Nordic Journal of Botany 33: 484–497, 2015

doi: 10.1111/njb.00659, ISSN 1756-1051

© 2015 The Authors. Nordic Journal of Botany © 2015 Nordic Society Oikos
Subject Editor and Editor-in-Chief: Torbjörn Tyler. Accepted 26 October 2014

Taxonomic status of Aster, Galatella and Tripolium (Asteraceae) in

view of anatomical and micro-morphological evidence

Dunja Karanovic’, Jadranka Lukovic’, Lana Zoric’, Goran Anačkov and Pal Boža

D. Karanović (dunja.karanovic@dbe.uns.ac.rs), J. Luković, L. Zorić, G. Anačkov and P. Boža, Dept of Biology and Ecology, Faculty of Science,
Univ. of Novi Sad, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia.

Using light and scanning electron microscopy (SEM), comparative analyses of anatomical and micro-morphological
characteristics of the leaf, stem and peduncle have been carried out on five species of the family Asteraceae (Aster amellus,
Galatella linosyris, G. cana, G. sedifolia and Tripolium pannonicum), previously included in Aster although more recent
morphological and phylogenetic studies indicate that Galatella and Tripolium should be considered as separate genera. The
aim of the present study was to establish whether these species could be differentiated by anatomical characters. Our results
will further inform the decision to separate the four aforementioned species from the genus Aster, as well as indicate whether
anatomical data confirm the proposed circumscription of the genera Galatella and Tripolium. The anatomical observations
and data analyses using discriminant and correspondence analysis showed that the combination of selected qualitative
and quantitative characters separated the species into three groups, corresponding to the three genera. The characters of
the leaf blade, its epidermis in particular, proved to be of the highest significance for discrimination at generic rank. The
specific qualitative features that characterize each of the species and the genera were emphasized. Our findings support the
attribution of the examined species to three genera, which proved to be anatomically distinguishable and well defined.

Asteraceae, comprising more than 1600 genera and 23 000
species, belongs to a group of the most versatile families
among flowering plants and has almost global distribution
(Anderberg et al. 2007). It includes plants that have various
life forms, and thus diverse anatomy. Anatomical character-
istics have a significant role in plant taxonomy, as they can
provide useful information for establishing relationships and
identifying plant taxa (Metcalfe and Chalk 1957, Scatena
et al. 2005, Zorić et al. 2012, Sosa et al. 2013). As anatomical
differences among closely related species are more quantita-
tive than qualitative, they are mainly useful for delimitation
of higher taxonomic ranks, such as genera and families. In
Asteraceae, anatomical features shown to have considerable
taxonomic value include secretory canals, laticiferous canals,
types of glandular and non-glandular hairs, anomalous sec-
ondary thickening as well as the occurrence of medullary and
cortical bundles (Metcalfe and Chalk 1957).
Our present work is focused on Galatella linosyris (L.)
Rchb. f., G. cana (Waldst. & Kit.) Nees, G. sedifolia (L.)
Greuter, Tripolium pannonicum (Jacq.) Dobrocz. subsp.
pannonicum and Aster amellus L. The taxonomic placement
of the examined species is much debated. The classical taxo-
nomical approach of ‘Flora of Europe’ and ‘Flora of Serbia’
includes them all in the genus Aster L. (Table 1). How-
ever, it is well known that the genus Aster is taxonomically
problematic when it comes to the delimitation of species,
especially Old World Aster (Nesom 1994a, 1994b). More
recent in-depth studies of phenotypic and genetic features,
as well as new phylogenetic studies, have revealed that Aster
should be more narrowly defined (Nesom 1994a, 1994b,
2008, Greuter 2003, Li et al. 2012). It has thus been sug-
gested that Galatella Cass. and Tripolium Nees. should be
excluded from Aster and several authors have treated them
as separate genera (Jones and Young 1983, Nesom 1994b,
Greuter 2003, Anderberg et al. 2007, Li et al. 2012). Our
research was motivated by the fact that studies on Aster and
its delimitation are mostly based on gross morphological and
cytogenetic, rather than anatomical investigations (Chatterji
1962, Jones and Young 1983, Semple et  al. 1983, Nesom
1994a, 1994b, Li et al. 2012). According to Jones and Young
(1983), Aster is a large and complex genus and the narrow
generic concept permits retaining in Aster only the species
closely related to the type (i.e. A. amellus). Guided by this
premise, A. amellus was selected as the representative species
of the genus Aster for our comparative analysis.
According to Nesom (1994a, 1994b), Galatella
and Tripolium together with Crinitina Soják form the
‘Galatella group’ of Asterinae s.s. Some authors have pre-
ferred to treat these genera as three sections within Aster while
others consider them as separate genera (Li et  al. 2012).
Nesom (1994b) stated that Galatella and Aster are closely
related, as they share a number of characteristics, as for
example: sessile-glandular leaves, disc style branches with
relatively short, papillate collecting appendages, and flat,

484
Table 1. Taxonomic treatments of the analyzed species.

Species Flora of Europe Flora of Serbia Habitat type

(Nesom 2008)
Galatella linosyris (L.) Rchb.
f. 1853
G. cana (Waldst. & Kit.)
Nees 1832
G. sedifolia (L.) Greuter
2003
Tripolium pannonicum
(Jacq.) Dobrocz. 1962
subsp. pannonicum
Asteramellus L. 1753
(Merxmüller and Schreiber
1976)
Aster linosyris (L.) Bernh.
1800
Aster sedifolius L. 1753
subsp. canus (Waldst. &
Kit.) Merxm. 1974
Aster sedifolius L. 1753
ssp. sedifolius
Aster tripolium L. 1753
subsp. pannonicus
(Jacq.) Soó 1925
Aster amellus L. 1753
(Boža and Vasic’ 1986,
Gajic’ 1975)
Aster linosyris (L.)
Bernh. 1800
Aster canus W. et K.
1802
Aster sedifolius L. 1753
Aster tripolium L. 1753
var. pannonicus
(Jacq.) Beck1770
Aster amellus L. 1753
(Boža and Vasic’ 1986, Gajic’ 1975,
Merxmüller and Schreiber 1976)
Rocky places, open grassland
Grassy, moist, bushy and saline
places
Saline places and dry, bushy
slopes
Saline places
Scrub, wood-margins, rocky, dry
grassy places

