- Email: [email protected]
Preparation of pollen for stable carbon isotope analyses
Chemical Geology 165 Ž2000. 339–344 www.elsevier.comrlocaterchemgeo
Technical Note
Preparation of pollen for stable carbon isotope analyses N.J. Loader a
a,)
, D.L. Hemming
a,b
The Godwin Institute for Quaternary Research, Department of Earth Sciences, Pembroke Street, UniÕersity of Cambridge, Cambridge, CB2 3SA, UK b Laboratory of Tree Ring Research, UniÕersity of Arizona, West Stadium a58, Tucson, AZ, 85719, USA Received 5 May 1999; received in revised form 6 August 1999; accepted 6 August 1999
Abstract Differential diagenesis of the chemical components of pollen in sub-fossil records necessitates the isolation of a single ‘resistant’ chemical component of pollen, prior to its isotopic analysis. As sporopollenin is particularly resistant to diagenesis and comprises typically 65%–80% of the pollen grain, it is a suitable component for this purpose. Conventional Žacetylation. methods of pollen preparation for palynological studies are reliant on the use of carbon-bearing compounds which introduce large errors to the pollen carbon isotopic composition Ž d13C.. We present results from a novel non-carbon containing acid extraction technique to isolate sporopollenin. Using this technique we demonstrate that the d13C signals in raw and processed pollen are highly correlated, but display an offset associated with the non-sporopollenin components. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Pollen; Carbon stable isotope; Palynology; C 13 rC 12 ; Sporopollenin; Pollen analysis
1. Introduction Stable isotopes have revolutionised the way in which we can interpret proxy environmental records and their use has enabled a more complete understanding of palaeoenvironmental change. At present, the majority of this quantitative work has been confined to the isotopic analysis of inorganic carbonates deposited in the deep oceans ŽShackleton and Opdyke, 1973; Jansen, 1989; Shackleton et al., 1990; Patience and Kroon, 1991; Duplessy et al., 1992.. While a number of terrestrial isotopic indicators of environmental change have been exploited, including measurements from tree-rings ŽEdwards et al., 1985; )
Corresponding author. Tel.: q44-1223-334870; fax: q441223-334871; e-mail: [email protected]
Leavitt and Long, 1991; Hemming et al., 1998., speleothems ŽBar-Matthews et al., 1998., ice ŽThompson et al., 1986., sub-fossil leaves ŽTurney et al., 1997. and lacustrine cores ŽGasse and Lin, 1988., none of these exhibit the geographical coverage and temporal range of the ocean record. Conventional palynology provides a large-scale indicator of vegetation dynamics operating over long Žgeological. timescales ŽBirks and Gordon, 1985; Birks and Line, 1993; Tzedakis and Bennett, 1995.. These analyses often involve considerable time-lags between environmental forcing and vegetation response, such that the precise timing of environmental changes, especially rapid ones, are difficult to identify. If the products of photosynthesis used to form pollen grains reflect variations in the carbon-dioxide and meteoric water utilised by the plant and its
0009-2541r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 5 4 1 Ž 9 9 . 0 0 1 7 6 - X
340
N.J. Loader, D.L. Hemmingr Chemical Geology 165 (2000) 339–344
metabolism, then the stable isotope ratios of pollen grains will indicate these changes. Therefore, comparison of existing, well dated, palynological records with corresponding pollen stable isotope data will help identify the timing of environmental changes and the magnitude and phase relationship of the associated vegetation response. The relative proportions of organic substances composing pollen grains varies with species and degree of diagenesis ŽMoore et al., 1991; Benner et al., 1987.. As these components exhibit different isotope compositions it is necessary to isolate a single chemical component for isotopic analysis. Sporopollenin ŽZetzsche, 1932. is particularly resistant to diagenesis and comprises typically 65%–80% of the pollen grain ŽShaw and Yeadon, 1964; Brooks and Shaw, 1968.. As such, it is the most suitable component for isotope analyses. Traditionally pollen has been concentrated from core samples using a suite of chemical oxidation techniques. However, Amundson et al. Ž1997. demonstrated that one of these processes, acetylation Ž‘acetolysis’, Erdtman, 1960., provided a significant source of carbon isotopic contamination. The reason for this is most likely related to the partial removal of glacial acetic acid used during processing, or incomplete removal of cellulose acetate, a by-product of acetylation. Consequently, d13 C measurements on acetylated pollen will not yield a reliable signal. To avoid carbon contamination a simple and inexpensive acid digestion technique to isolate sporopollenin was developed and its effectiveness assessed.
