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 (, Cryptophyta) from Korea

Bomi Choi1, Misun Son2, Jong Im Kim1 and Woongghi Shin1,* 1Department of Biology, Chungnam National University, Daejeon 305-764, Korea 2Yeongsan River Environment Research Center, Gwangju 500-480, Korea

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 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;

INTRODUCTION

The genus Cryptomonas, which belongs to the class display one or two morphotypes within a clonal culture. Cryptophyceae, was established by Ehrenberg (1831). It is In the cryptomorph, the inner periplast component (IPC) distributed in freshwater habitats worldwide. Cells can be is made of hexagonal to polygonal plates, whereas in the easily recognized by two unequal biflagella, olive-brown- camphylomorph the IPC is a sheet-like layer (Faust 1974, ish to olive-greenish in color, large ejectisomes lined in Brett and Wetherbee 1986, Hill 1991, Hoef-Emden and the furrow-gullet system, and a peculiar swaying swim- Melkonian 2003, Hoef-Emden 2007). ming behavior due to the asymmetric shape, dorsally flat- Since Ehrenberg (1831, 1832) described six Crypto- tened and ventrally concave in lateral view. Cells have two monas species, many additional species were added chloroplasts originated from , which contain the (Pascher 1913, Schiller 1925, 1929, 1957, Skuja 1939, 1948, accessory pigment 566 of 1956, Huber-Pestalozzi 1950, Starmach 1974). Tradition- (Hill and Rowan 1989, Clay et al. 1999, Deane et al. 2002, ally, Cryptomonas species have been characterized by Hoef-Emden and Melkonian 2003). Cryptomonas species 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- Corresponding Author ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted * non-commercial use, distribution, and reproduction in any medium, E-mail: [email protected] 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

onymized both genera Campylomonas and 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 Fig. 1. Distributions of the genus Cryptomonas species from Korea. species.

MATERIALS AND METHODS shape, and internal organization (Bourelly 1970). Howev- er, it is difficult to delimit Cryptomonas species due to the Algal cultures and microscopy paucity of morphological characters and the less-than- adequate visibility of living cells using light microscopy Specimens were collected from freshwater habitats in (Pringsheim 1968). For example, an early attempt to orga- Korea (Fig. 1). Live cells were isolated by Pasteur capillary nize photosynthetic taxonomy was carried pipette and were brought into a unialgal culture. The cells out by Ehrenberg (1838), who included several unrelated were cultivated in f/2 medium (Guillard and Ryther 1962, genera in the family Cryptomonadina with original short Guillard 1975) with soil extract. The clonal cultures were diagnosis. Later, Dujardin (1841) described two families maintained under a light : dark regime of 14 : 10 at 20- (Xanthodiscaceae and Chilomonaceae) within the or- 22°C using cool-white fluorescence lamps with illumina- der Cryptomonadineae. In his classification system, the tions of 30 µmol protons m-2 s-1. The morphology was ex- family Chilomonadaceae consisted of four genera (Chi- amined by differential interference contrast with a 100X lomonas, , Cryptomonas, and Cyanomonas). oil immersion lens (Carl Zeiss Co., Göttingen, Germany). These genera were characterized by nutritional mode, Calibration of magnification was done with grated mi- number and color of chloroplast. Butcher (1967) orga- crometer. The shape and length of cells, length of the nized main photosynthetic genera of the Cryptophyceae fullow-gullet system, color and number of chloroplasts, into three families (Hilleaceae, Hemiselmidaceae, and presence / absence and number of pyrenoids were exam- ) based on complexity of furrow- ined. Cellular dimensions were determined by measuring gullet system with or without ejectisome. He also used 20-25 cells of each taxon from photographic images. Light number of ejectisome rows in the furrow-gullet system to micrographs were taken with an Axio CamHRc (Carl Zeiss discriminate between each genus, and thus, described 12 Co.) photomicrographic system attached to the micro- new Cryptomonas species from salt water. scope. Hill (1991) revised the broad generic definition of the genus Cryptomonas recognized by Butcher (1967) and DNA isolation, polymerase chain reaction (PCR), erected four new genera based on their furrow-gullet and sequencing system, periplast structure, plastidial complex and rhi- zostyle; Campylomonas, , , and Approximately 10 mL of cultures in exponential growth Teleaulax. Recently, molecular work (Marin et al. 1998, were harvested by centrifugation (4,500 ×g, model 5415; Deane et al. 2002) has shown that Cryptomonas species Eppendorf, Hamburg, Germany) for 1 min at room tem- grouped together with species of Campylomonas and perature and washed three times with sterilized distilled Chilomonas. Hoef-Emden and Melkonian (2003) syn- water. Total genomic DNA was extracted from the pellet

http://dx.doi.org/10.4490/algae.2013.28.4.307 308 Choi et al. Taxonomy of the Genus Cryptomonas

