Proceedings of the 7th and 8th Symposia on Collection Building and Natural History Studies in Asia and the Pacific Rim,
edited by Y. Tomida et al., National Science Museum Monographs, (34): 163–170, 2006.

Molecular-cytotaxonomy of Cycas (Cycadales) Using
5S Ribosomal DNA Markers
Goro Kokubugata1 and Paul I. Forster2
1

Tsukuba Botanical Garden, National Science Museum, Tokyo, 4–1–1 Amakubo, Tsukuba,
Ibaraki 305–0005, Japan(e-mail: gkokubu@kahaku.go.jp)
2
Queensland Herbarium, Environmental Protection Agency, Brisbane Botanic Gardens,
Mt Coot-tha Road, Toowong, Queensland 4066, Australia.
(e-mail: paul.forster@epa.qld.gov.au)

Abstract Somatic chromosomes of fourteen species of Cycas (Cycadaceae, Cycadales; Stevenson,
1992) were compared by the fluorescence in situ hybridization (FISH) method using 5S ribosomal
DNA (5S rDNA) probes. The fourteen species of Cycas showed the same chromosome number of2n22, and a similar karyotype consisting of 4 st, 12 t, 4 sm and 2 m chromosomes. In the FISH
comparison, C. revoluta, C. taitungensis, C. wadei, C. diannanensis and C. micholitzii exhibited a 5S
rDNA site on two t chromosomes in a metaphase complement. On the other hand, C. siamensis,
C. circinalis, C. cairnsiana, C. couttsiana, C. media, C. megacarpa, C. micronesica and C. thouarsii
exhibited a 5S rDNA site on four t chromosomes in a metaphase complement. The two cytologicalgroups based on the number of 5S rDNA agreed with the taxonomic treatment and phylogenetic
hypothesis suggested by Hill (1996, 2004), but disagreed with those by De Laubenfels and Adema(1998). Recognition of the genus Epicycas as discrete from Cycas is not justified on the basis ofcytology.
Key words:

5S rDNA, Cycas, cytotaxonomy, FISH.

The genus Cycas (Cycadaceae, Cycadales; Stevenson, 1992) primarily occurs in Australia
and the eastern parts of mainland Asia, with small numbers of species extending to Japan,
Melanesia, Micronesia, Polynesia and Africa. This genus is the most widely distributed and thelargest in the Cycadales (Stevenson et al., 1995; Hill et al., 2004). Although taxonomical studies
of Cycas sensu lato based on morphological comparisons have been advancing (Hill, 1992,
1994a, 1994b, 1995, 1996, 2004; De Laubenfels and Adema, 1998; Chen et al., 2004), cytological studies have been poorly published on this genus.
Recently the molecular-cytological technique of the FISH method has been applied to cytotaxonomic comparisons in certain Cycadales taxa, and it has been shown to be a powerful
method for analyzing intergeneric- and infrageneric-chromosomal relationships in Zamiaceae(Kokubugata and Kondo, 1998; Kondo and Tagashira, 1998; Tagashira and Kondo, 2001), Bowenia and Stangeria (Kokubugata et al., 2000a; Kokubugata et al., 2002b) and in Cycas(Kokubugata et al. 2002b).
The specific aims of the present study are to compare the 5S rDNA sites on somatic chromosomes of fourteen Cycas species by the FISH method for considering interspecific-taxonomical
relationships in this genus. In particular, the cytological characteristics of species referred to the164

G. Kokubugata and P. I. Forster

genus Epicycas by De Laubenfels and Adema (1998) are compared to those that they left in thegenus Cycas.
Materials and Methods