2. Method To test if the d13 C signal present in raw pollen is retained in the isolated sporopollenin, the d13 C ratios
of raw and acid digested C3 and C4 pollen covering a wide isotopic range were compared. Samples of modern pollen from three species Ž Betula pendula, Pinus sylÕestris and Picea abies . were collected along a 1000 m altitudinal transect in southwest Switzerland. C4 Ž Zea mays . samples were obtained commercially ŽSigma, USA.. As the amount of C3 pollen collected for each sample was small, to allow suitable sample replication during technique development, additional C3 pollen Ž Populus tricocarpa. were also commercially obtained ŽSigma, USA. and a larger quantity of Pinus sylÕestris pollen donated by the Botanical Garden in Zagreb. The dry pollen was sieved and ca. 10 mg was placed into a test tube. 10 ml of concentrated sulphuric acid was added to each and the samples were covered and left to stir gently at room temperature using a magnetic ‘flea’. The influence of processing time was examined by subjecting samples to 0.5, 1, 2, 4, 8, 24, 48 and 168-h treatments. The solution was filtered using Whatman 3 ml Vectaspin 10 mm polypropylene mesh microcentrifuge tubes ŽWhatman International, UK. and the extracted pollen was retained in the filter insert. Samples were washed with cold de-ionised water and centrifuged at 3000 rpm for 5 min. This step was repeated until the filtrate was clear and no residue was evident in the holding tube after centrifuging. Samples were then vacuum dried in their tubes prior to d13 C analysis. Isotope measurements were carried out using a Fisons NA1500NC elemental analyser interfaced, via a cryogenic trap, to a SIRA II Isotope Ratio Mass Spectrometer. Results are expressed as per mil Ž‰. deviations from the PDB-1 standard ŽCoplen, 1995.. To ensure that the acid digestion technique removed all the cellulose from the pollen grains a differential Ž‘saffranin-fast green’. staining technique
Table 1 Reproduceability of pollen samples used in the development of the acid digestion method Note the large offset between the acetylated and acid treated samples Žsee also Fig. 1.. Sample species
d13 C PD B raw pollen Žunprocessed.
d13 C PDB acid digestion method Žthis study.
d13 C PDB ‘acetylation’ Žafter Erdtman, 1960.
C4 Z. mays C3 Populus tricocarpa C3 Pinus sylÕestris
y10.45 " 0.03 Ž n s 5. y23.97 " 0.05 Ž n s 5. y27.39 " 0.17 Ž n s 3.
y12.26 " 0.08 Ž n s 3. y27.52 " 0.23 Ž n s 3. y29.57 " 0.08 Ž n s 12.
y18.44 " 0.18 Ž n s 5. y33.27 " 0.13 Ž n s 5. –
N.J. Loader, D.L. Hemmingr Chemical Geology 165 (2000) 339–344
341
Plate 1. Ža. Raw Pinus sylÕestris pollen composite stained with safranin-fast green, highlighting the cellulose intine of the grains Žgreen.. Note: No cellulose is present in the saccae and the sporopollenin is only partially stained Žpink. due to the presence of a polysaccharide which can be removed by an alkali wash prior to composite staining. Žb. Acid treated Pinus sylÕestris pollen composite stained with safranin-fast green. The strong safranin staining of the sporopollenin exine Žpink. demonstrates the removal of the outer polysaccharide layer Žsee Plate 1a.. The cellulose intine has also been removed.
342
N.J. Loader, D.L. Hemmingr Chemical Geology 165 (2000) 339–344
ŽWaterhouse, pers. comm.. was used to identify the sporopollenin and cellulose components.
