Characterization of a Mineral Coating of the Plant Dyerophytum Indicum

Z. Naturforsch. 2015; 70(3-4)c: 59–64

Anatoly Chernyshev* and Khaled Melghit Characterization of a mineral coating of the plant Dyerophytum indicum

Abstract: The mineral coating of Dyerophytum indicum carbonates. The apparent discrepancy of the mineral Kuntze (Plumbaginaceae) was analyzed by isotope ratio composition with that reported in the literature led us mass spectrometry, X-ray diffraction, thermogravimetric to perform an initial chemical characterization of this and differential thermal analysis, and scanning electron coating. microscopy/energy-dispersive X-ray spectroscopy methods. Its composition was found to be similar to those of the carbonate mixtures isolated from rotting cacti and speleothems. The coating consisted of three major phases 2 Materials and methods

(monohydrocalcite, nesquehonite, and calcite), a minor The samples of mineral coating from the leaves of D. indicum were phase of hydromagnesite, and traces of silica and sylvite. collected from two sites in the Sultanate of Oman: the water-bearing This is the first time that the occurrence of monohydrocal- floor of Wadi Muaiden (N23° 0.0′; E57° 39.5′), and from a wadi near cite, nesquehonite, and hydromagnesite in a living higher Muscat (N23° 2 7.7 ′; E58° 20.5′), in December 2010. Average daily tem- plant has been reported. A possible mechanism of the for- peratures at the time of collection ranged from +15 °C to +27 °C. The coating was separated from the leaf surface with a soft brush, and mation of the coating is also discussed. did not undergo any further treatment to avoid solid phase reactions. The analyses were performed within a week from the collection of Keywords: Dyerophytum; plant biomineralization; samples. Plumbaginaceae. Isotope ratio mass spectrometry (IRMS) analysis of 13C and 18O was done on a GVI Isoprime dual inlet stable isotope mass spectrom- eter (GV Instruments, Manchester, UK) interfaced with a Multiprep DOI 10.1515/znc-2014-4170 sample preparation system. Measurements were calibrated against Received October 8, 2014; revised January 13, 2015; accepted April Vienna Pee Dee Belemnite (VPDB) and Vienna Standard Mean 12, 2015 Ocean Water (VSMOW) reference standards (IAEA, Vienna, Austria). Raw δ values for oxygen were further adjusted for carbonate acid fractionation factors and converted to the VPDB scale as described previously [2]. Powder X-ray analysis was performed on a PW1710 diffractom- 1 Introduction eter (Philips Analytical, Almelo, The Netherlands); scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analyses were performed on a JED-2300 energy dispersive Dyerophytum indicum Kuntze (Arabic: ‘mellah’ or salty) X-ray analyzer (JEOL, Tokyo, Japan). Thermal analyses were done belongs to the Plumbaginaceae family. It is an erect shrub on an SDT 2960 thermogravimetric analyzer (TA Instruments, New up to 2 m tall, and is distributed throughout the Arabian Castle, DE, USA), under nitrogen gas and with a heating rate of Peninsula and Western India. In Oman, it is a common 10 °C min–1. plant of wadis (desert and mountain erosion valleys of intermittent streams) at altitudes of up to 1800 m [1]. The leaves of D. indicum are covered with friable white mineral coating (Figure 1) which has been described in the 3 Results literature as a mere salt, even usable for cooking (see e.g. The phase composition of the D. indicum mineral [1]). However, based on our own experience in the field, coating was determined from two different samples by the coating is tasteless, insoluble in water, and contains X-ray diffraction (XRD) analysis. The first XRD test (not shown) revealed six inorganic phases in the coating: *Corresponding author: Anatoly Chernyshev, The Research Council, calcite (CaCO3 trigonal), 45 wt.%; aragonite (CaCO3 PO Box 1422, PC 130, Al Athaiba, Sultanate of Oman, orthorhombic), 2 wt.%; monohydrocalcite (CaCO ·H O), Phone: +968 24343336, Fax: +968 24509820, 3 2 E-mail: [email protected] 12 wt.%; dolomite [CaMg(CO3)2], 3 wt.%; nesquehonite

Khaled Melghit: Chemistry Department, Faculty of Exact Sciences, (Mg(HCO3)(OH)·2H2O, according to [3]), 38 wt.%; and University of Constantine, 1 Ain El Bey, Algeria traces of silica. 60 Chernyshev and Melghit: Mineral coating of Dyerophytum indicum

