Taxonomic Re-Examination of Chlamydomonas Strains Maintained in the NIES-Collection

Microbiol. Cult. Coll. 2（1）：19 ─ 12, 2013 Taxonomic re-examination of Chlamydomonas strains maintained in the NIES-Collection

Kosei Yumoto, Fumie Kasai and Masanobu Kawachi ＊

Microbial Culture Collection, Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, 16-2, Onogawa, Tsukuba, Ibaraki 305-8506, Japan

INTRODUCTION ments, and they also occur in extreme environments The genus Chlamydomonas (Volvocales, such as acidic ponds and snow surfaces. Some Chlorophyceae) comprises a large number of species Chlamydomonas strains are also used as models in (more than 600), and molecular phylogenetic analy- basic research in many fields, such as genomics, ses using 18S rRNA and chloroplast genes have genetics, physiology, and developmental biology shown it to be polyphyletic (Buchheim et al., 1997; (Harris, 2009). The NIES-Collection holds more than Hoham et al., 2002; Nozaki et al., 1998; Pröschold et 80 Chlamydomonas strains, most of which were al., 2001). In previous studies, the principal clades transferred from the University of Tokyo’s IAM within the genus were clearly, and for the most part Collection in 2007, when the collection was closed, consistently, distinguished, although the relation- and some were deposited as Chlamydomonas spe- ships among the clades have not always been well cies without species level identification. No molecu- resolved (Buchheim et al., 1997; Nakada et al., 2008; lar phylogenetic data are available for most of the Pröschold et al., 2001). On the basis of these lineages, Chlamydomonas strains in the NIES-Collection, Pröschold et al. (2001) performed several taxonomic although the 18S rDNA sequences of approximately revisions and proposed two new genera, but the 10 strains have been published (Nakada et al., 2010; completion of the taxonomic revision of the genus is Nakada & Tomita, 2011). Therefore, to better char- expected to take considerable time. Under such cir- acterize the Chlamydomonas strains in the NIES- cumstances, Nakada et al. (2008) performed compre- Collection taxonomically, we sequenced their 18S hensive molecular analyses of Volvocales, including rDNA, determined their phylogenetic positions, and Chlamydomonas species, based on the 18S rRNA assigned them to the subgroups defined by Nakada gene sequence, and adopted PhyloCode et al. (2008). Also of note, 18S rDNA sequences have (International Code of Phylogenetic Nomenclature, been published for 30 strains that originated from Cantino & de Queiroz, 2010) to explicitly define indi- the same strains as the NIES strains and have been vidual clades. In the PhyloCode system, names maintained in other culture collections. Therefore, applied to clades are defined in terms of phylogenet- we investigated the strain histories and the pub- ic relationships (Cantino & de Queiroz, 2010). lished sequence data to confirm the taxonomic iden- Accordingly, we consider the subgroups that were tity of these strains and to exclude the strains that phylogenetically defined by Nakada et al. (2008) by may have been replaced by other strains during following the PhyloCode to be the best way for indi- long-term subculturing. cating the appropriate taxonomic positions of individual Chlamydomonas strains maintained in the MATERIALS AND METHODS Microbial Culture Collection at the National Institute We determined the taxonomic positions of 79 for Environmental Studies, Tsukuba, Japan (NIES- Chlamydomonas strains maintained at the NIES- Collection). Collection by sequencing their 18S rDNA. Algal cul- Various Chlamydomonas species are familiar tures were incubated in test tubes, each containing organisms that contribute to the algal biodiversity in 10 ml of medium [AF-6, or C medium for freshwater soil and water (both freshwater and marine) environ- strains, and f/2, ESM, or STP medium for marine strains (Kasai et al., 2009)], at 20℃ under an irradi- *Corresponding author ance of 8–32 mmol photons m–2 s–1 provided by day- Email: [email protected] light fluorescent lamps in a 12:12-h light:dark cycle.

─ 1 ─ Taxonomic re-examination of NIES Chlamydomonas strains Yumoto et al.

