Variation of DNA amount in 47 populations of the subtribe Artemisiinae and related taxa (Asteraceae, Anthemideae): Karyological, ecological, and systematic implications

Abstract

Genome size has been estimated by flow cytometry in 47 populations of 40 species of the tribe Anthemideae (Asteraceae), mainly from Artemisia and other genera of the subtribe Artemisiinae and related taxa. A range of 2C values from 3.54 to 21.22 pg was found. DNA amount per basic chromosome set ranged from 1.77 to 7.70 pg. First genome size estimates are provided for one subtribe, 10 genera, 32 species, and two subspecies. Nuclear DNA amount correlated well with some karyological, physiological and environmental characters, and has been demonstrated as a useful tool in the interpretation of evolutionary relationships within Artemisia and its close relatives.

Full text

Variation of DNA amount in 47 populations of
the subtribe Artemisiinae and r elated taxa
(Asteraceae, Anthemideae): karyological,
ecological, and systematic implications
Sònia Garcia, María Sanz, Teresa Garnatje, Agnieszka Kreitschitz,
E. Durant McArthur, and Joan Vallès
Abstract: Genome size has been estimated by flow cytometry in 47 populations of 40 species of the tribe
Anthemideae (Asteraceae), mainly from Artemisia and other genera of the subtribe Artemisiinae and related taxa. A
range of 2C values from 3.54 to 21.22 pg was found. DNA amount per basic chromosome set ranged from 1.77 to
7.70 pg. First genome size estimates are provided for one subtribe, 10 genera, 32 species, and two subspecies. Nuclear
DNA amount correlated well with some karyological, physiological and environmental characters, and has been demon
-
strated as a useful tool in the interpretation of evolutionary relationships within Artemisia and its close relatives.
Key words: Artemisia, C value, ecology, evolution, flow cytometry, genome size, nuclear DNA amount variation, phy
-
logeny, polyploidy, systematics.
Résumé : La taille du génome de 47 populations de 40 espèces de la tribu Anthemideae (Asteraceae), principalement
du genre Artemisia et d’autres représentants de la sous-tribu Artemisiinae ou de groupes proches à celle-ci, a été es-
timée par cytométrie en flux. Les valeurs 2C sont comprises entre 3,54 et 21,22 pg. La quantité d’ADN par dotation
chromosomique de base est comprise entre 1,77 et 7,70 pg. La taille du génome a été déterminée pour la première fois
dans une sous-tribu, 10 genres, 32 espèces et deux sous-espèces. La quantité d’ADN nucléaire est en très bonne corré-
lation avec des caractères caryologiques, physiologiques et écologiques ; elle s’est avérée aussi utile pour
l’interprétation des relations évolutives chez Artemisia et ses taxons apparentés.
Mots clés : Artemisia, cytométrie en flux, écologie, évolution, phylogénie, polyploïdie, systématique, taille du génome,
valeur C, variation de la quantité d’ADN nucléaire.
Garcia et al. 1014
Introduction
The amount of nuclear DNA (C value) is a fundamental
biodiversity character, directly or indirectly related to many
phenotypic traits and other important factors such as repro
-
ductive biology, ecology, and plant distribution (Bennett
1998). More than 100 positive or negative correlations with
nuclear DNA amount have been documented. Measurements
of the amount of nuclear DNA, which initially focused on
cytogenetics, physiology, and ecology, have recently become
more important in systematic and phylogenetic research
(Kellogg 1998; Leitch et al. 1998). With the growing recog
-
nition of its relevance, there is a need for additional DNA C-
value assessments in plants (Bennett and Leitch 1995;
Bennett 1998; Hanson et al. 2001a, 2001b). Bennett and col
-
leagues have assembled six reference lists of nuclear DNA
amounts since 1976; these data are available through an in
-
ternet database (http://www.rbgkew.org.uk/cval/homepage.
html; Bennett and Leitch 2003), which facilitates compara
-
tive studies and other data-based research. Nevertheless, the
Genome 47: 1004–1014 (2004) doi: 10.1139/G04-061 © 2004 NRC Canada
1004
Received 9 January 2004. Accepted 2 June 2004. Published on the NRC Research Press Web site at http://genome.nrc.ca on
19 November 2004.
Corresponding Editor: J.H. de Jong.
S. Garcia, M. Sanz, and J. Vallès.
1
Laboratori de Botànica, Facultat de Farmàcia, Universitat de Barcelona. Av. Joan XXIII s/n,
08028 Barcelona, Catalonia, Spain.
T. Garnatje. Institut Botànic de Barcelona (CSIC-Ajuntament de Barcelona), Passeig del Migdia s/n, Parc de Montjuïc, 08038
Barcelona, Catalonia, Spain.
A. Kreitschitz. Division of Plant Morphology and Development, Institute of Plant Biology, University of Wroc»aw, ul. Kanonia
6/8, 50-328 Wroc»aw, Poland.
E.D. McArthur. Shrub Sciences Laboratory, Rocky Mountain Research Station, Forest Service, United States Department of
Agriculture, Provo, UT 84606, USA.
1
Corresponding author (e-mail: joanvalles@ub.edu).

existing data scarcely represent the global angiosperm flora
(Bennett and Leitch 1995): fewer than 2% of angiosperm
species have a known C value and more than 50% of angio
-
sperm families lack even a single estimate of nuclear DNA
amount for any species.
