Bull. Natl. Mus. Nat. Sci., Ser. B, 39(3), pp. 101–105, August 22, 2013

Morphology and Molecular Phylogenetics of Sphaerospermopsisoumiana (M.Watan.) Tuji et Niiyama Compared with
Sphaerospermopsis torques-reginae (Komárek) Werner et al.
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 15 May 2013; accepted 26 June 2013)

Abstract The morphology and the 16S rDNA sequences of Shaerospermopsis oumiana(M.Watan.) Tuji et Niiyama from Japan and S. torques-reginae (Komárek) Werner et al. fromSouth America are compared and their phylogenetic relationship are discussed. The results showthat S. oumiana and S. torques-reginae are distinct species.
Key words : Anabaena oumiana, Anabaena torques-reginae, 16S rDNA sequences, morphology,
Sphaerospermopsis oumiana, Sphaerospermopsis torques-reginae.

Introduction
Polyphasic study using the morphological,
ecological, physiological and molecular methods
is important to solve the phylogeny of cyanobacteria and several new genera have been established for planktonic taxa of traditional Anabaena and Aphanizomenon based on thepolyphasic approach (Komárek, 2005; Rajaniemi
et al., 2005; Tuji and Niiyama, 2010; Wacklin etal., 2009; Zapomĕlová et al., 2009).
Zapomĕlová et al. (2009, 2010) described a
new genus, Sphaerospermopsis, based on themorphological features and 16S rDNA sequences.
The trichome of Sphaerospermopsis is free-ﬂoating, solitary, coiled or straight, akinetes arespherical or nearly spherical and adjacent to heterocytes, and terminal cells elongated andtapered or undifferentiated. Zapomĕlová et al.
(2009) pointed out the necessity to conﬁrm theafﬁliation of Anabaena oumiana M.Watan. andA. torques-reginae Komárek to genus Sphaerospermopsis using analysis of 16S rDNA
sequences. A. torques-reginae was originallydescribed by Komárek (1984) from Cuba.
Komárek (2005) stated that A. torques-reginae
occurs in Cuba, Brazil and San Salvador. A.
oumiana was observed for the ﬁrst time in 1994
from Lake Biwa in Japan and described as a new
species by Watanabe (1996). The trichome morphology of A. torques-reginae and A. oumianacorresponds well to that of Sphaerospermopsis.
Tuji and Niiyama (2010, 2012) analyzed 16SrDNA and rbcLX sequences of many Japaneseplanktonic Anabaena strains and proposed thenew combination of Sphaerospermopsis oumiana(M.Watan.) Tuji et Niiyama for Anabaena oumiana based on both morphological characteristics(Watanabe et al., 2004) and 16S rDNA and rbcLX
DNA sequences. Werner and Laughinghouse IV(2009) reported A. oumiana for the ﬁrst timefrom South America. Later Werner et al. (2012)
reidentiﬁed this taxon as A. torques-reginae
according to the opinion of Komárek andZapomĕlová (2007), who treated Anabaena
oumiana as a synonym of Anabaena torquesreginae, and then transferred to Sphaerospermopsis torques-reginae (Komárek) Werner et al.

102

Akihiro Tuji and Yuko Niiyama

from the morphological and molecular studies.
The aim of this study is to compare the morphology and the 16S rDNA sequences of S.
oumiana from Japan and S. torques-reginae fromSouth America and discuss the phylogenetic relationship of these taxa.

tion Criterion) and the substitution nucleotide
matrix parameters were calculated by the software. One thousand bootstraps were generated.
Neighbor-joining (NJ) analysis was performed
using the same model as for the ML. Bootstrapping values for the NJ tree was also generatedusing 1000 replicates. All positions containing
gaps and missing data were eliminated.

Materials and Methods
The history of cultured strains of Japanese
Sphaerospermopsis oumiana used in this study
and the method of DNA extraction and ampliﬁcation are stated in Tuji and Niiyama (2010).
The Basic Local Alignment Search Tool(BLAST) at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and some data of our own strains wereused to ﬁnd sequences similar to the sequence ofS. oumiana and S. torques-reginae. Shortsequences in NCBI were not used. Molecular
evolutionary analyses for obtained sequenceswere conducted using MEGA 5 computer program (Tamura et al., 2011). Alignments werechecked manually. Maximum Likelihood (ML)tree was calculated using the software with thebest ﬁts model by BIC scores (Bayesian Informa-Results and Discussion
Sphaerospermopsis oumiana and S. torquesreginae have similar morphology as following,trichomes are free-ﬂoating and solitary, akinetes
are spherical and at one or both sides of the heterocytes and heterocytes are spherical (Komárek,1984; Watanabe, 1996). However, the trichomesof S. torques-reginae are irregularly coiled andtwo trichomes are arranged in parallel (Komárek,1984, 2005) or irregularly or regularly coiled(Werner and Laughinghouse IV, 2009; Werner etal., 2012), while those of S. oumiana are regularly coiled with thick mucilage but not arrangedin parallel (Watanabe, 1996; Watanabe et al.,2004) (see Table 1). The size of akinetes ofTable 1. Morphological comparison and locality of Sphaerospermopsis oumiana and S. torques-reginae according to Komárek (1984), Watanabe (1996), Watanabe et al. (2004), Werner and Laughinghouse IV (2009) andWerner et al. (2012)
S. oumiana

S. oumiana

S. torques-reginae

S. torques-reginae

S. torques-reginae

Reference

Watanabe (1996)

Watanabe et al. (2004) Komárek (1984)

Werner and Laughinghouse IV (2009)

Werner et al. (2012)