3. Results and discussion It is evident from Table 1 that multiple analyses on the raw and processed pollen Žusing both acid digestion and acetylation. result in d13 C ratios with similar reproduceability between treatments Žtypically - 0.1‰., but very different values. The large isotopic offset between the pollen processed using the acid digestion and acetylation techniques supports the findings of Amundson et al. Ž1997. and suggests contamination by carbon compounds during processing. As the acid digestion technique does not involve the addition of any carbon compounds, these values are believed to be a quantitative indicator of the d13 C value of sporopollenin. The C3 Populus pollen is considerably smaller than the other pollen analysed and, as a result, grains of this species tended to block the filter. This resulted in remnants of intine remaining in the sample during the acid digestion technique, which would account for the relatively poor reproduceability of the d13 C values of this species. Indeed, when these samples were examined under a microscope it was noted that remnants other than sporopollenin were present. For future analyses the choice of membrane porosity must therefore be considered.
Fig. 1. Relationship between sample processing time and d13 C composition of acid treated sporopollenin. Error bars of "1 sny 1 are shown.
Table 2 Comparison of d13 C values obtained from acid digestion processing of modern Pinus sylÕestris pollen for different time periods Processing time Žh.
Average d 13 C PD B Ž‰.
sny1
n
0.0 Žraw. 0.5 1.0 2.0 4.0 8.0 24.0 48.0 168.0 Ž1 week.
y27.39 y29.70 y29.67 y29.86 y29.56 y29.80 y29.55 y29.58 y29.32
0.17 0.10 0.25 0.26 0.07 0.04 0.05 0.08 0.09
3 3 3 3 3 2 3 3 3
Using differential staining of cellulose and sporopollenin the raw Žunprocessed. pollen ŽPlate 1. can be compared with pollen processed for 8 h ŽPlate 1.. It is apparent that cellulose Žstained green. is present in the central section of the raw Pinus grains Žnote: the saccae are cellulose free. and no cellulose is evident in the acid treated grains. The sporopollenin Žstained pink. is apparent on both raw and acid treated pollen and is believed to show less staining on the raw pollen as a result of the presence of an external ‘protective’ polysaccaride layer ŽShaw and Yeadon, 1964.. When the same staining technique was applied to untreated sub-fossil Žca. 10 ka BP. pollen some cellulose was identifiable in a number of the pollen grains. This test may also prove useful when examining palaeosequence records to determine the extent of diagenesis downcore, prior to isotopic analysis. Examining the influence of processing time on pollen d13 C ŽFig. 1., there appears to be no d13 C dependence on times over 30 min ŽTable 2.. We adopt a processing time of 8 h because this yielded consistent d13 C ratios and, when examined under a microscope, samples treated for less time showed some partially extracted grains while those processed for longer showed signs of mechanical degradation. Similarly, it was found that grains that had been stirred extremely violently became so fragmented that the filter becomes blocked, trapping non-sporopollenin fragments in the sample. The raw and processed d13 C ratios are extremely strongly correlated Ž r ) 0.9. and have a 1:1 relationship Ž m s 1.02. ŽFig. 2.. It is noted that this relationship is valid for both the C3 and C4 species. There-
N.J. Loader, D.L. Hemmingr Chemical Geology 165 (2000) 339–344
343
Fig. 2. Comparison of the d13 C composition of raw and acid treated pollen. C3 and C4 species are shaded grey and black respectively. The acid digestion treated pollen is shown by the grey diamonds — C3 Ž Betula pendula, Pinus sylÕestris and Picea abies ., grey circle — C3 Ž Populus tricocarpa. and black circle — C4 Ž Zea mays .. Pollen treated with acetolysis is shown by the grey square — C3 Ž Populus tricocarpa. and black square — C4 Ž Zea mays ..
fore, the C3:C4 ratio, which is an important source of information for past changes in vegetation balance, is preserved ŽTeeris and Stowe, 1976; StreetPerrot et al., 1998.. The fact that the d13 C ratios of the two acetylated samples, also plotted on Fig. 2, clearly lie off the regression line of the other samples, is support for the acid treatment technique.
