Karyotype and cytogeography of the genus Heracleum (Apiaceae

Transcription

JSE
jse_031
Dispatch: 5-22-2009 CE:
Journal MSP No. No. of pages: 14
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Journal of Systematics and Evolution
47 (4): 1–14 (2009)
PE: Rod
doi: 10.1111/j.1759-6831.2009.00031.x
Karyotype and cytogeography of the genus Heracleum (Apiaceae)
in the Hengduan Mountains
∗
Xian-Lan DENG Xing-Jin HE
Wei-Lue HE Yun-Dong GAO
Yu-Cheng ZHANG
Hai-Yan LIU
(Laboratory of Systematic and Evolutionary Botany, College of Life Science, Sichuan University, Chengdu 610064, China)
Abstract In the present study, the karyotypes of 34 populations belonging to 11 species and one variety of
Heracleum from the Hengduan Mountains in China were examined. Chromosome numbers and the karyotypes of
three species (H. souliei, H. kingdoni, and H.wenchuanense) are reported for the first time, as are the karyotypes
of H. moellendorffii and H. henryi (tetraploid). Populations of H. candicans, H. franchetii, and H. kingdoni in the
Hengduan Mountains were found to consist of a mixture of diploid and tetraploid plants. Except for four species of
Heracleum, namely H. candicans, H. franchetii, H. henryi, and H. kingdoni, which have both diploid and tetraploid
karyotypes, all other species of Heracleum are were found to be diploid. All karyotypes were found to belong to the
2A type of Stebbins, with the exception of H. candicans var. obtusifolium, which belongs to 2B, and H. hemsleyanum
and H. franchetii (Mt Dujuan, Daocheng, Sichuan, China), which belong to 1A. There was only a slight difference
in the karyotype asymmetry index, which suggests a close kinship for species of Heracleum and that the entire
phylogenetic development of Heracleum is relatively primitive. Species that exhibited advanced morphological
features were also more advanced in karyotype structure, with the order of karyotype evolution being 1A→2A→2B.
This phenomenon indicates that the species distributed in the Hengduan Mountains have not diverged completely and
that the Hengduan Mountains are a relatively young and active area for the evolution of Heracleum. Polyploidization
in Heracleum may be an important evolutionary mechanisms for some species, generating diversity. The biological
attributes, distribution range, and the geological history of the genus have all played a part in accelerating the
evolution through polyploidization or aneuploidization. It is known that as the distribution latitude of Heracleum
decreases from north to south, the chromosome number, ploidy level, and asymmetry structure appear to increase.
In the Hengduan Mountains, these tendencies are also evident. Finally, based on all the available cytogeographic
data, we speculate that the more advanced tetraplont or aneuploid species of Heracleum in India may be derived
from early diplont species that were distributed in the Caucasus region and Hengduan Mountains. The dispersal
of Heracleum was from Eurasia to India, because this correlates with the emergence of the Himalayan Mountains
through tectonic movement. Thus, the Hengduan Mountains are not only a center of diversity for Heracleum, but
also a center of active speciation in modern times.
Key words cytogeography, evolution, Heracleum, karyotype, polyploidization.
The genus Heracleum L. belongs to the Umbelliferae, tribe Peucedaneae, subtribe Tordylieae (Drude,
1898). It consists of approximately 70 species distributed throughout the world (Pu & Watson, 2005).
Heracleum species are distributed mainly in the Northern Hemisphere, with most species occurring in Asia
and Europe, only one species in North America, and a
few species in East Africa. The two main diversity centers for Heracleum are in the Caucasus and the SinoHimalayan region (Logacheva et al., 2008). Heracleum
is also widespread in the Hengduan Mountains of China,
with 26 species and two varieties belonging to four sec-
∗
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Received: 6 October 2008 Accepted: 7 March 2009
Author for correspondence: Xing-Jin HE, E-mail: [email protected];
Tel. 028-85415006; Fax: 028-85415006.
2009 Institute of Botany, Chinese Academy of Sciences
tions: Millefolia T. S. Wang & Shan, Wendia (Hoffm.)
Manden., Villosa Manden., and Heracleum L. (Shan,
1992). Nineteen of the species are endemic to China.
Heracleum species are perennial herbs and they are easily recognized by their radiant outer petals, strongly dorsally compressed fruit, and distinctly clavate vittae. The
classification of Heracleum is based mainly on the morphology of the leaves and the quantity and morphology
of the vittae.
