We described for the first time the freshwater dinoflagellate

Unruhdinium penardii var. robustum, collected from Paldang

Reservoir, Korea, on October 21, 2019. The overall shape was

pentagonal to rhombic. The cell size was 31 μm in length

(23~39 μm), and 29 μm in width (23~38 μm). Their plate

formula was determined to 4', 0a, 6'', 5''', 0p, and 2''''; and the

apical plate was symmetric. Two apical spines were prominent,

and approximately 6 robust hypothecal spines were observed.

Molecular comparisons of the 18S rDNA sequences showed

that our species was well-matched with the already-known U.

penardii (99.9% similarity); because U. penardii var. robustum

18S rDNA sequence was not available in the public database.

Additional 28S rDNA sequences had high similarity with

already-known U. penardii var. robustum (99.8% similarity).

These morphological and molecular results confirmed that our

dinoflagellate was clearly U. penardii var. robustum, and it was

recorded for the first time in Korean freshwaters.

Keywords: Unruhdinium penardii, 18S rDNA, freshwater dino-

flagellate, morphology, Paldang Reservoir

The dinoflagellates are a large group of primary producers in

aquatic environments. Most are marine plankton, but they are

also present in freshwater habitats. Morphologically, they are

divided into two forms (i.g., thecate and athecate); among the

thecate, plate formula is an important key character to identify

species morphologically. Until now, at least 3,561 species have

been reported in the dinoflagellates, and about 452 species are

described in freshwaters (Guiry and Guiry, 2020). The genus

Peridiniopsis Lemmermann are one of the major freshwater

dinoflagellates, and they are divided in nine groups by

morphological characteristics like plate formula (Popovsky

and Pfiester, 1990). Recently, the group Penardii was separated

to be formed a new genus, Unruhdinium Gottschling, since

they have distinct plate formula and diatom endosymbiont

unlike other Peridiniopsis (Gottschling et al., 2017).

In Korea, the dinoflagellates have been studied for long

time, because they are responsible for harmful algal blooms

(Lee, 1999). Most studies have been carried out targeting

marine toxic dinoflagellates, and extremely limited studies

have been conducted in freshwaters. Upon searching the latest

national database (National species list of Korea, 2019), we

identified about 24 freshwater dinoflagellates, including the

genus Ceratium Schrank, Peridiniosis Lemm, Parvodinium

Carty, Peridinium Ehrenberg. It was just 5% when compared to

the world records (Guiry and Guiry, 2020). Of them, Ceratium

Korean Journal of Microbiology (2020) Vol. 56, No. 2, pp. 146-151 pISSN 0440-2413DOI https://doi.org/10.7845/kjm.2020.0034 eISSN 2383-9902Copyright ⓒ 2020, The Microbiological Society of Korea

New record of the dinoflagellate Unruhdinium penardii var. robustum

(Dinophyceae) from Paldang Reservoir, Korea

Taehee Kim, Yeon-Su Lee, and Jang-Seu Ki*

Department of Life Science, Sangmyung University, Seoul 03016, Republic of Korea

팔당댐의 담수 와편모조류 Unruhdinium penardii var. robustum

(Dinophyceae)에 관한 미기록종 연구

김태희 ･ 이연수 ･ 기장서*

상명대학교 생명과학과

(Received April 10, 2020; Revised May 20, 2020; Accepted May 20, 2020)

*For correspondence. E-mail: kijs@smu.ac.kr;

Tel.: +82-2-2287-5449; Fax: +82-2-2287-0070

First record of U. penardii var. robustum from Korea ∙ 147

Korean Journal of Microbiology, Vol. 56, No. 2

hirundinella was firstly described from Han River (Chung et

al., 1968). Since then, several freshwater dinoflagellates have

been recorded via ecological surveys (Han et al., 1995; Kim,

1998; Kim et al., 2009), but their taxonomic identities have not

been examined morphologically. To our knowledge, only three

freshwater dinoflagellates had a morphologically accurate

identification, which are Peridinium bipes f. occultatum,

Peridinium aciculiferum, and Peridinium umbonatum (Ki and

Han, 2005, 2008; Ki et al., 2005).

