Most
Skeletonema
species have been identified under the name of
S. costatum
. To assess the morphological species diversity in the genus
Skeletonema
, we surveyed the nine localities in the coastal waters of South Korea. The light microscopy (LM) and scanning electron microscopy (SEM) observations in this study showed that ultrastructural features of genus
Skeletonema
discriminated among four species:
S. dohrnii
Sarno & Kooistra,
S. marinoi
Sarno & Zingone,
S. subsalsum
(Cleve) Bethge, and
S. tropicum
Cleve. In
S. dohrnii
, cell diameters were 3-6 μm and the pervalvar axes were 13-19 μm. One or two partial chloroplasts were visible in a cell. Cells of
S. marinoi
were 4-10 μm and the pervalvar axes were 8-18 μm. Each cell contained one chloroplast. Cells of
S. subsalsum
which contained 1-2 chloroplasts were 8-13 μm and the pervalvar axes were 11-20 μm. Cells of
S. tropicum
were 10-18 μm and the pervalvar axes were 4-9 μm. 2-4 chloroplasts were seen in each cell. Tip width of fultoportula in
S. dohrnii
and
S. marioni
was flared and flat, but that in
S. subsalsum
and
S. tropicum
was narrow. Morphological groups among them,
S. dohrnii
and
S. marinoi
were the most widely distributed in all seasons, while
S. tropicum
was only occurred in a summer season.
INTRODUCTION
Genus
Skeletonema
are the most important contributors to phytoplankton blooms in many coastal zones, worldwide (Karentz and Smayda 1984; Cloren
et al
. 1985; Estrada
et al
. 1985). In particular,
S. costatum
(Grev.) Cleve is the most conspicuous and widespread member of marine phytoplankton. Hasle (1973) reported that
S. costatum
was a cosmopolitan species, showing a wide range of tolerance for temperature and salinity, and that it exhibited extremely morphological variations in both size and shape. Moreover, this species is difficult to identify and easy to confuse with other
Skeletonema
species; in fact, Hasle wrote "To most marine phycologists, the name
Skeletonema
is synonymous with the specific name
S. costatum
." Other researchers also pointed out that
Skeletonema
costatum have high morphological variations (Gallagher 1980, 1982; Gallagher
et al
. 1984). It seems that many
Skeletonema
species might be hidden within the taxon.
The genus
Skeletonema
was established by Greville (1865) for a single species,
S. barbadensel
, recovered from the Barbados deposit. Characteristics given for the genus are cells joined by long marginal processes to form a filment. Plastids are disc-like or cup-shaped (Round
et al
. 1990). In the recent times, the 18 species of
Skeletonema
were recognized by various morphological characteristics and aquatic habits:
S. subsalsum
(Cleve) Bethge and
S. potamos
(Weber) Hasle in brackish and/or fresh-water regions (Hasle and Evensen 1975, 1976),
S. tropicum
Cleve in tropical regions (Hulburt and Guillard 1968), and
S. ardens
Sarno & Zingon (Sarno
et al
. 2007),
S. dohrnii
Sarno & Kooistra (Sarno
et al
. 2005), S. grethae Zingone & Sarno (Sarno
et al
. 2005),
S. grevillei
Sarno & Zingone (Zingone
et al
. 2005),
S. marinoi
Sarno & Zingone (Sarno
et al
. 2005),
S. menzelii
Guillard, Carpenter & Reimann (Guillard
et al
. 1974), and
S. pseudocostatum
Medline emend. Zingone et Sarno (Medlin
et al
. 1991) in oceanic area, respectively.
Skeletonema
blooms are frequently seen in Korean coastal waters throughout the year (e.g., Lee
et al
. 1997). Until now, these species have been usually reported as the name of
S. costatum
. Therefore, it is now needed to inform for species diversity in genus
Skeletonema
. The purpose of this study was to discriminate taxonomic features of four species in genus
Skeletonema
using light and scanning electron microscopy.
