全 文 ：Journal of Systematics and Evolution 46 (4): 490–498 (2008) doi: 10.3724/SP.J.1002.2008.08021
(formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com
A cytogenetic study of five species in the genus Osmunda
1Shou-Zhou ZHANG 1Zi-Can HE * 2Chen-Rui FAN 1Bin YAN
1(Shenzhen Fairylake Botanical Garden, Shenzhen 518004, China)
2(Shenzhen Middle School, Shenzhen 518001, China)
Abstract In the present study, obervation was made on chromosome morphology and behavior during meiosis
of spore mother cells (SMCs) for five species in the genus Osmunda: O. angustifolia Ching, O. japonica Thunb.,
O. vachellii Hook., O. banksiifolia (Presl) Kuhn., and O. mildei C. Chr. The chromosome number of root tip cells
of the five species is uniformly 2n=44. Chromosome pairing and synapsis were normal during meiosis and the
common configurations at metaphase I were circular bivalents in O. angustifolia, O. japonica, O. vachellii and O.
banksiifolia. Trivalents and univalents were occasionally observed in O. banksiifolia, while univalents at meta-
phase I, and chromosome bridges and fragments were observed at anaphase II in O. angustifolia. It is suggested
that translocation and inversion are responsible for the phenomenon observed. No chromosome pairing and syn-
apsis were observed in O. mildei from prophase I to metaphase I, and they resulted in abnormal chromosome
behavior: more than 80% of the SMCs showing lagging chromosomes and unequal segregation of chromosomes.
The spores produced were almost sterile because of abnormal chromosome constitution. Based on the departure
from the normal homologous chromosome pairing and synapsis, it is suggested that Osmunda mildei might be an
interspecific hybrid.
Key words chromosome behavior, meiosis, Osmunda.
Osmunda L. (Osmundaceae) is an ancient fern
genus that originated in the Triassic (Foster & Gifford,
1974). It comprises 15 species, of which eight are
distributed in China (Li et al., 2003). Shenzhen is
situated in southern subtropical region at 22˚27′–
22˚52′ N and 113˚46′–114˚37′ E. Five species are
recorded in this area: O. angustifolia Ching, O. ja-
ponica Thunb., O. vachellii Hook., O. mildei C. Chr.
and O. banksiifolia (Presl) Kuhn. Among them, O.
japonica and O. vachellii are widely distributed, while
the other three occur only in special habitats. Os-
munda mildei, a rare species, was discovered in Hong
Kong more than 100 years ago, but was mis-identified
as O. bipinnata L. by Hooker in 1857. However,
Christensen (1904) described it as a new species, O.
mildei C. Chr. (Li et al., 2003). It was believed to be
extinct, because it had not been found in its type
locality and neighboring areas until we found it on
three sites in Shenzhen among shrubs. It occurred at
an altitude of 200–400 m, and the total number of
individuals found was no more than 10. At the same
time, O. mildei was collected again near its type
locality. A new site with only one individual was
reported in Mt. Qiyun, Jiangxi Province where O.
mildei, O. japonica and O. vachellii occurred together.
Since Guignard (1899) reported 2n=44 for O.
regalis (Löve et al., 1977), the chromosome numbers
or karyomorphology of 15 taxa have been recorded,
all with 2n=44 (He et al., 2006). The chromosome
configuration of SMCs at metaphase I and the chro-
mosome number of somatic cells have been used for
identification of natural hybrids and artificial mo-
noploids, and for investigation of basic chromosome
numbers of homosporous ferns and the origin of
ancient polyploids (Klekowskj & Baker, 1966; Kle-
kowskj, 1973; Löve et al., 1977; Gastony & Darrow,
1983; Kawakami et al., 2003). The 2n=44 of O.
japonica and O. banksiifolia was reported from Japan
(Hirabayashi, 1963; Tatuno & Yoshida, 1966, 1967;
Mitui, 1968; Takei, 1988) and karyomorphology and
chromosome pairing of artificial monoploids were
also investigated (Takei, 1988; Kawakami et al., 1999;
Kawakami et al., 2003). All these studies were fo-
cused on the chromosome configuration of diakine-
sis/metaphase I to analyze chromosome pairing and
further to determine the chromosome number, without
studying the chromosome pairing behavior at pro-
phase I. Recently we studied the chromosome behav-
ior of Osmunda at meiosis and found that no pairing
of homologous chromosomes occurred at metaphase I
in O. mildei. It was different from the abnormal
meiosis of three types of SMCs in ferns, and was also
different from that of SMCs of artificial monoploids
of Osmunda (Kawakami et al., 2003). Was it because
synapsis of homologous chromosomes disappeared in

