Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 2003? 2003
142?
309320
Original Article
KARYOTYPE EVOLUTION IN
BORONIA
F. SHAN
ET AL.
Botanical Journal of the Linnean Society, 2003, 142, 309–320. With 17 figures
Karyotype evolution in the genus Boronia (Rutaceae)
FUCHENG SHAN, GUIJUN YAN* and JULIE A. PLUMMER
School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western
Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
Received August 2002; accepted for publication January 2003
New chromosome counts are reported for Boronia clavata 2n = 14, B. heterophylla ‘Near White’ 2n = 15, B. ‘Carousel’
2n = 16, B. deanei 2n = 22, B. chartacea 2n = 32, B. keysii 2n = 32, B. pilosa 2n = 44, B. anethifolia 2n = 36 and
B. citriodora 2n = 108. Studies in 20 genotypes of 18 species and one interspecific hybrid revealed that they are
highly complex in terms of chromosome number, ploidy level, chromosomal length, karyotype constitution and
asymmetry. Karyotype analysis indicated that Boronia taxa with high chromosome numbers are primitive and those
with lower numbers are derived. The basic chromosome number for this genus is suggested to be x = 18. Analysis of
chromosome number, variations of total chromosome length (TCL) and average chromosome length (ACL), Nombre
Fondamental (NF) and karyotype asymmetry suggest that dysploid reduction is the major mechanism in Boronia
karyotype evolution. Chromosomal rearrangements might also have been involved. Origin, chromosome number
changes and spread of Boronia are discussed in relation to the species divergence and the geological and climatic
changes of the Australian continent. © 2003 The Linnean Society of London, Botanical Journal of the Linnean
Society, 2003, 142, 309–320.
ADDITIONAL KEYWORDS: Australia – chromosomes – divergence – dysploidy – Gondwana.
INTRODUCTION
A karyotype is the summary of the overall morphological characters of the chromosome complement at
mitotic metaphase. It is described primarily by chromosome number, absolute and/or relative length of
chromosomes, position of primary and secondary constrictions within them and distribution of material
with different staining properties (Stebbins, 1971).
Karyotype analysis of related species can generate
information on chromosomal evolution within a group
(Stebbins, 1971; Gonzalez-Aguilera & FernandezPeralta, 1984; Dimitrova & Greilhuber, 2000).
Changes in these characters have played an important
role in the evolution of plant species (Tobgy, 1943;
Stace, 1978; Watanabe et al., 1995; Vanzela, Ruas &
Marin-Morales, 1997), since they produce and alter
structure of gene linkage groups, which comprise
adaptive clusters of interacting genes (Stebbins,
1971). Therefore karyotyping is a means of understanding relationships between species, the processes
*Corresponding author. E-mail: [email protected]
which have brought about evolutionary diversification, and the directions which evolution has taken
(Sherman, 1946; Stace, 1978; Gonzalez-Aguilera &
Fernandez-Peralta, 1984; De Melo Nationiel et al.,
1997; Vanzela et al., 1997; Das et al., 1999; Watanabe
et al., 1999; Dimitrova & Greilhuber, 2000; Pedrosa,
Schweizer & Guerra, 2000; Vilatersana et al., 2000).
The karyotype of Boronia Sm. (Rutaceae), a genus of
Australian native plants, has changed significantly
during its evolution. Its chromosome number varies
widely, ranging from 2n = 14, through 15, 16, 18, 22,
32, 36 to 2n = 72, which has attracted considerable
attention with regard to studies of genome evolution
(Stace, Armstrong & James, 1993; Astarini, Yan &
Plummer, 1999). There has been debate about whether
chromosome number has increased or decreased during evolution in the family Rutaceae, especially in
Boronia. The critical issue is whether x = 9 or x = 18 is
basic for Boronia, the tribe Boronieae and the entire
family Rutaceae (Smith-White, 1954; Stace et al.,
1993). Several approaches have been attempted,
including identification of chromosome numbers
(Smith-White, 1954, 1959; Stace & Armstrong, 1992;
Stace & Patrick, 1993), comparison of morphological
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
309
310
F. SHAN ET AL.
traits (Weston, Carolin & Armstrong, 1984; Stace
et al., 1993) and biochemical analysis of various components (da Silva, Gottlieb & Ehrendorger, 1988).
However, no consistent conclusion has been achieved
to date. Recent phylogenetic analysis using DNA
markers indicates that Boronia has evolved from high
chromosome number to low, which suggests x = 18 is
its basic chromosome number (F. Shan, G. Yan & J. A.
Plummer, unpubl. data). However, the mechanisms
responsible for chromosome number reduction and the
changes in karyotype in Boronia remain unknown.
The main purpose of this study is to determine karyotype evolutionary trends and the mechanism for
chromosome number change in Boronia. Original
counts of chromosome numbers are reported and karyological data of representative species are presented.
Ecological and distribution data are also incorporated
to provide a better understanding of the origin, phylogeny and mechanism of chromosomal evolution in
Boronia.