The Han River is an important water resource in Seoul and

the metropolitan area, and thus many studies have been

conducted on water quality managements (Yoo and Lim, 1990;

Kim et al., 2009). The Paldang Reservoir is where the three

tributaries (the North Han river, the South Han River and

Kyungan Stream) of the Han River join. In a recent study, we

found blooms of a freshwater dinoflagellate in the Paldang

Reservoir, and it was suspected to be Peridiniopsis sp. by 18S

metagenomics (Boopathi and Ki, 2016); however, their

taxonomic identities are still unknown to date.

In the present study, we examined morphological characters

of the bloom-forming dinoflagellate collected from Paldang

Reservoir, and determined their 18S and 28S rDNA sequences

using molecular cloning. We compared their morphological

and molecular key characters with available freshwater dino-

flagellates for their accurate taxonomy.

Materials and Methods

Water sampling and preservation

Water samples were collected on October 21 2019, at

Paldang Reservoir (GPS code: 37°39'15.63''N, 127°17'15.89''E;

Fig. 1) when a freshwater dinoflagellate bloomed. Water tem-

perature, pH and conductivity were measured from the

monitoring site in the Paldang Reservoir using the YSI 566

Multi Probe System (YSI). In addition, dissolved oxygen

(DO), total nitrogen (TN), and total phosphate (TP) data were

brought from the public database of the Water Environment

Information System (http://water.nier.go.kr).

For morphological observations, water samples were fixed

with Lugol’s solution at 1% final concentration. In addition, a

total of 300 ml water sample was filtered with 10 µm-pore sized

membrane filter (Cat. No. TCTP04700, 47 mm diameter,

Millipore), and this filter was stored at -20°C, until DNA

extraction with cetyltrimethylammonium bromide (CATB)

buffer.

Morphological observations

Morphological features of dinoflagellate cells were observed

with a light and epifluorescence microscope (Carl Zeiss Axioskop)

equipped with a ProgRes® CF scan CCD camera (Jenoptik).

Lugol’s solution fixed dinoflagellate cells were prepared by

calcofluor white staining method (Fritz and Triemer, 1985) and

the plate formula was determined using epifluorescence

microscope. Average body length, width and hypotheca spine

length were calculated by measuring 30 cells. Digital images

were taken with a ProgRes® CF scan CCD camera, and

analyzed with ProgRes CapturePro 2.10.0.1 software (Jenoptik).

DNA extraction and PCR

Genomic DNA of Unruhdinium was extracted from filtered

water samples, when the cells bloomed at Paldang Reservoir.

Total DNA on filtered membranes was extracted by the CTAB

method described by Richards et al. (1994). Nuclear 18S and

28S rDNA sequences were separately amplified by polymerase

Fig. 1. A map of the Paldang Reservoir, Korea. The black circle represents

the sampling site.

148 ∙ Kim et al.

미생물학회지 제56권 제2호

chain reaction (PCR) with newly designed dinoflagellate-

specific 18S primers (forward: Peri-18F340, 5'-GAC GGG

TAA CGG AGA ATT AG-3'; reverse: Peri-18R1690, 5'-ATT

CAC CGG ATC ACT CAA TC-3') and 28S primers (forward:

Fresh-dinos-28F80, 5'-AGT AAT GGC GAA TGA ACA

GG-3'; reverse: Fresh-28R910, 5'-CCT ATA CCC ATG TAT

GAC GAA C-3'). PCR was carried out in 20 μl reaction mixture

containing 11.8 μl of sterile distilled water, 2 μl of 10× Ex PCR

buffer (TaKaRa), 2 μl of a dNTP mix (4 mM each), 1 μl of each

primer (10 pmoles), 0.2 μl Ex Taq polymerase (2.5 U), and 2 μl

of template. PCR was performed on a Bio-Rad iCycler with the

following conditions: pre-denaturation at 94°C for 3 min;

followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and

72°C for 75 sec; with a final extension at 72°C for 10 min. The

resulting PCR products were subjected to electrophoresis in

1.0% agarose gel (Promega), stained with Midori Green, and

visualized under ultraviolet light on a transilluminator.