Artemisia (Asteraceae, Anthemideae), the principal focus
of the present study, is the largest of the tribe Anthemideae
and among the largest genera in the family Asteraceae. It
comprises from 200 to more than 500 taxa at the specific or
subspecific level, according to various authors (see Vallès
and McArthur 2001 and references therein). Five large
groups (Absinthium, Artemisia, Dracunculus, Seriphidium,
and Tridentatae) are classically considered within Artemisia,
at sectional or subgeneric levels (Torrell et al. 1999). How
-
ever, the present infrageneric classification does not repre
-
sent natural groups (Persson 1974; McArthur et al. 1981;
Vallès and McArthur 2001) and there is still no agreement
about the global treatment of the genus. Several genera have
been segregated from Artemisia (Vallès et al. 2003 and refer
-
ences therein): big ones, such as Seriphidium, and small, of
-
ten monotypic ones, such as Mausolea. In addition, other
genera, such as Ajania, are systematically close to Artemisia
or in taxonomic conflict with it. They are the basis for the
subtribe Artemisiinae (Bremer and Humphries 1993), and
have close relationships with genera belonging to the
subtribes Handeliinae, Tanacetinae, Leucantheminae and
Chrysantheminae. Molecular biology can shed light on the
real structure of this pool of genera and studies based on
DNA sequences have been and are being carried out to clar-
ify its systematics (Watson et al. 2002; Vallès et al. 2003),
which indicate the need of rearrangements to achieve a
phylogenetically based organization of the Artemisiinae.
Artemisia is a widely distributed genus in the northern
hemisphere, mainly in temperate areas, (Bremer 1994) and
is rather scarce in the southern hemisphere. It is better repre-
sented in Eurasia than in North America. Central Asia con
-
stitutes its main centre of speciation and diversification
(McArthur and Plummer 1978; Vallès and McArthur 2001).
The species of the genus can be found from sea level to high
mountains, frequently colonizing semiarid environments.
Some Artemisia species occur in isolation, but more com
-
monly they form extensive, landscape-dominant populations.
Most of the species are perennial, only around 15 are annual
or biennial. Polyploidy is a frequent phenomenon in the ge
-
nus, which also has dysploidy, with two basic chromosome
numbers (x = 8 and x = 9). Many Artemisia species have a
high economic value, in that thay have medical, food, for
-
age, or ornamental uses; on the other hand, some taxa are in
-
vasive weeds that can adversely affect agronomic harvests
(Vallès and McArthur 2001 and references therein; Wright
2002).
The present study also includes six additional Artemisiinae
and four genera belonging to three other Anthemideae subtribes,
as detailed in the Materials and methods. These taxa, particularly
those belonging to the Artemisiinae, are phylogenetically
close to Artemisia, up to the point that some of them had
been previously classified as members of Artemisia (the
proximity to this genus can be deduced from the complex
synonymy of many of these species presented in Table 1),
but alternatively these taxa are placed in other genera.
Materials and methods
Plant material
Table 1 shows the 47 populations studied, grouped by
subtribe, genus, and subgenus, along with their site of origin
and herbarium voucher information. The study material in
-
cludes 27 species and four subspecies from the genus Arte
-
misia, four from Dendranthema (including one subspecies
and one variety), and one each from Brachanthemum,
Filifolium, Kaschgaria, Mausolea, and Neopallasia (all from
subtribe Artemisiinae). Other Anthemideae taxa represented
in the study include one species each of Lepidolopsis
(Handeliinae), Nipponanthemum (Leucantheminae),
Hippolytia, and Tanacetopsis (both, Tanacetinae). The cho
-
sen populations represent, apart from different taxonomic
groups, distinct geographic areas, life forms, ploidy levels,
and chromosome numbers. Vouchers for most materials are
deposited in the herbarium of the Centre de Documentació
de Biodiversitat Vegetal de la Universitat de Barcelona
(BCN). Other vouchers are in the herbaria of the Shrub Sci
-
ences Laboratory, Provo, Utah (SSLP), the Botanical Insti
-
tute V.L. Komarov of the Russian Academy of Sciences,
Saint Petersburg (LE), and A. Kreitschitz, Wroc»aw. Some
species have been obtained from botanical gardens through
Index Seminum (Sapporo Botanic Garden, Hokkaido Uni-
versity, Japan; and Vladivostok Botanical Institute, Russian
Academy of Science, Russia), with known original location.
Young leaves used for flow cytometry assays were taken
from plants cultivated in pots. The achenes or adult plants
were collected in natural populations. Seeds of Pisum
sativum L. ‘Express Long’ and an adult Petunia hybrida
Vilm. ‘PxPc6’, both used as internal standards for flow cyto-
metric measurements, were obtained from the Institut des
Sciences du Végétal (CNRS, Gif-sur-Yvette, France).
Flow cytometry measurements
DNA 2C values of the tested species were estimated using
flow cytometry. Pisum sativum L. ‘Express Long’ and Petunia
hybrida Vilm. ‘PxPc6’ (2C = 8.37 and 2.85 pg, respectively;
Marie and Brown 1993) were used as internal standards to
cover the range of 2C values found. In one case, when the
peak of the unknown Artemisia absinthium and the internal
standard Pisum sativum overlapped, an Artemisia species
(A. abrotanum, 2C = 11.41 pg) that had previously been as
-
sessed with Pisum for nuclear DNA amount estimation, was
used as internal standard (Torrell and Vallès 2001); this ex
-
ceptional case was due to the lack of Petunia hybrida at that
moment. Young healthy leaf tissues from the species to be
studied and a calibration standard were placed together in a
plastic Petri dish and chopped with a razor blade in
Galbraith’s isolation buffer (Galbraith et al. 1983). The
amount of target species leaf (about 25 mm
2
) was approxi
-
mately twice that of the internal standard. The suspension of
nuclei in the isolation buffer was filtered through a nylon
mesh with a pore size of 70
µ
m and stained for 20 min with
propidium iodide (Sigma-Aldrich Química, Alcobendas,
Madrid, 60
µ
g/mL), the chosen fluorochrome standard
(Johnston et al. 1999); tubes were kept on ice during stain
-
ing and then left at room temperature until measurement.
