56 Northeastern Naturalist Vol. 16, Special Issue 5
Saxifraga gemmulosa Boiss. (Saxifragaceae),
an Endemic Nickel Bioindicator from Ultramafic Areas
of the Southern Iberian Peninsula
Blanca Díez-Garretas1, Alfredo Asensi1, Lourdes Rufo2, Nuria Rodríguez3,
Daniel Sánchez-Mata4, Ricardo Amils3,5, and Vicenta de la Fuente2,*
Abstract - The western Betic Mountain Range contains the largest ultramafic rock
area in the Iberian Peninsula. The predominant flora of this southern territory (over
two hundred taxa) was screened for Ni accumulation. Only two species showed important
concentrations of Ni in their tissues, Alyssum serpyllifolium subsp. malacitanum
(Brassicaceae), a Ni hyperaccumulator, and Saxifraga gemmulosa (Saxifragaceae).
Saxifraga gemmulosa is a rare endemic species restricted to the ultramafic outcrops
of Málaga (South Spain), mainly growing in basic or ultrabasic rock crevices, where
it appears with other serpentinophytes such as Asplenium adiantum-nigrum subsp.
corunnense (Aspleniaceae). Nickel and other representative elements present in Saxifraga
gemmulosa and its soils from Sierra Bermeja (Málaga) were studied by inductively
coupled plasma-mass spectrometry (ICP-MS). The structures of the plant were
micromorphologically analysed by scanning electron microscopy (SEM) coupled to
an Energy-Dispersive X-Ray Probe (EDX). The results showed the Ni hyperaccumulating
characteristics of S. gemmulosa. As observed in other Ni hyperaccumulator
plants, accumulation was mainly detected in leaf epidermis.
Introduction
The ultramafic flora and vegetation of the Iberian Peninsula are strongly
characterized by high endemicity due to their numerous specialized taxa,
known as serpentinophytes, both obligate (serpentine endemic) and facultative
(able to grow on serpentines and on other substrates) (Borhidi 1992,
Brooks 1998, Reeves et al. 1999). However, only two nickel hyperaccumulators
have been reported for this area in the literature: Alyssum serpyllifolium
subsp. lusitanicum T.R. Dudley & P. Silva and A. serpyllifolium subsp. malacitanum
Rivas Goday ex G. López (Asensi et al. 2004, Brooks et al. 1981).
Recently, more than 200 taxa from the ultramafic areas of the southern
Iberian Peninsula were tested for Ni hyperaccumulation using a semiquantitative
test. This test gave a positive reaction for Saxifraga gemmulosa Boiss.
1Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga,
E-29071 Málaga, Spain. 2Departamento de Biología, Facultad de Ciencias, Universidad
Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain. 3Centro de Astrobiología
(INTA-CSIC), E-28850 Torrejón de Ardoz, Madrid, Spain. 4Departamento
de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid,
E-28040 Madrid, Spain. 5Centro de Biología Molecular (UAM-CSIC), Universidad
Autónoma de Madrid, E-28049 Madrid, Spain. *Corresponding author - vicenta.
fuente@uam.es.
Soil and Biota of Serpentine: A World View
2009 Northeastern Naturalist 16(Special Issue 5):56–64
2009 B. Díez-Garretas et al. 57
(Rufo et al. 2004). Saxifraga gemmulosa was discovered and described by P.
E. Boissier in Sierra Bermeja, Málaga, Spain (Boissier 1838) and exquisitely
drawn in his magnificent Voyage Botanique (Boissier 1839–45).
Saxifraga gemmulosa is included in section Saxifraga, subsection Saxifraga,
series Biternatae (Engl. & Irmsch.) Gornall together with Saxifraga
bourgaeana Boiss. and Reuter and Saxifraga biternata Boiss. (Webb and
Gornall 1989). These species are endemic to the western Betic Range (Rivas-
Martínez et al. 1991) and are included in the Spanish Red List of Vascular
Flora, based on IUCN criteria (Moreno 2008) and the Red List of Andalusia
(Cabezudo et al. 2005). Saxifraga gemmulosa has been catalogued as vulnerable
(VU) in both lists.
