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Pollen morphology and localization of Ni in some Ni-hyperaccumulator taxa of Alyssum L. (Brassicaceae)

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Pollen morphology of seven Alyssum L. taxa growing on serpentine soils in different places in the European Mediterranean macrobioclimate territory were studied, described and compared. Cluster analysis was performed to show similarity between species and their populations. The shape of the pollen grains varies among the species and among the grains within the same anther. The pollen grains are 3-colpate, prolate, with long and narrow colpi reaching the poles. The ornamentation of the exine varies from micro-reticulate to reticulate between the species. Pollen sterility/fertility was also calculated. The highest percentage of sterile pollen (73.76%) was calculated for Alyssum murale subsp. murale and the lowest (9.54%) for A. bertolonii subsp. bertolonii. All species are representatives of sect. Odontarrhena (C.A.Meyer) Koch well known as Ni-hyperaccumulators. Nickel and other elements present in pollen and stamen were studied by inductively coupled plasma-mass spectrometry (ICP-MS). The stamen parts of all species were micromorphologically analyzed by scanning electron microscopy (SEM) coupled to an Energy-Dispersive X-Ray Probe (EDX). Accumulation of Ni was detected in the stamens of all studied species and rarely in the pollen grains. The distribution patterns of Ni were similar among species examined.
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Pollen morphology and localization of Ni in some Ni-
hyperaccumulator taxa of Alyssum L. (Brassicaceae)
D. Pavlovaa, V. De La Fuenteb, D. Sánchez-Matac & L. Rufob
a Department of Botany, Faculty of Biology, Sofia University, Bulgaria
b Department of Botany, Faculty of Sciences, Autonomous University of Madrid, Cantoblanco
(Madrid), Spain
c Department of Plant Biology II, Faculty of Pharmacy, Complutense University, Madrid,
Spain
Accepted author version posted online: 19 Nov 2014.Published online: 12 Dec 2014.
To cite this article: D. Pavlova, V. De La Fuente, D. Sánchez-Mata & L. Rufo (2014): Pollen morphology and localization of Ni
in some Ni-hyperaccumulator taxa of Alyssum L. (Brassicaceae), Plant Biosystems - An International Journal Dealing with all
Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, DOI: 10.1080/11263504.2014.989284
To link to this article: http://dx.doi.org/10.1080/11263504.2014.989284
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ORIGINAL ARTICLE
Pollen morphology and localization of Ni in some Ni-hyperaccumulator
taxa of Alyssum L. (Brassicaceae)
D. PAVLOVA
1
, V. DE LA FUENTE
2,
*,D.SA
´NCHEZ-MATA
3,
**, & L. RUFO
2,
1
Department of Botany, Faculty of Biology, Sofia University, Bulgaria;
2
Department of Botany, Faculty of Sciences,
Autonomous University of Madrid, Cantoblanco (Madrid), Spain and
3
Department of Plant Biology II, Faculty of Pharmacy,
Complutense University, Madrid, Spain
Abstract
Pollen morphology of seven Alyssum L. taxa growing on serpentine soils in different places in the European Mediterranean
macrobioclimate territory were studied, described and compared. Cluster analysis was performed to show similarity between
species and their populations. The shape of the pollen grains varies among the species and among the grains within the same
anther. The pollen grains are 3-colpate, prolate, with long and narrow colpi reaching the poles. The ornamentation of the
exine varies from micro-reticulate to reticulate between the species. Pollen sterility/fertility was also calculated. The highest
percentage of sterile pollen (73.76%) was calculated for Alyssum murale subsp. murale and the lowest (9.54%) for A. bertolonii
subsp. bertolonii. All species are representatives of sect. Odontarrhena (C.A. Meyer) Koch well known as Ni-
hyperaccumulators. Nickel and other elements present in pollen and stamen were studied by inductively coupled plasma-
mass spectrometry. The stamen parts of all species were micromorphologically analyzed by scanning electron microscopy
coupled to an energy-dispersive X-ray probe. Accumulation of Ni was detected in the stamens of all studied species and rarely
in the pollen grains. The distribution patterns of Ni were similar among the species examined.
Keywords: Nickel, Mediterranean, serpentine, stamen, sterile pollen
Introduction
In temperate areas of the world (especially Medi-
terranean Europe and Turkey) the family
Brassicaceae contains the largest number of Ni-
hyperaccumulators (Reeves & Adiguzel 2008), most
of them restricted to serpentine substrate. High
metal tolerance is not homogeneously distributed
over taxonomic groups and shows differences not
only within a taxonomic group, but even among
populations of the same species (van der Ent et al.
2013). Among the genera with the highest number of
species accumulating Ni is Alyssum L. About 48 taxa,
all from sect. Odontarrhena (C.A. Meyer) Koch, are
known to be Ni-hyperaccumulators, with .0.1% in
dry leaf tissue (Brooks et al. 1979) such as all studied
species. The capacity of some plants to bind metals is
a form of tolerating and storing heavy metals whose
physiological function is not well understood. The
process of hyperaccumulation seems to depend on
the characteristic patterns of metal translocation and
distribution, while their concomitant tolerance for
excess metal relies on different detoxication mech-
anisms (Serregin & Kozhevnikova 2009).
Nickel as essential trace element was suspected to
have an important metabolitic role in plants. The
toxicity of nickel has been attributed to a negative
impact on inactivation of biomolecules, changes in
permeability of the cell membrane, reactions of thiol
groups with cations and damage to photosynthesis
apparatus (Barket et al. 2009). In areas rich in nickel,
various abnormalities of vegetative growth such as
necrosis and chlorosis of leaves, reduction of biomass
were also found (Yusuf et al. 2011). Nickel and other
heavy metals can also affect plant reproduction because
of anomalies in gamete development, embryogenesis,
seed production (Mohsenzadeh et al. 2011) and polar
cell growth (Breygina et al. 2012).
Information regarding the presence of Ni in
generative plant organs is not frequently found
(Przybylowicz et al. 1995; Psaras & Manetas 2001;
Zhang et al. 2014). There are few studies related to
q2014 Societa
`Botanica Italiana
Correspondence: D. Pavlova, Department of Botany, Faculty of Biology, University of Sofia, 8 Dragan Tzankov Boulevard, 1164 Sofia, Bulgaria.
Email: pavlova@biofac.uni-sofia.bg
Plant Biosystems, 2014
http://dx.doi.org/10.1080/11263504.2014.989284
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heavy metal effect on plant sex organs and cells mostly
reporting the inhibitory effect of the metals in pollen
grains (Sawidis & Reiss 1995; Sawidis 1997; Sawidis
et al. in press; Tuna et al. 2002; Yousefi et al. 2011).
In relation tothe effect of nickel is thequestion of its
distribution within the plant body. Even a tiny plant
part such as the pollen grain can show a high variability
in species- and metal-specific concentrations of nickel
(Ernst 2006). Studies on nickel localization in
vegetative and reproductive organs of some hyper-
accumulator plants started to be published several
years ago including Berkheya coddii Roessler (Compo-
sitae) (Migula et al. 2007); Senecio coronatus (Thunb.)
Harv. (Compositae) (Przybylowicz et al. 1995); Senecio
anomalochrous Hilliard (Mesjasz-Przybylowicz et al.
2001); Sebertia acuminata Pierre ex Baill. (Sapotaceae)
(Perrier et al. 2004); Alyssum bertolonii Desf. and
A. serpyllifolium Desf. s.l. (Brassicaceae) (Marmiroli
et al. 2004; de la Fuente et al. 2007); Euphorbia helenae
Urb., Leucocroton linearifolius Britton, L. flavicans Mu
¨ll.
Arg., Phyllanthus orbicularis Kunth, P. discolor Poepp. ex
Spreng. and P. £pallidus (Euphorbiaceae) (Berazain
et al. 2007).
There is not enough information about pollen
sterility/fertility and pollen morphology of the Ni-
hyperaccumulators, including Alyssum species. Infor-
mation about pollen morphology of Alyssum is
available from some regional pollen morphological
studies and in several surveys of the family (Erdtman
1966; Inceoglu & Karamustafa 1977; Rollins &
Banerjee 1979;Dı
´ez & Ferna
´ndez 1987; Anchev &
Deneva 1997; Perveen et al. 2004). Pollen of Alyssum
species is ascribed to Hornungia-type by Faegri and
Iversen (1989). The basis for such conclusion is the 3-
zonocolpate pollen and the reticulate ornamentation
of the exine. This primary description of the pollen
morphology was subsequently complemented by
Moore et al. (1991), Reille (1992) and Beug (2004).
The main objective of this work was to gather
information on pollen morphology and localization
of Ni in stamens and pollen grains of eight Ni-
hyperaccumulator Alyssum taxa: A. murale Waldst. &
Kit. subsp. murale,A. murale subsp. pichleri (Velen.)
Stoj. & Stef., A. serpyllifolium Desf. subsp. lusitanicum
Dudley & P. Silva (A. pintodasilvae Dudley),
A. serpyllifolium Desf. subsp. malacitanum Rivas
Goday, A. markgrafii O.E. Schulz, A. bertolonii
Desv. subsp. bertolonii,A. bertolonii Desv.
subsp. scutarinum Nya
´r. and A. heldreichii Hausskn.,
all growing on serpentines in Mediterranean Europe.