Heracleum has been recognized as a natural group
since 1753 (Pu & Watson, 2005). Due to its complex morphology and incomplete information from type
specimens (most of the specimens lack basal leaves and
intact inflorescences), species identification has been
problematic. Moreover, phylogenetic relationships with
the genus have not been investigated adequately. Hence,
most of the infrageneric species relationships are still
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unresolved. Furthermore, there has been no study on
the speciation and biogeography of these species. Studies on Heracleum conducted so far include palynology (He & Pu, 1992), chemotaxonomy (Rao et al.,
1995), molecular systematics (Logacheva et al., 2008),
anatomy of the leaf stalk and fruit (He et al., 1995,
1998), and cladistics (Zhao et al., 2004). In these studies,
the infrageneric grouping problems of Heracleum were
mainly discussed, with few authors investigating the
interspecific relationships. Based on differences in the
cytotype and morphology between H. millefolium and
H. millefolium var. longilobum, He et al. (1994) took
H. millefolium var. longilobum as a separate species:
H. longilobum. On the basis of results of comparisons of chemical constituents, as well as morphological and anatomical evidence for H. millefolium and H.
longilobum, Rao et al. (1995) considered it is reasonable
to take H. longilobum as a variety: H. millefolium var.
longilobum as part of the species H. millefolium. As for
the phylogenetic relationships of Heracleum, Logacheva
et al. (2008) explored the phylogenetic relationships between west Asian Heracleum species and related taxa
using data from sequences of the internal transcribed
spacer region of nuclear ribosomal DNA. The results
indicated that Heracleum was a polyphyletic genus and
that the infrageneric classification of some species was
not considered by existing classification systems. Zhao
et al. (2004) studied the phylogenic relationship among
16 species of Heracleum using cladistic analysis. The
results indicated that all Heracleum plants studied were
divided into four sections, namely sect Millefolia, sect
Plurivittata, sect Villosa, and sect Heracleum. The possible features of the primitive ancestors and the direction
of evolution of the related characteristics were also discussed. Zhao et al. (2004) reported that the evolutionary
trend of characteristics in Heracleum was as follows:
pollen shape evolved from a rectangular to an equatorially constricted type; the V-type vascular bundle of the
leaf stalk evolved from the ring type; the ribs and lateral
wings of the mature fruit eveloved from an underdeveloped to developed form; and the ratio of the width of the
carpellum to its thickness evolved from small to large.
Chromosome numbers (2n) in Heracleum have
been reported to range from 22, 24, 38, 40, and 44
to 46 (Gurzenkov & Gorovoy, 1971; Moor, 1973;
Gagnidze, 1975; Hore, 1977; Pan & Qin, 1981; Pan
et al., 1985; Qin et al., 1989; Marhold, 2006; Rostovfseva, 1979, 1982; Goldblatt, 1981, 1984, 1985). Subramanian (1986) studied the karyotype of eight species
in south India and He et al. (1994) studied the karyotype of eight species and two varieties in the Hengduan
Mountains. Chromosome numbers have been reported
for only 13 species and two varieties in the Hengduan
2009
Mountains (Pan & Qin, 1981; Pan et al., 1985; Qin
et al., 1989; He et al., 1994; Marhold, 2006). However,
because the Hengduan Mountains region is recognized
as a biodiversity “hot spot” (Myers et al., 2000) and
because the Hengduan Mountains have a complex biogeographical history, a cytological study of Heracleum
species in the Hengduan Mountain is highly desirable.
In the present study, the chromosome number and
karyotypes of 34 populations belonging to 11 species
and one variety of Heracleum from the Hengduan
Mountains were investigated. The aim of the present
study was provide cytological evidence to answer the
following questions: (i) what are the genetic relationships among the Heracleum species in the Hengduan
Mountains; (ii) how has the karyotype of Heracleum
evolved in the Hengduan Mountains, as well as worldwide; and (iii) what are the evolutionary mechanisms
and cytogeographical patterns of Heracleum throughout the world?
1 Material and methods
All plants studied and their seeds were collected in
the field (Table 1). Plants were cultivated in pots and
seeds germinated readily in moist sand in a Petri dish
after 1 month of chilling at 4◦ C for cytological studies.
Voucher specimens were deposited in the Herbarium of
Sichuan University (SZ) and the voucher numbers are
listed in Table 1.
For each population, fresh roots were obtained from
at least three individual plants. Actively growing root
tips were collected between 08:00 and 10:00 hours and
were treated with 0.1% colchicine at ambient temperature (10–20◦ C) for 8–10 h before fixation in Carnoy
I (one part glacial acetic acid to three parts absolute
ethanol) at 4◦ C for 2–24 h. Samples were macerated in
1 mol/L HCl at 60◦ C for 6 min, and then stained and
squashed in carbolic acid fuchsin.