0.02 substitutions/site 0.02 substitutions/site ML NJ 99

100 Moewusinia Moewusinia

Radicarteria Arenicolinia Radicarteria Arenicolinia Stephanosphaerinia 74 66 100 79 81 99 <50 63 Stephanosphaerinia Chlorogonia 54 Chlorogonia 68 Characiosiphonia Characiosiphonia Dunaliellinia 63 Dunaliellinia Polytominia 98 Polytominia 96 99 100 Monadinia Monadinia Phacotinia Phacotinia Crucicarteria 99 Crucicarteria 62 100 ** * ** * 94 Chloromonadinia 96 Chloromonadinia

90 Oogamochlamydinia Oogamochlamydinia 98

Tatrensinia Hafniomonas

Reinhardtinia Reinhardtinia

Tatrensinia 98 97

100 Desmotetra 99 Desmotetra Hafniomonas Treubarinia Treubarinia Golenkinia Golenkinia

Fig. 1 Overview of the ML and NJ trees based on the aligned 1558 positions of 181 sequences of 18S rDNA. The tree is drawn to scale, with branch lengths measured for the number of substitutions per site. Golenkinia is arbitrarily placed at the base of the tree. Bootstrap values of more than 50% in the ML and NJ analyses are shown on each branch except for the inside of major clades, which are shown in Figs. 2 and 3.

Total DNA was extracted from the cells of 79 sequence, using the primers reported by Nakayama strains of Chlamydomonas by using a DNeasy Plant et al. (1998), in a CEQ8000 DNA sequencer (Beckman Mini Kit (Qiagen) according to the manufacturer’s Coulter). The 18S rDNA sequences of protocol. The 18S rDNA was amplified by PCR Chlamydomonas reported here have been submitted using the originally designed primers: 63F to the DDBJ with the accession numbers shown in (5'-ACGCTTGTCTCAAAGATTA-3') and 1818R Table 1. (5'-ACGGAAACCTTGTTACGA-3'). PCR conditions Additional 102 sequences (Figs. 2 & 3) were select- were as follows: 94℃ for 10 min; 30 cycles of 94℃ ed using the NCBI BLAST-bl2seq program (http:// for 1 min, 55℃ for 45 s, and 72℃ for 30 s; and a final www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) as extension at 72℃ for 5 min. All PCR amplifications well as from phylogenetic trees published by were carried out in a PCR thermal cycler (TaKaRa Nakada et al. (2008). In total, 181 sequences were PERSONAL, TaKaRa) in a total volume of 15 ml, con- aligned using ClustalX (Thompson et al., 1997) taining 0.2 mM of each dNTP, 0.5 mM of each primer obtained from the DDBJ, and manually refined on pair, 10× PCR buffer, 0.25 U Ex Taq DNA poly- MEGA5 software (Tamura et al., 2011), by referring merase (TaKaRa), and 0.5–5 ng of template DNA. to an alignment applied in Nakada et al. (2008). A PCR products were purified by using a QIAquik total of 1558 positions were used for the analyses PCR Purification Kit (Qiagen) according to the man- that followed. ufacturer’s protocol. The PCR products (1576–1680 We also used MEGA5 software to perform maxi- base pairs of 18S rDNA) were then used to mum likelihood (ML) and neighbor-joining (NJ) anal-

─ 2 ─ Microbiol. Cult. Coll. June 2013 Vol. 29, No. 1

Chlamydomonad sp. Pic6/3T-3w AY220588 Chlamydomonas nasuta NIES-2225 0.02 92 72 Chlorococcum hypnosporum PS-2678 AB488561 100 Cd leiostraca NIES-2217 A Chlamydomonas sp. CCAP 11/57 FR865595 58 63 Cc hypnosporum UTEX119 U41173 Cd moewusii var. rotunda NIES-2222 . rotunda clade