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

Table 1. PCR and sequencing primers Designation Sequence (5′ to 3′) PCR and sequencing primers for nuclear ITS2a 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 ofa,e Hoef-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 bined for phylogenetic analyses. Combined data were model from the combined data was estimated by the fol- analyzed by using Bayesian and RAxML methods. The lowing values; empirical base frequencies (A = 0.2701, C = resulting phylogenetic tree is shown in Fig. 2. The com- 0.2014, G = 0.2697, T = 0.2589), substitution rates (A ↔ C = bined sequence data analyzed in this study were 6,227 1.0745, A ↔ G = 3.9918, A ↔ T = 1.9262, C ↔ G = 0.6372, C nucleotides for 77 strains of Cryptomonas. The average ↔ T = 8.1268), proportion of invariable sites (0.6217), and nucleotide frequencies of informative positions were gamma distribution shape parameter (0.6929). 27.0% adenine, 20.1% cytosine, 27.0% guanine, and 25.9% BA was performed with MrBayes 3.1.2 (Huelsenbeck thymine. and Ronquist 2001). Each analysis was initiated from a For phylogenetic analyses, 79 strains were used (Table random starting tree, and the program was set to run four 2). Forty-seven new Cryptomonas strains were included, chains of Markov chain Monte Carlo iterations simulta- and two species (Teleaulax acuta and theta) neously for 2,000,000 generations. Trees and parameters were selected as outgroup. Our phylogenetic tree divided were sampled every 1,000 generations, and the burn-in into fourteen species and six unidentified strains with point was identified graphically by tracking the likeli- high support values (pp = 1.00, ML = 100). Three uniden- hoods (Tracer V.1.5; http://tree.bio.ed.ac.uk/software/ tified strains branched off basally without close relatives, tracer/), and then the first 800 trees were burned to en- and C. obovoidea, C. commutata, C. erosa, and Cryptomo- sure that they had stabilized. A majority rule consensus nas sp. CNUCRY75 strain had strong support values (pp = tree was calculated from the remaining trees to examine 1.00, ML = 100). Next was C. marssonii with three strains, the posterior probabilities of each clade. The maximum including a Korean CNUCRY4 strain. C. ovata, C. obovata, likelihood (ML) phylogenetic analyses were done using C. phaseolus, C. gyropyrenoidosa, C. paramecium, C. bo- the RAxML 7.0.4 program (Stamatakis 2006) with the gen- realis, C. lundii, and Cryptomonas sp. Okgeum121810C eral time reversible (GTR) model. We used 1,000 indepen- were recovered as a monophyletic group with only a dent tree inferences using the -# option of the program Bayesian support value (pp = 0.96). C. pyrenoidifera, C. to identify the best tree. Bootstrap values were calculated tetrapyrenoidosa, and C. curvata were grouped together using 1,000 replicates, using the same substitution model. with high support values (pp = 1.00, ML = 98). The Cryp- tomonas sp. CNUCRY284 was closely related to 15 strains Secondary structure prediction of C. curvata, with high supportives (pp = 1.00, ML = 100), while eight strains of C. tetrapyrenoidosa were sistered Nuclear ITS2 sequences were folded using the mfold with 14 strains of C. pyrenoidifera (pp = 1.00, ML = 99). server (http://mfold.rna.albany.edu/?q=mfold/RNA- Folding-Form) (Zuker 2003) and RNAfold server (http:// Secondary structure prediction of nuclear ITS2 rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) (Mathews 2004) with default values. A complete secondary struc- The lengths of most nuclear ITS2 sequences of the ture graph of the nuclear ITS2 of Cryptomonas sp. M1634 examined strains were between 330 (Cryptomonas sp. was previously published (Hoef-Emden 2007). Prediction CNUCRY284) and 590 nt (Cryptomonas sp. Sinjeong- of secondary structure was performed according to puta- bangjuk080611A). Several features that were reported by tive secondary structure graph of Hoef-Emden (2007) as Schultz et al. (2005) were also observed in the Cryptomo- a template. Structures inferred by mfold or RNAfold were nas nuclear ITS2 (Figs 3-9). For all ITS2 sequences, four- examined for common stems, loops, and bulges, and helix structures could be inferred with long third helices were identified by comparison to a previously published (Fig. 3). In all helices II, an unpaired U-U was found (Fig. sequence for Cryptomonas sp. M1634. 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. RESULTS (2005). Its position was downstream instead of upstream from the terminal loop (Figs 6-8). Phylogenetic analyses Across species, the helices I and II were quiet variable in their terminal parts and also varied in length (Figs 4 Nuclear ITS2, partial LSU rDNA, and SSU rDNA, and & 5). The numbers of internal loops differed from zero chloroplast psbA and LSU rDNA sequences of cultured (C. obovoidea, Cryptomonas sp. CNUCRY75, and Okgeum strains of the genus Cryptomonas from Korea were com- 121810C) to two (C. curvata and C. phaseolus) (Fig. 4).

