Diatoms of the family Rhizosoleniaceae De Toni are an important group of marine phytoplankton, which are obtained from net and water samples from a variety of habitats. The genera Rhizosolenia Brightwell, Neocalyptrella Hernandez-Becerril and Meave, Pseudosolenia Sundstrom and Proboscia Sundstrom are very common marine diatom species that often dominate marine diatom assemblages (Sundstrom 1986, Hernandez-Becerril and Meave del Castillo 1996, 1997). The marine diatom genus Rhizosolenia was established over a century ago(Ehrenberg 1843) with R. americana as the type species.Subsequently, a detailed description and differential diagnosis of the genus was provided, based mainly on morphological traits common to four species (R. styliformis,R. imbricata, R. setigera, and R. alata) that included the following characteristics: filamentous, frustules sub-cylindrical, hugely elongated, silicious, marked by transverse lines, extremities calyptriform, pointed with a bristle and commonly single-celled (Brightwell 1858).

In coastal waters of Korea, Shim (1994) reported 26 Rhizosolenia species and two related genera, and Lee(1995) listed up 19 Rhizosolenia species and four related genera in an examination of data from the early 1930’s to the end of 1995. Since then, Korean studies have been done sporadic, and species identification, synonymies and the phylogenetic system of the family Rhizosoleniaceae remain insufficient.

The present study was undertaken to provide a detailed survey of the genus Rhizosolenia species from coastal waters of Korea. This survey includes morphological details revealed by light microscopy (LM) and scanning electron microscopy (SEM), and a critical review of taxonomical and distributional data.

Rhizosolenia species were collected from September 2008 to February 2010 at 30 sites in Korean coastal waters (Table 1, Fig. 1). Specimens were collected at a depth of about 10 m using a manufactured 20 ㎛ mesh-sized plankton net by oblique or vertical towing. Net samples were immediately fixed with 4% neutralized formalin, 2.5% glutaraldehyde and Lugol's solution.

To remove cell organelles and organic matter, samples were prepared as previously described (Hasle and Fryxell 1970, Simonsen 1974) with some modifications. Briefly, each sample was extensively washed with distilled water using five cycles of centrifugation followed by decantation of as much water as possible, leaving the sample almost dry. An equal amount of saturated KMnO4 was added, and the sample was dispersed by agitation and left for 24 h. An equal volume of concentrated HCl was added, the sample was boiled gently, and rinsed / decanted repeatidly with distilled water as before.

LM observations of acid-cleaned natural samples were conducted using an Axioskop 40 light microscope (Carl Zeiss, Jena, Germany) equipped with a Axiocam MRc5 digital camera (Carl Zeiss). The cleaned materials were mounted to produce a permanent slide as previously described (Hasle and Fryxell 1970). Briefly, a drop of each cleaned sample was transferred to a cover slip that was then cleaned with 100% ethanol. The deposited material was dried over a gentle heat or left overnight until the resin became firm. Dehydrated sub-samples were immersed in Pleurax. The permanent slides were observed at a magnification of ×400 or ×1,000.

SEM was done using a model JSM-5600LV apparatus (Jeol, Tokyo, Japan). Acid-cleaned natural samples, or samples prepared as previously described (Hasle and Fryxell 1970) were attached to aluminum stubs and coated with cold-palladium. Other samples were prepared as described by Jung et al. (2010), where live cells were fixed with modified Parducz’s fixative (Parducz 1967) at 1 : 1 v/v for 10 min at room temperature. The fixative was a 2% solution of osmium tetroxide in filtered seawater and a saturated solution of mercuric chloride in distilled water mixed at a ratio of 5 : 1 v/v, respectively. Fixed cells were harvested by gravity filtration on a 2.0 ㎛ polycarbonate membrane (TTTP; Millipore, Billerica, MA, USA). To prevent the formation of NaCl crystals, any seawater remaining associated with the specimen was removed by washing for 2 min at room temperature with drops of distilled water followed by drops of 0.05 M C₂H₁₂AsNaO₅ buffer (pH 8.0). For dehydration, drops of diethyl ether (C₄H₁₀O) were continuously dripped on the samples for 10 min at 30°C. Following this procedure, several drops of hexamethyldisilane (C₆H₁₉NSi₂) were immediately dispensed on the membrane to complete the drying process.

Monographs of species belonging to family Rhizosoleniaceae were used for identification (Cupp 1943, Hendey 1964, Sundstrom 1986, Hasle and Syvertsen 1996). Terminology followed previously published general proposals (Ross et al. 1979, Sundstrom 1986, Round et al. 1990, Hernandez-Becerril 1995, Hasle and Syvertsen 1996).

Systematics and phylogenetics of members of the family Rhizosoleniaceae are suggested by Sundstrom (1986) as follows. The characters observed in the Rhizosolenia species presently-studied are summarized in Table 2. Each description includes references for identification,

by means of LM and SEM.

