全 文 :广义报春苣苔属 (苦苣苔科) 的染色体
新计数及其分类学意义∗
刘瑞瑞1ꎬ2ꎬ SKOG Laurence E.4ꎬ 廖景平1ꎬ 周太久3∗∗ꎬ 温 放3
(1 中国科学院华南植物园ꎬ 广东 广州 510650ꎻ 2 中国科学院大学ꎬ 北京 100049ꎻ 3 中国科学院广西壮族自治区
广西植物研究所ꎬ 广西 桂林 541006ꎻ 4 植物学系ꎬ 史密斯学会ꎬ 自然历史国家博物馆ꎬ 华盛顿)
摘要: 报道了广义报春苣苔属 14种 3变种和 7个未定名种的染色体数目ꎬ 并对其近缘属———广义石山苣
苔属的 3个种也进行了细胞学研究ꎮ 结合最近的分类处理和系统发育假设ꎬ 对所得结果和以前发表的染色
体数据进行了综合分析ꎬ 结果表明: 广义报春苣苔属的染色体数目为 2n = 36ꎬ 基数为 x = 18ꎬ 表现出高度
稳定性ꎮ 染色体相对较小ꎬ 以中间着丝粒和亚中间着丝粒染色体为主ꎮ 尽管一些类群的染色体大小之间存
在微小差异ꎬ 但是染色体形态的均一性和染色体数目的一致性有力地支持分子系统学的研究结果ꎮ 另外ꎬ
相同的染色体数目及相似的染色体形态也表明广义报春苣苔属和广义石山苣苔属的亲缘关系较近ꎬ 与分子
系统学的结果一致ꎮ
关键词: 染色体数目ꎻ 染色体形态ꎻ 广义石山苣苔属ꎻ 广义报春苣苔属ꎻ 分类处理
中图分类号: Q 942 文献标识码: A 文章编号: 2095-0845(2014)01-013-09
New Chromosome Counts and Their Taxonomic Implications
in Primulina sensu lato (Gesneriaceae)
LIU Rui ̄Rui1ꎬ2ꎬ SKOG Laurence E.4ꎬ LIAO Jing ̄Ping1ꎬ ZHOU Tai ̄Jiu3∗∗ꎬ WEN Fang3
(1 South China Botanical Gardenꎬ Chinese Academy of Sciencesꎬ Guangzhou 510650ꎬ Chinaꎻ 2 University of Chinese Academy
of Sciencesꎬ Beijing 100049ꎬ Chinaꎻ 3 Guangxi Institute of Botanyꎬ Guangxi Zhuangzu Autonomous Region and Chinese
Academy of Sciencesꎬ Guilin 541006ꎬ Chinaꎻ 4 Botany Departmentꎬ MRC ̄166ꎬ Smithsonian Institutionꎬ
National Museum of Natural Historyꎬ Washingtonꎬ D.C. U.S.A)
Abstract: Chromosome numbers can be helpful in understanding the taxonomy and phylogenetic patterns within
plant groups. Primulina sensu lato ( s. l.)ꎬ a genus that has recently been redefined and expanded to include all spe ̄
cies of Chiritopsisꎬ two of Wentsaiboea and all of section Gibbosaccus of Chiritaꎬ has not been well documented cyto ̄
logically. In the present study we determined the chromosome numbers of fourteen speciesꎬ three varieties and seven
undescribed taxa of Primulina s. l. using conventional rapid squash techniques. In additionꎬ three species of its clo ̄
sest allyꎬ Petrocodon sensu lato ( s. l.)ꎬ were also investigated cytologically. With the exception of Primulina taba ̄
cumꎬ all counts are new. A high stability of chromosome numberꎬ 2n= 36ꎬ based on x= 18ꎬ was recorded in Primu ̄
lina s. l. Chromosomes were relatively small in sizeꎬ with mostly metacentrics and submetacentrics predominating. Al ̄
though slight variation in chromosome size occurs among taxaꎬ the overall homogeneity of chromosome morphology
植 物 分 类 与 资 源 学 报 2014ꎬ 36 (1): 13~21
Plant Diversity and Resources DOI: 10.7677 / ynzwyj201413044
∗
∗∗
Funding: The National Natural Science Foundation of China (31200246ꎬ 31200176ꎬ and 30900089)ꎻ the Technologic Support System
Running Special for Strategic Biological Resources of Chinese Academy of Science ( CZBZX ̄1)ꎻ the Science and Technology
Tackles Key Problem Project of Guilin ( 20070311 )ꎻ Basic Research Funding of the Guangxi Academy of Sciences
(12YJ25ZW013)
Author for correspondanceꎻ E ̄mail: glztj@ sina. com
Received date: 2013-03-09ꎬ Accepted date: 2013-05-29
作者简介: 刘瑞瑞 (1985-) 女ꎬ 博士研究生ꎬ 主要从事苦苣苔科植物的细胞学研究ꎮ E ̄mail: mangory@ 163. com
and pattern of basic chromosome number conforms with the results of molecular phylogenetic studiesꎬ which strongly
support the monophyly of Primulina s. l.ꎬ and also recent taxonomic treatment. Interestinglyꎬ the possession of the
same chromosome numbers together with similar chromosome morphologyꎬ indicates that Primulina s. l. and Petroc ̄
odon s. l. are closely relatedꎬ which is consistent with the molecular phylogeny of these genera.
