Abstract
Although
karyologically well studied, the genus Tanacetum (Asteraceae) is poorly
known from the perspective of molecular cytogenetics. The prevalence of
polyploidy, including odd ploidy warranted an extensive cytogenetic
study. We studied several species native to Iran, one of the most
important centres of diversity of the genus. We aimed to characterise
Tanacetum genomes through fluorochrome banding, fluorescent in situ
hybridisation (FISH) of rRNA genes and the assessment of genome size by
flow cytometry. We appraise the effect of polyploidy and evaluate the
existence of intraspecific variation based on the number and
distribution of GC-rich bands and rDNA loci. Finally, we infer ancestral
genome size and other cytogenetic traits considering phylogenetic
relationships within the genus.
We report first genome size (2C) estimates ranging from 3.84 to 24.87 pg
representing about 11 % of those recognised for the genus. We found
striking cytogenetic diversity both in the number of GC-rich bands and
rDNA loci. There is variation even at the population level and some
species have undergone massive heterochromatic or rDNA amplification.
Certain morphometric data, such as pollen size or inflorescence
architecture, bear some relationship with genome size. Reconstruction of
ancestral genome size, number of CMA+ bands and number of rDNA loci
show that ups and downs have occurred during the evolution of these
traits, although genome size has mostly increased and the number of CMA+
bands and rDNA loci have decreased in present-day taxa compared with
ancestral values.
Tanacetum genomes are highly unstable in the number of GC-rich bands and
rDNA loci, although some patterns can be established at the diploid and
tetraploid levels. In particular, aneuploid taxa and some odd ploidy
species show greater cytogenetic instability than the rest of the genus.
We have also confirmed a linked rDNA arrangement for all the studied
Tanacetum species. The labile scenario found in Tanacetum proves that
some cytogenetic features previously regarded as relatively constant, or
even diagnostic, can display high variability, which is better
interpreted within a phylogenetic context.
The striking and unexpected cytogenetic diversity of genus Tanacetum L. (Asteraceae): a cytometric and fluorescent in situ hybridisation study of Iranian taxa
- Nayyereh Olanj,
- Teresa Garnatje,
- Ali Sonboli,
- Joan Vallès and
- Sònia GarciaEmail authorView ORCID ID profile
BMC Plant Biology201515:174
DOI: 10.1186/s12870-015-0564-8
© Olanj et al. 2015
Received: 17 April 2015
Accepted: 26 June 2015
Published: 8 July 2015
Abstract
Background
Although karyologically well studied, the genus Tanacetum
(Asteraceae) is poorly known from the perspective of molecular
cytogenetics. The prevalence of polyploidy, including odd ploidy
warranted an extensive cytogenetic study. We studied several species
native to Iran, one of the most important centres of diversity of the
genus. We aimed to characterise Tanacetum genomes through fluorochrome banding, fluorescent in situ
hybridisation (FISH) of rRNA genes and the assessment of genome size by
flow cytometry. We appraise the effect of polyploidy and evaluate the
existence of intraspecific variation based on the number and
distribution of GC-rich bands and rDNA loci. Finally, we infer ancestral
genome size and other cytogenetic traits considering phylogenetic
relationships within the genus.
Results
We report first genome size
(2C) estimates ranging from 3.84 to 24.87 pg representing about 11 % of
those recognised for the genus. We found striking cytogenetic diversity
both in the number of GC-rich bands and rDNA loci. There is variation
even at the population level and some species have undergone massive
heterochromatic or rDNA amplification. Certain morphometric data, such
as pollen size or inflorescence architecture, bear some relationship
with genome size. Reconstruction of ancestral genome size, number of
CMA+ bands and number of rDNA loci show that ups and downs have occurred
during the evolution of these traits, although genome size has mostly
increased and the number of CMA+ bands and rDNA loci have decreased in
present-day taxa compared with ancestral values.
Conclusions
Tanacetum
genomes are highly unstable in the number of GC-rich bands and rDNA
loci, although some patterns can be established at the diploid and
tetraploid levels. In particular, aneuploid taxa and some odd ploidy
species show greater cytogenetic instability than the rest of the genus.
We have also confirmed a linked rDNA arrangement for all the studied Tanacetum species. The labile scenario found in Tanacetum
proves that some cytogenetic features previously regarded as relatively
constant, or even diagnostic, can display high variability, which is
better interpreted within a phylogenetic context.
Keywords
5S 35S Aneuploidy Evolutionary cytogenetics Genomic instability L-type arrangement Polyploidy Odd ploidy Ribosomal DNABackground
Tanacetum L. is a genus of the family Asteraceae Bercht. & J. Presl and includes approximately 160 species [1]. It is one of the largest genera within the tribe Anthemideae Cass., together with genera such as Artemisia L., Achillea L. and Anthemis L. Commonly known as tansies, Tanacetum
species are native to many areas of the Northern Hemisphere, occupying
the temperate zones of Europe, Asia, North Africa and North America, but
particularly abundant in the Mediterranean and Irano-Turanian regions.
Although the presence of Tanacetum in the Southern Hemisphere is much less common [1, 2], some species are grown worldwide such as T. parthenium (L.) Sch. Bip., which can behave as a weed outside its native range.
Tanacetum
species are mostly perennial herbs, although the genus has some annuals
and some subshrubs. They usually form rhizomes and are aromatic plants.
Their capitula, solitary or arranged in more or less dense or loose
compound inflorescences, always contain disc flowers (flosculous,
yellow, numerous — up to 300), sometimes with ray flowers (ligulate,
white, yellow or pale pink). Tanacetum
is considered to hold a crucial position for understanding the
phylogenetic relationships within its tribe, but available phylogenetic
reconstructions show that these species form an imbroglio whose generic
relationships and infrageneric arrangement are still unsettled [3]. Many species of Tanacetum
are widely distributed and are used as sources of medicines, food or
forage. In particular, several studies have shown that essential oils
from T. parthenium [4, 5, 6] and T. balsamita L. [7, 8, 9] have strong antibacterial, cytotoxic, neuroprotective and antioxidant activity. T. balsamita has also shown anti-inflammatory properties [10]. West and central Asia are two important speciation centres of the genus [11],
and Iran is one of the main areas of speciation and diversification,
promoted by a unique combination of ecosystems. In Iran the genus is
represented by 36 species according to the most recent revisions,
including 16 endemic taxa [3, 12, 13, 14, 15, 16, 17].
The karyology of Tanacetum has been extensively studied, with chromosome counts known for a considerable number of its species [18, 19, 20, 21].
Its basic chromosome number is x = 9, as in most Anthemideae and
Asteraceae; indeed x = 9 is likely the ancestral basic number for the
family as a whole [22]. Ploidy levels are found up to 10× [23].
Recent work has added more karyological information for this genus; it
seems that polyploidy is an important evolutionary force and the
existence of odd ploidy, aneuploidy and presence of B-chromosomes is not
uncommon [18, 20].
Methods such as fluorochrome banding and fluorescent in situ
hybridisation (FISH) of 5S and 18S-5.8S-26S (35S) ribosomal RNA genes
(rDNA) provide chromosome markers, excellent tools to improve karyotype
description [24]. These methods have proven useful for comparing taxa at different levels, particularly in plants (see, for example, [25] on several Asteraceae genera; [26], on Fragaria L.; [27] on Thinopyrum Á. Löve). However broader cytogenetic information is largely missing for Tanacetum,
as happens for many wild species, unlike crops or other economically
interesting plants whose chromosomes have been more deeply investigated.
Genome size estimation, easily obtained by flow cytometry, has been
used in a similar way (see, for example, [28] on Tripleurospermum Sch. Bip.; [29] on Carthamus L.; [30] on Artemisia
L.). The combination of these methods can improve our understanding of
chromosome evolution and genome organisation processes in plants [31].
Moreover, molecular cytogenetic studies, together with genome size
evaluation, are also useful in a wide range of biological fields, from
taxonomy, evaluation and conservation of genetic resources, to plant
breading [24, 32, 33, 34].
Despite being a large and well-known genus, molecular cytogenetic studies of Tanacetum are limited to a single work reporting data on two species: T. achilleifolium (M. Bieb.) Sch. Bip. and T. parthenium [35]. That study described co-localisation of both 5S and 35S ribosomal RNA genes in Tanacetum, the so-called linked type (L-type) arrangement of rDNA, confirming preliminary findings for this genus [25].
This rDNA organisation is typical of several Asteraceae members,
particularly those belonging to tribes Anthemideae and the Heliantheae
Cass. alliance (see [25, 36]
for details). However, the most common rDNA organisation in plants, and
also in family Asteraceae, is that in which both rRNA genes are
separated (S-type arrangement). Remarkably, [35] found that one 35S rDNA locus was separated in T. achilleifolium,
while the other one remained co-localised with the 5S. This dual
organisation of rDNA in the same species (i.e. both L-type and S-type
coexisting) is exceptional.
Likewise, genome sizes for Tanacetum are only known for few species, reduced to three research works to our knowledge [37, 38, 39]. In this study, we establish a deeper knowledge of Tanacetum
genomes through molecular cytogenetic and genome size analysis. We
focus on several species native to Iran, since this area constitutes a
centre of speciation and diversification of the genus. All ploidy levels
previously reported for the genus (from 2x to 10x) exist in Iran [20],
many of the studied tansies grow there in polyploid series, and odd
stable ploidy, aneuploidy and presence of B-chromosomes have been found [3, 20]. Our specific goals were (1) to characterise the genomes of Tanacetum
species by flow cytometry, fluorochrome banding and FISH of rRNA genes,
and particularly, to observe the rDNA organisation in these species,
(2) to detect the karyotype and genome size patterns of the genus and
describe their typical models, if any, (3) to address the presence of
polymorphisms at the cytogenetic level, (4) to assess the impact of
polyploidy in Tanacetum
genomes, and (5) to reconstruct ancestral character states of genome
size and karyotype features such as number of rDNA loci and CMA+ bands
to infer genome evolution in the context of a phylogenetic framework of
the genus.
