Research Article

Algae 2013, 28(4): 307-330

http://dx.doi.org/10.4490/algae.2013.28.4.307
Open Access

Taxonomy and phylogeny of the genus Cryptomonas (Cryptophyceae,

Cryptophyta) from Korea
Bomi Choi1, Misun Son2, Jong Im Kim1 and Woongghi Shin1,*
Department of Biology, Chungnam National University, Daejeon 305-764, Korea
1

Yeongsan River Environment Research Center, Gwangju 500-480, Korea

2

The genus Cryptomonas is easily recognized by having two flagella, green brownish color, and a swaying behavior.
They have relatively simple morphology, and limited diagnostic characters, which present a major difficulty in differen-
tiating between species of the genus. To understand species delineation and phylogenetic relationships among Crypto-
monas species, the nuclear-encoded internal transcribed spacer 2 (ITS2), partial large subunit (LSU) and small subunit
ribosomal DNA (rDNA), and chloroplast-encoded psbA and LSU rDNA sequences were determined and used for phylo-
genetic analyses, using Bayesian and maximum likelihood methods. In addition, nuclear-encoded ITS2 sequences were
predicted to secondary structures, and were used to determine nine species and four unidentified species from 47 strains.
Sequences of helix І, ІІ, and ІІІb in ITS2 secondary structure were very useful for the identification of Cryptomonas spe-
cies. However, the helix ІV was the most variable region across species in alignment. The phylogenetic tree showed that
fourteen species were monophyletic. However, some strains of C. obovata had chloroplasts with pyrenoid while others
were without pyrenoid, which used as a key character in few species. Therefore, classification systems depending solely
on morphological characters are inadequate, and require the use of molecular data.

Key Words: Cryptomonas; Cryptophyta; morphology; phylogeny; taxonomy

INTRODUCTION

The genus Cryptomonas, which belongs to the class
Cryptophyceae, was established by Ehrenberg (1831). It is
distributed in freshwater habitats worldwide. Cells can be
easily recognized by two unequal biflagella, olive-brown-
ish to olive-greenish in color, large ejectisomes lined in
the furrow-gullet system, and a peculiar swaying swim-
ming behavior due to the asymmetric shape, dorsally flat-
tened and ventrally concave in lateral view. Cells have two
chloroplasts originated from red algae, which contain the
accessory pigment phycoerythrin 566 of phycobiliprotein
(Hill and Rowan 1989, Clay et al. 1999, Deane et al. 2002,
Hoef-Emden and Melkonian 2003). Cryptomonas species
display one or two morphotypes within a clonal culture.
In the cryptomorph, the inner periplast component (IPC)
is made of hexagonal to polygonal plates, whereas in the
camphylomorph the IPC is a sheet-like layer (Faust 1974,
Brett and Wetherbee 1986, Hill 1991, Hoef-Emden and
Melkonian 2003, Hoef-Emden 2007).
Since Ehrenberg (1831, 1832) described six Crypto-
monas species, many additional species were added
(Pascher 1913, Schiller 1925, 1929, 1957, Skuja 1939, 1948,
1956, Huber-Pestalozzi 1950, Starmach 1974). Tradition-
ally, Cryptomonas species have been characterized by
mainly morphological characters, such as cell size, cell

This is an Open Access article distributed under the terms of the Received August 20, 2013, Accepted December 5, 2013
Creative Commons Attribution Non-Commercial License (http://cre-
ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted
*Corresponding Author
non-commercial use, distribution, and reproduction in any medium, E-mail: shinw@cnu.ac.kr
provided the original work is properly cited. Tel: +82-42-821-6409, Fax: +82-42-822-9690

Copyright © The Korean Society of Phycology 307 http://e-algae.kr pISSN: 1226-2617 eISSN: 2093-0860
Algae 2013, 28(4): 307-330
Fig. 1. Distributions of the genus Cryptomonas species from Korea.
shape, and internal organization (Bourelly 1970). Howev-
er, it is difficult to delimit Cryptomonas species due to the
paucity of morphological characters and the less-than-
adequate visibility of living cells using light microscopy
(Pringsheim 1968). For example, an early attempt to orga-
nize photosynthetic cryptomonad taxonomy was carried
out by Ehrenberg (1838), who included several unrelated
genera in the family Cryptomonadina with original short
diagnosis. Later, Dujardin (1841) described two families
(Xanthodiscaceae and Chilomonaceae) within the or-
der Cryptomonadineae. In his classification system, the
family Chilomonadaceae consisted of four genera (Chi-
lomonas, Rhodomonas, Cryptomonas, and Cyanomonas).
These genera were characterized by nutritional mode,
number and color of chloroplast. Butcher (1967) orga-
nized main photosynthetic genera of the Cryptophyceae
into three families (Hilleaceae, Hemiselmidaceae, and
Cryptomonadaceae) based on complexity of furrow-
gullet system with or without ejectisome. He also used
number of ejectisome rows in the furrow-gullet system to
discriminate between each genus, and thus, described 12
new Cryptomonas species from salt water.
Hill (1991) revised the broad generic definition of the
genus Cryptomonas recognized by Butcher (1967) and
erected four new genera based on their furrow-gullet
system, periplast structure, plastidial complex and rhi-
zostyle; Campylomonas, Geminigera, Storeatula, and
Teleaulax. Recently, molecular work (Marin et al. 1998,
Deane et al. 2002) has shown that Cryptomonas species
grouped together with species of Campylomonas and
Chilomonas. Hoef-Emden and Melkonian (2003) syn-
http://dx.doi.org/10.4490/algae.2013.28.4.307
onymized both genera Campylomonas and Chilomonas
to the genus Cryptomonas. More recently, Hoef-Emden
(2007) revised the genus Cryptomonas again, and pro-
vided a secondary structure of the nuclear internal tran-
scribed spacer 2 (ITS2) as a good marker to identify Cryp-
tomonas species. In addition, she emended five species
based on molecular signatures as diagnostic characters.
In this study, we report unrecorded Korean Cryptomo-
nas species isolated from freshwaters. We infer phylo-
genetic relationships among species using a combined
nuclear-encoded ITS2, partial large subunit ribosomal
DNA (LSU rDNA), and small subunit ribosomal DNA
(SSU rDNA), and chloroplast-encoded psbA (photosys-
tem II protein D1) and LSU rDNA sequence data. We also
predicted the ITS2 secondary structure of Cryptomonas
species.
MATERIALS AND METHODS
Algal cultures and microscopy
Specimens were collected from freshwater habitats in
Korea (Fig. 1). Live cells were isolated by Pasteur capillary
pipette and were brought into a unialgal culture. The cells
were cultivated in f/2 medium (Guillard and Ryther 1962,
Guillard 1975) with soil extract. The clonal cultures were
maintained under a light : dark regime of 14 : 10 at 20-
22°C using cool-white fluorescence lamps with illumina-
tions of 30 µmol protons m-2 s-1. The morphology was ex-
amined by differential interference contrast with a 100X
oil immersion lens (Carl Zeiss Co., Göttingen, Germany).
Calibration of magnification was done with grated mi-
crometer. The shape and length of cells, length of the
fullow-gullet system, color and number of chloroplasts,
presence / absence and number of pyrenoids were exam-
ined. Cellular dimensions were determined by measuring
20-25 cells of each taxon from photographic images. Light
micrographs were taken with an Axio CamHRc (Carl Zeiss
Co.) photomicrographic system attached to the micro-
scope.
DNA isolation, polymerase chain reaction (PCR),
and sequencing
Approximately 10 mL of cultures in exponential growth
were harvested by centrifugation (4,500 ×g, model 5415;
Eppendorf, Hamburg, Germany) for 1 min at room tem-
perature and washed three times with sterilized distilled
water. Total genomic DNA was extracted from the pellet
308
 Choi et al. Taxonomy of the Genus Cryptomonas

