INTRODUCTION
Diatoms are an important floral component in the ecosystems of tidal flats, often representing the most abundant algal group of phytobenthos (Allan and Castillo 2007). For many diatom species, tidal flats are very harsh environments to live in. Being exposed periodically to large environmental variability, tidal flats are occasionally characterized by high turbidity at high tide and strong physicochemical variations at low tide (e.g. temperature, light, and nutrients) (Admiraal 1984; Underwood and Kromkamp 1999). On the other hand, different kinds of substrate existing in this region like muds, sands, gravels, rocks, seaweeds, and other artificial surfaces such as nets and broken buoys provide tremendous habitats for benthic diatoms. Thus tidal flats are regarded as a peculiar ecosystem where diverse floral constituents can be displayed in association with diatom assemblages adapted successfully to this environments. In addition to these various kinds of substrates mentioned above, the nature of tidal flats readily accessible by walk enable scientists to sample and analyze this diverse diatom taxa without great difficulties.
In Europe several diatom-based indices have been proposed and proved effective for assessing the biological qualities of water systems (Whitton et al. 1991; Whitton and Rott 1996; Prygiel et al. 1999). While researches on freshwater diatoms or phytoplankton in the sea have been done extensively, benthic diatoms attached to sediments and substrates of tidal flats have received little attention. Until two decades ago the diatom taxa inhabiting deposits of tidal flats had been informed rarely and their ecology had not been well-known. During the last decade this conditions have improved slightly with efforts of several scientists, for instance, those studying benthic diatoms of coral reefs in the Western Indian Ocean where a rather rich diatom flora was discovered with many new species (Riaux-Gobin and Compère 2009; Lobban and Jordan 2010; Riaux-Gobin et al. 2010a, 2011; Riaux-Gobin and Al-Handal 2012), but it is still insufficient. This situation is not much different in Korea. It is known that all marine diatoms including phytoplankton occupy only 40% (ca. 800 species) of the 2,000 species reported already in Korea up to date (NIBR 2015). Current state on the number of reported marine benthic diatoms might be mainly due to lacking research. It would be able to be changed in the near future.
As the ultrastructure of cell wall is getting more and more important in identifying diatoms, it is very hard to classify pennate diatoms into species level using light microscopy because of the optical resolution limit of microscope objectives. Consequently electron microscopy becomes essential tool in diatom taxonomy now. As shown in the case of Cocconeiopsis wrightii and Simonsenia delognei which had been classified into different genera Cocconeis and Nitzschia, respectively (Park et al. 2014; Witkowski et al. 2015), these species have been changed to the new genera with the help of electron microscopy (Witkowski 2000).
The aim of this study is to find and display the morphology of unrecorded diatom species in Korea using light and scanning electron microscopy (SEM). Especially, the find structures of cell wall observed by SEM is stressed. We presented the following information on each species; nomenclature, basionym, synonym, references, distribution in Korea and morphological characteristics.
MATERIALS AND METHODS
Study area locate in the region of Korean coast of Yellow sea and Jeju Island (Table 1). Sampling was conducted at 28 sites from February to December 2016. Diatoms were collected from rocks, gravels, sands and muds of tidal flats, and seaweeds attached to the substrates and floating in the seawaters. After the samples were brought to the Lab, each cell in the sample was isolated and transferred to 24-well culture plate (Corning Cell Culture Vessels). The strains cultured successfully in the 24-well plate were maintained in 125 mL PC bottles. For the preparation of light micro- scopic observation (Zeiss Axio Imager), the cell organelles and organic matters were removed by the method of Hasle and Fryxell (1970). Several aliquots of the cleaned material were mounted in Pleurax and observed at ×400 to ×1,000 magnification. Some portion of the materials was used for SEM observation. Diatom cells were filtered on 3.0 μm PC membrane, dried in air, attached on the stub, coated with platinum and examined with SEM (Hitach S-4800). Identification of diatoms was mainly based on Witkowski et al. (2000) and specific references described in each species.