Collection information ofSkeletonemaspecies used in the analyses
Collection information of Skeletonema species used in the analyses
MATERIALS AND METHODS
Skeletonema diatoms were collected with a 20 μm meshsizeplankton net from November 2008 to July 2009 in thecoastal waters of South Korea (Table 1). Morphologicaldifferences of Skeletonema species were assessed by lightmicroscopy (LM) (Axioskop; Zeiss, Jena, Germany)equipped with an MRc5 digital camera (Zeiss) and scanningelectron microscopy (SEM) (JSM-5600LV; Jeol,Tokyo, Japan). For removal of cell organelles, organicmatters in samples were removed according to Hasleand Fryxell’s method (1970). Samples were washed withdistilled water to remove NaCl. The rinsed samples werecleaned by adding an equal amount of KMnO4 and HClin boiling water bath until the sample becomes clear oronly slightly colored. Then, samples were again washedwith distilled water. Another preparation procedure wasaccording to Jung et al.’s method (2009). Live cells werefixed with modified Parducz’s fixative (Parducz 1967) at1:1 v/v for 10 min at room temperature: the fixative comprised2% solution of osmium tetroxide (75632; Sigma-Aldrich, St. Louis, MO, USA) in filtered seawater and asaturated solution of mercuric chloride (M1136; Sigma)in distilled water mixed in the ratio of 5:1 v/v, respectively.Fixed cells were harvested by gravity filtration(XX10 025 00; Millipore, Bedford, MA, USA) onto a 2.0μm polycarbonate membrane (TTTP; Millipore). To preventthe formation of NaCl crystals, any seawaterremaining associated with the specimen was removed bywashing for 2 min at room temperature with drops ofdistilled water followed by drops of 0.05 M sodiumcacodylate trihydrate buffer (pH 8.0; 20838, Sigma). Fordehydration, drops of tert-butanol (308250; Sigma) werecontinuously dripped on the specimen for 10 min at 30°C. Following this procedure, several drops of hexamethyldisilazane(HMDS, H4875; Sigma) were immediatelydispensed onto the membrane to complete the dryingprocess. Finally, the specimens were coated with goldpalladiumfor 3 min, and examined using a SEM (JSM-5600 LV; Jeol).
Diatom terminology followed the literatures of Anonymous (1975), von Stosch (1975) and Ross
et al
. (1979).
RESULTS
We recognized four
Skeletonema
species, whose characteristics are shown in
Table 2
and
Figs 1
-4
:
S. dohrnii
(Fig. 1)
,
S. marinoi
(Fig. 2)
,
S. subsalsum
(Fig. 3)
, and
S. tropicum
(Fig. 4)
.
Morphological characteristics of four species in genus Skeletonema in this study and comparison with Sarno et al.’s observation(2005)
Morphological characteristics of four species in genus Skeletonema in this study and comparison with Sarno et al.’s observation(2005)
Skeletonema dohrnii
Sarno & Kooistra. Cells 3-6 μm and the pervalvar axes 13-19 μm (16 μm in length). Short chains composed of 7-28 cylindrical cells, forming slightly curved
(Figs 1A, B)
; longer chains at over 20 cells were very rare. One or two partial chloroplasts were visible in each cell
(Fig. 1A)
. In SEM observation, the distances between sibling cells were 6-12 μm (9 μm). Numbers of the fultoportula processes (FPs) and distances between FPs were 8-10 and 0.6-0.9 μm, respectively. The bases of the terminal FPs (TFPs) were open
(Fig. 1E)
, and the tips of the TFPs were flat and flared, with slightly jagged margins
(Fig. 1F)
. The shape of the intercalary FPs (IFPs) was similar to that of the TFPs
(Figs 1F, G)
. Each IFP was connected with one or two processes of the adjoining cell, forming a slightly interdigitated linkage
(Figs 1B, D)
. The terminal rimoportula process (TRP) was located at the sub-central zone of a valve face and consisted of a long tubular process, which widened along its length and truncated at its apex like a trumpet
(Fig. 1F)
. The intercalary RP (IRP) was shorter than the IFPs and was located near the center of the valve, with a tube-shaped distal end
(Fig. 1G)
. The valves were convex
(Fig. 1E)
, and the areolae were clear, widely spaced and irregularly arranged
(Figs 1F, G)
.
Skeletonema marinoi
Sarno & Zingone. Cells 4-10 μm and the pervalvar axes 8-18 μm. A colony was straightly connected with 16-33 cells, and each cell contained one chloroplast
(Figs 2A, B)
. In SEM, distances between sibling cells 3-9 μm. The numbers of FPs and distances between FPs were 9-13 and 0.6-1.1 μm, respectively. In TFPs, the base was open and the end of split tube was flat and flared or sometimes jagged
(Fig. 2C)
. The shape of the IFPs was similar to that of the TFPs
(Figs 2B, F, H)
. IFPs between sibling cells formed 1 : 1 or 1 : 2 linkages, and the intercellular junctions were either interdigitated or simply connected
(Figs 2D, F, G, H)
. The TRP located in the valve center was long with a trumpet-shaped end
(Figs 2B, C)
. The IRP was a short tube located at the margin of the valve face
(Fig. 2F)
. The valves were flat or slightly convex
(Figs 2C, D)
. The areolae distributed from the valve center had clear tetragonal forms
(Fig. 2F)
.