using the Dokdo-Prep Blood Genomic DNA Purification
Kit (Elpis-Biotech Inc., Daejeon, Korea) following the
manufacturer’s blood sample protocol. PCR was per-
formed using specific primers for nuclear ITS2, nuclear
SSU rDNA, chloroplast psbA and chloroplast LSU rDNA
(Table 1). The PCR amplification was performed on a to-
tal volume of 25 µL, containing 0.15 µL of TaKaRa Ex Taq
DNA polymerase (TaKaRa Bio Inc., Otsu, Japan), 2 µL
of each dNTP, 2.5 µL of 10× Ex Taq buffer, 1 µL of each
primer, and 1-10 ng of template DNA. The nuclear ITS2,
nuclear SSU rDNA, chloroplast psbA, and chloroplast
LSU rDNA were amplified using a PTC-0150 Minicycler
(MJ Research, Perkin-Elmer Co., Norwalk, CT, USA) with
the following program: 94°C for 5 min, 30 cycles of 94°C
for 1 min, 37-55°C for 1 min, and 72°C for 4 min, 72°C for
10 min and a 4°C hold. The PCR products were ~1.5 kb
for nr ITS2 partial LSU rDNA, 1.7 kb for nr SSU rDNA, 1.0
kb for cp psbA, and 2.7 kb for cp LSU rDNA and were pu-
rified using the Dokdo-Prep PCR Purification Kit (Elpis-
Biotech Inc.) according to the manufacturer’s protocol.
The purified template was sequenced with internal prim-
ers of conserved regions using an ABI 3730xl sequencer
(Perkin-Elmer Applied Biosystems, Foster City, CA, USA).
The alignment for each gene sequence was aligned by the
eye, and was edited using the Genetic Data Environment
(GDE 2.4) program (Smith et al. 1994). Unalignable nucle-
otides were excluded from phylogenetic analyses.
Strain identification
Nuclear ITS2 is likely a suitable marker to identify spe-
cies according to its degree of conservation (Hoef-Emden
2007), and nuclear ITS2 as well as partial LSU rDNA se-
quences were used to examine groups of genetically iden-
tical strains and to identify species of the strains.
Phylogenetic analyses
Phylogenetic trees were constructed using Bayesian
analysis (BA). Before the BA, we performed a likelihood
ratio test using Modeltest, version 3.7 (Posada and Cran-
dall 1998) to determine the best model under the hier-
archical likelihood ratio tests (hLRTs) and Akaike Infor-
mation Criterion (AIC). Evolutionary best-fit model was
selected as the GTR + I + Γ model from a combined five
gene data (nr ITS2 and nr partial LSU rDNA, nr SSU rDNA,

Table 1. PCR and sequencing primers

Designation Sequence (5′ to 3′)
a
PCR and sequencing primers for nuclear ITS2
crITS_03F CGA TGA AGA ACG YAG CGA
crITS_05R TAC TTG TTC GCT ATC GGT CTC T
Partial LSU rDNA
crLSU_29Fb TGA ACT TAA GCA TAT CAA TAA GCG G
crLSU_942R GGA AAC TTC GGA GGG AAC
Nuclear SSU rDNA
18S_CrN1Fc CTG CCA GTA GTC ATA TGC TTG TCT
18S_826Fd GTC AGA GGT GAA ATT CTT GGA T
18S_956Rd GAT CGT CTT CGA TCC CCT
18S_BRKe GGA AAC CTT GTT ACG ACT TCT C
Chloroplast psbAf
psbA_F ATG ACT GCT ACT TTA GAA AGA CG
psbA_R2 TCA TGC ATW ACT TCC ATA CCT A
Chloroplast LSU rDNAf
23S_38F TTC AGA AGC GAT GAA GGG
23S_586F GAA GAA TGA GCC GGC GAC
23S_740R CAT GGT TAG ATC ATC CGG GTT C
23S_1260F ATG TCG GCT TGA GTA GCG
23S_1305R CGG GGC ATT GGA TTC TCA
23S_1937F CCG CAC GAA AGG CGT AAC
23S_2024R GTG CAG GTA GTC CGC ATC
23S_2742R GGG CTT CCT ACT TAG ATG CTT T
PCR, polymerase chain reaction; ITS2, internal transcribed spacer 2; LSU rDNA, large subunit ribosomal DNA; SSU rDNA, small subunit ribo-
somal DNA.
Modified primers of a,eHoef-Emden et al. (2002), Hoef-Emden (2007), b,cMarin et al. (1998), dKim et al. (2007), fSon (2009).