Karyotype formulae were based on measurements
of fine metaphase chromosomes taken from photographs. The chromosomes of at least 30 cells were
counted and measurements were made in at least five
cells. The nomenclature for the centromeric positions
of chromosome introduced by Levan et al. (1964) was
used in the present study: m, median centromeric chromosome with arm ratio of 1.0–1.7; sm, submedian centromeric chromosome with arm ratio of 1.7–3.0; st,
subterminal centromeric chromosome with arm ratio of
3.0–7.0. The karyotype classification of Stebbins (1971)
and the index of asymmetry (As.K%) defined by Arano
(1963) were adopted in the present study.
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DENG et al.: Karyotype and cytogeography of Heracleum
Table 1
Source of materials
Taxon
Heracleum candicans Wall. ex DC.
H. candicans var. obtusifolium
(Wallich ex de Candolle) F. T. Pu &
M. F. Watson
H. millefolium Diels
H. hemsleyanum Diels
H. souliei H. Boissieu
H. yungningense Handel-Mazzetti.
H. moellendorffii Hance
H. franchetii M. Hiroe
H. stenopterum Diels
H. wenchuanense F. T. Pu & X. J. He
H. henryi H. Wolff
H. kingdoni H. Wolff
Locality
Altitude(m)
Voucher (SZ)
Riwa village, Daocheng, Sichuan, China
Chitu village, Daocheng, Sichuan, China
Mt Chitu, Daocheng, Sichuan, China
Mt Julong, Daocheng, Sichuan, China
Zhongdian, Yunnan, China
Litang, Sichuan, China
Yajiang, Sichuan, China
Wenchuan, Sichuan, China
3200
3300
3375
3500
3300
3400
3900
4140
2100
X. L. Deng & Q. Wang DC003
Q. Z. Wang W200617
X. L. Deng & Q. Wang DL003
X. L. Deng & H. Y. Liu 200707004
Kangding, Sichuan, China
Daocheng, Sichuan, China
Zheduotang, Kangding, Sichuan, China
Mt Zheduo, Kangding, Sichuan, China
Daocheng, Sichuan, China
Mt Dujuan, Daocheng, Sichuan, China
Mt Julong, Sichuan, Daocheng, China
Julong village, Daocheng, Sichuan, China
Mt Tuer, Litang, Sichuan, China
Mt Kalazi, Litang, Sichuan, China
Mt Dujuan, Daocheng, Sichuan, China
Litang, Sichuan, China
Tengchong, Yunnan, China
Baoshan, Yunnan, China
Qushi, Tengchong, Yunnan, China
Mt Gaoligong, Tengchong, Yunnan, China
3100
3970
4040
3080
2750
3750
3590
2000
3600
3500
3720
3900
3800
4000
4000
3450
3600
3600
3920
3900
3500
1447
1514
1540
1729
X. L. Deng & Q. Wang YK004
X. L. Deng & F. D. Pu YK007
X. L. Deng & F. D. Pu DC012
X. L. Deng & H. Y. Liu 200707002
X. L. Deng & F. D. P u DC009
X. L. Deng & F.D. Pu DC014
X. L. Deng & F. D. Pu DL004
Y. Yu & H. Y. Liu dyy080816
X. L. Deng & Y.D. Gao YN008
X. L. Deng & Y. Yu YN009
X. L. Deng & Y. D. GaoYN007–6
X. L. Deng & H. Y. Liu YN007–7
2 Results
The karyotype results for all populations examined are given in Table 2, whereas Figs 1–6 show the
chromosome size and morphology of all populations
studied. Results for chromosome numbers and karyotypes of three species (H. souliei, H. kingdoni, and
H. wenchuanense) and the karyotypes of two tetraploid
species (H. moellendorffii and H. henryi) are reported
here for the first time. In addition, the mixoploidy of
H. candicans, H. franchetii, and H. kingdoni is reported
for the first time.
Eight populations of H. candicans were examined. The single population from Daocheng, Chitu village, has mixoploid cell types, where both diploid and
tetraploid cytotypes exist in the same population. Seven
populations were found to be diploid with 2n = 22
(Fig. 1), which is in accordance with the reports of He
et al. (1994) and Marhold (2006). Within the mixoploid
population, the percentage of diploid cells (2n = 22;
Fig. 1B, b) was 75%, the percentage of tetraploid cell
types (2n = 44; Fig. 4G) was 25%. The tetraploid chro
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mosomes were small and difficult to measure accurately.