obovate, 2-(4-)ribbed, often glandular achenes. Li et  al.
(2012) argue that these similarities are superficial, and have
evolved in parallel, and therefore Galatella should be sepa-
rated from Aster. Anyway, Galatella species possess certain
characteristics clearly separating them from the genus Aster,
such as glandular, single-veined leaves, broad, parallel-
nerved phyllaries, sterile ray flowers usually with non-coiling
ligules, relatively shallow disc corolla lobes and a uni-seriate
pappus. As noted by Tamamschjan (1959), Galatella and
Linosyris Cass., which differ significantly from Aster, have
been included arbitrarily in Aster by some authors. The
diagnostic features of the species of Aster, including its typi-
cal representative A. amellus, are loose corymbs with a few
heads, broad phyllaries mostly graduated in 3 series, tubular
disc corollas with a tube which is half the corolla length,
abruptly ampliate and with spreading-reflexed lobes, short
style appendages that are almost deltoid and closely papil-
late, strongly flattened, obovate achenes with two thick-
ened lateral ribs, commonly with sessile glands near the
apex, and pappus bristles in 2–3 series. As the morphology
of Aster achenes is very constant and, with the exception of
size, shows only small variations, it was previously used for
clarifying relationship at subtribal rank and defining mono-
phyletic groups (Grierson 1964, Nesom 1994b).
Tripolium pannonicum has been included in the
genus Aster at different taxonomic levels (Gajić 1975,
Merxmüller and Schreiber 1976). However, numerous other
authors have considered it as a representative of monotypic
genus Tripolium (Tamamschjan 1959, Zhang and Bremer
1993, Anderberg et al. 2007). According to Nesom (1994b),
given that Tripolium and Galatella share a number of
similarities, such as corymboid capitules, herbaceous, broadly
rounded, multi-nerved phyllaries, and tendency to rayless-
ness, these taxa may be closely related. A phylogenetic study
conducted by Li et al. (2012), where Galatella and Tripolium
constitute a well-supported clade, supports this conclusion.
The general morphological structure of Galatella, Tripo-
lium and Aster was described by Anderberg et al. (2007). On
the other hand, literature data on micro-morphological and
anatomical characteristics of the studied species are limited
(Pătrut et al. 2005, Bercu et al. 2012). In particular, more
detailed investigations are lacking, as most studies in this
field rely on the general description of leaf and stem anat-
omy of Asteraceae provided by Metcalfe and Chalk (1957).
While Galatella has mainly been investigated for its bio-
chemical aspects (Letchamo et al. 2004, Gođevac et al. 2012,
Adekenov et al. 2013), the ecological and physiological char-
acteristics of T. pannonicum has been described in numer-
ous papers (Perera et al. 1997, Sági and Erdei 2002, Grigore
and Toma 2006). Still, A. amellus has been most extensively
analyzed, addressing its ecological, morphological and
genetic characteristics (Raabová et al. 2007, Mandáková and
Münzbergová 2008, Münzbergová et al. 2011).
Given the controversial taxonomic status of the analyzed
species, and the paucity of data pertaining to their anat-
omy and micro-morphology, this work aimed to establish
whether a more precise classification can be obtained based
on the comparative analysis of qualitative and quantitative
characteristics of the vegetative organs. The objective was to
determine whether these species can be differentiated at the
anatomical level. The results would further assist in establish-
ing if the exclusion of G. linosyris, G. cana, G. sedifolia and
T. pannonicum from the genus Aster is warranted, as well
as whether the separation of Galatella and Tripolium may
be supported by anatomical and micro-morphological char-
acters. The analyzed characteristics are also discussed in the
light of their ecological and adaptive significance.
Material and methods
Plant material was collected during the flowering period,
and was selected from native populations found at different
localities in north Serbia. After the plants were identified,
their voucher specimens were deposited in BUNS (Table 2).
Segments from the middle part of the leaf blade, stem and
peduncle were separated and fixed in 50% ethanol. Micro-
morphological analysis of leaf blade epidermis was performed
using light and scanning electron microscopy (SEM). For
light microscopy, leaf blade adaxial and abaxial epidermal
prints were made. Leaf blade surfaces were covered with
liquid transparent varnish, and epidermal prints removed
using transparent adhesive tape. Stomata and trichomes were
counted within five randomly selected areas on both sur-
faces, excluding the main veins. The average value was used
to estimate the count per mm2 of the leaf blade surface. For