Cloning and sequencing

PCR products were purified with the QIAquick PCR

Purification Kit (Qiagen GmbH), ligated into pTOP TA V2

vector (TOPclonerTM TA Kit, Enzynomics), and transformed

into competent cells. DNA sequencing reactions of the clones

were run with the ABI PRISM® BigDye™ Terminator Cycle

Sequencing Ready Reaction Kit (PE Biosystems) using the

PCR products and PCR primers. After that, the remaining DNA

sequences were determined by primer walking. Labeled DNA

fragments were analyzed on an automated DNA sequencer

(Model 3700, Applied Biosystems).

Phylogenetic and sequence similarity analyses

For phylogenetic analysis, we constructed a data set of 18S

rDNA sequences, including sequences of our species and those

of other relatives obtained from the National Center for

Biotechnology Information (NCBI) database. These sequences

were aligned with MAFFT (Kuraku et al., 2013). Ambiguous

regions were removed using Gblocks server with the least

stringent settings (http://molevol.cmima.csic.es/castresana/G

blocks_server.html). Maximum-Likelihood (ML) analysis was

conducted with the same data matrix using the Tamura-Nei

model in MEGA X (Kumar et al., 2018).

Additional 28S rDNA sequence of Korean Unruhdinium

was analyzed by BLAST searches in the National Center for

Biotechnology Information (NCBI) and sequence similarity.

Results and Discussion

Environmental factors and biological data

In our field sampling, water temperature was recorded at

21.6°C, showing the fall characteristics of the temperate zone.

Additional environmental factors, such as pH, conductivity,

DO, TN, and TP were presented in Table 1. As for phyto-

plankton taxa, we identified 13 genera and 21 species, including

a dinoflagellate, 5 diatoms, 7 Chlorophyta, 5 Zygnematophyceae,

and 3 Euglenophyceae. The dinoflagellate Unruhdinium was

composed of about 35% of the total cells. Until now, there was

a little data on the appearance of the specific dinoflagellates in

the Paldang Reservoir; however, previous studies had reported

that certain dinoflagellates appeared mainly in the fall (Han et

al., 1995; Kim et al., 2009).

Color, cell size, and shape

Cell shape of the Korean Unruhdinium sp. was pentagonal to

rhombic (Fig. 2A), contrary to the oval shape of U. minimum

and U. jiulongense (Zhang et al., 2014; You et al., 2015). The

species presented a yellow-brownish color.

The cell was 31 μm in length (23~39 μm), and 29 μm in

width (23~38 μm) (Fig. 3). Therefore, the ratio of length and

width was approximately 0.94. Overall, these morphological

characters were well matched with those described in

Unruhdinium penardii var. robustum (Zhang et al., 2011).

However, the cell size of the Korean species was slightly

smaller than the original description from Chinese freshwaters

species.

Table 1. Environmental factors recorded Paldang Reservoir in October 21,

2019

Environmental factor Value

Water temperature (°C) 21.6

pH 8.1

Elecrical conductivity (μs/cm) 208

DO (mg/L) 8.6

TN (mg/L) 2.28

TP (mg/L) 0.03

First record of U. penardii var. robustum from Korea ∙ 149

Korean Journal of Microbiology, Vol. 56, No. 2

General description and thecal plate formula

The epitheca of the Korean Unruhdinium sp. had prominent

two apical spines and the plate pattern was symmetric (Fig. 2).