Eight populations of H. candicans shared the same type
(2A) and the As.K% was similar, ranging from 57.71%
to 59.99%. A pair of small satellites was observed on the
shorter arms of the 10th chromosome pair in the Yajiang
population (Fig. 1F, f). In contrast, one small satellite
was present in the Daocheng, Chitu (Fig. 1C, c) and
Yunnan, Zhongdian (Fig. 1G, g) populations, indicating
the phenomenon of satellite heterozygosis.
Eight populations of H. franchetii were examined in
the present study. The single population collected from
Mt Dujuan was a mixed population with both diploid
(2n = 22; Fig. 3B, b) and tetraploid (2n = 44; Fig. 4H).
The ratio of diploidy to tetraploidy levels in that population was 3 : 1 and the diploid karyotype type was 1A.
Conversely, the other seven populations were diploid
with 2n = 22 (Figs 3A, C–H, 4A) and belonged to the
2A type. These observations support the results reported
by He et al. (1994). Because the tetraploid chromosomes
were very small and difficult to measure, no karyotype
data were obtained. The As.K% of the eight populations ranged from 54.99% to 59.83%. A pair of small
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Table 2 Karyotype structure for each Heracleum taxon and population investigated
Taxon
Karyotype
Type
As.K (%)
Heracleum candicans
2n = 2x = 18m + 2sm + 2st
2n = 2x = 18m + 2sm + 2st
2n = 2x = 18m + 4st(1sat)
2n = 2x = 18m + 2sm + 2st
2n = 2x = 16m + 4sm + 2st
2n = 2x = 18m + 2sm + 2st(1sat)
2n = 2x = 18m + 2sm + 2st
2n = 2x = 16m + 6sm(2sat)
2A
2A
2A
2A
2A
2A
2A
2A
57.94
59.13
59.65
58.62
59.99
58.35
58.00
57.71
Q. Z. Wang W200617
1A, a
1B, b; 4G
1C, c
1D, d
1E, e
1F, f
1H, h
1G, g
H. candicans var. obtusifolium
2n = 2x = 18m + 4sm
2n = 2x = 16m + 2sm + 2st + 2T
2n = 2x = 18m + 2sm + 2st(2sat)
2n = 2x = 18m + 2sm + 2st(2sat)
2n = 2x = 20m + 2sm
2n = 2x = 20m + 2sm
2n = 2x = 16m + 4sm + 2st
2n = 2x = 18m + 2sm + 2st
2n = 2x = 18m + 2sm + 2st
2n = 2x = 20m + 2sm
2n = 2x = 20m + 2sm
2n = 2x = 18m + 2sm + 2st
2n = 2x = 14m + 6sm + 2st
2n = 2x = 16m + 4sm + 2st(2sat)
2n = 2x = 20m + 2sm
2n = 2x = 16m + 6sm
2n = 2x = 16m + 6sm
2n = 2x = 18m + 2sm + 2st(2sat)
2n = 2x = 16m + 4sm + 2st
2n = 2x = 20m + 2sm
2n = 2x = 18m + 4sm
2n = 2x = 18m + 4sm
2n = 4x = 36m + 8sm
2n = 2x = 36m + 8sm
2n = 4x = 36m + 8sm
2n = 4x = 36m + 8sm
2A
2B
2A
2A
1A
2A
2A
2A
2A
1A
2A
2A
2A
2A
2A
2A
2A
2A
2A
2A
2A
2A
2A
2A
2A
2A
55.68
61.74
59.56
59.26
55.82
57.15
60.79
58.41
58.46
55.59
54.99
59.83
59.97
59.79
56.74
59.82
58.81
59.21
59.33
55.66
57.97
57.25
57.81
57.82
56.95
57.87
X. L. Deng & H. Y. Liu 200707004
X. L. Deng & H.Y. Liu 200707002
X. L. Deng & F.D. Pu YK002
Y. Yu & H.Y. Liu dyy080816
X. L. Deng & Y. D. Gao YN008
X. Yu & Y. D. Gao YN009
X. L. Deng & Y. D. Gao YN007–6
X. L. Deng & Y. D. Gao YN007–7
2A, a
2B, b
2C, c
2D, d
2E, e
2F, f
2G, g
2H, h
3A, a
3B, b; 4H
3C, c
3D, d
3E, e
3F, f
3G, g
3H, h
4A, a
4B, b
4C, c
4D, d
4E, e
4F, f
5A, a
5B, b
5C, c; 4I
6A, a
H. millefolium
H. hemsleyanum
H. souliei
H. yungningense
H. moellendorffii
H. franchetii
H. stenopterum
H. wenchuanense
H. henryi
H. kingdoni
Voucher (SZ)
Figure
As.K, karyotype asymmetry index.