environmental carbonate source, which suggested the involvement of the plant in the formation of the mineral. To estimate the lifetime of the mineral coating on a living plant, the following experiment was performed. Two neighboring D. indicum plants were selected. On both plants, the coating was carefully removed from a few leaves using water and brush. Simultaneously, on a few other leaves, the existing coating was marked with a food colorant. The site was then visited regularly, over the course of 10 days, to check the state of the leaves. By the end of the experiment, we observed neither the formation of new mineral nor the disappearance of the marked mineral. Therefore, the residence time of the mineral on the plant must be more than 10 days, and the week elaps- ing between sample collection and XRD analysis should have not influenced its composition significantly. It has been reported previously that the XRD analysis of lansfordite could be successfully completed only with a fresh sample of the mineral [5], as it readily dehydrates into an X-ray amorphous substance. Consequently, despite an improvement in XRD refinement upon the inclusion of lansfordite phase, a prolonged residence of the coating on the plant made the presence of this mineral unlikely. The EDS analysis of the coating (Supplement A) confirmed the presence of the elements from the phases iden- tified by XRD analysis, i.e., Ca, Mg, C, and O. In some cases, such as in plates e–h of Suppl. A, the spectrum also exhib- ited peaks of K together with Cl, indicating the presence of KCl, most likely in the form of sylvite mineral. The EDS image on plate g of Suppl. A showed silica traces, which were also detected in the initial XRD test. The samples of Figure 1: D. indicum: Whole plant (top) and close-up of leaves (bottom), with visible whitish mineral coating (scales are coating were not treated chemically before analysis, so the approximate). possibility of contamination could be excluded. There is at least one previous report on sylvite and silica as products of plant biomineralization in Tradescantia pallida [6]. To rectify these results, we repeated the analysis Based on the critical analysis of XRD and EDS data, with Rietveld refinement, as implemented in the FullProf the composition of D. indicum mineral coating can be best program [4]. The best fit to the experimental data (Figure 2) described as having three major mineral phases: mono- was reached when the phases of monohydrocalcite, hydrocalcite (∼13 wt.%), nesquehonite (∼38 wt.%), and nesquehonite, and calcite were combined with lansfordite calcite (∼45 wt.%); a minor hydromagnesite phase; and

(MgCO3·5H2O) and hydromagnesite [Mg5(CO3)4(OH)2·4H2O]. traces of silica and sylvite. The presence of lansfordite, However, the inclusion of dolomite or aragonite did not aragonite and dolomite, though possible, requires addi- improve the refinement, so the presence of these minerals tional experimental evidence. in the coating remains to be confirmed. The plot of the TGA and DTA analyses (Figure 3) We performed IRMS analysis for 13C/12C and 18O/16O revealed three pronounced endotherms, which corre- isotope ratios in the mineral phase collected from two spond to the dehydration of nesquehonite to magnesite plant specimens, and compared the results with those (MgCO3) at 125 °C, followed by decomposition of the latter obtained for wadi water evaporite, surrounding mineral at 440 °C, and the decomposition of calcite at 745 °C [7]. The dust and calcareous rocks, as well as with literature data DTA curve does not contain the exotherm at 510 °C, which on atmospheric CO2 (Table 1). The concentration of either is typical for the decomposition of hydromagnesite [7]. 13C or 18O in the coating was higher than in any possible The absence of this peak indicates that nesquehonite Chernyshev and Melghit: Mineral coating of Dyerophytum indicum 61

Figure 2: Rietveld plot of D. indicum mineral coating. The upper trace shows the observed data as dots overlaid with the calculated pattern (solid line). The lower trace is the difference plot between the observed and calculated pattern. The vertical markers show the positions calculated for Bragg reflections for the following phases: I, MgCO3·5H2O; II, CaCO3.H2O; III, Mg5(CO3)4(OH)2.4H2O; IV, Mg(HCO3)(OH)·2H2O; V, CaCO3 (calcite).

Table 1: Delta values (δ) for 13C and 18O ratios for two samples of D. indicum coating, wadi water evaporite, local mineral dust, and calcite rocks (VPDB scale).

δ, Plant 1 δ, Plant 2 δ, Plant (average) δ, Water evaporite δ, Mineral dust δ, Calcite rock δ, CO2, atmospheric [2] 13C 3.71 4.96 4.34 −1.39 1.4 2.78 −7.5 18O 15.85 16.99 16.42 −4.70 −2.71 −1.16 6.9

dehydrates directly to magnesite. A small endotherm at dissolution and re-precipitation of carbonates from brine ca. 470 °C is close to the temperature of the decarboniza- in isolated pores. A comparably large isotope fractionation tion of hydromagnesite [8], and a similar endotherm at has also been observed in a chemical system when calcite 205 °C fits best with the reported dehydration of monohy- precipitated concurrently with monohydrocalcite [11]. drocalcite at 171 °C–206 °C [9]. The reported δ13C and δ18O values for monohydrocalcite and calcite in decaying saguaro [5] are much lower than in D. indicum coating. 4 Discussion The composition of the D. indicum coating bears a number of typical features of biomineralization as well A positive value of δ13C in biogenic carbonates has been as some uncommon ones. Almost the same combina- reported previously, but only for a few species of benthic tion of minerals has been observed in speleothems, i.e., algae [2]. Moreover, the δ13C of the D. indicum mineral cave mineral deposits [12, 13]. The microscopic morpho coating is unusually large by magnitude, and is compa- logy of the coating is also somewhat similar to that of the rable only to that of calcareous sediments associated described speleothem formations in that it consists of with seagrasses [10]. In the latter case, 13C enrichment of ‘knobs’ formed by microcrystalline concentrical layers of the sediments had been attributed to repetitive cycles of carbonates (Figure 4). 62 Chernyshev and Melghit: Mineral coating of Dyerophytum indicum

Figure 3: Thermogravimetric (TG, solid line) and differential thermal analysis (DTA, dashed line) curves of D. indicum mineral coating.