Key words: Chromosome numbersꎻ Chromosome morphologyꎻ Petrocodon sensu latoꎻ Primulina sensu latoꎻ Taxo ̄
nomic treatments
Primulina sensu stricto ( s. s.)ꎬ belonging to
Tribe Didymocarpeae (Gesneriaceaeꎬ Cyrtandroideae)ꎬ
was established formally by Hance ( 1883) with a
single speciesꎬ Primulina tabacum Hance. Howeverꎬ
two recent molecular studies of phylogenetic relation ̄
ships of Chirita D. Don and associated genera resul ̄
ted in some taxonomic changes (Wang et al.ꎬ 2011ꎻ
Weber et al.ꎬ 2011a). The monotypic Primulina
tabacumꎬ two species of Wentsaiboea D. Fang & D.
H. Qin and all of Chiritopsis W.T. Wang were nested
in a well ̄supported clade mainly with species of
Chirita sect. Gibbosaccus C.B. Clarke (Wang et al.ꎬ
2011ꎻ Weber et al.ꎬ 2011a). As a resultꎬ the previ ̄
ous taxonomic treatments were critically re ̄evaluated
and new taxonomic combinations were made to re ̄
flect evolutionary relationshipsꎬ which brought all
species of this group into the synonymy of Primulina
sensu lato ( s. l.). As newly definedꎬ Primulina s. l.
represents now a large genus of Old World Gesneri ̄
aceaeꎬ comprising approximately 130 + speciesꎬ
characterized by acaulescentꎬ perennial plants main ̄
ly with opposite decussate leaves in whirls of three
(Wang et al.ꎬ 1998ꎻ Li and Wangꎬ 2004ꎻ Wei et
al.ꎬ 2010ꎻ Wang et al.ꎬ 2011ꎻ Weber et al.ꎬ
2011aꎻ Liu et al.ꎬ 2011ꎻ Hong et al.ꎬ 2012ꎻ Huang
et al.ꎬ 2012ꎻ Li et al.ꎬ 2012ꎻ Wen et al.ꎬ 2012aꎬ
bꎬ cꎬ dꎻ Wu et al.ꎬ 2012aꎻ Wu et al.ꎬ 2012bꎻ Xu
et al.ꎬ 2012aꎬ b). All the species occur predomi ̄
nately in limestone areas of western and southern
Chinaꎬ with about a dozen species extending into or
being endemic to Vietnam. The Guangxi Province in
China is perhaps the center of species diversity for
this genusꎬ with high levels of species richness and
many endemism (Wang et al.ꎬ 1998ꎻ Li and Wangꎬ
2004ꎻ Wei et al.ꎬ 2010ꎻ Weber et al.ꎬ 2011a).
Although in recent years molecular data has be ̄
come the center of numerous phylogenetic investiga ̄
tionsꎬ cytological data still play an important role in
increasing our understanding of the genome evolution
when seen in the light of molecular phylogenies
(Weber and Burttꎬ 1998ꎻ Jong and Möllerꎬ 2000ꎻ
Liu et al.ꎬ 2001ꎻ Liuꎬ 2004ꎻ Liu and Yangꎬ 2011).
