全 文 ：二倍体铁破锣的核型及四倍体细胞型的首次发现
杨 亲 二
(中国科学院植物研究所系统与进化植物学开放研究实验室　北京 100093)
Correction of karyotype of diploid Beesia calthifolia
and discovery of a tetraploid cytotype
YANG Qin-Er
(Laboratory of Systemat ic and Evolut ionary Botany , Insti tute of Botany , the Chinese Academy of S ciences , Beijing 100093)
Abstract　In this paper , the chromosomes of Beesia calthifolia were re-examined.In the 40
plant individuals of the population f rom Xinning County , Hunan Province , central China ,
one w as found to be tet raploid with the karyotype formula of 2n=4x=32=16m+8sm +4st
+4t , and the remaining w ere all found to be diploids with the karyotype formula of 2n=2x
=16 =8m +4sm +2st +2t.All the 10 individuals of the population f rom Cang shan
M ountain , Dali City ,Yunnan Province , southwestern China , were unexpectedly found to be
tetraploids w ith the kary otype formula of 2n=4x =32=16m +8sm +4st+4t.Tetraploid
cyto type w as reported in this species for the fi rst time.Based on the results and those
previously reported , it is considered that there may exist some errors in the results of the
karyo type analysis of this species previously reported by Shang(1985).He might have at
least mistakenly recognized the centromere position of the fourth pai r of chromosomes.This
pair of chromosomes should have subterminal rather than median centromeres.Furthermo re ,
the karyotypic differences of B.cal thi folia and B .del tophyl la were analyzed and the
sy stematic position of the genus Beesia was discussed in detail.
Key words　Beesia calthifolia;Karyotype;Sy stematic position
Beesia Balf.et W.W.Smith is a small genus of the Ranunculaceae , including only tw o
very closely related species.B .calthi folia (Maxim.ex Oliv.)Ulbr.is w idely dist ributed in
China and no rthern Myanmar , while B .deltophylla C.Y.Wu ex Xiao is endemic to
southeastern Xizang s Medog County of China(Xiao , 1979).
Both species have been cy tologically studied and found to have the same chromosome
number of 2n=16(Yang et al., 1995 ;Shang , 1985), but their kary otypes reported by
those authors are somewhat dif ferent.Shang (1985) reported the karyo type of B .
calthi fol ia as consisting of 10 m-, 4 sm-and 2 t-chromosomes , w ith five large pairs(the f irst
to fif th)having median centromeres(the fif th pair having an arm ratio of 1.22), tw o pairs
(the six th and the eighth)having submedian centromeres , and one pair(the seventh pai r)
having subterminal cent romeres.Yang et al.(1995) reported the karyotype of B .
del tophyl la as consisting of 10 m-, 4 st- and 2 t-chromosomes , with f ive pairs(the first to
fifth)having median centromeres(the fifth pair having an arm ratio of 1.66), tw o pai rs(the
six th and seventh)having subterminal cent romeres , and one pair(the eighth)having
terminal centromeres.As pointed out in the paper on the karyotype of B.deltophy lla cited
中国科学院资源与环境重点项目资助的课题。
1998-08-24收稿 , 1998-11-06收修改稿。
植 物 分 类 学 报　37(1):1～ 9 (1999)
Acta Phytotaxonomica Sinica
Takhtajan A , 1987.Systema Magnoliophytorum.Sectio Leninopolitana, Lening rad:Officina Edito ria
“ Nauka”
Tamura M , 1966.Morpho logy , eco logy and phylo geny of the Ranunculaceae Ⅵ .Sci Rep Osaka Univ , 15:13
～ 35
Tamura M , 1990.A new classification of the family Ranunculaceae 1.Acta Phy totax Geobo t , 41:93～ 101
Tamura M , 1995.Ranunculaceae.In:Hiepko P.Die Natǜrlichen Pflanzenfamilien.Zwei Aufl , 17a(4).