http://dx.doi.org/10.4490/algae.2013.28.4.307 310 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 Algae 2013, 28(4): 307-330 A psb None None None KF907456 None None None KF907457 KF907458 KF907459 KF907460 KF907461 KF907462 KF907463 KF907464 KF907465 None KF907466 None None None KF907467 None None None LSU None None None KF907412 AM709636 None None KF907413 KF907414 KF907415 KF907416 KF907417 KF907418 KF907419 KF907420 KF907421 None KF907422 None None None KF907423 None None None GenBank accession No. GenBank SSU AM051188 AJ420696 AJ420697 KF907369 None AM051189 None KF907370 KF907371 KF907372 KF907373 KF907374 KF907375 KF907376 KF907377 KF907378 None KF907379 None None AM051201 KF907380 AJ421149 None None A and pt LSU rDNA sequences Nuclear psb None None AJ566165 KF907322 AJ566147 None AJ566150 KF907323 KF907324 KF907325 KF907326 KF907327 KF907328 KF907329 KF907330 KF907331 AJ566149 KF907332 AJ566164 AJ566162 AJ566163 KF907333 AJ566154 AJ566141 AJ566161 ITS2-LSU E E E E E E E E E E E ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ 23 44 10 10 10 44 48 30 21 57 06 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ N, 127°18 N, 126°54 N, 128°20 N, 128°20 N, 128°20 N, 126°37 N, 126°27 N, 126°17 N, 126°56 N, 127°05 N, 127°52 ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ 35 10 23 23 23 02 29 33 59 36 05 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ Origin Germany Cologne, Unknown freshwater A bog near mount Überling, Austria; USA Bigelow, freshwater England; Cornwall, Trenant., Germany Münster, Freshwater Korea Goheung, Jeonnam, Daesu, Pungcheonje, 34°45 Cheongyang, Chungnam, Korea Jeongsan, 36°16 Korea Gyeongnam, Haman, Beopsu, Daesong, 35°20 Korea Gyeongnam, Haman, Beopsu, Daesong, 35°20 Korea Gyeongnam, Channgyeong, Ibang, Okcheon, 35°20 Korea Jeju, Bonggae, Mulchat-Oreum, 35°25 Chungnam, Korea Taean, Cheongsan, Sinjang, 33°21 Chungnam, Korea Buyeo, Buyeoeup, Gungnamji, 36°41 Korea Gyeongnam, Namhae, Gohyeon, Chamyeon, 36°24 Germany; freshwater Brandenburg, Schlachtensee, Chungnam, Korea Cheondong, Gwangseok, Nonsan, 36°14 freshwater Senckenberg, Forschungsinstitut Germany, Unknown Überling, Austria Cheongyang, Chungnam, Korea Bibong, Jungmuk, 34°55 GermanyDörpetalsperne, Germany Cologne, Germany Cologne, used in this study and the GenBank accession numbers for their nr 5.8S-ITS2-LSU, nr SSU, pt nr SSU, their nr 5.8S-ITS2-LSU, numbers for used in this study and the GenBank accession Cryptomonas Strain M1083 (CCAC0113) SCCAP-K0063 M0739 (CCAC0109) S Begelow CCAC0006 CCAC0080 CCAP979/62 CNUCRY15 CNUCRY19 CNUCRY22 CNUCRY42 CNUCRY44 CNUCRY48 CNUCRY64 CNUCRY90 CNUCRY121 M1484 (CCAC1484) Sojungsoryuji032611 (M0788) CCAC0018 CCAP979/67 M0741 CNUCRY146 M1079 M1086 (CCAC0176) M0850 (CCAC0107) Strains of the genus Strains C. borealis C. borealis C. commutata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. curvata C. erosa C. erosa C. erosa C. gyropyrenoidosa C. gyropyrenoidosa C. gyropyrenoidosa C. lundii C. Taxon Cryptomonas Table 2. Table