RESULTS AND DISCUSSION
Twenty one species of benthic diatoms were newly recorded in Korean tidal flat and were comprised of 1 class, 9 order, 12 family, 16 genus and 21 species. The species are classified into as follows based on the species database of AlgaeBase (Guiry and Guiry 2017). We provide SEM images in addition to light microscopic images, basionym, synonym, reference, species information and distribution in Korea.
Division Bacillariophyta
Class Bacillariophyta
Order Bacillariales
Family Bacillariaceae
Nitzschia amabilisH. Suzuki 2010
Nitzschia andicola Lange-Bertalot & Rumrich 2000
Nitzschia perindistincta Cholnoky 1960
Tryblionella gaoanaWitkowski & Chunlian Li 2016
Order Biddulphiales
Family Biddulphiaceae
Lampriscus orbiculatus (Shadbolt) Peragallo & Peragallo 1902
Order Cocconeidales
Family Achnanthidiaceae
Planothidium quarnerensis (Grunow) Witkowski et al. 2000
Family Cocconeidaceae
Anorthoneis vortexSterrenburg 1988
Cocconeis convexa M. H. Giffen 1967
Cocconeis krammeri Lange-Bertalot & Metzeltin 1996Fig. 1
Cocconeis mascarenicaRiaux-Gobin & Compère 2008
Cocconeiopsis breviata (Hustedt) Witkowski 1966
Order Fragilariales
Family Fragilariaceae
Koernerella recticostata (Körner) M. P. Ashworth et al. 2011
Order Mastogloiales
Family Mastogloiaceae
Craspedostauros britannicus E. J. Cox 1999
Order Naviculales
Family Berkeleyaceae
Climaconeis undulata (Meister) Lobban, Ashworth & Theriot 2010
Family Diploneidaceae
Diploneis vacilans (A. Schmidt) Cleve 1959
Family Naviculaceae
Haslea feriarum M. A. Tiffany & F. A. S. Sterren- burg 2015
Haslea nipkowii (Meister) M. Poulin & G. Massé 2004
Gyrosigma tessimum var. angustissima Simonsen 1959
Family Pleurosigmataceae
Donkinia carinata (Donkin) Ralfs 1861
Order Rhaphoneidales
Family Rhaphoneidaceae
Neodelphineis silenda (M. H. Hohn & J. Hellerman) N. Desianti & M. Potapova 2015
Order Catenulaceae
Family Catenulaceae
Amphora helenensisGiffen 1973
1.Amphora helenensisGiffen 1973 (Fig. 2A-L)
References: Witon and Witkowski 2006. p. 210, fig. 194; Witkowski et al. 2000. p. 139, pl. 163, figs. 31-33.
Specimen examined: slide PJG2016001 in Kunsan University, Gunsan.
Distribution in Korea: A. helenensis was observed fifteen times all year round in 2016. The regions observed were Dodu port in Jeju, fish farm in Shinan-Gun, Baramarae beach, Beopsan-ri, Hagampo, and Mallipo in Taean-Gun.
Morphological characteristics: Valves are elliptical, one side is slightly curved and the other rounded. The overall shape is crescent. 13-17 μm long, 3-3.5 μm broad. Transapical striae 20-21 in 10 μm.
2.Anorthoneis vortex Sterrenburg 1977 (Fig. 3A-G)
Reference: Sterrenburg 1988. p. 375, figs. 8-15.
Specimen examined: slide PJG2016002 in Kunsan University, Gunsan.
Distribution in Korea: A. vortex was observed ten times all year round in 2016. The regions observed were Baramarae beach, Beopsan-ri, Hagampo, and Mallipo in Taean-Gun.
Morphological characteristics: valves are heterovalvar and elliptical. Raphe is slightly away from the central area of the valve. 13-16 μm long, 9-10.5 μm broad. Transapical striae 13-15 in 10 μm.
3.Climaconeis undulata (Meister) Lobban, Ashworth and Theriot 2010 (Fig. 3H-Q)
Basionym: Gomphocaloneis undulata Meister 1932.
Synonym: Gomphocaloneis undulata Meister 1932; Navicula scopulorum var. triundulata Meister 1934; Navicula scopulorum var. triundulata Meister 1934.
References: Lobban et al. 2010. pp. 296-297, figs. 5-8, 19-25.