309 http://e-algae.kr
Algae 2013, 28(4): 307-330
cp psbA, and cp LSU rDNA sequences). The GTR + I + Γ
model from the combined data was estimated by the fol-
lowing values; empirical base frequencies (A = 0.2701, C =
0.2014, G = 0.2697, T = 0.2589), substitution rates (A ↔ C =
1.0745, A ↔ G = 3.9918, A ↔ T = 1.9262, C ↔ G = 0.6372, C
↔ T = 8.1268), proportion of invariable sites (0.6217), and
gamma distribution shape parameter (0.6929).
BA was performed with MrBayes 3.1.2 (Huelsenbeck
and Ronquist 2001). Each analysis was initiated from a
random starting tree, and the program was set to run four
chains of Markov chain Monte Carlo iterations simulta-
neously for 2,000,000 generations. Trees and parameters
were sampled every 1,000 generations, and the burn-in
point was identified graphically by tracking the likeli-
hoods (Tracer V.1.5; http://tree.bio.ed.ac.uk/software/
tracer/), and then the first 800 trees were burned to en-
sure that they had stabilized. A majority rule consensus
tree was calculated from the remaining trees to examine
the posterior probabilities of each clade. The maximum
likelihood (ML) phylogenetic analyses were done using
the RAxML 7.0.4 program (Stamatakis 2006) with the gen-
eral time reversible (GTR) model. We used 1,000 indepen-
dent tree inferences using the -# option of the program
to identify the best tree. Bootstrap values were calculated
using 1,000 replicates, using the same substitution model.
Secondary structure prediction
Nuclear ITS2 sequences were folded using the mfold
server (http://mfold.rna.albany.edu/?q=mfold/RNA-
Folding-Form) (Zuker 2003) and RNAfold server (http://
rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) (Mathews
2004) with default values. A complete secondary struc-
ture graph of the nuclear ITS2 of Cryptomonas sp. M1634
was previously published (Hoef-Emden 2007). Prediction
of secondary structure was performed according to puta-
tive secondary structure graph of Hoef-Emden (2007) as
a template. Structures inferred by mfold or RNAfold were
examined for common stems, loops, and bulges, and
were identified by comparison to a previously published
sequence for Cryptomonas sp. M1634.
RESULTS
Phylogenetic analyses
Nuclear ITS2, partial LSU rDNA, and SSU rDNA, and
chloroplast psbA and LSU rDNA sequences of cultured
strains of the genus Cryptomonas from Korea were com-
http://dx.doi.org/10.4490/algae.2013.28.4.307
bined for phylogenetic analyses. Combined data were
analyzed by using Bayesian and RAxML methods. The
resulting phylogenetic tree is shown in Fig. 2. The com-
bined sequence data analyzed in this study were 6,227
nucleotides for 77 strains of Cryptomonas. The average
nucleotide frequencies of informative positions were
27.0% adenine, 20.1% cytosine, 27.0% guanine, and 25.9%
thymine.
For phylogenetic analyses, 79 strains were used (Table
2). Forty-seven new Cryptomonas strains were included,
and two species (Teleaulax acuta and Guillardia theta)
were selected as outgroup. Our phylogenetic tree divided
into fourteen species and six unidentified strains with
high support values (pp = 1.00, ML = 100). Three uniden-
tified strains branched off basally without close relatives,
and C. obovoidea, C. commutata, C. erosa, and Cryptomo-
nas sp. CNUCRY75 strain had strong support values (pp =
1.00, ML = 100). Next was C. marssonii with three strains,
including a Korean CNUCRY4 strain. C. ovata, C. obovata,
C. phaseolus, C. gyropyrenoidosa, C. paramecium, C. bo-
realis, C. lundii, and Cryptomonas sp. Okgeum121810C
were recovered as a monophyletic group with only a
Bayesian support value (pp = 0.96). C. pyrenoidifera, C.
tetrapyrenoidosa, and C. curvata were grouped together
with high support values (pp = 1.00, ML = 98). The Cryp-
tomonas sp. CNUCRY284 was closely related to 15 strains
of C. curvata, with high supportives (pp = 1.00, ML = 100),
while eight strains of C. tetrapyrenoidosa were sistered
with 14 strains of C. pyrenoidifera (pp = 1.00, ML = 99).
Secondary structure prediction of nuclear ITS2
The lengths of most nuclear ITS2 sequences of the
examined strains were between 330 (Cryptomonas sp.
CNUCRY284) and 590 nt (Cryptomonas sp. Sinjeong-
bangjuk080611A). Several features that were reported by
Schultz et al. (2005) were also observed in the Cryptomo-
nas nuclear ITS2 (Figs 3-9). For all ITS2 sequences, four-
helix structures could be inferred with long third helices
(Fig. 3). In all helices II, an unpaired U-U was found (Fig.
5). Three ‘UGGU’ motifs (UGGGU, UGG, GGU or similar)
with conserved positions across species were found in
helices III, but differed from the results of Schultz et al.
(2005). Its position was downstream instead of upstream
from the terminal loop (Figs 6-8).
Across species, the helices I and II were quiet variable
in their terminal parts and also varied in length (Figs 4
& 5). The numbers of internal loops differed from zero
(C. obovoidea, Cryptomonas sp. CNUCRY75, and Okgeum
121810C) to two (C. curvata and C. phaseolus) (Fig. 4).
310
 Choi et al. Taxonomy of the Genus Cryptomonas

Fig. 2. Bayesian tree of the nuclear internal transcribed spacer 2, partial large subunit ribosomal DNA (LSU rDNA), and small subunit rDNA, and
chloroplast psbA and LSU rDNA combined data set. The Bayesian posterior probability and maximum likelihood bootstrap value are shown above
or below the branches. Scale bar represents: substitution per site.

311 http://e-algae.kr
 Table 2. Strains of the genus Cryptomonas used in this study and the GenBank accession numbers for their nr 5.8S-ITS2-LSU, nr SSU, pt psbA and pt LSU rDNA sequences
GenBank accession No.
Taxon Strain Origin Nuclear Plastid
ITS2-LSU SSU LSU psbA
Cryptomonas
C. borealis M1083 (CCAC0113) Cologne, Germany None AM051188 None None
Algae 2013, 28(4): 307-330

C. borealis SCCAP-K0063 Unknown None AJ420696 None None

C. commutata M0739 (CCAC0109) A bog near mount Überling, Austria; freshwater AJ566165 AJ420697 None None
C. curvata Begelow S Bigelow, USA KF907322 KF907369 KF907412 KF907456
C. curvata CCAC0006 Trenant., Cornwall, England; freshwater AJ566147 None AM709636 None
C. curvata CCAC0080 Münster, Germany None AM051189 None None
C. curvata CCAP979/62 Freshwater AJ566150 None None None

http://dx.doi.org/10.4490/algae.2013.28.4.307
C. curvata CNUCRY15 Pungcheonje, Daesu, Goheung, Jeonnam, Korea KF907323 KF907370 KF907413 KF907457
34°45′35″ N, 127°18′23″ E
C. curvata CNUCRY19 Jeongsan, Cheongyang, Chungnam, Korea KF907324 KF907371 KF907414 KF907458
36°16′10″ N, 126°54′44″ E
C. curvata CNUCRY22 Daesong, Beopsu, Haman, Gyeongnam, Korea KF907325 KF907372 KF907415 KF907459
35°20′23″ N, 128°20′10″ E
C. curvata CNUCRY42 Daesong, Beopsu, Haman, Gyeongnam, Korea KF907326 KF907373 KF907416 KF907460
35°20′23″ N, 128°20′10″ E