http://dx.doi.org/10.4490/algae.2013.28.4.307 312 Choi et al. Taxonomy of the Genus Cryptomonas A psb None KF907468 None KF907469 KF907470 None None None KF907471 None KF907472 None None KF907473 None None KF907474 None KF907475 KF907476 KF907477 KF907478 KF907479 None Plastid LSU None KF907424 None KF907425 KF907426 None None None KF907427 None KF907428 None None KF907429 None None None None KF907430 KF907431 KF907432 KF907433 KF907434 None GenBank accession No. GenBank SSU AM051191 KF907381 None KF907382 None None None AM051200 KF907383 None KF907384 None AM051193 KF907385 None AJ420695 KF907386 None KF907387 KF907388 KF907389 KF907390 KF907391 None Nuclear AJ566155 KF907334 AJ566156 KF907335 KF907336 AJ566170 AJ566166 AJ566167 KF907337 AJ566169 KF907338 AJ566168 AJ566153 KF907339 AJ566151 AJ566152 KF907340 AJ566158 KF907341 KF907342 KF907343 KF907344 KF907345 AJ566157 ITS2-LSU E E E E E E E E E E E E ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ 9 36 38 31 00 10 54 44 23 43 02 21 0 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ 9 0 N, 126°49 N, 128°41 N, 126°46 N, 126°43 N, 128°20 N, 129°22 N, 126°12 N, 126°54 N, 128°39 N, 128°20 N, 126°56 N, 127° ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ 2 28 30 28 54 23 47 53 50 44 46 59 0 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ Nova Scotia, Canada Nova Origin Germany Münster, Korea Gyeongnam, Haman, Beopsu, Daesong, 36°26 Unknown Korea Jeonbuk, Jeongeup, Soseong, Girin, 35°40 Korea Jeonbuk, Jeongeup, Soseong, Girin, 35°34 Unknown Land, Germany;Schommelsnaaf, Bergisches freshwater drying up lake, Unknown Korea Dongbankdongsan, Chocheon, Jeju, 33°30 Unknown Chungnam, Korea Nonsan, Noseong, 35°20 Unknown Germany Spessart, Korea Gyeongbuk, Youngdeok, Ganggu, Deokgock, 36°24 Germany Spessart, Wiesen, Austria Burgenland, Japan Hokkaido, 36°51 Park, National Highland Breton Cape Lake, of Shore Warren Chungnam, Korea Nonsan, Sangwol, Hancheon, 36°04 Korea Gyeongbuk, Pohang, Buk, Singwang, Ugak, 35°39 Korea Gyeongnam, Haman, Beopsu, Daepyeong 35°19 Cheongyang, Chungnam, Korea Jeongsan, 36°24 Korea Gyeongbuk, Cheongdo, Iseo, Gakgye, 36°16 Unknown Strain CCAC0086 CNUCRY4 M1476 Hanjeong080611A Saenae080611D ASW09006 CCAC0031 CCAP979/46 CNUCRY76 M1088 Songgock032611 UTEX2194 CCAC0064 CNUCRY231 M1097 M1171 Mukawa100608B (M1303) CCAC0056 Angol032611 Changdang11311E CNUCRY5 CNUCRY20 Gakgae043011 SAG2013 Continued C. marssonii C. marssonii C. marssonii C. obovata C. obovata C. obovoidea C. obovoidea C. obovoidea C. obovoidea C. obovoidea C. obovoidea C. obovoidea C. ovata C. ovata C. ovata C. ovata C. ovata C. paramecium C. phaseolus C. phaseolus C. phaseolus C. phaseolus C. phaseolus C. phaseolus C. Taxon Table 2. Table