Class Bacillariophyceae Haeckel 1878

Order Centrales Hustedt 1930

Suborder Rhizosoleniineae Simonsen 1979

Family Rhizosoleniaceae De Toni 1890

Genus Rhizosolenia Brigtwell 1858

R. bergonii H. Peragallo 1892

R. formosa H. Peragallo 1888

R. hyalina Ostenfeld in Ostenfeld and Schmidt 1901

R. setigera Brigtwell 1858

Peragallo 1892, p. 15, Pl. 3, Fig. 5; Hustedt 1920, p. 318, Fig. 1-3, as R. amputata; Hustedt 1930, p. 575, Fig. 327;Cupp 1943, p. 81, Fig. 43a-g; Hendey 1964, p. 151, Pl. 3, fig. 4; Sundstrom 1986, p. 72, Fig. 32 & 33, 177-189; Hernandez-Becerril 1995, p. 256, Fig. 13-16; Hasle and Syvertsen 1996, p. 155, Pl. 29; Sunesen and Sar 2007, p. 629, Fig. 4-15.

Synonym: Rhizosolenia amputata Ostenfeld 1902

The cells of R. bergonii are usually solitary, cylindrical, bilaterally symmetrical, and circular in cross-section. Specimens found in Korean coastal waters are 7.5-18.6 ㎛ in diameter, and cells are 211.7-313.3 ㎛ long. Valves are acute and conical. The ventral part of valve is longer than the dorsal. The valve areolae are elongated in the pervalvar axis direction. Areolae are 20-25 per 10 ㎛, arranged in converging striae towards the apex 15 per 10 ㎛, trapezoidal in outline. The process is thick, straight, clefted at the tip, 3.2-16.1 ㎛ long, internal structure of the process is visible by LM, with basal lumen spindle-shaped, abruptly narrowed in a tubular canal and expanded into a funnel-shaped aperture at the tip. Contiguous area is visible by SEM, slightly depressed, not delimited by marginal ridges. Claspers and otaria are absent. Girdle segments are in two dorsiventral columns or several columns, very variable in size and shape, rhomboidal to trapezoid in outline. Segment with horizontal axis and perpendicular axis are each other 7.5-18.6 ㎛ and 8.1-12.2 ㎛ in length. Segment areolae, 22 in 10 ㎛, are arranged in striae parallel to the pervalvar axis, 20-22 per 10 ㎛, with a secondary quincuncial pattern.

Distribution: R. bergonii is a species from the warm water region (Sundstrom 1986, Hasle and Syvertsen 1996, Romero and Hensen 2002). During the present study, it was recorded in January and October 2009 in the Korea Strait and Yangyang, respectively. The species was frequently found in our samples.

Remarks: R. bergonii resembles R. temperei H. Peragallo and R. acuminata (H. Peragallo) H. Peragallo in general appearance, areola structure, and lack of otaria. Nevertheless, the former may be well separated, based on the shape of the process, with a cleft of tip.

Peragallo 1888, p. 83, Pl. 6, Fig. 43; Sundstrom 1986. p. 33, Fig. 12, 13, 88-93 & 96-99; Hernandez-Becerril 1995, p. 256, Fig. 25-30.

Synonym: Rhizosolenia styliformis f. latissima Brightwell in H. Peragallo 1892

The cells of R. formosa are usually solitary or rarely in pairs, cylindrical, bilaterally symmetrical, and circular in cross-section. Specimens are 93.3-206.3 ㎛ in diameter. The shallowly conical valves and cells are 493.3-700 ㎛ long. The ventral part of valve is much longer than the dorsal, medially extended into a tongue accommodating impression of sister-cell process. The valve areolae are elongated in pervalvar direction, 18-25 in 10 ㎛. The process is tapering from the base, resembling the tube, 3.8-27.7 ㎛ long, 1.9-3.9 ㎛ in diameter of the process. Contiguous area is visible with LM, distinct along the central part of ventral surface. Claspers and otaria are present and fairly prominent. Girdle segments are arranged in

two dorsiventral columns, usually of wing shape, median margins short, lateral margins very long. Segment horizontal axis and perpendicular axis are 93.3-206.3 ㎛ and 23.9-28.3 ㎛ length, respectively. Segments areolae, 10-43 per 10 ㎛, with a secondary quincuncial pattern.

Distribution: R. formosa is probably circumglobal within oceanic waters and the warm water region. It was observed in the waters surrounding Ko Phuket, Thailand through at the year, but rarely abundant (Sundstrom 1986). In this study, R. formosa is a newly recorded species in September 2008 in oceanic waters of the Yellow Sea in Korea.

Remarks: R. formosa is a very large species and very weakly frustules in the genus Rhizosolenia. At LM observations, R. formosa is distinguished by the shape of the girdle segments and by its large size. According to Sundstrom (1986), R. formosa resembles R. styliformis and R. formosa in Thailand. But areola patterns and structure of both valve and segments are indistinguishable among the two species (Sundstrom 1986).