312
C. curvata CNUCRY44 Okcheon, Ibang, Channgyeong, Gyeongnam, Korea KF907327 KF907374 KF907417 KF907461
35°20′23″ N, 128°20′10″ E
C. curvata CNUCRY48 Mulchat-Oreum, Bonggae, Jeju, Korea KF907328 KF907375 KF907418 KF907462
35°25′02″ N, 126°37′44″ E
C. curvata CNUCRY64 Cheongsan, Sinjang, Taean, Chungnam, Korea KF907329 KF907376 KF907419 KF907463
33°21′29″ N, 126°27′48″ E
C. curvata CNUCRY90 Gungnamji, Buyeoeup, Buyeo, Chungnam, Korea KF907330 KF907377 KF907420 KF907464
36°41′33″ N, 126°17′30″ E
C. curvata CNUCRY121 Chamyeon, Gohyeon, Namhae, Gyeongnam, Korea KF907331 KF907378 KF907421 KF907465
36°24′59″ N, 126°56′21″ E
C. curvata M1484 (CCAC1484) Schlachtensee, Brandenburg, Germany; freshwater AJ566149 None None None
C. curvata Sojungsoryuji032611 Cheondong, Gwangseok, Nonsan, Chungnam, Korea KF907332 KF907379 KF907422 KF907466
36°14′36″ N, 127°05′57″ E
C. erosa CCAC0018 (M0788) Germany, Forschungsinstitut Senckenberg, freshwater AJ566164 None None None
C. erosa CCAP979/67 Unknown AJ566162 None None None
C. erosa M0741 Überling, Austria AJ566163 AM051201 None None
C. gyropyrenoidosa CNUCRY146 Jungmuk, Bibong, Cheongyang, Chungnam, Korea KF907333 KF907380 KF907423 KF907467
34°55′05″ N, 127°52′06″ E
C. gyropyrenoidosa M1079 Dörpetalsperne, Germany AJ566154 AJ421149 None None
C. gyropyrenoidosa M1086 (CCAC0176) Cologne, Germany AJ566141 None None None
C. lundii M0850 (CCAC0107) Cologne, Germany AJ566161 None None None
 Table 2. Continued
GenBank accession No.
Taxon Strain Origin Nuclear Plastid
ITS2-LSU SSU LSU psbA
C. marssonii CCAC0086 Münster, Germany AJ566155 AM051191 None None
C. marssonii CNUCRY4 Daesong, Beopsu, Haman, Gyeongnam, Korea KF907334 KF907381 KF907424 KF907468
36°26′28″ N, 126°49′36″ E
C. marssonii M1476 Unknown AJ566156 None None None
C. obovata Hanjeong080611A Girin, Soseong, Jeongeup, Jeonbuk, Korea KF907335 KF907382 KF907425 KF907469
35°40′30″ N, 128°41′38″ E
C. obovata Saenae080611D Girin, Soseong, Jeongeup, Jeonbuk, Korea KF907336 None KF907426 KF907470
35°34′28″ N, 126°46′31″ E
C. obovoidea ASW09006 Unknown AJ566170 None None None
C. obovoidea CCAC0031 Schommelsnaaf, Bergisches Land, Germany; AJ566166 None None None
drying up lake, freshwater
C. obovoidea CCAP979/46 Unknown AJ566167 AM051200 None None
C. obovoidea CNUCRY76 Dongbankdongsan, Chocheon, Jeju, Korea KF907337 KF907383 KF907427 KF907471
33°30′54″ N, 126°43′00″ E
C. obovoidea M1088 Unknown AJ566169 None None None
C. obovoidea Songgock032611 Noseong, Nonsan, Chungnam, Korea KF907338 KF907384 KF907428 KF907472
35°20′23″ N, 128°20′10″ E

313
C. obovoidea UTEX2194 Unknown AJ566168 None None None
C. ovata CCAC0064 Spessart, Germany AJ566153 AM051193 None None
C. ovata CNUCRY231 Deokgock, Ganggu, Youngdeok, Gyeongbuk, Korea KF907339 KF907385 KF907429 KF907473
36°24′47″ N, 129°22′54″ E
C. ovata M1097 Wiesen, Spessart, Germany AJ566151 None None None
C. ovata M1171 Burgenland, Austria AJ566152 AJ420695 None None
C. ovata Mukawa100608B Hokkaido, Japan KF907340 KF907386 None KF907474
36°51′53″ N, 126°12′44″ E
C. paramecium CCAC0056 (M1303) Shore of Warren Lake, Cape Breton Highland National Park, AJ566158 None None None
Nova Scotia, Canada
C. phaseolus Angol032611 Hancheon, Sangwol, Nonsan, Chungnam, Korea KF907341 KF907387 KF907430 KF907475
36°04′50″ N, 126°54′23″ E
C. phaseolus Changdang11311E Ugak, Singwang, Buk, Pohang, Gyeongbuk, Korea KF907342 KF907388 KF907431 KF907476
35°39′44″ N, 128°39′43″ E
C. phaseolus CNUCRY5 Daepyeong Beopsu, Haman, Gyeongnam, Korea KF907343 KF907389 KF907432 KF907477
35°19′46″ N, 128°20′02″ E
C. phaseolus CNUCRY20 Jeongsan, Cheongyang, Chungnam, Korea KF907344 KF907390 KF907433 KF907478
36°24′59″ N, 126°56′21″ E
C. phaseolus Gakgae043011 Gakgye, Iseo, Cheongdo, Gyeongbuk, Korea KF907345 KF907391 KF907434 KF907479
36°16′02″ N, 127°09′09″ E
C. phaseolus SAG2013 Unknown AJ566157 None None None

http://e-algae.kr
Choi et al. Taxonomy of the Genus Cryptomonas
 Table 2. Continued
GenBank accession No.
Taxon Strain Origin Nuclear Plastid
ITS2-LSU SSU LSU psbA
C. pyrenoidifera CCAC0024 (M0923) Wahnbachtalsperre, Germany AJ566145 None None None
C. pyrenoidifera CCAC0032 (M1096) Wahner Heide, Cologne, Germany; shadowy puddle AJ566143 None None None
Algae 2013, 28(4): 307-330

C. pyrenoidifera CCAP979/61 Musikantenteich, Rlirschberg, Austria AJ566142 AJ421147 None None

C. pyrenoidifera CCMP152 Köln, Germany AJ566140 AJ421150 None None
C. pyrenoidifera CNUCRY27 Gyorae, Jocheoneup, Jeju, Korea KF907346 KF907392 KF907435 KF907480
35°19′58″ N, 128°19′54″ E
C. pyrenoidifera CNUCRY69 Yeongtapji, Gungdong, Yuseong, Daejeon, Korea KF907347 KF907393 KF907436 KF907481
53°26′14″ N, 126°43′47″ E
C. pyrenoidifera CNUCRY134 Surok, Baeksan, Gimje, Jeonbuk, Korea KF907348 KF907394 KF907437 KF907482

http://dx.doi.org/10.4490/algae.2013.28.4.307
36°22′86″ N, 127°20′42″ E
C. pyrenoidifera CNUCRY138 Andeok, Geumma, Iksan, Jeonbuk, Korea KF907349 KF907395 KF907438 KF907483
35°59′05″ N, 127°02′44″ E
C. pyrenoidifera CNUCRY152 Daepyeong, Beopsu, Haman, Gyeongnam, Korea KF907350 KF907396 KF907439 KF907484
35°19′46″ N, 128°20′02″ E
C. pyrenoidifera CNUCRY166 Bisan, Soi, Eumseong, Chungbuk, Korea KF907351 KF907397 None KF907485
36°24′31″ N, 126°31′35″ E
C. pyrenoidifera CNUCRY188 Buyeoeup, Buyeo, Chungnam, Korea KF907352 KF907398 KF907440 KF907486