MATERIAL AND METHODS
Boronia heterophylla F.Muell. ‘Red’, ‘Near White’ and
‘Cameo’ (Sunglow Flowers, 1990) were obtained from
Sunglow Flowers Pty Ltd (Perth) and B. anemonifolia
Cunn. from Bernawarra Gardens (Barrington). All
other species were collections held at the University of
Western Australia. Twenty genotypes of 18 species
and one interspecific hybrid with different chromosome numbers and ploidy levels were chosen for
karyotype analysis. They were B. anemonifolia,
B. anethifolia Endl., B. ‘Carousel’ (a hybrid between
B. heterophylla
and
B. molloyae),
B. chartacea
P.H. Weston, B. citriodora J.D. Hook., B. clavata
P.G. Wilson, B. crenulata Smith, B. deanei Maiden &
E.Betche, B. denticulata Smith, B. fraseri Hook.,
B. gracilipes F. Muell., B. heterophylla (three genotypes ‘Red’, ‘Near White’ and ‘Cameo’), B. keysii
Domin, B. megastigma Bartling, B. mollis Lindley,
B. molloyae J.R. Drumm., B. muelleri (Benth.) Cheel,
B. pilosa Labill. and B. serrulata Smith. The other
four species, B. coerulescens F. Muell., B. filifolia
F. Muell., B. ledifolia (Vent.) J. Gay and B. purdieana
Diels, were used only for chromosome number determinations. The distribution of the species is listed in
Table 1. All plants were kept in a shade house at the
University of Western Australia, Perth. Herbarium
specimens were deposited at the Western Australian
Herbarium of the Department of Conservation and
Land Management, Perth (for voucher numbers see
Table 1).
Root tips were collected from potted plants and were
treated with a saturated aqueous solution of
paradichlorobenzene for 4 h at room temperature and
fixed in Carnoy’s I fixative (95% ethanol:acetic acid,
3:1 v/v) for 24 h. Fixed root tips were stored at -20 ∞C
until required. Samples were hydrolysed in 1 M HCl at
60 ∞C for 8–10 min, stained in Feulgen solution for 2 h
and squashed in a drop of FLP (formic acid, lactic acid,
propionic acid)-orcein on a microscope slide. With the
cover slip in place, the slide was heated and then
pressed firmly to flatten cells. Some chromosome
spreads were prepared using the air-dried method
(Geber & Schweizer, 1988) and stained with FLPorcein. All observations were made under a ¥100 oil
immersion objective lens using a Zeiss Axioplan microscope (MC80). Photographs were taken on monochrome film under a ¥100 oil immersion objective
using a 35 mm camera.
Only metaphase cells in which individual chromosomes were clearly distinguishable were used for making counts. At least ten dividing cells were counted for
each sample to determine the chromosome number.
Photographs of selected cells were used to take measurements of the length of the short arm ( s) and long
arm (l) of each chromosome. The chromosome length
(t = l + s) and arm ratio (r = l/s) were then calculated.
Chromosomes were classified on the basis of arm ratio
using the standard nomenclature (Levan, Fredga &
Sandberg, 1964): m, median (r = 1.01–1.69); sm, submedian (r = 1.70–3.00); st, subterminal (r = 3.01–
7.00); and t, terminal (r = 7.01–8).
The Nombre Fondamantal (NF) of each chromosome
complement was calculated. NF is the fundamental
number of the karyotype (Schubert & Rieger, 1985),
i.e. the number of chromosome arms. Each median,
submedian and subterminal chromosome has two
chromosome arms. Each terminal chromosome has
one.
Karyotypes were classified using the categories proposed by Stebbins (1971), who suggested 12 types of
karyotype differing in degree of asymmetry. Karyotype asymmetry is determined by the centromeric
location and uniformity of chromosome sizes. Asymmetry increases from Type 1 to Type 4 as the proportion of chromosomes with an arm ratio higher than 2.0
increases. Asymmetry also increases from type A to
type C according to the size ratio between the largest
and smallest chromosomes.