Specimen examined: slide PJG2016003 in Kunsan University, Gunsan.
Distribution in Korea: C. undulata was observed two times mainly in May and June 2016. The regions observed were Beopsan-ri in Taean-Gun, and Jeung island in Shinan-Gun.
Morphological characteristics: C. undulata is rugged from the end of the cell and the girdle view is rectangular. 150- 160 μm long, 12.5-14 μm broad. Transapical striae 15-17 in 10 μm.
4.Cocconeiopsis breviata (Hustedt) Witkowski 2000 (Fig. 3R-X)
Basionym: Navicula breviata Hustedt 1966.
Reference: Witkowski et al. 2000. p. 172, pl. 67, fig. 22.
Specimen examined: slide PJG2016004 in Kunsan University, Gunsan.
Distribution in Korea: C. breviata was observed five times in March, August and November 2016. The regions observed were Baramarae beach, Beopsan-ri, Hagampo, Mallipo, and Pado-ri, in Taean-Gun.
Morphological characteristics: Valves are biraphid and elliptical. 10-13 μm long, 6-8 μm broad. Transapical striae 14-17 in 10 μm.
5.Cocconeis convexa MH Giffen 1967 (Fig. 4A-D)
Reference: Giffen 1967. p. 183, figs. 14-17.
Specimen examined: slide PJG2016005 in Kunsan University, Gunsan.
Distribution in Korea: C. convexa was observed two times in June 2016. The regions observed were Chagui Island and Dodu port in Jeju.
Morphological characteristics: Valves are heterovalvar and elliptical. Slightly wavy stirae in rapheless valve (RLV). Poroids maintain steady intervals. In raphe valve (RV) raphe length short, straight. 18-21 μm long, 15-16 μm broad. Transapical striae in RLV are 38-42 in 10 μm, 26-28 in RV.
6.Cocconeis krammeri Lange-Bertalot H and D Metzeltin 1996 (Fig. 4E-H)
References: Lange-Bertalot and Metzeltin 1996. p. 390, pl. 125, 2 tables; Beltrones et al. 2017. p. 45, fig. 38.
Specimen examined: slide PJG2016006 in Kunsan University, Gunsan.
Distribution in Korea: C. krammeri was observed one time in December 2016. The regions observed was Pado-ri in Taean-Gun.
Morphological characteristics: Valves are elliptical. 28- 33 μm long, 17-22 μm broad. External RLV are convex, have four slightly curved sternum, or gradually tapering sternum. Internal valve arranged constant pore, which on external sternum location. Raphe of external RV are wavy. Transapical striae are 24-26 in 10 μm. The internal raphe is also same the external. The helictoglossa at the end of raphe is protruding. Transapical striae are 24-25 in 10 μm.
7.Cocconeis mascarenicaRiaux-Gobin & Compère 2008 (Fig. 4I-O)
Reference: Riaux-Gobin and Compère 2008. p. 141, figs. 33-40.
Specimen examined: slide PJG2016007 in Kunsan University, Gunsan.
Distribution in Korea: C. mascrenica was observed five times in March and June 2016. The regions observed were Baramarae beach, Beopsan-ri, Hagampo, and Mallipo in Taean-Gun.
Morphological characteristics: Valves are elliptical. 4-5.5 μm long, 2.2-3.4 μm broad. External RLV are convex, have four slightly curved sternum, or gradually tapering sternum.
8.Craspedostauros britannicus Cox EJ 1999 (Fig. 4P-W)
Reference: Cox 1999. p. 137, figs. 26, 27.
Specimen examined: slide PJG2016008 in Kunsan University, Gunsan.
Distribution in Korea: This species attached to small stone was observed one time in March 2016. The regions observed was Pado-ri in Taean-Gun.
Morphological characteristics: Linear to narrowly lanceolate valves, 38 μm long, 7 μm broad. Transapical striae are 26-28 in 10 μm. areolae are similar size, with four or more pores per cribrum.
9.Diploneis vacilans (AWF Schmidt) Cleve 1894 (Fig. 5A-H)
Basionym: Navicula vacillans Schmidt AWF 1875.
Synonym: Navicula vacillans Schmidt AWF 1875.