485
Table 2. List of the species used in the study, with voucher data of the examined material.
Collecting site
Species Source area
Galatella linosyris Serbia, Rimski šanac
G. cana Serbia, Svilojevo
G. sedifolia Serbia, Žabalj
Tripolium pannonicum Serbia, Žabalj
subsp. pannonicum
Aster amellus Serbia, Beočin
SEM analysis, small pieces of dry leaves were sputter-coated
with gold for 180 s, at 30 mA (BAL-TEC SCD 005), and
subsequently viewed using an JEOL JSM-6460LV electron
microscope at an acceleration voltage of 20 kV.
Cross sections were obtained using a Leica CM 1850
cryostat at a cutting interval of 40 mm. All observations
and measurements were performed using a ZEISS light
microscope AxioVision Release 4.8.1. Based on the results,
stomatal index and index of pallisade tissue cells were cal-
culated as their length to width ratio. The main vein index
was calculated as the ratio of leaf blade thickness at the main
vein and leaf blade thickness at 1/4 of the leaf blade width.
Tissue percentages were calculated as proportions in relation
to the leaf blade thickness (taken as 100%) or the leaf blade,
stem and peduncle cross-section area (100%). To confirm
the presence of lignin, the acid-floroglucin test was used
and visualization of lipid components was performed using
Sudan III (Ruzin 1999).
Statistical data processing was performed using Statistica
for Windows (2011). Means and coefficients of variation
were calculated, and significance of differences in measured
parameters between the species was determined using Dun-
can's test (p  0.05). Specimen grouping was tested using
Discriminant analysis, based on parameters that differed
significantly among species. Qualitative characters were sub-
jected to Multiple Correspondence Analysis in order to deter-
mine the grouping tendency of the examined species. This
allowed the qualitative features that contributed the most to
the clear definition of the analyzed species to be specified.
Results
Leaf blade anatomical characteristics
Epidermal cells of all examined species were polygonal
to irregular in shape, and were characterized by ribbed
thickenings on the surface of the external periclinal walls
(Fig. 1). In the species of Galatella, epicuticular wax in the
form of flakes was observed (Fig. 2A). In addition, their epi-
dermal cells were small and anticlinal cell walls were straight
to slightly sinuous. The lateral cell walls of T. pannonicum
and A. amellus were more plicate, and the cells were consid-
erably larger compared to Galatella species. Epidermal thick-
ness was highest in A. amellus (Table 3).
486
Voucher
Coordinates Date number
45°21′50.56′′N 21 Oct 2008 2–1782
19°55′43.81′′E
45°39′43.78′′N 25 Oct 2008 2–1787
19002′42.50′′E
45°23′53.86′′N 21 Oct 2008 2–1783
20°12′01.10′′E
45°21′05.94′′N 21 Oct 2008 2–1784
20°02′39.78′′E
45°10′15.69′′N 29 Sep 2009 2–1786
19°42′31.03′′E
The leaf blades of all analyzed species were amphistoma-
tous, with anomocytic stomata (Fig. 1). The number of sto-
mata was lowest in A. amellus (Table 3), while T. pannonicum
had a small number of relatively large stomata. Larger num-
ber of small stomata was found in Galatella species. Stomatal
index values indicated that A. amellus and T. pannonicum
had more elongated stomata than Galatella species.
Densely distributed trichomes were recorded on epider-
mis of all the examined species, at both sides, apart from
T. pannonicum whose leaves were glabrous. SEM analysis
confirmed the presence of two types of multicellular, uni-
seriate, non-glandular trichomes, as well as multi-cellular,
bi-seriate glandular trichomes (Fig. 2). Large, multi-
cellular, bi-seriate glandular trichomes were present in
G. sedifolia and G. cana (Fig. 2B–C), whilst in other spe-
cies they were very rare or absent. Whip-like non-glandular
trichomes (type I) consisted of a short, upright stalk, com-
posed of several cells, and a very elongated, curled termi-
nal cell. While they occurred in all Galatella species, they
were most numerous in G. cana, forming a dense indu-
mentum giving the leaves a cobweb-like, stringy appear-
ance (Fig. 2C). Non-glandular upright trichomes (type II)
were recorded only in A. amellus. These trichomes were
composed of 3–4 large, thick-walled cells and a pointed
terminal cell with a base comprised of 6–8 raised epider-
mal cells, arranged in a form of rosette. Protuberances
were observed on the outer walls of the terminal cells
(Fig. 2D). Significantly, the lowest number of non-
glandular trichomes was recorded in A. amellus (Table 3).
In anatomical terms, the leaf blades of the examined spe-
cies had an isolateral structure (Fig. 3A–B). The palisade tis-
sue consisted of large cells, which were rich in chloroplasts
and were arranged in 2–3 and 1–3 layers, on the adaxial and
abaxial side, respectively. Palisade tissue was most developed
in T. pannonicum, and was characterized by looser arrange-
ment of palisade cells. These cells were significantly larger,
in comparison to those of other examined species (Table 4).
Index values indicated that, palisade cells were most
elongated in G. linosyris and this difference was statistically
significant. Large, individual oil bodies were clearly observed
in the palisade tissue cells of all the examined species
(Fig. 4A–B). In T. pannonicum, they were the particularily
large and had a granular structure, while homogeneous in
other species. Individual crystal druses were observed in the
spongy tissue cells of A. amellus.
Figure 1. Light micrographs of adaxial (A), (C), (E), (G), (I) and abaxial (B), (D), (F), (H), (J) epidermis of Galatella, Tripolium and
Aster species. Frontal view of epidermis. (A), (B) epidermal cells with anomocytic stomata, ribbed thickening on the surface of external
periclinal walls and straight anticlinal walls in G. linosyris, (C), (D) G. cana (E), (F) G. sedifolia and (G), (H) with sinuous anticlinal walls
in T. pannonicum and (I, J) A. amellus.

487
Figure 2. Scanning electron micrographs of adaxial (B) and abaxial (A), (C), (D) leaf blade epidermis of Galatella, Tripolium and Aster
species. (A) whip-like non-glandular trichome (type I) and epicuticular wax in the form of flakes on the abaxial leaf blade surface in G.
linosyris. (B) multi-cellular, bi-seriate glandular trichomes on the adaxial leaf blade surface in G. sedifolia. (C) whip-like non-glandular
trichomes (type I) and multi-cellular, bi-seriate glandular trichomes on the abaxial leaf blade surface in G. cana. (D) upright non-glandular
trichome (type II) on the abaxial leaf blade surface in A. amellus. Abbreviations: gt  glandular trichomes, ngt  non-glandular trichomes
(type I, II), w  epicuticular wax.