Material and methods
Plant sampling
Material for study (stamens, pollen grains) was
obtained from dry plants collected from natural
populations of the species. The edaphic properties of
their substrate correspond to typical characteristics of
ultramafic soils (basic pH, high concentrations of Mg ,
Ni and Fe, low quantities of Ca) (de la Fuente et al.
2007; Bani et al. 2010,2013; Tzonev et al. 2013).
The plant taxa studied, their localities and
characteristics of the sampling locations are pre-
sented in Supplementary material (Table SI).
Voucher specimens have been deposited in the
Herbarium of Sofia University, Bulgaria (SO),
Aristotle University at Thessaloniki, Greece (TAU),
the Herbarium of Tirana University, Albania, and
the Herbarium of the Faculty of Pharmacy,
Complutense University at Madrid, Spain (MAF).
Accepted taxonomy and nomenclature follow the
proposals of Euro þMed PlantBase (Marhold 2011)
except for the complex Alyssum ser pyllifolium s.l.
Microscopic analysis
The measurements and pollen descriptions are made
on acetolyzed pollen prepared in the standard way
(Erdtman 1966). Slides for light microscopy were
prepared by mounting the pollen in glycerol jelly, and
observations were made with OLYMPUS BX-51
microscope under E40, 0.65 and oil immersion
(E100, 1.25), using 10 £eye piece. Thirty measure-
ments of pollen grains were made by each character
for each taxon. The results of the measurements of
the polar diameter (P), equatorial diameter (E),
colpus length (L), mesocolpium (M), apocolpium
(A) and P/Eratio are shown in Table I. For scanning
electron microscopy (SEM), pollen grains suspended
in a drop of 95% ethanol were coated with gold and
examined with Jeol microscope JSM-5510.
To estimate percentage pollen viability anthers of
recently opened flowers were squashed, the pollen
stained with Alexander stain (Alexander 1969). The
viable (red) and inviable (green) pollen grains were
counted and the mean values for 10 anthers from 10
different flowers and species are presented (Sup-
plementary material, Figure S1). The pollen termi-
nology in general follows Faegri and Iversen (1989)
and Punt et al. (2007).
A SEM coupled to an energy-dispersive X-ray
probe (EDX) was used to micromorphologically
analyze stamens (filaments, anthers, anther’s connec-
tive) and pollen grains of Alyssum taxa. Sample
preparation followed the method of Psaras et al.
(2000) and Xiang et al. (2013). For each type of
tissue, four to five samples were analyzed from each
species. The samples were mounted onto conductive
graphite stubs and sputters and gold-coated in a BIO-
RAD SC 502 (Hertfordshire, UK) apparatus for
electrical conductivity and to prevent charging under
the electron beam. The samples were examined with a
Hitachi S-3000N (Tokyo, Japan) SEM using an
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acceleration voltage of 20 kV and a working distance
for analysis of 15 mm. The analysis was performed at
room temperature. The qualitative element compo-
sition of the samples was determined by energy-
dispersive X-ray microanalysis using an INCAx-sight
with an SiLi Detector (Oxford, UK), with a
detection limit of 10% of the main element. This
instrument is able to detect the lighter elements, C,
O and N, and the numerical data generated are
referenced as default to the higher peak obtained in
each spectrum, which in our case generally corre-
sponded to C.
Data analysis
Univariate and multivariate statistical procedures were
applied to examine variation among the populations on
the basis of pollen-morphological measurements.
Cluster analysis using Euclidean distance and
unweighted pair group means average was used as
the computational criteria to determine the similarities
between studied taxa (Supplementary material,
Figure S2). Mean values of the pollen features for the
species with more than population were used. Means,
standard deviations and standard errors were calcu-
lated for EDX analyses. Data were log-transformed
after being tested for normality with the Shapiro– Wilk
test ( p.0.05) and for homogeneity of variance with
the Levene test ( p.0.05). Means were compared by
one-way analysis of variance. Bonferroni corrections
between means were calculated only if an F-test was
significant at the 0.05 level of probability. All statistics
were performed using StatSoft – Statistica 7 program.
Results
Pollen morphology
The pollen grains are 3-zonocolpate, prolate and
subprolate. The ectocolpi are straight, very narrow,
pointing at the poles. The colpus margin is uneven, the
colpus membrane usually is invisible covered by the
margins of the colpus. The sexine is thicker than the
nexine. The columellae layer is composed of high,
straight, unbranched rod-shaped columellae. The
tectum is interrupted, twice thinner than the columel-
lae layer. The ornamentation varies from reticulate
(A. serpyllifolium s.l., A. heldreichii,A. markgrafii,
A. murale) with lumina different in shape to a fine
reticulate (A. bertolonii s.l.). In equatorial view, the
pollen grains are elongated, elliptic, in polar view they
are circular, triangular-obtuse.
Taking into account the dimensions of the pollen
grains, their shape in equatorial and polar view, two
pollen types and two subtypes could be recognized:
Table I. Taxa examined with measurements (mm) with mean (in brackets) and ranges of the pollen features: polar (P) and equatorial (E) axes, length of the colpus (L), mesocolpium (M) apocolpium
(A) and shape index P/E.
Sampled plants Site code PELMAP/E
A. murale subsp. murale 1 19.5–25.5 (22.26) 15–19.5 (16.98) 13.5–24 (18.9) 7.5– 15 (10.14) 3.0–6.0 (4.11) 1.31
2 22.5–30 (24.48) 18–21 (18.93) 18–25.5 (20.82) 9–13.5 (11.4) 3.0–4.5 (3.54) 1.3
3 15– 24 (20.91) 15–21 (16.74) 13.5–21 (18) 7.5– 12 (10.74) 3.0–4.5 (3.36) 1.25
A. murale subsp. pichleri 4 13.5– 24 (20.19) 13.5– 21 (17.61) 13.5– 19.5 (17.1) 10.5–16.5 (12.12) 3.0–7.5 (5.58) 1.15
A. serpyllifolium subsp. malacitanum 5 21– 27 (24.42) 15– 19.5 (17.79) 18– 25.5 (21.78) 7.5–13.5 (10.95) 3.0–6.0 (3.9) 1.38
A. serpyllifolium subsp. lusitanicum 6 19.5– 30 (25.02) 13.5– 19.5 (17.1) 15 22.5 (19.02) 7.5– 13.5 (10.08) 3.0– 6.0 (4.32) 1.47
A. bertolonii subsp. betrolonii 7 18–25.5 (21.96) 16.5–21 (17.7) 15 –22.5 (17.88) 7.5– 13.5 (10.38) 3.0– 6.0 (4.8) 1.24
A. bertolonii subsp. subsp. scutarinum 8 18– 25.5 (22.02) 18– 22.5 (19.32) 15– 24 (19.26) 9 13.5 (10.98) 4.5– 7.5 (5.4) 1.14
A. markgrafii 9 21– 30 (25.14) 15–21 (18.6) 19.5 27(23.34) 7.5–13.5 (10.44) 3.0–4.5 (3.12) 1.35
10 21– 30 (25.98) 13.5–21 (17.88) 18–27 (22.98) 7.5–15 (10.83) 3.0–4.5 (4.08) 1.45
A. heldreichii 11 18–19.5 (18.96) 15–19.5 (16.32) 15–18 (17.1) 7.5–12 (9.42) 3.0–6.0 (3.84) 1.16
Note: Site code corresponds to numbers in Table SI from the Supplementary material.
Pollen and localization of Ni in Alyssum 3
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Pollen type I (Figure 1(A)(C))
The polar diameter is less than 20 mm, the
ornamentation is reticulate.
The pollen grains are sub-prolate (A. heldreichii),
P/E¼1.16, dimensions P£E¼1819.5 £15
19.5 mm. In polar view, the pollen grains are
triangular-obtuse; in equatorial view, elongated to
elliptic. The ectocolpi are long, straight and narrow,
L¼15 –18 mm. The exine is 0.75 mm thick in the
mesocolpium. The ornamentation is reticulate with
difference in shape and size lumina. The ornamenta-
tion is the same in the apocolpium and mesocolpium.
Pollen type II (Figure 1(D)(I);Figure 2)
The polar diameter is more than 20 mm, the
ornamentation is reticulate to fine reticulate.
Subtype IIa (Figure 1(D) (I);Figure 2(A) (C))
The pollen grains are sub-prolate (A. murale
subsp. murale,A. murale subsp. pichleri,A. bertolonii
subsp. bertolonii and A. bertolonii subsp. scutarinum).
P/E¼1.141.31, dimensions P£E¼13.5 30
£13.5 –22.5 mm. In polar view, the pollen grains
are triangular-obtuse. The ectocolpi are long,
shallow, the colpus membrane is invisible.
L¼17.1 –20.82 mm. The exine is 1 –1.25 mm thick
in the mesocolpium. The columellae layer is 0.6 mm
thick, composed of long, straight, unbranched and
different in height columellae. The tectum is 0.3
0.5 mm thick, about twice thinner than the columel-
lae layer in mesocolpium. The ornamentation is
reticulate, the largest in size lumina are observed in
the intercolpia (1 –1.5 mm).