———————————
Received: 19 February 2008 Accepted: 30 May 2008
* Author for correspondence. E-mail: .
ZHANG et al.: A cytogenetic study of five species in Osmunda

491
advance or no homologous chromosomes existed at
all? Or was it a special meiosis like mitosis? A sys-
tematic study on cytology of Osmunda was carried out
to explore why no pairing of homologous chromo-
somes occurred in SMCs of O. mildei. The chromo-
some behavior at mitotic prophase and meiotic pro-
phase I was observed, and the studies on chromosome
morphology and behavior at mitosis have been pub-
lished (He et al., 2006). The goals of this study are:
(1) to observe the behavior of homologous chromo-
somes at leptotene to diakinesis in SMCs, and (2) to
reveal differences between the species in chromosome
behavior and morphology by studying the chromo-
some configuration during meiosis in SMCs.
1 Material and Methods
Samples of the five Osmunda species were all
collected from Shenzhen (Table 1). To obtain SMCs
at a proper stage for meiotic analysis, materials were
collected and observed in the first ten days of April.
The meiotic process was observed through the method
of cell-wall degradation using a hypotonic treatment,
followed by Giemsa staining (He et al., 2006). Each
slide was washed for several seconds and examined
using a Leitz compound microscope.
2 Results
2.1 Osmunda vachellii
The chromosome number of root tip cells at mi-
tosis was 2n=44 (Fig. 1). At prophase I slender twisted
unineme chromosomes gradually became thick,
straight and short (Fig. 2). At leptotene, chromatin
condensed into unineme chromosomes, which formed
a tangled reticulum with chromomeres easily seen
(Fig. 3). At zygotene, homologous chromosomes
began to pair simultaneously at several points along
the chromosome length (Fig. 4). At pachytene, 22
bivalents were not easily distinguished from each
other in morphology and structure, but it was easy to
observe the complex of bivalents due to the synapsis
of homologous chromosomes (Fig. 5). At diplotene, it
was difficult to distinguish some bivalents although
the chromosomes had thickened and shortened (Fig.
6). At diakinesis (Fig. 7) and metaphase I (Fig. 8),
chiasmas were present. The average chromosome
configuration was 22 II (Table 2). The leptotene-
zygotene lasted for 48 hours. At anaphase I, when
homologous chromosomes separated, 22 chromo-
somes appeared in the daughter cells (Fig. 9). At
telophase, when chromosomes uncoiled into chroma-
tin, nucleoli and nuclear membranes reappeared, and
dyads formed. After the first meiotic division, cell
walls formed, and cytokinesis was of a successive
type. At prophase II, chromatin in the daughter nuclei
of each dyad condensed and coiled into chromosomes
again. At metaphase II, 22 chromatids could be easily
seen (Fig. 10). During telophase II, when chromo-
somes uncoiled into chromatin, nucleoli and nuclear
membranes reappeared, and a tetragonal configuration
of tetrads formed.
2.2 Osmunda banksiifolia
The chromosome number for root tip cells of O.
banksiifolia was 2n=44 (Fig. 11). The chromosome
configuration at early prophase I (Fig. 12), diakinesis
and metaphase I indicated that the pairing and synap-
sis of homologous chromosomes were normal (Figs.
13, 14). The chromosome behavior was similar to that
of O. vachellii. During diakinesis and metaphase I,
bivalents were dominant, while trivalents were occa-
sionally observed (Fig. 14), indicating that transloca-
tions occurred between non-homologous chromo-
somes. The average chromosome configuration was
0.05I+21.77II+0.14III (Table 2), and n=22 was clearly
shown at anaphase I (Fig. 15) and II (Fig. 16).
2.3 Osmunda japonica
The chromosome number for root tip cells of O.
japonica was 2n=44 (Fig. 17). The chromosome
behavior during meiosis was similar to that of O.
vachellii. At zygotene (Fig. 18), pairing between
homologous chromosomes began simultaneously at
several points along the chromosome length, and the
centromeres were obvious. At diplotene, repulsion
occurred between homologues of the bivalents (Fig.

