Bull. Natl. Mus. Nat. Sci., Ser. B, 45(3), pp. 93–101, August 22, 2019

Microcoleus pseudautumnalis sp. nov. (Cyanobacteria, Oscillatoriales)
producing 2-methylisoborneol
Yuko Niiyama* and Akihiro Tuji
Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan* E-mail: 3ken@kahaku.go.jp(Received 13 May 2019; accepted 26 June 2019)

Abstract A new species, Microcoleus pseudautumnalis, producing both 2-methylisoborneol(2-MIB) and geosmin is described. We have conducted a systematic study of a bad-smelling,
2-MIB producing planktic Pseudanabaena species in Japan and described four new species (P.
foetida, P. subfoetida, P. cinerea, and P. yagii). In the course of this study, we found another kindof ﬁlamentous cyanobacteria with a bad smell in a plankton sample collected from a pond inJapan. The morphology of M. pseudautumnalis resembles that of M. autumnalis (Trevisan ex
Gomont) Strunecký, Komárek et Johansen (basionym: Phormidium autumnale Trevisan ex
Gomont). The sheath is thin and always contains only one trichome. Trichomes are immotile, grayish-green, not constricted at the cross-walls, not attenuated or attenuated towards the ends withtruncated or capitated apical cells, and sometimes with calyptrae that are relatively wider (6.9–
7.6 μm) than those of M. autumnalis. The phylogeny of the 16S rRNA gene of M. pseudautumnalis
revealed that it is in the clade of the genus Microcoleus and contains an 11-bp insert. Microcoleus
autumnalis s. str. is said to lack this insert. Microcoleus pseudautumnalis has four kinds of 2-MIB
genes, and the phylogeny of this taxon is different from those of Pseudanabaena sp. and Planktothricoides raciborskii.
Key words : 16S rRNA gene sequences, 2-MIB, 2-methylisoborneol, geosmin, Microcoleus
autumnalis, Microcoleus vaginatus.

Introduction
Bad-smelling drinking water supplied fromreservoirs and bad-smelling ﬁsh and shellﬁshcause genuine problems in Japan. The substancecausing the odor was identiﬁed as 2-methylisoborneol (2-MIB) or geosmin (Yagi, 1983), andodor-producing cyanobacteria have beenreported in several lakes, reservoirs, and ponds(cf. Morii et al., 1982; Yamada et al., 1985,
1986; Oikawa et al., 2000, Oikawa and Ishibashi,
2004; Tsunoda et al., 2014). The authors conducted a systematic study of the odor-producing
ﬁlamentous cyanobacteria that included a
detailed morphological description, ecologicalobservation, and phylogenetic analysis, and wehave clariﬁed that thin ﬁlamentous planktic cyanobacteria producing 2-MIB belong to the genus
Pseudanabaena. Four new Pseudanabaena species were described (P. foetida Niiyama, Tuji etIchise, P. subfoetida Niiyama et Tuji, P. cinerea
Tuji et Niiyama and P. yagii Tuji et Niiyama)(Niiyama et al., 2016; Tuji and Niiyama, 2018).
In the course of the above-mentioned study,
we observed another kind of cyanobacteria species producing 2-MIB, which showed clearly different morphological characteristics from Pseudanabaena but had some resemblance toPhormidium or Microcoleus species. In Japan, it
has been reported that some benthic or periphytic
cyanobacteria with a Phormidium-like morphology in streams or banks produce geosmin or