References: Cleve 1894. pp. 1-194, pl. 5; Witon and Witkowski 2006. p. 212, fig. 216.
Specimen examined: slide PJG2016009 in Kunsan University, Gunsan.
Distribution in Korea: D. vacilans attached to Gelidium sp. was observed one time in June 2016. The regions observed was Chagui Island in Jeju.
Morphological characteristics: Valves are elliptical to linear, become rounded toward apices. 30 μm long, 10-15 μm broad. Transapical striae are 15 in 10 μm.
10.Donkinia carinata (Donkin) Ralfs 1861 (Fig. 5I-P)
Basionym: Pleurosigma carinatum Donkin 1858.
Synonym: Pleurosigma carinatum Donkin 1858.
References: Pritchard 1861. pp. i-xii, 1-968, pl. 40; Hartley et al. 1996. pp. 1-601, pl. 290.
Specimen examined: slide PJG2016010 in Kunsan University, Gunsan.
Distribution in Korea: This species moves below sand grain and was observed one time in May 2016. The regions observed was Baeksajang beach in Taean-Gun.
Morphological characteristics: Valves are linear-lanceolate, 75 μm long, 9 μm broad. Transapical striae are 22-25 in 10 μm and pattern was diagonal line. Raphe was sigmoid.
11.Gyrosigma tenuissimum var. angustissima Simonsen 1959 (Fig. 6A-H)
Reference: Okamoto et al. 2003. pp. 498-505.
Specimen examined: slide PJG2016011 in Kunsan University, Gunsan.
Distribution in Korea: This species moves below sand grain and was observed one time in May 2016. The regions observed was Pado-ri in Taean-Gun.
Morphological characteristics: Valves of this species are sigmoid, 75-85 μm long, 6-7 μm broad. G. tenuissimum var. angustissima is similar to G. coelophilum. Two taxa differ in the density of the longitudinal striae. G. coelophilum has 44 striae in 10 μm. G. tenuissimum var. angustissima has 23-26.
12.Haslea feriarum Tiffany MA and Sterrenburg FAS 2015 (Fig. 6I-O)
Reference: Sterrenburg et al. 2015. p. 150, figs. 27-32.
Specimen examined: slide PJG2016012 in Kunsan University, Gunsan.
Distribution in Korea: This species attached to seaweed was observed one time in December 2016. The regions observed was Baeksajang beach in Taean-Gun.
Morphological characteristics: Valves are linea-lanceolate, asymmetric, pseudo-stauros in middle. Raphe located one-sided. 63.2-65.2 μm long, 8.3-8.6 μm broad. Striae are rectangle, transapical 23-24 in 10 μm.
13.Haslea nipkowii (Meister) Poulin M and G Massé 2004 (Fig. 7A-H)
Basionym: Gyrosigma nipkowii Meister 1932.
Synonym: Gyrosigma pallidum Riznyk 1973.
References: Poulin et al. 2004. pp. 181-196; Sterrenburg et al. 2015. p. 150, figs. 27-32.
Specimen examined: slide PJG2016013 in Kunsan University, Gunsan.
Distribution in Korea: This species attached to seaweed was observed two times in May and June 2016-2017. The regions observed were Beopsan-ri in Taean-Gun and fish farm in Shinan-Gun.
Morphological characteristics: Valves are sigmoid that linear to linear-lanceolate. Sigmoid raphe sternum, straight helictoglossa. 100-115 μm long, 13-16 μm broad. Striae are rectangle, transapical 23-24 in 10 μm, longitudinal 30- 35.
14.Koernerella recticostata (Körner) Ashworth MP, CS Lobban and EC Theriot 2011 (Fig. 7I-N)
Basionym: Asterionella notata var. recticostata H Körner 1971.
Synonym: Asterionella notata var. recticostata H Körner 1971.
Reference: Lobban et al. 2011. p. 184, figs. 21-25.
Specimen examined: slide PJG2016014 in Kunsan University, Gunsan.
Distribution in Korea: This species attached to Gelidium sp. was observed one time in June 2016. The regions observed was Chagui Island in Jeju.