Berlin:Duncker and Humblo t
Tanaka R, 1971.Types of resting nuclei in O rchidaceae.Bot Mag(Tokyo), 84:118～ 132
Tanaka R, 1977.Recent kary otype studies.In:Ogaw a K et al.Plant Cy tology.Tokyo:Asakura Shoten
Ulbrich E , 1929.Ranunculaceae novae vel criticae Ⅷ .Notizbl Bo t Gart Berl , 10:863 ～ 880
Wang W-T(王文采), 1979.Ranunculaceae.F l Reip Pop Sin.Vol 27.Beijing:Science Press
Xiao P-G(肖培根), 1979.Beesia.F l Reip Pop Sin.Vol 27.Beijing:Science P ress
Yang Q-E(杨亲二), 1995.On the chromosomes of Calathodes and its sy stematic positio n.Acta Phy to tax
Sin(植物分类学报), 33:453～ 460
Yang Q-E(杨亲二), Gong X(龚询), Gu Z-J(顾志建), Wu Q-A(武全安), 1993.A karyomorphological
study of five species in the Ranunculaceae from Yunnan , w ith a special consideration on systematic
positio ns of Asteropyrum and Calathodes.Acta Bo t Yunn , 15:179 ～ 190
Yang Q-E(杨亲二), Gu Z-J(顾志建), Sun H(孙航), 1995.The kary otype of Beesia deltophylla and its
systematic significance.Acta Phy to tax Sin(植物分类学报), 33:225 ～ 229
Ying T-S(应俊生), Zhang Y-L(张玉龙), 1994.The Endemic Genera of Seed Plants of China.Beijing:
Science Press
摘要　本文重新检查了铁破锣的核型。来自湖南新宁的 40 个个体中 , 1 个个体为四倍体 , 其核型公式
为 2n=4x=32=16 m+8sm+4st+4t ;其余个体为二倍体 , 其核型公式为 2n=2x=16=8m+4sm+2st
+2t。来自云南大理的 10 个个体全为四倍体 , 其核型公式为 2n=4x=32=16 m+8sm+4st+4t。据此
认为商效民(1985)报道的该种的核型分析结果有误。他至少将第 4 对染色体的着丝点位置辨认错了。
该对染色体不具有中部着丝点而实际上具有近端部着丝点。本文还比较了铁破锣和角叶铁破锣的核型
差异 , 并详细讨论了铁破锣属的系统位置。
关键词　铁破锣;核型;系统位置
1 期 杨亲二等:二倍体铁破锣的核型及四倍体细胞型的首次发现 9　
above , there may exist some errors in Shang s results of the karyotype analysis of B .
calthi fol ia.He might have at least mistakenly recognized the position of the centromeres of
the fourth pairs of chromosomes.This pair of chromosomes may have subterminal rather
than median centromeres , the satellites on the sho rt arms indicated by Shang being actually
short arms.Therefo re , it is quite necessary to re-examine the chromosomes of this species to
determine its karyotypic consti tut ion.The paper is to repo rt the results of the re-examination
and the unexpected discovery of a tetraploid cyto type in this species.
1　Materials and Methods
Two populations , one f rom Zhiyunshan Mountain , Xinning County , Hunan Province ,
central China , which includes 40 plant individuals , the other f rom Cangshan M ountain , Dali
City , Yunnan Province , southwestern China , which includes 10 plant individuals , were
collected from the field and then transplanted in the greenhouse of the Laborato ry of
Systematic and Evolutionary Bo tany , Insti tute of Botany , the Chinese Academy of Sciences
in Beijing and in that of the Botanic Garden , Kunming Institute of Bo tany , the Chinese
Academy of Sciences in Kunming , respectively.The voucher specimens , Luo Yi-bo 945 and
Yang Qin-er 9415 , were both deposited in the Herbarium , Institute of Botany , the Chinese
Academy of Sciences(PE).
For the observation of the chromosomes , actively growing roots w ere harvested and then
pret reated with 0.1% colchcine for 2.5 hours at room temperature.They were then f ixed in
Carnoy s f luid(absolute ethanol∶glacial acetic acid=3∶1)at 4℃ for 30 minutes.After being
macerated in the mix ture of 1 mol/ L HCl and 45% acetic acid(1∶1)at 60℃ fo r three
minutes , they were then stained in 1% aceto-orcein and squashed.
Karyomorphological classification of resting nuclei and mitotic prophase chromosomes
followed Tanaka(1977 , 1971).The symbols fo r the description of kary otypes follow ed
Levan et al.(1964).
2　Results
The two poulations studied were similar in karyomo rphology of resting nuclei.In
resting nuclei(Fig.2:A), numerous chromocenters were observed.The o ther regions w ere
also stained well but unevenly.Thus , the resting nuclei belonged to the complex
chromocenter type.