94

Yuko Niiyama and Akihiro Tuji

2-MIB. However, the classiﬁcation of suchorganisms is unclear. This study focuses on themorphology and genetic characteristics of our
newly found cyanobacteria that produces 2-MIB.
Materials and Methods
Sampling site and cultured strain
The sample was collected from Naka-numa
Pond, Ryugasaki City, Ibaraki Pref., Japan in
September 2016, using a plankton net. Nakanuma Pond is surrounded by paddy ﬁelds, and norivers or streams ﬂow in or out of this pond.
Naka-numa Pond has an almost round shape witha diameter of about 100 m, and its maximum
depth is about 14 m.
Isolation was done by the agar plate method(Tuji and Niiyama, 2014) with BG-11 medium(Waterbury and Stanier, 1981). Only one unialgal
strain with a bad odor, Ak1609, was obtained.
For the maintenance of the strain, 10 ml of modiﬁed C medium (Ichimura and Watanabe, 1977;
Niiyama et al., 2011) contained in a test tube wasused. The culture was illuminated by cool-whiteﬂuorescent lamps, with a photon ﬂux density ofca. 20 μmol/m2/sec, a photoperiod of 8 hours lightand 16 hours dark, and a temperature of 18°C.
Morphological observation was performed forthis cultured strain under a light microscope(BH-2, Olympus Corporation, Tokyo, Japan).
Microphotographs were taken with a Canon digital camera EOS Kiss X5 (Canon Inc., Tokyo,
Japan). The cultured strain is maintained in the
Department of Botany, National Museum of
Nature and Science. The specimens are housed in
the herbarium of TNS (Department of Botany,
National Museum of Nature and Science).
SPME-GC/MS analysis
The odor-producing substance in the culture
medium of Ak1609 was analyzed using the gas
chromatography/mass spectrometry combinedwith solid phase micro extraction (SPME-GC/
MS) method (JWWA, 2011) at Hiroshima Environment and Health Association.

Genomic DNA extraction, polymerase chain
reaction (PCR) ampliﬁcation, sequencing andassembling
A 1.5-ml volume of fresh cultured material
was centrifuged at 10,000 rpm for 5 min at room
temperature. The supernatant was removed, andthe cell pellets were kept in a freezer at −20°C
until extraction. Total genomic DNA wasextracted using an extraction kit (DNeasy Plant
Mini Kit, Qiagen) in accordance with the manufacturerʼs instructions. The region between the
16S rRNA gene and the internal transcribedspacer (ITS) was ampliﬁed using four primers
sets, set G (PLG1.3 and pits-CyanR), set J (PS16S-27f and pits-CyanR), set K (PS-16S-27f and
16S-1492R), and set L (PLG1.3 and 16S-1492R)(Table 1). The region between rbcL and rbcX
was ampliﬁed using primer set M (cx-b andcw-b). The region of the gene cluster for 2-MIB
was ampliﬁed using primer sets N (1609-mtf-r1
and MIB-cnbA-852f), O (1609-mtf-r2 and MIBcnbA-736fb), P (PS-cnbA-start and 1609-mtfr1), Q PS-cnbA-start and 1609-mtf-r2, R (MIBSAM-F2 and 1609-mtf-r1), S (MIB-SAMF2
and 1609-mtf-r2), T (MIB-SAMF2 and MIBSAMR1), and U (MIB-CnbA-852f and MIBSAMR1). These primers for MIB genes were
designed using Wang et al. (2011) and modiﬁed
during this study.
PCR was performed on a thermal cycler (Veriti Thermal Cycler, Thermo Fisher Scientiﬁc)using 0.25 μl polymerase (Phusion Hot Start Flex
DNA Polymerase, BioLabs), 5 μl GC Buffer inthe polymerase kit, 2 μl 2.5 mM dNTPS, 14.25 μl
sterile deionized water, 0.25 μl each of 10 pM
concentrations of the forward and reverse primers, and 1 μl of DNA template. The temperature
cycling program used the following conditions:
16S rRNA to ITS regions; 98°C for 30 sec; 35
cycles of 98°C for 10 sec, 54°C for 15 sec, and72°C for 60 sec, and the ﬁnal elongation step was
72°C for 7 min; rbcLX regions; 98°C for 30 sec;
10 cycles of 98°C for 10 sec, 60°C to 50°C(−1.0°C per cycle) for 15 sec, 72°C for 60 sec;
35 cycles of 98°C for 10 sec, 50°C for 15 sec, and72°C for 60 sec, and the ﬁnal elongation step wasMicrocoleus pseudautumnalis sp. nov
Table 1.