Plant Materials
In the present study, we investigated chromosomes of six species of Cycas sensu lato, C. diannanensis Z. T. Guan et G. D. Tao (Epicycas diannanensis (Z. T. Guan et G. D. Tao) De
Laubenfels), C. micholitzii Dyer (E. micholitzii (Dyer) De Laubenfels), C. cairnsiana F. Muell,
C. couttsiana K. D. Hill, C. megacarpa K. D. Hill and C. ophiolitica K. D. Hill by the fluorescence in situ hybridization (FISH) using 5S ribosomal DNA (5S rDNA) probes (Table 1). The 5S
rDNA distribution patterns on somatic chromosomes of the six species were compared withthose of eight species previously examined by Kokubugata et al. (2002b, Table 1), namely C. revoluta Thunb., C. taitungensis C. F. Shen, K. D. Hill, C. H. Tsou et C. J. Chen, C. wadei Merr., C.
siamensis Miq. (E. siamensis (Miq.) De Laubenfels), C. circinalis L., C. media R. Br., C. micronesica K. D. Hill and C. thouarsii R. Br.
Plants of six Cycas species cultivated in the Tsukuba Botanical Garden were investigated in
the present study (Table 1). Young leaflets of the cultivated plants were collected and pretreatedin 2 mM 8-hydroxyquinoline at 4°C for 20 h, and fixed in acetic ethanol (1 : 3) at 4°C for 24 hthen stored in 70% ethanol under 0°C for the present FISH experiments.
Probes and Labeling of PCR-amplified 5S rDNA for FISH
5S rDNA sequences were amplified by total genomic DNA of C. revoluta cultivated in the
Tsukuba Botanical Garden (accession no. TBG 140356) by the polymerase chain reaction (PCR)
Table 1.

Fourteen species Cycas sensu lato and their cytological characters compared in the present study.

De Laubenfels& Adema (1998)

Hill (1996, 2004)
Species

Cycas revoluta
Cycas revoluta
Cycas taitungensis Cycas taitungensis
Cycas wadei
Cycas wadei
Epicycas
Cycas diannanensis
diannanensis
Epicycas micholitzii Cycas micholitzii

Epicycas siamensis Cycas siamensis
Cycas cairnsiana Cycas cairnsiana
Cycas circinalis
Cycas circinalis
Cycas couttsiana Cycas couttsiana
Cycas media
Cycas media
Cycas megacarpa Cycas megacarpa
Cycas micronesica Cycas micronesica
Cycas ophiolitica Cycas ophiolitica
Cycas thouarsii
Cycas thouarsii

No.
Reference*
5S rDNA

Distribution

2nKaryotypeJapan
TaiwanPhilippines
China,
Laos,
VietnamStangerioides China,
Laos,
VietnamIndosinenses Thailand
Cycas
Australia
Cycas
India
Cycas
Australia
Cycas
Australia
Cycas
Australia
Cycas
Guam
Cycas
Australia
Cycas
Madagascar

22
22
22
22

12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m

2
2
2
2

a
a
a
b

22

12 t4 st4 sm2 m

4

b

22
22
22
22
22
22
22
22
22

12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m
12 t4 st4 sm2 m

4
4
4
4
4
4
4
4
4

a
b
a
b
a
b
a
b
a

SectionAsiorientales
Asiorientales
Wadeanae
Stangerioides*a. Kokubugata et al. (2002b). b. the present study.

Cytotaxonomy in Cycas

165

following Hizume (1995). The PCR-amplified 5S rDNA was labeled with digoxigenin(DIG-)14-dATP by the nick translation method. The labeled probe was dissolved in 50% formamide and 10% dextran sulfate (w/v) in 2X SSC. This hybridization mixture was denatured at
75°C for 10 min, then immediately chilled in cold water for 10 min and stored at 0°C.
Fluorescent in situ Hybridization (FISH)
The stored leaflets in 70% ethanol were macerated in an enzyme solution with 2% cellulase“Onozuka” RS (Yakult) and 1% pectoryase “Y-23” (Seisin) in distilled water (w/v; pH 4.5) at
36°C for 20 min before being washed in distilled water and 45% acetic acid at room temperaturefor 5 min. The macerated leaflet was placed on a slide and squashed in 45% acetic acid with acover slip by the standard squash method. The slide was dried at 36°C for 30 min after removing
the cover slip with a dry-ice block. It was digested with 0.1% RNase in 2X SSC at 36°C for 1 h,washed in 2X SSC for 10 min, treated in 4% paraformaldehyde in phosphate-buffered saline(PBS) for 5 min and washed in distilled water for 10 min. The slide was then dehydrated in 75%,
85% and 100% ethanol for 3 min each before being dried at 36°C for 20 min. The dried slide received 10 m l of the hybridization mixture and was covered with a cover slip and sealed with rubber cement. The probe DNA and chromosomal DNA on the slide were then denatured at 75°C
for 15 min on a hot plate and immediately incubated at 37°C in a humid chamber overnight for
DNA hybridization to occur. Following the hybridization, the cover slip was removed and theslide was twice washed in 4X SSC at 40°C for 10 min. The hybridized probes on chromosomal
DNA were detected with 20 m g/ml Anti-digoxigenin-fluorescein, Fab-fragmentavidin in 1%
bovine serum albumin in 4X SSC at 37°C for 1 h. The slide was twice washed in 4X SSC atroom temperature for 10 min before receiving 30 m l of a mixed solution of 10% PBS, 90% glycerol with 0.1g/ml 1,4-Diazabicyclo [2.2.2.] octan and 1 m g/ml propidium iodide (PI). The slide
was then mounted with a cover slip for counter staining and held at 4°C for 2 h at least.
The hybridization signals were made visible by Anti-digoxigenin-fluorescein, Fab-fragmentavidin as yellow fluorescence using the single band pass excitation filter (Zeizz filter set no. 2).
Non hybridized regions were made visible by PI as reddish fluorescence using the single band
pass excitation filter (Zeizz filter set no. 15). The two fluorescences were respectively captured bya Pixera Penguin 600 CL (DiRactorTM) camera as digital images. The two digital images wereoverlapped after being modified to the best contrast between signal and PI-counterstain using thesoftware of Lumina Vision OL (Mitani), and then the overlapped-color image was converted to a
black and white image. The signals were visible as lighter bands or dots in the black and white
images.
Description of Chromosomes
The chromosomes at mitotic metaphase were classified by arm ratio (Rlong arm length/
short arm length) following Levan et al. (1964). Median-centromeric (1.0R1.7), submediancentromeric (1.8R3.0), subterminal-centromeric (3.1R7.0), and terminal-centromericchromosomes (7.1R) are respectively abbreviated as m, sm, st and t chromosomes in this paper.
Results and Discussion