314
36°50′30″ N, 127°45′05″ E
C. pyrenoidifera CNUCRY189 Buyeoeup, Buyeo, Chungnam, Korea KF907353 KF907399 KF907441 KF907487
36°50′30″ N, 127°45′05″ E
C. pyrenoidifera M1077 Überling, Austria AJ566144 AM051201 None None
C. pyrenoidifera Sumeun041911 Gwangnyeong, Aewoleup, Jeju, Korea KF907354 KF907400 KF907442 KF907488
35°33′55″ N, 126°46′30″ E
Cryptomonas sp. CNUCRY75 Seonheul, Chocheoneup, Jeju, Korea KF907355 KF907401 KF907443 KF907489
37°16′41″ N, 126°59′15″ E
Cryptomonas sp. CNUCRY284 Yeonhwa, Haga, Aewoleup, Jeju, Korea KF907356 KF907402 KF907444 KF907490
33°30′54″ N, 126°43′00″ E
Cryptomonas sp. Dumo2 100310C Dumo2 Hangyeong, Jeju, Korea KF907357 KF907403 KF907445 KF907491
35°33′55″ N, 126°46′30″ E
Cryptomonas sp. Okgeum121809C Jenam, Yeosan, Iksan, Jeonbuk, Korea KF907358 None KF907446 KF907492
33°27′18″ N, 126°20′50″ E
Cryptomonas sp. Sinjeong080611A Bohwa, Soseong, Jeongeup, Jeonbuk, Korea KF907359 None KF907447 KF907493
33°23′10″ N, 126°11′33″ E
Cryptomonas sp. Yeonra043011B Hwayangeup, Cheongdo, Gyeongbuk, Korea KF907360 KF907404 None KF907494
36°03′33″ N, 127°05′04″ E
C. tetrapyrenoidosa CNUCRY123 Pallack, Changnyeong, Gyeongnam, Korea KF907361 KF907405 KF907448 KF907495
35°31′13″ N, 128°24′07″ E
C. tetrapyrenoidosa CNUCRY127 Seoho, Suwon, Gyeonggi, Korea KF907362 KF907406 KF907449 KF907496
37°16′41″ N, 126°59′15″ E
 Table 2. Continued
GenBank accession No.
Taxon Strain Origin Nuclear Plastid
ITS2-LSU SSU LSU psbA
C. tetrapyrenoidosa Deokam032610 Deogam, Yeonsan, Nonsan, Chungnam, Korea KF907363 KF907407 KF907450 KF907497
35°05′56″ N, 128°12′34″ E
C. tetrapyrenoidosa Du-ung022611D Sindu, Wonbuk, Taean, Chungnam, Korea KF907364 KF907408 KF907451 KF907498
34°54′41″ N, 129°39′27″ E
C. tetrapyrenoidosa Hudong12610D Jeongsan, Cheongyang, Chungnam, Korea KF907365 KF907409 KF907452 KF907499
36°13′54″ N, 127°11′05″ E
C. tetrapyrenoidosa Onsu032611C Odong, Yeongo, Goseong, Gyeongnam, Korea KF907366 KF907410 KF907453 KF907500
36°50′10″ N, 126°11′25″ E
C. tetrapyrenoidosa Sinchon101709C Sinchon, Bannam, Naju, Jeonnam, Korea KF907367 KF907411 KF907454 KF907501
33°22′43″ N, 126°27′59″ E
C. tetrapyrenoidosa Yeongildong111310B Ugak, Singwang, Buk, Pohang, Gyeongbuk, Korea KF907368 None KF907455 KF907502
35°42′41″ N, 128°36′33″ E
Outgroup
Guillardia theta Unknown None X57162 NC000926 NC000926
Teleauiax acuta MUCC088 None AF508275 None None
New sequences are indicated in bold type.
ITS2, internal transcribed spacer 2; LSU, large subunit; SSU, small subunit; rDNA, ribosomal DNA.

315
Strains labeled, CCAC, Culture collection of Algae at the University of Cologne, Germany; CCAP, Culture Collection of Algae at the Protozoa, UK; CCMP, Center for Culture of Marine Phytoplankton;
KR, Culture collection of the Chungnam National University, Woongghi’s Lab, Korea; M, Culture Collection Melkonian at the University of Cologne, Germany; MUCC, Melbourne University Culture
Collection; SAG, Sammlung von AlgenKulturen der Universität Göttingen, Germany; SCCAP, Scandinavian Culture Center for Algae and Protozoa, Denmark; UTEX, UTEX, Culture Center of Algae,
Austin, TX, USA.

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Choi et al. Taxonomy of the Genus Cryptomonas
Algae 2013, 28(4): 307-330
Fig. 3. Putative secondary structure of the nuclear internal
transcribed spacer 2 of the Cryptomonas obovoidea Songgock032611
(from 5’ to 3’ terminus in clockwise direction). In total a sequence of
352 nucleotides were submitted to the mfold server. The presumably
correct structure with the four helix domains was found without
applying any force options. Helices were numbered in Roman
numerals.
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The shortest helix I was found in C. obovoidea and Cryp-
tomonas sp. Okgeum121810C, whereas the longest helix
I was found in C. pyrenoidifera (Fig. 4). The shortest helix
II was found in C. pyrenoidifera CNUCRY27, whereas the
longest was found in C. obovata (Fig. 5). In helix II, the
proximal parts and an internal loop with a U-U mismatch
were highly conserved across species, whereas all were
different in length, with various additional internal loops
found in the distal parts (Fig. 5). Among the helix III (Figs
6-8), the shortest one was found in C. pyrenoidifera (Fig.
7). The longest helix III was found in Cryptomonas sp. Sin-
jeong080611A (Fig. 8). It averaged about twice the length
of other helices, which also differed markedly between
each other (Figs 6-8). Although remarkable differences in
length were observed, the structures of the terminal parts
were highly conserved. Close to the terminal loop, a high-
ly conserved region across species was found, consisting
of an alternating upstream CUCUCU motif paired with a
GAGAGGA downstream motif, and included an internal
loop (region IIIa in Figs 6-8). A combination of three in-
ternal loops with the two other loops protruding to the 5′
side and one symmetrical internal loop in the middle was
also found in all helix III (IIIb in Figs 6-8). Two UGGU mo-
tifs were always found on the 3′ side opposite to the two
outer loops of the IIIb part, whereas a third ‘UGGU’ motif
was present upstream from the other two (arrows in Figs
6-8). In the proximal part, the helices were less conserved
and showed various numbers and sizes of internal loops
apart from one large internal loop which seemed to be
present in all helices, but was not conserved in primary
sequence across all clades (Figs 6-8). Cryptomonas sp.
Yeonra042011B, Dumo2 100311C, and Sinjeong080611A
had a branch structure at its middle part but IIIa and IIIb
parts were conserved. The nuclear ITS2 sequences were
generally not aligned across species in helix IV, which
caused difficulty in predicting their structure (Fig. 9). The
longest helix IV was found in C. gyropyrenoidosa. C. cur-
vata Sojung032611and Cryptomonas sp. CNUCRY284 had
identically short helices IV. Cryptomonas sp. CNUCRY284
strain showed sister relationships with C. curvata clade.
Cryptomonas sp. Yeonra043011B and Dumo2 100310C
strains included in the same clade was similar helices IV.
Morphology of Cryptomonas species from Korea
Cell shape and length, length of the fullow-gullet sys-
tem, color and number of chloroplasts, and the pres-
ence / absence and number of pyrenoids were examined.
The results are summarized in Table 3 and illustrated in
Figs 10-12.
316
 Choi et al. Taxonomy of the Genus Cryptomonas

Fig. 4. Predicted secondary structures of nuclear internal transcribed spacer 2 helix I (clockwise from 5’ to 3’ termini). Parts of the sequences
which differ among strains of the same species are labeled by rectangular boxes. In helix I, the proximal parts are marked in brackets and were
highly conserved across strains.