95

Primers for PCR and sequences used in this study.

TargetPrimer name

16S rRNA-ITS

16S-27f
PLG1.3 (CYA108F)
pits-CyanR
F3 L
16S-1492r

AGAGTTTGATCMTGGCTCAG
ACGGGTGAGTAACRCGTRA
CTCTGTGTGCCAAGGTATCGTCCCGCAACGAGCGCAAC
GGTTACCTTGTTACGACTTLane 1991
Urbach et al. 1992
Ernst et al. 2003
Hiraishi et al. 1994
Turner et al. 1999

ReferencerbcL–rbcX

cx-b
cw-b

GGGGCARGTARGAAAGGRTTTCGTACGTAGCTTCYGGTGGTATCCACGT

Tuji & Niiyama 2018
Tuji & Niiyama 2018

MIB genes

MIB-SAMF2
cnba-852f
MIB-cnba-736fb
PS-mtf-start
MIB-mtf-278f
MIB-mtf-446r
PS-mtf-end
PS-mic-start
MIB-mic-400f
PS-mic-end
PS-cnbB-start
MIB-cnbB-681f
MIB-cnbB-890r
1609-cnbB-1300fb
PS-cnbB-end
MIB-SAMR1
1609-mtf-r1
1609-mtf-r2

GAVTTCCTSVTGGRCCACCTCG
GMRYTGCGBGARCGYCARGARYACGATTGTCGAYTACGARACCTCKCCRCG
ATGTCAACGCCCCAAAMTATCACTGC
GGCGGTTCYGGTCGCGGCGG
CGCATGGCTCCCGTCTCGAAGCC
TTACCGAATGATGCGGTCAGCAACGATGAAAGATACCAACYTGGATAATACGACCCAKMTCGGCTGTTGATTTAGGCTAGTGATTGTGAATCTGGC
ATGACCCAAGACTTTAACTCCCATGGCGCCCGCCAAAAGCCCAAGATACGCTCGCGCAACTCATGCACAGTCTATCCTTGTCCCCGACGCCGTCGGCATTCCTACCGCCCGATCTCGACATCCTCG
TCSACGTACATGSTSGACTCGT
TGGTGGTAATAGCCGTCAACTTGGCCGAGC
AGTTGGCAACCGACTTCTGATATTCGCTGCG

Wang et al. 2011
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Wang et al. 2011
This study
This study

72°C for 7 min; MIB genes region; 98°C for
30 sec; 35 cycles of 98°C for 10 sec, 55°C for
15 sec, 72°C for 180 sec, and the ﬁnal elongation
step was 72°C for 7 min.
The concentrations of the ampliﬁed products
were veriﬁed on a 1% agarose gel. Direct
sequencing of the PCR products was undertakenusing the primers presented in Table 1 with Big
Dye Terminator Chemistry and an ABI 3130xl
Genetic Analyzer (Applied Biosystems, Foster
City, CA). The obtained sequences were assembled using Chromas PRO (Technelysium Pty
Ltd, Tewantin, Australia). The assembled results
were checked manually after automatic assembling by Chromas Pro.

checked manually. A maximum likelihood (ML)tree was calculated using MEGA software withthe best ﬁt model determined by Akaike Information Criterion (AIC) corrected scores, and thesubstitution nucleotide matrix parameters were
calculated by the software. A tree using 1000
bootstrap replicates was generated. Each codon
position was partitioned and analyzed for
16S rRNA. Neighbor-joining (NJ) trees with
1000 bootstrap replicates were also calculatedusing MEGA software. All positions containing
gaps and missing data were partially deleted (sitecoverage cutoff 50%). Planktothricoides raciborskii (strain: NIES-207) was used for the outgroup.