Subtype IIb (Figure 2(D)(I))
The pollen grains are prolate (A. markgrafii,
A. serpyllifolium s.l.). P/E¼1.35 1.47. Large in
size pollen grains with P£E¼2130 £13.5
21 mm. In polar view, the pollen grains are
triangular-obtuse; in equatorial view, elliptical
elongated to rectangular-obtuse. The ectocolpi are
long, narrow and almost closed. L¼17.88
21.78 mm. The exine is 11.5 mm thick in the
mesocolpium. The columellae are straight, different
in height. The tectum is uneven, 0.5 mm thick, up to
two times thinner than the columellae layer. The
ornamentation is reticulate, with lumina different in
size and shape, without perforations around the
apertures. The diameter of the largest lumina is
variable (1 –1.5 mm).
Figure 1. LO and SEM micrographs of pollen grains of Alyssum heldreichii,A. murale subsp. murale and A. murale subsp. pichleri. (A)
A. heldreichii LO micrographs of a colpus; (B) SEM section of the exine; (C) ornamentation of a pollen; (D) A. murale subsp. murale
equatorial view (LO); (E) mesocolpium (SEM); (F) ornamentation of a pollen; (G) A. murale subsp. pichleri LO micrographs of a colpus; (H)
ornamentation of a pollen; (I) ornamentation in the apocolpium.
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The dendrograms obtained by the hierarchical
cluster analysis (Supplementary material, Figure S2)
reveals A. heldreichii as an outlier (cluster A) with the
smallest pollen grains. The second group (cluster B)
includes all other species, separated in two subgr oups.
The species A. murale subsp. pichleri is related to
A. bertolonii subsp. scutarinum by showing similar
values for the polar diameter unlike A. serpyllifolium
subsp. malacitanum and A. markgrafii.Highest
similarity between A. murale subsp. murale and
A. bertolonii subsp. bertolonii (linkage distance 2.1)
and A. murale subsp. pichleri and A. bertolonii
subsp. bertolonii (linkage distance 2.3) was also
shown. Statistical analysis using Euclidean distance
separates these taxa on the bases of differences in
pollen shape (P/Eindex).
Pollen viability
The percentage of fertile/sterile pollen in all studied
populations is also presented (Supplementary
material, Figure S1). The species Alyssum murale
subsp. pichleri has 6.42% sterile pollen, the lowest from
all studied populations and a little above 5%, a limit
considered as a normal abortion (Mic
ˇieta & Murin
1996). The highest percentage of sterile pollen
(38.65%) was calculated for A. markgrafii from Serbia,
followed by A. serpyllifolium s.l., A. heldreichii,etc.
Ni-localization
Several elements were detected: C, O, Ca, Mg, Ni,
K, S, Cl and P (Figure 3). Our study was focused on
the main elements (Ca, Mg and Ni) related also with
the serpentine habitat (Table II).
Calcium was nearly always present in both
stamens and pollen. It was found in sample ranks
(from 0.13% to 11.92% in the stamens and from
0.09% to 7.95% in pollen). The mean Ca
concentrations of stamens were significantly higher
than pollen concentrations in A. markgrafii from
Serbia, A. murale subsp. murale from Chernichevo,
A. heldreichii from Greece and A. bertolonii
subsp. bertolonii. No differences were found in the
rest of the studied taxa. Regarding the same species
from different localities, significant differences were
found between the mean Ca concentration of
stamens of A. murale subsp. murale from Fetler and
G. Kamenjane sites. Also, Ca concentration of pollen
from both subspecies of A. bertolonii is significant.
As for the whole set of species, significant differences
were detected for the mean Ca concentrations of
stamens (A. murale subsp. pichleri ,A. markgrafii,
A. murale subsp. murale,A. heldreichii) and pollen
(A. bertolonii subsp. scutarinum ,A. serpyllifolium
subsp. malacitanum,A. murale subsp.murale,
A. markgrafii).
Figure 2. LO and SEM micrographs of pollen grains of Alyssum bertolonii s.l., A. markgrafii and A. serpyllifolium s.l. (A) A. bertolonii s.l.
mesocolpium (SEM); (B) SEM section of the exine; (C) ornamentation of a pollen; (D) A. markgrafii equatorial view (LO); (E)
ornamentation of a pollen (SEM); (F) polar view and ornamentation; (G) A. serpyllifolium s.l. equatorial view (LO); (H) ornamentation of a
pollen; (I) ornamentation and colpus.
Pollen and localization of Ni in Alyssum 5
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Figure 3. SEM micrographs and energy-dispersive X-ray microanalysis. (A) A. heldreichii pollen; (B) A. serpyllifolium s.l. – pollen;
(C) A. markgrafii pollen; (D) A. murale subsp. murale pollen; (E) A. serpyllifolium s.l. – filament.
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Occasionally, accumulations of Ca were observed
in pollen and stamens (Figure 3(B),(E)), some of
them in a crystallized form (Figure 3(C)). The
concentrations of Ca varied from 4% to 10%.
Magnesium was found always in lower concen-
trations than Ca, and it was not always detected. The
measurements of Mg varied among 0.08% and
6.06% in stamens and from 0.10% to 1.12% in
pollen grains. It was not detected in the pollen of
A. murale subsp.muralefrom Chernichevo.
No significant differences were found among the
mean Mg values except between concentrations of
stamens and pollen of A. heldreichii and A. murale
subsp. pichleri.
Nickel was detected in lower concentrations than
Ca. The values observed varied from 0.08% to
3.02% in stamens and from 0.15% to 0.59% in
pollen. It was detected in all analyzed species in the
stamens but it was not observed by EDX technique
in the pollen of A. serpellyfolium subsp. lusitanicum,
A. markgrafii from Serbia, A. murale subsp. murale
from Chernichevo and Kamenjane and A. bertolonii
subsp. scutarinum. In the rest of the samples, Ni was
detected in pollen but not in all measurements.
Significant differences were only found between the
mean concentrations of stamen and pollen of
A. serpyllifolium subsp. malacitanum and
A. heldreichii.
Discussion
Brassicaceae is considered as a stenopalynous family
(Erdtman 1966). The group of studied Alyssum taxa
is homogenous in pollen morphology which con-
firmed the description presented by Faegri and
Iversen (1989) and Moore et al. (1991).
The pollen shape varies from sub-prolate to
prolate and this confirms previous data provided for
different species of Alyssum (Inceoglu & Karamustafa
1977; Anchev & Deneva 1997; Orcan & Bunzet
2003; Perveen et al. 2004). The shape of the grains
varies among the species and among the species
Table II. Mean (M) Ca, Mg and Ni concentrations and other statistical parameters (Min: minimum; Max: maximum; SD: standard
deviation; N: number of analysis) for stamens and pollens from the studied samples obtained by EDX and expressed in percentage (%).
Stamen Pollen
Taxa Site code Element NMMin Max SD NMMin Max SD
A. murale subsp. murale 1 Ca 4 1.59 0.65 3.71 1.43 4 0.78 0.27 1.73 0.66
Mg 4 0.68 0.27 1.21 0.47 4 0.23 0.13 0.31 0.07
Ni 3 0.82 0.29 1.66 0.74 1 1.19
2 Ca 4 5.31 2.93 7.72 1.98 3 1.06 0.65 1.50 0.43
Mg 2 0.36 0.19 0.52 0.23 3 n.d.
Ni 3 0.93 0.47 1.73 0.70 3 n.d.
3 Ca 4 1.32 0.32 2.35 0.85 7 1.57 0.10 3.13 1.32
Mg 3 0.45 0.34 0.57 0.12 7 0.30 0.11 0.86 0.26
Ni 2 0.48 0.47 0.48 0.01 7 n.d.
A. murale subsp. pichleri 4 Ca 8 0.67 0.13 1.76 0.51 4 0.56 0.36 0.83 0.23
Mg 8 0.52 0.37 0.88 0.18 4 0.28 0.21 0.35 0.07
Ni 5 0.17 0.08 0.25 0.07 3 0.16 0.15 0.17 0.01
A. serpyllifolum subsp. malacitanum 5 Ca 11 2.77 0.17 11.92 3.22 10 1.39 0.36 3.04 0.95
Mg 11 1.00 0.15 6.06 1.75 8 0.31 0.18 0.64 0.15
Ni 7 0.61 0.33 0.86 0.23 3 0.26 0.16 0.37 0.11
A. serpyllifolum subsp. lusitanicum 6 Ca 5 1.29 0.27 2.81 0.97 6 1.75 0.26 7.95 3.04
Mg 5 0.71 0.31 1.20 0.35 6 0.43 0.19 0.83 0.24
Ni 2 0.65 0.64 0.66 0.01 6 n.d.
A. bertolonii subsp. bertolonii 7 Ca 14 1.14 0.30 2.29 0.67 14 0.39 0.09 1.23 0.35
Mg 15 0.41 0.15 0.94 0.22 13 0.34 0.10 1.12 0.26
Ni 5 0.54 0.17 1.06 0.36 2 0.46 0.33 0.58 0.18
A. bertolonii subsp. scutarinum 8 Ca 6 0.63 0.47 0.92 0.23 3 1.10 0.50 1.56 0.54
Mg 6 0.42 0.22 0.52 0.13 3 0.29 0.12 0.41 0.15
Ni 1 0.55 3 n.d.