The major component of the coating, calcite, is a fre- The magnesium carbonates nesquehonite and lans- quent biogenic mineral in animals, but is seldom seen in fordite (MgCO3·5H2O) have previously been found in plant tissues. The remarkable, albeit sparse, examples of decomposing saguaro [5], but neither of them is formed higher plant calcification are cystoliths in the leaves of in the living plant. In decaying saguaro, nesquehonite some plant families [14, 15], rhizoliths, i.e. formations of was the most abundant mineral. Similarly, in D. indicum, calcareous cementation on the surface of roots [16, 17]. the nesquehonite content (38%) is high but not as high There are also reports for Cactaceae as well as for some as that of calcite (45%). To our knowledge, there are no algae, fungi, and bryophytes [18, 19]. other published cases of magnesium carbonates formed Aragonite is a metastable phase of calcium car- by plants. These minerals form in the presence of hydro- bonate, but its transformation to calcite is negligibly carbonate anions and magnesium cations, producing slow and may take up to thousands of years [20] under primarily lansfordite at below +4 °C, or nesquehonite at normal conditions. Therefore, it cannot be considered higher temperatures [5, 23]. Hydromagnesite [Mg5(CO3)4 as a transient mineral on a pathway leading to calcite, (OH)2·4H2O] can precipitate from solution at either high and if any aragonite had been formed by the plant, it temperatures (above 52 °C) or at lower temperatures in should have remained as such in the coating. There are the presence of bacteria [13, 24–27]. It is also a metasta- at least two chemical mechanisms facilitating aragonite ble phase, which can be transformed into more stable crystallization from complex carbonate mixtures: either dolomite in the presence of Ca2+ [27]. The average daily through partial monohydrocalcite dehydration in a wet temperatures at the time of the formation of the mineral atmosphere [21], or in the presence of elevated concen- samples studied in the present work (15 °C –27 °C) were far trations of Mg2+ [22]. The nesquehonite content of the below 52 °C required for the purely chemical precipitation coating indicates significant amounts of magnesium in of hydromagnesite. This points to the involvement of the the plant exudates; however, calcium carbonate is pre- plant in this process. The absence of dolomite and arago- sumably present as calcite rather than aragonite. The nite from the samples, together with the major presence absence of aragonite from the coating suggests that there of nesquehonite and calcite, allows us to hypothesize that are no conditions favoring its formation during plant the plant exudate changes its composition periodically. It mineralization. might contain high concentrations of either Ca2+ or Mg2+, Chernyshev and Melghit: Mineral coating of Dyerophytum indicum 63

fragments, similar to those of cyanobacteria or fungal hyphae, but their actual origin has not been determined. Based on our data and those that have been previously published, we suggest the following scheme for D. indicum biomineralization (Figure 5). First, the plant produces an exudate with elevated concentrations of calcium or magnesium, and hydrocarbonate. If the major cation is Ca2+, monohydrocalcite precipitates first, according to the mechanism suggested by Fischbeck and Müller [13], which leads to an increased Mg2+/Ca2+ ratio. This ratio facilitates the precipitation of nesquehonite (or lansfordite at low temperatures) and small amounts of aragonite; moreover, Marschner reports that contact with air is essential for monohydrocalcite formation [21]. When brine contained high concentrations of Mg2+, the major precipitate was nesquehonite, in the same proportion as in the D. indicum mineral coating, i.e., 38%. Monohydro calcite then gradually dehydrates either to calcite or to aragonite [31]. Aragonite forms in wet atmosphere, at temperatures ranging from −2 °C to +50 °C. In dry atmosphere, or in wet atmosphere containing ammonia, the major product of dehydration is calcite [21]. Further maturation of the mineral mixture might involve such reactions as transformation of nesquehonite to hydromagnesite [24], dissolution of nesquehonite in the exudate and subse- quent crystallization of monohydrocalcite and dolomite [25], and repetitive recrystallization of the participating Figure 4: SEM of mineral coating of D. indicum: general morphology mineral phases leading to heavy isotope enrichment [11]. of calcareous ‘knobs’ (top), and nesquehonite prismatic crystals The total water content of the coating, according to embedded in the mineral matrix (bottom). the TGA analysis is 18%. This water could be important for the plant during drought periods. In addition, the coating but not both simultaneously. An indirect indicator of such can accumulate water both mechanically and chemically, periodical changes is the layered structure of the coating (Figure 4). Another component of the coating, monohydrocalcite, is a common satellite mineral of nesquehonite [21]. This couple of minerals has also been found in rotten saguaro [5] and in limestone caves [13]. An uncommon feature of the coating is that it does not contain oxalates, the widespread products of plant biomineralization. Specifically, oxalate crystals are often observed in desert plants [5, 18, 28], and in association with calcite cystoliths [15]. A possible explanation could be that leaf surface-associated microorganisms partici- pate in mineral formation. The oxalate anion could either be degraded by these microorganisms into the carbonate, or not formed at all. 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