65 Cd moewusii var. rotunda NIES-2223 B var Cd moewusii var. rotunda NIES-2577 100 Cd moewusii CCAP11/64B FR865602 C 94 Cd moewusii var. rotunda NIES-2578 Cd moewusii SAG11-61a JN903982 B C. moewusii 100 Cd moewusii NIES-2220 D Cd moewusii CCAP11/16F FR865565 D Cd moewusii var. rotunda NIES-2221 C Cd applanata NIES-2205 E Cd moewusii CCAP11/16G FR865566 G clade Cd simplex NIES-2241 F 100 Cd simplex CCAP11/26 FR865572 F Cd moewusii NIES-2576 Cd moewusii NIES-2219 G Cd moewusii SAG 11-11 U70786 C. moewusii Cd moewusii CGC CC-1419 U41174 Cd noctigama NIES-1048 Cd noctigama NIES-2229 H Cd noctigama CCAP11/17 FR865568 H Cd noctigama UTEX1339 AF008241 I Cd noctigama NIES-2228 I 96 Cd dorsoventralis CCAP11/4 FR865582 J Cd dorsoventralis SAG36.72 JN903979 100 Cd noctigama NIES-2579 Cd dorsoventralis NIES-2213 J Cd noctigama SAG6.73 AF008238 Cd noctigama SAG33.72 U70782 H Moewusinia 68 Cd parkeae NIES-1733 100 Cd parkeae NIES-1022 93 Chlamydomonas sp. NIES-2324 Chlamydomonas sp. NIES-2323 Chlamydomonas sp.MBIC10473 AB058351 100 Cd parkeae MBIC 10599 AB05837 Cd parkeae SAG24.89 DQ009747 100 Cd parkeae NIES-440 100 Cd eustigma NIES-2499 Cd acidophila CCAP 11/133 AJ783841 Carteria radiosa NIES-432 D86500 Radicarteria 66 Chlorosarcinopsis arenicola UTEX1697 AB218701 Arenicolinia Stephanosphaera sp. UTEX2409 U70798 Cd debaryana CCAP11/1 FR865523 K Stephanosphaerinia 100 100 Cd debaryana NIES-2211 K 88 Chlorogonium elongatum NIES-1357 (=SkCl-2) AB278624 74 67 Cg elongatum IAM C-293 AB278621 90 Cg euchlorum SAG12-2a AJ410441 74 Cg capillatum NIES-692 AB278611 Cd perpusilla var. perpusilla SkCr-10 (=NIES-1849) AB290339 L Cd perpusilla var. perpusilla NIES-1849 L Chlorogonia 100 Cd perpusilla var. perpusilla NIES-1848 Haematococcus lacustris UTEX16 DQ009774 100 Brachiomonas sp. MBIC 10757 AB183637 Chlamydomonas sp. NIES-2322 Characiosiphon rivularis UTEX LB 1763 AF395437 Characiosiphonia Dunaliella salina EF195157 Dunaliellinia 68 Cd applanata CCAP11/9 FR865616 M Cd applanata NIES-2206 M 66 Cd applanata CCAP11/2 FR865570 authentic N 88 Cd applanata NIES-2204 N Cd applanata UTEX2399 U13985 E Cd applanata NIES-2202 Polytominia 96 Cd leiostraca CCAP11/49 FR865590 A Polytoma ellipticum SAG62-18 U22933 99 Po uvella SAG62-2M U22942 98 81 Cd monadina SAG31.72 U57694 68 Cd monadina SAG8.87 AY220559 73 Cd monadina var. monadina NIES-438 Chlamydomonas sp. NIES-2318 Cd kuwadae NIES-968 O Monadinia Cd kuwadae NIES-968 AB451190 O 100 100 Cd coccoides NIES-1021 Chlamydomonas sp. NIES-2315 Chlamydomonas sp. NIES-2314 62 Phacotus lenticularis 97801-21 AY009896 Phacotinia Carteria crucifera NIES-421 D86501 Crucicarteria Cd transita NIES-2245 * Cd pseudococcum NIES-2232 100 * Chlamydomonas sp. PS-2677 * Fig. 2 Details of the ML tree obtained based on 18S rDNA sequences. Moewusinia, Stephanosphaerinia, Chlorogonia, Polytominia, and Monadinia are shown with representative strains of Radicarteria, Arenicolinia, Characiosiphonia, Dunaliellinia, Phacotinia, and Crucicarteria. Bootstrap values of more than 50% are shown on each branch. Sequences analyzed in the present study are indicated in filled circles. The same alphabetical characters (A–O) indicate strains of the same origin.

─ 3 ─ Taxonomic re-examination of NIES Chlamydomonas strains Yumoto et al.