For each population, five individuals were analyzed; two
© 2004 NRC Canada
Garcia et al. 1005

samples of each individual were extracted and measured in
-
dependently. Measurements were made at the Serveis
Cientificotècnics generals de la Universitat de Barcelona us
-
ing an Epics XL flow cytometer (Coulter Corporation,
Hialeah, Fla.). The instrument was set up with the standard
configuration: excitation of the sample was done using a
standard 488-nm air-cooled argon-ion laser at 15 mW
power. Forward scatter (FSC), side scatter (SSC), and red
(620 nm) fluorescence for propidium iodide were acquired.
Optical alignment was based on optimized signal from 10-
nm fluorescent beads (Immunocheck, Epics Division, Coul
-
ter Corporation). Time was used as a control of the stability
of the instrument. Red fluorescence was projected on 1024
monoparametrical histograms. Gating single cells by their
area versus peak fluorescence signal excluded aggregates.
Acquisition was automatically stopped at 8000 nuclei. The
total nuclear DNA content was calculated by multiplying the
known DNA content in Pisum or Petunia by the quotient be
-
tween the 2C peak positions of the target species and the
chosen internal standard in the histogram of fluorescence in
-
tensities for the 10 runs, based on the assumption that there
is a linear correlation between the fluorescence signals from
stained nuclei of the unknown specimen and the known in
-
ternal standard and the DNA amount. Mean values and stan
-
dard deviations were calculated based on the results for the
five individuals.
Statistical analyses (analysis of variance, means compari-
son by least significant difference test) were carried out to
evaluate the relationships between the studied variables
(DNA content, DNA per basic chromosome set, altitude, and
life cycle, among others). All the analyses were performed
with the program Statgraphics Plus 5.0 (Statistical Graphics
Corp., Rockville, Md.). In addition to the data obtained in
the present study (Table 2), those from a previous paper on
Artemisia genome size (Torrell and Vallès 2001) were also
used for the statistical analyses of the present work.
Results and discussion
The results of flow cytometric assessment of the nuclear
DNA content of 47 populations of 40 species belonging to
the tribe Anthemideae are presented in Table 2, together
with genome size data in megabase pairs (1 pg = 978 Mbp,
Doleñel et al. 2003), other karyological characters, and in
-
formation on life cycle and on the internal standard used for
each estimation. The analyses were of good quality (mean
half peak coefficient of variation (HPCV) = 3.06%). This
second study of Artemisia DNA by flow cytometry also in
-
cludes some related genera. It expands the flow cytometry
database by a factor of three — the earlier work reported 21
Artemisia species (Torrell and Vallès 2001). In addition to
the flow cytometry work, nuclear DNA content had been es
-
timated for only seven Artemisia species by cyto
-
densitometry after Feulgen staining (Nagl and Ehrendorfer
1974; Geber and Hasibeder 1980; Greilhuber 1988; Bennett
and Smith 1991; S.R. Band, personal communication;
Dbrowska 1992).
For the genera Brachanthemum, Dendranthema,
Filifolium, Hippolytia, Kaschgaria, Lepidolopsis, Mausolea,
Neopallasia, Nipponanthemum, and Tanacetopsis the DNA
content values presented in this paper are the first estimates
(Bennett and Leitch 2003). Likewise, the DNA content as
-
sessment reported here is the first record for the subtribe
Handeliinae, 32 species (13 of the 10 above-cited genera and
19 of Artemisia) and three Artemisia subspecies (Table 2).
When all the Artemisia species with available genome
size data — those from this paper, those reported by Torrell
and Vallès 2001, and those from the papers cited in the first
paragraph of this section, noted in the Bennett and Leitch
(2003) database — are taken into account, variations are, re
-
spectively, 7.33- and 4.40-fold for DNA amount and DNA
amount per basic chromosome set. The variation is 3.04 fold
for 2C value and 3.53 fold for DNA per basic chromosome
set in the other genera studied.
Relationships with karyological characters
As might be expected, 2C value means are significantly
different (p < 0.005) for chromosome number and ploidy
level. Both minor and major differences are even found be
-
tween 2n =16and2n = 18 taxa (Table 2 and Torrell and
Vallès 2001). However, there are exceptions to this positive
relationship; diploid A. abrotanum has only 5.78 pg of nu
-
clear DNA amount with 18 chromosomes, whereas A.
leucodes has 15.39 pg with the same chromosome number
and, surprisingly, A.×wurzellii, with 34 chromosomes, has
8.60 pg. Similar results have been seen in other groups of
Asteraceae e.g., Siebera pungens with a 2C value of 16.98
pg and 20 chromosomes and Amphoricarpus neumayeri with
1.73 pg and 24 chromosomes (Garnatje et al. 2004). Never-
theless, the general trend is an increase of nuclear DNA
amount with the increase of chromosome number (Papaver,
Srivastava and Lavania 1991; Achillea,Dbrowska 1992).
Although genome size and ploidy level are highly corre-
lated, nuclear DNA amount per basic chromosome set de-
creases with polyploidy. Analysis of variance (ANOVA)
shows a significant difference (p < 0.05) in nuclear DNA
amount mean values between diploids and tetraploids, the
latter having less nuclear DNA amount per basic chromo
-
some set than the former; we did not perform analyses with
other ploidy levels, because we had only minimal represen
-
tation of each one. This supports the Grant’s (1969) hypoth
-
esis that there is a decrease in nuclear DNA amount in
polyploids associated to an adaptive response for the stabili
-
zation of the higher polyploids (dodecaploids) in Betula.Nu
-
clear DNA loss per basic chromosome set in polyploids has
also been reported in many other taxa (Bennett 1972;
Murray et al. 1992; Ohri 1996; Dimitrova and Greilhuber
2000; Friedlender et al. 2002).
Aneusomaty may be another source of genome size varia
-
tion. Some of the highest standard deviation values in the
plants studied, such as those of Artemisia campestris subsp.
sericea (Table 2) or A. dracunculus (Torrell and Vallès 2001)
correspond to aneusomatic populations (Kreitschitz 2003;
Kreitschitz and Vallès 2003). Similar variations have been
reported in aneusomatic Helianthus annuus (Cavallini and
Cremonini 1985; Michaelson et al. 1991).