dddddddddddddddddd
ddd ddd ddd ddd ddd ddd
(dddddddddd, ddddddddd dd 610064)
d d dddddddddddd Heracleum dd11d1dd 34dddddddddddd; dddddd H. soulieiddddd H. wenchuanense ddddd H. kingdoni ddddddddddddddd H. moellendorffiiddddd H. henryi (ddd)ddddddddd; ddd
d Heracleum candicansddddd H. franchetii dddddddddddddddddddddd: 1) dddddddddddddddd
dddddddddddddddddd, ddddddddddddddddddd H. candicans var. obtusifolium dd Stebbins d 2B d
ddd H. hemsleyanumddddd(ddddddddd)ddStebbinsd 1A dd,ddddd 2A d,ddddddddddddd,dddd
ddddddddddddddddddd, ddddddddddddddddddddd2) dddddddddd 1A→2A→2B dd
dd, dddddddddddddddd, dddddddddddddddddddddddddd, dddddddddddddd
dddddddd3) dddddddddddddddddddddddd, dddddddddddddddddddddddddd
dddddddddddddddddddddddddddddddd4) dddddddd, ddddddddddd, ddddddd
dddddddddddddddddddddddddd; ddddd, dddddddd5) ddddddddddddddddddd
d,dddddddddddddddddddddddddddddddddddddddddddddddddddddddddd;
dddddddddddddddddddddddddddddddddddddddddddddddddddddd,dddddd
ddddddd
d d d ddd;dd;ddd;dd;ddddd
satellites was observed on the shorter arms of the 11th
chromosome pair in the population collected from Mt
Tuer in Litang (Fig. 3F).
Two populations of H. henryi were examined in
the present study. Both were tetraploid with 2n = 44
(Fig. 5A, a, B, b), confirming the report of Qin et al.
(1989), but contradicting the results of Pimenov et al.
reported by Marhold (2006). The karyotype both belonged to 2A of Stebbins (1971), and the As.K%
was similar for the two populations (57.81% for the
Tengchong population and 57.82% for the Baoshan
population).
Two populations of H. kingdoni were also examined in the present study. In the Qushi population, both
diploid and tetraploid plants were found. The percentage of diploid plants (2n = 22) was 30% (Fig. 4I).
The chromosomes of the diploid plants were very small
and difficult to measure, so no karyotype data were
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Fig. 1. Karyograms of Heracleum candicans (arrows indicate satellite chromosomes). A, a, Daocheng, Riwa village. B, b, Daocheng, Chitu village
(2n = 22, XQ DC004). C, c, Daocheng, Mt Chitu. D, d, Daocheng, Chitu village (XQ DC006). E, e, Daocheng, Mt Julong. F, f, Yajiang. G, g, Zhongdian.
H. h, Litang.
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Fig. 2. Karyograms of Heracleum species (arrows indicate satellite chromosomes). A, a, H. candicans var.obtusifolium (Wenchuan). B, b, H. candicans
var.obtusifolium (Kangding). C, c, H. millefolium (Daocheng). D, d, H. millefolium (Yajiang). E, e, H. hemsleyanum. F, f, H. souliei (Kangding,
Zheduotang). G, g, H. souliei (Kangding, Mt Zheduo). H, h, H. yungningense.
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Fig. 3. Karyograms of Heracleum species (arrows indicate satellite chromosomes). A, a, H. moellendorffii. B, b, H. franchetii (2n = 22; Daocheng,
Mt Dujuan). C, c, H. franchetii (Daocheng, Chitu village). D, d, H. franchetii (Daocheng, Mt Julong). E, e, H. franchetii (Daocheng, Julong village). F,
f, H. franchetii (Litang, Mt Tuer). G, g, H. franchetii (Litang, Mt Kalazi). H, h, H. franchetii (Yajiang).
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Fig. 4. Karyograms of Heracleum species (arrows indicate satellite chromosomes). A, a, H. franchetii (Kangding). B, b, H. stenopterum (Daocheng,
Mt Dujuan). C, c, H. stenopterum (Daocheng, Mt Julong, XQ DC011). D, d, H. stenopterum (Daocheng, Mt Julong; XQ DC019). E, e, H. stenopterum
(Litang). F, f, H. wenchuanense. G, g, H. candicans (Daocheng, Chitu village ; XQ DC004 ; 2n = 44). H, h, H. franchetii (Daocheng, Mt Dujuan;
2n = 44). I, i, H. kingdoni (Qushi; 2n = 22).
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Fig. 5. Karyograms of Heracleum species (arrows indicate satellite chromosomes). A, a, H. henryi (Tengchong). B, b, H. henryi(Baoshan). C, c, H.
kingdoni (Qushi; 2n = 44).