To describe karyotype asymmetry more precisely,
the degrees of asymmetry (A) and length heterogeneity (A2) were also estimated. To deal with karyotypes
with odd chromosome numbers, equation Equationn 1
modified from Equation 2 (Watanabe et al., 1999) was
used to calculate A1:
2n
A1 = (1 / 2n)Â
i=1
n
A1 = (1 / n)Â
i=1
li - si
ti
li - si
ti
(1)
(2)
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
KARYOTYPE EVOLUTION IN BORONIA
311
Table 1. Chromosome numbers available in Boronia species from published papers or our own observations, distribution
of species and notes on synonyms and varietal names. Author abbreviation, S-W: Smith-White (1954); S-W2: Smith-White
(1959); SP: Stace & Patrick (1993); SA: Stace & Armstrong (1992); AYP: Astarini et al. (1999); SYP: this study
Chromosome number
Taxon
n
algida
anemonifolia
10
18
anethifolia
barkerana
chartacea
citriodora
clavata
coerulescens
crassifolia
crenulata
Author
Distribution by state1
16
15
15*
14
SA
S-W
SYP
SYP
S-W
SYP
SYP
SYP
S-W
S-W; SYP
S-W
S-W
SYP
SYP
S-W; SA
SYP
S-W
SA
SYP
S-W
S-W
SYP
S-W
SYP
AYP; SYP
SYP
AYP; SYP
NSW, VIC, TAS
NSW, VIC, TAS
NSW, VIC, TAS
QLD, NSW
NSW
NSW
VIC, TAS
WA
WA, SA, NSW, VIC
WA, SA, NSW, VIC
WA
WA
WA
NSW
WA
WA
WA
SA, VIC
SA, VIC
NSW
NSW
NSW
WA
WA
WA
WA
WA
14
AYP; SYP
WA
32*
SYP
S-W
QLD
QLD, NSW, TAS
SYP
S-W
SYP
QLD, NSW, TAS
WA
WA
S-W
S-W
SYP
S-W
QLD, NSW
NSW
NSW
WA
SYP
SA
SYP
SA
S-W
S-W
WA
NSW, VIC, TAS
NSW, VIC, TAS
SA, QLD, NSW, VIC, TAS
WA
SA, QLD, NSW, VIC, TAS
2n
36
36*
9
32*
108*
14*
18
36
9, 18
9, 18
18
22*
deanei
denticulata
9
fastigiata
filifolia
9
9
floribunda
fraseri
11
16
gracilipes
8
18
18
32
heterophylla
keysii
ledifolia
16
32
megastigma
7
14
microphylla
mollis
11
16
32
molloyae
8
muelleri
11
nana
nematophylla
parviflora
18
9
9
16
22
Voucher
number2
Comments
2 genotypes examined
06253083
06253091
06253105
06253113
06253121
06253148
06253156
Polyploid
Polyploid
Polyploid
Polyploid
‘Dean’s Boronia’
06253164
06253172
06253180
06253202
06253199
2 genotypes examined
‘Red’
‘Near White’
‘Cameo’, registered
variety (Sunglow
Flowers, 1990)
‘Moonglow’, registered
variety (Sunglow
Flowers, 1990)
06253210
Also as B. triphylla
(Armstrong, 1981)
06253075
06253229
06253008
As B. elatior (Wilson,
pers. comm.)
06253067
06253237
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
312
F. SHAN ET AL.
Table 1. Continued
Chromosome number
Taxon
n
pilosa
11
pinnata
polygalifolia
pulchella
purdieana
ramosa
rigens
serrulata
Author
Distribution by state1
Voucher
number2
44*
S-W
SYP
SA, VIC, TAS
SA, VIC, TAS
06253245
‘Rose Blossom’,
Polyploid
22
S-W
SYP
NSW
NSW
06253016
06253253
2 genotypes examined
S-W
S-W
S-W; SYP
S-W
S-W
S-W
SYP
S-W
S-W
QLD, NSW
WA
WA
WA
NSW
NSW
NSW
WA
WA
SP
S-W
S-W
S-W
SYP
WA
NSW
NSW
WA
WA
2n
11
18
7
9
18
18
11
22
spathulata
tenuis
thujona
triphylla
viminea
‘Carousel’
9
9
16
11
16
9
16*
Comments
06253024
06253032
Possible
misidentification
(Stace et al., 1993)
06253040
06253059
Hybrid between
B. heterophylla and
B. molloyae (Elliot
& Jones, 1980)
1
Data from Hnatiuk (1990): abbreviations: NSW, New South Wales; QLD, Queensland; SA, South Australia; TAS,
Tasmania; VIC, Victoria; WA, Western Australia.
2
Deposited at the Western Australian Herbarium of the Department of Conservation and Land Management, Perth.
*Denotes first cytological report for the taxon.
A1 ranges from zero (completely symmetrical) to one
(completely asymmetrical). The somatic chromosome
number of a genotype is 2n, while n in Equation 2 is
the haploid chromosome number of an individual or
taxon. The lengths of the long arm, the short arm and
the chromosome length of the ith chromosome are
expressed as li, si and ti, respectively, in the equation
above.
A2 was defined as a coefficient of variation (standard
deviation/average) of chromosome length in a cell
(Watanabe et al., 1999). A2 was calculated as:
A2 = s / t ¥100%
where s is the standard deviation and t is the mean
chromosome length for each genotype. We calculate
s:
2n
when 2n < 30:
s=
2
 (ti - t )
i=1
2n - 1
2n
and when 2n > 30:
s=
2
 (ti - t )
i=1
2n
Neither A1 nor A2 depends on chromosome number or
chromosome size.