Saxifraga gemmulosa is an interesting endemic species distributed
mainly throughout the ultramafic territories of Málaga, Spain, covering Sierra
Bermeja and adjacent mountains (Real, Parda, Palmitera, Aguas). It is
found at 600–1400 m, growing in rock crevices with other serpentinophytes
such as Asplenium adiantum-nigrum subsp. corunnense (Christ) Rivas Mart.
and Notholaena marantae (L.) Desv. (Asensi et al. 2004). It also colonizes
ultramafic rock crevices in shady slopes in thermo- and mesomediterranean
subhumid-humid territories with bryophytes and ferns such as Anogramma
leptophylla (L.) Link. and Selaginella denticulata (L.) Link. (Pérez et al.
1989). Two peculiar phytosociological associations, from the Bermejan
biogeographical subsector, Rondean sector, Betic province (Rivas-Martínez
et al. 1997, 2002), were proposed to frame these habitats: Asplenio corunnensis-
Saxifragetum gemmulosae (chasmophytic ultramafic vegetation) and
Selaginello denticulatae-Saxifragetum gemmulosae (bryo-pteridophytic
ultramafic vegetation).
The main objective of this work was to gather information on the concentration
and localization of Ni in S. gemmulosa to establish its accumulator
status and to compare it with other Ni accumulator plants.
Methods
Plant and soil samples
Plant samples from two populations of S. gemmulosa were collected
from the ultramafic areas of Sierra Bermeja (Estepona). Sampled localities
were “Puerto de Peñas Blancas” (Universal Transverse Mercator coordinates
(UTM): 30SUF0242) and “Camino de los Pinsapos” (UTM coordinates:
30SUF0240), both in the Spanish Sierra Bermeja (Málaga: Genalguacil).
Herbarium voucher specimens are preserved in the Faculty of Pharmacy,
Complutense University at Madrid (MAF 167907) and personal collections.
Complete individuals of each population were collected together with their
corresponding soil samples. For each sample, data corresponding to location,
bioclimatology, edaphology, and phytosociological inventories were
recorded. The bioclimatic regime of both localities is mesomediterranean
and humid. Plants grow in ultramafic crevices. Edaphic properties of this
substrate correspond to typical characteristics of ultramafic soils (basic pH,
58 Northeastern Naturalist Vol. 16, Special Issue 5
high concentrations of Mg, Ni, and Fe, and low quantities of Ca; Aguilar
et al. 1998, Rufo et al. 2005). Phytosociological data correspond to the Asplenio
corunnensis-Saxifragetum gemmulosae community (Rivas-Martínez
et al. 2001, 2002).
Total metal analysis
For elemental analysis (Na, Mg, Ca, P, K, Cr, Mn, Fe, Co, Ni, Cu, Zn,
Rb, Sr), small portions (200 mg) of samples, carefully cleaned with a
brush, and dried fragments of leaves and petioles were placed in borosilicate
test tubes and ashed at 660 ºC overnight. Ashes were digested with
2M hydrochloric acid in a HACH heater-block for 6 h at 150 ºC. Aliquots
of the different samples were analyzed by inductively coupled plasma–
mass spectrometry (ICP-MS) using an ELAN-6000 PE-Sciex (Toronto,
ON, Canada) instrument.
Sample preparation for scanning electron microscopy
A scanning electron microscope (SEM) coupled to an energy-dispersive
X-ray probe (EDX) was used to micromorphologically analyzed S.
gemmulosa roots, stems, leaves (blades and petioles), flowers, and seeds.
Leaf and stem cross-section samples were treated to enhance SEM visualization
of the different plant tissues. Dry leaf and stem samples were
fixed in situ with formyl acetic alcohol (FAA). After washing with a 0.1
M phosphate buffer (pH 7.4), they were dehydrated through a graded
ethanol series. Then they were cut with a sharp blade and mounted onto
stubs.
For the semiquantitative SEM-EDX analysis, the analyzed tissues consisted
of the following: 1) from leaves (blades and petioles)—upper and
lower epidermal cells, mesophyll, and vascular bundle; 2) from stems—
epidermis, cortex, central cylinder, and pith; 3) from roots—epidermis,
cortex, central cylinder, and pith; and 4) from flowers—sepal, petal,
stamen, pollen, and seeds. Sample preparation followed the method
described by Psaras et al. (2000). For each type of tissue four to five
samples were analyzed from each population. Cross sections of leaf and
stem were cut with a sharp blade and mounted flat on the surfaces of conductive
graphite stubs and sputters and then gold-coated in a BIO-RAD
SC 502 apparatus for electrical conductivity and to prevent charging under
the electron beam. Samples were examined with a Hitachi S-3000N
(Japan) SEM using an acceleration voltage of 20 kV and a working
analysis distance of 15 mm. During analysis, the sample stage was at
room temperature. The qualitative element composition of samples was
determined by EDX microanalysis using a INCAx-sight with a Si-Li 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 quantitative numerical data of the obtained spectra are referenced as
default to the highest peak obtained in each spectrum, which in our case
generally corresponded to C.