Results
The chromosome counts here represent most ploidy levels found in Tanacetum to present, all x = 9-based. We found B-chromosomes in one of the populations of T. pinnatum and in T. fisherae, and some of the populations investigated, such as those of T. archibaldii and T. aureum
(Lam.) Greuter, M.V.Agab. & Wagenitz, presented mixed ploidy. In
addition, several of the studied taxa are odd polyploids, such as the
case of triploid T. joharchii Sonboli & Kaz. Osaloo and T. kotschyi (Boiss.) Grierson, and the pentaploid T. fisherae
Aitch. & Hemsl. which is also a hypoaneuploid since it has lost one
chromosome out of the 45 expected. More detailed karyological
information is in Table 1.
Table 1
Provenance and
voucher number from the Medicinal Plants and Drug Research Institute
Herbarium (MPH), Shahid Beheshti University (Tehran) of the populations
of Tanacetum studied, together with genome size, number of CMA+ bands and number of rDNA sites
Species
|
Population
|
PL1
|
2n2
|
2C3
|
2C4
|
SD5
|
1Cx6
|
HPCV7
|
CMA8
|
rDNA9
|
---|---|---|---|---|---|---|---|---|---|---|
T. archibaldii Podl.
|
Mazandaran: Pole Zangoleh road (1790)
|
2
|
18
|
8.77
|
8577
|
0.04
|
4.39
|
1.77
|
56itc (50, 54, 66)
|
4
|
T. balsamita L.
|
Mazandaran: Pole Zangooleh road (1788)
|
2
|
18
|
10.38
|
10152
|
0.09
|
5.19
|
1.13
|
40tc (24, 30, 34, 36, 40, 42, 44)
|
4
|
T. budjnurdense (Rech.f) Tzvel.
|
Khorasan: Bujnourd (1477)
|
2
|
18
|
10.13
|
9907
|
0.19
|
5.07
|
1.77
|
4t
|
4
|
T. canescens DC.
|
Zanjan: Soltanieh (1912)
|
2
|
18
|
9.3
|
9095
|
0.13
|
4.65
|
1.68
|
4, 6 and 8tc
|
6 (8)
|
T. aureum (Lam.) Greuter, M.V.Agab. & Wagenitz
|
Urmia: Meyab (1848)
|
4
|
36
|
17.08*
|
16704
|
1.38
|
4.27
|
2.62
|
28tc (26, 32, 34)
|
10 (8)
|
T. aureum (Lam.) Greuter, M.V.Agab. & Wagenitz
|
Urmia: Suluk Waterfall (1861)
|
4
|
36
|
15.47*
|
15130
|
0.36
|
3.87
|
2.79
|
6 and 10t (3, 4, 5)
|
10
|
T. heimerlii (Nabělek) Parsa
|
Urmia: Sero road, Golsheykhan (1227)
|
2
|
18
|
8.25
|
8069
|
0.06
|
4.13
|
2.09
|
4t (2, 3, 5, 6)
|
4 and 6
|
T. oligocephalum (DC.) Sch.Bip.
|
Urmia: Chaldoran (1914)
|
2
|
18
|
7.67
|
7501
|
0.05
|
3.84
|
2.53
|
6t (4)
|
6
|
T. oligocephalum (DC.) Sch.Bip.
|
Urmia: Naghadeh (1868)
|
4
|
36
|
17.57*
|
17183
|
0.62
|
4.39
|
2.2
|
22t(10, 12, 14, 20, 24)
|
12 (8, 10)
|
T. oligocephalum (DC.) Sch.Bip.
|
Urmia: Mamakan (1911)
|
4
|
36
|
14.87*
|
14543
|
0.28
|
3.72
|
3.02
|
10t (8, 9)
|
10
|
T. fisherae Aitch. & Hemsley.
|
Kerman Mehr mountain, north and east slopes (1916)
|
5
|
44A
|
17.11*
|
16734
|
0.27
|
3.42
|
2.69
|
30 tc (8, 14, 22, 24, 28)
|
10 (5, 7, 6, 12, 15)
|
T. hololeucum (Bornm.) Podl.
|
Mazandaran: Pole Zangoleh road (1791)
|
2
|
18
|
8.45
|
8264
|
0.2
|
4.23
|
1.61
|
14 and 16t (18, 20, 22)
|
6
|
T. joharchii Sonboli & Kaz.Osaloo
|
Khorasan, Chenaran, (1620)
|
3
|
27
|
11.31*
|
11061
|
0.11
|
3.77
|
0.92
|
24itc (32 and 36)
|
6 (5, 8)
|
T. kotschyi (Boiss.) Grierson
|
Urmia, Anhar road, Suluk (1129)
|
3
|
27
|
10.04*
|
9819
|
0.07
|
3.35
|
1.63
|
24tc (20, 28, 32, 34)
|
6
|
T. kotschyi (Boiss.) Grierson
|
Tabriz: Mishodagh (1339)
|
3
|
27
|
10.72*
|
10484
|
0.12
|
3.57
|
1.83
|
44tc (28, 32, 42, 44, 48)
|
6
|
T. kotschyi (Boiss.) Grierson
|
Zanjan: Ghidar (1419)
|
3
|
27
|
8.58*
|
8391
|
0.09
|
2.86
|
1.89
|
18tc (20, 22, 26)
|
4
|
T. parthenifolium (Willd.) Sch.Bip.
|
Urmia: Suluk Waterfall (1127)
|
2
|
18
|
4.68
|
4577
|
0.09
|
2.34
|
3.07
|
4t
|
4
|
T. parthenium (L.) Sch.Bip.
|
Tehran: Tochal (1483)
|
2
|
18
|
3.84
|
3756
|
0.04
|
1.92
|
2.46
|
2t (3, 4)
|
2 (3, 4)
|
T. parthenium (L.) Sch.Bip.
|
Tehran: Shahid Beheshti University, agricultural field of research. Cultivated (1633)
|
2
|
18
|
4.51
|
4411
|
0.04
|
2.26
|
3.06
|
14tc (8, 10)
|
6
|
T. parthenium (L.) Sch.Bip.
|
Hamadan: Dare Morad Beig (1903)
|
2
|
18
|
4
|
3912
|
0.04
|
2.00
|
3.02
|
3t (2,4)
|
2(3, 4)
|
T. persicum (Boiss.) Mozaff.
|
Chahar Mahal & Bakhtiari: Sabz Kuh (1502)
|
2
|
18
|
4.4
|
4303
|
0.69
|
2.20
|
2.49
|
4t
|
4
|
T. pinnatum Boiss.
|
Hamadan: Asad Abad (1895)
|
2
|
18B
|
13.19
|
12900
|
0.06
|
6.60
|
2.09
|
4t
|
4
|
T. pinnatum Boiss.
|
Hamadan: Malayer (1896)
|
2
|
18
|
13.18*
|
12890
|
0.08
|
6.59
|
2.75
|
4t (6)
|
4
|
T. pinnatum Boiss.
|
Hamadan: Razan (1894)
|
4
|
36
|
24.87*
|
24323
|
0.58
|
4.15
|
1.45
|
6t (3, 4, 5)
|
4 and 6 (8)
|
T. polycephalum Sch.Bip. ssp. argyrophyllum (K.Koch) Podlech
|
Urmia: Meshkin Shahr (1884)
|
2
|
18
|
9.26
|
9056
|
0.14
|
4.63
|
1.3
|
6t (5, 7, 8, 10)
|
6 (7, 8)
|
T. polycephalum Sch.Bip. ssp. argyrophyllum (K.Koch) Podlech
|
Urmia: Ghasemloo Valley (1866)
|
4
|
36
|
17.88*
|
17487
|
0.84
|
4.47
|
2.84
|
8 and 10t (5, 6, 13)
|
12 (14)
|
Species
|
Population
|
PL1
|
2n2
|
2C3
|
2C4
|
SD5
|
1Cx6
|
HPCV7
|
CMA8
|
rDNA9
|
T. polycephalum Sch.Bip. ssp. argyrophyllum (K.Koch) Podlech
|
Urmia: Oshnaviyeh (1867)
|
4
|
35
|
16.82*
|
16450
|
0.4
|
4.21
|
2.94
|
6, 10, 20 and 24t
|
14 (10, 11, 12, 13, 15)
|
T. polycephalum Sch.Bip. ssp. argyrophyllum (K.Koch) Podlech
|
Urmia: Marand (1856)
|
4
|
36
|
17.89*
|
17496
|
0.16
|
4.47
|
2.4
|
32 and 36t (8, 20)
|
12 (14)
|
T. polycephalum Sch.Bip.ssp. azerbaijanicum Podlech
|
Urmia: Ghishchi (1212)
|
4
|
36
|
18.24*
|
17839
|
0.31
|
4.56
|
2.4
|
16t (8, 14)
|
12
|
T. polycephalum Sch.Bip. ssp. duderanum (Boiss.) Podlech
|
Mazandaran: Pole Zangoleh road (1795)
|
4
|
36
|
17.63*
|
17242
|
0.53
|
4.41
|
3.22
|
14tc (18, 20, 22, 24)
|
12 (11)
|
T. polycephalum Sch.Bip. ssp. farsicum Podlech
|
Hamadan: Kabudar Ahang (1901)
|
6
|
54
|
24.12**
|
23589
|
0.39
|
4.02
|
3.46
|
22 and 24t (18, 20, 26)
|
13 (14, 17)
|
T. polycephalum Sch.Bip. ssp. heterophyllum (Boiss.) Podlech
|
Mazandaran: Pole Zangoleh road (1797)
|
4
|
36
|
18.10*
|
17702
|
0.29
|
4.53
|
2.48
|
18 and 22t (16, 18, 20, 30, 32)
|
12 (9, 10, 11)
|
T. polycephalum Sch.Bip.ssp. heterophyllum (Boiss.) Podlech
|
Hamadan: Asad Abad (1899)
|
6
|
54
|
22.99**
|
22484
|
0.56
|
3.83
|
2.88
|
8t (10, 12, 14, 16)
|
18 (15, 16, 17)