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Algae 2013, 28(4): 307-330

Fig. 5. Predicted secondary structures of nuclear internal transcribed spacer 2 helix II (clockwise from 5’ to 3’ termini). Parts of the sequences
which differ among strains of the same species are labeled by rectangular boxes. In helix II, the proximal parts marked in brackets were highly
conserved across strains. In all helices, the conserved un-paird U-U motif was found.

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 Choi et al. Taxonomy of the Genus Cryptomonas

Fig. 6. Secondary structure predictions of helix domains III of Cryptomonas curvata Sojung032611, C. gyropyrenoidosa CNUCRY146, C. marssonii
CNUCRY4, C. obovoidea Songgock032611, C obovata Hanjeong080611A and C. ovata CNUCRY231 (clockwise from 5’ to 3’ termini). Region IIIa
comprised the most conserved part of the nuclear internal transcribed spacer 2. The highly conserved internal three-loop structures of all IIIb
regions were accompanied by three UGGU motifs (arrows) which were consistently observed across species.

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Algae 2013, 28(4): 307-330

Fig. 7. Secondary structure predictions of helix domains III of Cryptomonas phaseolus Gakgae 043011, C. pyrenoidifera CNUCRY138, C.
tetrapyrenoidosa CNUCRY123, Cryptomonas sp. CNUCRY75, Cryptomonas sp. CNUCRY284, and Cryptomonas sp. Okgeum121810C (clockwise from
5’ to 3’ termini). Region IIIa comprised the most conserved part of the nuclear internal transcribed spacer 2. The highly conserved internal three-
loop structures of all IIIb regions were accompanied by three UGGU motifs (arrows) which were consistently observed across species.

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 Choi et al. Taxonomy of the Genus Cryptomonas

Fig. 8. Secondary structures predictions of helix domains III of Cryptomonas sp. Yeonra043011B, Cryptomonas sp. Dumo2 100311C, and
Cryptomonas sp. Sinjeong080611A (clockwise from 5’ to 3’ termini). Region IIIa comprised the most conserved part of the nuclear internal
transcribed spacer 2. The highly conserved internal three-loop structures of all IIIb regions were accompanied by three UGGU motifs (arrows)
which were consistently observed across species. Helix III of the three strains differed from the helices of the other species, in that it has middle
branch.

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Algae 2013, 28(4): 307-330

Fig. 9. Presumed secondary structures of helices IV of the nuclear internal transcribed spacer 2 (ITS2). This part of the nuclear ITS2 was the most
variable region across species in the alignment. It was also difficult to define 5’ and 3’ termini of this helix.

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 Choi et al. Taxonomy of the Genus Cryptomonas

A B C D

E F G H

Fig. 10. Light micrographs of genus Cryptomonas. (A & B) C. curvata Begelow S. (C & D) C. gyropyrenoidosa CNUCRY146. (E & F) C. marsonii
CNUCRY4. (G & H) C. ovata CNUCRY231. C, contractile vacuole; Cp, chloroplast; E, ejectisome; F, flagellum; G, gullet; MO, maupas oval; Py, pyrenoid; S,
starch. Scale bars represent: A-H, 10 μm.

Table 3. Morphological points of comparisons among Korean Cryptomonas species

Taxa Shape Length (μm) Gullet length/Cell body Chloroplast Pyrenoid Maupas ovals
C. curvata Ovoid 18-20 2/3 2 2 2
C. obovata Ovoid 26-36/29-38 2/3-3/4 2 2/× 2
C. obovoidea Sigmoid 11-14 3/4 2 2 ×
C. phaseolus Ovoid 12-16 2/3 2 2 ×
C. pyrenoidifera Ovoid 18-20 2/3 2 2 2
C. tetrapyrenoidosa Ovoid 16-22 3/4 2 4 ×
Cryptomonas sp. Ovoid 13-17 1/2-2/3 2 2 ×
Okgeum121809C
Cryptomonas sp. Ovoid 18-21 3/4 2 2 2
CNUCRY284
Cryptomonas sp. Ovoid 14-18 1/2-3/4 2 3 ×
Dumo2 100310C
Cryptomonas sp. Ovoid 14-18 2/3 2 2 ×
Yeonra043011B

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A B C D

E F G H

Fig. 11. Light micrographs of genus Cryptomonas. (A & B) C. obovata Saenae080611D. (C & D) C. obovata Hanjeong080611A. (E & F) C. obovoidea
Songgock032611. (G & H) C. phaseolus Gakgae043011. C, contractile vacuole; Cp, chloroplast; E, ejectisome; F, flagellum; G, gullet; MO, maupas
oval; Py, pyrenoid; S, starch. Scale bars represent: A-H, 10 μm.

Cryptomonas curvata (Ehrenberg) Hoef-Emden vacuoles were placed near the gullet. Two flagella were
et Melkonian 2003 inserted in a vestibulum of the cell invagination.

Specimens examined. Sojungsoryuji, Namsan, Korea; Cryptomonas gyropyrenoidosa Hoef-Emden et

Seohojeosuji, Suwon, Korea; Daepyeong swamp, Haman,
Korea; Jjokji pond, Changyeong, Korea; 1100goji, Seog-
wipo, Korea; Chungsan, Taean, Korea; Gungnamji, Buyeo,
Korea.
Light microscopy. Cryptomonas curvata was an ovoid-
elliptical shape in the ventral view (Fig. 10A & B). The cells
were 18-20 μm in length. The length of the gullet with
rows of ejectisomes was 2/3 of cell length. Cells had two
brown-chloroplasts, each with a pyrenoid. Contractile
Melkonian 2003
Specimens examined. Jungmukji, Cheongyang, Korea.
Light microscopy. Cryptomonas gyropyrenoidosa was
broad elliptical shape and was flattened in ventral view
(Fig. 10C & D). The cells were 20-23 μm in length. The
length of the gullet with rows of small ejectisomes was 1/2
of cell length. Cells had brown chloroplasts surrounded
by many starch grains. Two red maupas ovals were locat-

