Bull. Natl. Mus. Nat. Sci., Ser. B, 42(3), pp. 83–89, August 22, 2016

The Identity and Phylogeny of Pseudanabaena Strain, NIES-512,
Producing 2-methylisoborneol (2-MIB)
Akihiro Tuji* and Yuko Niiyama
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan* E-mail: tuji@kahaku.go.jp(Received 19 May 2016; accepted 22 June 2016)

Abstract A Pseudanabaena strain, NIES-512, has a morphological character that is intermediate
between P. foetida and P. subfoetida. Although the 16S rRNA shows very high similarity among
these species, the secondary structures of the 16S rRNA-23S rRNA spacer region were different
between NIES-512 and these two species. Because of the morphological similarity and high similarity of the 16S rRNA region, we assigned these differences to the variety level. P. foetida var.
intermedia Tuji et Niiyama is here newly described based on NIES-512, and a new combination P.
foetida var. subfoetida is proposed.
Key words: 2-MIB, Pseudanabaena foetida var. intermedia, Pseudanabaena foetida var.
subfoetida, secondary structure.

Introduction
A Pseudanabaena strain, NIES-512 maintained at the National Institute for Environmental
Studies (NIES), was isolated from Nagoya castle,
Nagoya, Aichi Prefecture, Japan by N. Yamada
on 1 November 1981 as Phormidium tenue
Gomont (Watanabe et al., 2000). It was re-identiﬁed by T. Honma as Pseudanabaena galeata
Böcher, and is now distributed by NIES using
this identiﬁcation (Kasai et al., 2009). Since thestrain produces a musty-odor compound, 2-MIB,
it has been used in a great deal of research(Yamada et al., 1985; Kasai et al., 2009; Iwase
and Abe, 2010) and has become a model strain
for 2-MIB producing cyanobacteria.
Recently, we described two new Pseudanabaena species that produce 2-MIB (Niiyama etal., 2016). Ecological characters are important on
species level (Anagnostidis and Komárek, 1988;
Komárek and Anagnostidis, 1999). Pseudanabaena galeata is ﬁrst observed on the muddy

bottom of shallow blackish lake (Böcher, 1949)
and it is epiphytic or endogloeic (Komárek andAnaganostidis, 2005). Böcher (1949) describesthat P. galeata is able to slowly move. Niiyama
et al. (2016) point out the morphological similarity of P. foetida and P. galeata and differences of
their habitat and motility. The identiﬁcation of
NIES-512 needed to be re-examined considering
the morphology, habitat, motility and genetic
characteristics. In this paper, we clarify the identity and phylogeny of NIES-512.
Materials and Methods
Cultured strains, genomic DNA extraction,
PCR ampliﬁcation and sequencing
The strains were provided by NIES and maintained under the conditions described in Niiyama
et al. (2016). The methods of micro-photographing and ﬁxing followed those used by Niiyama etal. (2016), and information on other strains was
also obtained from the previous report. The

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Akihiro Tuji and Yuko Niiyama

methods of genomic DNA extraction, PCR
ampliﬁcation and sequencing, were also followed
those used by Niiyama et al. (2016).
Secondary structure models of ITS regions
The putative secondary structures of the 16S
rRNA-23S rRNA spacer region were predictedwith the Mfold Web server (Zuker, 2003), withdefault settings including folding temperature set
at 37°C. Four parameters of constraint for Mfold,were set for D1-D1ʼ, RNA-Ile, RNA-Ala, and
Box-B regions.
Results and Discussion
The micro-photographs of NIES-512 andrelated taxa are presented in Fig. 1. The cell sizesof NIES-512 were intermediate between those of
Pseudanabaena foetida Niiyma, Tsuji et Ichise(strain PTG) and P. subfoetida Niiyma et Tuji(strain Ak1318). The cell width of NIES-512 was
1.6–2.2 μm and thicker than that of PTG (1.0–
1.5 μm) and thiner than that of Ak1318 (2.1–
2.9 μm). These sizes also overlapped. The cell
lengths of NIES-512 was 3.0–8.3 μm, almost
identical to that of Ak1318 (2.5–8.5 μm) andslightly shorter than that of PTG (3.9–11.0 μm).
The cell lengths of these strains also overlapped(Table 1). The color of the trichomes and cells
was bright blue-green in all three strains. Other
morphological characteristics were also shared
among these three strains (Table 2).
The phylogeny of 16S rRNA revealed similarity between NIES-512 (LC153790) and two new
species described in Niiyama et al. (2016).
NIES-512 and dqh15 (JF429939.1) are almost
identical. Differences were found near the 5′ and3′ ends, and these might simply be sequencing
errors.
NIES-512 and PTG have differences of 5 basepositions (0.3% of 1467 bp), and NIES-512 andAk1318 have differences of 12 base positions(0.8% of 1467 bp). These similarities showed that
NIES-512 should be included in the P. foetida –subfoetida species complex.
Niiyama et al. (2016) divided this complex

