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Bryology and Lichenology |
2Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602 USA; 3Department of Botany and Range Science, Brigham Young University, Provo, Utah 84602 USA; 4Department of Physics and Astronomy, Brigham Young University, Provo, Utah 84602 USA; 5Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California 94550 USA
Received for publication October 20, 2000. Accepted for publication March 22, 2001.
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ABSTRACT |
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Key Words: calcium oxalate • lichen • mycobiont • Parmeliaceae • proton microprobe PIXE
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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; Palmqvist, 2000
). The mycobiont in turn uses these resources to construct and maintain a complex, living "habitat" that effectively addresses many of the environmental and nutritional requirements of the photobiont (Nash, 1996a
; Palmqvist, 2000
). Many of the specific attributes and dynamics of this complex symbiotic relationship are still unclear, including the hypothesized role of the mycobiont in acquiring and delivering mineral nutrients to the photobiont (Palmqvist, 2000
). Since the photobiont is effectively isolated from mineral nutrient resources by the fungal "habitat," a mycobiont-mediated delivery mechanism likely exists. Quantitative analysis of the distribution of mineral elements in the stratified layers of a foliose lichen could provide evidence for such a mechanism. Furthermore, such information might also provide valuable insights into the dynamic metabolic interactions that underlie and sustain the lichen symbiosis. In this paper the authors describe and discuss the results of proton microprobe PIXE analysis of thin sections of the foliose, vagrant soil lichen Xanthoparmelia chlorochroa.
Scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDX) spectroscopy has been widely used to obtain qualitative element information on the microscopic level in biological samples. Several studies of lichens have been conducted using SEM-EDX (Garty, Galun, and Kessel, 1979
; Jackson, 1981
; Jones, Wilson, and McHardy, 1981
; Wadsten and Moberg, 1985
); however, these data were not quantitative. Much of this work focused on dense, inorganic crystal deposits on the exterior (pruina) and interior of the thallus where element concentrations are relatively high. While this technique is adequate for major and some minor element concentrations, its lack of sensitivity reduces its usefulness in element analyses for concentrations below several hundred parts per million (ppm). However, SEM imaging and semi-quantitative EDX analysis of structures has served an important complementary role in this research.
Another analytical technique is microbeam proton induced x-ray emission (PIXE) spectroscopy, which yields two-dimensional, quantitative element information with micron-level spatial resolution. Instead of using electrons, PIXE uses protons accelerated to energies of 2–5 MeV (megaelectron volts) to generate the characteristic x-rays of elements in a sample. The use of protons gives microbeam PIXE several distinct advantages over SEM-EDX analysis. Energetic protons produce less Bremsstrahlung background relative to SEM-EDX, enabling quantification of trace elements in the low ppm range. In addition, proton beams have a much greater range through biological material, and exhibit minimal spatial broadening compared with electron beams (Watt, 1997
). The greater proton range permits analysis of thicker samples.
A wide variety of biological samples have been analyzed using proton microprobe PIXE (Watt et al., 1991
; Watt and Landsberg, 1993
), including higher plants (Reiss et al., 1985
; Hughes et al., 1988
; Mesjasz-Przybylowicz et al., 1994
; Przybylowicz et al., 1996
), fungi (Gadd et al., 1988
), and bryophytes (Watkinson and Watt, 1992
). The stratified nature of foliose lichens makes them particularly well suited to microbeam analysis. Preliminary analysis of the foliose lichen Xanthoparmelia chlorochroa was recently reported (Clark et al., 1999
). The results of this study showed significant element partitioning in the thallus. Nuclear microprobe analysis of another individual of the same species is reported herein, along with analytical data from several other techniques (SEM, thermogravimetry, and fluorometry). The purpose of this study was to obtain quantitative element distribution patterns within a stratified lichen thallus at micrometer-level resolution and very low element concentrations. It was expected that detailed element measurements would provide basic insights into the dynamics of mineral nutrient transfer between the mycobiont and photobiont.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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). Additional samples were thin-sectioned with a glass knife using a cryo-microtome at –65°C. Thallus sections (20 µm thick) were then transferred onto a thin nylon foil, free of trace metals, and freeze dried. Sections were analyzed at the nuclear microprobe facility in the Center for Accelerator Mass Spectrometry (CAMS) at Lawrence Livermore National Laboratory. A 3 MeV proton beam was scanned across the sample using electrostatic deflection. Protons passed completely through the sample. Samples were first analyzed using scanning transmission ion microscopy (STIM), which measured proton energy loss at each pixel in the scan. These data were used to calculate the areal density (or thickness) of the sample in milligrams per square centimeter. The second measurement, along the same scan path, was a microbeam PIXE scan, which generated data for the element maps.