Cd applanata NIES-2203 0.02 95 Cd augustae var. ellipsoidea NIES-158 94 100 Chloromonas augustae SAG 9.87 AJ410453 Cm augustae SAG5.73 AJ410452 100 Cd chlorococcoides NIES-2575 P Cm carrizoensis (Cd chlorococcoides) SAG46.72 AJ410446 P 97 Cd actinochloris NIES-2201 Q 100 Cd actinochloris SAG 1.72 AJ410445 Q 115Cd mutabilis UTEX 578 U57695 R 73 Cm actinochloris SAG34.72 JN904007 R Cd mutabilis NIES-2224 R Cm rosae UTEX1337 U70796 Cm clathrata UTEX LB 1970 U70791 Chloromonadinia 100 Cd subangulosa NIES-2243 Cd gerloffii NIES-2215 S 70 Cd subtilis NIES-2244 87 Cd gerloffii CCAP11/72 FR865610 S 100 Cd ulvaensis NIES-2247 100 Cd inflexa CCAP11/42 FR865584 Chlamydomonas sp. CCAP11/158 FR865552 Cd pluvinata NIES-2233 57 Lobochlamys culleus SAG 18.72 AJ410462 T 94 Cd culleus NIES-2209 T 92 Cd culleus NIES-2210 Lb culleus SAG17.73 AJ410461 Oogamochlamydinia 100 Cd fimbriata NIES-2214 U 85 Lb segnis SAG 17.72 AJ410464 U Lb segnis SAG 1.79 AJ410457 80 90 74 Cd segnis NIES-2240 V 91 Lb segnis CCAP11/13 JN982286 V 100 Lb segnis SAG 52.72 AJ410456 Oogamochlamys gigantea SAG 21.72 AJ410468 100 Og gigantea SAG 44.91 AJ410465 Og gigantea UTEX 1753 AJ410467 Og gigantea SAG 9.84 AJ410466 Chlamydomonas sp. NIES-2320 100 Chlamydomonas sp. NIES-2321 54 Chlamydomonas sp. NIES-2319 Og zimbabwiensis UTEX 2213 AJ410471 100 Chlamydomonadaceae sp. Tow8/18T-9w AY220578 Chlamydomonas sp. NIES-2317 87 Chlamydomonas sp. NIES-2316 Og ettlii UTEX 2218 AJ410469 Cd reinhardtii UTEX 1061 M32703 (J02949) Cd reinhardtii NIES-2239 Cd reinhardtii CCAP 11/32CW15 FR865576 W Cd reinhardtii NIES-2237 W Cd reinhardtii NIES-2235 (=IAM C9) 93 Cd reinhardtii PS-2708 Cd reinhardtii NIES-2238 X Cd reinhardtii SAG 11-32a AB511835 X Cd reinhardtii NIES-2463 Y Cd reinhardtii KkS0801B1 AB511836 Y 56 Cd reinhardtii UTEX 90 AB511834 Z Cd reinhardtii NIES-2236 Z Cd globosa TkS0803C2 AB511838 a 88 Cd globosa SAG 81.72 AY781663 Cd incerta SAG 7.73 AY781664 Reinhardtinia 50 Cd globosa NIES-2462 a Volvox carteri f. nagariensis UTEX 1885 X53904 99 Cd orbicularis SAG 11-19 AB511839 b Cd orbicularis NIES-2230 b Cd zebra SAG10.83 U70792 100 Cd zebra NIES-2248 Cd sphaeroides NIES-2242 100 Cd debaryana var. cristata NIES-884 d Cd debaryana NIES-2212 Cd debaryana CCAP 11/70 FR865608 98 Cd debaryana SAG 26.72 AF008240 Cd debaryana SAG 15.72 AB542922 d Chlamydomonad sp. Pic 8/18 P-1w AY220086 Cd fasciata NIES-437 99 Cm oogama SAG 9.79 U70793 Cd inflexa NIES-2216 79 Cd rapa NIES-2234 100 Cd rapa SAG 48.72 U70790 Fasciculochloris boldii UTEX 1451 AB244240 Cd asymmetrica SAG 70.72 U70788 Cd asymmetrica CCAP11/41 FR865583 100 Cd asymmetrica NIES-2207 Cd asymmetrica NIES-2208 e 93 60 60 Cd asymmetrica CCAP11/7 FR865607 e Cd asymmetrica CCAP11/7 JN903983 e Cd mexicana CCAP11/55A FR865592 97 Cd mexicana SAG 11-60a AF395434 98 65 Cd mexicana NIES-2218 100 Hormotila blennista UTEX 1239 U83123 Heterotetracystis akinetos UTEX 1675 AB244242 Cd proteus NIES-2231 Eimeriidae environmental sample Amb_18S_473 EF023182 Cd typica NIES-2246 Desmotetra stigmatica UTEX 962 AB218711(DQ009760) Desmotetra Chlorococcum cf. tatrense CCCryo 101-99 AF514407 Tatrensinia Fig. 3 Details of the ML tree obtained based on 18S rDNA sequences. Chloromonadinia, Oogamochlamydinia, and Reinhardtinia are shown with representative strains of Desmotetra, and Tatrensinia. Bootstrap values of more than 50% are shown on each branch. Sequences analyzed in the present study are indicated in filled circles. The same alphabetical characters (P–e) indicate strains of the same origin.