Fig. 1. Micro-photographs of holotype specimens.
Scale bar＝50 μm. A. Pseudanabaena foetida
var. foetida (TNS-AL-57781 from PTG). B.
Pseudanabaena foetida var. subfoetida (TNS-AL58650 from Ak1318). C. Pseudanabaena foetida
var. intermedia (TNS-AL-58658 from NIES-512).

NIES-512(Ak1363)
NIVA-CYA276/6(Ak1189)
PTB(Ak1201)
Ak1319P. foetida var. intermedia

1.1–1.5

0.9–1.3

2.0–3.0

1.6–2.2

2.1–2.9

1.0–1.5

1.28(0.153)2.42(0.204)1.92(0.138)2.52(0.282)1.03(0.131)1.38(0.113)mean μm(std.)

Cell widthmin–maxμm

1.5–5.0

2.5–7.1

3.0–8.0

3.0–8.3

2.5–8.5

3.9–11.0

6.18(1.803)
4.61(1.391)
5.09(1.255)5.35(1.489)4.47(1.276)3.20(0.947)

mean μm(std.)

Cell lengthmin–max
μm

Cell width and length (μm). L/W＝cell length/cell width; std＝standard deviation; N＝50.

P. limnetica

P. limnetica

P. sp.

P. foetida var. subfoetida

PTG(Ak1200)
Ak1318StrainPseudanabaena foetida var. foetidaTaxon

1.1–4.2

2.5–7.0

1.0–3.6

1.6–4.6

0.9–4.0

2.7–8.5

min–max

L/W

4.90(1.477)1.94(0.664)2.65(0.677)2.15(0.666)
4.39(1.269)2.34(0.735)mean(std.)

Table 1. Cell size of cultured strains PTG, Ak1318, NIES-512, NIVA-CYA276/6, PTB and Ak1319

58649

57780

58651

58658

58650

57781

LC016775

LC016774

LC016776

LC153790

LC016779

LC016773

Specimen no. Accession number
TNS-AL
16S rRNA

Pseudanabaena Strain - NIES-512
85

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Akihiro Tuji and Yuko Niiyama

Fig. 2. Phylogenetic position of strains PTG, Ak1318, NIES-512 and related strains determined by Maximum
Likelihood (ML) method using 16S rRNA gene. Accession numbers are followed by taxonomic names. Numbers at branches indicate NJ (Neighbor Joining) / ML bootstrap support values (only values higher than 60 are
shown).

into two species, P. foetida and P. subfoetida,based on the secondary structure of the 16S
rRNA-23S rRNA spacer region. Since the secondary structures of RNA-Ile and RNA-Ala, presented in Niiyama et al. (2016), have problems,four constraint for the D1-D1ʼ, RNA-Ile, RNAAla, and Box-B regions were set in the Mfold
software. The secondary structures of theD1-D1ʼ, RNA-Ile, V2, RNA-Ala and Box-B
regions were clearly recognized in these threestrains (Fig. 3). The structures of RNA-Ile and
BOX-B are almost identical within these threestrains. The structure of the D1-D1ʼ region in
NIES-512 agree with the structure of Ak1318
and differ from that of PTG. The structure of theV2 region in NIES-512 agrees with that of PTG.