|
T. sonbolii Mozaff.
|
(305) Urmia: Takab
|
2
|
18
|
9.17
|
8968
|
0.19
|
4.59
|
2.12
|
5t (4, 6, 8)
|
8
|
T. tabrisianum (Boiss.) Sosn. & Takht.
|
Urmia: Ahar (1905)
|
6
|
54
|
23.56**
|
23042
|
1.12
|
3.93
|
2.59
|
20 and 26t (14, 16, 27)
|
14 and 16 (10, 12)
|
T. tabrisianum (Boiss.) Sosn. & Takht.
|
Urmia: Ahar (1906)
|
6
|
54
|
24.01**
|
23482
|
0.16
|
4.00
|
1.96
|
50t (28, 40)
|
16 (14, 15, 26)
|
T. tenuisectum (Boiss.) Podl.
|
Tehran: Damavand (863)
|
2
|
18
|
7.68
|
7511
|
0.13
|
3.84
|
1.11
|
32, 34 and 46tc
|
6 (8, 10)
|
T. tenuissimum (Trautv.) Grossh.
|
Urmia: Jolfa (1855)
|
4
|
36
|
16.26*
|
15902
|
1.33
|
4.07
|
2.74
|
16 and 22 tc
|
9
|
Genome size
Table 1
presents holoploid genome size data (2C), together with other
karyological features of the studied species, as well as information on
some closely related taxa for comparison. We analysed 38 populations of
20 species and five subspecies of Tanacetum, including ploidy from 2x to 6x. Genome sizes (2C) ranged from 3.84 pg (belonging to one of the diploid populations of T. parthenium) to 24.87 pg (from a tetraploid population of T. pinnatum
Boiss.), an overall 6.47-fold range, and a 3.29-fold range at the
diploid level. Mean 2C at diploid level is 8.05 pg. The low Half Peak
Coefficient of Variation (HPCV) mean value (2.29 %) indicates good
quality of the flow cytometric assessments. Fluorescence histograms from
the flow cytometer are presented in Fig. 1 to illustrate the accuracy of measurements with all internal standards used.
We
found intraspecific genome size differences in most cases in which
several populations were assessed, reaching 22.18 % in the triploid T. kotschyi, 16.04 % in the diploid T. parthenium, 9.43 % in the tetraploid populations of T. aureum, 8.10 % in the tetraploid T. polycephalum Sch. Bip., 1.89 % in the hexaploid T. tabrisianum (Boiss.) Sons. & Takht., and negligible variability (<0.1 %) among diploid T. pinnatum populations.
Genome size (2C) and total karyotype length (TKL) were significantly (p < 0.0001)
and positively correlated with ploidy, but monoploid genome size (1Cx)
did not decrease with ploidy. Nevertheless, when data of the same
species at different ploidy levels was compared, there was a trend to
genome downsizing i.e. reduction of monoploid genome size in T. polycephalum and T. pinnatum,
whose 4× and 6× polyploids present, respectively, 6.07 % and 17.96 %
less genome size than expected from the genome size in their diploid
populations. In addition, genome size is positively correlated with TKL (p = 0.003), with the number of rDNA signals (p < 0.0001) and with pollen morphometric characters such as polar axis (p = 0.03) and equatorial diameter (p = 0.02). Species with different compound inflorescences have significantly different genome sizes (p = 0.009);
species with solitary capitula have the smallest genome compared to
species presenting corymbs of capitula, which have the greatest amounts
of DNA (5.54 pg vs 13.2 pg at the diploid level).
GC-rich regions
Table 1 shows the results of fluorochrome banding with chromomycin and FISH assays, and Figs. 2 and 3 present selected representative Tanacetum metaphases. For the sake of clarity, only three chromosomal locations have been considered both for chromomycin A3 (CMA) and rDNA signals, following the treatment used in the www.plantrdnadatabase.com.
These are: (peri)centromeric, interstitial and (sub)terminal. Results
of chromomycin banding, which stains GC-rich DNA portions, are highly
variable within and between Tanacetum species and even among individuals in some cases. In only four species is the number of bands always constant (the diploids T. parthenifolium Sch. Bip., T. persicum (Boiss.) Mozaff., T. pinnatum and T. budjnurdense (Rech.f.) Tzvelev) and low — four, see picture of T. pinnatum (Fig. 2a). However, from a minimum of two CMA+ bands in a wild population of the diploid T. parthenium (Fig. 3g) to a maximum of 66 bands for the diploid T. archibaldii Podlech (Fig. 3a)
there are myriad variations. In most cases, however, there is a
considerable range of variability within a species. The preferred
position is usually (sub)terminal, and sometimes detached or terminal
decondensed DNA (probably rDNA) is clearly seen with this staining (see
Fig. 3k). Several species also present pericentromeric bands, and in two species (T. archibaldii and T. joharchii), several intercalary signals are also visible (Fig. 3a and 3k).
Pericentromeric (and to a lesser extent, intercalary) bands appear in
species that already present a high number of GC-rich bands.
Several taxa of different ploidy (different populations from T. aureum, T. heimerlii (Nábělek) Farsa, T. parthenium, T. polycephalum Sch. Bip. subsp. argyrophyllum (K.Koch) Podlech, T. pinnatum, T. sonbolii Mozaff. and T. tabrisianum) show odd numbers of bands in different individuals (Table 1). Intensity and size differences of chromomycin signals are clearly visible in several species, such as T. kotschyi (Fig. 2d), T. oligocephalum (DC.) Sch. Bip. (Fig. 2g), T. balsamita (Fig. 3c) and T. joharchii (Fig. 3k).
There
is no significant relationship between ploidy and the most commonly
found number of signals for a given species, nor with genome size. In
addition, the number of GC-rich bands is positively correlated with the
altitude at which species occur, considering all taxa (p = 0.04) and only diploids (p = 0.006).
rDNA loci
The FISH assays of a large sample representing genus Tanacetum
show a totally homogeneous L-type organisation of ribosomal RNA genes.
The number of signals within a species (even within a population) and
between species at the same ploidy is usually heterogeneous although not
as heterogeneous as the number of CMA+ bands. The minimum number of
signals found was two (one locus) for one population of T. parthenium and the maximum was 26 (13 loci) for some individuals of one population of T. tabrisianum (although most T. tabrisianum had eight loci, see Fig. 2n).
In all cases, rDNA signals occupied terminal or subterminal positions,
always coincidental with CMA+ signals, and sometimes appearing as
decondensed (as T. joharchii in Fig. 3d, l arrows). Species such as T. fisherae and T. tabrisianum (Fig. 2k, n,
asterisks), presented locus size differences, but in general, this was
homogeneous. The number of rDNA signals was positively and significantly
correlated with ploidy and genome size (p < 0.0001
for both), but there was no reduction in number of loci, as the number
of signals per haploid genome did not diminish significantly with
increasing ploidy. However, a reduction in the number of signals was
detected in individual polyploid series for T. pinnatum and three out of four of T. polycephalum.
In all other cases there was additivity; that is, the tetraploid had
exactly twice as many signals as the diploid, except in the case of one
tetraploid T. polycephalum population, in which there was upsizing by one locus.
The
heterogeneity in the number of signals for a given species (that is,
the different number of rDNA loci that could be found in metaphases
coming from the same species) was positively correlated with ploidy (p < 0.0001)
which means that with increasing ploidy there was a tendency to
instability in the number of rDNA signals. In particular, the
hypoaneuploid T. fisherae (2n = 5x = 44) and T. polycephalum var. argyrophyllum (2n = 4x = 35) were the most unstable with respect to the number of rDNA signals.
Phylogenetic relationships among species and ancestral characters
Statistical
analyses at the genus level should consider phylogenetic relationships
among taxa to be as unbiased as possible. However, due to lack of enough
data, these comparisons could not be done in most cases. Still, we
detected significant and positive correlations using the phylogenetic
generalised least squares method (PGLS) between genome size (2C),
ploidy, and number of rDNA signals (p < 0.0001),
i.e. all parameters increase/decrease in concert. The reconstruction of
character evolution into the phylogeny (Fig. 4),
based on diploid taxa, provides ancestral 2C values ranging from 7.98
to 8.84 pg, from 10 to 13 for CMA+ bands, and from 4 to 6 rDNA signals
for Tanacetum species.
Discussion
All species investigated present x = 9 as the basic chromosome number confirming previous research [20, 23]. In contrast to other Anthemideae, in which other basic chromosome numbers have been found (e.g. Artemisia presents x = 7, 8, 9, 10, 11; Pentzia Thunb., x = 7, 8, 9, Lasiospermum Fisch., x = 9, 10 [40]) x = 9 it is the only found in Tanacetum until present [41].
To our knowledge, genome size was available for only four species of the genus, the diploid T. vulgare (mean 2C = 8.85 pg, [37]), a tetraploid population of T. cinerariifolium (Trevir.) Sch. Bip. (2C = 14.53 pg, [38]) and some hexaploid populations of T. balsamita and T. corymbosum (L.) Sch. Bip. (2C = 21.44 pg and 2C = 19.95 pg, respectively, [39]). Therefore this research contributes new genome sizes for all species and subspecies studied here (with the exception of T. balsamita), representing approximately 11 % of the recognised species of the genus. The amount of nuclear DNA is mostly intermediate in Tanacetum. According to the genome size categories in plants established by [42],
three of the 20 species we studied (17.65 %) have small genome sizes
(2.8 ≤ 2C < 7 pg), whereas the remaining have intermediate genome
sizes (7 ≤ 2C < 28 pg), including all ploidy levels. Mean genome size
of the diploid taxa studied (8.35 pg) was coincidental with the mean of
the tribe Anthemideae (8.30 pg) and of the family Asteraceae
(2C = 8.20 pg), according to data from the Genome Size in Asteraceae
Database (www.asteraceaegenomesize.com). Closely related diploid genera, such as Artemisia, have similar mean genome sizes (2C = 7.75 pg) whereas the majority of diploid Tanacetum allies present remarkably lower mean 2C values (2C = 5.9 pg for Achillea, 2C = 6.4 pg for Anacyclus L., 2C = 5.12 for Anthemis, 2C = 5.71 for Matricaria L., 2C = 5.13 for Tripleurospermum). The comparatively larger mean genome size of Tanacetum
could be because our sample lacks annual representatives (as does most
of the genus) which, quite often — though not always — tend to present
lower genome sizes than their counterparts [43].