2009 B. Díez-Garretas et al. 59
Results
Soils
The Ni concentration in soils (Table 1) corresponded to those typically
found in ultramafic soils, within the range of 1532–4254 mg kg-1 reported by
Rufo et al. (2005). Similarly, soil concentration values for Ca, Mg, Cr, and Co
were in the range corresponding to ultramafic soils. An important characteristic
of the soils from this area is its high concentration of Fe. Elemental nutrients,
such as P and K, among others, presented extremely low concentrations.
Plants
Micromorphological analysis by SEM/EDX. Saxifraga gemmulosa is an
evergreen perennial species with numerous long stalked basal leaves, with
simple or divided blades covered by fairly long glandular hairs (Webb and
Gornall 1989). Figure 1 shows some micromorphological characteristics of
the taxon. In the leaf blade, a higher density of stomas can be detected in
the abaxial than in the adaxial surfaces (Figs. 1E and 1F). The stomas are
anomocytic, lacking subsidiary cells. In both; blade and petioles (Fig. 1A),
long glandular hairs can be observed (Fig. 1B). Seeds are ellipsoidal, medium
brown, and completely covered (including the conspicuous raphe) with
relatively coarse tubercles (Fig. 1G). The pollen grains show the granular
surface typical of the Saxifraga section (Fig. 1H; Webb and Gornall 1989).
The semiquantitative EDX analysis of the tissues of the different plant
structures only gave detectable Ni concentrations in leaf (blades and petioles)
epidermal cells (Figs. 1C and 1D). Accumulation of white material on
the irregular surface of the leaf blade epidermis was observed, which corresponded
to high concentrations of Ca and Mg according to the EDX analysis
(Fig. 1C).
Total concentration of Ni and other elements in leaves (blades and petioles).
Table 1 shows the total content of the analyzed elements in leaves,
blades, and petioles of the two selected samples of S. gemmulosa compared
with their corresponding soils. The Ni content in both populations was higher
than 0.1% D.W., implying that this species is a bioindicator that reaches
levels of hyperaccumulation.
Since SEM data are semiquantitative and Ni values are on occasion under
the limit of the microanalysis detector, it was not possible to compare the Ni
content of the adaxial and abaxial epidermis of the parts of the leaf (blades
and petioles), as in other species with higher content, such as A. serpyllifolium
subsp. malacitanum or different species of Phyllanthus (Berazaín et al.
2007, Fuente et al. 2007).
Calcium and Mg were found in high concentrations. In the case of Ca, the
values for this element were much higher than the values in the soils. In plants,
the Ca/Mg ratio was high, the reverse of that detected in soils. Similarly to Ca,
the values obtained for P and K in the plant were much higher than those in the
soils, indicating an efficient active transport for these elements in the plant.
Interestingly enough, while most of the heavy metals detected in the soil have
60 Northeastern Naturalist Vol. 16, Special Issue 5
Figure 1. SEM micrographs of micromorphological details of different structures of
Saxifraga gemmulosa and EDX microanalysis. A: Transverse section of leaf petiole. B:
Detail of glandular hairs of a leaf blade. C: EDX spectrum showing the elemental composition
of leaf blade epidermis. D: Leaf blade epidermis. E: Detail of the abaxial surface
of a leaf blade with stomata. F: Detail of the adaxial surface of a leaf blade. G: SEM
micrograph of a seed. H: Detail of pollen grains of S. gemmulosa inside the stamen.
much lower concentrations than in the plant (Cr, Mn, Fe, Co, Cu, and Zn), Rb
and Sr showed higher concentrations (Table 1).
2009 B. Díez-Garretas et al. 61
Discussion
A complete list of taxa of about 200 species from southern Iberian
Peninsula ultramafics had been previously screened for Ni accumulation.