Phylogenetic reconstruction
Phylogenetic and molecular evolutionary analyses for the sequence of Ak1609 and similar
sequences retrieved from National Center for
Biotechnology information (NCBI) were conducted using the MEGA 7 computer program(Kumar et al., 2016). The alignments were

Results and Discussion
The morphology of the cultured strain,
Ak1609, resembles that of Microcoleus autumnalis (Trevisan ex Gomont) Strunecký, Komárek
et Johansen (≡Phormidium autumnale Trevisan
ex Gomont) (Figs. 1–10, Table 2). Ak1609 and96

Yuko Niiyama and Akihiro TujiFigs. 1–10.

Light microscopy photographs of Microcoleus pseudautumnalis sp. nov. Scale bars＝10 μm.

1
1

1

1
1
many
many
many
many

M. pseudautumnalis
M. autumnalis

M. autumnalis

M. autumnalis
M. autumnalis
M. vaginatus
M. vaginatus
M. vaginatus
M. vaginatus

4–6
4.3–8.1(2.5)3–7
3.5–7(4)5–7
1.5–9.8(3.5)4–7

6.9–7.6(8.3)
4–7(9)

cell widthμm

1.3–8.5

1.3–3.8
2–5(6.7)

2–4(5)

2.0–5.0
2–5(7)

cell length
μm
habitat

0.5–1.5
0.3–1

0.3–1

0.5

periphytic on submersed substrate,
in streams
epipelic
periphytic
subaerophytic
on moist soil
epipelic
on various kinds of soils or walls,
stream periphyton

0.5–2.5
planktic
0.5 or more on wet soil, mud, walls, rocks in rivers

cell L/W

＋
＋

−
−
−
−
−
−

−
−

＋
＋
＋
＋
＋
＋

＋
−
−

＋

−

bad
smell

＋

11 bp
insertmotility

this study
Whitton 2002 (as “Phormidium autumnale”)
Komárek & Anagnostidis 2005 (as “P.
autumnale”)
Hašler et al. 2012 (as “P. autumnale”)
Strunecký et al. 2013
Komárek & Anagnostidis 2005
Whitton 2002
Hašler et al. 2012
Strunecký et al. 2013reference

M. vaginatusmany

1 Microcoleus pseudautumnalis Ak1609
2 Microcoleus vaginatus ISBAL M6
3 Microcoleus vaginatus SRS1-KK2
4 Microcoleus cf. vaginatus Ru-6-12
5 Microcoleus vaginatus E1 1
6 Microcoleus vaginatus E17
7 Microcoleus vaginatus ISBAL M14
8 Microcoleus vaginatus ISBAL M22
9 Microcoleus vaginatus ISBAL M2
10 Microcoleus vaginatus KZ-23-1
11 Microcoleus vaginatus K25 08
12 Phormidium autumnale sv30
13 Phormidium cf. autumnale CCALA 145
14 Microcoleus vaginatus SNM1-KK1
15 Microcoleus sp. PET 11 7
16 Microcoleus vaginatus ISBAL M10
17 Oscillatoria amoena CCAP 1459/39
18 Microcoleus sp. MUM 11 5
19 Phormidium autumnale JR1220 Phormidium autumnale CCALA 154
21 Phormidium sp. isolate: 2008
22 Phormidium autumnale LCR Cyant3a
23 Phormidium autumnale Arct-Ph5
24 Phormidium autumnale LCR-CYANT11
25 Phormidium autumnale SAG 78.79
26 Planktothricoides raciborskii NIES-207(3.8)4.5–5.5