Table 1 The origin of materials investigated
Species Locality Voucher
Osmunda banksiifolia (C. Presl.) Kuhn Mt. Wutong, Shenzhen, China (深圳梧桐山) Z. C. Chen & B. Yan (陈珍传, 闫斌) 011815
O. vachellii Hook. Mt. Wutong, Shenzhen, China (深圳梧桐山) Z. C. Chen & B.Yan (陈珍传, 闫斌) 011819
O. angustifolia Ching Meishajia, Shenzhen, China (深圳梅沙尖) B. Yan & G. D. Wang (闫斌, 王国栋) 050012
O. japonica Thunb. Mt. Wutong, Shenzhen, China (深圳梧桐山) Z. C. Chen & B. Yan (陈珍传, 闫斌) 010905
O. mildei C. Chr. Mt. Tianxin, Shenzhen, China (深圳田心山) Z. C. Chen & B. Yan (陈珍传, 闫斌) 0383

Journal of Systematics and Evolution Vol. 46 No. 4 2008 492
Table 2 The chromosome configuration at M1 in spore-mother cell
of five Osmunda species
Chromosome configuration
Species Cell number I II III
O. angustifolia 22 0.18 21.91
O. japonica 25 22.00
O. vachellii 25 22.00
O. banksiifolia 22 0.05 21.77
O. mildei 24 43.83 0.08