The plate formula of the epitheca was composed of 4 apicals, 0

intercalary, and 6 presingular plates (4', 0a, 6'') (Fig. 2B and C).

The plate 1' had a rhombic shape, and plates 2' and 4' were

pentagonal. However, plate 3' was hexagonal, since this plate

connected the plates 2', 4', 2'', 3'', 4'', and 5'' (Fig. 2H). In a view

of the presingular plate’s shape, plate 1'', 3'', 4'', and 6'' were

trapezoid, but plate 2'' and 5'' were trapezoid with 5 sides (Fig.

2H). The cingulum was composed of 5 plates (c1~c5), and c1

was very short compared to the others (Fig. 2B and H). In

addition, upper and lower cingulum had some tooth shaped

extension (Fig. 2H).

In addition, hypotheca plates of the Korean Unruhdinium sp.

comprised 5 postcingular and 2 antapical plates (5''', 0p, 2'''') as

shown in the antapical view (Fig. 2D and E). Plates 1''', 2''', 4''',

and 5''' were quadrangular, but plate 3''' was pentagonal

because it connected the plates 2''', 4''', 1'''', 2'''' and the

cingulum (Fig. 2E). Plate 1'''' and 2'''' were pentagonal and

connected with 1''' and 5''', respectively. Approximately 6

robust hypothecal spines were observed around the plates 1''''

and 2'''' (Fig. 2A and D). The length of the hypothecal spines

was 2 to 4.6 μm. All these plate formulas completely matched

with those of Unruhdinium members, particularly U. penardii

(Lemmermann, 1910).

Moreover, in an earlier study, Zhang et al. (2011) reported a

new Peridiniopsis species (= Unruhdinium penardii var.

robustum) in a comparison with U. penardii. The plate formula

of both species is the same; however, U. penardii var. robustum

has two prominent apical spines and numerous robust

hypothecal spines (Zhang et al., 2011). In addition, upon

comparisons of Unruhdinium species spines (e.g., U. kevei, U.

niei, and U. penardii), we found that U. penardii var. robustum

has the largest number of spines, and other 3 species have only

0 to 4 spines (Grigorszky et al., 2001; Liu et al., 2008; Zhang et

al., 2011). Although the cell size of Korean Unruhdinium sp.

was a little small, the plate formula, the presence of epical

spines and the large number hypothecal spines support that our

Korean Unruhdinium was morphologically attributed to U.

penardii var. robustum.

(A) (B)

(C)

(D)

(E)

(H)

Fig. 2. Light, Epifluorescence micrographs and drawing of the Korean

Unruhdinium penardii var. robustum collected from Paldang Reservoir.

(A) Entire cell shape. (B) epitheca and cingulum in ventral view. (C)

epitheca in dorsal views. (D) hypotheca in ventral view. (E) hypothecain

dorsal view. (H) The line drawing was based on the epifluorescence

micrographs of (B) and (C). Light microscope; (A). Epifluorescence

microscope; (B), (C), (D), (E). Blue and red arrows represent apical and

hypothecal spines, respectively. Ps=posterior sulcal. Scale bar = 10 µm.

Fig. 3. Body length and width of Korean Unruhdinium penardii var.

robustum. Each was determined by calculating the average measurements

of 30 cells.

150 ∙ Kim et al.

미생물학회지 제56권 제2호

Molecular affiliation of Korean Unruhdinium by 18S

and 28S rDNA

With molecular cloning, we determined 1,286 bp of 18S

rDNA sequences from 5 clones (GenBank accession nos.

MT273080 - MT273084). Upon comparisons, we found that

all the 18S rDNA sequences were nearly identical (more than

99.9% similarity among our sequences). In addition, BLAST

searches showed that our Korean Unruhdinium 18S rDNA

sequences had top-hits with species from the same genus:

Unruhdinium penardii (AB353771 and HM596543), followed

by U. minimum (JQ639767) and U. niei (HM596542) with high

similarities (> 98.9%). Additional phylogenetic analysis of our

18S rDNA sequences and other freshwater dinoflagellates

showed that the Korean Unruhdinium clustered with other

Unruhdinium species and formed a sisterhood clade with two

different sequences of U. penardii available in the public

database (AB353771, HM596543).