Interactive Data Language software (Research Systems, Boulder, Colorado, USA) was used to generate element maps from the PIXE data. Specific areas of interest were then selected from the x-ray map and analyzed using spectral analysis code PIXEF to obtain x-ray counts for each element. Average proton energy loss through the same area was used to calculate average areal density. Thin film standards of known element mass for Ca, Ti, Cr, Fe, Cu, and Sr were used to calibrate the x-ray detector response. Quantitative calibration factors were calculated for specific areas of the sample using the detector calibration and the sample areal density. The sample element matrix was assumed to be typical of biological material except when modifications were necessary for larger particles and the calcium layer. Element concentrations (ppm) were calculated by multiplying x-ray counts in a given area by quantitative calibration factors. This method assumes element concentrations are relatively consistent in a given area. Values for Al and Si are only semiquantitative due to a lack of calibration standards in this x-ray region. A more detailed discussion of sample preparation and experimental techniques and parameters is contained in Clark (2001)
.
Electron micrographs were generated using a JEOL 840 scanning electron microscope (Jeol, Peabody, Massachusetts, USA) equipped with an energy dispersive x-ray detector. Data from microbeam PIXE element maps prompted additional analysis using the following instruments: thermal gravimetric analysis (TGA) using a Seiko Instruments SSC/5200 TG/DTA 220 (Seiko Instruments Inc., Torrance, California, USA), UV fluorescence analysis using a Perkin Elmer LS 50B luminescence spectrometer (Perkin Elmer, Inc., Wellesley, Massachusetts, USA) with a 495 nm long pass filter to eliminate higher order diffraction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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100 µm. The region from 140 µm to 200 µm is the algal layer, with the medulla extending from 200 µm to just beyond 500 µm. The lower cortex is located between 520 and 530 µm. Each point on the concentration profile is 3 pixels long (15 µm) and the full width (15 pixels or 45 µm) along the short axis of the scan. Element concentrations were calculated in each increment under the assumption that element concentrations were consistent across the short axis. With the exception of a few areas in the upper cortex, the element maps substantiate the above assumption. The observed distribution and concentration of calcium prompted further analysis to identify the molecular composition of the calcium layer. Thermal gravimetric analysis confirmed the presence of calcium oxalate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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; Clark et al., 1997
). However, thallus concentrations for calcium (9.22% mass) and arsenic (26 ppm) represent some of the highest values reported for the Intermountain Area. In spite of the overall variability in element concentrations along the scan path, several distinctive distribution patterns emerged. For example, phosphorus, sulfur, chlorine, and arsenic are distributed similarly, with elevated concentrations on the upper and lower surfaces, lower concentrations in the algal layer, and even lower concentrations in the medulla (Fig. 1). These four elements likely occur as anions (PO43–, SO42–, Cl–, and AsO43–), which share similar transport mechanisms. This most likely explains their similar distribution patterns. The highest levels of potassium (18%) are on the lower cortex, with elevated concentrations also on the upper cortex with decreasing concentrations in the medulla. In contrast, the highest concentrations of calcium are in the medulla, with lower concentrations in the algal layer and lower cortex. The transition metals Mn, Cu, and Zn, show an intermittent distribution in the thallus with the lowest levels in the medulla (Fig. 3). The metals Ti, Cr, Fe, and Ni (Figs. 4, 5) show generally low, but relatively uniform concentrations across the thallus except in well-defined particles on the lower cortex. The crustal elements Al, Si, Ti, and Fe (Figs. 4, 5) show large concentrations on the lower cortex, suggesting a strong substrate connection for these elements.
Since the mycobiont makes up the bulk of the thallus and directly interfaces with the external environment, it has generally been assumed that mineral nutrients are acquired and delivered to the photobiont by the mycobiont (Palmquist, 2000
). Data from 25 microbeam PIXE scans reported by Clark et al. (1999)
and Clark (2001)
for 12 lichen species clearly demonstrate that several key nutrient elements are specifically partitioned in relation to the algal layer. These data strongly support the assumption that there is a mycobiont-mediated mechanism for specifically acquiring and delivering essential mineral nutrients to the photobiont layer. The specific location of these elements, interior or exterior to the cell walls of the symbionts, is still uncertain and suggests the need for additional research.
