Rikunomizu（Limnology in Tokai Region of Japan）70 : 19 - 24（2015）

ReportZygospore formation of a Spirogyra variformis TRANSEAU (Zygnemataceae)
collected from an irrigation canal of rice fields at Mikkabi, Hamamatsu, Japan
Kentaro NOZAKI 1）
Abstract
Zygospore formation of a Spirogyra variformis TRANSEAU collected from an irrigation canal of rice fields
on 26 March 2014 was described using microscopic photographs of the vegetative and reproductive
cells, and physical and chemical characteristics of their sampling site were shown in order to understandthe habitable conditions of the S. variformis. Vegetative cell of the S. variformis was 40-50μm wide
and 60-100 μm long with plane and end cell walls. One chloroplast was making 2-5 turns in eachcell. Conjugation was scalariform-type, and tubes elongated both sides of the S. variformis filaments.
Reproductive cells were mostly cylindrical, sometimes enlarged or inflated. Characteristics of fullyripened zygospores were ellipsoidal, 40-50 μm in wide, 50-60 μm in long, smooth medium spore wall andbrownish color. Water temperature at noon, electric conductivity and pH were 11.8°C, 26.2 mS m-1 and6.5, respectively. Dissolved inorganic nitrogen (DIN) was 1.14 mg L-1 (NO3--N accounted for 97 %), andreactive phosphorous (PO43--P) was 0.004 mg L-1.
Key words: conjugation, Spirogyra variformis TRANSEAU, springtime, zygospore(Received: 26 March 2015, Accepted: 30 April 2015)
of conjugation in laboratory experiments (Yamashita andSasaki, 1979; Simons et al., 1984; Ikegaya et al., 2012;
Zwirn et al., 2013; Nozaki, 2015b). Previous studies ofSpirogyra classification have been almost entirely carriedout using samples collected from natural environment,
making the identification of the exact species very difficult.
Consequently, ecological studies of Spirogyra were almost
unidentified as a species (Graham et al., 1995; Berry andLembi, 2000; Nozaki, 2001; Nozaki and Mitamura, 2002).
In addition, only a few studies about taxonomic description
have showed the physical and chemical parameters of thesampling site (e.g. Simons and van Beem, 1990; Nozaki,2013). Thus, little is known about the abiotic habitableconditions of each species of Spirogyra from previousdescriptions.
In the present report, zygospore formation of a Spirogyra
collected from an irrigation canal of rice fields in springtime
was described using microscopic photographs of theIntroduction
Filamentous green alga genus Spirogyra (Zygnemataceae)
which often constructs a mat forming community in lake
littoral, pond and slow-flowing streams, is a common genera
found in freshwaters. Nearly 400 species of Spirogyra weredescribed in monographs (Hoshaw and McCourt, 1988), and90 species were found in Japan (Yamagishi, 1966, 1977).
The formation process of conjugation and zygospores ofSpirogyra is a suitable teaching material introducing sexual
reproduction in biology learning (Nozaki, 2014, 2015a).
Spirogyra species have been classified by the shape andcolor of conjugation and fully-ripened zygospores formedin sexual reproduction (Transeau, 1938; Yamagishi, 1966,1977). Although conjugation and zygospore developmentof Spirogyra are a well-known phenomenon, little isunderstood about their detailed processes and mechanism,
because of the difficulty in artificial reproducible induction
1)

School of Education, Sugiyama Jogakuen University, Moto-machi 17-3, Hoshigaoka, Chikusa, Nagoya 464-8662, Aichi, Japan(E-mail: ken@sugiyama-u.ac.jp)

19Kentaro NOZAKI

vegetative and reproductive cells, and physical and chemical
characteristics of their sampling site were shown in order tounderstand the habitable conditions of the Spirogyra. This
research was supported by a Grant-in-Aid for Scientific
Research (C) from the Japan Society for the Promotion ofScience (No. 24501114) to Kentaro NOZAKI.