Genome downsizing and polyploidy in Tanacetum
Polyploidy
and hybridisation are important evolutionary forces shaping plant
genomes and underlying the huge angiosperm diversity. Both can confer
evolutionary advantages [44, 45, 46]
attributed to the plasticity of plant genomes and to increased genetic
variability, generating individuals capable of exploiting new niches [47].
Polyploidy is linked to numerous epigenetic/genomic changes such as
chromosome rearrangements, transposable element mobilisation, gene
silencing or genome downsizing [48, 49, 50]. Certainly, genome downsizing would be a widespread biological response to polyploidisation [51]. This may lead to diploidisation of the polyploid genome [52, 53, 54]. There is no evidence of genome downsizing across Tanacetum ploidy levels. However, there are genome size trends within separately polyploid series of particular species. Tetraploid T. pinnatum presents up to 6.07 % lower 1Cx than expected from the 1Cx of the diploid populations, and hexaploid and tetraploid T. polycephalum
present, respectively, 17.96 % and 4.28 % lower 1Cx than expected from
the 1Cx of the diploid population. This is consistent with previous
observations of more pronounced genome downsizing with higher ploidy [30, 45, 55, 56, 57]. Recent work [57]
has demonstrated erosion of low copy-number repetitive DNA in
allopolyploids, sometimes counteracted by expansion of a few repeat
types. Age and genomic similarity of the parental genome donors of the
polyploids play a role in the extent of genome size change with
polyploidy [56] and a deeper understanding of the likely hybridogenic origin of some of the Tanacetum polyploids studied would allow more robust hypotheses on the balancing genomic processes these taxa may have undergone.
Small genome size and invasiveness
Tanacetum parthenium appears listed in several countries as an invasive weed [58, 59].
Its genome size was the smallest obtained in our study (three
populations were analysed whose mean was 2C = 4.12 pg). This is
consistent with previous findings [60],
which detected that many weeds (including those in family Asteraceae)
had smaller amounts of DNA than closely related (non-weedy) species.
This relationship is supported by recent work [61, 62]. The other species with small genome sizes in our sample (T. parthenifolium and T. persicum)
have not, however, been recorded as weeds. Therefore a small genome
size (particularly, smaller than that of closely related species) is a
necessary but not sufficient condition for a plant to become a weed. A
recent review [63]
concluded that invasive species were characterised by small and very
small genomes, yet this conclusion may be biased by the general trend of
land plants to small genome sizes as a whole [42].
Intraspecific instability and massive amplification of GC-rich DNA occur in several Tanacetum species
We found that ribosomal DNA is always CMA+ in Tanacetum (see Discussion on rDNA loci below), common to other studies [45, 64, 65] although exceptions are found [66].
For most of the studied populations, the number of CMA+ bands
significantly exceeded that of rDNA signals and there was no apparent
relationship with ploidy or with genome size (Table 1).
The number of CMA+ bands is neither stable within a species nor within a
population. The presence of odd and of non-homologous signals was
occasionally observed, for example in T. aureum and in T. oligocephalum (Table 1),
where a single chromosome with two CMA+ bands at each end was observed
instead of the two identical chromosomes expected. Odd ploidy species,
such as T. fisherae (5x) and T. kotschyi
(3x), were particularly labile with respect to the number of CMA+
bands. However, the greatest variability in number of CMA+ bands
corresponded to the diploid T. balsamita, in which sevendifferent numbers of signals were found (Table 1 and Fig. 3c).
Such instability in the number of GC-rich bands was unexpected and has
seldom been reported. Only the highly variable CMA+ banding pattern
previously found in Citrus L. and close genera [67] is similar to the variability found in Tanacetum, probably as a consequence of amplification or reduction in satellite sequences known to be particularly GC-rich [68]. It is possible that some as yet undescribed satellite DNA type, specific to Tanacetum, is in part responsible for these karyotype features.
Another characteristic of the CMA+ banding pattern in Tanacetum was the striking number of signals found in certain species, particularly in diploid taxa (Table 1, Fig. 3a, 3c, 3i, 3k). This contrasts with previous work on genus Artemisia [69, 70],
in which a large number of CMA+ bands was only detected in some
polyploids, while the only CMA+ bands in diploids were those exactly
corresponding to rDNA loci. In other Asteraceae genera, such as Cheirolophus Cass., a large number of CMA+ bands was also reported, mostly coincidental with 35S rDNA signals [71]; this was also the case for Filifolium [72]. In Centaurea L. [73] the number of CMA+ bands was the same as or smaller than the number of 35S rDNA signals, while in some Xeranthemum L. [74], Galinsoga Ruiz & Pav. and Chaptalia Vent. [75], few additional GC-rich bands were observed.
While
most bands are in terminal position, pericentromeric GC-rich
heterochromatin was detected in several species, some of them closely
related, such as T. polycephalum, T. aureum and T. canescens DC. on one hand (Table 1), and T. fisherae (Fig. 2j), T. kotschyi (Fig. 2d), T. tenuisectum Sch.Bip. and T. joharchii (Fig. 3k) on the other. In fact, in Arabidopsis thaliana (L.) Heynh., centromeres are one of the most GC-rich genomic regions [76].
Differences in total GC% among eukaryotes are largely driven by the
composition of non-coding DNA of which retrotransposons are the most
abundant (for example, LTR Huck elements contain more than 60 % GC, [77]). Possibly, some centromere-specific LTR could have undergone amplification in these closely related Tanacetum genomes.
What
can this fluctuating distribution of CMA+ bands mean, and what are the
implications? It is feasible that a specific satellite and/or
retroelement family may be expanded or contracted in Tanacetum
genomes. Although the number and the distribution of CMA+ bands are
thought to be relatively constant features of plant karyotypes [24, 70],
our results strongly argue against this view, since variability was
found even within a population. In addition, there were few evident
ecological or geographic patterns in Tanacetum,
that is, few significant relationships were found between the number or
variability of GC-rich signals and geographical distribution, weedy
behaviour, or soil features. The only significant association is with
altitude: Tanacetum species living at higher altitudes tend to present more GC-rich DNA. In line with this hypothesis, [78] found a large number of heterochromatic bands (both GC- and AT-rich) in species from the Asteraceae genus Myopordon
Boiss. inhabiting high mountain areas. These authors related the
development of such heterochromatic bands in terminal regions with an
adaptation to protect telomere function from UV radiation, a major
genome-damaging agent, particularly in high mountains. Heterochromatin
expansion in terminal regions (as in Tanacetum) has also been suggested to enhance chromosomal pairing during cell division [79].
Genomic organisation of rDNA and typical distribution pattern of Tanacetum
Our
cytogenetic study confirmed that both the 5S and the 35S rRNA genes are
co-localised (L-type arrangement) in all chromosomes. Such organisation
was found in Artemisia for the first time in higher plants [36], and subsequently inferred for at least 25 % of Asteraceae species [25]. In the latter study, Southern blot hybridisation was performed on a sample of T. parthenium,
and the profile obtained also suggested L-type organisation for its
rDNA. Prior to our study, the only evidence of this particular rDNA
organisation directly in chromosomes was from T. achilleifolium and T. parthenium [35]. Curiously, these authors found one unlinked 5S locus additional to two regular L-type loci in T. achilleifolium, while T. parthenium
showed L-type arrangement in all loci. Within the sample studied we
could not find a single species with unhomogenised rDNA (i.e. that both
kinds of rDNA arrangement, linked and separated, were present in the
same species), since both rDNA probes invariably overlapped in all loci.
Nevertheless, possible incomplete homogenisation of rRNA genes may also
be present in other close genera such as Achillea and Chrysanthemum L. [72, 80].
Besides, in some metaphases decondensed rDNA signals are detected.
These probably correspond to active nucleolar organizer regions (NORs),
i.e. rDNA that is being actively transcribed, visible in T. balsamita (Fig. 3d, one signal) and in T. joharchi (Fig. 3l, two signals). Decondensed rDNA, however, is not always detected during metaphase.
Unexpected variation in number of rDNA loci
The
number of rDNA signals was always smaller and less variable than that
of CMA+ bands, as found previously in other closely related species (in Artemisia, [45, 70]) and even in other families (genus Ipomoea from Convolvulaceae, [81]).
In particular, the most common number of rDNA loci at the diploid (with
two to three loci) and tetraploid (with five to six loci) levels was
relatively constant and consistent with previous data for Tanacetum [35, 82] or for the closely related genera Matricaria and Tripleurospermum [25].
However, taxa with odd, higher ploidy or aneuploid levels often
displayed higher intraspecific polymorphism in the number of signals. Of
these, the hypoaneuploid population of T. polycephalum var. argyrophyllum was particularly striking, since metaphases with 10, 11, 12, 13, 14 and 15 rDNA signals were observed; the hypoaneuploid T. fisherae (2n = 5x = 44) showed a similar condition (Table 1). Thus, processes of hypoaneuploidy could affect genomic stability producing this variation in number of loci.