Particle entrapment
Element maps show small regions of elevated element concentrations consistent with inorganic particles imbedded in the thallus. This is not unexpected, since particle entrapment has been recognized as a nutrient accumulation mechanism in lichens (Edwards, Farwell, and Seaward, 1991
). Four such particles, near the upper cortex, are apparent in Figs. 3 and 5. The particle size, chemical composition, and position vary. Element concentrations in these four particles are summarized in Table 2. Particle 1 (10–15 µm in diameter) is located in the center, 90 µm from the left edge of the scan. Particle 2 is immediately above particle 1 on the top edge of the scan and has approximately the same diameter. Particle 3 is located just to the right of particle 2 and is 15 µm in diameter. Particle 4 (25–30 µm in diameter) is 140 µm from the left and in the lower part of the scan. SEM scans of the region showed that these particles were not on the surface of the sample section, but were imbedded in the thallus. A fifth particle (25–35 µm in diameter), seen in the titanium map in Fig. 4, is located near the bottom of the scan at 300 µm.
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150 ppm iron and 2500 ppm Ti. This Fe to Ti ratio, 0.06, is unusual given that the lithosphere Fe/Ti ratio is 6.5 (Poldervaart and Green, 1959
). The absence of other elements in this region raises the possibility that the particle is TiO2, a common white compound used in paint and paper. In the lower cortex, the following elements have peak concentrations in the increment centered at 510 µm: Al, Si, S, K, Ti, Fe, and Cu. These elements account for >30% of the mass in this region. The lower cortex likely contains soil particles smaller than the 5 µm resolution. In the increment centered at 495 µm, P, Cl, Zn, and Mn have peak concentrations. Mn also has a peak at 465 µm due to a particle with a considerable amount of Fe. These elements seem to be moving into the thallus more readily than the previously listed elements. In contrast, Ca and Sr have distinctly lower concentrations in the lower cortex. The calcium pattern also shows a void region between the medulla and upper cortex. This pattern suggests that calcium is transported efficiently through the upper and lower cortices into the medulla.
Fe/Ti ratios for particles 2, 3, and 4 varied from 16 to 40+. In contrast, the Fe/Ti ratio in the lower cortex is 1.4, while the Fe/Ti ratio in particle 5 is 0.060. Overall, the Fe/Ti ratios are distributed on either side of the ratios reported for the bulk material (7.9) and the lithosphere (6.5). These elements may be in different mineral phases as they enter the lichen from the substrate or from dust fall. The mycobiont may also be moving these elements within the thallus at different rates.
Particle entrapment by Xanthoparmelia chlorochroa appears to be common. Particle origin, whether from the atmosphere or substrate, is uncertain. Some particles show decreasing element concentrations with distance from their center, which may indicate particle dissolution. However, we cannot discount the possibility that these profiles are an artifact resulting from particle-detector geometry.
Calcium chemistry and possible functions
The most striking feature of the element maps is the large band of calcium in the medulla below the algal layer. The average calcium concentration in the sample is 9.22% mass (Table 1). Figure 2 shows clearly that calcium is not homogeneously distributed across the thallus, and in several areas it comprises more than 10% of the sample by mass. Since these levels far exceed nutrient requirements, an alternative explanation for this distribution pattern is required. A likely chemical form might be an inorganic calcium salt of carbonate or oxalate. This seems consistent with the elemental data, since concentrations of phosphorus, sulfur, and chlorine in the medulla are several orders of magnitude lower than calcium, suggesting the absence of significant amounts of Ca3(PO4)2, CaSO4, and CaCl2. Wadsten and Moberg (1985)
and Purvis (1984)
have reported that the most common inorganic form of calcium in lichens is calcium oxalate (CaC2O4). In addition, Fourier Transform Raman spectroscopic studies have documented the extensive accumulation of calcium oxalate in lichens (Edwards, Farwell, and Seaward, 1991;
Edwards et al., 1992;
Seaward and Edwards, 1995;
Edwards, Farwell, and Seaward, 1997;
Seaward, 1997
).