Table 1　Parameters of chromosomes in Beesia calthifolia from Xinning County , Hunan Province(Luo Yi-bo 945)
Chromosome
No.
Diploid
(2n=2x=16=8m+4sm+2st+2t)
Tet raploid
(2n=4x=32=16m+8sm+4st+4t)
Relat ive length Arm rat io Type Relat ive length Arm rat io Type
1 9.61+7.61=17.22 1.26 m 9.72+7.97=17.69 1.22 m
2 8.40+7.46+15.86 1.13 m 8.28+7.32=15.60 1.13 m
3 8.10+6.47=14.57 1.25 m 6.88+6.67=13.55 1.03 m
4 7.28+5.32=12.60 1.37 m 6.99+5.58=12.57 1.25 m
5 8.43+3.26+11.69 2.58 sm 7.28+3.57=10.85 2.04 sm
6 7.07+3.26=10.33 2.16 sm 7.15+3.07=10.22 2.33 sm
7 7.09+2.05=9.14 3.45 st 8.15+2.02=10.17 4.03 st
8 7.65+0.93=8.58 8.22 t 8.30+1.05=9.35 7.90 t
2　 植　物　分　类　学　报 37 卷
Fig.1　Phytomicrographs of metataphase chromosomes in diploid(A , C)and tet raploid(B , D)
Beesia ca lth ifolia from Xinning County , Hunan Province
The tw o populations studied w ere also similar in karyomorphology of mi totic prophase
chromosomes.In prophase chromosomes(Fig.2:B), hetero- and euchromatic segments w ere
distinguishable but their boundaries were no t distinct and the heterochromatic segments w ere
distributed in the distal and interstitial as well as proximal regions.Thus , the prophase
chromosomes belonged to the intersti tial type.
1 期 杨亲二等:二倍体铁破锣的核型及四倍体细胞型的首次发现 3　
Fig.2　Phytomicrographs of resting nuclei(A), prophase chromosom es(B)and metaphase ch romosomes(C , D)
in tet raploid Beesia ca lthi folia f rom Dali County , Yunnan Province
4　 植　物　分　类　学　报 37 卷
In the populat ion f rom Xinning County , Hunan Province , 39 individuals w ere found to
be diploids w ith the chromosome number of 2n=16(Fig.1:A , C), ranging in length from
11.89μm to 5.93μm , but one individual w as tet raploid with the chromosome number of 2n
=32(Fig.1 :B , D), ranging in length f rom 13.07 μm to 6.91μm.The diploid plants had
8 m-, 4 sm -, 2 st- and 2 t-chromosomes(Table 1).The only tetraploid plant had 16 m-, 8
sm-, 4 st- and 4 t-chromosomes(Table 2).
All the 10 individuals of the populat ion from Cang shan Mountain , Dali City , Yunnan
Province , were found to be tet raploids w ith the chromosome number of 2n=32(Fig .2 :C ,
D), ranging in leng th f rom 10.34 to 5.66μm.The karyotype was formulated as 2n=32=
16m+8sm+4st+4t(Table 2).
Table 2　Parameters of chromosomes in Beesia calthifolia from Dali City , Yunnan Province(Yang Qin-er 9415)
Chromosome
No.
2n=4x=32=16m+8sm+4st+4t
Relative length Arm ratio Type
1 9.11+7.61=16.72 1.20 m
2 8.95+7.11=16.06 1.26 m
3 7.24+6.53=13.77 1.11 m
4 6.80+5.55=12.35 1.23 m
5 8.01+3.19=11.20 2.51 sm
6 6.92+3.25=10.17 2.13 sm
7 8.34+2.21=10.55 3.77 st
8 8.04+1.13=9.17 7.12 t
Fig.3　Dist ribut ion of the two species in Beesia and their ploidy
△ B.del tophyl la 　○ B.cal thi fol ia
D=diploid;T=tetraploid
1 期 杨亲二等:二倍体铁破锣的核型及四倍体细胞型的首次发现 5　
3　Discussions
3.1　Karyotype of the diploid Beesia calthifolia
The present results of the karyo type analysis of the diploid Beesia calthifolia are
somewhat dif ferent f rom those reported by Shang(1985).Shang reported the karyotype as
consisting of 10 m-, 4 sm- and 2 t-chromosomes , the fif th pair having an arm rat io of 1.22.