Systematic implications: intraspecific and interspecific
variation
Nuclear DNA amount can be useful in the interpretation
of evolutionary relationships. C value may increase or de
-
crease with evolution and comparisons between the different
© 2004 NRC Canada
1006 Genome Vol. 47, 2004

© 2004 NRC Canada
Garcia et al. 1007
Taxa Origin of materials Herbarium voucher
Subtribe Artemisiinae
Genus Artemisia
Subgenus Absinthium
A. absinthium L. Czeszów, Lower Silesia, Poland Hb. A. Kreitschitz
A. aschurbajewii C. Winkler Asku-Zhabagli nature reserve, Zhambul district, Kazakhstan BCN 11693
A. austriaca Jacq. Swieta Katarzyna, Lower Silesia, Poland Hb. A. Kreitschitz
A. frigida Willd. Almond, Colo. BCN 11568
A. glacialis L. Valmontey, Aosta valley, Italy BCN 11566
A. lagocephala (Fischer ex Besser) DC. Snezhnaya mountain,USSR I. S. Vladivostok N53, BCN S-805
A. persica Boiss. Asku-Zhablagli nature reserve, Zhambul district, Kazakhstan BCN 11696
A. sieversiana Ehrh. in Willd. Khanatalap, Almaty district, Kazakhstan BCN 11692
Subgenus Artemisia
A. abrotanum L. Wroc»aw (Tarnogaj), Lower Silesia, Poland Hb. A. Kreitschitz
A. abrotanum L. Wroc»aw (Kozanów), Lower Silesia, Poland Hb. A. Kreitschitz
A. afra Jacq. Transwaal, Makapan, South Africa BCN 11570
A. princeps Pampan. Nopporo forest park, Ebetsu, Sapporo, Japan BCN S-812
A. santolinifolia Turcz ex H. Kraschen. Asku-Zhabagli nature reserve, Zhambul district, Kazakhstan BCN 11694
A. stelleriana Besser Glazkovka, USSR I. S. Vladivostok N55, BCN S-813
A. tournefortiana Reichenb. Karapalpakstan, Uzbekistan BCN 11630
A. vulgaris L. Chrzstawa Ma»a, Lower Silesia, Poland Hb. A. Kreitschitz
A. vulgaris L. Staniszów, Lower Silesia, Poland Hb. A. Kreitschitz.
A. vulgaris L. Lhasa, Tibet, People’s Republic of China BCN 11590
A. × wurzellii C. M. James & Stace in C. M. James, Wurzell & Stace Northumberland park, London, UK BCN 11670
Subgenus Dracunculus
A. arenaria DC. Volgograd, USSR LE (Korobkov)
A. campestris L. Wayne County, Utah SSLP (McArthur 2777)
A. campestris L. Konotop, Wielkopolska Region, Poland Hb. A. Kreitschitz
A. campestris L. Zagan, Ziemia Lubuska Region, Poland Hb. A. Kreitschitz
A. campestris L. subsp. sericea (Fr.) Leuwke & Rothm. Hel, Helska sandbank, Poland Hb. A. Kreitschitz
A. filifolia Torrey Mohave County, Ariz. SSLP (McArthur 2784)
A. scoparia Waldst. & Kit. Sultanuizdag Mountains, Karkalpakstan, Uzbekistan BCN 11628
Subgenus Seriphidium
A. leucodes Schrenk Dgizak, Uzbekistan BCN 11631
Subgenus Tridentatae
A. arbuscula Nutt. Millard County, Utah SSLP (McArthur 2779)
A. bigelovii A. Gray Emery County, Utah SSLP (McArthur 2778)
A. cana Pursh. subsp. viscidula (Osterhout) Beetle Wasatch County, Utah SSLP (McArthur 2775)
A. nova Nelson Nye County, Nev. SSLP (McArthur 2739)
A. pygmaea A. Gray Emery County, Utah SSLP (McArthur 2780)
A. tridentata Nutt. subsp. tridentata Juab County, Utah SSLP (McArthur U-79)
A. tridentata Nutt. subsp. vaseyana (Rydb.) Beetle Juab County, Utah SSLP (McArthur 2507)
Genus Brachanthemum
Brachanthemum titovii H. Kraschen. Aktogai, Almaty district, Kazakhstan BCN 11690
Table 1. Provenance of the populations of Anthemideae studied.

© 2004 NRC Canada
1008 Genome Vol. 47, 2004
Taxa Origin of materials Herbarium voucher
Genus Dendranthema
D. arcticum Tzvelev subsp. maekawanum (Kitam.) H. Koyama Sapporo, Japan I.S. Sapporo, BCN S-815
D. indica Des Moul. var. coreanum Levl. & Van. Sapporo, Japan I.S. Sapporo, BCN S-809
D. maximowiczii (Komarov) Tzvelev Glazkovka, USSR I.S. Vladivostok N70, BCN S-810
D. zawadskii (Herbich) Tzvelev Sapporo, Japan I.S. Sapporo, BCN S-814
Genus Filifolium
F. sibiricum (L.) Kitam. (Artemisia sibirica(L.) Maxim., Tanacetum
sibiricum L.)
Oktyabrsky, USSR I.S. Vladivostok N76, BCN S-806
Genus Kaschgaria
K. brachanthemoides (C. Winkl.) Poljakov, Artemisia
brachanthemoides C. Winkl., Tanacetum brachanthemoides (C.
Winkl.) H. Kraschen.)