Lichenized fungi have been shown to secrete oxalic acid as a metabolic by-product (Syers and Iskandar, 1973
; Jones, Wilson, and Tait, 1980
). The SEM image in Fig. 7 shows fungal hyphae in the calcium-enriched area heavily encrusted with small crystals. Calcium produced the most prominent EDX signal in this region. There is a distinct difference between these hyphae (Fig. 7) and those in Fig. 8, which are located in the medulla near the lower cortex, which show essentially no crystal development. The EDX spectrum in this region (near the lower cortex) showed much lower calcium levels than the region shown in Fig. 7.
Calcium oxalate is hygroscopic and exists in several hydration states, the two most common being monohydrate, CaC2O4·H2O (whewellite) and dihydrate, CaC2O4·(2+x)H2O (weddellite). The presence of CaC2O4·(2+x)H2O in our samples was confirmed using thermal gravimetric analysis (TGA), which has been used previously to analyze lichens (Syers, Birnie, and Mitchell, 1967
). Calcium oxalate is a TGA standard that is well characterized. The TGA results (mass loss as a function of increasing temperature) are shown in Fig. 9.
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and Edwards, Farwell, and Seaward (1997)
hypothesized that calcium oxalate may serve some role in water regulation, but neither presented any specific data or discussion supporting the hypothesis. The dihydrate crystal structure has pores, which can accommodate up to an additional 0.5 mole of water per mole of CaC2O4 (Frey-Wyssling, 1981
). This phenomenon may provide an explanation for the proximity of the calcium oxalate band to the algal layer. Calculations from the TGA data showed that 13.7 µg of zeolitic water were released when 1.704 mg of lichen was heated from 25°C to 40°C, yielding a total of 8 mg of water per gram. These temperatures match with summer conditions at the Mercur site. The zeolitic water could be replenished by dewfall during early morning hours and then released at high temperatures later in the day. This may explain how the lichen is able to maintain minimal but essential water levels for photobiont activity during the prolonged dry periods typical of the hot summer months.
Furthermore, the TGA data indicate that 24.4 µg of water were released in the temperature range characteristic of the dihydrate decomposition, with 25.0 µg released at the temperature range characteristic of the monohydrate decomposition. The ratio of water mass released in the three transitions (zeolitic :monohydrate : dihydrate) is 0.5 : 1 : 1, suggesting that most, if not all, of the hydrated calcium oxalate is CaC2O4·(2+x)H2O. Wadsten and Moberg (1985)
, Prieto et al. (1999)
, and Modenesi et al. (2000)
reported that lichens from humid climates produced only calcium oxalate monohydrate, while lichens from drier climates contained CaC2O4·(2+x)H2O. The Mercur site is an arid desert location, and predictably monohydrate crystals are essentially absent. This provides further evidence supporting a possible water regulation role for the calcium oxalate band.
Under hot summer conditions the water released would be in the vapor phase, which is significant considering green algal photobionts are able to use water vapor for photosynthesis (Nash, 1996a). This mechanism could supply sufficient water vapor to the algal layer so that it is consistently able to maintain some photosynthetic activity, as the CaC2O4·(2+x)H2O band alternately traps and releases zeolitic water. The size of the crystals and their proximity to the algal layer would be important factors in the operation of this mechanism. Figure 7 shows that the CaC2O4 crystals are generally 0.1–0.5 µm in diameter, much smaller than those reported by Wadsten and Moberg (1985)
on the cortical surface. The small crystal size maximizes crystal surface area and thus zeolitic water transfer.
Calcium accumulation and localization in the medulla directly beneath the algal layer has also been reported for another specimen of Xanthoparmelia chlorochroa from Montrose, Colorado, USA (Clark et al., 1999
). In addition, similar calcium distribution patterns and calcium oxalate accumulations have also been reported for several additional lichen taxa including Xanthoparmelia cumberlandia, Rhizoplaca melanophthalma, Rhizoplaca chrysoleuca, Rhizoplaca haydenii, and Rhizoplaca marginalis (Clark, 2001
). However, other foliose lichens, three from the genus Umbilicaria (torrefacta, virginis, and americana), Lasallia papulosa, and Dermatocarpon reticulatum failed to show any significant calcium accumulation (Clark, 2001
).
An additional benefit of the location of the calcium oxalate band relative to the photobiont layer may relate to photosynthetic efficiency. The number of algal cells within the lichen is regulated by the mycobiont (Palmqvist, 2000
). To increase photosynthetic output, increased opportunity for light absorption may be as important as chlorophyll concentration or algal cell density. Modenesi et al. (2000)
suggested that crystals can possibly act as radiation reflectors, but did not report any data confirming this idea. To explore the possible role of calcium oxalate in light regulation, the reflection properties of calcium oxalate were calculated and studied using a fluorometer.