In addition to this, we determined 823 bp of 28S rDNA from

Unruhdinium penardii clones (GenBank no. MT453902). A

BLAST search showed that our sequence had top-hit with

Unruhdinium penardii var. robustum (HM596558) with

99.8% DNA similarity. This clearly supported that the Korean

Unruhdinium belonged to U. penardii var. robustum genetically.

In conclusion, we examined morphological and molecular

traits of Korean U. penardii var. robustum. Based on cell shape,

plate formula, spines and sequence similarity, we report the

first record of U. penardii var. robustum, in Korean freshwaters.

적 요

본 연구는 2019년 10월 21일 한국 팔당댐으로부터 채집한

담수 와편모조류인 Unruhdinium penardii var. robustum을 최

초로 기술하였다. 전체적인 모양은 마름모꼴의 오각형이다.

세포크기는 31 μm (23~39 μm)의 길이와 29 μm (23~38 μm)의

너비이다. 각판 배열은 4', 0a, 6'', 5''', 0p, 2''''로 관찰되었다. 상

각 각판은 대칭이다. 두개의 정단 가시(apical spines)는 뚜렷

하게 관찰되며, 하각 가시(hypothecal spines)는 약 6개가 관

찰되었다. U. penardii var. robustum은 기존에 보고된 U.

penardii의 18S rDNA 염기서열과 높은 유사도(99.9%)를 보

였고, 분자계통학적으로 근연관계를 형성하였다. 추가적인

28S rDNA 염기서열 분석에서 기존에 보고된 U. penardii var.

Fig. 4. A phylogenic tree of Unruhdinium penardii var. robustum 18S rDNA with other freshwater dinoflagellates. Highlighted node represents high

similarity with our species (MT273080) in bold and the genus Unruhdinium.

First record of U. penardii var. robustum from Korea ∙ 151

Korean Journal of Microbiology, Vol. 56, No. 2

robustum과 높은 유사도(99.8%)를 보였다. 이러한 형태적, 분

자적 결과는 팔당댐의 와편모조류가 U. penardii var. robustum

이라는 것을 명확하게 제시하며, 해당 종은 국내 담수에서의

최초 기록이다.

Acknowledgments

We thank to Dr. S Abassi for English correction and proof

reading of the final draft. This work was supported by the

National Research Foundation of Korea (NRF) grant funded by

the Korea government (MSIT) (No. 2020R1A2C2013373).

References

Boopathi T and Ki JS. 2016. Unresolved diversity and monthly

dynamics of eukaryotic phytoplankton in a temperate freshwater

reservoir explored by pyrosequencing. Mar. Freshwater Res.

67, 1680–1691.

Chung YH, Kay ES, and Park DW. 1968. A study on the microflora of

the Han River (II). Korean J. Bot. 11, 1–70.

Fritz L and Triemer RE. 1985. A rapid simple technique utilizing

calcofluor white M2R for the visualization of dinoflagellate

thecal plates 1. J. Phycol. 21, 662–664.

Gottschling M, Čalasan AŽ, Kretschmann J, and Gu H. 2017. Two new

generic names for dinophytes harbouring a diatom as an

endosymbiont, Blixaea and Unruhdinium (Kryptoperidiniaceae,

Peridiniales). Phytotaxa 306, 296–300.

Grigorszky I, Vasas F, and Borics G. 2001. Preridiniopsis Kevei SP.

nov., A new freshwater dinoflagellate species (peridiniceae,

dinophyta) from Hungary. Acta Bot. Hung. 43, 163–174.

Guiry MD and Guiry GM. 2020. AlgaeBase. World-wide electronic

publication, National University of Ireland, Galway. https://

www.algaebase.org; searched on 16 March 2020.