Collateral closed vascular bundles, surrounded by paren-
chymatous sheath, were arranged in a row, in the central leaf
blade plane (Fig. 3). In the main vein, one vascular bundle
was present, with a strongly developed surrounding sheath,
extending to both epidermises. The main vein was the
most prominent in A. amellus and this finding was statisti-
cally significant. Regular series of conductive elements were
observed in the main vein xylem, although regularity was
less expressed in T. pannonicum (Fig. 3C–D). Schizogenous
secretory ducts, with lumen lined with a layer of secretory
epithelial cells, were observed above the phloem of larger
bundles in all examined species (Fig. 4C). High percentage
of vascular bundles in leaf blade cross section characterized
the species of Galatella, whilst the lowest percentage were
observed in T. pannonicum (Table 4).
Stem anatomical characteristics
A polygonal stem cross-sections, typically with five clearly
pronounced ribs, was observed in Galatella species (Fig. 5).
Young stem parts of T. pannonicum had wavy cross-section
margins with pronounced invaginations, while the older parts

Table 3. Micro-morphological characters of leaf blade epidermis of Aster, Galatella, and Tripolium species. Data displayed as mean value
(coefficient of variation in percent).

G. linosyris G. cana G. sedifolia T. pannonicum A. amellus

Adaxial epidermis
Thickness (%) 8.0 (9.4)bc 7.0 (17.5)c 8.1 (9.3)b 7.6 (12.1)bc 11.3 (17.0)a
Cell cross-section area (mm2) 822 (28.8)c 361 (35.3)d 464 (28.9)d 1170 (26.8)a 982 (44.8)b
Cuticle thickness (%) 3.8 (14.7)a 2.4 (36.3)c 2.2 (26.0)c 1.5 (31.2)d 3.1 (13.3)b
No. of non-glandular trichomes/mm2 33 (52.1)c 112 (41.3)a 56 (27.4)b ABSENT e 11 (80.1)d
No. of stomata/mm2 113 (23.4)c 133 ( 33.4)b 163 (20.2)a 96 (28.2)d 62 (20.2)e
Stomata length (mm) 40.3 (9.0)c 32.5 (11.8)d 31.6 (8.4)e 44.0 (11.2)b 47.6 (12.7)a
Stomata width (mm) 35.0 (12.3)b 25.5 (13.6)e 26.3 (9.5)d 33.0 (13.7)c 35.4 (13.7)a
Stomatal index 1.2 (12.9)c 1.3 (14.1)b 1.2 (12.0)c 1.4 (16.0)a 1.3 (15.5)b
Abaxial epidermis
Thickness (%) 8.5 (15.7)a 6.7 (23.7)c 8.3 (13.7)ab 8.0 (14.2)bc 9.6 (23.7)a
Cell cross-section area (mm2) 827 (30.2)c 379 (40.4)d 452 (30.0)d 1420 (34.5)a 1101 (130.1)b
Cuticle thickness (%) 4.1 (20.1)a 2.1 (34.4)b 2.6 (19.4)b 1.4 (17.4)c 2.7 (14.7)b
No. of non-glandular trichomes/mm2 39 (51.0)c 112 (32.1)a 63 (31.7)b ABSENT d 7 (124.9)d
No. of stomata/mm2 86 (33.1)c 211 (25.0)a 189 (23.3)b 87 (27.1)c 85 (23.4)c
Stomata length (mm) 42.0 (10.1)c 33.0 (11.6)d 30.0 (11.1)e 48.1 (12.6)a 46.5 (13.2)b
Stomata width (mm) 35.2 (15.4)a 26.0 (12.1)c 25.4 (13.3)c 33.1 (13.3)b 34.0 (12.2)b
Stomatal index 1.2 (13.3)d 1.3 (14.1)c 1.2 (13.0)d 1.5 (15.0)a 1.4 (14.1)b

Notes: Different superscripts indicate statistically significant differences between analyzed species according to Duncan’s test (p  0.05).

488
Figure 3. Light micrographs of leaf blade cross sections of Galatella, Tripolium and Aster species. (A) isolateral leaf blade structure and
compactly arranged palisade cells in G. linosyris. (B) isolateral leaf blade structure with loose arrangement of palisade cells in T. pannonicum.
(C) detail of main vein cross section of A. amellus showing collateral vascular bundle with regular series of xylem conductive elements and
a schizogenous secretory duct above the phloem. (D) detail of main vein cross section of T. pannonicum illustrating a collateral vascular
bundle with less expressed regularity of the series of xylem conductive elements. Abbreviations: sd  secretory duct, x  xylem elements.

of the stem were characterized by a more regular, rounded
shape, similarly to A. amellus. Epidermal cells were oval to
isodiametric in shape in all analyzed species, with the excep-
tion of A. amellus, in which they were tangentially elongated
(Fig. 5E). The epidermis consisted of tiny cells with thick-
ened walls, covered with a relatively thick, wrinkled cuticle.
Trichomes of the type found on the leaf blade were pres-
ent on the stem epidermis, albeit in much smaller number.
A subepidermal layer in the form of one row of regularly
arranged cells was visible only in G. linosyris (Fig. 5F).
The cortex was composed of collenchyma and chlor-
enchyma, which interspersed alternately. Collenchyma,
in the form of 1–4 layers of cells, was placed in the ribs in
Galatella species and in A. amellus. In T. pannonicum, col-
lenchyma was less developed and was located in the recesses
of the stem, while chlorenchyma was loose and present in the
ribs (Fig. 5D). While individual oil bodies in the chloren-
chyma cells were observed in all examined species, they were
particularily large and most numerous in G. sedifolia and
A. amellus (Fig. 5E).
Vascular bundles were arranged in one circle within
the central cylinder. Secretory ducts were observed in the
cortex above the phloem (Fig. 4D). Sclerification of the cen-
tral cylinder was very pronounced due to the presence of

Table 4. Anatomical characteristics of leaf blade of Aster, Galatella and Tripolium species. Data displayed as mean value (coefficient of
variation in percent).