19), and pairing and synapsis were normal. At diaki-
nesis and metaphase I, 22 circular bivalents were
easily seen (Fig. 20), of which some were occasionally
interlocked (Fig. 21). At anaphase I and anaphase II,
the behaviour of chromosomes was normal, but
micronuclei and chromosome fragments were occa-
sionally observed (Fig. 22). At anaphase II, n=22 was
easily seen (Fig. 23). Micronuclei, lagging chromo-
somes and chromosome fragments occurred in some
SMCs, which might be associated with interlocked
bivalents from prophase I to metaphase I.
2.4 Osmunda angustifolia
The chromosome number of somatic cells of O.
angustifolia was 2n=44 (Fig. 24). Chromosome paring
and synapsis at leptotene, pachytene (Fig. 25), dip-
lotene (Fig. 26), diakinesis and metaphase I were
normal. At metaphase I, circular bivalents were
dominant, but a small number of rod bivalents (Fig.
27) and univalents were infrequently observed, with
the average chromosome configuration 0.18I+21.91II
(Table 2). At anaphase I, when homologous chromo-
somes separated, micronuclei and chromosome frag-
ments were occasionally observed (Fig. 28). At ana-
phase II, 22 chromosomes were clearly seen (Fig. 29),
and chromosome bridges and fragments were occa-
sionally found (Fig. 30). The spores with 1–3 micro-
nuclei accounted for 27%, indicating abnormalities
and abortion occurring during spore development of
O. angustifolia (Figs. 31, 32).
2.5 Osmunda mildei
The chromosome number of root tip cells was
2n=44 (Fig. 33). It took 48 hours for SMCs to finish
prophase I and metaphase I, as in the other four
species; the thread-like unineme chromosomes formed
a reticulum (Fig. 34). No chromosome pairing and
synapsis were observed (Figs. 35–39) during meiotic
prophase I to metaphase I.
The chromosomes did not exhibit motions such
as pairing, synapsis and repulsion. At metaphase I,
there were usually 44 univalents, but occasionally one
rod bivalent was observed (Fig. 39). The 44 univalents
randomly moved to two poles, and many lagging
chromosomes, difficult to discern in number, appeared
in 80% of the SMCs (Figs. 40–42). At anaphase I two
daughter cells formed with unequal numbers of chro-
mosomes, and some micronuclei were present at
anaphase II. However, occasionally morphologically
normal daughter cells were formed. When sister
chromatids separated in meiosis II, more than 80% of
cells had lagging chromosomes, chromosome bridges
or fragments. Micronuclei were observed (Figs. 43,
44), and besides tetrads, polyads were also formed
(Fig. 45). The spores were irregular in size and form
(Fig. 46), and thus could not germinate under normal
breeding conditions.
3 Discussion
The chromosome numbers of the five species
were uniformly 2n=44. This is consistent with the
previous reports (Tatuno & Yoshida, 1966; Takei,
1988). Studies on chromosome morphology and
behavior during meiosis of SMCs of four widespread
species indicate that their meiosis was normal, except
that trivalents were present in O. banksiifolia, possibly
due to translocation. One or two bivalents sometimes
completed synapsis in advance and subsequently
became univalents in O. angustifolia, which suggests
that some differences occur between the homologues.
The suggestion is supported by the presence of chro-
mosome bridges and fragments in anaphase II. In O.
mildei, however, no chromosome pairing occurred
from prophase I to metaphase I, which obviously
departs from the synapsis and pairing of homologous
chromosomes. It resulted in abnormal chromosome
behavior: lagging chromosomes and unequal segrega-
tion of chromosomes in more than 80% of the SMCs.
The resulted spores were almost sterile because of
abnormal chromosome constitution.
No chromosome pairing was observed in SMCs
of Asplenium aethiopicum, where meiosis ceased
when restitution nuclei formed. The SMCs later split
into two unreduced spores (Braithwaite, 1964). The
abnormal meiosis in A. aethiopicum is distinctly
different from that of O. mildei. The observation on
meiosis of SMCs of the artificial monoploid of O.
japonica showed that meiosis took place in its SMCs,
producing dyads. The meiosis was believed (Kawa-
kami et al., 2003) to be same as that in SMCs of some
ferns: the meiosis ceases in restitution nuclei, cytoki-
nesis does not take place, while fertile dyads are
produced in meiosis II (Braithwaite, 1964). In the
study of Kawakami et al. (2003), tetrads were ob-
served in some SMCs, but the chromosome behavior
ZHANG et al.: A cytogenetic study of five species in Osmunda

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Figs. 1–8. Chromosomes of root tip cells and meiosis of SMCs in Osmunda vachellii. 1, 2. Mitosis of root tip cells. 3–8. Meiosis of SMCs. 3.
Leptotene. 4. Zygotene. Thick arrowhead indicates a bivalent which is not synapsis, and thin arrowheads indicate synaptic bivalents. 5. Pachytene. 6.
Diplotene. 7. Diakinesis. 8. Metaphase I.
Journal of Systematics and Evolution Vol. 46 No. 4 2008 494


















































Figs. 9–20. Chromosomes of root tip cells and meiosis of SMCs in Osmunda vachellii, O. banksiifolia, and O. japonica. 9, 10. Meiosis of SMCs
in O. vachellii. 9. Anaphase I. 10. Anaphase II. 11–16. O. banksiifolia. 11. Mitosis of root tip cells. 12–16. Meiosis of SMCs. 12. Zygotene. 13, 14.
Diakinesis. Arrowheads show trivalents. 15. Anaphase I. 16. Anaphase II. 17–20. O. japonica. 17. Mitosis of root tip cells. 18–20. Meiosis of SMCs.
18. Zygotene. 19. Early Diplotene. 20. Diakinesis.
ZHANG et al.: A cytogenetic study of five species in Osmunda