Ostenfeld and Schmidt 1901, p. 160,Fig. 11; Hustedt 1920, Pl. 319, Fig. 11-13; Sundstrom 1986, p. 76, Fig. 34, 190-194; Hernandez-Becerril 1995, p. 258, Fig. 32-35; Hasle and Syvertsen 1996, p. 151, Pl. 28; Sunesen and Sar

2007, p. 631, Fig. 16-24.

Synonym: Rhizosolenia pellucida Cleve 1901

The cells of R. hyalina are usually solitary, chain forming, cylindrical, bilaterally symmetrical, and circular in cross-section. Specimens found in Korean coastal waters are 20-63.3 ㎛ in diameter. The sub-conical valves and cells are 225.7-300 ㎛ long. The ventral part of valve is longer than the dorsal. Valve outline is characteristically undulated. The valve areolae are elongated in pervalvar direction, 43-45 per 10 ㎛. The process is thin, straight or slightly curved, long, tapering needle-shaped at the apex, and 26.7-80 ㎛ long. Contiguous areas clearly visible only near the margin where the sister-cell’s processes begin, claspers are clearly founded. Otaria extend along the process, narrow, rounded at the tip, parallel to long axis of process. Girdle segment columns are 2n (n = 2, 3, … 8), cells with only two girdle segment columns were not found in the present study, rhomboidal to trapezoidal, with subrectangular to rounded areolae. Segment horizontal axis and perpendicular axis are 23.8-26.2 ㎛ and 10.5-14.3 ㎛ in length, respectively. Segment areolae, 24-36 per 10 ㎛, with a secondary quincuncial pattern.

Distribution: R. hyalina is a warm water species (Sundstrom 1986, Hasle and Syvertsen 1996). It was reported for the first time from the South Atlantic Ocean in 1982 from marine waters of Rio Grande do Sul by Rosa (1982) and subsequently quoted in several papers from other coastal waters of Brazil (Moreira Filho et al. 1999). During this study, R. hyalina was observed in September 2008 in the oceanic waters of the Yellow Sea.

Remarks: R. hyalina was clearly and concisely described by Ostenfeld (Ostenfeld and Schmidt 1901). This species shows the characteristics outlined above. There was very little variability observed. Only a few floristic studies include R. hyalina (Hustedt 1920, Gaarder 1951, Sourina 1968). It was probably contained within Allen and Cupp's (1935) description of R. clevei, which it resembles superficially. However, R. clevei apparently always contain the endobiotic cyanobacterium Richelia intracellularis (Sundstrom 1984), that R. hyalina never contains.

Hustedt 1930, p. 588, Fig. 336; Navarro 1981, p. 430, Fig. 46 & 47; Sundstrom 1986, p. 104, Fig. 286-288; Hernandez-Becerril 1995, p. 264, Fig. 44 & 45; Hasle and Syvertsen 1996, p. 157, Pl. 30; Sunesen and Sar 2007, p. 633, Fig. 25-34.

Synonym: Rhizosolenia japonica Castracane 1886

The cells are solitary or in pairs, cylindrical, bilaterally symmetrical, circular in cross-section, 3.3-16.7 ㎛ in diameter, and 125-350 ㎛ long. The valve is sharply sub-conical, elongated. Valve areolae are poroid; Fig. 4. areolae with slit-like pores, 20-50 per 10 ㎛, with a secondary orthostic pattern. Scattered pores are circular, irregularly distributed among areolae or taking the place of some areolae, distinguishable in external view. The process is very long needle shape, almost straight, wider at the base and gradually tapering along the apex, 33.3-130.7 ㎛ long. Contiguous area is observed, groove-shaped, deeper at the basal region than at the terminal region. Otaria are absent, claspers are poorly noticeable. Segment with horizontal axis and perpendicular axis are 23.8-26.2 ㎛ and 10.5-14.3 ㎛ in length respectively. Girdle segments are rhomboidal to trapezoidal and arranged in two dorsiventral columns. Segment areolae are rounded, 30-50 per 10 ㎛, arranged in striae oriented in pervalvar direction, with a secondary quincuncial pattern.

Distribution: R. setigera is a cosmopolitan taxon, probably absent from polar waters according to Hasle and Syvertsen (1996). This form has frequently been reported from Argentinian waters (Vouilloud 2003) as variety in some cases. The species was observed from September 2008 to September 2009 in the Korea Strait, around Jeju Island, Daecheon, Wolseung, Kanghwa Island, Geoje Island, Namhae, Sacheon and Tongyeang.

Remarks: R. setigera resembles R. antennata f. semispina and R. hebetata f. semispina in morphology of valve and process. However, this species is distinguished from the latter two species by the lack of otaria. Additionally, R. setigera is a cosmopolitan taxon, the other species occur in cold water regions.