All the studied specimens were collected by the Spanish botanist Salvador
Rivas Goday and collaborators and are preserved in the MAF Herbarium
(Faculty of Pharmacy, Universidad Complutense University at Madrid). The
screening was carried out by a semiquantitative chemical test. Only two
species of the almost two hundred tested gave a positive reaction: Alyssum
serpyllifolium subsp. malacitanum (Brassicaceae) and Saxifraga gemmulosa
(Saxifragaceae) (Rufo et al. 2004).
Total Ni concentrations measured in this work are between 1532 and
4254 mg kg-1, a range reported in soils from Sierra Bermeja (Rufo et al.
2005). The Ca/Mg rations reported here are between 0.02 and 0.33 (Table 1),
which corroborate the values reported in the same study, underscoring the Ca
deficiency existing in these soils.
The Ca/Mg ratios obtained in this work, 0.88–1.29 (Table 1), are close to
the range found in other hyperaccumulator plants (Rufo et al. 2005). As other
authors have stated regarding the ecology of serpentine soils, the Ca/Mg
ratio in plant tissues is much lower for plants growing on serpentines than
on most other substrata. In general, this pattern is observed in the published
data from serpentinicola flora (Reeves et al. 1999).
Due to its Ni concentration, S. gemmulosa should be considered a new
Ni hyperaccumulator species. Previously, Vergano and Gabbrielli (1981)
mentioned the existence of other species of Saxifraga (S. aizoon Jacq. and S.
exarata Vill.) in the Aosta Valley that were able to accumulate high concentrations
of Ni, a characteristic that was cited subsequently (Brooks 1987). Both
species are taxa with wide distributions and are not restricted to ultramafic
areas. As these data have not been confirmed (Vergnano et al. 1982), S. gem-
Table 1. Total elemental content of leaves (blades and petioles) of two populations of Saxifraga
gemmulosa (in mg kg-1) and their corresponding soils, calculated by ICP-MS technique. Ca/Mg
= molar Ca/Mg ratio.
Element Plant1 Plant2 Soil1 Soil2
Na 1273 1219 255 71.3
Mg 12,555 13,956 10,572 97,610
Ca 26,991 20,657 5882 3697
P 1913 2171 202 140
K 27,658 26,565 1074 527
Cr 19.6 23.7 768 538
Mn 82.4 95.2 1442 1226
Fe 1143 638 22,231 67,988
Co 8.10 8.01 126 132
Ni 1014 1516 1765 2066
Cu 9.40 4.72 18.3 17.8
Zn 21.7 18.4 64.2 59.4
Rb 25.5 54.8 4.74 7.20
Sr 107 85.8 5.97 13.9
Ca/Mg 1.29 0.888 0.334 0.023
62 Northeastern Naturalist Vol. 16, Special Issue 5
mulosa should be considered the first Ni hyperaccumulator and bioindicator
species of the genus Saxifraga. It remains to be seen whether other Saxifraga
species from ultramafic floras are also Ni hyperaccumulators.
The Ni distribution pattern for S. gemmulosa is similar to the pattern described
for A. serpyllifolium subsp. malacitanum (Fuente et al. 2007) and
other species from diverse ultramafic areas (Broadhurst et al. 2004, Küpper et
al. 2001), in which Ni preferentially concentrates in epidermal cells of leaves.
However, in contrast to A. serpyllifolium subsp. malacitanum (Fuente et al.
2007), the trichomes of S. gemmulosa lack calcifications or Ni concentrations
that can be detected by the techniques used in this work.
The high concentration of Ca and Mg in these plants (especially Ca, at
almost one order of magnitude higher concentration than in soils) is especially
noteworthy. Calcium accumulation is found mainly in the epidermal
cells from leaves (Fig. 1C). A similar characteristic has been observed in the
ecologically related species of S. gemmulosa such as A. serpyllifolium subsp.
malacitanum (Fuente et al. 2007).
The concentration of Rb and Sr in this plant is rather peculiar, probably
as a result of the use of the same transport systems for two critical elements
for plants, K and Ca, respectively. Unfortunately, there is sparse information
concerning the accumulation behavior for these cations in the literature
and its possible correlation with Ni accumulation. Further research should
clarify whether this is a general trend for plants growing in ultramafic soils
or is a unique characteristic of this species.
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