Kurtagai canyon, Almaty district, Kazakhstan BCN 11691
Genus Mausolea
M. eriocarpa (Bunge) Poljakov (Artemisia eriocarpa Bunge) Gazli, Bukhara, Uzbekista. BCN 11629
Genus Neopallasia
N. pectinata (Pall.) Poljakov (Artemisia pectinata Pall.) Southern slope of eastern Tien-Shan, Republic of Xingjan-
Uigur, People’s Republic of China
LE
Subtribe Handeliinae
Genus Lepidolopsis
L. turkestanica (Regel & Schmalh.) Poljakov (Crossostephium
turkestanicum Regel & Schmahl., Artemisia turkestanica (Regel &
Schmalh.) Franch., Tanacetum turkestanicum (Regel & Schmalh)
Poljakov)
Sostube, Chimkent district, Kazakhstan BCN S-807
Subtribe Leucantheminae
Genus Nipponanthemum
N. nipponicum (Franchet ex Maxim.) S. Kitamura (Chrysanthemum
nipponicum (Franchet ex Maxim.) Sprenger, Ch. nipponicum
Matsum., Leucanthemum nipponicum Franchet ex Maxim.)
Higashi-Hiroshima, Japan BCN S-811
Subtribe Tanacetinae
Genus Tanacetopsis
T. goloskokovii (Poljakov) Karmysch. Sogeti Mountains, Almaty district, Kazakhstan BCN S-808
Genus Hippolytia
H. megacephala (Rupr.) Poljakov (Artemisia megacephala Rupr.) Asku-Zhabagli nature reserve, Zhambul district, Kazakhstan BCN 11695
Note: Most of the vouchers are deposited in the herbarium of the Centre de Documentació de Biodiversitat Vegetal, Universitat de Barcelona (BCN). Some others are in the herbarium of the Rocky
Mountain Research Station, Provo, Utah (SSLP), in the herbarium of the Botanical Institute V.L. Komarov of the Russian Academy of Sciences, Saint Petersburg (LE), or in the herbarium of A.
Kreitschitz (Wroc»aw). I.S. indicates that the achenes have been obtained through an Index Seminum.
Table 1 (concluded).

© 2004 NRC Canada
Garcia et al. 1009
Taxa
Life
cycle
a
2C ± SD (pg)
b
2C (Mbp)
c
2n
d
Ploidy
level
DNA per basic
chromosome set Standard
e
Subtribe Artemisiinae
Genus Artemisia
Subgenus Absinthium
A. absinthium P 9.06±0.07 8860.7 18
(1)
2x 4.53 A. abrotanum
A. aschurbajewii* P 10.36±0.29 10132.1 36
(2)
4x 2.59 Petunia
A. austriaca* P 5.95±0.15 5819.1 16
(3)
2x 2.98 Pisum
A. frigida * P 5.25±0.06 5134.5 18
(4)
2x 2.63 Pisum
A. glacialis * P 8.52±0.15 8332.6 16
(4)
2x 4.26 Petunia
A. lagocephala * P 6.75±0.06 6601.5 18
(4)
2x 3.38 Petunia
A. persica* P 6.55±0.02 6405.9 18
(2)
2x 3.28 Pisum
A. sieversiana A 6.17±0.07 6034.3 18
(2)
2x 3.09 Petunia
Subgenus Artemisia
A. abrotanum * (Tarnogaj) P 11.41±0.11 11159.0 36
(1)
4x 2.85 Pisum
A. abrotanum * (Kozánow) P 5.78±0.07 5652.8 18
(1)
2x 2.89 Pisum
A. afra* P 6.31±0.34 6171.2 18
(4)
2x 3.16 Pisum
A. princeps * P 14.60±0.24 14278.8 52
(4)
6x 2.43 Pisum
A. santolinifolia * P 4.62±0.07 4518.4 18
(4)
2x 2.31 Pisum
A. stelleriana * P 6.10±0.07 5965.8 18
(4)
2x 3.05 Petunia
A. tournefortiana A/B 7.06±0.07 6904.7 18
(5)
2x 3.53 Pisum
A. vulgaris (Tibet) P 12.15±0.52 11882.7 36
(4)
4x 3.04 Pisum
A. vulgaris (Mala) P 6.23±0.04 6092.9 16
(3)
2x 3.12 Pisum
A. vulgaris (Staniszów) P 6.49±0.32 6347.2 16
(3)
2x 3.25 Pisum
A. × wurzellii* P 8.60±0.22 8410.8 34
(4)
4x 2.15 Petunia
Subgenus Dracunculus
A. arenaria * P 10.29±0.15 10063.6 36
(4)
4x 2.57 Petunia
A. campestris (Utah) P 6.38±0.05 6239.6 18
(4)
2x 3.19 Petunia
A. campestris (Konotop) P 9.78±0.13 9564.8 36
(3)
4x 2.45 Pisum
A. campestris (Zagan) P 9.92±0.18 9701.8 36
(3)
4x 2.48 Pisum
A. campestris ssp. sericea* P 10.61±0.45 10376.6 36
(1)
4x 2.65 Pisum
A. filifolia * P 7.14±0.18 6982.9 18
(4)
2x 3.57 Petunia
A. scoparia * A 3.54±0.05 3462.1 16
(5)
2x 1.77 Petunia
Subgenus Seriphidium
A. leucodes * A 15.39±0.43 15051.4 18
(5)
2x 7.70 Pisum
Subgenus Tridentatae
A. arbuscula * P 9.22±0.11 9017.2 18
(5)
2x 4.61 Petunia
A. bigelovii * P 15.49±0.10 15149.2 36
(6)
4x 3.87 Pisum
A. cana ssp. viscidula* P 8.54±0.09 8352.1 18
(6)
2x 4.27 Petunia
A. nova* P 6.37±0.14 6229.9 18
(6)
2x 3.19 Petunia
A. pygmaea* P 11.54±0.18 11286.1 18
(6)
2x 5.77 Pisum
A. tridentata ssp. tridentata* P 8.17±0.08 7990.3 18
(6)
2x 4.09 Petunia
A. tridentata ssp. vaseyana* P 8.66±0.07 8469.5 18
(6)
2x 4.33 Petunia
Genus Brachanthemum *
B. titovii * P 6.98±0.08 6826.4 18
(2)
2x 3.49 Pisum
Genus Dendranthema *
D. arcticum ssp. maekawanum* P 20.03±1.30 19589.3 72
(4)
8x 2.50 Petunia
D. indica var. coreanum* P 12.14±0.11 11872.9 36
(4)
4x 3.04 Pisum
D. maximowiczii * P 15.78±0.17 15432.8 54
(4)
6x 2.63 Pisum
D. zawadskii * P 21.22±0.52 20753.2 72
(4)
8x 2.65 Pisum
Genus Filifolium *
F. sibiricum * P 9.44±0.31 9232.3 18
(4)
2x 4.72 Petunia
Genus Kaschgaria *
K. brachanthemoides * P 14.09±0.31 13780.0 18
(2)
2x 7.05 Pisum
Genus Mausolea *
M. eriocarpa * P 13.79±0.13 13486.6 36
(5)
2x 3.45 Pisum
Genus Neopallasia *
N. pectinata * A 10.56±0.21 10327.7 36
(4)
2x 2.64 Pisum
Table 2. Nuclear DNA content and other karyological characters of the populations studied.