─ 4 ─ Microbiol. Cult. Coll. June 2013 Vol. 29, No. 1

yses. An ML tree was constructed by applying a Polytominia, 6 to Monadinia, 8 to Chloromonadinia, ‘General Time Reversible’ model for nucleotide sub- 11 to Oogamochlamydinia, and 21 to Reinhardtinia stitution with gamma-distributed among-site rate (Table 1). variation and invariant sites (GTR+G+I). An NJ tree We were unable to re-identify three strains, was constructed by applying a ‘Kimura-2- Chlamydomonas transita NIES-2245, Cd. pseudococ- parameter’ model. Bootstrap analyses were per- cum NIES-2232, and Chlamydomonas sp. PS-2677 formed to assess the robustness of the trees (100 (indicated by asterisks in Figs. 1 & 2), based on our replicates for ML analysis, 1000 replicates for NJ results, because they were not included in any of analysis). the subgroups defined by Nakada et al. (2008). To measure sequence similarity among the NIES However, we found several sequences that were strains and strains of the same origin (but held in most similar to (not the same as) the sequences of other culture collections), we used“Identities” the above-mentioned three NIES strains by the obtained by aligning two (or more) sequences apply- Nucleotide BLAST search, and that these most simi- ing the NCBI BLAST-bl2seq program. These values lar sequences belonged to Sphaeropleales, not to show how many positions are identical among the Volvocales. For example, the sequence of Cd. transi- query sequence and the portion of the subject ta NIES-2245 was most similar to six sequences sequence(s) when two (or more) sequences are including the sequence of Asterarcys quadricellulare aligned. Aligned positions were 1584–1666 that cov- strain KNUA020 (JQ043183) [Identity=99.2% ered 99–100% of the query sequences (i.e., sequences (Identical position=1637/total position=1650)]. It was of the NIES strains).“Identities” were also used to also similar to the sequence of Scenedesmus vacuola- infer the taxonomic identity of three NIES strains tus SAG 211-8b (X56104) [I=99.2% (1636/1650)]. The that were not included in the subgroups defined by sequence of Cd. pseudococcum NIES-2232 was the Nakada et al. (2008). most similar to four sequences including the sequence of Scenedesmaceae sp. Tow 9/21 P-1w RESULTS AND DISCUSSION (AY197641) [I=99.6% (1643/1650)]. It was also similar P h y l o g e n e t i c p o s i t i o n a n d s u b g r o u p s o f to the sequence of Scenedesmus obliquus SAG 276-3a Chlamydomonas strains (X56103) [I=99.5% (1641/1650)]. The sequence of In both the ML and NJ analyses, each major clade, Chlamydomonas sp. PS-2677 was most similar to including representative species of the clade as that of Scenedesmus armatus var. subalternans defined by Nakada et al. (2008), was reproduced CCAP 276/4A (FR865727) [I=99.7% (1572/1576)]. On with high bootstrap support (Fig. 1, 90–100% in the the other hand, the sequence of Cd. transita NIES- ML tree; 96–99% in the NJ tree), except for the clade 2245, which positioned most basally within a clade including Chlorogonium euchlorum (i.e., the sub- the three species constituted, was less similar to the group Chlorogonia, <50% and 63% bootstrap values sequence of Carteria crucifera NIES-42 [I=95.0% in the ML and NJ trees, respectively). We assigned (1572/1654)] compared to the sequences of the strains included in individual clades to the corre- Sphaeropleales. Therefore, we strongly suspect that sponding subgroups defined by Nakada et al. (2008) these three NIES strains have been replaced or mis- (Table 1), based on the results of our phylogenetic identified, and will not submit the DNA sequences of analyses (Figs. 2 & 3) as well as on their relation- these strains until detailed morphological and com- ships to the species analyzed by Nakada et al. (2008). parative studies have been performed. Although the ML and NJ topologies differed from The sequences of 49 strains, among the 79 NIES one another (Fig. 1), major clades were similarly sup- strains analyzed in the present study, were newly ported with high bootstrap values in the ML and NJ determined (Table 1). These strains seemed to analyses. Therefore, we showed only the ML tree include interesting ones, which created new inde- (Figs. 2 & 3), while the bootstrap values in the NJ pendent clades without any closely related strains analysis were shown only in the text, if necessary. that have been registered. We therefore speculate All NIES Chlamydomonas strains, except for that these strains may belong to new taxa; i.e., four three (see below), were assigned to eight subgroups: strains, NIES-1022, 1733, 2323 and 2324, which 23 strains belonged to Moewusinia, 1 to belonged to Moewusinia (among these four strains, Stephanosphaerinia, 3 to Chlorogonia, 3 to two strains had been identified as Cd. parkeae, but

─ 5 ─ Taxonomic re-examination of NIES Chlamydomonas strains Yumoto et al.