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Karyograms of Heracleum kingdoni (Mt Gaoligong).
obtained. The percentage of the tetraploid plants, with
karyotype 2n = 44 was 70% (Fig. 5C, c), the same as the
chromosome number of the population collected from
Gaoligongshan (Fig. 6A). The karyotypes of the Qushi
and Gaoligongshan populations were 2A (Table 2), and
they had similar As.K% values of 56.95% and 57.87%,
respectively.
Fourteen populations of the other seven species
and the one variety examined were found to be diploid
(2n = 22; Figs. 2–4). The karyotype of H. hemsleyanum
belonged to 1A, H. candicans var. obtusifolium from
Kangding was 2B; the remaining species and the one
variety were all 2A. Two populations of H. millefolium
(Fig. 2C, c, D, d) and the population of H. stenopterum
collected from Mt Dujuan in Daocheng (Fig. 4B, b)
had a pair of small satellites chromosome, no satellite
chromosomes were observed in the other populations.
The distribution of As.K% values for the seven species
and one variety was comparatively large, ranging from
55.66% to 61.74% (Table 2).
3 Discussion
3.1 Genetic relationship and karyotype evolution
The karyotypes of the 13 species and two varieties
of Heracleum in the Hengduan Mountains have been
studied previously (He et al., 1994), as well as in the
present study. Stebbins (1971) pointed out that the evolutionary trend of karyotypes in the vegetable kingdom
was from symmetry to asymmetry. Thus, relatively ancestral or primitive plants in systematic evolution have
relatively symmetric karyotypes, an asymmetric kary-
otype often seen in derivative or more advanced plants.
Based on the classification system of Stebbins (1971),
the karyotypes obtained from the plants in the present
study can be classified into three groups, namely 1A,
2A, and 2B.
The 1A group included H. hemsleyanum, H.
franchetii (Mt Dujuan, Daocheng, Sichuan, China), H.
wolongense (He et al., 1994), and H. stenopterum (He
et al., 1994), the chromosomes of which were all metacentric or submetacentric and the karyotype structure of
which was slightly symmetrical. All these species belonged to the section Heracleum defined by Shan (1992)
and all species had rectangular pollen. The ratio of the
length of the polar axis to that of the equatorial axis was
small, the germinal furrow was long, and the germinal
pore was large (He & Pu, 1992). For these species, the
bundle types of the leaf stalk were the or ring forms
(He et al., 1995). All these morphological characteristics suggest that these species are a relatively primitive
group within the genus Heracleum, which agrees with
the results of our karyotypic analysis.
The 2A group was characterized by subtelocentric chromosomes with an asymmetric karyotype structure. Most of the species examined in the present study
belonged to this group and they were classified under
the sections Villosa, Millefolia, or Heracleum according to Shan (1992). These species had developed an
equatorially constricted type pollen, as well as an intermediate type between the rectangular and equatorially
constricted pollen (He & Pu, 1992). In addition, the ribs
and lateral wings of the mature fruit were relatively developed, and the ratio of the width of the carpellum to its
thickness was high (He et al., 1998). The bundle types
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of the leaf stalks in these species were of the ring or V
forms, and the V form is relatively developed (He et al.,
1995). These cytological and morphological features indicate that there is a mixture of ancestral and advanced
groups, as well as intermediate groups, in these species.
The 2B group consisted only H. candicans var. obtusifolium (Kangding population), with a chromosome
type that was metacentric, submetacentric, subtelocentric, and T chromosome. The karyotype structure was
much more asymmetric than in the other groups and the
As.K% was 61.74%. The cytological features of this
species were congruent with various other morphological features, such as mature fruit characteristics, with a
ratio of the width of the carpellum to its thickness > 7.2
and well-developed lateral wings (He et al., 1998). The
equatorially constricted pollen type was most developed
in this species (He & Pu, 1992) and the special leaf stalk
bundle was a star-scattered type (He et al., 1995). Furthermore, this population of plants was found to be quite
hairy. On the basis of the pattern of karyotype evolution proposed by Stebbins (1971) and the phylogenetic
analyses of cladistics (Zhao et al., 2004) in Heracleum,
the data indicate that H. candicans var. obtusifolium is
relatively advanced. This species belong to sect Villosa,
as proposed by Shan (1992).
On the basis of the results reported above, we can
conclude that species in the genus Heracleum that were
more advanced morphologicallyy were also more advanced in terms of their karyotype, and that the karyotype evolutionary trend in this genus is also from symmetry to asymmetry, as put forward by Stebbins (1971).