2

99.90
—
1
1
3
3
1
3
2
4
3
2
2
4
4
4
6
7
8
7
8
10
14
13
19
83

1

—
1
2
2
2
2
2
2
3
3
3
3
3
5
5
5
5
6
7
8
9
11
13
14
18
83

4

5

6

7

8

9
99.71
99.81
99.90
99.90
99.71
99.71
99.90
99.71
—
4
5
2
2
6
4
4
6
9
8
9
8
8
14
15
19
83

10
99.71
99.61
99.71
99.71
99.90
99.90
99.71
99.90
99.61
—
4
2
4
8
8
4
4
9
6
9
8
10
16
17
20
81

on arid soils

99.81 99.81 99.81 99.81
99.71 99.71 99.90 99.71
99.81 99.81 100.00 99.81
99.81 99.81 100.00 99.81— 100.00 99.81 100.00—
099.81 100.00
—
2
2
99.81
—
0
0
2
3
3
1
3
1
1
3
1
5
5
4
5
1
1
1
1
3
3
1
3
7
7
5
7
7
7
5
7
3
3
3
3
3
3
5
3
8
8
8
8
5
5
7
5
10
10
8
10
7
7
7
7
9
9
9
9
15
15
15
15
16
16
14
16
20
20
20
20
81
81
82
81

2–5(6.7)

99.81 99.81
99.90 99.90— 100.00
—
0
2
2
2
2
0
0
2
2
1
1
3
3
4
4
1
1
1
1
5
5
5
5
3
3
5
5
8
8
7
7
8
8
7
7
9
9
15
15
14
14
20
20
82
82

3
99.71
99.71
99.61
99.61
99.52
99.52
99.61
99.52
99.52
99.61
—
5
5
7
7
7
8
9
10
8
11
13
16
16
20
82

11
99.71
99.81
99.90
99.90
99.90
99.90
99.90
99.90
99.81
99.81
99.52
—
2
6
6
2
4
9
6
9
6
8
16
15
21
81

12
99.71
99.81
99.90
99.90
99.71
99.71
99.90
99.71
99.81
99.61
99.52
99.81
—
6
6
4
6
9
8
9
8
10
16
15
21
83

13
99.52
99.61
99.52
99.52
99.32
99.32
99.52
99.32
99.42
99.23
99.32
99.42
99.42
—
8
8
10
11
12
11
12
14
18
17
23
87

14

＋＋

99.52
99.61
99.52
99.52
99.32
99.32
99.52
99.32
99.61
99.23
99.32
99.42
99.42
99.23
—
8
10
9
12
7
10
10
14
15
19
83

15
99.52
99.61
99.71
99.71
99.71
99.71
99.71
99.71
99.61
99.61
99.32
99.81
99.61
99.23
99.23
—
6
7
8
11
4
6
14
13
19
83

16

＋

99.52
99.42
99.52
99.52
99.71
99.71
99.52
99.71
99.42
99.61
99.23
99.61
99.42
99.03
99.03
99.42
—
11
2
7
10
12
16
13
17
83

17

−

99.42
99.32
99.23
99.23
99.23
99.23
99.23
99.23
99.13
99.13
99.13
99.13
99.13
98.94
99.13
99.32
98.94
—
13
14
5
7
9
10
14
85

18

20
99.23
99.32
99.23
99.23
99.03
99.03
99.23
99.03
99.13
99.13
99.23
99.13
99.13
98.94
99.32
98.94
99.32
98.65
99.13
—
15
16
19
13
18
83

21
99.13
99.23
99.32
99.32
99.32
99.32
99.32
99.32
99.23
99.23
98.94
99.42
99.23
98.84
99.03
99.61
99.03
99.52
98.84
98.55
—
4
10
11
15
83

22
98.94
99.03
99.13
99.13
99.13
99.13
99.13
99.13
99.23
99.03
98.75
99.23
99.03
98.65
99.03
99.42
98.84
99.32
98.65
98.46
99.61
—
12
9
13
81