A. markgrafii 9 Ca 6 1.76 0.67 3.34 1.10 5 1.40 0.90 1.91 0.51
Mg 6 0.48 0.16 0.93 0.29 3 0.32 0.26 0.45 0.11
Ni 6 0.98 0.35 1.66 0.57 4 0.41 0.23 0.59 0.21
10 Ca 5 3.03 0.64 4.60 1.72 4 0.74 0.34 1.36 0.43
Mg 5 0.37 0.08 0.53 0.18 4 0.26 0.12 0.45 0.16
Ni 5 0.59 0.47 0.78 0.14 4 n.d.
A. heldrechii 11 Ca 20 2.09 0.31 4.65 1.19 9 0.73 0.15 2.53 0.71
Mg 18 0.53 0.26 1.17 0.22 9 0.27 0.16 0.44 0.09
Ni 12 1.03 0.35 3.02 0.73 2 0.30 0.27 0.33 0.04
Note: n.d.: not detected.
Pollen and localization of Ni in Alyssum 7
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populations. According to the number and position
of the apertures the pollen grains belong to the 3-
zonocolpate pollen class. The colpi in all species are
long, shallow and gradually narrowing towards the
poles. The exine is 11.5 mmthickandthe
ornamentation is reticulate with difference in shape
and size lumina. The diameter of the largest
isodiametric lumina in the mesocolpium is around
1mm. The muri of the reticulum are supported by
columellae with free or merged tips. We confirm the
observations of Rollins and Banerjee (1979) and
Anchev and Deneva (1997) for differences in pollen
shape and ornamentation between acetolyzed and
non-acetolyzed pollen. The ornamentation of the
acetolyzed pollen is always appearing reticulate to
fine reticulate, while untreated pollen grains have
typical reticulum and an elliptical elongated shape
(pollen class prolate, P/E.1.41). The perforated
reticular sculpture of pollen in Alyssum reported by
Anchev and Deneva (1997) was not found for the
selected Alyssum species.
The investigated populations of A. murale subsp.
murale show a considerable diversity in its pollen
morphology by the dimensions and shape of the
grains in equatorial and polar views unlike the
populations of A. markgrafii. We suggest that their
shape was changed because of the heavy metal effect
on the pollen development process as it was proved
for other metalliferous plants (Mohsenzadeh et al.
2011). Variation in pollen size between studied
populations could be the result of polyploidy which is
already mentioned for the species of Brassicaceae
(Anchev & Deneva 1997). The analysis of the
relationship between the studied morphological
features present A. heldreichii as a distinct outlier
characterized by its lower values for the polar and
equatorial diameters (P/E¼1.16) and all measured
pollen characters. The taxa Alyssum murale subsp.
murale,A. bertolonii subsp. bertolonii,A. murale
subsp. pichleri and A. bertolonii subsp. scutarinum
have similar pollen shape and are grouped together in
the pollen type IIa. The pollen grains are sub-prolate
(P/E¼1.141.33) with long narrow colpi and
highest values of the mesocolpium in comparison
with all other taxa. Similarity in pollen shape,
ornamentation and size in this group of taxa is
higher between A. murale subsp.muraleand
A. bertolonii subsp. bertolonii and they are clustered
together. Although both subspecies of A. bertolonii
are geographically well isolated (Cecchi et al. 2010,
2013), the differences in their pollen morphology are
insignificant. Both taxa can not be recognized easily
by their pollen. Similarities in pollen ornamentation
between the species could be a result of their
pollination strategies. The closely related A. murale
subsp. murale and A. markgrafii according to their
molecular and morphological evidence (Cecchi et al.
2010) are placed in different clusters on the basis of
their pollen size and shape. The differences in pollen
size could be a result of polyploidy established for
some species populations (Anc
ˇev 1991; Cecchi et al.
2013) which needs future investigations. The high
percentage of sterile pollen found for most of the
studied populations also confirms our suggestions.
These data could be a result of the negative effect of
high concentrations of heavy metals in serpentine
soils and confirm Albooghobaish and Zarinkamar
(2011) and Mohsenzadeh et al. (2011) that high
concentrations of heavy metals decrease the number
of pollen grains and change their shape. Also
excessive amounts of heavy metals adversely affect
plant growth and development causing abnormalities
during the pollen developmental process. Therefore,
abnormal pollen can directly affect the fertilization
and reproduction of the plants (Mohsenzadeh et al.
2011).
Generally, pollen morphological data received in
this study support evolutionary trends outlined by
molecular and morphological studies of the investi-
gated Mediterranean species from the genus Alyssum
(Cecchi et al. 2013) but pollen features are not
enough for precise determination of the species.
Although previous studies have indicated the fact
that flowers of Ni-hyperaccumulator species contain
considerable amounts of Ni, no attention has been
paid to Ni distribution in the different parts of the
flowers. Flowers are composed of sterile protective
parts (petals and sepals) and fertile parts. It is known
that several Ni-hyperaccumulator species concentrate
Ni in their petals and sepals, but little is known about
the presence of Ni in the fertile parts of the flowers.
In this regard, this is the first report concerning Ni
localization in stamen and pollen in some of the south-
European Ni-hyperaccumulators of Alyssum. Most of
the Ni values obtained by SEM-EDX are within the
error range associated to this technique (,1%). Due
to the limitations of the technique Ni was not always
observed. The non-detection does not imply its
absence. In fact, it is very probable that the low Ni
concentration, especially compared with the high C
and O concentrations, avoids an easy detection of this
metal by EDX technique. Anyway, Ni was more easily
detected in stamens than in pollen in all the analyzed
species. Although significant differences were found
only for A. serpyllifolium subsp. malacitanum and
A. heldreichii, this fact may indicate the presence of
higher concentrations of this metal in stamens than in
pollen grains in all the studied species. Stamens are
reproductive organs but derive from vegetative parts
(modified leaves). The vegetative organs of Alyssum
species are known to have high Ni concentrations,
especially the epidermis of leaves and the base of
the trichomes (Psaras et al. 2000;Ku
¨pper et al. 2001;
Psaras & Manetas 2001; Broadhurst et al. 2004;dela
8D. Pavlova et al.
Downloaded by [Pavlova Dolja] at 12:37 15 December 2014
Fuente et al. 2007). This metal has also been found
in the testa and endosperm of seeds of A. serpyllifolium
subsp. malacitanum and A. serpyllifolium subsp.
lusitanicum (de la Fuente et al. 2007).
In A. bertolonii, a specific pattern of nickel
distribution was detected, with the highest concen-
trations present in parenchyma and sclerenchyma
cells for the roots; in the shoots, the highest amounts
of nickel were found in the stem epidermis, the leaf
epidermal surface and the leaf trichome base.
The presence of Ni in pollen in Ni-hyperaccu-
mulators has been recently reported in the Califor-
nian endemic annual Streptanthus polygaloides
(Sa
´nchez-Mata et al. 2013a,2013b). To the best of
our knowledge, this is so far the only report on the
presence of Ni in pollens. The localization of Ni on
pollen wall could also be related with the capacity of
cell wall for binding metals and determines plant
tolerance towards higher levels of nickel in the
serpentine soil (Serregin & Kozhevnikova 2009).
As many hyperaccumulators store Ni in covering and
conducting tissues (Serregin & Kozhevnikova 2009)
such localization would protect plants against
pathogens, herbivores and drought (Boyd 2007).
Our analyses detect the localization of Ni in
tissues of the filaments, anthers and pollen grains of
the Ni-hyperaccumulating Alyssum species and
contribute towards the clarification of the distri-
bution of this metal in the male sex organ of plants.
Most probably, physiological mechanisms in plants
and the metabolic status of plant tissues are
responsible for the reduced quantity of the metals
in pollen grains in comparison with the filaments and
anthers. The same mechanisms decrease the negative
metal effect on pollen tube growth and reproduction
of the species (Breygina et al. 2012; Mohsenzadeh
et al. 2011).
The range of values obtained for Mg concen-
trations, another important element of serpentine
soils, is similar to those obtained for Ni. Never-
theless, Mg was an element more easily detected by
EDX and its presence seems to be more or less
constant in stamens and in pollen of almost all the
analyzed species. This element has been also
observed in vegetative organs and seeds of Ni-
hyperaccumulator species of the Alyssum genus
(Marmiroli et al. 2004; de la Fuente et al. 2007).
Regarding Ca, the concentrations obtained were
always higher than those of Ni and Mg. Its presence
is constant in stamens and pollen. When significant
differences were found, its concentration was always
higher in stamens than in pollen. In addition, Ca
accumulations, occasionally in crystalline forms,
were observed in the stamens and in the pollen
grains. This was recently reported for the Californian
Ni-hyperaccumulator Streptanthus polygaloides (Sa
´n-
chez-Mata et al. 2013a).