Turbidity of water samples was measured with the water
analyzer (WA1, Nippon Denshoku Co.) using pre-filteredwater. Water sample was transferred to a glass fiber filter(GF-75, Advantec Co.) in preparation for the analysisof water color and nutrient concentrations. Water color
was also measured with a water analyzer (WA1, NipponDenshoku Co.). Nutrient analyses were carried out on
NH 4+-N, NO 2--N, NO 3--N and PO 43--P concentrations,respectively. Nutrient analysis procedures followed thetextbook of the Tokai Branch of the Japanese Society ofLimnology (2014).
Spirogyra sample in this experiment were using dominant
type (>90 % in cell numbers, unpublished data) in thesampling site (Fig. 2A). Spirogyra filaments were placed ina 10 cm glass Petri dish filled with filtered water in samplingsite and kept in a growth chamber (MLR-351H, Sanyo Co.)at 15°C under 170 μmol m-2 s-1 (approximately 20000 lux)
on a 12:12 hours of light and dark cycle. Spirogyra filaments
were observed under an optical microscope (BX 51,Olympus Co.), and microscopic photographs of conjugationand zygospores were taken by digital camera (CamediaC-5060, Olympus Co.). Temperature of the growth chamber
was set somewhat higher than that in the sampling site
during the incubation period, because the increase of watertemperature seemed to be a trigger inducing the conjugationof Spirogyra (Simons et al., 1984; Berry and Lembi, 2000;Nozaki, 2013; Nozaki, 2015b).

Methods
Spirogyra samples were collected from an irrigation canal ofrice fields on 26 March 2014, located at latitude 34°46’48”Nand longitude 137°32’23”E near the Ona Station of TenryuHamanako Railway, Mikkabi, Hamamatsu City in the TokaiRegion of Japan (Fig.1). Water temperature, pH (WAK-pH,Kyoritsurika Co.) and electric conductivity (CM21P, TOADDK Co.) were measured at 11:00 AM in the samplingsite. Spirogyra and water samples were stored in a box with
ice and were returned to the laboratory within 3 hours aftersampling.

Results and Discussion
Zygospores formation of the Spirogyra filaments is
illustrated in Figure 2A-E. Vegetative cells of the Spirogyra
measured 40-50 μm wide and 60-100 μm long with planeend cell walls. One chloroplast was making 2-5 turns ineach cell (Fig. 2A). Sexual reproduction of the Spirogyra
was started by formation of papilla on cells opposite eachother. After elongation of the papilla, 2 filaments aligned andconjugation tubes were formed between cells. Conjugationof the Spirogyra was scalariform-type (Yamagishi, 1999)
and tubes elongated both sides of the filaments (Fig. 2B).
Reproductive cells were mostly cylindrical, sometimes
enlarged or inflated. Zygospores matured in approximately
20-30 days after sampling. Characteristics of fully-ripened
zygospores were ellipsoidal, 40-50 μm in wide, 50-60 μmin long, smooth medium spore wall and brownish color(Fig. 2C-E). The Spirogyra in this report is identified tobe S. variformis TRANSEAU according to previous studies

AB

Fig. 1. Sampling
Spirogyrasitesite(A)Fig. 1. Sampling(A) and andSpirogyra
community (B)community
on 26 March 2014 (B) on 26(Ona, Mikkabi, Hamamatsu City).
March 2014
(Ona, Mikkabi, Hamamatsu City).

20

Zygospore formation of a Spirogyra variformis TRANSEAU

A

B

C

50 mm

Fig. 2. Microscopic photographs of zygospore formation of Spirogyra variformis TRANSEAU. A. Vegetative cells on 26 March,B.
Reproductive cells during conjugation on 27 March, C. Zygospores on 11 April, D. Crushed zygospore on 19 March, E. Wellripened zygospores on 26 April.

21

Kentaro NOZAKI

D50 mm

50 mm

EFig. 2. Continued.

Fig. 2. Microscopic photographs of zygospore formation of Spirogyra variformis TRANSEAU.
A. Vegetative cells on 26 March,B. Reproductive cells during conjugation on 27 March, C. Zygosporeson 11 April,Crushed1966;
zygosporeon
191977,
March,zygospores
26 April.
(Transeau,1938;D.
Yamagishi,
Yamagishi,KimE.et Well-ripened
conductivity
and pH aton13:17
PM were 9.3°C, 25.9 mS
m-1 and 6.5. NO3--N and PO43--P concentrations were 1383
and 4.5 μg L-1. These environmental parameters were very
similar to those of this study site. Thus, the possible abiotic
habitable conditions of S. variformis are about 10°C in watertemperature, 25 mS m-1 in electric conductivity and 1 mg L-1
in NO3--N concentration.
Results of this study suggest that S. variformis might beal., 2004; Nozaki, 2013). Morphological characteristics of S.
variformis are shown in Table 1.
Physical and chemical parameters of the water sample
collected from sampling site are shown in Table 2. Nozaki(2013) described formation processes of conjugation andzygospores of S. variformis collected from a Japanese lowland
marsh in early spring. At that time, water temperature, electric22