Chromosome Number and Karyotype
Six species of Cycas sensu lato (s. l.), namely C. diannanensis (E. diannanensis), C. mi-

166

G. Kokubugata and P. I. Forster

Fig. 1. FISH detected chromosomes of six species of Cycas sensu lato investigated in the present study. A. C. diannanensis (Epicycas diannanensis). B. C. micholitzii (E. micholitzii), C. C. cairnsiana. D. C. couttsiana.
E. C. megacarpa. F. C. ophiolitica. Lighter fluorescences indicated by allows are 5S rDNA sites. Scale bar indicates 10 m m.

cholitzii (E. micholitzii), C. cairnsiana, C. couttsiana, C. megacarpa and C. ophiolitica
observed in the present study showed the same chromosome number of 2n22. The chromosome number for C. ophiolitica confirmed that previously reported by Kokubugata et al. (2002c),
while those of C. diannanensis (E. diannanensis), C. micholitzii (E. micholitzii), C. cairnsiana, C. couttsiana and C. megacarpa are reported for the first time in the present study. The
chromosome numbers of the six species examined in the present study corresponded with thoseof eight species of Cycas s. l. previously reported, namely C. revoluta, C. taitungensis, C. wadei,
C. siamensis (E. siamensis), C. circinalis, C. media, C. micronesica and C. thouarsii (e.g.
Ishikawa, 1916; Ohri and Khoshoo, 1986; Peng et al., 1986; Paiva and Leitao, 1987; Kondo etal., 1995; Kokubugata et al., 2000b, 2002b, 2002c).
At mitotic metaphase karyotypes of the six species of Cycas s. l. consisted of four long st,
twelve t, four short sm (or m) and two short m chromosomes. The karyotypes of the six species
were similar to those of the other eight species previously reported (Hizume et al., 1992;
Kokubugata and Kondo, 1994; Hizume, 1995; Kondo et al., 1995; Hizume et al., 1998;
Kokubugata et al., 2000b, 2002b, 2002c).
In the present study C. diannanensis (E. diannanensis) and C. micholitzii (E. micholitzii)
exhibited a 5S rDNA site on the interstitial region near the terminal of two t chromosomes (Figs.
1A, B & 2D, E). On the other hand, C. cairnsiana, C. couttsiana, C. megacarpa and C. ophiolitica exhibited a 5S rDNA site on the interstitial region near the terminal in two t chromosomes andon the internal region near the centromere of the other two t chromosomes (Figs. 1C, D, E, F &2G, I, K, M).
Previously Kokubugata et al. (2002b) reported C. revoluta, C. taitungensis, C. wadei as
exhibiting a 5S rDNA site on the interstitial region near the terminal of two t chromosomes (Fig.
2A, B, C) similar to C. diannanensis (E. diannanensis) and C. micholitzii (E. micholitzii)(Fig. 2D, E). They also reported C. siamensis (E. siamensis), C. circinalis, C. media, C. mi-Cytotaxonomy in Cycas