A long insertion (53 bp) in the V2 region is also
identical between NIES-512 and PTG, and does
not exist in Ak1318. Several secondary structuraldifferences were seen between NIES-512 and theother two strains. NIES-512 should be in a different taxon from P. foetida (PTG) and P. subfoetida(Ak1318) based on the secondary structure of the
16S rRNA-23S rRNA spacer region. The secondary structure of this region has been widely used
to recognize the taxon (Iteman et al., 2000;
Boyer et al., 2001; Siegesmund et al., 2008).
However, the 16S rRNA region shows very highsimilarity among these three strains (＞99% similarity). Also, the morphologies of these threestrains are very similar, and their dimensions
mostly overlap. These three strains commonlyPseudanabaena Strain - NIES-512

Fig. 3. Secondary structure of the 16S rRNA-23S rRNA spacer region. a. Strain PTG. b. Strain NIES-512.
c. Strain Ak1318.

produce 2-MIB. Because of these similarities andto avoid superﬂuous taxonomic confusion inapplied environmental biology, we will divide
these strain at the variety level on the basis of

their differences.

87

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Akihiro Tuji and Yuko Niiyama
Table 2.

Morphological characteristics of strains, PTG, Ak1318 and NIES-512

Pseudanabaena foetida var. foetida
PTG (Ak1200)

P. foetida var. subfoetida
Ak1318P. foetida var. intermedia
NIES-512 (Ak1363)

trichome color
sheathmotility of trichomebright blue-green—
rarely tremblingbright blue-green—
rarely trembling

cell color
cell morphology

bright blue-green
long and thin cylindricalapical celllong cylindrical with rounded end

bright blue-green—
rarely tremblingor rarely slowly go
forwardbright blue-green
isodiametricto longer than wide
isodiamtetric to cylindricalwith rounded end
＋
＋
＋

Taxon
Strainpoler aerotope＋
constriction at cross-wall ＋
musty odor＋

Taxonomic description
Pseudanabaena foetida Niiyama, Tuji et Ichisein Niiyama et al., Fottea 16: 4-5. f. 6, 7, 14.
2016. (Fig. 1A)
Holotype: A formalin ﬁxed specimen, TNSAL-57781 in TNS, from cultured strain PTG.
Type strain: PTG maintained in the Lake Biwa
Environmental Research Institute, Japan.
Type locality: Lake Biwa, Shiga Pref., Japan.
Habitat: Plankton in lakes.
Pseudanabaena foetida var. subfoetida (Niiyama
et Tuji) Tuji et Niiyama comb. nov.
Basionym:
Pseudanabaena
subfoetida
Niiyama et Tuji in Niiyama et al., Fottea 16: 5. f.
10, 11, 15. 2016. (Fig. 1B)
Holotype: A formalin ﬁxed specimen, TNSAL-58650 in TNS, from cultured strain Ak1318(PS1306) maintained in the Department of Botany, National Museum of Nature and Science.
Type locality: Lake Kasumigaura, Ibaraki
Pref., Japan.
Habitat: Plankton in lakes.
Pseudanabaena foetida var. intermedia Tuji et
Niiyama, var. nov. (Fig. 1C)
Description: Trichomes solitary, straight, brightblue-green colored, with conspicuous constrictions at cross-walls, 1.6–2.2 μm wide, without
mucilage or sheath, not attenuated nor differentiated at the ends, without calyptra, infrequently