Zygospore formation of a Spirogyra variformis TRANSEAU
Table 1. Morphological characteristics of Spirogyra variformis TRANSEAU according to previous studies.
Refernce

Collecting site

Transeau (1938)

Cape Town, Africa

Vegetative cells
width
length(μm)
(μm)
43-53
(70-)108-140(-200)

1Zygospores
widelong(μm)
(μm)
45-54
58-90

Yamagishi (1966)

Kanagawa, Japan43-5060-140

145-5458-90

Yamagishi (1977)

Kanagawa, Japan
Deoksan,Yesan-gun,
Chungcheongnam-do, Korea
Fukui, Tsuruga, Japan

43-50

60-140145-54

58-90

46-54

197-695

1-3

42-49

62-74

40-50

60-100140-5050-60

Kim et al. (2004)
Nozaki (2013)

propagating in spring and forming zygospores in late spring
to early summer. Seasonal periodicity of the reproductive
stage of the Spirogyra species varied with the filament
width shown in previous studies. Berry and Lembi (2000)
found that unidentified Spirogyra species with 45 µm
width and one or two chloroplasts such as S. variformis
dominated from March to April in a shallow artificial lake,
in Columbus, Indiana of the United States. Large numbersof zygospores were observed to be produced during crash of
the Spirogyra community. On the other hand, Simmons andvan Beem (1990) reported that Spirogyra species having
filaments wider than 50 µm produced zygospores in summer
in The Netherlands. However, the mechanism of filament
width affecting seasonal varieties in the reproductive stage
is still not clear and further research is required.

(Chlorophyta) population in a midwestern lake (U.S.A.).
Journal of Phycology, 36: 841-851.
Graham, J. M., C. A. Lembi, H. L. Adrian and D. F. Spencer(1995): Physiological responses to temperature andirradiance in Spirogyra (Zygnematales, Charophyceae).
Journal of Phycology, 31: 531-540.
Hoshaw, R. W. and R. M. McCourt (1988): The
Zygnemataceae (Chlorophyta): a twenty-year update ofresearch. Phycologia, 27: 511-548.
Ikegaya, H., T. Nakase, K. Iwata, H. Tsuchida, S. Sonobeand T. Shimmen (2012): Studies on conjugation ofSpirogyra using monoclonal culture. Journal of Plant
Research, 125: 457-464.
Kim, J.-H., Y. H. Kim and I. K. Lee (2004): Morphotaxonomyof the Genus Spirogyra (Zygnemataceae, Chlorophyta) inKorea. Algae, 19: 91-105.
Nozaki, K. (2001): Abrupt change in primary productivity
in a littoral zone of Lake Biwa with the development of afilamentous green-algal community. Freshwater Biology,
46: 587-602.
Nozaki, K. (2013): Formation process of conjugation andzygospores of a filamentous green alga, Spirogyra species
collected from a lowland marsh, Naka-ikemi, Tsuruga,Fukui, Japan. Rikunomizu (Limnology in Tokai Region ofJapan), 60: 35-39.
Nozaki, K. (2014): Filamentous algae Genus Spirogyra(Chlorophyceae) as a useful teaching material of thesexual reproduction in biological education. Journal of
the School of Education, Sugiyama Jogakuen University,
7: 295-299 (in Japanese).
Nozaki, K. (2015a): Learning of sexual reproduction using
the formation process of conjugation of filamentous green
algae Genus Spirogyra (Zygnemataceae). Journal of theSchool of Education, Sugiyama Jogakuen University, 8:
159-167 (in Japanese).