Table 1 List of 79 NIES strains, which were used to 18S rRNA gene sequencing Subgroup Strain no. Species (revised species are underlined) History (depositor, deposit year) Accession names < Original designation Strain number in other culture collections, no. defined by boldface indicating strains DNA sequences of Nakada et al which have been published, with sequence (2008) identities1, if appropriate, in parentheses Moewusinia NIES-2499 Chlamydomonas eustigma Ettl <(Higuchi, 2009) AB701493 NIES-2217 Chlamydomonas leiostraca (Strehlow) Ettl

Table 1 Continued Subgroup Strain no. Species (revised species are underlined) History (depositor, deposit year) Accession names < Original designation Strain number in other culture collections, no. defined by boldface indicating strains DNA sequences of Nakada et al which have been published, with sequence (2008) identities1, if appropriate, in parentheses NIES-2221 Withdrawal

─ 7 ─ Taxonomic re-examination of NIES Chlamydomonas strains Yumoto et al.

─ 8 ─ Microbiol. Cult. Coll. June 2013 Vol. 29, No. 1

─ 9 ─ Taxonomic re-examination of NIES Chlamydomonas strains Yumoto et al.

reidentified to Chlamydomonas sp. in the present may occur through independent DNA substitutions study as mentioned below in the‘Revisions’ sec- in different subcultures, which were held in different tion), and three strains of Chlamydomonas sp., NIES- culture collections for a long time. Two examples of 2319, 2320 and 2321, which belonged to sequence comparisons among the three strains of Oogamochlamys. The identification of these strains the same origin may support this assumption. The may contribute to further understand sequence of Chlamydomonas noctigama NIES-2229 Chlamydomonas classification. differed in one and a total of 12 distinct positions from CCAP 11/17 and SAG 33.72, respectively, both Comparison of DNA sequences of the NIES of which originated from the same strain as NIES- strains and strains of the same origin 2229 (Fig. 2, H). Chlamydomonas mutabilis NIES- 18S rDNA sequences have been previously pub- 2224 differed in one and a total of 22 distinct posi- lished for 30 strains that originated from the same tions from SAG 34.72 (as Cd. actinochloris) and strains as the NIES strains, which were analyzed in UTEX 578, respectively, both of which originated the present study and are maintained in a depositor from the same strain as NIES-2224 (Fig. 3, R). or other culture collections (Table 1, strains shown Although these different positions were not always in boldface). Using“Identities,” we compared simi- included in the phylogenetic analyses because they larities between these published sequences and were sometimes outside of the region used for the sequences of the corresponding NIES strains to con- analyses, these strains were included in their respec- firm the taxonomic identity. Among 33 sequences tive, strongly supported clades. registered for the 30 strains, two sequences were On the other hand, a comparison of sequences 100% identical to the corresponding sequences of from NIES-2221 and CCAP 11/64B showed differ- the NIES strains, 24 sequences differed in 1–4 posi- ences in 91 positions and both strains originated tions (Identities=99.9–99.8%), and 4 sequences dif- from the same strain Tsubo 24 (-). In addition, NIES- fered in 8–22 positions (Identities=99.5–99.3%). On 2221 and CCAP 11/64B belonged to markedly differ- the other hand, three sequences differed in more ent clades within Moewusinia (Fig. 2, C). NIES-2221 than 90 positions (Identities=94.5–92.0%). did not belong to the originally identified Cd. We assume that the differences were produced as moewusii var. rotunda clade, but to the Cd. a consequence of different sequencers used and/or a moewusii clade. In addition, sexually complementary degree of accuracy in analyses when differences strains of NIES-2221 and CCAP 11/64B, which are were small such as in less than 4 positions. In the NIES-2223 and SAG 11-61a respectively, and both case with differences in 8–22 positions, in addition to originated from the same strain Tsubo 24 (+), belong the aforementioned reasons, sequence incongruity to the Cd. moewusii var. rotunda clade (Fig. 2, B).