Calcium in pollen exine in all species was higher
than Ni. Both elements demonstrate negative
correlation and probably this is some kind of
protection against the Ni effect on the pollen tube
germination. As previously stated (Breygina et al.
2012), Ni almost completely blocks pollen growth at
an early stage and demonstrates toxic effect after
germination.
In addition to Ni, Ca and Mg, other elements
such as K were detected in some of the analyses.
Potassium has been found in several analyses
sometimes in considerable concentration. This is an
essential element in plants; therefore, its presence
could be expected. However, it is important to point
out the usual deficiency of this element in serpentine
soils as it occurs with other nutrients.
The results obtained show that the male generative
plant organs are preserved and high levels of Ni were
not observed in all studied Alyssum hyperaccumula-
tors. The distribution of nickel depends on the species
organ, tissue and its metabolic activity. Nickel
distribution in stamen parts in the studied hyper-
accumulators exhibits a pattern similar to the one
reported for other plant parts (Psaras et al. 2000;
Bhatia et al. 2004). Further physiological analyses will
add new data to be correlated with pollen morpho-
logical and SEM/EDX observations.
Acknowledgements
The authors are thankful to Prof. Eleni Eleftheriadou
from Greece and Prof. Aida Bani from Albania for
kindly providing material for this study.
An anonymous reviewer presented critical comments
on the manuscript.
Funding
The research was realized within Project TK-02/39
supported by the National Research Council at the
Ministry of Education and Science in Sofia,
Bulgaria.
Supplemental data
Supplemental data for this article can be accessed at
10.1080/11263504.2014.989284.
Notes
*Email: vicenta.fuente@uam.es
**Email: dsmata@ucm.es
Email: lourdes.rufo@uam.es
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Pollen and localization of Ni in Alyssum 11
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... Little information is available for the localisation of metals in flower parts of serpentine plants, especially stamens, pollen and pistil Sánchez-Mata et al. 2014;Pavlova et al. 2016b). There are no data about metal localisation and accumulation in parasite plant generative organs. ...
... The presence of Ni on the pollen wall was previously detected in some Ni-hyperaccumulator species (Sánchez-Mata et al. 2014;Pavlova et al. 2016b;Meindl and Ashman 2017). Nickel was not found in all our samples for pollen and anther, suggesting that detection of this metal by EDX technique is not sensitive enough when Ni concentrations are low compared with high C and O concentrations. ...
... In the case of O. nowackiana, the mechanism by which Ni accumulates in the plant body is still not clear. In addition to Ni, Ca and Mg were detected to be more constant in studied plant parts similarly to the results obtained for Ni hyperaccumulating plants (Sánchez-Mata et al. 2014;Pavlova et al. 2016b). ...
Article
The holoparasite flowering plant Orobanche nowackiana Markgr. is a rare endemic plant that parasitises the Ni hyperaccumulator species Alyssum murale Waldst. and Kit. in Komjan Mt. (Albania). The purpose of this study was to establish baseline data concerning aspects of its pollen biology. To achieve this goal three objectives were addressed: (1) describe pollen morphology; (2) study pollen production and fertility/sterility; (3) study the localisation of metals in anthers and pollen. Pollen morphology was investigated with light microscope and scanning electron microscope (SEM) observations. The pollen grains are 3-colpate, most often oblate-spheroidal, with long colpi reaching the poles. The ornamentation is microreticulate. The anther and pollen grains were micromorphologically analysed by SEM coupled with an energy-dispersive X-ray probe (SEM-EDX). Low concentration of Ni was recorded for anthers (0.38%) and pollen (0.1–5.6%). Variationin pollen production was found fortheflowers ofthe sameindividual. The mean pollen production per flower and stamen was 59 365 and 14 938 pollen grains respectively. The sterile pollen was above the limit considered as a normal abortion and was between 10.1 and 38.0%. From a palynological point of view our results are important for taxonomy and support keeping the species in the genus Phelipanche. Additional keywords: holoparasite, Ni localisation, Orobanche nowackiana, pollen morphology, pollen production.
... Also, the higher amount of Brassicaceae pollen found in the Albanian samples can be correlated positively with the higher level of Ni found in these samples, bearing in mind the accumulation abilities of O. muralis (7-34,690 mg kg −1 Ni, Global Hyperaccumulator Database, http://hyperaccumulators.smi.uq.edu.au/collection) [46] and the localization of Ni in the pollen grains of Odontarrhena species [47]. ...
... This result may be due to the accidental transfer of soil particles by bees and collected pollen with localized nickel from Ni hyperaccumulator plants such as Odontarrhena muralis cultivated around the beehives. Nickel localization on pollen grains of Ni hyperaccumulator and parasitic plants were previously found [47,60] and in addition to Ni, also Ca, Mg, and K were elements detected in the pollen wall confirming its capacity for binding metals similarly to the cell wall. ...
Article
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Beebread from serpentine localities in Albania and Bulgaria were characterized based on their pollen and chemical element content (macroelements K, Ca, Mg, P and microelements Cd, Co, Cr, Cu, Fe, Mn, Na, Ni, Pb, Zn) aiming to (1) evaluate the effect of serpentine soil on the quality of beebread; (2) compare elemental concentrations in samples from serpentine areas in Albania and Bulgaria; and (3) compare the differences in pollen spectra. Chemical element content was determined using microwave digestion of beebread samples followed by ICP-OES measurements. The analytical procedure developed was validated by added/found method. Analytical figures of merit of analytical method proposed were presented. The melissopalynological analysis was applied for pollen characterization. The results demonstrate clear difference in the pollen spectra between the two sets of samples, confirming differences in local serpentine flora in both countries, but specific pollen type is difficult to be suggested. The pollen content is related to the flowering period, climatic conditions, and bees forage preferences. The samples vary in their elemental concentrations depending on the pollen type and year of collection. The highest average concentrations found for K, Ca, Mg, and P are close to values reported in the literature. However, elevated concentrations observed for Ni, Cr, Mn, and Fe in beebread, especially from Albania, are in line with the serpentine characteristics of studied areas. The concentrations of Cd and Pb for all beebread samples are below permissible limits. The results should be taken into consideration in future specific food safety regulations at national and international level.
... In addition fruit shape and size can be useful characteristic for determining in species. Investigated the pollen morphology and microsculpturing in species of and provided evidence for the close relationships among various species [27,28,30]. The general aperture form of is tricolporate but colpus length and width highly different in taxa. ...
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In the current study, macro- and micromorphological characters of fruits, seeds and pollen in Alyssum L. (14 to 19 species) were examined by stero, light (LM) and scanning electron microscopy (SEM) to evaluate the taxonomic significance of these characters. Detailed description of features of Turkish Alyssum species are provided with illustrations. Pollen grains are generally radially symmetrical and isopolar. The fruit surface is glabrous or stellate trichome. The seeds are ovate, spheroidal or oblong in shape among the studied species. The colors of the seed vary between light brown, dull yellow brownish, bright red brownish, dark brownish, dull light reddish, bright brownish, bright red brownish, dull brownish and dull yellowish. The surface of the seed is reticulate (normal reticulate, reticulate-foveate, reticulate-lineolate, reticulate-rugose), ruminate, rugose, lineolate, blister and colliculate in SEM. The pollen grains of the genus are subprolate or prolate. The pollen grains are tricolpate. Exine ornamentation are mainly reticulate. Ornamentations, pollen shape and size, aperture type, colpus length and width, fruit and seed shape, size and color, seed anti and periclinal walls have been observed as significant morphological characters.
... In addition fruit shape and size can be useful characteristic for determining in species. Investigated the pollen morphology and microsculpturing in species of and provided evidence for the close relationships among various species [27,28,30]. The general aperture form of is tricolporate but colpus length and width highly different in taxa. ...
Article
In the current study, macro- and micromorphological characters of fruits, seeds, and pollen in Alyssum L. (14 to 19 species) were examined by stereo, light (LM) and scanning electron microscope (SEM) to evaluate the taxonomic significance of these characters. Detailed description of features of Turkish Alyssum species are provided with illustrations. Generally, the pollen grains are radially symmetrical and isopolar. The fruit surface is glabrous or stellate trichome. The seeds are ovate, spheroidal or oblong in shape among the studied species. The colors of the seed vary between light brown, dull yellow brownish, bright red brownish, dark brownish, dull light reddish, bright brownish, bright red brownish, dull brownish and dull yellowish. The surface of the seed is reticulate (normal reticulate, reticulate-foveate, reticulate- lineolate, reticulate-rugose), ruminate, rugose, lineolate, blister and colliculate in SEM. The pollen grains of the genus are subprolate or prolate. The pollen grains are tricolpate. Exine ornamentations are mainly reticulate. Ornamentations, pollen shape and size, aperture type, colpus length and width, fruit and seed shape, size and color, seed anti and periclinal walls have been observed as significant morphological characters.
... It was identified in the study on taxa of Alyssum that pollen morphology was homogenous and pollen shape varied from subprolate to prolate (İnceoglu and Karamustafa, 1977;Faegri andIversen, 1989, Moore et al., 1991;Anchev and Deneva, 1997;Orcan and Binzet, 2003;Perveen et al., 2004). Pavlova et al., (2016) determined that colpi of all species was long, shallow, and got narrower towards poles. Additionally, exine is 1-1.5 µm-thick and was determined to vary in terms of ornamentation and size lumen. ...