4. Conclusions Partial diagenesis of cellulose in the sub-fossil record requires the isolation of a single chemical component of pollen prior to its d13 C analysis. We have demonstrated a rapid and inexpensive technique for the preparation of pollen samples for d13 C analysis, which does not introduce contaminating carbonbearing compounds into the sample. By isolating the sporopollenin we are able to demonstrate a consistent d13 C signal between both raw and processed pollen. This means that pollen d13 C records may be utilised, together with conventional palynology, to assess plant responses to environmental changes. Great potential exists for such analyses, and while undoubtedly modifications and refinements may be
necessary in the future, this work represents an important first step towards quantitative isotopic palynology. [PD]
Acknowledgements We thank John Parker, Nick Shackleton, Roy Switsur, Tony Carter, Adam Gardner, Monni Kotilainen, Iain Robertson and Kathy Willis for their advice and support. Thanks also to Mike Hall for running the mass spectrometer, Mr. Sci Mato Jurkovic for supplying pollen for this study and to John Waterhouse, Rebecca Sharman and Abrar Jawaid ŽAnglia Polytechnic-University. for assistance with the cellulose staining technique. This work was partially funded by the British Ecological Society.
References Amundson, R., Evett, R.R., Jahren, A.H., Bartoleme, J., 1997. Stable carbon isotope composition of Poaceae pollen and its potential in palaeovegetational reconstructions. Rev. Palaeobot. Palyn. 99, 17–24.
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Bar-Matthews, M., Ayalon, A., Kaufmann, A., 1998. Palaeoclimate evolution in the eastern Mediterranean region during the last 58000 years as derived from the stable isotopes of speleothems ŽSoreq Cave, Israel. ŽIAEA-SM-349r17. in Isotope Techniques in the study of Environmental Change, Ed. IAEA Vienna, 673–682. Benner, R., Fogel, M.L., Sprague, E.K., Hodson, R.E., 1987. Depletion of 13 C in lignin and its implications for stable carbon isotope studies. Nature 329, 708–710. Birks, H.J.B., Gordon, A.D., 1985. Numerical Methods in Quaternary Pollen Analysis. Academic Press, USA, 317 pp. Birks, H.J.B., Line, J.M., 1993. In: Cramer, J. ŽEd.., Glacial Regfugia of European Trees — A matter of Chance? Festschrift Zoller, Dissertaziones Botanicae Vol. 196. Borntraeger Verlagsbuchandlg, Berlin-Stuttgart, pp. 283–291. Brooks, J., Shaw, G., 1968. Identity of sporopollenin with older kerogen and new evidence for the possible biological source of chemicals in sedimentary rocks. Nature 220, 678–679. Coplen, T.B., 1995. Discontinuance of SMOW and PDB. Nature 373, 285. Duplessy, J.-C., Labeyrie, L., Juillet-LeClerc, A., Du Prat, J., 1992. A new method to reconstruct sea surface salinity. In: Bard, E., Broeker, W. ŽEds.., The last deglaciation. Absolute and Radiocarbon chronologies, NATO series 1–2. SpringerVerlag, Berlin, pp. 201–207. Edwards, T.W.D., Aravena, R.O., Fritz, P., Morgan, A.V., 1985. Interpreting palaeoclimate from 18 O and 2 H in plant cellulose: comparison with evidence from fossil insects and relict permafrost in southwest Ontario. Can. J. Earth. Sci. 22, 1720– 1726. Erdtman, G., 1960. The acetolysis method. Svensk. Bot. Tdskr. 54, 561–564. Gasse, F., Lin, R., 1988. Lacustrine isotope archives of past climate and environmental changes in the tropics and subtropics: some examples from Africa. In Isotope techniques in the study of environmental change, Ed. IAEA Vienna, 519–532. Hemming, D.L., Switsur, V.R., Waterhouse, J.S., Heaton, T.H.E., Carter, A.H.C., 1998. Climate variation and the stable carbon isotope composition of tree ring cellulose: an intercomparison of Quercus robur, Fagus sylÕatica and Pinus silÕestris. Tellus 50 B Ž1., 25–33.