Although
it would be expected that the number of signals remain relatively
constant for a given species, cases of intraspecific polymorphism in the
number of signals are increasingly reported. As for Tanacetum, diversity in the number of rDNA signals for a given species has been found in Fragaria vesca L. [26] and in Phaseolus vulgaris L. [83], for example. However, what is exceptional in Tanacetum
is that these polymorphisms happen even at the population level and,
albeit very rarely, sometimes within the same individual. All this,
together with the unexceptional situation of odd numbers of signals in
many taxa (which otherwise is rare) illustrates how dynamic Tanacetum genomes are.
Given
these fluctuations, the constantly terminal position of rDNA signals in
all the species studied could be considered surprising. However, this
is so in most plants: [84]
argued that there seems to be a strong positive selection favouring the
location of 35S rDNA at chromosome ends, probably as a result of
homologous recombination constraints.
As
with the number of CMA+ bands, there was no global reduction in the
number of signals per haploid genome with increasing ploidy. Similarly,
the number of rDNA loci did not show any apparent relationship with
genome size.
Our
analyses have allowed us to distinguish some interesting relationships
between several of the traits studied. As others have found [85, 86] morphological data regarding pollen size are tightly linked with genome size in Tanacetum, i.e. pollen size reflects genome size in this genus. In addition, species of Tanacetum
with solitary capitula have smaller genome sizes than those with
capitula organised in complex inflorescences. It is known that sometimes
polyploids tend to present larger reproductive organs and more flowers
per inflorescence than their diploid relatives [87],
but few studies have approached the relationship of genome size or
polyploidy with natural patterns, such as inflorescence architecture [88].
Suggested that the shift in inflorescence phyllotaxis from spiral to
distichous would have occurred at the same time as the expansion of
genome size characterising several groups of grasses [89], though admitting no clear reason why genome size as such should affect inflorescence architecture.
In
addition, the reconstruction of ancestral cytogenetic traits brings
evidence that these characters have followed increases and decreases
during evolution in Tanacetum (Fig. 4).
In general, it seems that genome size and the number of rDNA loci have
increased, while the number of CMA+ bands has decreased in most present
taxa. Few studies have specifically approached the evolution of
cytogenetic traits within a temporal and phylogenetic perspective and,
while events favouring increase in genome size and number of rDNA
signals during evolution have been detected [56], there is no discernible pattern in the direction of these changes. For example, [90] found a decrease in number of rDNA loci during the evolution of Hypochaeris L. The overall decrease of GC-rich DNA could also respond to depletion of certain repeated DNA sequences during evolution in Tanacetum.
Conclusions
This work is the first extensive cytogenetic report on Tanacetum species. We have confirmed linkage of both rDNAs in all chromosomal loci. Tanacetum
stands out as variable, particularly in the number of rDNA sites and
CMA+ bands. These vary widely even within a given population. In
particular, aneuploid and odd ploidy taxa appear more unstable. The
observed intrapopulation differences are likely a reflection of genomic
differentiation which could complement further population biology
studies. Besides, the number of GC-rich DNA bands found in certain
species is striking and deserves more study. A possible cause is the
amplification of repeat families or TEs in these species compared to
others showing utterly different profiles. Polyploidy and aneuploidy are
important evolutionary forces in this genus. Several of the studied
populations present spontaneous mixed ploidy, another sign of its
current genomic dynamism.
It
is difficult to set general patterns in the evolution of genome size,
number of rDNA loci or heterochromatin in plants. Yet, studies such as
ours contribute to the knowledge of these cytogenetic features at a
larger scale. Finally, the particularly labile cytogenetic scenario
observed in Tanacetum is
uncommon and has been seldom reported. Both chromosomal markers (rDNA
loci and GC-rich bands) tend to be relatively constant at the species
level, a feature that has allowed their use in biosystematics. Still,
even at the population level, these traits can be variable in Tanacetum and this variation is better understood considering evolutionary relationships between species.
Methods
Plant materials
Seeds of 38 populations of Tanacetum species were collected from the wild for molecular cytogenetics and genome size assessments (Table 1).
Specimen vouchers of the studied materials have been deposited at the
Medicinal Plants and Drug Research Institute Herbarium (MPH) of the
Shahid Beheshti University, Tehran.
Chromosome preparations
Root
tip meristems were obtained by germinating achenes on moist filter
paper in Petri dishes at room temperature in the dark. They were
pre-treated with 2 mM 8-hydroxyquinoline at room temperature for
3–3.5 h. Subsequently, the material was fixed in 3:1 v/v absolute
ethanol:glacial acetic acid and stored at 4 °C for 24 h, and then stored
in 70 % ethanol at 4 °C until use. For fluorochrome banding and
fluorescence in situ hybridisation (FISH), the chromosome spreads were obtained using the air-drying technique of [91],
with modifications. Fixed root tips were washed three times in
distilled water with shaking and later in citrate buffer (0.01 M citric
acid-sodium citrate, pH 4.6) for 30 min, excised and incubated for
20–35 min at 37 °C in an enzymatic mixture [4 % cellulase Onozuka R10
(Yakult Honsha), 1 % pectolyase Y23 (Sigma) and 4 % hemicellulase
(Sigma)]. Digested root tips were placed on a slide, excess enzymatic
solution was removed and protoplasts were obtained by applying gentle
pressure in a drop of 45 % acetic acid. The metaphase plates were
evaluated using a phase contrast microscope and slides were frozen for
at least 24 h at -80 °C. Later, the coverslip was quickly removed, the
slide rinsed with absolute ethanol and then air dried for at least two
days protected from dust.
Fluorochrome banding
In order to reveal GC-rich bands, the chromosomes were stained with the fluorochrome chromomycin A3 (CMA), according to [24, 92] with slight modifications. The slides were incubated in McIlvaine buffer pH 7, MgSO4 (0.1 g/L in McIlvaine buffer, pH 7) for 15 min, stained with CMA3 (0.2 mg/ml in McIlvaine buffer pH 7 MgSO4)
for 90 min in the dark, rinsed in McIlvaine buffer pH 7, and
counterstained with methyl green (0.1 % in McIlvaine buffer pH 5.5) for
10 min; rinsed in McIlvaine buffer pH 5.5, dried briefly at room
temperature, also in the dark, and mounted in two small drops of
Citifluor AF1 (glycerol/PBS solution).
Labelling of rDNA probes and FISH
For
hybridisation experiments we mostly used the same slides as for
fluorochrome banding with CMA after destaining with fixative,
dehydration through an ethanol series (70 %, 90 % and 100 %) and drying
for two days. The probe used for 35S rDNA localisation was a plasmid
carrying a 2.5 kb insert of 26S rRNA gene from Lycopersicum esculentum
Mill. labelled with Cy3 (Jena Biosciences) using the Nick Translation
Mix (Roche). The 5S rDNA probe was an approximately 0.7 kb-long trimer
of 5S rRNA genes from Artemisia tridentata
Nutt., labelled with Green dUTP using the Nick Translation Mix (Abbott
Molecular). This probe contained three units of the 5S rRNA gene
(120 bp) and the non-coding intergenic spacers (about 290 bp). Both
probes have been used following previous research [25, 65]. FISH was carried out according to [24]
with slight modifications. Slides were incubated in 100 μg/ml
DNase-free RNase in 2 × SSC (0.03 M sodium citrate and 0.3 M sodium
chloride) for 1 h at 37 °C, washed in 2xSSC three times for 5 min with
slow shaking, rinsed in 0.01 N HCl for 2 min and incubated in pepsin
(0.1 mg/ml in 0.01 N HCl) for 15 min at 37 °C, washed in 2xSSC for 5 min
twice, dehydrated in an ethanol series (70 %, 90 % and 100 %, for 3 min
in each) and air dried. The probe hybridisation mixture contained
25–100 ng/μl rDNA probes, formamide, 50 % (w/v) dextran sulphate, and
20 × SSC. This was denatured at 75 °C for 10 min and chilled on ice for 5
min. A volume of 30 μl was loaded onto slides and covered with plastic
coverslips. The preparations were denatured at 75 °C for 10 min and
transferred at 55 °C for 5 min. Hybridisation was carried out for more
than 18 h at 37 °C in a humidified chamber. Following hybridisation, the
slides were washed with shaking in 2 × SSC, 0.1 × SSC and 2 × SSC at 42
°C for 5 min twice each, and then once in 2 × SSC for 5 min, once in
4 × SSCT for 7 min, briefly rinsed in 1 × PBS and dried.
Samples
were counterstained with Vectashield (Vector Laboratories, Inc.,
Burlingame, CA, USA), a mounting medium containing 500 ng/μl of
4’,6-diamidino-2-phenylindole (DAPI). The fluorescence signals were
analysed and photographed using a digital camera (AxioCam HRm, Zeiss)
coupled to a Zeiss Axioplan microscope; images were analysed with
Axiovision HR Rev3, version 4.8 (Zeiss) and processed for colour
balance, contrast and brightness uniformity in Adobe Photoshop. A
minimum of 10 metaphase plates per population were analysed. Graphics
were assembled with PowerPoint 2010 (Microsoft). The data were submitted
to the Plant rDNA database, a database compiling information on rDNA
signal number, position and organisation [93, 94].
Flow cytometric measurements
For
flow cytometric measurements of leaf tissue, seedlings were obtained
from seeds grown in pots in the greenhouse of the Faculty of Pharmacy,
University of Barcelona. Five individuals per population of the
different Tanacetum species were studied, and of these, two samples of each were individually processed. Petunia hybrida Vilm. ‘PxPc6’ (2C = 2.85 pg), Pisum sativum L. ‘Express Long’ (2C = 8.37 pg) and Triticum aestivum L. ‘Chinese Spring’ (2C = 30.9 pg) from [95]
were used as the internal standards. Fresh leaf tissue for the standard
and the target species were chopped up together in 600 μl of LB01
buffer (8 % Triton X-100; [96])
supplemented with 100 μg/ml ribonuclease A (RNase A, Boehringer,
Meylan, France) and stained with 36 μl of 1 mg/ml propidium iodide
(Sigma-Aldrich, Alcobendas, Madrid, 60 μg/ml) to a final concentration
of 60 μg/ml, and kept on ice for 20 min. The fluorescence measurements
were performed using an Epics XL flow cytometer (Coulter Corporation,
Miami, FL, USA) at the Centres Científics i Tecnològics, University of
Barcelona. More details about the method are in [55]. The data have been submitted to the GSAD (Genome Size in Asteraceae Database) [97, 98].