In Gesneriaceaeꎬ the cytologically investigated taxa
frequently possess small chromosomesꎬ ranging from
less than 1 to 2 μmꎬ and as a result few karyotypes
have been published so farꎬ mainly in Chinese taxa
(Wang et al.ꎬ 1998ꎻ Wang and Guꎬ 1999ꎻ Zhou et
al.ꎬ 2004ꎻ Yang et al.ꎬ 2012) and two for African
taxa (Möller and Kiehnꎬ 2004). Howeverꎬ even the
establishment of ploidy levels or the exact chromo ̄
some numbers can be of taxonomic significanceꎬ
since these data can be very useful in supporting tax ̄
onomic decisions. For instance in the dissection of
Didymocarpus Wall.ꎬ a genus originally composed of
over 250 species but split into three different generaꎬ
Didymocarpus s. s.ꎬ Hovanella A. Weber & B. L.
Burtt and Henckelia Spreng. ( Weber and Burttꎬ
1998). In addition to morphological and biogeo ̄
graphical dataꎬ they used differences in chromosome
number and chromosome morphology to support their
decisions (Möller and Kiehnꎬ 2004). Another ex ̄
ample is the genus Streptocarpus Lindl. Based on
vegetative morphologyꎬ this genus was divided into
two subgeneraꎬ Streptocarpella Fritsch and Strepto ̄
carpus (Hilliard and Burttꎬ 1971). Jong and Möller
(2000) found that there was a strong correlation be ̄
tween basic number and growth form in the two sub ̄
genera of the genus on the African mainland.
Despite the potential taxonomic importance of
chromosomal dataꎬ the newly defined Primulina s. l.
41 植 物 分 类 与 资 源 学 报 第 36卷
is poorly known cytologically. The first chromosome
number in this genus was determined for P. dryas
( =Chirita sinensis) with 2n = 36 (Ratter and Pren ̄
ticeꎬ 1964). During the following forty yearsꎬ no
further numbers were added. Until recentlyꎬ due
mainly to the work of Christie et al. (2012) and Liu
et al. (2012)ꎬ the number of counts increased sig ̄
nificantly. To dateꎬ chromosome data are available
for 59 species out of ca. 130+ species recognized in
Primulina s. l.. The earlier investigations indicated
that the somatic chromosome numbers for Primulina
s. l. are highly conservativeꎬ with 2n= 36 being prev ̄
alentꎬ apart from two dubious counts and one
polyploidy event ( Cao et al.ꎬ 2003ꎻ Zhou et al.ꎬ
2004ꎻ Christie et al.ꎬ 2012ꎻ Liu et al.ꎬ 2012ꎻ Yang
et al.ꎬ 2012). Howeverꎬ except for P. tabacumꎬ all
the available counts are for species previously as ̄
signed to Chirita sect. Gibbosaccusꎬ while those of
erstwhile Wentsaiboea and Chiritopsis had not yet
been studied cytologically to date. Thusꎬ the present
survey was undertaken to contribute more chromo ̄
somal data. Additionallyꎬ three species of Petrocodon
sensu lato ( s. l.) were included in this study for
comparison of the chromosomal characteristics be ̄
tween these closely related genera. Petrocodon has
also recently been remodeled taxonomically (Weber
et al.ꎬ 2011b) and is the closest relative of Primuli ̄
na s. l. (Möller et al.ꎬ 2011)ꎬ and the chromosome
count in this genus is only available for P. hanceiꎬ
with 2n= 20 (Cao et al.ꎬ 2003).