Han MS, Auh YY, Ryu JK, Yoo KI, and Choi YK. 1995. Ecological

studies on Pal'tang river - reservoir system in Korea 2. Changes

in phytoplankton community structure. Korean J. Limnol. 28,

335–344.

Ki JS, Cho SY, and Han MS. 2005. Morphological characteristics of

Peridinium bipes f. occultatum (Dinophyceae) isolated from

three geographically segregated aquatic systems of Korea.

Korean J. Limnol. 38, 1–7.

Ki JS and Han MS. 2005. First record and morphological features of

the dinoflagellate Peridinium aciculiferum Lemm. (Dinophyceae)

in Korean freshwater. Korean J. Environ. Biol. 23, 323–327.

Ki JS and Han MS. 2008. New record of the freshwater dinoflagellate

Peridinium umbonatum Stein (Dinophyceae) from Togyo

Reservoir, Korea. Algae 23, 115–118.

Kim YJ. 1998. Ecological characteristics of phytoplankton community

in Lake Paltang Dam. Korean J. Limnol. 31, 225–234.

Kim JK, Lee SH, Bang HH, and Hwang SO. 2009. Characteristics of

algae occurrence in Lake Paldang. J. KSEE 31, 325–331.

Kumar S, Stecher G, Li M, Knyaz C, and Tamura K. 2018. MEGA X:

molecular evolutionary genetics analysis across computing

platforms. Mol. Biol. Evol. 35, 1547–1549.

Kuraku S, Zmasek CM, Nishimura O, and Katoh K. 2013. aLeaves

facilitates on-demand exploration of metazoan gene family trees

on MAFFT sequence alignment server with enhanced interactivity.

Nucleic Acids Res. 41, 22–28.

Lee JH. 1999. A review on red - tides and phytoplankton toxins in the

coastal waters of Korea. Korean J. Environ. Biol. 17, 217–232.

Lemmermann, E. 1910. Kryptogamenflora der Mark Brandenburg:

Algen I (Schizophyceen, Flagellaten, Peridineen): Algen I

(Schizophyceen, Flagellaten, Peridineen). pp. 497–712. Verlag

von Gebrüder Borntraeger.

Liu GX, Pei GF, and Hu ZY. 2008. Peridiniopsis niei sp. nov.

(Dinophyceae), a new species of freshwater red tide dinoflagellates

from China. Nova Hedwigia 87, 487–499.

National species list of Korea. 2019. National Institute of Biological

Resources, online at http://kbr.go.kr, accessed on (data of access)

Popovsky J and Pfiester LA. 1990. Süßwasserflora von Mitteleuropa.

Vol. 6. Dinophyceae (Dinoflagellida). pp. 1–272. Jena & Stuttgart,

Gustav Fischer.

Richards E, Reichardt M, and Rogers S. 1994. Preparation of genomic

DNA from plant tissue. Curr. Protoc. Mol. Biol. 27, 2–3.

Yoo KI and Lim BJ. 1990. On the phytoplankton community and water

pollution indication in the Lower Han River System. Korean J.

Limnol. 23, 267–277.

You X, Luo Z, Su Y, Gu L, and Gu H. 2015. Peridiniopsis jiulongensis,

a new freshwater dinoflagellate with a diatom endosymbiont

from China. Nova Hedwigia 101, 313–326.

Zhang Q, Liu G, and Hu Z. 2011. Morphological differences and

molecular phylogeny of freshwater blooming species, Peridiniopsis

spp.(Dinophyceae) from China. European J. Protistol. 47, 149–

160.

Zhang Q, Liu G, and Hu Z. 2014. Description of a new freshwater

bloom-forming dinoflagellate with a diatom endosymbiont,

Peridiniopsis minima sp. nov. (Peridiniales, Dinophyceae) from

China. Algol. Stud. 145–146, 119–133.