Boyer et al. 2002

99.32
99.23
99.32
99.32
99.52
99.52
99.32
99.52
99.23
99.42
99.03
99.42
99.23
98.84
98.84
99.23
99.81
98.75
—
9
12
14
16
11
15
81

19

98.75
98.65
98.55
98.55
98.55
98.55
98.55
98.55
98.65
98.46
98.46
98.46
98.46
98.26
98.65
98.65
98.46
99.13
98.46
98.17
99.03
98.84
—
13
13
90

23

98.65
98.75
98.65
98.65
98.46
98.46
98.65
98.46
98.55
98.36
98.46
98.55
98.55
98.36
98.55
98.75
98.75
99.03
98.94
98.75
98.94
99.13
98.75
—
6
83

24

98.26
98.17
98.07
98.07
98.07
98.07
98.07
98.07
98.17
98.07
98.07
97.97
97.97
97.78
98.17
98.17
98.36
98.65
98.55
98.26
98.55
98.75
98.75
99.42
—
85

25

91.99
91.99
92.08
92.08
92.18
92.18
92.08
92.18
91.99
92.18
92.08
92.18
91.99
91.60
91.99
91.99
91.99
91.80
92.18
91.99
91.99
92.18
91.31
91.99
91.80
—

26

Table 3. The similarity (%) and the number of base differences per partial 16S rRNA sequence (1036 positons) from 26 strains between sequences are shown.
All positions containing gaps and missing data were eliminated.

trichome(s)in sheath

Taxa

Table 2. Comparison of morphological, ecological and genetic characteristics of Microcoleus pseudautumnalis, M. autumnalis, and M. vaginatus. Taxon names are identical with thorse shown in Fig. 11.

Microcoleus pseudautumnalis sp. nov
97

98

Yuko Niiyama and Akihiro TujiFig. 11. Maximum likelihood tree based on 16S rRNA gene sequences. The general time reversible with gamma
distribution and invariable sites model (GTR＋G＋I) was used. Sequences retrieved from NCBI are used forphylogenetic analysis, and the OTUs are shown with their registered name, culture number and accession
number. Numbers at nodes indicate NJ (Neighbor Joining)/ML bootstrap support values (only values higher
than 70 are shown). ◇: lacking11-bp insert.

its cultured solution have a strong bad smell.
Both 2-MIB and geosmin were detected from thecultured solution by the SPME-GC/MS. Ak1609
is in the Microcoleus clade including M. autumnalis and M. vaginatus for the phylogenetic treeof the 16S rRNA gene (Fig. 11, Table 3). How-ever, it is immotile and planktic and produces
2-MIB and geosmin, and these characteristics
differ from those of M. autumnalis and M. vaginatus. Thus, we propose a new species, M. pseudautumnalis, as follows.

Microcoleus pseudautumnalis sp. nov

Microcoleus pseudautumnalis Niiyama et Tuji,
sp. nov.
Description: Filaments mostly straight, without any type of branching, combine to make a
black colored thin membranous colony in a test
tube. Sheath thin, ﬁrm, colorless, always containing one trichome. Trichomes immotile, with orwithout sheath, grayish-green to pale yellowishgreen colored, 6.9–7.6 μm in width, not constricted at the cross-walls, somewhat granulated
at the cross-walls, not attenuated at the ends withrounded apical cells or attenuated towards thecurved ends with truncated or capitated apicalcells, sometimes with calyptra. Cells shorter or
longer than wide, cell width to length ratio 0.5–
2.5, without gas vesicles. Thallus has the extreme
musty odor that comes from 2-MIB and geosmin.
Holotype: A formalin-ﬁxed specimen, TNSAL-61750 in TNS (Department of Botany,
National Museum of Nature and Science), from
cultured strain Ak1609 maintained in TNS.
Isotype: A formalin-ﬁxed specimen, TNSAL61748 in TNS.
Type locality: Naka-numa pond, Ibaraki Pref.,Japan.
Habitat: Plankton in ponds.
Table 2 shows a comparison of the morphological, ecological, and genetic traits and thepresence or absence of a bad smell for M. autumnalis (P. autumnale), M. vaginatus, and the new
species reported in this study, M. pseudautumnalis. M. pseudautumnalis always has one trichomein the sheath, just like M. autumnalis. Microcoleus pseudautumanalis has very closed sequence
to M. autumnalis and M. vaginatus in 16S rRNA(table 3). It is noted that M. vaginatus has 11
base pairs inserted into the 16S rRNA gene(Boyer et al., 2002; Siegesmund et al. 2008;
Hašler et al., 2012; Strunecký et al., 2013). On
the other hand, M. autumnalis (P. autumnale s.
str.) lacks this 11-bp insert (Hašler et al., 2012;
Strunecký et al., 2013) and forms a monophyletic group in the genus Microcoleus (Strunecký
et al., 2013). Microcoleus pseudautumnalis is in
the same clade as M. vaginatus based on an analysis of the 16S rRNA gene and has the 11-bp