Materials and methods
The species with both current and previously
applied namesꎬ origin informationꎬ and voucher
specimens are listed in Table 1. Plants of 24 taxa of
Primulina s. l. were collected from wild populations
growing on limestone karsts mainly of Guangxi Prov ̄
inceꎬ with a few from Guangdong and Hainan Prov ̄
inces of Chinaꎬ and cultivated in pots in the green ̄
house of the Guangxi Institute of Botany of Chinese
Academy of Sciences in Guilinꎬ China. Actively
growing root tips were harvested and pretreated with
distilled water at 0 ℃ for 24 h before being fixed in
Farmer’s solution ( absolute alcohol: glacial acetic
acid 3∶1) at 4 ℃ for at least 30 minꎬ then stored in
70% aqueous ethanol until studied. After being mac ̄
erated in a 1∶1 mixture of 1 molL-1 HCl and 45%
acetic acid at 60 ℃ for 3 minutesꎬ the root tips were
rinsed in distilled waterꎬ stainedꎬ and squashed in
Carbol Fuchsin. The best metaphase plates were
photographed using an Olympus BX51 microscope
(Tokyoꎬ Japan) with an Olympus DP71 camera at ̄
tachment. The chromosome numbers were counted in
at least ten cells with well ̄spread chromosomes from
five different root tips from one plant for each spe ̄
cies. The chromosome lengths were measured from at
least three metaphase cells using the software pack ̄
age Image J (Media Cyberneticsꎬ Bethesdaꎬ USA).
For the species treatment and generic circumscrip ̄
tionꎬ we followed Li and Wang (2004) and Weber
et al. (2011aꎬ b). Voucher specimens are deposited
in the herbarium at the Guangxi Institute of Botany
(IBK)ꎬ Guangxi Zhuangzu Autonomous Region and
Chinese Academy of Sciences.
Results
Chromosome numbers were obtained for 24 taxa
of Primulina s. l. ( including eight previously as ̄
signed to Chiritopsisꎬ six of Chirita sect. Gibbosac ̄
cusꎬ two of Wentsaiboeaꎬ along with Primulina taba ̄
cum and seven undescribed taxa)ꎬ as well as three
species of Petrocodon s. l. (two formerly in Lagaroso ̄
len and one in Didymocarpus) ( Table 1). All of
these are illustrated in Figs.1-2 to show chromosome
morphology and size. The Primulina taxa studied are
highly uniform in chromosome numberꎬ all with 2n=
36ꎬ a number also observed in three species of Pet ̄
rocodon s. l.. Except for the confirmatory count for
Primulina tabacumꎬ all the counts are presented here
for the first time. One or two chromosome satellites
were observed in ten Primulina taxaꎬ with one satel ̄
lite being predominant and occurring in eight taxaꎬ
whereas just a single satellite was found in one Pet ̄
rocodon species. The chromosome size in both genera
511期 LIU Rui ̄Rui et al.: New Chromosome Counts and Their Taxonomic Implications in Primulina sensu lato
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61 植 物 分 类 与 资 源 学 报 第 36卷
Fig. 1 1-15. Somatic chromosomes of Primulina s. l.. 1. P. bicolorꎻ 2. P. bipinnatifidaꎻ 3. P. cordifoliaꎻ 4. P. glandulosa var. yangshuoensisꎻ
5. P. heterotrichaꎻ 6. P. lobulataꎻ 7 P. luochengensisꎻ 8. P. luzhaiensisꎻ 9. P. pteropodaꎻ 10. P. renifoliaꎻ 11. P. repandaꎻ
12. P. repanda var. guilinensisꎻ 13. P. ronganensisꎻ 14. P. sp. nov. 1ꎻ 15. P. sp. nov. 2. Open arrowsꎬ
overlapping / touching chromosomesꎻ solid arrowsꎬ satellites. Bar = 10 μm
711期 LIU Rui ̄Rui et al.: New Chromosome Counts and Their Taxonomic Implications in Primulina sensu lato
is small according to the classification of Lima ̄de ̄
faria (1980). The taxa in Primulina s. l.ꎬ previously
assigned to Chiritopsisꎬ Chirita sect. Gibbosaccus and
Wentsaiboeaꎬ show sight variation in chromosome
sizeꎬ mostly falling into the range of 0.6 to 2.4 μmꎬ
while those in Petrocodon s. l. possess relatively large
chromosomesꎬ with lengths of 1. 3 to 3. 3 μm. Al ̄
though in both genera the small chromosomes don’t
allow a detailed karyotype analysisꎬ it was possible
to observe from the photographs of the well ̄spread
and somewhat larger chromosomes that the karyo ̄
types are symmetrical and very similar to one anoth ̄
erꎬ consisting mainly of metacentric and submeta ̄
centric chromosomes (Fig.1: 13ꎻ Fig.2: 3ꎬ 10).