Trichomemorphology
width
distancemucilage

regularly coiled

irregulaly coils, parallel ﬁlaments in coils

slightly irregular coils

28–46 μm
10–20 μm
thick

regular or slightly
irregular coils
20–40 μm
10–30μm

20–46 μm(8.3) 9–20 μm
thick (1–10.2 μm)

irregular or regularcoils(17.5) 20–54.5 μm
9–22.5 μm
thick (1–10.2 μm)

Cellmorphology
widthlengthsubspherical or barrelshaped
6.0–7.1 μm
3.0–6.5 μm

spherical

spherical

roundedrounded5.0–7.5 μm
3.8–7.5 μm

5.5–7.6 μm

5–7.6 μm
4.5–7.5 μm(4) 4.3–7 (8) μm
4–6.4 (8) μm

Heterocyte
morphology
widthlengthspherical
6.8–8.5 μm
6.4–8.0 μm

spherical
7.5–8.8 μm

spherical
6.8–10 μm

rounded(6) 7–9 (9.5) μm
6.8–8 μm

rounded(4–5) 5.4–9.5 μm(5) 5.5–9 μm

Akinetemorphology
widthspherical
10–12 μm

spherical
9.5–12.5 μm

spherical
14–14.5 μm

rounded
9–13 μm

rounded(7) 7.9–13 μm

Locality

JapanJapan

Cuba

BrazilColonbia, Brazil,
Argentina

Sphaerospermopsis oumiana and S. torques-reginae

103

Fig. 1. Phylogenetic position of Shaerospermopsis oumiana and S. torques-reginae and related taxa determinedby Maximum Likelihood (ML) method applied to 16S rDNA sequences. Originally deposited scientiﬁc names
were used. The analysis involved 46 nucleotide sequences. All positions containing gaps and missing data
were eliminated. There were a total of 1239 positions in the ﬁnal dataset. Numbers at branches indicate NJ(Neighbor Joining)/ML bootstrap support values. Deposited taxonomic names in gene bank, were used.

1 AB551454.1|_Anabaena_oumiana_TAC464
2 GU197605.1|_Anabaena_oumiana_CHAB510
3 GU197604.1|_Anabaena_oumiana_CHAB338
4 AB551437.1|_Anabaena_kisseleviana_TAC34
5 AB551458.1|_Anabaena_reniformis_TAC478
6 AB551459.1|_Anabaena_reniformis_TAC481
8 AB551475.1|_Anabaena_reniformis_TAC543
9 FM161348.1|_Anabaena_reniformis_06–01
13 AY701558.1|_Anabaena_kisseleviana_TAC34
14 AY701573.1|_Anabaena_ﬂos-aquae_NIES-73
15 AF317628.1|_Anabaena_ﬂos-aquae_strain_AWQC_264A
19 GU197620.1|_Dolichospermum_ﬂos-aquae_CHAB634
21 FJ830569.1|_Anabaena_aphanizomenoides_CENA188
22 AB551453.2|_Anabaena_aphanizomenoides_TAC456
26 GU197621.1|_Sphaerospermopsis_aphanizomenoides_CHAB2378
30 FM177480.1|_Anabaena_sphaerica_f._conoidea_1LT27S01
31 GQ859609.1|_Anabaena_sphaerica_var._tenuis_PMC188.03
39 FM177479.1|_Anabaena_sphaerica_f._conoidea_2LT27S09
40 HQ730086.1|_Sphaerospermopsis_torques-reginae_ITEP-024
41 HQ730087.1|_Sphaerospermopsis_torques-reginae_ITEP-026
42 GU197626.1|_Anabaena_eucompacta_CHAB915
43 GU197627.1|_Anabaena_eucompacta_CHAB464
45 AF516737.1|_Cylindrospermopsis_raciborskii_strain_Bal_5
0400310103374335812121423401400310103374335812121423402447545477118779121616182744303101033743358121214234043101033743358121214234052343444144291111112037612122632247111113223981033743358121214234091448544691313152441133374335812121423401428344291313132039158544491313152239

195686131111131834

2155110121210193822469131313223726659915223730

111313111837311414202742

39

222
19344022193441

938423143

Table 2. Estimates of evolutionary divergence among the 16S rDNA gene sequences of Shaerospermopsis oumiana and S. torques-reginae and related taxa. The numbers of base differences per sequence from between sequences are shown. The analysis involved 46 nucleotide sequences. All positions containing gaps and missing
data were eliminated. There were a total of 1239 positions in the ﬁnal dataset.
45104
Akihiro Tuji and Yuko Niiyama

Sphaerospermopsis oumiana and S. torques-reginae

S. torques-reginae is 14–14.5 μm (Komárek,1984) or 7.9–9–13 μm (Werner and Laughinghouse IV, 2009; Werner et al., 2012) and those ofS. oumiana is 9.5–12.5 μm (Table 1). The maximum size of akinetes of S. torques-reginae islarger than those of S. oumiana. The cells of S.
oumiana are spherical or barrel-shaped and thecells of S. torques-reginae are spherical. Distribution of both taxa is also so different, as S.
oumiana is found only in Japan, while S.
torques-reginae is found from Central and SouthAmerica.
Phylogenetic tree based on 16S rDNA (Fig. 1)
suggests that S. oumiana is genetically far fromS. torques-reginae in the genus Sphaerospermopsis though supported values on this tree arealmost very low. The number of base differences
between these two species are bigger than thedifferences between S. oumiana and otherSphaerospermopsis species such as S. kisseleviana (Elenkin) Zapomĕlová et al. and S. aphanizomenoides (Forti) Zapomĕlová et al. These morphological (Table 1) and molecular (Fig. 1 andTable 2) results indicate that S. oumiana and S.
torques-reginae are not synonyms and should bedifferentiated at species level.
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