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ed in the center of each cell.
Cryptomonas marssonii (Skuja) Hoef-Emden et
Melkonian 2003
Specimens examined. Daesong, Haman, Gyeongnam,
Korea.
Light microscopy. Cryptomonas marssonii was a sig-
moid shape to asymmetrical in ventral view (Fig. 10E &
F). The cells were 20-23 μm in length. Cells had two brown
chloroplasts and many starch grains around chloroplasts.
Each chloroplast has a pyrenoid. A contractile vacuole
was located in the anterior of the cell.
Cryptomonas ovata (Ehrenberg) Hoef-Emden et
Melkonian 2003
Specimens examined. Songcheonji, Iksan, Korea; Mu-
kawa, Muroran, Hokkaido, Japan.
Light microscopy. Cryptomonas ovata has long-ovoid
of elliptical shape in ventral view (Fig. 10G & H). The cells
were 32-37 μm in length. The length of gullet with rows
of ejectisomes was 3/5 that of cells. Cells had two brown
chloroplasts surrounded by many starch grains. Contrac-
tile vacuole was located in apical cell and three maupas
ovals were located in the center of the cell.
Cryptomonas obovata Czosnowski 1948
Specimens examined. Saenaebangjuk, Jeongeup, Ko-
rea; Hanjeongje, Jeongeup, Korea.
Light microscopy. Cryptomonas obovata had two
marked cells which were characterized by presence / ab-
sence of pyrenoids (Fig. 11A-D). The cells were 26-36 μm
in length. Cells had two brown chloroplasts, each with a
terminal pyrenoid. The other cell had biconvex shape in
right or left lateral view (Fig. 11C). The cells were 29-38 μm
in length. Cells had two red-brown chloroplasts without
pyrenoid. Both had ovoid shape in ventral view (Fig. 11B
& D). The length of gullet with rows of ejectisomes was
2/3 or 3/4 that of cells. Contractile vacuole located in api-
cal part of the cell and two maupas ovals located in the
center of the cell. Two flagella inserted in a vestibulum of
the cell invagination.
Cryptomonas obovoidea (Pascher) Hoef-Emden
2007
Specimens examined. Songgock, Nonsan, Korea; Mok-
malji, Taean, Korea; Dongbackdongsan, Jeju, Korea.
325
Choi et al. Taxonomy of the Genus Cryptomonas
Light microscopy. Cryptomonas obovoidea had ventral
reflex shape in right or left lateral view and ovoid with
broad end in ventral view (Fig. 11E & F). The cells were
11-14 μm in length. The length of gullet with rows of ejec-
tisomes was 3/4 that of cells. Cells had two green-brown
chloroplasts, each with a pyrenoid. Contractile vacuole
located in apical cell. Maupas oval absent. Two flagella
inserted in a vestibulum of the cell invagination.
Cryptomonas phaseolus (Skuja) Hoef-Emden 2007
Specimens examined. Angolji, Nonsan, Korea; Gak-
gaeji, Cheongdo, Korea; Jangdangji, Pohang, Korea; Dae-
pyeong swamp, Haman, Korea; Jeongsan, Cheongyang,
Korea.
Light microscopy. Cryptomonas phaseolus had bicon-
vex shape in right or left lateral view and long ovoid with
narrow end in ventral view (Fig. 11G & H). The cells were
12-16 μm in length. The length of gullet with rows of ejec-
tisomes was 2/3 that of cells. Cells had two brown chloro-
plasts, each with a pyrenoid. The cell didn’t have maupas
oval when observed in culture. Two flagella were inserted
in a vestibulum of the cell invagination.
Cryptomonas pyrenoidifera (Geitler) Hoef-Emden
et Melkonian 2003
Specimens examined. Gyorae, Jeju, Korea; Yeongtapji,
Daejeon, Korea; Wonseongje, Surok, Baeksan, Korea;
Andeokjeosuji, Iksan, Korea; Soiji, Bisan, Eumseong, Ko-
rea; Gungnamji, Buyeo, Korea; Sumeunmulbaengdeui,
Aewol, Jeju, Korea.
Light microscopy. Cryptomonas pyrenoidifera was bi-
flattened in right or left lateral view and ovoid-elliptical
shape in ventral view (Fig. 12A & B). The cells were 18-20
μm in length. The length of gullet with rows of ejectisomes
was 2/3 that of cells. Cells had two brown-chloroplasts,
each with an obvious pyrenoid. Contractile vacuole was
placed at above two maupas ovals. Two flagella were lo-
cated in a vestibulum of the cell invagination.
Cryptomonas tetrapyrenoidosa (Skuja) Hoef-
Emden et Melkonian 2003
Specimens examined. Sinchonje, Naju, Korea; Du-
ungseupji, Taean, Korea; Onsusoryuji, Goseong, Korea;
Deokamji, Nonsan, Korea; Yeongildongji, Pohang, Korea;
Hudongje, Cheongyang, Korea; Seohojeosuji, Suwon, Ko-
rea.
Light microscopy. Cryptomonas tetrapyrenoidosa was
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Algae 2013, 28(4): 307-330

A B C D

E F G H

I J K L

Fig. 12. Light micrographs of genus Cryptomonas. (A & B) C. pyrenoidifera Sumeun041911. (C & D) C. tetrapyrenoidosa Onsu032611C. (E &
F) Cryptomonas sp. Okgeum121810C. (G & H) Cryptomonas sp. CNUCRY284. (I & J) Cryptomonas sp. Dumo2 100310C. (K & L) Cryptomonas sp.
Yeonra043011B. C, contractile vacuole; Cp, chloroplast; E, ejectisome; F, flagellum; G, gullet; MO, maupas oval; Py, pyrenoid; S, starch. Scale bars
represent: A-L, 10 μm.