Through somatic chromosomal studies on 13
species and two varieties, the karyotype evolutionary
order was found to be 1A→2A→2B, which followed
the pattern of Stebbins (1971) with 2B being the most
advanced in evolution. However, most of these karyotypes were either 1A or 2A, and the chromosomes
were almost metacentric or submetacentric, which indicated that those species of Heracleum were relatively
primitive in in terms of the entire phylogenetic development in Heracleum. Different species of Heracleum
in the Hengduan Mountains appeared to have distinct
As.K% values, but the differences were not marked, with
values ranging from from 54.99% to 61.74%. This reflects the fact that the 13 species and two varieties in the
Hengduan Mountains have a somewhat close kinship.
Heracleum millefolium var. longilobum exhibited
aneuploidy (He et al., 1994), indicating that it should be
a more advanced species; a similar trend was observed
with pollen morphology. H. henryi and H. kingdoni were
found to be tetraploid, with 2n = 44 and chromosomes
that are slightly asymmetric. Sharma and Sarkar (1970)
reported that the chromosome number of H. nepalense
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D. Don was 2n = 24, 44 (as cited in He et al., 1994).
Subramanian (1986) examined the karyotype of eight
species of Heracleum distributed throughout south India
and observed cell types with 2n = 40, 44, and 46. This
indicates that these species are tetraploid or aneuploid.
All exhibited a more asymmetric structure (Subramanian, 1986). Furthermore, the morphological features
of these species suggest that they are are advanced.
Pimenov et al., as reported in Marhold (2006), found
that the chromosome number of H. candicans and H.
pinnatum in India was 2n = 22.
From the above analysis, we can conclude that there
are two evolutionary pathways in the Heracleum species,
especially in karyotype structure and the tendency for
asymmetry and polyploidy or aneuploidy.
3.2 Polyploidy and evolutionary mechanisms
Polyploidy is an important driving force for diversification and speciation in plants (Levin, 2002). Recent
estimates suggest that 70% of all angiosperms have
undergone one or more episodes of polyploidization
(Masterson, 1994). Several studies have shown that certain Heracleum species are polyploids, and species such
as H. wallichii (Hore, 1977), H. candicans (Figs 1,
4G), H. franchetii (Figs 3, 4H), H. henryi (Fig. 5A,
B; Marhold, 2006), and H. kingdoni (Figs 4I, 5C, 6A)
have intraspecific polyploidization phenomena. Examples were seen for H. candicans and H. franchetii from
Daocheng, Sichuan, and for H. kingdoni from Qushi,
Yunnan, in which both diploid and tetraploid cytotypes
can be found in a single population. The diploid cytotype was dominant in the two populations from Sichuan,
but the opposite was true for the population of H. kingdoni from Yunnan. When polyploids have diverged sufficiently, they differ morphologically from their progenitors (Greilhuber & Ehrendorfer, 1988). Because these
species have not diverged completely (they are morphologically similar), they exist as mixoploids. Polyploidy
as a new cytotype is the manifestation of evolution.
This indicates that Heracleum in the Hengduan Mountains differentiates continually to polyploidy. Most of
the polyploids in nature are derived from the union
of unreduced gametes (Ramsey & Schemske, 1998).
Therefore, formation of tetraploids is possibly due to the
abnormal mitotic divisions of certain cells in the diploid,
which produce progeny with a doubling of chromosome
number. The frequency of diploid gametes in nature is
dependent on several factors, such as a plant’s own biological characteristics, heterozygotism, the environment
etc. (Ramsey & Schemske, 1998). Apparently, heterozygotism promotes the formation of unreduced gametes
(Ramsey & Schemske, 1998). Moreover, a harsh environment can also accelerate this phenomenon. All these
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factors likely contribute to the production of multiploids
in nature (Ramsey & Schemske, 1998).
Heracleum is a genus of temperate perennial herbs.
Several populations of different species can be found
growing together in the wild and most of these coexisting populations occur at high altitude. These features
demonstrate that Heracleum has a tendency to undergo
polyploidization in its evolution. Meanwhile, the history of geographical changes indicates that Himalayan
tectogenesis, beginning from the Neogene, has had an
extensive influence on the paleoclimate and geomorphy of India, Nepal, and southwestern China (Sun & Li,
2003). Along with the uplifting of the mountains, a complex and highly variable environment has been formed
that has provided a suitable ecological and evolutionary niche for the survival and development of polyploids. Therefore, polyploidization may be an important
evolutionary mechanism of some Heracleum species
that has accelerated the species diversity within this
genus.