─ 10 ─ Microbiol. Cult. Coll. June 2013 Vol. 29, No. 1

Therefore, we concluded that NIES-2221 had been inal isolation can probably be excluded. Therefore, replaced by another strain after it was transferred we retained these two strains in our collection list to Japan. The data also indicate that organisms iden- with a revision of the species names. Two strains of tified as Cd. moewusii var. rotunda are clearly dis- Cd. parkeae, NIES-1022 and 1733, were downgraded tinguished from those identified as Cd. moewusii to Chlamydomonas sp. (Table 1), because the phylo- (Fig. 2) by phylogenetic analysis of 18S rDNA, genetic analysis results showed that, along with two although the species name Cd. moewusii has some- other strains, NIES-2323 and 2324, they constituted a times been used for both organisms. distinctly different subclade from Cd. parkeae (100% Larger sequence differences were observed in the bootstrap value in the ML tree, Fig. 2, and 99% in following cases: 132 positions between the NJ tree). Chlamydomonas applanata NIES-2205 and UTEX Stephanosphaerinia: Chlamydomonas debaryana 2399 (Fig. 2, E), and 128 positions between NIES-2211 was revised to Chlamydomonas sp. The Chlamydomonas leiostraca NIES-2217 and CCAP species Cd. debaryana are included in the subgroup 11/49 (Fig. 2, A). Each strain belonged to a different Reinhardtinia (Nakada et al., 2008; cf.‘Reinhardtii- subgroup defined by Nakada et al. (2008): Cd. appla- clade’ in Pröschold et al., 2001); however, this strain nata NIES-2205 in Moewusinia whereas UTEX 2399 was not classified as such. In addition, the published in Polytominia (Fig. 2, E); Cd. leiostraca NIES-2217 in sequence of CCAP 11/1, which originated from the Moewusinia whereas CCAP 11/49 in Polytominia same strain as NIES-2211, is highly similar to the (Fig. 2, A). Since Cd. applanata was not identified to NIES-2211 sequence (Fig. 2, K, Table 1). Therefore, belong to the subgroup Polytominia, as its authentic we concluded the possibility of errors, such as misla- strain CCAP 11/2, which belongs to Polytominia, we beling, since its original isolation could probably be concluded that NIES-2205 had been replaced by excluded. The strain was added to our collection list another strain after it was transferred to Japan. with revision, although a specific name for the strain As a result, we withdrew two strains, Cd. appla- could not be determined. nata NIES-2205 and Cd. moewusii var. rotunda Monadinia: Chlamydomonas coccoides NIES-1021 NIES-2221 from our collection list. Cd. leiostraca was revised to Cd. kuwadae because this strain was NIES-2217 could not be reidentified due to the shown to be included in a subclade that consisted of unavailability of the authentic strain of the species Cd. kuwadae with high bootstrap support (100% in in culture collections. Detailed phylogenetic, morpho- the ML tree, Fig. 2, and 99% in the NJ tree) based logical, and comparative studies are required. on the results of phylogenetic analyses. Oogamochlamydinia: Recently, Pröschold et al. Revisions (2001) established two new genera, Oogamochlamys Among the strains that belong to the eight sub- and Lobochlamys, in this subgroup and redefined groups, 15 strains were revised (depicted as under- the species included in these genera. In this revision, lined words in Table 1) as follows: two strains, Lobochlamys segnis SAG 17.72 and Lb. Moewusinia: Two strains, Chlamydomonas sim- culleus SAG 18.72, that originated from the same plex NIES-2241 and Cd. dorsoventalis NIES-2213 strains as NIES-2214 and 2209, respectively, were were revised to Cd. moewusii and Cd. noctigama, included. According to this revision (Pröschold et al. respectively (Table 1), based on the phylogenetic 2001) and based on the phylogenetic analyses per- analysis results that classified them in the subclades formed in the present study, the four NIES strains Cd. moewusii and Cd. noctigama, respectively, with were revised, as follows (Table 1): Both Cd. segnis a high bootstrap support (100% and 96%, respective- NIES-2240 (Fig. 3, V) and Cd. fimbriata NIES-2214 ly, in the ML tree, Fig. 2; 99% for both in the NJ (Fig. 3, U) were revised to Lb. segnis, and two tree). In addition, published sequences of CCAP strains of Cd. culleus, NIES-2209 (Fig. 3, T) and 2210, 11/26 and CCAP 11/4 that originated from the same were revised to Lb. culleus. strains as NIES-2241 and 2213, respectively, also Chloromonadinia: Pröschold et al. (2001) emend- showed that these strains belonged to Cd. moewusii ed the genus Chloromonas and revised several spe- and Cd. noctigama, respectively (Fig. 2, F & J). cies (including subspecies) within the genus. In this These findings indicate the possibility of errors, revision, Chlamydomonas augustae, and two variet- such as mislabeling, during the time since their orig- ies of the species, Cd. augustae var. eupapillata (the