RESULTS
CHROMOSOME
NUMBER
Chromosome complements are illustrated in
Figures 1–12. Numbers were determined for the first
time in B. clavata (2n = 14), B. heterophylla ‘Near
White’ (2n = 15), B. ‘Carousel’ (2n = 16), B. deanei
(2n = 22), B. chartacea (2n = 32), B. keysii (2n = 32),
B. pilosa (2n = 44), B. anethifolia (2n = 36) and
B. citriodora (2n = 108). Other chromosome numbers
were confirmed for B. megastigma (2n = 14),
B. heterophylla ‘Red’ (2n = 15), B. heterophylla ‘Cameo’
(2n = 14), B. heterophylla ‘Moonglow’ (2n = 14),
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
KARYOTYPE EVOLUTION IN BORONIA
313
Figures 1–12. Somatic chromosomes of some Boronia species. Spreads in Figs 1,3 and 6 were prepared using an air-dry
method (Geber & Schweizer, 1988) and stained with FLP-orcein. Others were prepared by a normal squash method and
stained with FLP-orcein. Fig. 1. B. clavata (2n = 14). Fig. 2. B. heterophylla ‘Cameo’ (2n = 14). Fig. 3. B. heterophylla ‘Near
White’ (2n = 15). Fig. 4. B. gracilipes (2n = 16). Fig. 5. B. ‘Carousel’ (2n = 16). Fig. 6. B. denticulata (2n = 18). Fig. 7.
B. deanei (2n = 22). Fig. 8. B. pilosa (2n = 44). Fig. 9. B. keysii (2n = 32). Fig. 10. B. chartacea (2n = 32). Fig. 11.
B. anethifolia (2n = 36). Fig. 12. B. citriodora (2n = 108). Scale bars = 5 mm.
B. molloyae
(2n = 16),
B. gracilipes
(2n = 16),
B. denticulata (2n = 18), B. crenulata (2n = 18),
B. purdieana
(2n = 18),
B. filifolia
(2n = 18),
B. serrulata (2n = 22), B. muelleri (2n = 22), B. mollis
(2n = 32), B. fraseri (2n = 32), B. ledifolia (2n = 32),
B. anemonifolia
(2n = 36)
and
B. coerulescens
(2n = 72).
KARYOTYPE
CONSTITUTION
The karyotypic constitutions of Boronia species
were highly variable between species and even
within the same species, e.g. B. heterophylla
(Fig. 13, Table 2). Boronia karyotypes consisted of
median, submedian, subterminal and terminal
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
314
F. SHAN ET AL.
Table 2. Karyotype parameters for 20 genotypes in 18 Boronia species and one interspecific hybrid. Abbreviations: PMC
= proportion of median chromosomes; SKC = Stebbins’ karyotype classification; TCL = total chromosome length; ACL =
average chromosome length; LCL = longest chromosome length; SCL = shortest chromosome length; A1 = degree of
asymmetry; A2 = length heterogeneity; NF = Nombre Fondamental; KF = karyotype formulae
Taxon
2n
Ploidy
level
PMC
(%)
SKC
TCL
(mm)
ACL
(mm)
LCL
(mm)
SCL
(mm)
A1
A2
NF
KF
B. clavata
B. megastigma
B. heterophylla 1a
B. heterophylla 2b
B. heterophylla 3c
B. molloyae
B. gracilipes
B. ‘Carousel’
B. denticulata
B. crenulata
B. serrulata
B. muelleri
B. deanei
B. pilosa
B. chartacea
B. keysii
B. mollisd
B. fraserid
B. anemonifoliad
B. anethifoliad
B. citriodorad
14
14
15
15
14
16
16
16
18
18
22
22
22
44
32
32
32
32
36
36
108
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
4x
2x
2x
2x
2x
2x
2x
–
29
29
20
33
29
25
25
13
44
78
73
73
82
82
69
75
NA
NA
NA
NA
NA
3A
2B
3A
3A
3B
3A
3B
3A
2A
2A
2B
2B
2B
2B
2B
2A
NA
NA
NA
NA
NA
49.92
46.50
53.97
51.44
49.68
46.91
56.84
46.42
61.14
61.17
54.95
63.89
50.06
124.84
58.18
64.72
63.22
66.86
50.80
52.84
199.74
3.57
3.32
3.60
3.41
3.55
2.93
3.55
2.90
3.40
3.40
2.50
2.90
2.28
2.84
1.82
2.02
1.98
2.09
1.63
1.47
1.85
4.57
4.61
4.93
4.77
4.87
3.90
4.84
3.47
4.27
4.61
3.73
4.63
3.55
4.34
2.96
2.83
3.20
2.83
2.03
1.89
2.62
2.92
2.26
2.57
2.41
2.25
2.07
2.22
2.20
2.58
2.49
1.58
1.76
1.54
1.84
1.02
1.51
1.28
1.49
1.33
1.24
1.18
0.35
0.36
0.37
0.36
0.38
0.35
0.35
0.37
0.32
0.19
0.22
0.21
0.18
0.21
0.23
0.19
NA
NA
NA
NA
NA
15.00
21.71
17.08
19.63
21.93
20.04
20.08
15.24
14.63
19.90
24.68
22.24
28.70
21.09
21.23
16.44
19.40
19.26
11.19
12.00
11.33
28
26
30
30
27
32
32
32
34
36
44
44
42
82
58
60
NA
NA
NA
NA
NA
4 m + 8sm + 2st
4 m + 8sm + 2t
3 m + 6sm + 6st
5 m + 6sm + 4st
4 m + 4sm + 5st + 1t
4 m + 8sm + 4st
4 m + 8sm + 4st
2 m + 14sm
8 m + 8sm + 2t
14 m + 4sm
16 m + 6sm
16 m + 6sm
18 m + 2sm + 2t
36 m + 2sm + 6t
22 m + 4sm + 6t
24 m + 2sm + 2st + 4t
NA
NA
NA
NA
NA
a
B. heterophylla ‘Red’ bB. heterophylla ‘Near White’ cB. heterophylla ‘Cameo’ dDue to difficulty of identification of centromeres
in small chromosomes the karyotype formulae, PMC, KCS, A1 and NF are not presented.