3.3 Cytogeography problem
Based on all reported cytological data, chromosome numbers have been described for nearly 60 species
of Heracleum. All 14 species studied in the Caucasus
region are diploid, with 2n = 22 (Gurzenkov & Gorovoy,
1971; Gagnidze, 1975; Rostovfseva, 1979, 1982;
Goldblatt, 1981, 1984, 1985). The chromosome numbers of H. wallichii, a native of Nepal, is 2n = 22, 38,
and 44 (Hore, 1977). Eleven species have been studied
in India (Subramanian, 1986; He et al., 1994; Marhold,
2006); except for two species (H. candicans and H. pinnatum) with 2n = 22 (Marhold, 2006), all other nine
species have 2n = 24, 40, 44, and 46, and their karyotypes exhibit a greater asymmetry than those of other
regions (Subramanian, 1986).
A total of 16 species and two varieties have been
karyotyped in China (Pan & Qin, 1981; Pan et al.,
1985; Qin et al., 1989; He et al., 1994; Marhold, 2006;
present study). Except for four species (H. candicans, H.
franchetii, H. henryi, and H. kingdoni) that have diploid
and tetraploid types, and H. millefolium var. longilobum,
which has diploid and aneuploid cytotypes, all other 12
species and one variety are 2n = 22. The karyotype
structure of these species varies from slight asymmetry
to asymmetry. From these data, we can draw the following conclusions. First, there are several cell types
in Heracleum: diploid (2n = 22), tetraploid (2n = 44),
and aneuploid (2n = 24, 38, 40, and 46). Second, as the
latitude changes from north to south (from the Caucasus region to south India), the chromosome numbers of
Heracleum appear to increase from diploid to tetraploid
or aneuploid, and the ploidy level also tends to increase
2009
from north to south. In the Hengduan Mountains, these
patterns are also evident. Third, the structural modifications in karyotype also change with latitude, ranging
from slight asymmetry in the north (i.e. China) to asymmetry in the south (i.e. south India).
There are two geographical distribution centers for
Heracleum. One is the Caucasus region, which has 30
species (Logacheva et al., 2008), and the other is the
Hengduan Mountains, with 26 species and one variety (Shan, 1992). The climate of the Caucasus region
is continental temperate. The chromosome type of all
14 species in the Caucasus region is diploid. Moreover, these species are marked by their slight external
morphological differentiation. The Hengduan Mountains are geologically young and have a complex terrain.
During the Quaternary glacial period, the region was not
completely covered by the glacier, making it a refuge for
many plant species (Xu, 2003). This resulted in abundant primitive and many relatively advanced species,
as determined by cytological and external morphological analyses. Of the Heracleum species in the Hengduan Mountains, 73% are endemic to China, but polyploidization of these endemic species is not common.
Moreover, the chromosomes of the polyploid species are
all metacentric or submetacentric, and their karyotypes
are slightly asymmetrical. Thus, the species in the region
were generated from diploid progenitors, which may be
due to the uplifting of the Himalayas and associated climatic changes that brought about rapid diversification of
the species. Twenty Heracleum species are distributed
throughout India. Most (especially those in south India)
are tetraploid and aneuploid (Subramanian, 1986), their
chromosomes are more subtelocentric and T chromosome, and their karyotypes are all more asymmetrical
than those in the Hengduan Mountains. According to
analyses of ploidy level and karyotype structure (asymmetry and chromosome type) of Heracleum species in
these three regions, we think that the species distributed
in India are more advanced than the species in China and
the Caucasus. From the cytogeographic and geobotanic
features mentioned above and on the basis of the hypothesis that ploidy variation is irreversible (Stebbins,
1971), we speculate that the more advanced tetraplont or
aneuploid species of Heracleum in India may be derived
from early diplont species distributed in the Caucasus
region and Hengduan Mountains. It is not difficult to
imagine that the dispersal of Heracleum species from
Eurasia to India was correlated with the substantial
Himalayan tectogenic geological event. The more advanced taxa in systematic evolution in Heracleum were
regenerating taxa that differentiated from the Himalayas
and Hengduan Mountains region after collision of the
India plate and the Asian continent. These taxa evolved
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into polyploids or aneuploids to adapt to their different
harsh environmental conditions and began differentiating and dispersing to other parts of Asia. Thus, the
Hengduan Mountains, with a large number of Heracleum species, is not only a center of diversity for this
genus, but is also a center of active speciation in modern
times.
Acknowledgements The authors are grateful to Professor Fa-Ding PU for species identification and to
Qiang WANG and Yan YU for assistance during our
collecting trip. The authors also thank Professor Sun LI
for revising the manuscript. This work was supported
by the National Natural Science Foundation of China
(grant no. 30670146) and the National Infrastructure of
Natural Resources for Science and Technology (grant
no. 2005DKA21403).
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Karyotype and cytogeography of the genus Heracleum (Apiaceae

Transcription

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