Fig. 2 1-12. Somatic chromosomes of Primulina s. l. and Petrocodon s. l.. 1. Primulina sp. nov. 3ꎻ 2. P. sp. nov. 4ꎻ 3. P. sp. nov. 5ꎻ
4. P. sp. nov. 6ꎻ 5. P. sp. nov. 7ꎻ 6. P. subulataꎻ 7. P. subulata var. yangchunensisꎻ 8. P. tabacumꎻ 9. P. villosissimaꎻ
10. Petrocodon hechiensisꎻ 11. Pet. jingxiensisꎻ 12. Pet. niveolanosus. Open arrowsꎬ overlapping / touching
chromosomesꎻ solid arrowsꎬ satellites. Bar = 10 μm
81 植 物 分 类 与 资 源 学 报 第 36卷
Discussion
The present study was carried out to survey ex ̄
tensively the chromosome numbers in Primulina s. l.ꎬ
with samples spanning all transferred genera and
covering most of the morphological range. Chromo ̄
some numbers were obtained for eight taxa previously
assigned to Chiritopsisꎬ six of Chirita sect. Gibbosac ̄
cus and two of Wentsaiboeaꎬ as well as the monotype
species P. tabacum and seven undescribed taxa. Ex ̄
ception for P. tabacumꎬ all the taxa were counted for
the first time. The taxa examined have the same
chromosome number of 2n= 36ꎬ and the number ob ̄
tained for P. tabacum by Christie et al. (2012) and
Yang et al. (2012) were confirmed here. Chromo ̄
some numbers we obtainedꎬ together with previous
reports of another 59 species (Ratter and Prenticeꎬ
1964ꎻ Cao et al.ꎬ 2003ꎻ Zhou et al.ꎬ 2004ꎻ Christie
et al.ꎬ 2012ꎻ Liu et al.ꎬ 2012ꎻ Yang et al.ꎬ 2012)ꎬ
has provided us with reliable chromosome numbers
for 76 of the ca. 130+ total species currently recog ̄
nized in this genus. With the exception of P. tamiana
(B. L. Burtt) Mich. Möller & A. Weber (2n= 32 in
Christie et al. 2012) and P. longgangensis (W. T.
Wang) Y. Z. Wang (2n= 28 in Cao et al. 2003 and
2n= 72 in Christie et al. 2012)ꎬ the somatic chro ̄
mosome number of Primulina s. l. is highly con ̄
servedꎬ with 2n = 36. It should be noted thatꎬ al ̄
though P. tamiana has been placed in Primulina s. l.
(Weber et al.ꎬ 2011a)ꎬ the preliminary molecular
data suggest that the species might not belong to this
genus (Christie et al. 2012). As for P. longgangen ̄
sisꎬ the count in Cao et al. (2003) is dubious since
the figure shows a poorly reproduced or prepared
prometaphaseꎬ while the counts of 2n = 72 in Chris ̄
tie et al. (2012) and 2n = 36 in Liu et al. (2012)
indicate that this species might be a polyploidy with
two ploidy levels. This would make Primulina uni ̄
formly 2n= 36 with one polyploidy. As all the Primu ̄
lina species currently recognized are morphologically
very similar to each otherꎬ we can postulate that the
basic chromosome number for the genus is x = 18ꎬ as
well as by Liu et al. (2012). This is supported by the
fact that its closest allyꎬ Petrocodon s. l.ꎬ has predom ̄
inantly also 2n=36 chromosomes (see below).
In Gesneriaceaeꎬ small chromosome size fre ̄
quently prevents detailed analysis of mitotic meta ̄
phase nuclei beyond mere establishment of chromo ̄
some numbers (Möller and Kiehnꎬ 2004). A very
similar situation was observed in Primulina s. l.. The
chromosomes of taxa previously assigned to Chirita
sect. Gibbosaccusꎬ Chiritopsisꎬ and Wentsaiboea as
well as the monotypic P. tabacum have similar sizesꎬ
most falling in the range of 0. 6 to 2. 4 μm. Only
slight variations occur among all the taxa where chro ̄
mosomal data are availableꎬ including previously re ̄
ported data (e.g. Christie et al.ꎬ 2012ꎻ Liu et al.ꎬ
2012ꎻ Yang et al.ꎬ 2012). The occurrence of chro ̄
mosome satellites is also a common phenomenon in
Primulina s. l. (Christie et al.ꎬ 2012ꎻ Liu et al.ꎬ
2012ꎻ Yang et al.ꎬ 2012). Of the 76 species with a ̄
vailable chromosomal dataꎬ 23 species possess at
least one or two satellites. They are relatively small
in sizeꎬ frequently residing on the ends of the larger
chromosomes. Thereforeꎬ it is necessary to exercise
great care when exceptionally large satellites are
presentꎬ for satellites may tend to detach and incor ̄
rect counts can be the result of such unrecognized
such ‘satellites’ . From well ̄spread metaphase chro ̄
mosomesꎬ the complements are most often consisting
of small ̄sized metacentrics and submetacentrics. If we
disregard the differences in the presence or absence
and number of satellitesꎬ taxa of Primulina s. l. are
quite similar with respect to chromosome morphologyꎬ
albeit with slight variations in chromosome size.