Jansen, E., 1989. The use of stable oxygen and carbon isotope stratigraphy as a dating tool. Quat. Intl. 1, 151–166. Leavitt, S.W., Long, A., 1991. Seasonal stable-carbon isotope variability in tree rings: possible paleoenvironmental signals. Chemical Geology ŽIsotope Geosciences Section. 87, 59–70. Moore, P.D., Webb, J.A., Collinson, M.E., 1991. Pollen Analysis, 2nd edn. Blackwell Scientific Publishing, Oxford, UK. Patience, A.J., Kroon, D., 1991. Oxygen isotope chronostratigraphy. In: Smart, Frances, ŽEds.., Quaternary Dating Methods Technical Guide 4 Quaternary Research Association. Cambridge, UK. Shaw, G., Yeadon, A., 1964. Chemical studies on the constitution of some pollen and spore membranes. Grana Palynologica 5 Ž2., 247–252. Shackleton, N.J., Opdyke, N.D., 1973. Oxygen isotope and palaeomagnetic stratigraphy of the equatorial Pacific core V28-238: oxygen isotope temperature and ice volumes on a 10 5 and 10 6 year scale. Quat. Res. 3, 39–55. Shackleton, N.J., Berger, A., Peltier, W.R., 1990. An alternative astronomical calibration of the lower Pleistocene timescale. Trans. Roy. Soc. Edin. 81, 251–261. Street-Perrot, F.A., Huang, Y., Perrott, R.A., Eglington, G., Barker, P., Ben Khelifa, L., Harkness, D.D., Olago, D., 1998. Impact of lower atmospheric carbon dioxide on tropical mountain ecosystems. Science 278, 1422–1426. Thompson, L.G., Moseley-Thompson, E., Dansgaard, W., Grootes, P.M., 1986. The Little Ice Age as recorded in the stratigraphy of the Quelccaya ice cap. Science 234, 364–365. Teeris, J.A., Stowe, L.G., 1976. Climatic patterns and the distribution of C4 grasses in North America. Oecologia 23, 1–12. Turney, C.S.M., Beerling, D.J., Harkness, D.D., Lowe, J.J., Scott, E.M., 1997. Stable carbon isotope variations during the last glacial-interglacial transition. Journal of Quaternary Sciences 12 Ž4., 339–344. Tzedakis, P.C., Bennett, K.D., 1995. Interglacial vegetation succession: a view from southern Europe. Quaternary Science Reviews 14, 967–982. Zetzsche, F., 1932. Sporopollenine. In Handbuch der Pflanzen Analyses, 3rd edn. G. Klein, Vienna.
Technical Note
Preparation of pollen for stable carbon isotope analyses N.J. Loader a
a,)
, D.L. Hemming
a,b
The Godwin Institute for Quaternary Research, Department of Earth Sciences, Pembroke Street, UniÕersity of Cambridge, Cambridge, CB2 3SA, UK b Laboratory of Tree Ring Research, UniÕersity of Arizona, West Stadium a58, Tucson, AZ, 85719, USA Received 5 May 1999; received in revised form 6 August 1999; accepted 6 August 1999
Abstract Differential diagenesis of the chemical components of pollen in sub-fossil records necessitates the isolation of a single ‘resistant’ chemical component of pollen, prior to its isotopic analysis. As sporopollenin is particularly resistant to diagenesis and comprises typically 65%–80% of the pollen grain, it is a suitable component for this purpose. Conventional Žacetylation. methods of pollen preparation for palynological studies are reliant on the use of carbon-bearing compounds which introduce large errors to the pollen carbon isotopic composition Ž d13C.. We present results from a novel non-carbon containing acid extraction technique to isolate sporopollenin. Using this technique we demonstrate that the d13C signals in raw and processed pollen are highly correlated, but display an offset associated with the non-sporopollenin components. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Pollen; Carbon stable isotope; Palynology; C 13 rC 12 ; Sporopollenin; Pollen analysis
1. Introduction Stable isotopes have revolutionised the way in which we can interpret proxy environmental records and their use has enabled a more complete understanding of palaeoenvironmental change. At present, the majority of this quantitative work has been confined to the isotopic analysis of inorganic carbonates deposited in the deep oceans ŽShackleton and Opdyke, 1973; Jansen, 1989; Shackleton et al., 1990; Patience and Kroon, 1991; Duplessy et al., 1992.. While a number of terrestrial isotopic indicators of environmental change have been exploited, including measurements from tree-rings ŽEdwards et al., 1985; )
Corresponding author. Tel.: q44-1223-334870; fax: q441223-334871; e-mail: [email protected]