chromosomes in varying numbers for different
karyotypes. Generally there were fewer medians
with decreasing chromosome number in a karyotype. The proportions of medians in the karyotype
(Table 2) were 75% in B. keysii and 69% in
B. chartacea (2n = 32), 73–82% in species with
2n = 22 or 44, 78% in B. crenulata and 44% in
B. denticulata (2n = 18), 13–25% in species with
2n = 16, 20–33% in B. heterophylla (2n = 14 or 15)
and 29% in species with 2n = 14.
NOMBRE
FONDAMENTAL
Nombre fondamental (NF) generally decreased as
chromosome number reduced (Table 2). In species
with 2n = 32, 22, 18 and 14, different species with
the same chromosome numbers have different NF
values. In species with 2n = 16 or 15, the NF in different species with the same chromosome numbers
were the same. However, within B. heterophylla the
NF in the 2n = 14 genotype was three fewer than the
2n = 15 genotypes. In species with 2n = 22, the NF in
B. muelleri and B. serrulata was 44, slightly differ-
ent from the 42 in B. deanei. The NF was 82 in
B. pilosa.
CHROMOSOME
LENGTH
Boronia chromosomes were small in size, ranging
from 1.02 to 4.93 mm in length. Average chromosome
length (ACL) tended to increase as chromosome number decreased (Fig. 14). Chromosomes were smallest
in species with 2n = 36, their ACL ranging from 1.47 to
1.63 mm. In species with 2n = 32, the ACL ranged from
1.82 to 2.09 mm. Those in species with 2n = 22 or 44
ranged from 2.28 to 2.90 mm. In most species with
2n = 18 or fewer the ACL was more than 3.32 mm but
less than 3.60 mm. The exceptions were B. ‘Carousel’
where it was 2.90 mm and B. molloyae where it was
2.93 mm.
Total chromosome length (TCL) in diploid species
varied from 46.42 mm to 66.86 mm (Table 2). A very
weak relationship was found between TCL and chromosome number (Fig. 15). Average TCL in diploid
species was 55.24 mm. In B. pilosa the TCL was
124.84 mm and it was 199.74 mm in B. citriodora.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
KARYOTYPE EVOLUTION IN BORONIA
315
Figure 13. Idiograms of 16 genotypes in 13 Boronia species and one interspecific hybrid. Scale bar = 2 mm.
KARYOTYPE
ASYMMETRY
According to the karyotype classification of Stebbins
(1971), species with 2n ≥ 18 had 2A to 2B karyotypes.
Species with 2n £ 16 had 3A or 3B karyotypes. The
exception was B. megastigma, which had a 2B
karyotype.
Degree of asymmetry A1 increased as chromosome
number decreased as a general trend (Fig. 16). Species
with 2n = 18 had A1 ranging from 0.35 to 0.38, while
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
316
F. SHAN ET AL.
4.00
80.00
TCL = 0.38 (2n) + 46.82
R 2= 0.22 P = 0.04
ACL = -0.09 (2n ) + 4.66
R 2 = 0.90 P = 0.00
3.50
70.00
TCL (mm)
ACL (mm)
3.00
2.50
60.00
50.00
2.00
40.00
1.50
30.00
1.00
12 14 16 18 20 22 24 26 28 30 32 34 36 38
12 14 16 18 20 22 24 26 28 30 32 34 36 38
2n
2n
Figure 14. Relationships between average chromosome
length (ACL) and chromosome number (2n) of diploid species in Boronia. Significant negative relationship was found
between average chromosome length and somatic chromosome number (P < 0.01). Most proportion of the average
chromosome length variation is due to changes of somatic
chromosome number (r2 = 0.90).
Figure 15. Relationships between total chromosome
length (TCL) and chromosome number (2n) of diploid species in Boronia. The relationship between total chromosome length and somatic chromosome number is weak
(P = 0.04), and only a small proportion of the total chromosome length variation is due to changes of somatic chromosome number (r2 = 0.22).