The reflectance, 
, at a calcium oxalate-air interface of radiation normal to the interface plane may be calculated using the Fresnel equation:
and air are the indices of refraction of calcium oxalate and air, respectively. The reflectance is 4% at each interface and would increase at any angle of incidence greater or less than 90°. Since each crystal represents an interface, the combined reflectivity of the calcium oxalate deposit in Xanthoparmelia chlorochroa is very large. Assuming a mean crystal diameter of 0.5 µm, 400 different crystal surfaces could be present if the deposit is 200 µm thick. The fluorescence spectrum of calcium oxalate was measured using a fluorometer. When 200 nm uv light was directed at the crystals, a broad wavelength shift was observed between 350 and 700 nm (Fig. 9). The intensity of the shift decreased dramatically when the excitation wavelength was changed to 250 nm (Fig. 10), and no shift at all occurred at 300 nm. Consequently, the spectral shift appears to be wavelength dependent and suggests that calcium oxalate shifts uv light into the visible region of the electromagnetic spectrum, thus making at least some uv energy available for photosynthesis. In view of these data, the role of calcium oxalate as a radiation reflector seems reasonable. This role could also explain the propensity of a number of lichens to accumulate substantial calcium oxalate deposits (pruina) on the upper cortex. These deposits would effectively decrease the amount of radiation reaching the algal layer. However, in Xanthoparmelia chlorochroa (from the Mercur and Montrose sites) and Rhizoplaca spp. the calcium oxalate deposits are located below the algal layer. With this orientation the calcium oxalate crystals would increase radiation to the photobiont layer. However, during cool seasons (spring and fall) and on overcast days in the summer, when light might be limiting, the calcium oxalate band could effectively concentrate light into the algal layer. Ultraviolet light is not attenuated on overcast days as much as visible light and would constitute a larger fraction of the radiation reaching the lichen. Under these circumstances the lichen could benefit from a uv to visible light shift near the algal layer.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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Some of this chemical partitioning may promote photosynthetic activity by creating a supportive environment for the photobiont. Element maps and SEM images show dense calcium oxalate bands at the interface between the medulla and algal layer. The strategic location of the calcium oxalate band below the algal layer suggests it may play several roles, including release of water vapor at higher ambient temperatures and light regulation. Regulation of water vapor and light by calcium oxalate may increase photosynthetic activity without an increase in algal cell density, allowing the lichen to maximize photosynthetic output without expending energy and resources to increase light harvesting ability. We are not the first to propose either mechanism, but our data provides new evidence to support these ideas.
Over the years lichenologists have argued for a biotrophic (parasitic) interpretation of the lichen association (Honegger, 1991
; Nash, 1996b
; Palmqvist, 2000
) and certainly for some species this conclusion has merit. However, in lichens with complex, highly evolved thalli the evidence is compelling for a mutualistic interpretation of the relationship (Honegger, 2001
). For example, in the structurally more complex lichens the data clearly show that the mycobiont actively addresses several fundamentally important environmental requirements of the photobiont, including organization and mechanical support of photobiont cells in a defined section of the thallus (Bûdel and Scheidegger, 1996
), regulation of the quality and quantity of light reaching the algal layer (Kappen, 1988
), chemical control of herbivory (Reutimann and Scheidegger, 1987
), the operation of a CO2 concentrating mechanism in water saturated cyanolichens (Palmqvist, 1993
; Palmqvist et al., 1994
), and a mycobiont-mediated mechanism for delivery of water and solutes from thallus surfaces to the algal layer (Honegger, 1997
). Furthermore, the lichen symbiosis accommodates a significant increase in the ecological range of the photobiont partner (Honegger, 2001
). Proton microprobe data, which have clearly documented well-defined element distribution patterns in some foliose lichens, provides additional support for a mutualistic interpretation of at least some lichen associations. By definition, mutualism involves interactions between symbionts where all partners are benefited. Clearly, in many lichen species both bionts derive substantial benefits from the association. Collectively, these data require rethinking of the way we view the lichen symbiosis.
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FOOTNOTES |
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6 Author for reprint requests (Tel.: +1 801 378 3668; Fax: +1 801 378 5474; e-mail: nolan_mangelson{at}byu.edu
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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