In this study , the karyotype w as found to consist of 8 m-, 4 sm-, 2 st- and 2 t-
chromosomes , the fifth pai r of chromosomes of this species having an arm ratio of 2.58.In
the karyotype of Beesia deltophyl la , although the fif th pair of chromosomes is categorized as
m-chromosomes , their arm ratio has reached 1.66.As seen from the chromosome figures in
Shang s paper , the fourth pair of chromosomes should have subterminal cent romeres , the
satellites as recognized by Shang actually being the sho rt arms.If so , the karyotype of the
materials studied by Shang actually also consists of 8 m-, 4 sm-, 2 st- and 2 t- chromosomes ,
which is basically the same as that of the materials studied here.The results are further
confirmed to g reat degree by those of the karyo type analyses of the tetraploid plants.The
only tet raploid plant f rom the same population of Hunan as the diploid plants and the 10
tetraploid plants f rom the population of Yunnan all have 16 m-, 8 sm-, 4 st- and 4 t-
chromosomes.More importantly , the chromosome parameters of the diploid and tetraploid
plants are quite near to each o ther.Thus , their uniformi ty in kary otype consti tut ion should
not have resulted f rom artificial erro rs in the measurement of chromosomes.
3.2　Main differences of the karyotype of the genus Beesia and those of the other genera in
the tribe Cimicifugeae
There exist some dif ferences betw een the kary otypes of B .deltophy lla and B .
calthi fol ia.Fo r example , the fifth chromosome pair in the fo rmer species belongs to m-
chromosomes , even though the arm ratio has reached 1.66(Yang et al., 1995), while the
fifth pair in the latter belong to sm-chromosomes , with the arm rat io being over 2.00(Table
1).Furthermo re , there are tw o very small satelli tes on the short arms of the eighth
chromosome pair in B .deltophylla , while no satellites are observed on the counterparts in
B .calthi folia .Nevertheless , basically , the karyo types of the tw o species under question
are very similar in comparison w ith the other groups in the Ranunculaceae.
Compared with the other genera in the tribe Cimicifugeae , the genus Beesia is
karyo typically distinguished mainly in the fifth chromosome pair.This pair in Beesia has an
arm ratio of 1.66(in B.deltophy lla)o r usually over 2.00(in B .calthi folia), while in the
remaining genera of the t ribe this pair belong s to typical m-chromosomes , with the arm ratio
being around 1.20(Yang et al., 1993;Hasegaw a , Peng , 1991;Blair , 1975;Hasegaw a ,
1970b;Kaw ano et al.,1966;Kurita , 1957).Thus , this pair of chromosomes may serve as
the chromosomal marker of the genus Beesia , by which it is cytologically distinguishable
from the other genera in the t ribe Cimicifugeae.
3.3　Polyploidy in the genus Beesia
Polyploidy is very rare and might have played an unimportant role in the speciation of
the t ribe Cimicifugeae.Up to date , tetraploid cy totype has been repo rted only in Cimici fuga
foet ida f rom Thimphu of Bhutan(Hasegaw a , 1969).So this is the second time to record a
tetraploid cyto type in this t ribe.Although in the 40 plant individuals from the population of
Xinning County , Hunan Province , only one individual is tet raploid , all the 10 individuals
from the population of Dali Ci ty , Yunnan Province , have stable chromosome number of 2n=
6　 植　物　分　类　学　报 37 卷
4x=32 , and thus are all tet raploids.Gross-morphologically , the diploid plants show no
signif icant differences f rom tet raploid ones , though there may exist some dif ferences in their
pollen g rain size and stomatal size , as has been observed in Cim ici fuga foetida by Hasegawa
(1970a , 1969).Beesia calthi folia is w idely distributed in China s northern Yunnan ,
Sichuan , Guizhou , northern Guangxi , western Hunan , western Hubei , southern Shanxi and
southern Gansu , and northern Myanmar(Fig .3)(Ying &Zhang , 1994;Xiao , 1979).As
shown in Fig .3 , the tet raploid cyto type occurs on the southw estern margin of the
distribution area of Beesia cal thi folia , which is si tuated wi thin the Hengduan Mountain
region , where the seed plants in many g roups show strong gross-morphological
differentiat ion.It should be of great interest to use a cy togeographical approach to address
the chromosomal dif ferent iation of Beesia calthi folia in reference to it s gross-morphological
differentiat ion.