Skeletonema subsalsum
(Cleve) Bethge. Cells 8-13 μm and the pervalvar axes 11-20 μm. A colony was straightly connected with 12-46 cells, and each cell contained 1-2 chloroplasts
(Figs 3A, B)
. In SEM, the numbers of FPs and distances between FPs were 15-24 and 0.4-0.9 μm, respectively. An external larger pore on the base of the tube of TFPs was open and the tips of them were pointed at the end
(Fig. 3D)
. Sibling cells were joined by IFPs, which were generally short at a mean of 5 μm
(Figs 3B, G)
. Each IFP was connected to one or two processes (1 : 1 or 1 : 2 linkages) of the adjoining cell, forming an interdigitated linkage
(Figs 3B, G)
. The TRP on the center was long and had a hook-like end
(Fig. 3F)
. The marginal IRP was a curved tube with a trumpet-shaped end and was longer than the IFP
(Figs 3C, E)
. The valves were convex
(Fig. 3D)
or flat
(Fig. 3E)
. The areolae of the valve face were rectangular to triangular and ran radially from the center
(Figs 3C, E)
. In the mantle, the areolae were paral-lel to the pervalvar axis
(Fig. 3C)
.
Skeletonema dohrnii. Light microscopy (A); Scanning electron microscopy (B-G). (A) Colony in girdle view. (B) Colony in girdleview connected with the the intercalary FPs (IFPs) (arrow). (C) Scale-covered the IFPs. (D) Intercalary valves with IFPs joined ina 1 : 1 or 1 : 2 fashion (arrow). (E) Convex valve view with loculate areolae. Note the IFPs entirely split and open at their base(arrow). (F) Terminal valve with the long and tubular terminal rimoportula process (arrow). (G) Short IFPP (arrow) and theflared tips and jagged margins of IFPs (arrowhead). Scale bars: A = 20 μm; B = 5 μm; C, D, E, G = 1 μm; F = 2 μm.
Skeletonema marinoi. Light microscopy (A); Scanning electron microscopy (B-H). (A) Colony in girdle view. (B) Colony in girdleview connected with the intercalary FPs (IFPs) and the terminal rimoportula process (TRP) (arrow). (C) The TRP (arrow) and flatand flared tips of the terminal FPs (arrowhead) in convex valve view. (D, F) Intercalary valves with 1 : 1 (arrow in Fig. D) or 1 : 2(Fig. F) IFP junctions and short the intercalary RP (arrowhead in Figs D, F). (E) Cingular band with rows of pores. (F) The tetragonalareolae distributed from the valve center. (G, H) Two IFP junctions of different forms. Scale bars: A , B = 10 μm; C, E, G, H =1 μm; D, F = 2 μm.
Skeletonema subsalsum. Light microscopy (A); Scanning electron microscopy (B-G). (A) Colony in girdle view. (B) Scale-coveredcolony in convex valve view connected with the intercalary FPs (IFPs). (C) The marginal long intercalary RP (IRP) (arrow) in theintercalary valve. (D) The pointed terminal FPs in convex valve view. (E) the marginal IRP (arrow) in the flat valve view. (F)Terminal valve with the subcentral hook-shaped terminal rimoportula process (arrow). (G) The intercalary valve with 1 : 1 shortIFP junctions. Scale bars: A = 20 μm, B, E, G = 2 μm, C, D, F = 1 μm.
Skeletonema tropicum. Light microscopy (A, B); Scanning electron microscopy (C-H). (A, B) Colony in girdle view with slightlycurved joint (A) and straight joint (B). (C) Colony in girdle view connected with the intercalary FPs (IFPs). (D, F) The trumpetshapedterminal rimoportula process (arrowhead) and claw-shaped terminal FPs (arrow). (E, G) The intercalary valve with 1 : 1IFP interdigitated junctions. (H) The short marginal intercalary RP (arrow) and the tetragonal areolae distributed from the valvecenter. Scale bars: A , C = 20 μm; B = 10 μm; D = 2 μm; E, F = 5 μm; G, H = 1 μm.