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Figs. 21–33. Chromosomes of root tip cells and meiosis of SMCs in Osmunda japonica, O. angustifolia, and O. mildei. 21–23. Meiosis of SMCs
in O. japonica. 21. Diakinesis. Arrowhead shows interlocking of two bivalents. 22. Anaphase I. Arrowheads show lagging chromosomes. 23.
Anaphase II. 24–32. O. angustifolia. 24. Mitosis of root tip cells. 25–31. Meiosis of SMCs. 25. Pachytene. 26. Diplotene. 27. Early metaphase I. 28.
Anaphase I. Arrowhead shows lagging chromosome. 29, 30. Anaphase II. Arrowhead shows lagging chromosome. 31. Tetrad. 32. Abnormal spores.
33. Mitosis of root tip cells in O. mildei.
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Figs. 34–46. Meiosis of SMCs in Osmunda mildei. 34–36. Prophase I. 37–39. Metaphase I. Arrowhead indicates a rod bivalent. 40–42. Anaphase
I. 43, 44. Anaphase II. Arrowhead shows lagging chromosomes. 45. Telophase II. 46. Sterile spores.

ZHANG et al.: A cytogenetic study of five species in Osmunda

497
was not mentioned. The absence of chromosome
pairing in SMCs of O. mildei at metaphase I is similar
to that in the material investigated by Braithwaite
(1964) and in SMCs of the artificial univalents of O.
japonica, but the study of O. japonica failed to ob-
serve the prophase I. The abnormal cytogenetics is a
character of interspecific hybrid F1, and therefore O.
mildei is probably F1 of a natural interspecific hybrid,
which completes its life history in a particular habitat.
The North American Osmunda × ruggii is a natural
hybrid of O. cinnamomea and O. regalis (Tryon,
1940; Wagner 1974; Wagner & Wagner, 1978; Li &
Haufler, 1994). Osmunda lancea var. latipinnula is a
natural hybrid of O. lancea and O. japonica based on
the analysis of karyotypes (Tatuno & Yoshida, 1967).
Therefore, interspecific hybridization likely happens
in this genus.
Osmunda mildei has sporadic distribution and
coexists with O. vachellii and O. japonica. It was
suggested in a previous study that O. mildei might be
a hybrid of O. angustifolia and O. japonica based on
analysis of karyotype and leaf morphology of the three
taxa (He et al., 2006). The new locality of O. mildei in
Mt. Qiyun, Jiangxi Province casts doubt on this
conclusion since O. angustifolia is distributed only in
southern subtropical region. Careful field explorations
and comparative studies on morphology of the five
species indicate that O. mildei might be a sterile
interspecific hybrid between O. japonica and O.
vachellii. The hypothesis is supported by allozyme
analysis and chloroplast DNA sequences (unpublished
data). If O. mildei is an interspecific hybrid, it is
another example of homoploid hybrids. The origin of
O. mildei as an interspecific hybrid may explain why
few individuals were reported in the field.
Acknowledgements We thank Prof. Jian-Qiang LI
at Wuhan Botanical Garden for the constructive
comments on an early version of the manuscript; Prof.
De-Yuan HONG is gratefully acknowledged for his
helpful comments.
References
Braithwaite AF. 1964. A new type of apogamy in fern. New
Phytologist 63: 293–305.
Foster AS, Gifford EM. 1974. Comparative morphology of
vascular plants. California: W. H. Freeman and Company.
Gastony GJ, Darrow DC. 1983. Chloroplastic and cytosolic
isozymes of the homosporous fern Athyrium filix-femina
L. American Journal of Botany 70: 1409–1415.
He Z-C (何子灿), Li Y (李勇), Yan B (闫斌), Zheng M (郑苗),
Zhang S-Z (张寿洲). 2006. Karyotype analysis of five
species in Osmunda (Osmundaceae). Acta Phytotaxono-
mica Sinica (植物分类学报) 44: 617–626 .
He Z-C (何子灿), Zhang S-Z (张寿洲). 2005. Behavioral
particularity on meiosis of Osmunda mildei C. Chr.
Fairylake (仙湖) 12: 15–17.
Hirabayashi H. 1963. Chromosome numbers in Japanese
Osmunda. Journal of Japanese Botany 38: 332–333.
Kawakami SM, Kawakami S, Kondo K, Kato J, Ito M. 2003.
The sporogenesis in haploid sporophytes of Osmunda
japonica (Osmundaceae). International Journal of Plant
Sciences 164: 527–534.
Kawakami SM, Kondo K, Kawakami S. 1999. Analysis of
nucleolar organizer constitution by fluorescent in situ
hybridization (FISH) in diploid and artificially produced
sporophytes of the fern Osmunda japonica
(Osmundaceae). Plant Systematics and Evolution 216:
325–331.
Klekowskj EJ Jr. 1973. Sexual and subsexual in homosporous
pteridophytes: a new hypothesis. American Journal of
Botany 60: 535–544.
Klekowskj EJ Jr, Baker HG. 1966. Evolutionary significance of
polyploidy in Pteridophyta. Science 153: 305–307.
Li JW, Haufler CH. 1994. Phylogeny and population biology of
Osmunda species: insights from isozymes. American Fern
Journal 84 (4): 105–114.
Li WTC, Chau LKC, Wu H. 2003. Flora of Hong Kong
Pteridophyta. Hong Kong: Kadoorie Farm & Botanic
Garden.
Löve A, Löve D, Pichi Sermolli REG. 1977. Cytotaxonomical
atlas of the Pteridophyta. Vaduz: J Cramer.
Mitui K. 1968. Chromosomes and speciation in ferns. Science
Reports of the Tokyo Daigaku Section B 13: 285–333.
Takei M. 1988. Numeral stability and morphological instability
of chromosomes in spore calli of Osmunda japonica. Plant
Tissue Culture Letter 5: 61–65.
Tatuno S, Yoshida H. 1966. Karyologische untersuchungen
über Osmundaceae. I. Chromosomen der gattung Os-
munda aus. The Botanical Magazine (Tokyo) 79: 244– 252.
Tatuno S, Yoshida H. 1967. Karyological studies on
Osmundaceae II. Chromosome of the genus Osmunda-
strum and Plenasium. The Botanical Magazine (Tokyo)
80: 130–138.
Tryon RM Jr. 1940. An Osmunda hybrid. American Fern
Journal 30: 65–68.
Wagner FS. 1974. A problematic royal fern hybrid, Osmunda ×
ruggii (Abstr.). American Journal of Botany 61 (suppl.):
39.
Wagner FS Jr, Wagner DH. 1978. New observation on the royal
fern hybrid Osmunda × ruggii. Rhodora 80: 92–106.