0.50
35.00
0.45
A1 = -0.01 (2n) + 0.49
R 2 = 0.58 P = 0.00
0.40
25.00
0.35
20.00
0.30
A2
A1
A2 = -0.15 (2 n ) + 22.32
2
R = 0.08 P = 0.23
30.00
0.25
15.00
0.20
10.00
0.15
5.00
0.10
0.05
12
14
16
18
20
22
24
26
28
30
32
34
36
0.00
12 14 16 18 20 22 24 26 28 30 32 34 36 38
2n
Figure 16. Relationships between degree of asymmetry
(A1) and chromosome number (2n) of diploid species in
Boronia. A strong negative relationship was found between
degree of asymmetry and somatic chromosome number
(P < 0.01). A large proportion of variation in the degree of
asymmetry is due to changes of somatic chromosome number (r2 = 0.58).
species with 2n = 18 had A1 of 0.19–0.23, with the
exception that B. denticulata (2n = 18) had A1 = 0.32
(Table 2).
No relationship was found between length heterogeneity A2 and chromosome number (Fig. 17). Length
heterogeneities were high in species with 2n = 22,
ranging from 22.24 to 28.70 (Table 2). They were low
in species with 2n = 36 and 108, ranging from 11.19
to 12.00. All other species had A2 ranging from 14.63
2n
Figure 17. Relationships between length heterogeneity
(A2) and chromosome number (2n) of diploid species in
Boronia. No regression was found between length heterogeneity and somatic chromosome number (r2 = 0.08,
P = 0.23).
to 21.93 and this was not correlated with chromosome
number.
DISCUSSION
BASIC
CHROMOSOME NUMBER AND
CYTO-EVOLUTIONARY TRENDS IN
BORONIA
One hypothesis of Boronia cyto-evolution is that the
basic chromosome number is x = 9. Species with x = 11,
10, 8 and 7 are suggested to be derived from x = 9 by
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
KARYOTYPE EVOLUTION IN BORONIA
aneuploid change. Species with x = 18 and 16 are products of the polyploidization of entities with x = 9 and x
= 8 (Smith-White, 1954). The alternative explanation
is that the basic chromosome number is x = 18, with
the chromosome number changed during evolution
through dysploid reduction (Stace et al., 1993).
Results in this study support the basic chromosome
number of x = 18. In general, symmetrical karyotypes
are regarded as more primitive and asymmetrical
karyotypes as more specialized (Stebbins, 1971). The
karyotype asymmetry is shown here to increase during the evolution of Boronia species where chromosome number descended. This indicates that the more
primitive species have the higher chromosome number. This evolutionary trend is observed in many
plants, such as in the genera Dalechampia (Euphorbiaceae; Vanzela et al., 1997), Brachyscome (Asteraceae;
Watanabe et al., 1999), Echinops (Asteraceae; Sheidai,
Nasirzadeh & Kheradnam, 2000) and the species Matricaria chamomilla (Asteraceae; Bara, Caraghin &
Truta, 1988). The support also comes from other evidence. Phylogenetic analysis using DNA markers indicates that Boronia has evolved from high to low
chromosome numbers (F. Shan et al. unpubl. data).
Interpretation of chromosome number and morphological characteristics favours a basic chromosome number of x = 18 (Stace et al., 1993). Boronia is within the
tribe Boronieae, which may have originated from an
ancestor with x = 18 (da Silva et al., 1988; Stace et al.,
1993). All this evidence suggests that x = 18 is possibly
the basic chromosome number for Boronia and chromosome number reduction is its evolutionary trend.
In the hypothesis of x = 9, polyploidization would be
an essential mechanism for the origin of species with
high chromosome numbers. Evidence was sought for
the occurrence of polyploidization during Boronia evolution. If polyploidization had occurred, the species
with x = 18 would have twice the total chromosome
length of species with x = 9. In addition, multivalents
might be expected in species with x = 16 or 18 if autopolyploids were produced. However, species with x =
18 and 9 had similar total chromosome length
(Table 2, Fig. 15) and no multivalents were observed
at metaphase I of meiosis in Boronia species with n =
18 or 16 (Shan, Yan & Plummer, unpublished). Thus
no evidence was found for polyploidization from x = 9
to x = 18 in Boronia.
Polyploidization has played an important role in the
evolution of the angiosperms (Stebbins, 1971; Gatt,
Hammett & Murray, 1999; Soltis & Soltis, 2000). Polyploids generally maintain higher levels of heterozygosity than their diploid progenitors and exhibit less
inbreeding depression than their diploid parents.
These benefits contribute to their success in nature.
However, this is not the case in Boronia. Polyploids
exist in five out of 42 Boronia species (Table 1), but
317
since low chromosome numbers have been found in all
these species except B. citriodora, these polyploids are
most likely to be autopolyploids. Although addition of
a whole set of chromosome (polyploidization) does
occur, it is within a species. Therefore, we suggest that
polyploidizaiton is not an important process for the
speciation of Boronia.
If we exclude polyploidization as a major mechanism in Boronia speciation, chromosome number
could also increase via aneuploidy. The result of this
process would be that total chromosome length would
increase significantly as chromosome number
increased but average chromosome length would
remain unchanged. However, total chromosome
lengths of all diploid species with either high or low
chromosome numberswere similar but average chromosome length decreased as chromosome number
increased. This indicated that aneuploid increase
could not have occurred in Boronia.