Phylogenetic analyses and reconstruction of character evolution
The nuclear ITS1 + ITS2 and chloroplast trnH-psbA sequences (listed in Additional file 1) were edited by BioEdit v. 7.1.3.0 [99] followed by manual adjustment. Artemisia taxa were considered as outgroups [3].
All taxa used for the phylogenetic analysis were diploid in order to
avoid the effect of polyploidy in the estimated nuclear DNA contents,
number of rDNA sites or GC-rich bands. Bayesian phylogenetic analysis
was performed in MrBayes 3.1.2 [100] using a SYM + G model determined from jModeltest v. 2.1.3 [101] under the Akaike information criterion (AIC; [102]),
to ascertain phylogenetic relationships. The Markov chain Monte Carlo
(MCMC) sampling approach was used to calculate posterior probabilities
(PPs). Four consecutive MCMC computations were run for 2,000,000
generations, with tree sampling every 100 generations. Data from the
first 1000 generations were discarded as the burn-in period. PPs were
estimated through the construction of a 50 % majority-rule consensus
tree.
The
ancestral character reconstructions (genome size, number of rDNA sites
and number of CMA+ bands) were conducted using unordered maximum
parsimony as implemented for continuous and meristic characters in
Mesquite v. 3.02 software [103]
using the 50 % majority-rule consensus tree resulting from the Bayesian
inference analysis as the input tree file. The output trees were edited
with Mesquite v. 3.02.
Statistical analyses
Analyses of regression, one-way ANOVA, X
2, Shapiro-Wilk test for normality
and Barlett’s test for equality of variances were performed with
RStudio, v.0.98.1078. In addition, the phylogenetic generalised least
squares (PGLS) algorithm as implemented in the nlme
package for R (Version 3.1-118) was used to analyse variation of genome
size, number of rDNA sites and number of CMA+ bands in a phylogenetic
context. Data on genome size and ribosomal DNA loci for the
complementary and outgroup species were extracted from the Plant rDNA
database [93].
Availability of supporting data
The data sets supporting the results of this article are available in the TreeBase repository, ID 17805 and http://purl.org/phylo/treebase/phylows/study/TB2:S17805 [104].
Abbreviations
- 1Cx:
-
Monoploid Genome Size
- 2C:
-
Holoploid Genome Size
- CMA:
-
Chromomycin A3
- FISH:
-
Fluorescent in situ Hybridisation
- NOR:
-
Nucleolar Organizer Region
- PGLS:
-
Phylogenetic Generalised Least Squares
- rDNA:
-
Ribosomal DNA (or ribosomal RNA genes)
- rRNA:
-
Ribosomal RNA
- TKL:
-
Total Karyotype Length
Declarations
Acknowledgments
This
work was supported by the Dirección General de Investigación Científica
y Técnica, Government of Spain (CGL2010-22234-C02-01 and 02/BOS and
CGL2013-49097-C2-2-P) and the Generalitat de Catalunya, Government of
Catalonia ("Ajuts a grups de recerca consolidats", 2009SGR0439 and
2014SGR514). SG benefitted from a Juan de la Cierva postdoctoral
contract from the Ministry of Economy and Competitiveness, Government of
Spain. NO benefitted from a fellowship from the Science, Research and
Technology Ministry of Iran. Aleš Kovařík is acknowledged for supplying
the rDNA probes and Spencer C. Brown for supplying internal standards
for flow cytometry. We thank the technical staff of the Medicinal Plants
and Drugs Research Institute, Shahid Beheshti University, who helped us
with fieldwork. Ricard Àlvarez, Jaume Comas, Chari González and Sonia
Ruiz are acknowledged for their assistance in flow cytometric analyses.
We acknowledge support of the publication fee by the CSIC Open Access
Publication Support Initiative through the Unit of Information Resources
for Research (URICI).
References
- Oberprieler C, Himmelreich S, Vogt R. A new subtribal classification of the tribe Anthemideae (Compositae). Willdenowia - Ann Bot Gard Bot Museum Berlin-Dahlem. 2007;37:89–114.View ArticleGoogle Scholar
- Oberprieler C, Himmelreich S, Källersjö M, Vallès J, Watson L, Vogt R. Tribe Anthemideae Cass. In: Funk V, Stuessy T, Bayer R, editors. Systematics, Evolution and Biogeography of the Compositae. Washington: IAPT; 2009. p. 631–66.Google Scholar
- Sonboli A, Stroka K, Kazempour Osaloo S, Oberprieler C. Molecular phylogeny and taxonomy of Tanacetum L. (Compositae, Anthemideae) inferred from nrDNA ITS and cpDNA trnH–psbA sequence variation. Plant Syst Evol. 2011;298:431–44.View ArticleGoogle Scholar
- Smith RM, Burford MD. Supercritical fluid extraction and gas chromatographic determination of the sesquiterpene lactone parthenolide in the medicinal herb feverfew (Tanacetum parthenium). J Chromatogr A. 1992;627:255–61.View ArticleGoogle Scholar
- Awang DVC. Prescribing therapeutic feverfew (Tanacetum parthenium (L.) Schultz Bip., syn. Chrysanthemum parthenium (L.) Bernh.). Integr Med. 1998;1:11–3.View ArticleGoogle Scholar
- Salamci E, Kordali S, Kotan R, Cakir A, Kaya Y. Chemical compositions, antimicrobial and herbicidal effects of essential oils isolated from Turkish Tanacetum aucheranum and Tanacetum chiliophyllum var. chiliophyllum. Biochem Syst Ecol. 2007;35:569–81.View ArticleGoogle Scholar
- Bagci E, Kursat M, Kocak A, Gur S. Composition and antimicrobial activity of the essential oils of Tanacetum balsamita L. subsp. balsamita and T. chiliophyllum (Fisch. et Mey.) Schultz Bip. var. chiliophyllum (Asteraceae) from Turkey. J Essent Oil Bear Plants. 2008;11:476–84.View ArticleGoogle Scholar
- Yousefzadi M, Ebrahimi SN, Sonboli A, Miraghasi F, Ghiasi S, Arman M, et al. Cytotoxicity, antimicrobial activity and composition of essential oil from Tanacetum balsamita L. subsp. balsamita. Nat Prod Commun. 2009;4:119–22.PubMedGoogle Scholar
- Esmaeili MA, Sonboli A, Ayyari Noushabadi M. Antioxidant and protective properties of six Tanacetum species against hydrogen peroxide-induced oxidative stress in K562 cell line: A comparative study. Food Chem. 2010;121:148–55.View ArticleGoogle Scholar
- Karaca M, Özbek H, Akkan HA, Tütüncü M, Özgökce F, Hi̇m A, et al. Anti-inflammatory activities of diethyl-ether extracts of Helichrysum plicatum DC. and Tanacetum balsamita L. in rats. Asian J Anim Vet Adv. 2009;4:320–5.View ArticleGoogle Scholar
- Vallès J, Garnatje T, Garcia S, Sanz M, Korobkov AA. Chromosome numbers in the tribes Anthemideae and Inuleae (Asteraceae). Bot J Linn Soc. 2005;148:77–85.View ArticleGoogle Scholar
- Mozzafarian V. Notes on the tribe Anthemideae (Compositae), new species, new records and new combinations for Iran. Iranian J Bot. 2005;11:115–27.Google Scholar
- Djavadi S. Three new records of Tanacetum for the flora of Iran. Rostaniha. 2008;9:23–32.Google Scholar
- Sonboli A, Kazempour Osaloo S, Riahi H, Mozaffarian V. Tanacetum joharchii sp. nov. (Asteraceae-Anthemideae) from Iran, and its taxonomic position based on molecular data. Nord J Bot. 2010;28:74–8.View ArticleGoogle Scholar
- Sonboli A, Oberprieler C. Insights into the phylogenetic and taxonomic position of Tanacetum semenovii Herder (Compositae, Anthemideae) based on nrDNA ITS sequences data. Biochem Syst Ecol. 2012;45:166–70.View ArticleGoogle Scholar
- Kazemi M, Sonboli A. A taxonomic reassessment of the Tanacetum aureum (Asteraceae, Anthemideae) species group: insights from morphological and molecular data. Turkish J Bot. 2014;38:1259–73.View ArticleGoogle Scholar
- Kazemi M, Sonboli A, Maivan HZ, Osaloo SK, Mozaffarian V. Tanacetum tarighii (Asteraceae), a new species from Iran. Ann Bot Fenn. 2014;51:419–22.View ArticleGoogle Scholar
- Chehregani A, Hajisadeghian S. New chromosome counts in some species of Asteraceae from Iran. Nord J Bot. 2009;27:247–50.View ArticleGoogle Scholar
- Inceer H, Hayirlioglu-Ayaz S, Guler H. Karyological studies of some representatives of Tanacetum L. (Anthemideae-Asteraceae) from north-east Anatolia. Plant Syst Evol. 2012;298:827–34.View ArticleGoogle Scholar
- Olanj N, Sonboli A, Riahi H, Osaloo SK. Karyomorphological study of nine Tanacetum taxa (Asteraceae, Anthemideae) from Iran. Caryologia. 2013;66:321–32.View ArticleGoogle Scholar
- Ghasemkhani T, Ahmadi M, Atri M. Variation of chromosome numbers in 14 populations of Tanacetum parthenium and eight populations of T. polycephalum in Hamedan Province, Iran. Chromosom Bot. 2013;8:103–8.View ArticleGoogle Scholar