Morphological characteristics: The valve outline is almost linear except for basal pore field, almost triangular. The basal pore field striae radiate like a fountain. Valve margin has spine that sequentially toward head pole from basal pore field. 74-76 μm long, 7-7.5 μm base broad and 2-2.2 μm head broad. Transapical striae is 28-30 in 10 μm.
15.Lampriscus orbiculatus (Shadbolt) Peragallo & Peragallo 1902 (Fig. 8A-F)
Basionym: Triceratium orbiculatum Shadbolt 1854.
Synonym: Triceratium orbiculatum Shadbolt 1854.
References: (Shadbolt) Peragallo and Peragallo 1902. pl. 106, figs. 2, 3; Rodriguez 2003. p. 213, fig. 3A-D; Nikolaev et al. 2001. pp. 1-119, pl. 39.
Specimen examined: slide PJG2016015 in Kunsan University, Gunsan.
Distribution in Korea: This species attached to Gelidium sp. was observed one time in June 2016. The regions observed was Chagui Island in Jeju.
Morphological characteristics: L. orbiculatus is connected to two or more cells. Girdle view rectangle, valves are circular. Valve face has four protrude ocellus. 70-75 μm long, 5 (short axis)-8 μm (long axis) broad in elliptical rim.
16.Neodelphineis silenda (Hohn MH and J Hellerman) N Desianti and M Potapova 2015 (Fig. 8G-M)
Basionym: Fragilaria silenda Hohn MH and J Hellerman 1966.
Synonym: Fragilaria silenda Hohn MH and J Hellerman 1966; Neodelphineis pelagica Takano 1982; Delphineis livingstonii AKSK Prasad 1986.
Reference: Desianti et al. 2015. p. 113, figs. 174-182.
Specimen examined: slide PJG2016016 in Kunsan University, Gunsan.
Distribution in Korea: This species moves below sand grain and was observed six times all year round in 2016. The regions observed were Baeksajang beach, Baramarae, and Hagampo in Taean-Gun.
Morphological characteristics: Valves are elliptical, and has thick and long sternum. 19-22 μm long, 10-14 μm broad, transapical striae 6-7 in 10 μm.
17.Nitzschia amabilisSuzuki H, T Nagumo and J Tanaka 2010 (Fig. 8N-U)
Synonym: Nitzschia laevis Hustedt 1923.
Reference: Suzuki et al. 2010.
Specimen examined: slide PJG2016017 in Kunsan University, Gunsan.
Distribution in Korea: This species attached to Gelidium sp. was observed eleven times all year round in 2016. The regions observed were Gimnyeong, Dodu port, Ojo-ri in Jeju, Baramarae beach, Hagampo, and Beopsan-ri in Taean- Gun.
Morphological characteristics: Valves are broadly elliptical, ending rounded. 8-14 μm long, 5-6 μm broad, transapical striae 32-38 in 10 μm, fibulae 10-14. N. amabilis is similar with N. laevis that 12-25 μm long, transapical striae 22-24, fibulae 10-14 in 10 μm.
18.Nitzschia andicola Lange-Bertalot & Rumrich 2000 (Fig. 9A-H)
References: Lange-Bertalot H. Annotated diatom micrographs. Vol. 9, p. 193, taf. 158, figs. 3-9, taf. 159, figs. 1-6.
Specimen examined: slide PJG2016018 in Kunsan University, Gunsan.
Distribution in Korea: This species was observed one time in tidal flat in June 2016. The regions observed was Ojo-ri in Jeju.
Morphological characteristics: Girdle view are sigmoid. Valves are linear, rapidly thin toward rim like needle form, has central nodule. 108-120 μm long, 4-6 μm broad, transapical striae 13-19, fibulae 7-10 in 10 μm.
19.Nitzschia perindistincta Cholnoky 1960 (Fig. 10A-H)
Synonym: Nitzschia fontifuga Cholnoky 1962; Nitzschia fundi Cholnoky 1960; Nitzschia zululandica Cholnoky 1968.
Reference: Witkowski et al. 2000. p. 399, pl. 208, figs. 12- 16.
Specimen examined: slide PJG2016019 in Kunsan University, Gunsan.