167

Fig. 2. Ideograms of fourteen species of Cycas sensu lato compared in the present study. A. C. revoluta. B. C.
taitungensis. C. C. wadei. D. C. diannanensis (Epicycas diannanensis). E. C. micholitzii (E. micholitzii). F.
C. siamensis (E. siamensis). G. C. cairnsiana. H. C. circinalis. I. Cycas couttsiana. J. C. media. K. C.
megacarpa. L. C. micronesica. M. C. ophiolitica. N. C. thouarsii. Opened areas indicated long arms. Grayish
areas indicated short arms. Solid bands indicated 5S rDNA sites.

cronesica and C. thouarsii as exhibiting a 5S rDNA site on the interstitial region near the terminal in two t chromosomes and on the internal region near the centromere of the other two t chromosomes (Fig. 2F, H, J, L, N) similar to C. cairnsiana, C. couttsiana, C. megacarpa and C. ophiolitica.

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Taxonomic Background
Hill (1996, 2004) suggested a classification of genus Cycas into five sections based on morphological characters. According to Hill (1996, 2004), C. revoluta and C. taitungensis were classified into section Asiorientales with another Chinese species viz. C. panzihuaensis L. Zhou & S.
Y. Yang; C. wadei was classified into section Wadeanae with another Philippine species viz. C.
curranii (Schuster) K. D. Hill; C. diannanensis and C. micholitzii were classified into section
Stangerioides with another 26 species occurring from China to India; C. siamensis was classified
into section Indosinenses with thirteen other species occurring from China to India; and C. circinalis, C. cairnsiana, C. couttsiana, C. media, C. megacarpa, C. micronesica, C. ophiolitica andC. thouarsii were classified into section Cycas with 46 species occurring in Australia, Melanesia,
Micronesia, Polynesia and Africa. Moreover, Hill (2004) recognized that there were two major
phylogenetic groups in the genus Cycas based on molecular and morphological characters: one
consisted of sections Asiorientales, Wadeanae and Stangerioides, and the other consisted of sections Indosinenses and Cycas.
On the other hand, De Laubenfels and Adema (1998) described a new genus Epicycas De
Laubenfels with the type of Epicycas micholitzii (Dyer) De Laubenfels. They distinguished thetwo genera Cycas and Epicycas on the basis of the former with well developed trunks and the latter with underground trunks. According to De Laubenfels and Adema (1998), C. diannanensis,
C. multipinnata and C. siamensis sensu Hill (2004) would be classified into the genus Epicycas
and respectively treated as E. diannanensis, E. multipinnata and E. siamensis. The other eleven
species would remain classified into the genus Cycas. However, Chen et al. (2004) suggested thatgenus Epicycas was not a natural group based on morphological characters and the genus has notgained taxonomic recognition.
Taxonomic Conclusions
In the present FISH comparison, the fourteen species can be classified into two cytologicalgroups: one is characterized by two 5S rDNA sites in a mitotic complement, and consists of C.
revoluta, C. taitungensis, C. wadei, C. diannanensis (E. diannanensis) and C. micholitzii (E.
micholitzii); the other is characterized by four 5S rDNA sites in a mitotic complement and consists of C. siamensis (E. siamensis), C. circinalis, C. cairnsiana, C. couttsiana, C. media, C.
megacarpa, C. micronesica, C. ophiolitica and C. thouarsii. The two cytological groups based on
the number of the 5S rDNA site agree with the two phylogenetic groups based on morphologicaland molecular data suggested by Hill (2004). The two cytological groups do not support therecognition of two genera suggested by De Laubenfels and Adema (1998) because C. diannanensis (E. diannanensis) and C. multipinnata (E. multipinnata) are cytologically close to C. revoluta, C. taitungensis and C. wadei; and C. siamensis (E. siamensis) is cytologically close to C.
circinalis, C. cairnsiana, C. couttsiana, C. media, C. megacarpa, C. micronesica, C. ophiolitica
and C. thouarsii.
In conclusion, the present FISH study does not support the taxonomic treatment of De
Laubenfels and Adema (1998), but does support the phylogenetic hypothesis of Hill (1996,2004).
Acknowledgements
The Authors are grateful to Prof. K. Kondo (Hiroshima Univ.) for critical comments. This

Cytotaxonomy in Cycas

169

study was conducted as a part of “Collection Building and Natural History Studies in Asia andthe Pacific Rim” fundamentally supported by the National Science Museum, Tokyo.
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