G. linosyris G. cana G. sedifolia T. pannonicum A. amellus

Leaf blade
Cross-section area (mm2) 0.8 (10.7)c 1.4 (37.4)b 0.8 (19.5)c 1.3 (19.4)b 4.0 (19.8)a
Epidermis (%) 18.3 (12.3)a 21.3 (64.2)a 19.7 (8.5)a 16.7 (8.5)a 20.4 (12.9)a
Palisade tissue (%) 48.9 (6.8)b 50.1 (28.8)ab 55.5 (23.5)ab 59.3 (4.2)a 54.8 (16.7)ab
Spongy tissue (%) 28.5 (12.2)a 25.0 (42.5)ab 25.3 (32.1)ab 20.7 (9.9)b 21.3 (45.0)ab
Vascular bundles (%) (without bundle sheath) 4.1 (18.2)ab 3.5 (30.3)abc 4.2 (15.7)a 3.1 (10.4)c 3.4 (24.5)bc
Leaf blade thickness at the main vein (mm) 521 (5.9)b 366 (5.5)c 383 (7.8)c 537 (10.1)b 590 (13.1)a
Leaf blade thickness at 1/4 width (mm) 481 (12.2)a 251 (9.1)c 270 (5.7)bc 469 (14.5)a 305 (9.3)b
Main vein index 1.1 (9.6)c 1.4 (14.2)b 1.4 (7.2)b 1.1 (11.3)c 1.9 (12.6)a
Adaxial palisade tissue
Palisade tissue thickness (%) 33.8 (7.3)c 37.6 (8.3)ab 36.1 (6.6)bc 38.0 (7.0)ab 40.1 (10.6)a
Cross-section area of palisade cells (mm2) 976 (27.9)c 741 (147.1)c 613 (20.6)c 1823 (32.0)a 1256 (36.8)b
Palisade cells index 4.5 (23.4)a 4.1 (22.5 )b 3.7 (20.5)bc 3.6 (23.7)c 3.7 (23.9)bc
Abaxial palisade tissue
Palisade tissue thickness (%) 31.6 (14.9)a 28.5 (18.8)ab 28.1 (11.6)ab 31.8 (10.9)a 26.3 (12.0)b
Cross-section area of palisade cells (mm2) 1030 (26.7)b 512 (22.7)c 573 (19.1)c 1422 (26.2)a 1082 (24.1)b
Palisade cells index 4.5 (20.6)a 3.3 (28.1)b 3.3 (16.9)b 3.4 (26.2)b 3.1 (25.5)b

Notes: Different superscripts indicate statistically significant differences between analyzed species according to Duncan's test (p  0.05).

489
Figure 4. Light micrographs of leaf blade and stem cross sections of Galatella, Tripolium and Aster species. (A) cross section at the level of
1/4 of the leaf blade width of A. amellus showing individual homogeneous oil bodies in the palisade tissue cells. (B) details of the leaf blade
cross section of T. pannonicum illustrating loose arrangement of palisade cells and oil bodies with granular structure. Oil bodies stained red
with Sudan III. (C) cross section of the main vein of G. cana with a secretory duct above the phloem. (D) details of cross section of stem of
G. sedifolia showing secretory duct in the cortex above the phloem. Abbreviations: ob  oil bodies, sd  secretory duct.

pericyclic sclerenchyma fibers and highly developed xylem,
which formed a thick ring. The central part of the stem was
filled with the large pith parenchyma cells. Galatella cana,
G. sedifolia and A. amellus contained a large number of crys-
tals, which were predominantly prismatic, but also took
rhomboid and rod-like forms. In G. cana, G. sedifolia and
T. pannonicum, crystal druses were also observed.
The stem cross section area was significantly larger in
A. amellus than in the other species. Moreover, this species
was characterized by the highest percentage of parenchyma
pith and a high percentage of xylem. Among other statisti-
cally significant results were the highest percentage of col-
lenchyma in G. sedifolia and the lowest in T. pannonicum, as
well as the highest percentage of xylem in G. linosyris, and
the lowest in T. pannonicum (Table 5).
Peduncle anatomical characteristics
The peduncle cross-section was regularly rounded to polygo-
nal in shape, or with pronounced lobes (Fig. 6). Its anatomy
was very similar to the stem.
While the values of peduncle cross-section area were not
statistically different among Galatella species, its size, as well
as the percentages of pith parenchyma and epidermis, was
highest in A. amellus (Table 5). Tripolium pannonicum was
distinguished by the highest percentage of chlorenchyma and
cortex parenchyma, and the lowest share of central cylinder.
Statistical analysis of anatomical characters
Discriminant analysis of quantitative characters showed that
those pertaining to the leaf blade contributed the most to
the discrimination of the studied species, which, along with
the cross section areas of peduncle and percentage of epider-
mis, loaded on the first axis (Table 6). As shown in Fig. 7,
the species of the genus Galatella are clearly separated from
T. pannonicum and A. amellus by the second discriminant
axis, primarily due to the leaf blade epidermis characteristics.
In fact, all examined species were clearly separated on the
basis of their quantitative characters.
Qualitative characters and the states that characterized
individual species were subjected to correspondence analysis
(Table 7). Because peduncle anatomy was highly correlated
to the stem anatomy, its characters were not included in this
analysis, in order to avoid redundancy.
The Correspondence Analysis results showed that
Galatella species were clearly distinguished from T.
pannonicum along the first correspondent axis (44.38%)
(Fig. 8). The specific qualitative features that character-
ize T. pannonicum are sinuous anticlinal walls of the epi-
dermal cells, absence of wax and trichomes on the leaf
blade epidermis, loose arrangement of chlorenchyma cells
in all examined organs, fine-grained structure of oil bod-
ies, weakly expressed regularity in the arrangement of
xylem elements within the vascular bundles, and stem
collenchyma located between the ribs. Characters that
defined the second correspondent axis, and contributed
to the total separation by 34.32%, clearly distinguished
A. amellus from the remaining examined species. These
were the presence of upright, multicellular, non-glandular
trichomes on all examined organs, a prominent main leaf-
vein, the presence of crystal druses in spongy tissue cells
and tangentially elongated stem epidermal cells.