Distribution in Korea: This species was observed two times on stone surface in May and December 2016. The regions observed was Pado-ri in Taean-Gun.
Morphological characteristics: Valves are long elliptical, 11-12 μm long, 3-4 μm broad, transapical striae 58-60 in 10 μm, fibulae 17-18. Densely spaced striae are differentiated from other species.
20.Planothidium quarnerensis (Grunow) Witkowski A, H Lange-Bertalot and D Metzelin 2000 (Fig. 10I-O)
Basionym: Rhaphoneis quarnerensis Grunow 1862.
Synonym: Cocconeis quarnerensis (Grunow) AWF Schmidt 1874; Cocconeis clavigera O’Meara 1867; Cocconeis lanceolate Heiden 1900; Heteroneis allmanniana E. Joergensen 1895; Navicula ovulum A. Schmidt 1874.
Reference: Witkowski et al. 2000. p. 123, pl. 55, figs. 2-7. Specimen examined: slide PJG2016020 in Kunsan University, Gunsan.
Distribution in Korea: This species moves below sand grain and was observed five times in March, June and August 2016. The regions observed were Baramarae beach, Beopsan-ri, Hagampo, and Mallipo in Taean-Gun.
Morphological characteristics: Planothidium are monoraphid diatom. Valves are elliptical and has broadly lanceolate sternum. 22-24 μm long, 12-18 μm broad. Transapical striae 8-9 in 10 μm, Radial striae.
21.Tryblionella gaoanaWitkowski A and C Li 2016 (Fig. 10P-T)
Reference: Witkowski et al. 2016. p. 190, fig. 15a-d.
Specimen examined: slide PJG2016022 in Kunsan Univer- sity, Gunsan.
Distribution in Korea: This species was observed one time on small stone surface in January 2017. The regions observed was Ojo-ri in Jeju.
Morphological characteristics: Valves are nitzschioid, narrow central and ending rounded. Raphe exist one-sided. 19-21 μm long, 6.6-6.9 μm broad. Transapical striae 27- 28 in 10 μm, fibulae 12-13. Girdle view has several open bands.
CONCLUSION
A total of 21 diatom species new to Korea were discovered from Korean coast of Yellow Sea and Jeju Island. Ten species, Anorthoneis vortex, Cocconeis krammeri, C. mascarenica, Craspedostauros britannicus, Donkinia carinata, Gyrosigma tenuissmum var. angustissima, Haslea feriarum, Neodelphineis silenda, Nitzschia perindistincta, Planothidium quarnerensis were observed only in Taean area located in the upper eastern coast of Yellow Sea. Six species, Cocconeis convexa, Diploneis vacilans, Koernerella recticostata, Lampriscus orbiculatus, Nitzschia lorenziana, Tryblionella gaoana were discovered only in Jeju Island. Three species, Amphora helenensis, Cocconeiopsis breviata, Nitzschia amabilis occurred in both region, and two species, Climaconeis undulate, Haslea nipkowii from both upper and lower eastern coast of Yellow Sea.
Craspedostauros and Koernerella are newly introduced genera in Korea. Craspedostauros comprise eight species at present, of which seven have been accepted taxonomically. C. britannicus is known to be distributed mostly in Europe including UK, Mediterranean and Baltic Sea. Koernerella consists of two species taxonomically accepted, of which K. recticostata was detected only in Pacific island, Guam. The occurrence of the species around Jeju Island with another tropical species, L. orbiculatus, which usually appears in tropical sea areas, might show the evidence of global warming in sea water temperature. Thus these species needs to be observed carefully and monitored continuously.
In spite of many benthic diatoms detected, still plenty of species from tidal flats of Korea are undiscovered and waiting for being discovered. This is mainly attributable to scarce studies on diatom species of tidal flats. In addition, pennate diatoms mostly occurring in tidal flats are difficult to be distinguished under light microscope. The chloroplast of living diatoms can be used to identify some genus, but not species. Recently, electron microscopy or molecular biology enhances the capability of classifying pennate diatoms. Therefore, through researches undertaken actively on unrecorded species, and continuing to expand monitoring and research regions, the number of the species newly recorded would be able to be increased.