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The present study evaluated the taxa A. linifolium Stephan ex. Willd. var. teheranicum Bornm., A. simplex Rudolph, A. trichocarpum T.R. Dudley & Hub.-Mor., A. armenum Boiss., A. praecox Boiss. & Bal., A. lepidoto-stellatum (Hausskn. &Bornm.) T. R. Dudley, A. sulphureum T. R. Dudley & Hub. - Mor., A. murale Waldst. & Kit. subsp. murale, A. pateri Nyar. subsp. pateri of the genus Alyssum L. from the family Brassicaceae in terms of palynological characteristics. Samples of these taxa were collected in field studies. 9 taxa from this genus were examined using light microscope (LM) and scanning electron microscope (SEM). Results of the study revealed some common characteristics such as tectate pollens, subprolate, prolate shapes of pollens, heteropolar symmetry. Aperture type was tricolpate. SEM microphoto also indicated that ornamentation structures were reticulate.
... It was identified in the study on taxa of Alyssum that pollen morphology was homogenous and pollen shape varied from subprolate to prolate (İnceoglu and Karamustafa, 1977;Faegri andIversen, 1989, Moore et al., 1991;Anchev and Deneva, 1997;Orcan and Binzet, 2003;Perveen et al., 2004). Pavlova et al., (2016) determined that colpi of all species was long, shallow, and got narrower towards poles. Additionally, exine is 1-1.5 µm-thick and was determined to vary in terms of ornamentation and size lumen. ...
Article
The present study evaluated the taxa A. linifolium Stephan ex. Willd. var. teheranicum Bornm., A. simplex Rudolph, A. trichocarpum T.R. Dudley & Hub.-Mor., A. armenum Boiss., A. praecox Boiss. & Bal., A. lepidoto-stellatum (Hausskn. &Bornm.) T. R. Dudley, A. sulphureum T. R. Dudley & Hub. - Mor., A. murale Waldst. & Kit. subsp. murale, A. pateri Nyar. subsp. pateri of the genus Alyssum L. from the family Brassicaceae in terms of palynological characteristics. Samples of these taxa were collected in field studies. 9 taxa from this genus were examined using light microscope (LM) and scanning electron microscope (SEM). Results of the study revealed some common characteristics such as tectate pollens, subprolate, prolate shapes of pollens, heteropolar symmetry. Aperture type was tricolpate. SEM microphoto also indicated that ornamentation structures were reticulate.
... The effect of this edaphic factor on plant reproduction and adaptation using the flowering response of Ni-treated plants concluded that Ni stimulates flowering and may increase plant fitness on serpentine soils (Ghasemi et al. 2014). Few studies of the effects of Ni on pollen of plants growing on ultramafic soils exist, with the exception of some fragmentary data related to Ni localisation in pollen grains in some Ni hyperaccumulator plants (Sánchez-Mata et al. 2014; Meindl et al. 2014; Pavlova et al. 2014). In most cases, these studies are related to Ni hyperaccumulating representatives of genus Allysum (Brasscaceae). ...
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In this work we studied and compared the toxic effect of nickel (Ni) on pollen germination and pollen tube length in Arabis alpina L. collected from serpentine and non-serpentine populations distributed in the Rila mountains, Bulgaria. Pollen grains were treated with prepared standard solutions of 100, 300, 500, and 700 mM Ni as NiCl2 in distilled water. A nutritional medium was also used to assess pollen germination. Nickel inhibited pollen germination and pollen tube elongation in both serpentine and non-serpentine plants. The percentage of germinated pollen in serpentine plants treated with Ni was higher than in non-serpentine plants but there was no difference in pollen tube elongation between groups. However, pollen tubes showed abnormalities such as coiling and swelling of the tip, or burst, and varied considerably among the samples. A complete break of pollen tube elongation is due to such abnormalities. Also, decreased pollen fertility in both populations was found. The plants from serpentines were less sensitive to (i.e. more tolerant of) elevated Ni concentrations commonly found in serpentine soils.
... Also, some essential elements (P, Fe, Al, Mg, Cu, Mn, Si, Ca, K, and Na) naturally distributed in the soil are included in the nectar transported via plant's root system [2]. A lot of data demonstrated the localization of metals in pollen grains as well [3][4][5][6]. ...
Article
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Honey samples collected during 2007-2010 from serpentine and non-serpentine localities in the Eastern Rhodopes Mt. (Bulgaria) were characterized on the basis of their pollen content by qualitative melissopalynological analysis and physicochemical composition. Water content, pH, electrical conductivity, macroelements-K, Ca, Mg, P, and microelements-As, Cd, Co, Cr, Cu, Fe, Mn, Na, Ni, Pb, and Zn were determined after the Harmonised Methods of the International Honey Commission and ICP-AES method. The results from serpentine honey samples were compared with data from bee pollen collected from the same serpentine area. Different elements have different concentrations in honey from the same botanical type even collected from the same geographical region, same locality, and same beehive but in different vegetation season. The elements Mg, Mn, Ni, and P contribute mostly for separation of the serpentine honey samples based on measured elemental concentrations and performed principal component analysis. The element concentrations were higher in bee pollen and above the permissible limits for the toxic metals Cd and Pb. No specific indicator plant species was found for identification of the geographical origin of serpentine honey in relation to the forage of bees.
Thesis
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SUMMARY Ultramafic outcrops represent less than 1% of the terrestrial surface. They have attracted the attention of many scientists for decades because of their unusual geochemical composition. Serpentine soils originate from ultramafic rocks and are characterized by the lack of essential elements for plants such as K, N, Ca and P). For soil scientists (Alexander 2009), ultramafic soils have also been of particular interest because the pedogenic processes of their formation are more diverse than those occurring on normal soils. Serpentines and peridotites are a group of silicate rocks with less than 45% silica (SiO2) and a low ratio of Si/Mg because they consist of olivine, pyroxene and serpentine clays. They are also characterized by a high concentration of Fe, Mg, Ni, Co, and Cr. The Ca / Mg ratio in ultramafic soils is less than 1. Peridotites and serpentines have the same chemical composition, except that serpentines have 13% (130 g / kg) more water in their composition (Coleman, 1971). The serpentine areas located in the Balkan Peninsula are widespread and form a belt that runs down from Bosnia to Greece. The ultramafic outcrops in the territory of Kosovo are dated from the cretaceous age, as most of the other outcrops in the neighboring countries (Dilek and Furnes 2009). The ultramafic outcrops comprise 4.48% of the entire territory of Kosovo of which 142.8 km2 (or 1.31% of the territory) bear soils developed on serpentines and 344.32 km2 (or 3.16%) bear soils developed on harzburgite (peridotite) The largest ultramafic complexes are located in the Ibri Valley (North), followed by Golesh Massif (Central Kosovo) and lastly in the southwestern part of Kosovo. Additionally, patchy ultramafic areas are present in other parts of the territory as well. The ultramafic soils in the Balkans Peninsula region are mainly covered with meadows or shrubby vegetation although it varies with altitude. The serpentine flora of the Balkans is characterized by a relatively high degree of endemism (Stevanovic´ et al. 2003). The greatest concentration of serpentine endemic species in the Balkans is found in the mountains of the western part of the peninsula in the territories of Bosnia, Serbia, Albania, Kosovo and Northern Greece. This aided in the facilitation of the Balkan Peninsula being an important refuge for plants during the Quaternary glaciations (Tatic´ and Veljovic´ 1990). Among this rich and diversified ultramafic flora, metal hyperaccumulation has evolved in quite a number of species, including many species of the genera Odontarrhena C.A.Mey (syn. Alyssum) (Brassicaceae), Noccaea (Thlaspi) (Brassicaceae) and Bornmuellera (Brassicaceae) which are known to be Nickel hyperaccumulators (Reeves and Brooks, 1983). Recently, significant hyperaccumulation of Ni have been reported in northern Greece in families other than Brassicaceae: i.e. Centaurea thracica (Compositae) and Viola vourinensis (Violaceae) (Psaras and Constantinidis 2009). These plants often show high levels of some metal elements like Ni and Co, compared to the same plants in other types of soil. In some cases, very high concentrations (> 1000 mg kg-1) of Ni in the serpentine plant tissues have been observed. Accumulation of Nickel over 1000 mg kg-1 has been observed for a long time in the Alyssum murale Waldst species. & Kit. In the serpentines of Mediterranean Europe, Turkey, and neighboring countries. An extraordinary contribution to these plants has recently been given in Albania and there is some progress in Kosovo as well. This study focused on the recognition and assessment of the characteristics of ultramafic and serpentine flora of Kosovo and the ecotoxicological risk for the food chain. The scientific objectives of this study were: • Assessing the level and of heavy metals in the studied Kosovo serpentine areas. • Determination of the total and exchangeable macronutrients (Ca, Fe, Mg) in the serpentine soils and the evaluation of the Ca / Mg ratio. • Inventoriate the serpentine flora occurring on Kosovo serpentine soils • Identification of accumulating and hyperaccumulating flora of Kosovo serpentine soils • Identification of the most efficient hyperaccumlators species. • To study of the relationship between the metal uptake from plant species and the availability of metals in corresponding soils. • Evaluating the concentration of Nickel in fresh milk from livestock grazing on serpentine soils and the possibility of the Ni entry into the food chain. • Assessment of the concentration of Nickel on the pollen of these plants and the possibility for these metals to enter into the food chain through