490
Figure 5. Light micrographs of stem cross sections of Galatella, Tripolium and Aster species. (A) stem cross sections of G. cana, (B)
G. sedifolia and (C) A. amellus showing collenchyma placed in the ribs and chlorenchyma located in the recesses, (D) stem cross
sections of T. pannonicum illustrating collenchyma placed in the invaginations and chlorenchyma located in the ribs, (E) detail of
stem cross section of A. amellus illustrating tangentially elongated epidermal cells and individual oil bodies in the chlorenchyma cells,
(F) detail of stem cross section of G. linosyris illustrating isodiametric epidermal cells, collenchyma, chlorenchyma and subepidermal
layer in the form of regularly arranged cells. Abbreviations: sl  subepidermal layer, c  collenchyma, ch  chlorenchyma , ec  epider-
mal cells, ob  oil bodies.

Table 5. Stem and peduncle anatomical characteristics of Aster, Galatella and Tripolium species. Data displayed as mean value (coefficient
of variation in percent).

G. linosyris G. cana G. sedifolia T. pannonicum A. amellus

Stem
Cross-section area (mm2) 3.9 (19.3)b 1.4 (40.2)c 1.6 (25.7)c 1.9 (19.8)c 5.0 (21.7)a
Collenchyma (%) 2.3 (33.7)b 2.8 (25.0)b 3.7 (22.9)a 0.7 (47.7)c 2.7 (16.0)b
Xylem (%) 30.8 (14.8)a 21.5 (16.2)b 19.2 (16.2)bc 16.8 (31.1)c 28.4 (16.6)a
Pith parenchyma (%) 13.5 (15.3)c 15.9 (13.6)bc 17.6 (10.5)b 17.1 (22.7)b 31.1 (18.4)a
Peduncle
Cross-section area (mm2) 0.6 (19.3)c 0.6 (31.4)c 0.6 (28.8)c 0.9 (33.4)b 1.5 (29.9)a
Epidermis (%) 11.4 (29.5)a 9.3 (15.7)b 9.9 (11.4)ab 10.4 (16.3)ab 7.3 (23.7)c
Chlorenchyma (%) 20.4 (47.5)b 18.6 (19.2)b 17.2 (15.7)b 34.0 (24.9)a 15.2 (42.4)b
Cortex (%) 41.5 (15.8)a 34.8 (11.7)b 34.8 (17.2)b 47.2 (21.8)a 28.8 (19.4)b
Central cylinder (%) 48.4 (17.5)b 60.4 (7.4)a 56.7 (8.3)a 31.2 (36.4)c 63.6 (15.2)a
Pith parenchyma (%) 5.9 (44.0)c 9.9 (24.2)b 7.7 (18.7)bc 8.1 (38.1)bc 23.3 (24.4)a

Notes: Different superscripts indicate statistically significant differences between analyzed species according to Duncan's test (p  0.05).

491
Figure 6. Light micrographs of peduncle cross sections of Galatella, Tripolium and Aster species. (A) polygonal shape of peduncle cross sec-
tion with collenchyma placed in the ribs and chlorenchyma located in the recesses in G. cana and (B) A. amellus, (C) peduncle cross section
illustrating chlorenchyma placed in the ribs and collenchyma located in the invaginations in T. pannonicum, (D) cross section of peduncle
of G. linosyris showing secretory duct above the phloem. Abbreviations: c  collenchyma, ch  chlorenchyma, sd  secretory duct.

Taxonomic key for examined species and genera on Discussion

the basis of the leaf, stem and peduncle anatomical
characteristics:
1. Anticlinal walls of leaf blade epidermal cells straight;
epicuticular wax and whip-like non-glandular trichomes
(type I) present; stem cross section with pronounced ribs
……...……………………………………………… 2
– Anticlinal walls of leaf blade epidermal cells sinuous;
epicuticular wax and whip-like non-glandular trichomes
(type I) absent; stem cross section without pronounced
ribs ………………………………………………… 4
2. Glandular, bi-seriate trichomes present on leaf, stem and
peduncle; subepidermal layer in stem absent ............... 3
– Glandular, bi-seriate trichomes absent on leaf, stem
and peduncle; subepidermal layer in stem present
G. linosyris
3. Number of whip-like non-glandular trichomes per mm2
greater than 100 ..…………………………… G. cana
– Number of whip-like non-glandular trichomes per
mm2 less than 70 …….......……………… G. sedifolia
4. Upright non-glandular trichomes (type II) present;
arrangement of xylem elements in the main vein regular;
oil bodies homogeneous; stem and peduncle collenchyma
located in the ribs; arrangement of chlorenchyma cells in
leaf, stem and peduncle compact.…………… A. amellus
– Upright non-glandular trichomes (type II) absent;
arrangement of xylem elements in the main vein irregular;
oil bodies granular; stem and peduncle collenchyma located
in the recesses; arrangement of chlorenchyma cells in leaf,
stem and peduncle loose …….………… T. pannonicum
For taxonomy and determination of many genera and
species, leaf blade features have proved to be very significant
(Cilliers and Kruger 1993, Aliero et  al. 2006, Milan et  al.
2006, Firetti-Leggieri et al. 2013), especially characteristics
of the leaf blade epidermis (Yang and Lin 2005, Adedeji and
Jewoola 2008, Zorić et al. 2009). The data obtained in this
study also indicated that the greatest differences between
the examined species were in the anatomical and micro-
morphological characteristics of the leaf blades. Certain
characteristics of the leaf blade epidermis, such as the outline
of the anticlinal walls and the shape of the external periclinal
wall of the epidermal cells, may be caused by environmental
factors. However, according to Barthlott (1981), the micro-
morphological characteristics of the leaf blade epidermis,
such as the sinuous appearance of the anticlinal walls of the
epidermal cells, as well as the cuticle ornamentation, are
taxonomically significant and can thus be used for the
separation of taxa at the genus or even species level. In
the species of Galatella, the anticlinal walls of the adaxial
and abaxial epidermal cells are straight to slightly sinuous,
while T. pannonicum and A. amellus exhibit a more pro-
nounced plication of side walls. According to Pătrut et  al.
(2005), ribbed thickenings of the cuticle, which are observed
on the epidermis surface of all the examined species, are signs
of adaptation, aimed at reducing evapotranspiration.
In addition to the cuticle ornamentation, the composi-
tion of the epicuticular wax may also serve as an important
taxonomic features (Barthlott 1981). Irregularly shaped,

492
Table 6. Quantitative characters of leaf blade, stem and peduncle and the loads of the first three axes of the discriminant analysis. Presented
characters showed statistical significance in the explanation of differences between the species (Bold values are  0.700; adea  adaxial
epidermis, abeb  abaxial epidermis).