3.4　Systematic position of the genus Beesia
The systematic position of the genus Beesia has long been a controversial mat ter.When
Balfour and W.W.Smith(1915)established this genus based on B.cordata Balfour and
W.W.Smith , they considered that it is related to Glaucidim and Hydrast is.This
viewpoint was never accepted.Hutchinson(1923)thought that Beesia might be actually
closely allied to Souliea and Cimici fuga and established a subtribe Cimicifuginae to
accommodate them , and simultaneously pointed out that B .cordata is a later synonym of
Cimici fuga calthaefolia Maxim.ex Oliv..Ulbrich(1929)agreed w ith Hutchinson and
formally proposed that the correct scientific name should be spelled as B.calthi folia
(Maxim.ex Oliv.)Ulbr., and pointed out that Beesia and Cimici fuga are tw o closely
related but qui te distinct genera.This opinion has been basically accepted by most of the later
authors , such as Janchen(1949), Buchheim(1964)and W.T .Wang(1979).Tamura
(1966), however , put Beesia together w ith Caltha , Trollius , Megaleranthis and
Calathodes in the t ribe T rollieae under the subfamily Helleboroideae , and regarded that
Beesia may represent the intermediate type between the t ribe Trollieae and the t ribe
Cimicifugeae(including Anemonopsis , Souliea , Cim ici fuga , Actaea in the sense of
Tamura).Very recent ly , Tamura(1995 , 1990)made a new classif icat ion of the family
Ranunculaceae.He subdivided the subfamily Helleboroideae into 4 tribes , i.e.Helleboreae
(including 3 subtribes , Calthinae , Beesinae and Helleborinae), Cimicifugeae , Nigellae and
Delphineae.He separated Beesia as an independent subtribe and put it betw een Calthinae
(including Caltha , Calathodes , Trol lius and Megaleranthis)and Helleborinae.Takhtajan
(1987)had nearly the same opinion as Tamura that Beesia might be close to Caltha ,
Calthodes and Megaleranthis.
Karyomorphologically , Beesia appears to be much more closely akin to Cim ici fuga and
i ts allies , such as Actaea and Souliea , than to Caltha and its possible allies , i.e.Calthodes ,
Trollius and Megaleranthis.In chromosome size , the chromosomes of Beesia are very
similar to those of Cimici fuga but signif icantly larger than those of Caltha , Calthodes ,
Trollius and Megaleranthis(Yang , 1995;Yang et al., 1995 , 1993;Lee &Yeau , 1985;
Blair , 1975;Hasegaw a , 1970b;Kawano et al., 1966;Kurita ,1965 , 1960 , 1958 , 1957).
In chromosome morphology , although there exist some differences betw een Beesia and
Cimici fuga and its allies as emphasized above , basically their chromosome mo rphology is
quite similar to each o ther but most different f rom that of Caltha , Calathodes , Troll ius and
Megaleranthis(Yang , 1995;Yang et al., 1995 , 1993;Lee & Yeau , 1985;Blai r , 1975;
1 期 杨亲二等:二倍体铁破锣的核型及四倍体细胞型的首次发现 7　
Hasegawa , 1970b;Kawano et al., 1966;Kurita , 1965 , 1960 , 1958 ,1957).In Beesia and
o ther genera of the tribe Cimicifugeae , the four largest chromosome pairs have median
centromeres , the fif th pair is also quite large and usually has median(in Anemonopsis ,
Souliea , Cimici fuga , Actaea)or rarely submedian centromeres(in Beesia calthi fol ia), the
tw o moderately large pai rs have submedian or subterminal cent romeres depending on the
species , and the smallest pair has very short but always clearly visible sho rt arms and
terminal cent romeres(Beesia , Anemonopsis , Souliea and Cim ici fuga), or simply has no
short arms(only in Actaea).In the Ranunculaceae , such combinations of karyotypic
characters are only found in this tribe.From karyomorphological evidence , therefore , i t
seems that Beesia might find i ts best sy stematic position in the tribe Cimicifugeae.
Acknowledgements　 I am very g rateful to Dr.Luo Yi-bo of Laboratory of Systematic and Evolutionary
Botany , Institute of Bo tany , the Chinese Academy of Sciences , fo r collecting living materials.
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