313 http://e-algae.kr Algae 2013, 28(4): 307-330 A psb None None None None KF907480 KF907481 KF907482 KF907483 KF907484 KF907485 KF907486 KF907487 None KF907488 KF907489 KF907490 KF907491 KF907492 KF907493 KF907494 KF907495 KF907496 Plastid LSU None None None None KF907435 KF907436 KF907437 KF907438 KF907439 None KF907440 KF907441 None KF907442 KF907443 KF907444 KF907445 KF907446 KF907447 None KF907448 KF907449 GenBank accession No. GenBank SSU None None AJ421147 AJ421150 KF907392 KF907393 KF907394 KF907395 KF907396 KF907397 KF907398 KF907399 AM051201 KF907400 KF907401 KF907402 KF907403 None None KF907404 KF907405 KF907406 Nuclear AJ566145 AJ566143 AJ566142 AJ566140 KF907346 KF907347 KF907348 KF907349 KF907350 KF907351 KF907352 KF907353 AJ566144 KF907354 KF907355 KF907356 KF907357 KF907358 KF907359 KF907360 KF907361 KF907362 ITS2-LSU E E E E E E E E E E E E E E E E E ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ 54 47 42 44 02 35 05 05 30 15 00 30 50 33 04 07 15 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ N, 128°19 N, 126°43 N, 127°20 N, 127°02 N, 128°20 N, 126°31 N, 127°45 N, 127°45 N, 126°46 N, 126°59 N, 126°43 N, 126°46 N, 126°20 N, 126°11 N, 127°05 N, 128°24 N, 126°59 ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ ″ 58 14 86 05 46 31 30 30 55 41 54 55 18 10 33 13 41 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ Origin Germany Wahnbachtalsperre, puddle Germany; shadowy Cologne, Heide, Wahner Austria Rlirschberg, Musikantenteich, Köln, Germany Korea Jeju, Jocheoneup, Gyorae, 35°19 Korea Daejeon, Yuseong, Gungdong, Yeongtapji, 53°26 Korea Jeonbuk, Gimje, Baeksan, Surok, 36°22 Korea Jeonbuk, Andeok, Geumma, Iksan, 35°59 Korea Gyeongnam, Haman, Beopsu, Daepyeong, 35°19 Chungbuk, Korea Eumseong, Soi, Bisan, 36°24 Chungnam, Korea Buyeo, Buyeoeup, 36°50 Chungnam, Korea Buyeo, Buyeoeup, 36°50 Überling, Austria Korea Jeju, Aewoleup, Gwangnyeong, 35°33 Korea Jeju, Chocheoneup, Seonheul, 37°16 Korea Jeju, Aewoleup, Haga, Yeonhwa, 33°30 Korea Jeju, Hangyeong, Dumo2 35°33 Korea Jeonbuk, Iksan, Yeosan, Jenam, 33°27 Korea Jeonbuk, Jeongeup, Soseong, Bohwa, 33°23 Korea Gyeongbuk, Cheongdo, Hwayangeup, 36°03 Korea Gyeongnam, Changnyeong, Pallack, 35°31 Korea Gyeonggi, Suwon, Seoho, 37°16 Strain (M0923) CCAC0024 (M1096) CCAC0032 CCAP979/61 CCMP152 CNUCRY27 CNUCRY69 CNUCRY134 CNUCRY138 CNUCRY152 CNUCRY166 CNUCRY188 CNUCRY189 M1077 Sumeun041911 CNUCRY75 CNUCRY284 100310C Dumo2 Okgeum121809C Sinjeong080611A Yeonra043011B CNUCRY123 CNUCRY127 sp. sp. sp. sp. sp. sp. Continued C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. pyrenoidifera C. Cryptomonas Cryptomonas Cryptomonas Cryptomonas Cryptomonas Cryptomonas tetrapyrenoidosa C. tetrapyrenoidosa C. Taxon Table 2. Table

http://dx.doi.org/10.4490/algae.2013.28.4.307 314 Choi et al. Taxonomy of the Genus Cryptomonas A psb KF907497 KF907498 KF907499 KF907500 KF907501 KF907502 NC000926 None Plastid LSU KF907450 KF907451 KF907452 KF907453 KF907454 KF907455 NC000926 None GenBank accession No. GenBank SSU KF907407 KF907408 KF907409 KF907410 KF907411 None X57162 AF508275 Nuclear KF907363 KF907364 KF907365 KF907366 KF907367 KF907368 None None ITS2-LSU E E E E E E ″ ″ ″ ″ ″ ″ 34 27 05 25 59 33 ′ ′ ′ ′ ′ ′ N, 128°12 N, 129°39 N, 127°11 N, 126°11 N, 126°27 N, 128°36 ″ ″ ″ ″ ″ ″ 56 41 54 10 43 41 ′ ′ ′ ′ ′ ′ Origin Chungnam, Korea Nonsan, Yeonsan, Deogam, 35°05 Chungnam, Korea Taean, Wonbuk, Sindu, 34°54 Cheongyang, Chungnam, Korea Jeongsan, 36°13 Korea Goseong, Gyeongnam, Yeongo, Odong, 36°50 Korea Jeonnam, Naju, Bannam, Sinchon, 33°22 Korea Gyeongbuk, Pohang, Buk, Singwang, Ugak, 35°42 Unknown Strain Deokam032610 Du-ung022611D Hudong12610D Onsu032611C Sinchon101709C Yeongildong111310B MUCC088 Continued C. tetrapyrenoidosa C. tetrapyrenoidosa C. tetrapyrenoidosa C. tetrapyrenoidosa C. tetrapyrenoidosa C. tetrapyrenoidosa C. theta Guillardia acuta Teleauiax Taxon Outgroup New sequences are indicated in bold type. in bold type. indicated are New sequences small subunit; rDNA, ribosomal DNA. subunit; SSU, large 2; LSU, spacer transcribed internal ITS2, of Marine Phytoplankton; Culture for Center UK; CCMP, the , at Collection of Algae Culture Germany; CCAP, of Cologne, collectionthe University at of Algae Culture CCAC, labeled, Strains Melbourne Germany; Culture University MUCC, Collection Melkonian of Cologne, the University at M, Culture Korea; Lab, Woongghi’s University, collection National of the Chungnam KR, Culture of Algae, Center Denmark; UTEX, Culture and Protozoa, Algae for Center Culture Scandinavian Göttingen, Germany; der Universität SCCAP, AlgenKulturen Sammlung von Collection; SAG, TX,Austin, USA. Table 2. Table