genome sizes provide a natural explanation of phylogenetic
relationships and systematics of many taxonomic groups
(Ohri 1998). Our nuclear DNA results agree with the molec-
ular phylogeny of Artemisia and other genera of
Artemisiinae (Torrell and Vallès 2001; Vallès et al. 2003),
as in other Asteraceae groups (Godelle et al. 1993; Zoldos et
al. 1998; Cerbah et al. 1999).
Highly significant statistical differences (p < 0.005) have
been detected in DNA amount per basic chromosome set in the
five subgenera of Artemisia, particularly between Seriphidium
and Dracunculus, and between Tridentatae on the one hand
and Artemisia and Dracunculus on the other (Table 3).
Moreover, subgenus Tridentatae is endemic to North Amer
-
ica and also forms a well supported clade in the molecular
phylogeny based on ITS analysis (Vallès et al. 2003). These
data support standing of the subgenus Tridentatae as an in
-
dependent group rather than its inclusion in Serphidium.
An important taxonomic character in subtribe Artemisiinae
is pollen grain exine ornamentation. Genera belonging to
subtribe Artemisiinae (Bremer and Humphries 1993) can be
separated, on the basis of exine ornamentation, in two
groups: one with Artemisia pollen type (with small spines)
and another with Anthemis pollen type (with longer spines)
(Martín et al. 2001, 2003). The genera Brachanthemum and
Dendranthema and other phylogenetically close genera from
other subtribes (Hippolytia, Lepidolopsis) present the
Anthemis pollen type, while members of Artemisia and other
Artemisiinae genera such as Filifolium, Kaschgaria,
Mausolea and Neopallasia present the Artemisia pollen type.
Pollen morphology is an indicator that the traditional classi
-
fication of subtribe Artemisiinae is unnatural (Martín et al.
2001, 2003). Genome size data also support separation of
the groups by pollen type: species with Artemisia pollen
type have significantly (p < 0.01) less nuclear DNA than
species with Anthemis pollen type. Genome size variation
supports the established correlation between pollen grain or-
namentation and the ITS phylogeny (Vallès et al. 2003).
Of the traditional subgeneric classification, the subgenus
Artemisia is less supported by molecular phylogeny than are
the subgenera Dracunculus, Seriphidium and Tridentatae. Its
species are dispersed in the phylogenetic tree (Vallès et al.
2003). Furthermore, subgenus Artemisia is the most hetero-
geneous in terms of morphological, chemical, ecological,
and karyological data (Ehrendorfer 1964; Torrell et al.
1999). Additionally, in the phylogenetic analysis of Vallès et
al. (2003) 5 out of the 10 taxa that were not included in any
clade belong to subgenus Artemisia, and members from this
subgenus appear distributed in four of the eight clades, con
-
firming again that the present infrageneric classification does
not represent natural groups (Persson 1974; Vallès and
McArthur 2001). Nuclear DNA amount analysis is thus quite
useful in support of molecular phylogeny and pollen data.
Further support of the heterogeneous nature of the subgenus
Artemisia is that it has the highest ratio between maximum
and minimum nuclear DNA amount per basic chromosome
set (Table 4). Conversely, subgenus Dracunculus, the most
homogeneous according to the molecular phylogeny (Vallès
et al. 2003), is the one that presents the lowest genome size
variability (the lowest ratio of all subgenera). Nuclear DNA
amount per basic chromosome set of Artemisia leucodes
(7.70 pg) is markedly different from the mean value of the
subgenus to which this species belongs, Seriphidium (3.89
pg). Similarly, Torrell and Vallès (2001) found a nuclear
DNA amount per basic chromosome set for Artemisia
judaica of 5.76 pg, far different from the mean value of its
subgenus, Artemisia (2.96 pg). In both cases, these taxa are
placed out of their respective traditional subgenera by ITS
phylogeny (Vallès et al. 2003). This confirms the value of
nuclear DNA content as a systematic marker and agrees with
the striking interspecific variation in genome size that occurs
© 2004 NRC Canada
1010 Genome Vol. 47, 2004
Taxa
Life
cycle
a
2C ± SD (pg)
b
2C (Mbp)
c
2n
d
Ploidy
level
DNA per basic
chromosome set Standard
e
Subtribe Handeliinae*
Genus Lepidolopsis *
L. turkestanica * P 11.14±0.34 10894.9 18
(2)
2x 5.57 Petunia
Subtribe Leucantheminae
Genus Nipponanthemum *
N. nipponicum * P 11.87±0.17 11608.9 18
(4)
2x 5.94 Pisum
Subtribe Tanacetinae
Genus Tanacetopsis *
T. goloskokovii * P 9.73±0.27 9515.9 18
(4)
2x 4.87 Petunia
Genus Hippolytia *
H. megacephala * P 12.47±0.19 12195.7 18
(2)
2x 6.24 Pisum
Note: The taxa for which genome size has been estimated for the first time are marked with an asterisk (*).
a
Life cycle: A, annual; B, biennial; P, perennial).
b
2C nuclear DNA content (mean value ± standard deviation of 10 samples).
c
1 pg = 978 Mbp (Doleñel et al. 2003).
d
Somatic chromosome number. (1) Kreitschitz and Vallès (2003); (2) Vallès et al. (2001b); (3) Kreitschitz (2003); (4) unpublished counts performed by
the present authors; (5) Vallès et al. (2001a); (6) McArthur and Sanderson (1999). All counts have been carried out in the populations studied in the pres
-
ent paper.
e
Internal standard used in each case (see text for details about Pisum and Petunia; for A. absinthium, the standard used was another Artemisia, A.
abrotanum, previously measured (2C = 11.41 pg, Torrell and Vallès 2001) against Pisum).