Root 1 Root 2 Root 3

Leaf blade Cross-section area 1.1225 1.24437 0.22652
Thickness of the leaf blade at 1/4 width 2.8775 0.00167 0.27240
No. of stomata/mm2 adea 0.9222 0.73715 0.16659
Stomata width ade 1.5777 1.74670 0.26715
Stomata length ade 0.9789 1.65190 0.52115
Stomatal index ade 0.5579 1.52609 0.72660
No. of stomata/mm2 abeb 0.8566 0.13777 0.43737
Stomata width abe 12.5217 5.03439 3.48636
Stomata length abe 12.8357 6.04285 4.20579
Stomatal index abe 11.9455 5.24144 2.90476
No. of non-glandular trichomes/mm2 abe 1.6424 1.47996 0.02330
Palisade cells index ade 1.2732 0.13569 0.36300
Ade thickness (%) 1.5864 0.62784 0.44225
Cuticle thickness ade (%) 0.0560 0.73649 0.16251
Palisade tissue thickness ade (%) 0.8516 0.44330 0.51815
Abe thickness (%) 3.7337 0.35900 1.09813
Cuticle thickness abe (%) 0.1798 0.51084 0.87189
Spongy tissue thickness (%) 1.2148 0.47704 0.33695
Stem Cross-section area 0.4517 0.55362 1.29390
Peduncle Cross-section area 1.0206 0.15467 0.24682
Epidermis (%) 1.1349 0.42300 0.18525
Chlorenchyma (%) 0.2946 0.12125 0.99951
Central cylinder (%) 0.6234 0.72699 0.54226
Cumulative percentages of the vectors 0.5109 0.79512 0.93580

more or less densely distributed wax flakes of different sizes
were observed on the leaf blade epidermis of Galatella spe-
cies, but not in the other examined genera. According to
Baker (1982), although wax type is a genetically determined
characteristic, its quantity and distribution can be affected
by environmental factors. In line with this argument, we
consider the presence of wax on leaf blade surfaces of Gala-
tella species as one of the features separating them from the
remaining species examined in this work.
Indumentum characteristics, composition, distribution
and density of trichomes, as well as stomata characteristics,
represent particularly informative features in the taxonomic
studies of some genera (Selvi and Bigazzi 2001, Aliero et al.
2006, Zorić et al. 2009). The results obtained in the present
study indicated that micro-morphological characteristics of
the leaf blades epidermis may be useful diagnostic charac-
teristics of the examined species. Whip-like non-glandular
trichomes were observed in all three species of the genus

Figure 7. Results of a discriminant analysis showing the position of centroids of Galatella, Tripolium and Aster species in the space of the
first two discriminant axes, based on the quantitative anatomical characters.

493
Table 7. Selected qualitative characters of leaf blade and stem of of Aster, Galatella and Tripolium species (  represents presence of
character.   represents absence of character; lin  G. linosyris. can  G. cana. sed  G. sedifolia. pan  T. pannonicum. ame  A. amellus).

lin can sed pan ame

Leaf blade
Isolateral leaf blade structure     
Anticlinal walls of epidermal cells Straight     
Sinuous     
Epicuticular wax     
Stomata type anomocytic     
Non-glandular trichomes Whip-like     
Upright     
Glandular trichomes     
Palisade tissue Compact     
Loose     
Crystal druses in spongy tissue cells     
Oil bodies Homogeneous     
Granular     
Bundle sheath extending to epidermis     
Xylem elements arrangement in the Regular     
main vein Irregular     
Secretory ducts in the leaf blade     
Prominent main vein     
Stem
Shape Polygonal     
Ribs not pronounced     
Epidermal cells Isodiametric     
Tangentially elongated     
Subepidermal layer     
Collenchyma In the ribs     
In the recesses     
Chlorenchyma Compact     
Loose     
Secretory ducts in the cortex     
Solitary crystals in the pith parenchyma     
Crystal druses in the pith parenchyma     

Figure 8. Results of a correspondance analysis showing the positions of centroids of Galatella, Tripolium and Aster species in the space of
the first two axes, based on qualitative anatomical characters.