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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 Fig. 3. Putative secondary structure of the nuclear internal transcribed spacer 2 of the Cryptomonas obovoidea Songgock032611 Cell shape and length, length of the fullow-gullet sys- (from 5’ to 3’ terminus in clockwise direction). In total a sequence of tem, color and number of chloroplasts, and the pres- 352 nucleotides were submitted to the mfold server. The presumably ence / absence and number of pyrenoids were examined. correct structure with the four helix domains was found without applying any force options. Helices were numbered in Roman The results are summarized in Table 3 and illustrated in numerals. Figs 10-12.

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

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ed in the center of each cell. Light microscopy. Cryptomonas obovoidea had ventral reflex shape in right or left lateral view and ovoid with Cryptomonas marssonii (Skuja) Hoef-Emden et broad end in ventral view (Fig. 11E & F). The cells were Melkonian 2003 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 Specimens examined. Daesong, Haman, Gyeongnam, chloroplasts, each with a pyrenoid. Contractile vacuole Korea. located in apical cell. Maupas oval absent. Two flagella Light microscopy. Cryptomonas marssonii was a sig- inserted in a vestibulum of the cell invagination. moid shape to asymmetrical in ventral view (Fig. 10E & F). The cells were 20-23 μm in length. Cells had two brown Cryptomonas phaseolus (Skuja) Hoef-Emden 2007 chloroplasts and many starch grains around chloroplasts. Each chloroplast has a pyrenoid. A contractile vacuole Specimens examined. Angolji, Nonsan, Korea; Gak- was located in the anterior of the cell. gaeji, Cheongdo, Korea; Jangdangji, Pohang, Korea; Dae- pyeong swamp, Haman, Korea; Jeongsan, Cheongyang, Cryptomonas ovata (Ehrenberg) Hoef-Emden et Korea. Melkonian 2003 Light microscopy. Cryptomonas phaseolus had bicon- vex shape in right or left lateral view and long ovoid with Specimens examined. Songcheonji, Iksan, Korea; Mu- narrow end in ventral view (Fig. 11G & H). The cells were kawa, Muroran, Hokkaido, Japan. 12-16 μm in length. The length of gullet with rows of ejec- Light microscopy. Cryptomonas ovata has long-ovoid tisomes was 2/3 that of cells. Cells had two brown chloro- of elliptical shape in ventral view (Fig. 10G & H). The cells plasts, each with a pyrenoid. The cell didn’t have maupas were 32-37 μm in length. The length of gullet with rows oval when observed in culture. Two flagella were inserted of ejectisomes was 3/5 that of cells. Cells had two brown in a vestibulum of the cell invagination. chloroplasts surrounded by many starch grains. Contrac- tile vacuole was located in apical cell and three maupas Cryptomonas pyrenoidifera (Geitler) Hoef-Emden ovals were located in the center of the cell. et Melkonian 2003

Cryptomonas obovata Czosnowski 1948 Specimens examined. Gyorae, Jeju, Korea; Yeongtapji, Daejeon, Korea; Wonseongje, Surok, Baeksan, Korea; Specimens examined. Saenaebangjuk, Jeongeup, Ko- Andeokjeosuji, Iksan, Korea; Soiji, Bisan, Eumseong, Ko- rea; Hanjeongje, Jeongeup, Korea. rea; Gungnamji, Buyeo, Korea; Sumeunmulbaengdeui, Light microscopy. Cryptomonas obovata had two Aewol, Jeju, Korea. marked cells which were characterized by presence / ab- Light microscopy. Cryptomonas pyrenoidifera was bi- sence of pyrenoids (Fig. 11A-D). The cells were 26-36 μm flattened in right or left lateral view and ovoid-elliptical in length. Cells had two brown chloroplasts, each with a shape in ventral view (Fig. 12A & B). The cells were 18-20 terminal pyrenoid. The other cell had biconvex shape in μm in length. The length of gullet with rows of ejectisomes right or left lateral view (Fig. 11C). The cells were 29-38 μm was 2/3 that of cells. Cells had two brown-chloroplasts, in length. Cells had two red-brown chloroplasts without each with an obvious pyrenoid. Contractile vacuole was pyrenoid. Both had ovoid shape in ventral view (Fig. 11B placed at above two maupas ovals. Two flagella were lo- & D). The length of gullet with rows of ejectisomes was cated in a vestibulum of the cell invagination. 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 Cryptomonas tetrapyrenoidosa (Skuja) Hoef- center of the cell. Two flagella inserted in a vestibulum of Emden et Melkonian 2003 the cell invagination. Specimens examined. Sinchonje, Naju, Korea; Du- Cryptomonas obovoidea (Pascher) Hoef-Emden ungseupji, Taean, Korea; Onsusoryuji, Goseong, Korea; 2007 Deokamji, Nonsan, Korea; Yeongildongji, Pohang, Korea; Hudongje, Cheongyang, Korea; Seohojeosuji, Suwon, Ko- Specimens examined. Songgock, Nonsan, Korea; Mok- rea. malji, Taean, Korea; Dongbackdongsan, Jeju, Korea. Light microscopy. Cryptomonas tetrapyrenoidosa was