References
Berry, H. A. and C. A. Lembi (2000): Effects of temperatureand irradiance on the seasonal variation of a Spirogyra

Table 2. Physical and chemical parameters at sampling site on
26 March 2014.
Sampling time
Water temperature (℃ )Electric conductivity (mS m-1)
pH
Turbidity (degree)
Color (degree)
NH4+-N (μgN L-1)
NO2--N (μgN L-1)
NO3--N (μgN L-1)
Dissolved inorganic nitrogen (mgN L-1)
PO43--P (μgP L-1)

Chloroplast

11:00
11.8
26.2
6.5
0.3
4.3
30.1
4.9
1101
1.1
3.7

23

Kentaro NOZAKI

Nozaki, K. (2015b): Study on factors inducing conjugationof a filamentous green alga Spirogyra sp. (Zygnemataceae)
collecting from Ona, Mikkabi, Hamamatsu, Shizuoka,
Japan. Journal of the Sugiyama Jogakuen University,Section Natural Sciences, 46: 21-29 (in Japanese).
Nozaki, K. and O. Mitamura (2002): Seasonal change in afilamentous green-algal community in the littoral zoneof Lake Biwa: Examination of temperature effect onits summer decline. Verhandlungen der Internationale
Vereinigung für Theoretische und Angewandte Limnologie,
28: 1739-1744.
Simons, J., A. P. Van Beem and P. J. R. Devries (1984):
Induction of conjugation and spore formation in speciesof Spirogyra (Chlorophyceae, Zygnemataceae). Acta
Botanica Neerlandica, 33: 323-334.
Simons, J. and A. P. van Beem (1990): Spirogyra species
and accompanying algae from pools and ditches in The
Netherlands. Aquatic Botany, 37: 247-269.
Tokai Branch of Japanese Society of Limnology (2014): Mijika
Na Mizu No Kankyokagaku (Methods for Environmental
Studies of Inland Waters). Asakura Publishing, Tokyo (inJapanese).
Transeau, E. N. (1938): Notes on Zygnemataceae. American
Journal of Botany, 25: 524-528.
Yamagishi, T. (1966): Studies on the Genus Spirogyra
collected in Japan. Scientific Report of Tokyo Kyoiku
Daigaku, Section Biology, 12: 73-105.
Yamagishi, T. (1977): Family Zygnemataceaea. In Illustrationsof the Japanese freshwater algae, Hirose H. and T. Yamagishi(eds.): 416-461. Uchida Roukakuho Publishing Co., Ltd.,Tokyo (in Japanese).
Yamagishi, T. (1999): 11. Study of Genus Spirogyra. In
Introduction to Freshwater Algae, T. Yamagishi (ed.): 455462. Uchida Roukakuho Publishing Co., Ltd., Tokyo (inJapanese).
Yamashita, T. and K. Sasaki (1979): Conditions for theinduction of the mating process and changes in contents ofcarbohydrates and nitrogen compounds during the matingprocess of Spirogyra. Journal of Faculty of Science,
Hokkaido University, Series V (Botany), 11: 279-287.
Zwirn, N., C. Chen, B. Uher and M. Schagerl (2013):
Induction of sexual reproduction in Spirogyra clones-does
an universal trigger exist ? Fottea, Olomouc, 13: 77-85.
(Guest Editor: Dr. Akihiro TUJI,
National Museum of Nature and Science)摘要

静岡県浜松市三ヶ日町の水田用水路から採集された
アオミドロ属 Spirogyra variformis TRANSEAU の接合胞子形成
野崎健太郎
2014年3月26日に静岡県浜松市三ヶ日町の水田用水路から採集されたアオミドロ属 Spirogyra variformis TRANSEAU の接合胞子形成を顕微鏡写真と採集地点の水環境とともに記述した。細胞は，幅40～50 μm，長さ60～100 μm で，細胞間の隔膜は平板状であった。葉緑体は1本で，2～5回転していた。
接合体は，並び合った2本の糸状体の両方から接合管が伸び，梯子状に形成された。接合体は大部分が円筒状であったが，時には拡張していた。熟した接合胞子は，楕円形で幅40～50μm，長さ50～60 μm，胞子中層膜は黄褐色で平滑であった。
南中時の水温は11.8℃，電気伝導度は26.2 mS m-1，pH は6.5であった。溶存無機態窒素濃度は1.14 mg L-1で，およそ97%は硝酸態窒素であった。リン酸態リン濃度は，0.004 mg L-1であった。
キーワード：接合体，Spirogyra variformis TRANSEAU，春季，接合胞子（椙山女学園大学教育学部 〒464-8662愛知県名古屋市千種区
星ヶ丘元町17-3，E-mail：ken@sugiyama-u.ac.jp）

24