494
Galatella, and were most abundant in G. cana. While glan-
dular trichomes were very rare or absent in G. linosyris, they
were more numerous in G. cana, and were present in G. sedi-
folia in a large number, especially on the adaxial side, giving
the leaf blade a glandular, dotted appearance. Aster amellus
was characterized possessing only by sharp, multi-cellular,
uni-seriate non-glandular trichomes, whilst the leaves of T.
pannonicum were glabrous.
According to Nesom (2008), G. linosyris has previously
alternatively been included in the genera Chrysocoma
(C. linosyris L.), Crinitaria (C. linosyris (L.) Less.), Linosyris
(L. vulgaris DC.) and Crinitina (C. linosyris (L.) Soják).
Our results show that G. linosyris can be distinguished
from the other two Galatella species by the absence of
glandular trichomes, presence of stem subepidermal layer
and absence of crystals in stem pith parenchyma. However,
these differences proved not to be statistically significant,
and based on anatomical data the three species formed
a coherent group indicating that all of them should be
treated as Galatella species.
Tripolium pannonicum is a typical halophyte (Nesom
1994b, Anderberg et al. 2007). Anatomical features, such
as epidermis without protective structures on its surface,
smaller number of stomata per mm2 of leaf blade surface
and stomata unprotected and leveled with the epidermis,
may be associated with its mesomorphic structure, whilst
large epidermal and palisade tissue cells indicate the halo-
morphic structure of this species. The halophyte cells are
voluminous, due to the high salt concentration in the soil
where this species grows (Dickison 2000). As salts inhibit
cell division, without limiting their expansion, organs
become fleshy.
Secretory structures are of particular taxonomic impor-
tance and their distribution has major diagnostic value. Rep-
resentatives of Asteraceae are characterized by two types of
secreting systems – glandular trichomes and secretory ducts.
While the former appear at the surface of organs, the latter
form within the plant body, and are located in the stem cortex
and leaf blade mesophyll, often as a part of the bundle sheath
(Bartoli et al. 2011). In the leaf blade of all analyzed species,
presence of secretory ducts was noted above the phloem of
the main vein and in some larger lateral bundles. In Aster-
aceae, essential oils, lipids, resins, sesquiterpene lactones,
alkaloids, pectin-like substances, tannins and flavonoids are
the main secretion products of ducts and glands (Bartoli
et al. 2011). While large oil bodies were easily observed in
all examined species, their fine-grained structure clearly dis-
tinguishes T. pannonicum from the other analyzed species.
According to Lersten et al. (2006), these oil bodies typically
occur individually, and almost always within palisade cells,
which is in accordance with our results.
Stem anatomy of the examined species conforms to the
description of the stem structure provided by Metcalfe and
Chalk (1957) for the family Asteraceae. A subepidermal
layer in the form of a single row of regularly arranged cells
was observed only in G. linosyris. The formation of the
subepidermal layer is a genetically determined characteris-
tic and is independent of the environment (Moraes et al.
2011). The cortex of T. pannonicum is better developed
than that of the other examined species. This is another
halophyte characteristic that provides certain protection in
the early stages of vascular tissue development (Dickison
2000). Grigore and Toma (2006) posited that the presence
of aerenchyma is a characteristic of halophytic species,
which explains the appearance of loose tissues (palisade in
the leaf blade and chlorenchyma in the stem and pedun-
cle) in T. pannonicum. Anderberg et  al. (2007) reported
that, in several genera of Astereae, crystals occurr in the
stem parenchyma cells, as confirmed by our results for G.
sedifolia, G. cana and A. amellus. The presence of crystals
is a common characteristic of higher plants, linked with
the physical protection, removal of oxalate from metabolic
processes, storage of calcium and regulation of light during
photosynthesis in plants found in shaded habitats (Moraes
et al. 2011). Nevertheless, in taxonomy, the presence, dis-
tribution and type of crystals can provide important diag-
nostic characters. Druses in the spongy tissue cells were
noted only in A. amellus, while their presence in the stem
parenchyma core was confirmed in G. cana, G. sedifolia
and T. pannonicum.
The anatomical structure of the peduncle of the analyzed
species has not been previously described in the literature.
However, as found by us, its anatomy corresponds to the
stem anatomy and its characteristics could be taxonomi-
cally significant, as they remain relatively constant under the
influence of environmental factors.
Discriminant analysis revealed significant differentiation
among the studied species, allowing their clear definition
and separation on the basis of the quantitative characters.
The correspondent analysis of qualitative characters and
their states also indicated that the analyzed species were
clearly delimited in the area defined by the first and the
second correspondent axes, and formed three separate
groups. The largest, compact group comprised three Gala-
tella species, while the centroids of T. pannonicum and A.
amellus were clearly detached and distant from each other.
Straight to slightly sinuous epidermal anticlinal cell walls,
presence of epicuticular wax and whip-like non-glandular
and bi-seriate glandular trichomes could be singled out as
the characters that separates Galatella from Tripolium and
Aster, which is in accordance with Barthlott’s (1981) list
of characters that may serve to characterize genera. Even
though Nesom (1994b) and Li et  al. (2012) emphasized
the close relationship between Galatella and Tripolium our
results indicate that the anatomical and micro-morpholog-
ical characteristics of representatives of these two genera
were not significantly related and as far as anatomy and
micro-morphology are concernes they are best considered
as different genera.
The results of our anatomical and micro-morphological
analyses of leaf blades, stem and peduncle highlighted signif-
icant differences among the examined species. These results
support the separation of species, all of which were previously
classified in the same genus Aster, into three separate gen-
era. Qualitative and quantitative anatomical characteristics,
together with previously defined morphological and genetic
characters, support and complement this classification, and
we believe that a more comprehensive anatomical analysis of
further representatives of these three genera could contribute
to a better understanding of their taxonomic relationships.
495
Conclusions
The presented results on anatomical and micro-morpho-
logical characteristics of G. linosyris, G. cana, G. sedifolia,
T. pannonicum and A. amellus, all of which were previously
referred to the broadly circumscribed genus Aster, revealed
a number of qualitative and quantitative characters of taxo-
nomic importance. Based on these characters, the examined
species were clearly separated into three well-defined groups
that corresponded to three genera: Aster, Galatella and
Tripolium. Galatella species could be singled out from
Tripolium and Aster by straight to slightly sinuous epider-
mal anticlinal cell walls, presence of epicuticular wax and
whip-like non-glandular and bi-seriate glandular trichomes.
Tripolium pannonicum is characterized by sinuous anticlinal
walls of epidermal cells, absence of leaf wax and trichomes,
loose arrangement of chlorenchyma cells, fine-grained struc-
ture of oil bodies, weakly expressed regularity in the arrange-
ment of xylem elements, and stem collenchyma located
between the ribs. Anatomical data complemented the extant
knowledge on the biological properties of the analyzed
species, and followed and supported their newly defined
taxonomical status, previously based on morphological
and genetic data only. We thus conclude that separation of
Galatella and Tripolium from the genus Aster is reasonable
from an anatomical point of view.
Acknowledgements – The authors would like to thank Mr Milos
Bokorov from Univ. Center for Electron Microscopy, Novi Sad, for
his technical assistance and SEM microscopy. This work was finan-
cially supported by the Ministry of Education, Science and Techno-
logical Development, Republic of Serbia, grant no. 173002.
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