Leavitt and Long, 1991; Hemming et al., 1998., speleothems ŽBar-Matthews et al., 1998., ice ŽThompson et al., 1986., sub-fossil leaves ŽTurney et al., 1997. and lacustrine cores ŽGasse and Lin, 1988., none of these exhibit the geographical coverage and temporal range of the ocean record. Conventional palynology provides a large-scale indicator of vegetation dynamics operating over long Žgeological. timescales ŽBirks and Gordon, 1985; Birks and Line, 1993; Tzedakis and Bennett, 1995.. These analyses often involve considerable time-lags between environmental forcing and vegetation response, such that the precise timing of environmental changes, especially rapid ones, are difficult to identify. If the products of photosynthesis used to form pollen grains reflect variations in the carbon-dioxide and meteoric water utilised by the plant and its
0009-2541r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 5 4 1 Ž 9 9 . 0 0 1 7 6 - X
340
N.J. Loader, D.L. Hemmingr Chemical Geology 165 (2000) 339–344
metabolism, then the stable isotope ratios of pollen grains will indicate these changes. Therefore, comparison of existing, well dated, palynological records with corresponding pollen stable isotope data will help identify the timing of environmental changes and the magnitude and phase relationship of the associated vegetation response. The relative proportions of organic substances composing pollen grains varies with species and degree of diagenesis ŽMoore et al., 1991; Benner et al., 1987.. As these components exhibit different isotope compositions it is necessary to isolate a single chemical component for isotopic analysis. Sporopollenin ŽZetzsche, 1932. is particularly resistant to diagenesis and comprises typically 65%–80% of the pollen grain ŽShaw and Yeadon, 1964; Brooks and Shaw, 1968.. As such, it is the most suitable component for isotope analyses. Traditionally pollen has been concentrated from core samples using a suite of chemical oxidation techniques. However, Amundson et al. Ž1997. demonstrated that one of these processes, acetylation Ž‘acetolysis’, Erdtman, 1960., provided a significant source of carbon isotopic contamination. The reason for this is most likely related to the partial removal of glacial acetic acid used during processing, or incomplete removal of cellulose acetate, a by-product of acetylation. Consequently, d13 C measurements on acetylated pollen will not yield a reliable signal. To avoid carbon contamination a simple and inexpensive acid digestion technique to isolate sporopollenin was developed and its effectiveness assessed.
2. Method To test if the d13 C signal present in raw pollen is retained in the isolated sporopollenin, the d13 C ratios
of raw and acid digested C3 and C4 pollen covering a wide isotopic range were compared. Samples of modern pollen from three species Ž Betula pendula, Pinus sylÕestris and Picea abies . were collected along a 1000 m altitudinal transect in southwest Switzerland. C4 Ž Zea mays . samples were obtained commercially ŽSigma, USA.. As the amount of C3 pollen collected for each sample was small, to allow suitable sample replication during technique development, additional C3 pollen Ž Populus tricocarpa. were also commercially obtained ŽSigma, USA. and a larger quantity of Pinus sylÕestris pollen donated by the Botanical Garden in Zagreb. The dry pollen was sieved and ca. 10 mg was placed into a test tube. 10 ml of concentrated sulphuric acid was added to each and the samples were covered and left to stir gently at room temperature using a magnetic ‘flea’. The influence of processing time was examined by subjecting samples to 0.5, 1, 2, 4, 8, 24, 48 and 168-h treatments. The solution was filtered using Whatman 3 ml Vectaspin 10 mm polypropylene mesh microcentrifuge tubes ŽWhatman International, UK. and the extracted pollen was retained in the filter insert. Samples were washed with cold de-ionised water and centrifuged at 3000 rpm for 5 min. This step was repeated until the filtrate was clear and no residue was evident in the holding tube after centrifuging. Samples were then vacuum dried in their tubes prior to d13 C analysis. Isotope measurements were carried out using a Fisons NA1500NC elemental analyser interfaced, via a cryogenic trap, to a SIRA II Isotope Ratio Mass Spectrometer. Results are expressed as per mil Ž‰. deviations from the PDB-1 standard ŽCoplen, 1995.. To ensure that the acid digestion technique removed all the cellulose from the pollen grains a differential Ž‘saffranin-fast green’. staining technique
Table 1 Reproduceability of pollen samples used in the development of the acid digestion method Note the large offset between the acetylated and acid treated samples Žsee also Fig. 1.. Sample species
d13 C PD B raw pollen Žunprocessed.