Table 2 (concluded).

in many, though not all, major taxonomic groups (Hanson et
al. 2001a, 2001b).
Amount of nuclear DNA per basic chromosome set statis
-
tically differs (p < 0.005) between Artemisia and its related
genera from other subtribes (Anthemideae not Artemisiinae).
Nuclear DNA amount per basic chromosome set of
Artemisiinae (Artemisia excluded) also differs (p < 0.05)
from those genera belonging to subtribes Tanacetinae,
Leucantheminae and Handeliinae. On the other hand, there
is no statistically significant difference in nuclear DNA
amount per basic chromosome set between genus Artemisia
and the other Artemisiinae analysed. In fact, many of the
non-Artemisia Artemisiinae studied here had been previ
-
ously included in Artemisia, and subsequently separated in
different genera, often new and with only one or two spe-
cies. DNA sequence analysis of these plants (Vallès et al.
2003) demonstrate most of these genera tightly embedded in
the Artemisia clade; this could be interpreted to support the
elimination of these new genera, and their species returned
again to Artemisia. The absence of statistically significant
difference in nuclear DNA amount per basic chromosome
set between these groups also supports this hypothesis. In
summary, all these results indicate that nuclear DNA amount
is an important tool in the analysis of phylogenetic relation-
ships.
Within the studied Artemisia taxa, in the present paper
and an earlier one (Torrell and Vallès 2001), different popu
-
lations have been analysed for some species. The differences
detected in nuclear DNA amount give a low degree of vari
-
ability in most of these species. This can be illustrated by
the comparison between the very similar 2C values obtained
in the present study and in Torrell and Vallès (2001) for
A. absinthium (9.06 in the present study / 8.52 in Torrell and
Vallès 2001), A. vulgaris (6.23, 6.49 / 6.08), A. campestris
(diploid: 6.38 / 5.87; tetraploid: 9.78, 9.92 / 11.00), and dif
-
ferent subspecies of A. tridentata (8.17, 8.86 / 8.18). Artemi
-
sia abrotanum also constitutes a case of nuclear DNA
amount constancy: although the analysed populations have
different ploidy levels (diploid and tetraploid), nuclear DNA
amount per basic chromosome set of both species only dif
-
fers in 1.40%. This fact is also interesting because
polyploids ordinarily have significantly less nuclear DNA
per basic chromosome set than corresponding diploids.
However, in this case both specimens show quite a similar
nuclear DNA amount per basic chromosome set and both
A. abrotanum specimens came from the same geographic
area (Wroc»aw, Poland), a circumstance that could partially
explain this homogeneity. Another possibility could be an
autopolyploid origin of the tetraploid population, which has
almost exactly double DNA amount of the diploid. Further
cytogenetic studies on A. abrotanum are necessary to con
-
firm this hypothesis. Additionally, tetraploid A. campestris
could have the same origin (2x, 5.87 pg; 4x, 11.0 pg; Torrell
and Vallès 2001). Our results support that although nuclear
DNA amount or C value is considered constant within a spe
-
cies, it is almost sure that a certain degree of genuine
intraspecific variation exists; the processes or mechanisms
that are usually able to cause it are duplications, deletions,
chromosomal polymorphisms, the existence of B chromo
-
somes, or the presence of transposable elements or repetitive
sequences (Greilhuber 1998; Ma»uszy½ska 1999).
Ecology and life cycle
No statistically significant relationship exists between life
cycle and C value among the species studied. However, the
taxon with the lowest nuclear DNA amount, A. scoparia,is
annual, and those with the highest C values are perennial, as
was the case in a previous report in other Artemisia species
(Torrell and Vallès 2001) or in other genera, including some
Anthemideae (Bennett 1972; Nagl and Ehrendrofer 1974;
Rees and Narayan 1981). It is generally assumed that a low
C-value correlates with a high rate of development; in other
words: if less nuclear DNA is duplicated, the cell cycle is
faster, and the developmental rhythm, consequently, is more
intense. This is specially useful for annual or ephemeral
plants, which have only limited time to carry out their life
cycle. The studied A. scoparia population inhabited an inter
-
mittently dry river bed, and its low C value (3.54 pg, the
lowest of the present study) promotes a fast life cycle that is
rapidly completed before the seasonal summer or fall floods.
This case supports the premise that annual species have a
smaller amount of nuclear DNA than perennials. In contrast,
however, Artemisia leucodes, another annual species, has
one of the biggest genomes (2C = 15.39 pg) of all the dip
-
loid species studied; its karyotype is made up of large chro
-
mosomes (Vallès et al. 2001a) and its high C value, despite
its annual life cycle, is supported by the Nagl and
Ehrendrofer (1974) explanation that large chromosomes
could have a higher metabolic rate that facilitates an increase
in RNA synthesis. This would increase the synthesis of the
necessary proteins to permit a faster life cycle. Although
many authors have found a positive correlation between ge
-
nome size and life cycle duration, numerous exceptions sug
-
gest that it is not so clear as initially thought. Some authors
have reported even a negative relationship between those pa
-
rameters (e.g., Pennisetum, Martel et al. 1997), whereas oth
-
ers have found no relationship (Grime and Mowforth 1982).