99

insert as does M. vaginatus (Fig. 12). Microcoleus autumnalis is said to be an epipelic or epiphytic species in aquatic habitats, and M. vaginatus is thought to be a soil or aquatic species, andboth are motile, especially M. vaginatus (Boyeret al. 2002). However, M. pseudautumnalis is animmotile and planctic species. No studies havebeen reported on Microcoleus species producing
2-MIB.
The 4507-bp sequence of the gene cluster for
2-MIB including the cnbA, mtf, mic, and cnbB
genes was obtained from M. pseudautumnalis(cultured strain Ak1609: accession no.:LC486303). These genes and their order are thesame as Pseudanabaena sp. dqh15 (HQ830028)
and Planktothricoides raciborskii CHAB3331(HQ830029) presented in Wang et al. (2011).
The gene cluster sequences excluding ITS
regions (4079 bp) were compared. Microcoleus
pseudautumnalis is closer to Pl. raciborskii(216 bp differences) than Pseudanabaena sp.
dqh15 (429 bp differences). Pseudanabaena sp.
dqh15 and Pl. raciborskii had 508 bp differences.
These large differences between M. pseudautumnalis and other 2-MIB-producing taxa suggestthat these 2-MIB-producing genes evolved separately and do not exhibit lateral gene transfer,which is found in many genes producing secondary metabolites such as geosmin (Hayashi et al.,2019).
Tsunoda et al. (2014) reported a 2-MIB producing peryphytic Phormidium sp. from theTama River in Japan, and they proposed that thisspecies may be P. autumunale or P. favosum
based on their microscopic observation. Because
the 16S rRNA sequence of the 2-MIB-producing
cultured strain presented in Tsunoda et al. (2014)(accession no.: AB820727) lacks the 11-bp insert(see Fig. 12) and is close to P. autumnale ArctPh5 in the phylogenetic tree (Fig. 11), it is different from M. pseudautumnalis and should be M.
autumnalis (P. autumnale s. str.) (Hašler et al.,2012; Strunecký et al., 2013).
Microcoleus pseudautumnalis produces not
only 2-MIB but also geosmin. Although many
musty-odor-producing cyanobacteria strains are

100

Yuko Niiyama and Akihiro TujiFig. 12. Variable positions of the rRNA regions between Phormidium autumnale (＝Microcoleus autumnalis), M.
pseudautumnalis and M. vaginatus showing 11-bp insert presented in Hašler et al. (2012) and Strunecký et al.
(2013).

reported in Japan, each cultured strain produces
only one kind of odor-producing substance(Yagi, 1983; Oikawa and Ishibashi, 2004). The
present study is the ﬁrst to report the productionof both 2-MIB and geosmin by one strain of cyanobacteria.
Acknowledgments
We express special thanks to the Hiroshima
Environment and Health Association for analyzing off-odor substances using SPME-GC/MS.
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