Robertsonian fission was another way to increase
chromosome number, as recorded in animals and some
plants (Schubert & Rieger, 1985). The result of
this process would make the karyotypes of species
with higher chromosome numbers more asymmetrical, but the NF would remain the same. However,
asymmetry of Boronia species decreased as chromosome number increased and NF varied. Species with
higher chromosome numbers were more symmetrical,
ruling out the possibility of Robertsonian fission.
Therefore there is strong evidence against all possible
routes towards chromosome number increase in Boronia evolution. It is thus almost impossible for the basic
chromosome number of Boronia to be x = 9.
MECHANISM
FOR KARYOTYPE EVOLUTION
IN
BORONIA
The processes that can make chromosome numbers
decrease include dysploid reduction, aneuploid
decease and Robertsonian fusion. Aneuploid decrease
is a process of individual chromosome loss, which
would make total chromosome length shorter as chromosome numbers decrease. Dysploid reduction is a
process of unequal translocation involving centromere
loss after transferring chromosome components from
one chromosome to the other (Stace & James, 1996).
This process is thought to be the main mechanism for
plant evolution (James, 1981) and has been proposed
for Boronia cyto-evolution (Stace et al., 1993). Dysploid reduction would not change total chromosome
length much but would increase average chromosome
length. Total chromosome lengths of all Boronia diploid species are similar at around 55.24 mm, but average chromosome length increased as chromosome
number decreased (Table 2, Figs 14,15). For example,
average chromosome length was about 1.5 mm in spe-
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
318
F. SHAN ET AL.
cies with 2n = 36, but more than 3 mm in most species
with 2n = 18 or fewer. This indicated that most of the
chromosomal segments had been kept in the Boronia
genome during the reduction of its chromosome number. This result rejected the possibility of aneuploid
decrease in Boronia cyto-evolution. Overall, the general trend was consistent with dysploid reduction,
which is therefore the most probable mechanism
responsible for major changes leading to Boronia chromosome number reduction.
Robertsonian fusion of two telocentric or acrocentric
chromosomes results in a decrease in chromosome
number. This leads to an increase in symmetry of the
karyotype but keeps the NF consistent (Jones, 1998).
In higher plants Robertsonian fusion seems to be relatively uncommon, though it is the most important
means of karyotype change in animals (Jones, 1998).
These changes were not observed in the Boronia
species studied, so this type of chromosomal change
seems unlikely to be responsible for Boronia cytoevolution.
Structural rearrangements of chromosomes may
also have happened during Boronia karyotype change.
This karyotype change keeps the chromosome number
constant but variations occur among karyotypes (Schubert & Rieger, 1985). This was observed frequently in
Boronia. All species with the same chromosome number had different karyotypes in terms of karyotype
organization, karyotype asymmetry, average chromosome length and NF (Table 2). Differences in total chromosome length between different species with the
same chromosome number may indicate duplication or
deletion of chromosome segments. Variation in asymmetry may indicate segment translocation, evidence
for which is also seen in the variation of rDNA site
number and location in the Boronia genome (F. Shan,
G. Yan & J. A. Plummer, unpubl. data). Variation of
rDNA sites in number from two to 11 indicates deletion
or duplication during karyotype evolution in Boronia.
The shift of rDNA locations from terminal to interstitial sites indicates chromosome structure rearrangements by fusion, breakage, translocation or inversion,
with rDNA sequences involved in Boronia. The variation in rDNA size at a particular site may indicate that
rDNA gene copy numbers increased or decreased
through gene amplification or deletion.
Our results have indicated that dysploid reduction
might be the major mechanism for Boronia karyotype
evolution. Chromosome structural rearrangements
would also add to the diversity of the Boronia
genomes.
ORIGIN
AND EVOLUTION OF
BORONIA
There are currently about 140 recognized species in
Boronia (Duretto, 1999). So far, chromosome numbers
have been determined in 42 species (Table 1). They
comprise 30% of all species in Boronia and represent
karyotypes with chromosome numbers ranging from
2n = 14, through 15, 16, 18, 22, 32, 36, 44, 72 to
2n = 108. Nearly all species with chromosome numbers higher than n = 9 are distributed in the eastern
states of Australia (Table 3) and species with n = 18
are distributed mainly in eastern Australia. Species
with n = 7 and 8 are exclusively discovered in Western
Australia and most species with n = 9 are found in the
south-western Australia as well. As x = 18 is suggested as the basic chromosome number for Boronia,
the south-eastern region of Australia, where most species with n = 18 are found, is suggested to be the first
centre of origin for Boronia and south-western Australia a secondary one.