Journal of Systematics and Evolution Vol. 46 No. 4 2008 498
五种紫萁属植物的细胞遗传学研究
1张寿洲 1何子灿* 2范晨瑞 1闫 斌
1(深圳市仙湖植物园 深圳 518004)
2(深圳市深圳中学 深圳 518001)

摘要 对紫萁属Osmunda五种植物: 狭叶紫萁O. angustifolia Ching、紫萁 O. japonica Thunb.、华南紫萁O. vachellii Hook.、粗
齿紫萁O. banksiifolia (Presl) Kuhn和粤紫萁O. mildei C. Chr.的体细胞染色体形态和孢子母细胞减数分裂时染色体的行为进行
了研究。五种紫萁属植物的体细胞染色体数目均为2n=44, 孢子母细胞减数分裂过程中, 狭叶紫萁、紫萁、华南紫萁和粗齿
紫萁染色体配对和联合行为正常, 中期I染色体构型多为环状二价体, 粗齿紫萁偶尔可观察到三价体和单价体, 狭叶紫萁中
期I偶可观察到1–2个提早分离的单价体, 后期II可观察到染色体桥和断片, 据此推测易位和倒位等染色体畸变作用在紫萁属
植物物种形成和演化过程中具有重要意义。粤紫萁是华南分布的一个特有珍稀种, 孢子母细胞减数分裂前期到中期无染色体
配对和联会, 导致染色体后期行为异常, 80%的孢子母细胞有落后染色体和不均等分离现象, 形成的孢子几乎完全败育, 基
于粤紫萁减数分裂显著偏离正常的同源染色体配对和联会现象, 结合核型方面和形态学方面证据, 认为粤紫萁是一个杂交
种。
关键词 染色体行为; 减数分裂; 紫萁属