- Semple J, Watanabe K. A review of chromosome numbers in Asteraceae with hypotheses on chromosomal base number evolution. In: Funk V, Stuessy T, Bayer R, editors. Systematics, Evolution and Biogeography of the Compositae. Washington: IAPT; 2009. p. 61–72.Google Scholar
- Chehregani A, Mehanfar N. New chromosome counts in the tribe Anthemideae (Asteraceae) from Iran. Cytologia (Tokyo). 2008;73:189–96.View ArticleGoogle Scholar
- Siljak-Yakovlev S, Pustahija F, Vicic V, Robin O. Molecular cytogenetics (FISH and fluorochrome banding): resolving species relationships and genome organization. Methods Mol Biol. 2014;1115:309–23.PubMedView ArticleGoogle Scholar
- Garcia S, Panero JL, Siroky J, Kovarik A. Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family. BMC Plant Biol. 2010;10:176.PubMed CentralPubMedView ArticleGoogle Scholar
- Liu B, Davis TM. Conservation and loss of ribosomal RNA gene sites in diploid and polyploid Fragaria (Rosaceae). BMC Plant Biol. 2011;11:157.PubMed CentralPubMedView ArticleGoogle Scholar
- Mahelka V, Kopecky D, Baum BR. Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae). Mol Biol Evol. 2013;30:2065–86.PubMedView ArticleGoogle Scholar
- Garcia S, Inceer H, Garnatje T, Vallès J: Genome size variation in some representatives of the genus Tripleurospermum. Biologia Plantarum. 2005, 49:381–387
- Garnatje T, Garcia S, Vilatersana R, Vallès J. Genome size variation in the genus Carthamus (Asteraceae, Cardueae): systematic implications and additive changes during allopolyploidization. Ann Bot. 2006;97:461–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Pellicer J, Garcia S, Canela MA, Garnatje T, Korobkov AA, Twibell JD, et al. Genome size dynamics in Artemisia L. (Asteraceae): following the track of polyploidy. Plant Biol (Stuttg). 2010;12:820–30.View ArticleGoogle Scholar
- Maghuly F, Schmoellerl B, Temsch EM, Laimer M. Genome size, karyotyping and FISH physical mapping of 45S and 5S genes in two cherry rootstocks: Prunus subhirtella and Prunus incisa xserrula. J Biotechnol. 2010;149:88–94.PubMedView ArticleGoogle Scholar
- Bennett M. Nuclear DNA amounts in angiosperms and their modern uses—807 new Estimates. Ann Bot. 2000;86:859–909.View ArticleGoogle Scholar
- Mortreau E, Siljak-Yakovlev S, Cerbah M, Brown SC, Bertrand H, Lambert C. Cytogenetic characterization of Hydrangea involucrata Sieb. and H. aspera D. Don complex (Hydrangeaceae): genetic, evolutional, and taxonomic implications. Tree Genet Genomes. 2009;6:137–48.View ArticleGoogle Scholar
- De Jesus ON, de OE SS, Amorim EP, Ferreira CF, de Campos JMS, Silva G de G, et al. Genetic diversity and population structure of Musa accessions in ex situ conservation. BMC Plant Biol. 2013;13:41.PubMed CentralPubMedView ArticleGoogle Scholar
- Abd El-Twab M, Kondo K. Physical mapping of 5S and 45S rDNA in Chrysanthemum and related genera of the Anthemideae by FISH, and species relationships. J Genet. 2012;91:245–9.PubMedView ArticleGoogle Scholar
- Garcia S, Lim KY, Chester M, Garnatje T, Pellicer J, Vallès J, et al. Linkage of 35S and 5S rRNA genes in Artemisia (family Asteraceae): first evidence from angiosperms. Chromosoma. 2009;118:85–97.PubMedView ArticleGoogle Scholar
- Keskitalo M, Lindén A, Valkonen JPT. Genetic and morphological diversity of Finnish tansy (Tanacetum vulgare L., Asteraceae). Theor Appl Genet. 1998;96:1141–50.View ArticleGoogle Scholar
- Siljak-Yakovlev S, Pustahija F, Šolić EM, Bogunić F, Muratović E, Bašić N, et al. Towards a genome size and chromosome number database of Balkan Flora: C-values in 343 taxa with novel values for 242. Adv Sci Lett. 2010;3:190–213.View ArticleGoogle Scholar
- Garcia S, Hidalgo O, Jakovljević I, Siljak-Yakovlev S, Vigo J, Garnatje T, et al. New data on genome size in 128 Asteraceae species and subspecies, with first assessments for 40 genera, 3 tribes and 2 subfamilies. Plant Biosyst - An Int J Deal with all Asp Plant Biol. 2013;147:1219–27.View ArticleGoogle Scholar
- Funk V, Stuessy T, Bayer R. Systematics, Evolution, and Biogeography of Compositae. Washington: IAPT; 2009.Google Scholar
- Sonboli A, Kazempour Osaloo S, Vallès J, Oberprieler C. Systematic status and phylogenetic relationships of the enigmatic Tanacetum paradoxum Bornm. (Asteraceae, Anthemideae): evidences from nrDNA ITS, micromorphological, and cytological data. Plant Syst Evol. 2011;292:85–93.View ArticleGoogle Scholar
- Leitch IJ, Soltis DE, Soltis PS, Bennett MD. Evolution of DNA amounts across land plants (Embryophyta). Ann Bot. 2005;95:207–17.PubMed CentralPubMedView ArticleGoogle Scholar
- Garcia S, Sanz M, Garnatje T, Kreitschitz A, Mcarthur ED, Vallès J: Variation of DNA amount in 47 populations of the subtribe Artemisiinae and related taxa (Asteraceae, Anthemideae): karyological, ecological, and systematic implications. Genome. 2004, 1014:1004–1014.
- Soltis DE, Albert VA, Leebens-Mack J, Bell CD, Paterson AH, Zheng C, et al. Polyploidy and angiosperm diversification. Am J Bot. 2009;96:336–48.PubMedView ArticleGoogle Scholar
- Garcia S, Garnatje T, Pellicer J, McArthur ED, Siljak-Yakovlev S, Vallès J. Ribosomal DNA, heterochromatin, and correlation with genome size in diploid and polyploid North American endemic sagebrushes (Artemisia, Asteraceae). Genome. 2009;52:1012–24.PubMedView ArticleGoogle Scholar
- Marques I, Draper D, Riofrío L, Naranjo C. Multiple hybridization events, polyploidy and low postmating isolation entangle the evolution of neotropical species of Epidendrum (Orchidaceae). BMC Evol Biol. 2014;14:20.PubMed CentralPubMedView ArticleGoogle Scholar
- Leitch AR, Leitch IJ. Genomic plasticity and the diversity of polyploid plants. Science. 2008;320:481–3.PubMedView ArticleGoogle Scholar
- Parisod C, Holderegger R, Brochmann C. Evolutionary consequences of autopolyploidy. New Phytol. 2010;186:5–17.PubMedView ArticleGoogle Scholar
- Parisod C, Senerchia N. Responses of transposable elements to polyploidy. In: Grandbastien MA, Casacuberta JM, editors. Plant Transposable Elements. Berlin Heidelberg: Springer; 2012. p. 147–68.View ArticleGoogle Scholar
- Tayalé A, Parisod C. Natural pathways to polyploidy in plants and consequences for genome reorganization. Cytogenet Genome Res. 2013;140:79–96.PubMedView ArticleGoogle Scholar
- Leitch IJ, Bennett MD. Genome downsizing in polyploid plants. Biol J Linn Soc. 2004;82:651–63.View ArticleGoogle Scholar
- Otto SP, Whitton J. Polyploid incidence and evolution. Annu Rev Genet. 2000;34:401–37.PubMedView ArticleGoogle Scholar
- Wolfe KH. Yesterday’s polyploids and the mystery of diploidization. Nat Rev Genet. 2001;2:333–41.PubMedView ArticleGoogle Scholar
- Soltis PS, Soltis DE. Polyploidy and Genome Evolution. Berlin Heidelberg: Springer; 2012.View ArticleGoogle Scholar
- Garcia S, Canela MÁ, Garnatje T, Mcarthur ED, Pellicer J, Sanderson SC, et al. Evolutionary and ecological implications of genome size in the North American endemic sagebrushes and allies (Artemisia, Asteraceae). Biol J Linn Soc. 2008;94:631–49.View ArticleGoogle Scholar
- Leitch IJ, Hanson L, Lim KY, Kovarik A, Chase MW, Clarkson JJ, et al. The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae). Ann Bot. 2008;101:805–14.PubMed CentralPubMedView ArticleGoogle Scholar
- Renny-Byfield S, Kovařík A, Chester M, Nichols RA, Macas J, Novák P, et al. Independent, rapid and targeted loss of highly repetitive DNA in natural and synthetic allopolyploids of Nicotiana tabacum. PLoS One. 2012;7:e36963.PubMed CentralPubMedView ArticleGoogle Scholar
- Hadjikyriakou G, Hadjisterkotis E. The adventive plants of Cyprus with new records of invasive species. Z Jagdwiss. 2002;48:59–71.Google Scholar
- Mito T, Uesugi T. Invasive alien species in Japan: the status quo and the new regulation for prevention of their adverse effects. Glob Environ Res. 2004;8:171–93.Google Scholar
- Bennett M. DNA Amounts in two samples of angiosperm weeds. Ann Bot. 1998;82:121–34.View ArticleGoogle Scholar
- Knight CA, Ackerly DD. Variation in nuclear DNA content across environmental gradients: a quantile regression analysis. Ecol Lett. 2002;5:66–76.View ArticleGoogle Scholar
- Pandit MK, White SM, Pocock MJO. The contrasting effects of genome size, chromosome number and ploidy level on plant invasiveness: a global analysis. New Phytol. 2014;203:697–703.PubMedView ArticleGoogle Scholar