The statistical analysis carried out on the species of this
study did not reveal any significant difference between the
© 2004 NRC Canada
Garcia et al. 1011
Subgenus Mean (pg) Homogeneous groups
Dracunculus 2.668 a
Artemisia 3.050 ab
Absinthium 3.563 bc
Seriphidium 3.892 bc
Tridentatae 4.088 c
Table 3. Comparison of means of DNA amount per
basic chromosome set in the subgenera of Artemisia.
Subgenus Maximum Minimum Ratio max/min
Absinthium 4.53 2.59 1.75
Artemisia 5.76 1.44 3.27
Dracunculus 2.93 1.77 1.66
Seriphidium 7.69 2.67 2.88
Tridentatae 5.79 3.21 1.80
Table 4. Maximum, minimum, and ratio (maximum/ minimum)
of nuclear DNA amount per basic chromosome set (pg) in the
subgenera of Artemisia .

studied populations of higher or lower altitudes. The tetra
-
ploid A. vulgaris studied in this paper has a 24.7% differ
-
ence with the one studied by Torrell and Vallès (2001) even
though both populations are tetraploid. These populations
grow in geographically and ecologically distinct conditions.
The population with the higher nuclear DNA amount is a Ti
-
betan population growing at 3650 m. An adaptation to alti
-
tude could at least partly explain the difference. Many
studies on this subject have reported that species inhabiting
arctic or high mountain areas tend to present larger
genomes, and are most frequently polyploids (Gregory and
Hebert 1999, and references therein). Some authors have
concluded that natural selection favours the modulation of
nuclear DNA content under certain weather conditions,
mainly linked with altitude or latitude (Bennett 1976). The
high taxonomic complexity of the A. vulgaris group may
also contribute to an explanation of this difference. Simi
-
larly, A. glacialis, found in Italy at an altitude of 2300 m has
a larger C value than the mean of the analysed diploid spe
-
cies of the genus, conforming to what other authors have
stated about the positive correlation between DNA amount
and altitude (Caceres et al. 1998). Nevertheless, similar stud
-
ies have found a negative correlation or even no relationship
between altitude and C value, (Creber et al. 1994; Reeves et
al. 1998; Vilhar et al. 2002), similar to the relationship be-
tween life cycle and genome size, suggesting again that the
link between the two is not clear.
It seems likely then that genome size variation in Artemi-
sia species does not depend on altitude. Nonetheless, it ap-
pears to be a response to other kinds of selective pressures,
such as adaptation to arid environments. Sanderson et al.
(1989) and McArthur and Sanderson (1999) found a better
adaptation to arid habitats in polyploid rather than in diploid
Artemisia and Atriplex in North American semi-desert habi-
tats, and Vallès et al. (2001a, 2001b) detected that tetraploid
species of Artemisia were more widely distributed in arid
lands than the related diploids. Both studies support the hy
-
pothesis that nuclear DNA amount increases — especially
by means of polyploidy — in plants adapted to extreme en
-
vironments. Our results agree with this idea, and show that
there can be a DNA amount increase even in diploids. Arte
-
misia leucodes and A. pygmaea, the two diploid taxa with
the highest DNA amount per basic chromosome set of the
Artemisia species analysed, inhabit desert or semi-desert re
-
gions of central Asia and North America, and are well
adapted to the extreme conditions of high temperature and
drought that characterize these environments. Moreover, dip
-
loid A. filifolia, another colonizing plant of sandy North
American deserts, shows the highest nuclear DNA amount
per basic chromosome set of its subgenus, Dracunculus.
Artemisia absinthium is a nitrophilous species usually
grown in ruderal zones. As the presence of high concentra
-
tions of nitrogen in the soil can also be considered as a diffi
-
cult, if not extreme, environmental condition, it is interesting
to observe that our A. absinthium population shows a higher
nuclear DNA amount per basic chromosome set than the
mean for the genus. Torrell and Vallès (2001) reported the
same relationship in another ruderal Artemisia species,
A. thuscula, taxonomically related to A. absinthium. In both
cases, the high nuclear DNA amount could be interpreted as
a response to the presence of nitrogen; this would support
Evans (1968), who detected a 10% increase in nuclear DNA
amount in varieties of Linum usitatissimum growing in
strongly nitrogenated soils and at high temperatures.
Concluding statement
C values in the subtribe Artemisiinae, including the large
genus Artemisia and related taxa, are a useful adjunct in par
-
allel or in correlation to other kinds of data, e.g., chromo
-
some number, life form, pollen grain exine patterns,
systematic placement, and ecology, in determining evolu
-
tionary relationships within this group of plants.
Acknowledgements
The authors gratefully thank Spencer C. Brown (Institut
des Sciences du Végétal, CNRS, Gif-sur-Yvette) for supply
-
ing the seeds to grow Pisum sativum and an adult plant of
Petunia hybrida, both used as internal standards, and for his
comments concerning technical aspects and results interpre
-
tation; Sonja Siljak-Yakovlev (Université de Paris-Sud,
Orsay) for fruitful discussions on the subject of the manu
-
script; Jaume Comas, Ricard Álvarez, Màrius Mumbrú
(Universitat de Barcelona), and Oriane Hidalgo (Institut
Botànic de Barcelona) for technical support in flow
cytometry; Miguel Ángel Canela (Universitat de Barcelona)
for advice on statistics; Aleksandr A. Korobkov (Botanical
Institute of the Russian Academy of Sciences, Saint Peters-
burg), Katsuhiko Kondo (Hisoshima University), and person-
nel from the Sapporo and Vladivostok Botanical Gardens for
supplying some materials of the species studied. This work
was supported by project BOS2001-3041-C02-01 of the
Spanish government. Two of the authors (S.G. and M.S.) re-
ceived predoctoral grants from the Spanish government and
one of the authors (A.K.) received a grant from the Polish
government (KBN 2396/W/IB/01).
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