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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 Light microscopy. Cryptomonas sp. Yeonra043011B were 16-22 μm in length. The length of gullet with rows had flattened shape in right of left lateral view and ovoid of ejectisomes was 2/3 that of cells. Cells had two brown- shape in ventral view (Fig. 12K & L). The cells were 14- chloroplasts, and each chloroplast had two or three pyre- 18 μm in length. The length of gullet with rows of ejec- noids facing each other in the left and right chloroplast tisomes was 2/3 of cell length. Cells had a green-brown lobes. Contractile vacuole was located in apical and two chloroplasts with a pyrenoid. The cell had two maupas flagella inserted in a vestibulum of the cell invagination. ovals. Two flagella inserted in a vestibulum of the cell in- Cells had two maupas ovals. vagination.

Cryptomonas sp. Okgeumji121810C DISCUSSION Specimens examined. Okgeumji, Iksan, Korea. Light microscopy. Cryptomonas sp. Okgeumji121810C Light microscopy has biconvex shape with narrow ends in right or left lat- eral view and broad ovoid shape in ventral view (Fig. 12E Although cryptomonad genera were delimited by ul- & F). The cells were 13-17 μm in length. The length of gul- trastructral features, such as periplast and flagellar ap- let with rows of ejectisomes was 1/2 or 2/3 of cell length. paratus, all Cryptomonas species have been described Cells had a brown chloroplast with a pyrenoid. Two fla- exclusively by light microscopy (Huber-Pestalozzi 1950, gella inserted in a vestibulum of the cell invagination. Butcher 1967). Pringsheim (1944, 1968) established clonal cultures, and examined morphological characters of the Cryptomonas sp. CNUCRY284 genus Cryptomonas. He recognized that morphological characters cannot be used satisfactorily for delimitation Specimens examined. Yeonhwaji, Jeju, Korea. of Cryptomonas species. Most species of the genus Cryp- Light microscopy. Cryptomonas sp. CNUCRY284 had tomonas from Korea are ovoid or sigmoid in shape, and biconvex shape with narrow end in right or left lateral cell length ranges from 11 to 38 μm. The ejectisomes were view and ovoid shape in ventral view (Fig. 12G & H). Cells arranged along a gullet located at vestibular region of the were 18-21 μm in length. Length of gullet with rows of cell. The length of gullet with rows of ejectisomes was 1/2- ejectisomes was 3/5 that of cells. Cells had a greenish- 3/4 that of cells. All cells had two chloroplasts with vari- brown chloroplast with an obvious pyrenoid. Contractile able color; reddish brown, brown, or green brown. How- vacuole above two maupas ovals. Two flagella inserted in ever, the number of pyrenoids was 2 to 6 per cell, but C. a vestibulum of the cell invagination. obovata Hanjeong080611A has chloroplasts without py- renoid. The maupas ovals were either present or absent. Cryptomonas sp. Dumo2 100310C However, distinction based on morphological characters is unclear for species of the genus Cryptomonas, as sug- Specimens examined. Dumojeosuji, Jeju, Korea. gested by Javornický (2003) and Hoef-Emden (2007). For Light microscopy. Cryptomonas sp. Dumo2 100310C example, C. obovoidea had a sigmoid cell shape, but be- had biconvex with narrow end in right or left lateral view longed to the species group with ovoid cell shape in our and ovoid shape in ventral view (Fig. 12I & J). The cells tree. Therefore, species with ovoid cell shape were not were 14-18 μm in length. The length of gullet with rows monophyletic. Other morphological characters, such as of ejectisomes was 1/2 or 3/4 of cell length. Cells had a the presence / absence of pyrenoid, were found across brown chloroplasts with a pyrenoid. Contractile vacuole clades. Saenae080611A strain of C. obovata clade has py- located in apical cell. Cells didn’t have maupas oval when renoid, but Hanjeong080611A strain lacked pyrenoid, in we established it in culture. Two flagella located in a ves- spite of being located in the same species clade. Incon- tibulum of the cell invagination. gruence between morphospecies concept and molecular phylogeny has been reported recently (Hoef-Emden and Cryptomonas sp. Yeonra043011B Melkonian 2003, Hoef-Emden 2007). Therefore, due to lack of clear species-specific morphological characters, Specimens examined. Yeonra, Hwayangeup, Cheon- new species may have to be defined by molecular signa- gdo, Gyeongbuk, Korea. tures, as suggested by Hoef-Emden (2007).