─ 11 ─ Taxonomic re-examination of NIES Chlamydomonas strains Yumoto et al. authentic strain, SAG 5.73) and Cd. augustae var. Kasai, F., Kawachi, M., Erata, M., Mori, F., Yumoto, ellipsoidea (the authentic strain, SAG 9.87) were K., Sato, M. & Ishimoto, M. (eds.) 2009. NIES- revised together to Chloromonas augustae; both Cd. Collection List of Strains, 8th Edition. Jpn. J. actinochloris (represented by SAG 1.72) and Cd. Phycol.(Sôrui) 57 (Suppl.): 1-350. mutabilis (represented by SAG 34.72) were emended Nakada, T., & Nozaki, H. 2009. Taxonomic study of to Chloromonas actinochloris; Cd. chlorococcoides two new genera of fusiform green flagellates, (represented by SAG 46.72) was revised to Tabris gen. nov. and Hamakko gen. nov. Chloromonas carrinzoensis. According to these revi- (Volvocales, Chlorophyceae). J. Phycol. 45: 482-492. sions, and based on our phylogenetic analysis, four Nakada, T., Misawa, K. & Nozaki, H. 2008. Molecular NIES strains included in this subgroup were revised systematics of Volvocales (Chlorophyceae, Chloro (Table 1): Cd. augustae var. ellipsoidea NIES-158 was phyta) based on exhaustive 18S rRNA phylogenet- revised to Cm. augustae. Chlamydomonas acti- ic analyses. Mol. Phylogenet. Evol. 48: 281-291. nochloris NIES-2201 (Fig. 3, Q) and Cd. mutabilis Nakada, T., Shinkawa, H., Ito, T. & Tomita, M. 2010. NIES-2224 (Fig. 3, R) were revised to Cm. actinochlo- Recharacterization of Chlamydomonas reinhardtii ris. Chlamydomonas chlorococcoides NIES-2575 (Fig. and its relatives with new isolates from Japan. J. 3, P) was revised to Cm. carrizoensis (Fig. 3, P). In Plant Res. 123: 67-78. addition, Chlamydomonas applanata NIES-2203 (= Nakada, T. & Tomita, M. 2011. Chlamydomonas neo- IAM C-216) was revised and downgraded to planoconvexa nom. nov. and its unique phylogenet- Chloromonas sp. (Table 1), because authentic strain ic position within Volvocales (Chlorophyceae). of C. applanata (CCAP 11/2) is included in Phycol. Res. 59: 194-199. Polytominia (Fig. 2). It is not unlikely that NIES-2203 Nakayama, T., Marin, B., Kranz, H.D., Surek, B., has been replaced by another strain. However, we Huss, V.A.R., Inouye, I. & Melkonian, M. 1998. The included this strain in our collection list as basal position of scaly green flagellates among the Chloromonas sp., because the sequence is unique at green algae (Chlorophyta) is revealed by analyses present and no relevant sequences have been pub- of nuclear-encoded SSU rRNA sequences. Protist lished. 149: 367-380. Nozaki, H., Ohta, N., Morita, E. & Watanabe, M.M. ACKNOWLEDGMENTS 1998. Toward a natural system of species in We are grateful to Dr. T. Nakada for his kind Chlorogonium (Volvocales, Chlorophyta): A com- advice on the phylogenetic analyses and for check- bined analysis of morphological and rbcL gene ing our sequence data, and critical review. sequence data. J. Phycol. 34: 1024-1037. Pröschold, T., Marin, B., Schlösser, U.G. & Melkonian, REFERENCES M. 2001. Molecular phylogeny and taxonomic revi- Buchheim, M.A., Buchheim, J.A. & Chapman, R.L. sion of Chlamydomonas (Chlorophyta). I. 1997. Phylogeny of Chloromonas (Chlorophyceae): Emendation of Chlamydomonas Ehrenberg and A study of 18S ribosomal RNA gene sequences. J. Chloromonas Gobi, and description of Phycol. 33: 286-293. Oogamochlamys gen. nov. and Lobochlamys gen. Cantino, P.D. & de Queiroz, K. 2010. PhyloCode: nov. Protist 152: 265-300. International Code of Phylogenetic Nomenclature Tamura, K., Peterson, D., Peterson, N., Stecher, G., Version 4c. http://www.ohio.edu/phylocode/ Nei, M. & Kumar, S. 2011. MEGA5: molecular evo- PhyloCode4c.pdf. lutionary genetics analysis using maximum likeli- Harris, E. 2009. The Chlamydomonas Sourcebook, hood, evolutionary distance, and maximum parsi- second edition, vol. 1, Academic Press, Oxford. mony methods. Mol. Biol. Evol. 28: 2731-2739. Hoham, R.W., Bonome, T.A., Martin, C.W. & Thompson, J.D., Gibson, T.J., Plewniak, F., Leebens-Mack, J.H. 2002. A combined 18S rDNA Jeanmougin, F. & Higgins, D.G. 1997. The ClustalX and rbcL phylogenetic analysis of Chloromonas Windows interface: flexible strategies for multiple and Chlamydomonas (Chlorophyceae, Volvocales) sequence alignment aided by quality analysis emphasizing snow and other cold-temperature tools. Nucleic Acids Research 24: 4876-4882. habitats. J. Phycol. 38: 1051-1064.

─ 12 ─