times, while Australian and Japanese species branches once or not at all (Yoshida 1998; Huisman 2015). However, other vegetative features are shared with D. minima. Although sporangia and gametophytes were not observed, the Korean specimens appear to be referred to D. minima based on vegetative morphology. This is also supported by molecular data.

Derbesia indica was originally described from India based on gametophytes (Patel and Isabella 1978). So, at that time, it was established as Halicystis indica, but later transferred to Derbesia (Silva et al. 1996). This species is characterized by the combined features of tufted sporophytic thalli arising from rhizoidal base, sparsely subdichotomously branched filaments with a basal septum and each spherical chloroplast with one pyrenoid (the present study). D. indica is similar to D. minima, D. attenuata, D. tenuissima (Moris & De Notaris) P. Crouan & H. Crouan, and D. boergesenii (M.O.P. Iyengar & Ramanathan) Mayhoub in having one pyrenoid per chloroplast. However, D. indica is readily distinguished from D. minima and D. attenuata by thallus size. D. indica shows a relatively large thallus (about 2 cm), while the latter two species have thallus height of about 2 mm and 5 mm, respectively. D. tenuissima differs from D. indica in lacking a cross septum (Wormersley 1984; Kim and Klochkova 2010) or in occasionally having a basal septum in lateral branches (Abbott and Huisman 2004; Huisman 2015). D. boergesenii is also distinguished from D. indica in branching pattern and size of lateral filaments. The former species shows irregular branching and a relatively small size of 15-36 μm diameter (Mayhoub 1976) rather than sparsely subdichotomously branching and large size of 50-80 μm diameter. More importantly, it is distinguished from D. indica in being fusiform rather than spherical chloroplast (Mayhoub 1976).

In a phylogenetic tree based on rbcL sequence, the Korean algae nest in the same clades as D. minima and D. indica, respectively (Fig. 3). According to Woolcott et al. (2000, Table 4), the genetic distance for rbcL sequence between Derbesia and Pedobesia is 13.9%. In the present study, the interspecific genetic distance in Derbesia was calculated as 1.4-11.9%. Based on these data, the genetic divergence between the sequences within the clades, which was 0.5-0.8%, is considered to be included in the intra-specific range for the Derbesia.

These morphological and molecular analyses indicate that the two siphonous Korean algae are identified as Derbesia minima and D. indica originally described from Indonesia and India, respectively. This study reports the first occurrence of the species in Korea.