bright blue-green
cylindricalcylindrical with rounded
end
＋
＋
＋

move. Cells cylindrical with rounded ends, brightblue-green, longer than wide, 3.0–8.3 μm long,ratio of width to length ca. 1.6–4.6, with aerotopes at both ends of cells, differentiated in centro- and chro-matoplasmic regions. Cell divisionperpendicular to the longitudinal axis of a trichome. Trichomes separate between two neighboring cells or by fragmentation without necridic
cells. Heterocytes are not known. Thallus has anextreme musty odor that comes from 2-methylisoborneol.
Holotype: A formalin ﬁxed specimen, TNSAL-58658 in TNS (Department of Botany,
National Museum of Nature and Science), from
cultured strain NIES-512.
Type strain: NIES-512 maintained in National
Institute for Environmental Studies (NIES).
Type locality: Nagoya castle, Aichi Pref.,Japan.
Habitat: Plankton in lakes and ponds.
Synonyms:
Phormidium tenue Gomont sensu N.Yamada
in Watanabe et al. (2000).
Pseudanabaena galeata Böcher sensu T.
Honma in Kasai et al. (2009).
Acknowledgments
This work was supported by KAKENHI(Grant Number 15K00574) from the Japan Society for the Promotion of Science.

Pseudanabaena Strain - NIES-512

References
Anagnostidis, K. and Komárek, J. 1988. Modern
approach to the classiﬁcation system of cyanophytes 3—Oscillatoriales. Algological Studies 50–53: 327–472.
Böcher, T. W. 1949. Studies on the sapropelic ﬂora of thelake Flyndersø with special reference to the Oscillatoriaceae. Kongelige Danske videnskabernes selskab, Biologiske Meddelelser 21: 1–46.
Boyer, S. L., Flechtner, V. R. and Johansen, J. R. 2001. Is
the 16S–23S rRNA internal transcribed spacer region agood tool for use in molecular systematics and population genetics? A case study in cyanobacteria. Molecular
Biology and Evolution 18: 1057–1069.
Iteman, I., Rippka, R., Tandeau de Marsac, N. and Herdman, M. 2000. Comparison of conserved structuraland regulatory domains within divergent 16SrRNA–23S rRNA spacer sequences of cyanobacteria.
Microbiology 146:1275–1286.
Iwase, S. and Abe, T. 2010 Identiﬁcation and change inconcentration of musty-odor compounds during frowthin blue-green algae. Journal of the School of Marine
Science and Technology, Tokai University 8: 27–33.
Kasai, F., Kawachi, M., Erata, M., Mori, F., Yumoto, K.
Sato, M. and Ishimoto, M. 2009. NIES-Collection Listof Strains, 8th Edition. Japanese Journal of Phycology
57 (1) Suppl.: 1–350.
Komárek, J. and Anagnostidis, K. 1999. Cyanoprokaryota. 1. Chroococcales. In: Ettl, H., Gärtner, G.,

89

Heynig, H. and Mollenhauer, D. (eds.), Süßwasserﬂora
von Mitteleuropa. Band 19/1, pp. 548, Elsevier, Heidelberg & Berlin.
Komárek, J. and Anagnostidis, K. 2005. Cyanoprokaryota. 2. Teil: Oscillatoriales. In: Büdel, B., Gärdner,G., Krienitz, I. and Schagerl. M. (eds.), Süswasserﬂora
von Mitteleuropa, vol. 19/2. pp. 759 pp, Elsevier,München.
Niiyama, Y., Tuji, A., Takemoto, K. and Ichise, S. 2016.
Pseudanabaena foetida sp. nov. and P. subfoetida sp.
nov. (Cyanophyta/Cyanobacteria) producing 2-methylisoborneol from Japan. Fottea 16: 1–11.
Siegesmund, M., Johansen, J. R., Karsten, U. and Friedl,
T. 2008. Coleofasciculus gen. nov. (Cyanobacteria):
morphological and molecular criteria for revision of thegenus Microcoleus Gomont. Journal of Phycology 44:
1572–1585.
Yamada, N., Aoyama, K., Yamada, M. and Hamamura, N.
1985. Studies on earthy-musty odor in natural water (1)Growth characteristics and 2-methylisoborneol production of Phormidium tenue. Japan Journal of Water Pollution Research 8: 515–521.
Watanabe, M. M., Kawachi, M., Hiroki, M. and Kasai, F.
2000. NIES-Collection List of strains. Sixth edition.
Microalgae and protozoa. 159 pp. National Institute forEnvironmental Studies, Environment Agency, Tsukuba.
Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids
Research 31: 3406–3015.