the honey To achieve the study objectives, ten sampling sites were selected across the entire territory of Kosovo, which represent the most representative situations in terms of climate and ultramafic bedrock characteristics. Collection of soil and plants’ samples was carried out from mid-May to the second half of June of 2014. Twelve pedons were excavated at the 10 sites and 27 soil samples were taken from the different horizons, one for each horizon of these pedons. For each plant sample, 5–7 individual plants were randomly collected (not less than three individuals per species). Plants with scarce presence (less than 3 individuals) were not considered. Flora was collected in a selected area of 300–500 m2 and the species and family name were determined at the time of sample procurement. Plants were identified using Flora Europaea (Tutin et al. 1993). Each plant that originally collected was subjected to the presence/absence test of Ni with filter paper impregnated with Ni-dimethylglyoxime The individuals of a same plant species were then mixed altogether to form a composite plant sample. Through these 10 representative serpentine areas, we have aimed to include the entire map of Kosovo serpentines in the most representative way, to provide more comprehensive information about their physico chemical composition, then about serpentine flora, and its inventory and their capabilities to accumulate or hyperaccumulate the metals from the soil. The corresponding regions lie at different altitudes and have different geographical characteristics. Kosovo is affected by both the Mediterranean mild climate and the European continental climate. The lowest area altitude studied in this thesis is that of Radoniq with a height of 455 m, continuing with the Qafa e Prushit of 483 m. The other seven areas lie between the altitude of 587 and 967 m, while the site ten on the map, which is Kaçandoll lies at an altitude of 1101 m. Soil samples were analyzed for basic physico chemical characteristics. Initially, physical composition was defined by determining its texture. Texture has been determined using the hydrometer method. As far as chemical soil analysis is concerned, following parameters were performed: total concentrations of Zn, Cd, Co, Cu, Ni, Pb, Mn, Cr, Fe, Ca, Mg, using the USEPA 3050B. Reading is performed on the spectrometer 4200 MP-AES. In addition exchangeable metals such as Ca, Mg and Ni metals are determined, to see their availability in soil and Ca/Mg ratio. Exchangeable Ca. Mg and Ni were determined using Mehlich-3 method (Schroder et al. 2010). Soil pH was measured in a solution of 1:2.5 soils: water suspensions (Vogel 1994). Before pH measurements, the pH-meter was calibrated with buffer solutions at pH 4, 7 and 9. Soil conductivity was determined by measuring out 1/2 of a cup of the dried soil and put into a glass beaker and by measuring out 1/2 of a cup of distilled water that was also put this into the same beaker with the soil. Then the beakers were put in a shaker for 30 min. After mixing the soil–water suspension stood for 30 min to settle down. For measuring soil electric conductivity (EC), EC- meter was calibrated with calibrating solution 1000 ± 10 μS cm-1at 25oC. The physical characteristic of these soils are mainly rocky with granular texture The pH value of the serpentine soil shows mainly the basic character in most samples and varies from moderately alkaline (7.58) to typical alkaline (8.64) and in only two samples the pH was slightly acidic, i.e. below 7.00 (6.83 and 6.86). Soil electric conductivity (EC) likewise varied at a substantial rate from sample to sample, the results obtained from the lowest sample was at site 6 (59 μS cm-1) and the highest was 492 μS cm-1 site 2. Serpentine soil studied in 10 sites (12 profiles) are generally characterized by high levels of heavy metals such as Ni, Mn, Co and Cr, that directly derived from ultramafic rocks (Whittaker 1954, Brooks 1987, Bani et al., 2010, 2014), while Cu concentrations (except for a profile), Zn, Cd and Pb are at normal soil levels. Nickel as one of the most representative metals, which representing the typical character of ultramafic soil is found in all soil samples ranging from 872 mg kg-1 (zone 2) to 3338 mg kg-1 (zone 2-horizon B), while the available Ni fraction was in the range of 33 mg kg-1 (zone 1) to 154 mg kg-1 (zone 2). Pertaining to the concentrations of major elements (Fe, Ca, and Mg) contained within the soil samples collected, the soils demonstrated typical ultramafic characteristics with high concentration of available Mg and from low to moderately high concentration of Ca. A very important element for ultramafic soil characteristics is also the Ca/Mg ratio. The Ca/Mg quotient obtained from 19 soil samples were relatively low (0.08–0.99), while in seven serpentine soil samples (site 4-horizon A, site 6-horizon A, site 7-horizon A, B, C, site 9-horizon A, and site 10-horizon B) higher values of bioavailable Ca compared to Mg (Ca/Mg ratios 1.00–2.38) were present, excluding site 4 (horizon B) where the Ca/Mg ratio was the highest (3.18). In ultramafic soils of Kosovo, the Ca/Mg ratio in some samples displayed higher values of bioavailable Ca compared to Mg, indicating lower Ca deficiency stress for the plants. The higher Ca concentration compared to Mg in those soil samples is probably due to weathering and leaching processes combined with biological activity(i.e. biogeochemical cycling of Ca) at these locations (Alexander et al. 2007; Echevarria 2018). Principal Component Analysis (PCA) on soil characteristics of the 27 soil horizon samples revealed several properties of ultramafic soils of Kosovo. According to the PCA the first two principal factors explain 46.7% of the total fluctuation of all the factors studied in this paper. The Factor (F1) (horizontal axis) was significantly correlated to Cu, Conductivity, Ni CEC, and Mg CEC. Soil pH was negatively correlated to all the other contributing variables, which were in the positive range. Meanwhile only Ca and Ca CEC significantly contributed to the second factor F2 (vertical axis) and they both contributed positively to this axis. Regarding serpentine flora, 162 plant taxa present, belonging to 24 families were collected Amongst the samples of plant species collected, 27 species were reoccurring at multiple sampling sites (i.e. in two or more sites). 16 species were reoccurring at three or more sites. Only, two plant species were identified as reoccurring at all sampling sites. This occurrence of specific species located across all sites were from Odontarrhena genera, whether murale or markgrafii. Unfortunately, Bornmuellera dieckii could not be found, although it was reported to occur on serpentine sites located in the Mushtisht region (Millaku et al. 2008). Plant samples also are analyzed in the following parameters: Zn, Cd, Mn, Ni, Cu, Pb, Cr, Co, Ca and Mg. Analyzes were conducted using the methods applied in the analytical laboratory of the Kosovo Agricultural Institute. In all 10 sites there was Odontarrhena muralis species found. Alyssum murale is a well-known Ni hyperccumulating plant in the Balkans. The total quantity of Ni in tissues of all ten. Odontarrhena samples studied here was moderately high 1586 mg kg-1 to high (up to 7564 mg kg-1), which is generally lower than in the same species of plants found in Albania (Shallari et al., 1998; Bani et al., 2014). In A. murale tissues (site 5) where was the lowest concentration of Ni in this species among the 10 sites, Ni concentration was 41 times higher than in corresponding soil, while in the case of site 2, which was the highest concentration, the ratio between concentration of Ni in A. murale tissues and in the corresponding soil was 65:1. In all 162 plants we can conclude that these plants are able to uptake sufficient amounts of Ca from the serpentine soils, even the content of this essential element is low in the corresponding soil. In this study, the majority of the plant species did not demonstrate metal concentrations > 1000 mg kg-1 excluding Odontarrhen) and Noccaea species, which met the criterion to be classified as hyperaccumulating plants. As noted above despite the low Ca content in soil samples, the Ca concentration in the aerial part of all the Alyssum populations was many times higher. Compared to Ca content, the concentration of available Mg in soil samples is followed in most plants, but not at all. In some cases it was double to three times higher, but there were also cases when the intake of Mg was lower than Mg which was bioavailable in the soil. Besides Odontarrhena species, there was another Ni hyperaccumulating species, which is outstretched in Balkans region as well. Noccaea ochroleuca (Boiss and Heldr.) F.K.Mey. species were found at three sites (site 2, 6 and 9). The Ni concentrations in N. ochroleuca ranged from 798.7 to 5888 mg kg-1 and is more or less similar to the findings found in Noccaea in Albania, Greece and Bulgaria, Bani et al. (2010) and Kazakou et al. (2010). The amount of Zn in the species of Noccaea ochroleuca found in one of the areas does not meet the criteria to be as hyper-accumulating plants of Zn, but considering the low concentration of Zn in the corresponding soil, then the plant/soil ratio is 19: 1. This plant has been able to uptake 19 times more Zn than it had in the corresponding soil. In conclusion, this species can be considered as very suitable for cultivation in polluted regions for soil phyto-rehabilitation purposes. With regard to the accumulation capacity of Ca, all species of Alyssum have accumulated Ca from the corresponding soil with factor > 1. This is due to the fact that Ca concentration in the aerial part of Alyssum is very high, as a consequence of the extraordinary ability of this plant to accumulate large amounts of Ca in its tissues, even in high ratio of Mg/Ca soil (these are characteristics of ultramafic soils). Based on the obtained results of heavy total metals in soil and plants, we also obtained the bioaccumulation factor for each plant found in these areas. Bioaccumulation factor (BF) was calculated for each plant based on the concentration of heavy metals in aerial part of the plant divided by the heavy metal concentration in soil (Zu et al.2005) and is given in equation as following: BF = [Metal] plant / [Metal] soil. For BF evaluation, the total Ni concentration in