Skeletonema tropicum
Cleve. Cells 10-18 μm and the pervalvar axes 4-9 μm, rarely exceeding twice the cell diameter. A colony was straightly connected with 12-52 cells, and 2-4 chloroplasts were seen in each cell
(Figs 4A-C)
. In SEM, 18-22 FPs per valve were seen, and the distances between FPs were 0.6-0.9 μm. The TFPs were open along the full length of the tubes, the ends of which were slightly broadened and the tips of which were clawshaped
(Figs 4D, F)
. The IFPs were open at both the ends and the bases, and centers were closed
(Fig. 4D)
. The intercellular junctions between sibling cells were intricate, tight, and knuckle-like, and were usually joined with 1 : 1 linkage
(Figs 4E, G)
. Sibling IFPs were not vertical to the valve face but were slightly deflective
(Fig. 4E)
. The tubular TRP was long and was located between the sub-central valve face, with a slightly dilated trumpet- like end
(Fig. 4F)
. The TRP diameter was longer than those of the TFPs. The short IRP, such as a spine, was located in the marginal valve
(Fig. 4H)
. The valves were flat or sometimes slightly convex
(Fig. 4E)
. Tetragonal areolae in straight or curved tangential rows were radially arranged and tended to fasciculate. The areolae of the mantle were round
(Fig. 4H)
.
DISCUSSION
Identification of genus
Skeletonema
is according to the key characteristics: cylindrical cells with a ring of long processes emerging from the edge of the valve face. However, exact identification of species in the genus
Skeletonema
is very difficult due to varying expression (Castillo
et al
. 1995).
S. dohrnii
and
S. marinoi
are morphologically involved in a same group (Sarno
et al
. 2005) and differ from other species in that their FPs possess flat and flare tips with dentate margins. They commonly appear the 1 : 2 linkages with displaced IFPs. The only morphological distinction between two species is the ultrastructure of the cingular bands (Sarno
et al
. 2005).
S. subsalsum
has a thick frustule with a pseudoloculate structure and short IFPs, which are displaced in mainly 1 : 1 junction. Somewhat similar IFPs forms of
S. subsalsum
are found in those of
S. costatum
(Sarno
et al
. 2005): description of
S. subsalsum
in Hasle and Evensen (1975) is rather similar to the
S. costatum
in
Fig. 3G
in Zingone
et al
. (2005). However, the short and irregular TFPs of
S. subsalsum
look quite different from the straight and narrow clawshaped ones of
S. costatum
(Sarno
et al
. 2005). Cell shapes of
S. tropicum
are similar with those of
S. costatum
. Hasle (1973) demonstrated that
S. costatum
and
S. tropicum
were not distinguishable from the morphological patterns of the frustules in LM, even though
S. tropicum
has a range of cell diameter that includes some sizes larger than
S. costatum
. We could identify four species of the genus by taking into account as many characters as possible, but some forms showed a certain overlap in some characters, mainly the shape of the external part of the fultoportula and rimoportula, and the number of chloroplasts.
Distribution of
Skeletonema dohrnii, S. japonicum
and
S. tropicum
in Korean coastal areas was reported by Kooistra
et al
. (2008).
S. marinoi
and
S. subsalsum
were the first species demonstrated in this study.
S. tropicum
, a warm water species, was collected in summer in the South and Yellow Sea.
S. dohrnii
and
S. marinoi
were observed in all sampling sites.
S. subsalsum
was collected in the Kyung-gi and Gang-mok Bay, both of which have low salinity (< 25 psu). S. japonicum, a cold water species, was not collected in these investigations.
S. costatum
has a wide geographical distribution and presented in all seasons (Castillo
et al
. 1995). However, the species was not collected in this study.
More than four
Skeletonema
species (
S. dohrnii
,
S. marinoi
,
S. subsalsum
and
S. tropicum
) may occur in Korean coastal waters. The shape and size of the chains are useful taxonomic characters for species identification as described above. In LM, it is possible to use these characters to observe identification keys. Fine structures such as number and position of the processes (both rimo- and fultoportulae), and number of areolae between processes seen only by electron microscopy are important keys (Medlin
et al
. 1991). To overcome this identification confusion, many ultra-structural key characters can be estimated using SEM. For increase of species diversity, specimens are carefully observed. Furthermore, to estimate the increase of biodiversity, physiological factors such as life cycle based on laboratory, field, and molecular studies are needed.
Acknowledgements
This work was supported by a grant of Sangmyung University and The survey of Indigenous Biology of Korea from National Institute of Biological Resources (NIBR) of Korea.
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