The distribution patterns of Boronia indicate that
the ancestor with n = 18 might have evolved into spe-
Table 3. Distribution of Boronia chromosome numbers* in Australia. Abbreviations: E, eastern states of Australia, NSW,
New South Wales; QLD, Queensland; SA, South Australia; TAS, Tasmania; VIC, Victoria; WA, Western Australia
Number of species distributed in eastern Australia
Haploid chromosome
number
Number of species
distributed in WA
Total in E
NSW
VIC
QLD
SA
TAS
n=7
n=8
n=9
n = 10
n = 11
n = 16
n = 18
n = 54
4
4
8
–
–
1
2
–
–
–
3
1
8
5
6
1
–
–
2
–
7
4
6
–
–
–
2
–
2
–
3
1
–
–
1
–
1
2
3
–
–
–
2
1
1
–
2
–
–
–
1
–
2
1
2
1
*If there are polyploid genotypes within the same species, only the lower chromosome number is presented. Some species
may be distributed in more than one state. The numbers of these species are duplicated in the table.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 309–320
KARYOTYPE EVOLUTION IN BORONIA
cies with n = 16, 11, 10, 9 through dysploid reduction
during evolution in the eastern states of Australia.
Some species with n = 9 may have spread to the southwestern region of Western Australia and evolved further into species with n = 8 and 7. At the same time,
primitive species with n = 18 and maybe n = 16 might
have spread to Western Australia as well. Perhaps
because of the very limited number of species with n =
18 or 16 in Western Australia, no species with n = 11
and 10, as found in eastern Australia, have been found
in Western Australia. Species with n = 8 and 7 were
found only in Western Australia, indicating they are
derived from species with n = 9 there. No species with
n = 8 or 7 have been found in eastern Australia, suggesting that either species with n = 9 stopped evolving
to species with n = 8 and 7, possibly due to an unfavourable climate change, or they have yet to be discovered.
Integration of information from chromosome number, distribution, specific divergence time (F. Shan
et al. unpublished), and geographical and climatic
changes in the Australian continent is required to
develop a hypothesis on the origin and evolution of
Boronia. We propose that Boronia originated in the
south-east region of the Australian continent resulting
in the most primitive species, with n = 18, which are
still found in this area. The Australian continent is
one part of Gondwana which consisted of India, Australia, Antarctica, Africa and South America. Australia separated from Africa and South America 120–
148 Myr (Nei & Kumar, 2000), from India 130–
140 Myr and from Antarctica and New Zealand some
80 Myr (Nei, 1987). Boronia is endemic to Australia
and has not been found in New Zealand or any other
continent. Therefore Boronia might have appeared on
the Australian continent after 80 Myr. Before 57 Myr
it dispersed through eastern Australia and chromosome numbers decreased from n = 18 to n = 16 and perhaps as low as n = 9. During this period eastern
Australia was separated from the western shield of
the Australian continent by the Tambo Sea, which
Boronia species could not cross to the western part of
the continent. During the Eocene (57–35 Myr) a cool
climate favoured a more extensive Nothofagus forest
(Cookson, 1946). This resulted in further cytoevolution and spread of Boronia in the eastern shield.
During this period a relatively narrow land bridge
formed between the western shield and eastern region
of the continent (David, 1950). It was then possible for
Boronia to start to cross the previous barrier and disperse westwards, with most species having n = 9 and a
few species with n = 18 and perhaps 16. The primitive
species with n = 18 or 16 perhaps had little or no chromosome number change, although chromosome
structural rearrangement might have taken place, or
derivatives of n = 18 or 16 which have not been discovered might have arisen. This would explain why
319
most species distributed in Western Australia have
low chromosome numbers and only a few have numbers of n = 18 and 16, which are more primitive (for
example B. tenuis; F. Shan et al., unpublished). In the
Miocene (23–5 Myr) a warm moist climate favoured
the extension of rain forest vegetation (Crocker &
Wood, 1947), which may have encouraged the expansion of Boronia much further south and west (F. Shan
et al., unpublished). During this time the barrier
between east and west was recreated by the oncoming
aridity due to the Nullarbor Gulf extension far to the
north of the present Bight coastline. The barrier has
been maintained by aridity, reinforced by the calcareous soils of the Nullarbor Plain in the Pliocene period
(5 Myr) and Quaternary Era (1.6 Myr). In the cooler
and drier conditions of the Pliocene (since 5 Myr) the
rain forest flora contracted and rain forests gradually
disappeared. These changes stopped Boronia dispersal between the eastern and western Australian
continent. Thereafter Boronia underwent separate
karyotype evolution in the west and east. All these factors may have attributed to the distribution pattern of
Boronia today.
ACKNOWLEDGEMENTS
The authors thank Dr Paul Wilson, taxonomist at the
Western Australian Herbarium of the Department of
Conservation and Land Management for the identification and confirmation of genotypes in this research,
advice on preparation and lodgement of Herbarium
specimens at the Western Australian Herbarium of
the Department of Conservation and Land Management, Perth, Dr Helen Stace of the University of Western Australia for helpful discussions, Sunglow Flowers
Pty Ltd in Western Australia, Bernawarra Gardens in
Tasmania and WildTech Nursery Pty Ltd in Victoria
for some Boronia species. Fucheng Shan was supported by an International Postgraduate Research
Scholarship and a University Postgraduate Award
from the University of Western Australia, Perth.
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