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). 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