- Suda J, Meyerson LA, Leitch IJ, Pyšek P. The hidden side of plant invasions: the role of genome size. New Phytol. 2015;205:994–1007.PubMedView ArticleGoogle Scholar
- Cabral JS, Felix LP, Guerra M. Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae). Genet Mol Biol. 2006;29:659–64.View ArticleGoogle Scholar
- Gouja, H., Garnatje, T., Hidalgo, O., Neffati, M., Raies, A., & Garcia, S. (2014). Physical mapping of ribosomal DNA and genome size in diploid and polyploid North African Calligonum species (Polygonaceae). Plant Systematics and Evolution, 301: 1569-1579
- Carvalho R, Soares Filho WS, Brasileiro-Vidal AC, Guerra M. The relationships among lemons, limes and citron: a chromosomal comparison. Cytogenet Genome Res. 2005;109:276–82.PubMedView ArticleGoogle Scholar
- Da Silva AEB, Marques A, dos Santos KGB, Guerra M. The evolution of CMA bands in Citrus and related genera. Chromosome Res. 2010;18:503–14.View ArticleGoogle Scholar
- Beridze T, Tsirekidze N, Roytberg M. On the tertiary structure of satellite DNA. Biochimie. 1992;74:187–94.PubMedView ArticleGoogle Scholar
- Torrell M, Cerbah M, Siljak-Yakovlev S, Vallès J. Molecular cytogenetics of the genus Artemisia (Asteraceae, Anthemideae): fluorochrome banding and fluorescence in situ hybridization. I. Subgenus Seriphidium and related taxa. Plant Syst Evol. 2003;239:141–53.View ArticleGoogle Scholar
- Garcia S, Garnatje T, Hidalgo O, McArthur ED, Siljak-Yakovlev S, Vallès J. Extensive ribosomal DNA (18S-5.8S-26S and 5S) colocalization in the North American endemic sagebrushes (subgenus Tridentatae, Artemisia, Asteraceae) revealed by FISH. Plant Syst Evol. 2007;267:79–92.View ArticleGoogle Scholar
- Garnatje T, Hidalgo O, Vitales D, Pellicer J, Vallès J, Robin O, et al. Swarm of terminal 35S in Cheirolophus (Asteraceae, Centaureinae). Genome. 2012;55:529–35.PubMedView ArticleGoogle Scholar
- Abd El-Twab MH, Motohashi T, Fujise A, Tatarenko E, Kondo K, Kholboeva SA, et al. Characterization of chromosome complement in Filifolium sibiricum (L.) Kitamura by aceto-orcein, CMA, DAPI and FISH 5S and 45S rDNA. Chromosome Bot. 2011;6:75–80.View ArticleGoogle Scholar
- Dydak M, Kolano B, Nowak T, Siwinska D, Maluszynska J. Cytogenetic studies of three European species of Centaurea L. (Asteraceae). Hereditas. 2009;146:152–61.PubMedView ArticleGoogle Scholar
- Garnatje T, Vallès J, Vilatersana R, Garcia-Jacas N, Susanna A, Siljak-Yakovlev S. Molecular cytogenetics of Xeranthemum L. and related genera (Asteraceae, Cardueae). Plant Biol (Stuttg). 2004;6:140–6.View ArticleGoogle Scholar
- Vanzela ALL, Ruas CF, Oliveira MF, Ruas PM. Characterization of diploid, tetraploid and hexaploid Helianthus species by chromosome banding and FISH with 45S rDNA probe. Genetica. 2002;114:105–11.PubMedView ArticleGoogle Scholar
- Zhang R, Zhang C-T. Isochore structures in the genome of the plant Arabidopsis thaliana. J Mol Evol. 2004;59:227–38.PubMedView ArticleGoogle Scholar
- Meyers BC, Tingey SV, Morgante M. Abundance, distribution, and transcriptional activity of repetitive elements in the maize genome. Genome Res. 2001;11:1660–76.PubMed CentralPubMedView ArticleGoogle Scholar
- Hidalgo O, Garcia-Jacas N, Garnatje T, Romashchenko K, Susanna A, Siljak-Yakovlev S. Extreme environmental conditions and phylogenetic inheritance: systematics of Myopordon and Oligochaeta (Asteraceae, Cardueae-Centaureinae). Taxon. 2008;57:769–78.Google Scholar
- Siljak-Yakovlev S, Cartier D. Heterochromatin patterns in some taxa of Crepis praemorsa complex. Caryologia. 1986;39:27–32.View ArticleGoogle Scholar
- Abd El-Twab MH, Kondo K. FISH physical mapping of 5S, 45S and Arabidopsis-type telomere sequence repeats in Chrysanthemum zawadskii showing intra-chromosomal variation and complexity in nature. Chromosome Bot. 2006;1:1–5.View ArticleGoogle Scholar
- Srisuwan S, Sihachakr D, Siljak-Yakovlev S. The origin and evolution of sweet potato (Ipomoea batatas Lam.) and its wild relatives through the cytogenetic approaches. Plant Sci. 2006;171:424–33.PubMedView ArticleGoogle Scholar
- Honda Y, Abd El-Twab MH, Ogura H, Kondo K, Tanaka R, Shidahara T. Counting sat-chromosome numbers and species characterization in wild species of Chrysanthemum sensu lato by fluorescent in situ hybridization using pTa71 probe. Chromosom Sci. 1997;1:77–81.Google Scholar
- Pedrosa-Harand A, de Almeida CCS, Mosiolek M, Blair MW, Schweizer D, Guerra M. Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution. Theor Appl Genet. 2006;112:924–33.PubMedView ArticleGoogle Scholar
- Roa F, Guerra M. Distribution of 45S rDNA sites in chromosomes of plants: structural and evolutionary implications. BMC Evol Biol. 2012;12:225.PubMed CentralPubMedView ArticleGoogle Scholar
- Knight C, Clancy R, Götzenberger L: On the relationship between pollen size and genome size. J Bot 2010, 2010. http://www.hindawi.com/journals/jb/2010/612017/abs/.
- Bainard JD, Husband BC, Baldwin SJ, Fazekas AJ, Gregory TR, Newmaster SG, et al. The effects of rapid desiccation on estimates of plant genome size. Chromosome Res. 2011;19:825–42.PubMedView ArticleGoogle Scholar
- Robertson A, Rich TCG, Allen AM, Houston L, Roberts C, Bridle JR, et al. Hybridization and polyploidy as drivers of continuing evolution and speciation in Sorbus. Mol Ecol. 2010;19:1675–90.PubMedView ArticleGoogle Scholar
- Kellogg EA, Camara PEAS, Rudall PJ, Ladd P, Malcomber ST, Whipple CJ, et al. Early inflorescence development in the grasses (Poaceae). Front Plant Sci. 2013;4:250.PubMed CentralPubMedView ArticleGoogle Scholar
- Kellogg EA, Bennetzen JL. The evolution of nuclear genome structure in seed plants. Am J Bot. 2004;91:1709–25.PubMedView ArticleGoogle Scholar
- Cerbah M, Coulaud J: rDNA organization and evolutionary relationships in the genus Hypochaeris (Asteraceae). Journal of Heredity. 1998:312–318.
- Geber G, Schweizer D. Cytochemical heterochromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads. Plant Syst Evol. 1987;158:97–106.View ArticleGoogle Scholar
- Schweizer D. Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma. 1976;58:307–24.PubMedView ArticleGoogle Scholar
- Garcia S, Garnatje T, Kovařík A. Plant rDNA database: ribosomal DNA loci information goes online. Chromosoma. 2012;121:389–94.PubMedView ArticleGoogle Scholar
- Garcia S, Gálvez F, Gras A, Kovařík A, Garnatje T. Plant rDNA database: update and new features. Database (Oxford). 2014;2014:bau063.View ArticleGoogle Scholar
- Marie D, Brown SC. A cytometric exercise in plant DNA histograms, with 2C values for 70 species. Biol Cell. 1993;78:41–51.PubMedView ArticleGoogle Scholar
- Loureiro J, Rodriguez E, Dolezel J, Santos C. Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Ann Bot. 2006;98:679–89.PubMed CentralPubMedView ArticleGoogle Scholar
- Garnatje T, Canela MÁ, Garcia S, Hidalgo O, Pellicer J, Sánchez-Jiménez I, et al. GSAD: a genome size in the Asteraceae database. Cytometry A. 2011;79:401–4.PubMedView ArticleGoogle Scholar
- Garcia S, Leitch IJ, Anadon-Rosell A, Canela MÁ, Gálvez F, Garnatje T, et al. Recent updates and developments to plant genome size databases. Nucleic Acids Res. 2014;42:D1159–66.PubMed CentralPubMedView ArticleGoogle Scholar
- Hall T. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41:95–8.Google Scholar
- Huelsenbeck J, Ronquist F, Nielsen R, Bollback J. Bayesian inference of phylogeny and its impact on evolutionary biology. Science. 2001;294:2310–4.PubMedView ArticleGoogle Scholar
- Darriba D, Taboada G, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012;9:772–2.
- Akaike H. A Bayesian extension of the minimum AIC procedure of autoregressive model fitting. Biometrika. 1979;66:237–42.View ArticleGoogle Scholar
- Maddison WP, Maddison DR: Mesquite: a modular system for evolutionary analysis. Version 3.02. 2015. http://mesquiteproject.org.
- Garcia S. The striking and unexpected cytogenetic diversity of genus Tanacetum L. (Asteraceae): a cytometric and fluorescent in situ hybridisation study of Iranian taxa. TreeBase 2015.
- Sonboli A, Olanj N, Pourmirzaei A. Biosystematics and phylogeny of Tanacetum fisherae, a new record from Iran. Rostaniha. 2011;12:165–75.Google Scholar
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