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Molecular phylogenetic analyses (Schultz et al. 2005, Hoef-Emden 2007). The helix І, ІІ, and ІІІb were less conserved regions than In the molecular phylogenetic tree based on nuclear helix ІІІa (the distal part of helix ІІІ). The proximal part of 18S rDNA, the genus Cryptomonas is known as mono- helix І and ІІ had similar sequences among species, but phyletic (Deane et al. 2002). Although nuclear 18S rDNA were variable in their terminal parts. Specific structures data has been used for resolving phylogenetic relation- of helix ІІІb appeared in all strains, despite the differences ships among genera of Cryptophyceae (Marin et al. 1998, among sequences. Sequences of helix І, ІІ, and ІІІb in ITS2 Deane et al. 2002), the tree did not show obvious resolu- secondary structure were very useful for the identifica- tion within the genus Cryptomonas. The phylogenetic tion of Cryptomonas species. The helix ІV was varied from relationships among Cryptomonas species were likely to species to species because of sequence diversity. Through involve fast-evolving genes which contain more phyloge- deduction of the secondary structure, molecular signa- netic information (Deane et al. 2002). Our phylogenetic tures to identify lower rank of genus or species could be tree based on combined nuclear SSU, partial LSU, and determined. 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- ACKNOWLEDGEMENTS logenetic relationships among Cryptomonas species, but there remain areas of uncertainty. For example, the posi- This research was the survey of Indigenous Biological tions of some species (C. ovata, C. obovata, C. phaseolus, Resources of Korea from National Institute of Biological C. gyropyrenoidosa, C. paramecium, C. borealis, and C. Resources and supported by the National Research Foun- lundii) were not clearly resolved in the tree. The topolo- dation Program funded by the Korea Government/MEST gies of our trees obtained by ML and Bayesian analyses (NRF-C1ABA001-2010-0020700). 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 REFERENCES LSU and SSU rDNA data (Hoef-Emden et al. 2002, Hoef-Emden 2007). Especially our tree is better Bourelly, P. 1970. Les algues d’eau douce: initiation à la sys- supported in internal nodes than that of the previous one tematique. Tome III: Les algues blues et rouges. Les (Hoef-Emden 2007). Eugléniens, Peridinienset Cryptomonadines. Editions N. Boubée & Cie, Paris, 512 pp. Nuclear ITS2 secondary structures Brett, S. J. & Wetherbee, R. 1986. A comparative study of peri- plast structure in Cryptomonas cryophila and C. ovata The nuclear ITS2 in Cryptomonas species showed the (Cryptophyceae). Protoplasma 131:23-31. typical conserved secondary structure with four helices Butcher, R. W. 1967. An introductory account of the smaller which are found in most lineages (Schultz et algae of British coastal waters. Part IV: Cryptophyceae. al. 2005). Molecular characters were derived from the Fishery Investigations, Series IV. Ministry of Agriculture, nuclear ITS2; 1) the highly conserved regions were eas- Fisheries and Food, Her Majesty’s Stationary Office, ily aligned among Cryptomonas species, 2) the less con- London, 54 pp. served regions provided enough signal to identify syn- Clay, B. L., Kugrens, P. & Lee, R. E. 1999. A revised classifica- apomorphies or species-specific unique combinations tion of the Cryptophyta. Bot. J. Linn. Soc. 131:131-151. of sequences for clades (species), and 3) the highly vari- Deane, J. A., Strachan, I. M., Saunders, G. W., Hill, D. R. A. & able regions were aligned within but not between species McFadden, G. I. 2002. Cryptomonad evolution: nuclear (Hoef-Emden and Melkonian 2003). Most conserved re- 18S rDNA phylogeny versus cell morphology and pig- gions in all strains were located in the distal part of helix mentation. J. Phycol. 38:1236-1244. ІІІ. The sequences of the distal parts were not very use- Dujardin, F. 1841. Histoire naturelle des Zoophytes. Infusoires, ful for species identification, but seem to be available to comprenant la physiologie et la classification de ces Ani- distinguish cryptomonad genera as molecular signatures. maux, et la maniere de les etudier a l’aide du microscope. Compare to other eukaryotic ITS2 secondary structures, Librairie Encyclopedique de Roret, Paris, 295 pp. the UGGU motifs have shifted from the 5′ part to 3′ part Ehrenberg, C. G. 1831. Symbolae physicae seu icones et de- of the helix ІІІ in previous studies, as well as in this study scriptiones animalium evertebratorum sepositis insectis

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