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an ovoid shape in ventral view (Fig. 12C & D). The cells
were 16-22 μm in length. The length of gullet with rows
of ejectisomes was 2/3 that of cells. Cells had two brown-
chloroplasts, and each chloroplast had two or three pyre-
noids facing each other in the left and right chloroplast
lobes. Contractile vacuole was located in apical and two
flagella inserted in a vestibulum of the cell invagination.
Cells had two maupas ovals.
Cryptomonas sp. Okgeumji121810C
Specimens examined. Okgeumji, Iksan, Korea.
Light microscopy. Cryptomonas sp. Okgeumji121810C
has biconvex shape with narrow ends in right or left lat-
eral view and broad ovoid shape in ventral view (Fig. 12E
& F). The cells were 13-17 μm in length. The length of gul-
let with rows of ejectisomes was 1/2 or 2/3 of cell length.
Cells had a brown chloroplast with a pyrenoid. Two fla-
gella inserted in a vestibulum of the cell invagination.
Cryptomonas sp. CNUCRY284
Specimens examined. Yeonhwaji, Jeju, Korea.
Light microscopy. Cryptomonas sp. CNUCRY284 had
biconvex shape with narrow end in right or left lateral
view and ovoid shape in ventral view (Fig. 12G & H). Cells
were 18-21 μm in length. Length of gullet with rows of
ejectisomes was 3/5 that of cells. Cells had a greenish-
brown chloroplast with an obvious pyrenoid. Contractile
vacuole above two maupas ovals. Two flagella inserted in
a vestibulum of the cell invagination.
Cryptomonas sp. Dumo2 100310C
Specimens examined. Dumojeosuji, Jeju, Korea.
Light microscopy. Cryptomonas sp. Dumo2 100310C
had biconvex with narrow end in right or left lateral view
and ovoid shape in ventral view (Fig. 12I & J). The cells
were 14-18 μm in length. The length of gullet with rows
of ejectisomes was 1/2 or 3/4 of cell length. Cells had a
brown chloroplasts with a pyrenoid. Contractile vacuole
located in apical cell. Cells didn’t have maupas oval when
we established it in culture. Two flagella located in a ves-
tibulum of the cell invagination.
Cryptomonas sp. Yeonra043011B
Specimens examined. Yeonra, Hwayangeup, Cheon-
gdo, Gyeongbuk, Korea.
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Choi et al. Taxonomy of the Genus Cryptomonas
Light microscopy. Cryptomonas sp. Yeonra043011B
had flattened shape in right of left lateral view and ovoid
shape in ventral view (Fig. 12K & L). The cells were 14-
18 μm in length. The length of gullet with rows of ejec-
tisomes was 2/3 of cell length. Cells had a green-brown
chloroplasts with a pyrenoid. The cell had two maupas
ovals. Two flagella inserted in a vestibulum of the cell in-
vagination.
DISCUSSION
Light microscopy
Although cryptomonad genera were delimited by ul-
trastructral features, such as periplast and flagellar ap-
paratus, all Cryptomonas species have been described
exclusively by light microscopy (Huber-Pestalozzi 1950,
Butcher 1967). Pringsheim (1944, 1968) established clonal
cultures, and examined morphological characters of the
genus Cryptomonas. He recognized that morphological
characters cannot be used satisfactorily for delimitation
of Cryptomonas species. Most species of the genus Cryp-
tomonas from Korea are ovoid or sigmoid in shape, and
cell length ranges from 11 to 38 μm. The ejectisomes were
arranged along a gullet located at vestibular region of the
cell. The length of gullet with rows of ejectisomes was 1/2-
3/4 that of cells. All cells had two chloroplasts with vari-
able color; reddish brown, brown, or green brown. How-
ever, the number of pyrenoids was 2 to 6 per cell, but C.
obovata Hanjeong080611A has chloroplasts without py-
renoid. The maupas ovals were either present or absent.
However, distinction based on morphological characters
is unclear for species of the genus Cryptomonas, as sug-
gested by Javornický (2003) and Hoef-Emden (2007). For
example, C. obovoidea had a sigmoid cell shape, but be-
longed to the species group with ovoid cell shape in our
tree. Therefore, species with ovoid cell shape were not
monophyletic. Other morphological characters, such as
the presence / absence of pyrenoid, were found across
clades. Saenae080611A strain of C. obovata clade has py-
renoid, but Hanjeong080611A strain lacked pyrenoid, in
spite of being located in the same species clade. Incon-
gruence between morphospecies concept and molecular
phylogeny has been reported recently (Hoef-Emden and
Melkonian 2003, Hoef-Emden 2007). Therefore, due to
lack of clear species-specific morphological characters,
new species may have to be defined by molecular signa-
tures, as suggested by Hoef-Emden (2007).
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Algae 2013, 28(4): 307-330
Molecular phylogenetic analyses
In the molecular phylogenetic tree based on nuclear
18S rDNA, the genus Cryptomonas is known as mono-
phyletic (Deane et al. 2002). Although nuclear 18S rDNA
data has been used for resolving phylogenetic relation-
ships among genera of Cryptophyceae (Marin et al. 1998,
Deane et al. 2002), the tree did not show obvious resolu-
tion within the genus Cryptomonas. The phylogenetic
relationships among Cryptomonas species were likely to
involve fast-evolving genes which contain more phyloge-
netic information (Deane et al. 2002). Our phylogenetic
tree based on combined nuclear SSU, partial LSU, and
ITS2 and plastid psbA and LSU rDNA data have a topol-
ogy similar to recently published phylogenies (Hoef-
Emden 2007). However, our data further clarify the phy-
logenetic relationships among Cryptomonas species, but
there remain areas of uncertainty. For example, the posi-
tions of some species (C. ovata, C. obovata, C. phaseolus,
C. gyropyrenoidosa, C. paramecium, C. borealis, and C.
lundii) were not clearly resolved in the tree. The topolo-
gies of our trees obtained by ML and Bayesian analyses
are very similar, although the level of support for indi-
vidual nodes varies considerably. The tree also differs
slightly from a tree based on combined nuclear partial
LSU and nucleomorph SSU rDNA data (Hoef-Emden et
al. 2002, Hoef-Emden 2007). Especially our tree is better
supported in internal nodes than that of the previous one
(Hoef-Emden 2007).
Nuclear ITS2 secondary structures
The nuclear ITS2 in Cryptomonas species showed the
typical conserved secondary structure with four helices
which are found in most eukaryote lineages (Schultz et
al. 2005). Molecular characters were derived from the
nuclear ITS2; 1) the highly conserved regions were eas-
ily aligned among Cryptomonas species, 2) the less con-
served regions provided enough signal to identify syn-
apomorphies or species-specific unique combinations
of sequences for clades (species), and 3) the highly vari-
able regions were aligned within but not between species
(Hoef-Emden and Melkonian 2003). Most conserved re-
gions in all strains were located in the distal part of helix
ІІІ. The sequences of the distal parts were not very use-
ful for species identification, but seem to be available to
distinguish cryptomonad genera as molecular signatures.
Compare to other eukaryotic ITS2 secondary structures,
the UGGU motifs have shifted from the 5′ part to 3′ part
of the helix ІІІ in previous studies, as well as in this study
http://dx.doi.org/10.4490/algae.2013.28.4.307
(Schultz et al. 2005, Hoef-Emden 2007).
The helix І, ІІ, and ІІІb were less conserved regions than
helix ІІІa (the distal part of helix ІІІ). The proximal part of
helix І and ІІ had similar sequences among species, but
were variable in their terminal parts. Specific structures
of helix ІІІb appeared in all strains, despite the differences
among sequences. Sequences of helix І, ІІ, and ІІІb in ITS2
secondary structure were very useful for the identifica-
tion of Cryptomonas species. The helix ІV was varied from
species to species because of sequence diversity. Through
deduction of the secondary structure, molecular signa-
tures to identify lower rank of genus or species could be
determined.
ACKNOWLEDGEMENTS
This research was the survey of Indigenous Biological
Resources of Korea from National Institute of Biological
Resources and supported by the National Research Foun-
dation Program funded by the Korea Government/MEST
(NRF-C1ABA001-2010-0020700).
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tematique. Tome III: Les algues blues et rouges. Les
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