the A horizon is taken as a reference, since the most herbaceous plants have their root system only in the first 20 cm of soil depth. The bioaccumulation factor for Ni, Zn and Cu is calculated only for plants that have this factor greater than one (> 1). The highest Ni BF was 6.81 in A. murale (site 2). Also it was higher than one for other species of Odontarrhena and Noccaea genera at 8 sites (1, 2, 4, 6–10). Among the plants collected, 8 plants in 2 sites (6 and 8) were identified with Copper BF > 1. Copper concentrations in the plants ranged from 1.24 mg kg-1 (Odontarrhena muralis - site 5) up to 14.0 mg kg-1 in A. murale site 8 and falls in normal range. Zinc BF greater than 1 was recorded for 25 species at 9 sites (site 1–4, 6–10). The highest BF for Zn was 18.7 in N. ochroleuca (site 6). BF for the rest of metals was < 1, and that’s why those data were not reported in this studyat all. These plant species may prove highly beneficial for growth in industrially polluted areas, for phytoremediationpurposes, as they accumulate considerable quantities of heavy metals from the soil with their root system. These plant species could potentially be utilized for cleaning heavy metal in soils (i.e. phytoextraction). Phytoremediation potential of these plants species need to be further researched. One of the objectives of this study was to analyze the concentration of Ni in fresh cow’s milk coming from livestock grazing on serpentine soils and determining the concentration of Ni on the pollen of these plants to see the possibility of this metal entryto the food chain, through cow’s milk and honey from bees. For the determination of Ni in milk, sampling was conducted into two stages: we completed the first stage in the end of June 2016 when we took 6 samples of milk from 6 different cows, which represents 10% of this herd and this milk straight from the udder was then analyzed for its nickel content. At the same time we took fresh milk samples from three other cows that were grazing on other common pastures (from non-serpentine regions). The second stage of sampling took place in January 2017, when six more samples were taken from the same cows to see whether the Ni quantity had diminished when cows were no longer grazing on those pastures due to the cold wintertime, being kept inside the barns and fed on dried feed originating from the non-serpentine regions. It should be mentioned that the milk was poured straight from the udder into the PVC flask with the purpose of eliminating any contact of milk with utensils galvanized with nickel. The samples were then placed into a portative fridge in order to secure the temperature of 4oC and sent to the lab for determination of their Ni quantities. Ni content varies in its amount from 1.76 to 3.16 mg kg-1, whereas the average amount of Ni in those six samples reaches a value of 2.39 mg kg-1.In the second stage of sampling conducted six months later, when the cows stayed inside the barn, the Ni content was much lower varying from 0.52 to 1.36 mg kg-1with the average value at only 0.80 mg kg-1. There is no other Ni source within the studied region that could have a direct impact on the Ni amount found in the raw cow milk samples since there is no other source of Ni in the area or its vicinity coming from a potential smelter or mine. Consequently we have eliminated all other factors at the very outset of this study and our main focus was how an amount of Ni found in the serpentine flora could enter the food chain through the milk of the cows grazing on the aforementioned pastures. Serpentine soil of Mushtisht is a potentially toxic contaminated source to surrounding environment due to the high content of total and exchangeable Nickel. Present data suggest that the plant species collected at the site exhibited different concentrations in metals. Also, the present flora shows higher levels of Ni than flora coming from normal soils. The results from the study show that, the Ni content found in the fresh cow’s milk is much higher during the grazing period outnumbering for many times the amount of Ni found in non-serpentine pastures or during winter when livestock is kept and fed inside the barn. Our first results from this study area will guide us for a more detailed study in the future to confirm the dangerous Ni presence in cow’s raw milk and subsequently the food chain. To further searching the Ni entry in the food chain we analyzed the amount of Ni concentration in a bee farm located in another serpentine area. The serpentine region where the honey was collected lies in the south-eastern part of Kosovo with geographical latitude and longitude of 42o8.7426’N; 21o15.54696’E respectively and altitude of 650 m above the sea level with Rezhance being the closest settlement to this location. This serpentine region covers an area of 215 hectares containing several kinds of low herbal plants able to accumulate or hyperaccumulate. The objective of this paper was to determine: 1) The Ni quantity in these serpentine plants and 2) The possibility of the Ni entry into the food chain through the honey. Initially sampling was conducted in the end of May 2014 by digging out a soil profile and taking the soil samples and collecting the entire flora present therein at the time of sampling. Since the results showed the high presence of nickel in both soil and plants, in September 2014 we carried out with the sampling of honey from the hives which were in the vicinity of this area. For three consecutive years, in 2014, 2015 and 2016 the honey samples were taken from the same apiary consisted of 10 hives to determine the level of Ni in honey produced by bees from nectar collection in serpentine multi-floral location. According to the beekeeper he had a total of 10 hives and honey is harvested only once a year, in August, so the taken sample was very representative and was considered as a composite sample. The hill, which is located just 200 m from the apiary, is covered with Alyssum murale which is a hyperaccumulator plant of Ni in Balkans and can be attractive to honeybees to collect the nectar from it as well. In addition, three samples were collected in other non-serpentine regions to compare the results of Ni found in honey coming from both serpentine and non-serpentine flora. The sampling in non-serpentine areas is performed with a radius greater than 10 km from the serpentine soils, as it is assumed that bees travel for up to 10 km to collect nectar from flowers. The samples were taken in the plastic container and sent to the lab for determination of Ni levels. Ash contents were determined by heating 10 g of honey precedence 100 oC to moisture amount decrease, after 500 oC to constant weight dry up with an infra-red lamp to prevent foaming (AOAC, 1984). Nickel (Ni) was measured using Perkin Elmer 3110 Atomic Absorption Spectrophotometer (AAS). From the obtained results it is seen that the level of Nickel in Rezhance is typically of serpentine environment, which means that this area is naturally occurring contaminated with Ni and other heavy metals. The average total amount of Ni in all three horizons was 1744 mg kg-1, whereas the available Ni in soil was 87 mg kg-1, thusly this might have adverse effect on entire environmental pollution. Ni content found in the honey exceeding for twice at least the amount of Ni found in the honey of non-serpentine flora. The Ni content varies from 3.36 to 3.92 mg kg-1, whereas the average amount of Ni in those three samples from serpentine flora reaches a value of 3.71 mg kg-1, while the Ni levels found in the three samples coming from non-serpentine flora are considerably lower than the levels found in the honey coming from serpentine flora. These values vary from1.36 to1.92 mg kg-1, with an average of 1.66 mg kg-1 which is generally similar with findings from Poland (Formicki G. et al., 2013), but those are higher than outcomes found in Eastern Rhodopes mountain of Bulgaria (Atanassova J. et al., 2016) The high Ni content is concerning because large quantity of Ni in the body poses a health threat. The Nickel level in the honey is so far higher compared to other foods documented. Nickel levels in food are generally in the range 0.01-0.1 mg kg-1, but there are large variations (Booth J., 1990 and Jorhem L, Sundström B., 1990). As a conclusion the high content of Nickel in the honey coming from serpentine regions could be from the hyper accumulating plant sources (growing in serpentine soil) where the pollen was taken from, or could be also as a result of dust generated from naturally metalliferous soils such as serpentine soils of Rezhance. There was no other source that could have direct impact on Ni level on honey. The beekeepers should be informed about possible negative effect of naturally metalliferous soils on the quality of honey and bee pollen and should pay attention to the environmental characteristics of the locality where they place beehives. A strict control on bee pollen used for medication is recommended in spite of further investigations in this aspect (Atanassova J. et al., 2016).
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Botanical exploration of serpentine soils in Turkey and neighbouring countries has shown that the region includes at least 59 taxa capable of hyperaccumulating nickel (to >0.1% of plant dry weight). These hyperaccumulators belong to the Brassicaceae (Aethionema R.Br., Alyssum L., Bornmuellera Hausskn., Pseudosempervivum (Boiss.) Grossh. (Cochlearia L.), and Thlaspi L. s.l.) and the Asteraceae (Centaurea L.). We review present knowledge of the hyperaccumulators and provide additional data recently obtained. Some species are serpentine-endemic and invariably Ni hyperaccumulating; others show more complex distribution and Ni-accumulating behaviour. Many are good subjects for biochemical studies on the Ni-accumulation and sequestering processes. There is potential in Turkey for exploiting Ni hyperaccumulation for remediation of Ni-contaminated soils ('phytoremediation') and for economical selective extraction of metal compounds by cropping hyperaccumulators ('phytomining'). However, there is a need for further exploration of the natural resources and some further taxonomic work by traditional and DNA methods. Attention must be paid to conservation issues, as some of the relevant species are quite rare.
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