Up to nowꎬ the available chromosome counts of
Primulina s. l. have covered 57% species of Chirita
sect. Gibbosaccusꎬ 53% of Chiritopsis and 100% of
Wentsaiboeaꎬ including their type speciesꎬ P. drays
( =Chirita sinensis)ꎬ P. repanda ( =Chiritopsis repan ̄
da) and P. renifolia ( =Wentsaiboea renifolia). As a
wholeꎬ the high uniformity of chromosome numbers
and similarity of chromosome morphology are congru ̄
ent with the results of molecular phylogenetic studies
(Wang et al.ꎬ 2011ꎻ Weber et al.ꎬ 2011a)ꎬ strong ̄
911期 LIU Rui ̄Rui et al.: New Chromosome Counts and Their Taxonomic Implications in Primulina sensu lato
ly support the monophyly of Primulina s. l. as cur ̄
rently constructed and the taxonomic decision of
transferring all species of Chiritopsis and two of
Wentsaiboea to the originally monotypic Primulinaꎬ
together with all of Chirita sect. Gibbosaccus.
On the other handꎬ the close relationship of
Primulina s. l. and Petrocodon s. l. revealed by Möller
et al. (2009ꎬ 2011)ꎬ is supported by the chromo ̄
somal data we obtained here. Three species of Petro ̄
codon s. l. possess the same somatic chromosome
number of 2n= 36 as Primulina s. l.ꎬ and their chro ̄
mosome morphology are also very similar to each
otherꎬ with metacentrics and submetacentrics pre ̄
dominating. It is noteworthy that the chromosomes of
taxa in Petrocodon s. l. appear to be larger than those
of Primulina s. l.ꎬ which somewhat supports their
status as two independent genera. Neverthelessꎬ fur ̄
ther investigations are very desirable on this genusꎬ
particularly since another species in the extended
Petrocodon s. l. has been counted with 2n = 20 chro ̄
mosomes (Cao et al.ꎬ 2003)
As aforementionedꎬ a high stable somatic chro ̄
mosome number 2n = 36 appears to be predominant
in Primulina s. l.. Christie et al. (2012) argued that
at first this number might be a straightforward dupli ̄
cation from an ancestor with 2n = 18 chromosomes.
Howeverꎬ Christie et al. (2012) further argued thatꎬ
given that members of other clades around Primulina
s. l. include genera with high somatic chromosome
numbersꎬ e.g.ꎬ Loxostigma C. B. Clarke (2n= 34)ꎬ
Hemiboea C. B. Clarke (2n= 32ꎬ 36)ꎬ Petrocosmea
Oliv. (2n = 34)ꎬ and Oreocharis Benth. (2n = 34)
(Möller et al.ꎬ 2002 onwards)ꎬ chromosome dupli ̄
cation in Primulina s. l. might have occurred long be ̄
fore the diversification of this genus. Our findings that
some species of the closest allyꎬ Petrocodon s. l.ꎬ
shared the same chromosome number with Primulina
s. l. supported their deduction. Although at present
there are insufficient data to speculate on the age of
the genus Primulina s. l.ꎬ it may be relatively young
given the uniformity in its chromosome numbers and
chromosome morphology. Howeverꎬ much more work
remains to be done.
Acknowledgements: We greatly appreciate Dr. KS Mao and
Prof. M Möller for critical reading and invaluable comments
on the manuscript. We are grateful to Dr. M Kang and B Pan
for collecting the materials.
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