d13 C PDB acid digestion method Žthis study.
d13 C PDB ‘acetylation’ Žafter Erdtman, 1960.
C4 Z. mays C3 Populus tricocarpa C3 Pinus sylÕestris
y10.45 " 0.03 Ž n s 5. y23.97 " 0.05 Ž n s 5. y27.39 " 0.17 Ž n s 3.
y12.26 " 0.08 Ž n s 3. y27.52 " 0.23 Ž n s 3. y29.57 " 0.08 Ž n s 12.
y18.44 " 0.18 Ž n s 5. y33.27 " 0.13 Ž n s 5. –
N.J. Loader, D.L. Hemmingr Chemical Geology 165 (2000) 339–344
341
Plate 1. Ža. Raw Pinus sylÕestris pollen composite stained with safranin-fast green, highlighting the cellulose intine of the grains Žgreen.. Note: No cellulose is present in the saccae and the sporopollenin is only partially stained Žpink. due to the presence of a polysaccharide which can be removed by an alkali wash prior to composite staining. Žb. Acid treated Pinus sylÕestris pollen composite stained with safranin-fast green. The strong safranin staining of the sporopollenin exine Žpink. demonstrates the removal of the outer polysaccharide layer Žsee Plate 1a.. The cellulose intine has also been removed.
342
N.J. Loader, D.L. Hemmingr Chemical Geology 165 (2000) 339–344
ŽWaterhouse, pers. comm.. was used to identify the sporopollenin and cellulose components.
3. Results and discussion It is evident from Table 1 that multiple analyses on the raw and processed pollen Žusing both acid digestion and acetylation. result in d13 C ratios with similar reproduceability between treatments Žtypically - 0.1‰., but very different values. The large isotopic offset between the pollen processed using the acid digestion and acetylation techniques supports the findings of Amundson et al. Ž1997. and suggests contamination by carbon compounds during processing. As the acid digestion technique does not involve the addition of any carbon compounds, these values are believed to be a quantitative indicator of the d13 C value of sporopollenin. The C3 Populus pollen is considerably smaller than the other pollen analysed and, as a result, grains of this species tended to block the filter. This resulted in remnants of intine remaining in the sample during the acid digestion technique, which would account for the relatively poor reproduceability of the d13 C values of this species. Indeed, when these samples were examined under a microscope it was noted that remnants other than sporopollenin were present. For future analyses the choice of membrane porosity must therefore be considered.
Fig. 1. Relationship between sample processing time and d13 C composition of acid treated sporopollenin. Error bars of "1 sny 1 are shown.
Table 2 Comparison of d13 C values obtained from acid digestion processing of modern Pinus sylÕestris pollen for different time periods Processing time Žh.
Average d 13 C PD B Ž‰.
sny1
n
0.0 Žraw. 0.5 1.0 2.0 4.0 8.0 24.0 48.0 168.0 Ž1 week.
y27.39 y29.70 y29.67 y29.86 y29.56 y29.80 y29.55 y29.58 y29.32
0.17 0.10 0.25 0.26 0.07 0.04 0.05 0.08 0.09
3 3 3 3 3 2 3 3 3
Using differential staining of cellulose and sporopollenin the raw Žunprocessed. pollen ŽPlate 1. can be compared with pollen processed for 8 h ŽPlate 1.. It is apparent that cellulose Žstained green. is present in the central section of the raw Pinus grains Žnote: the saccae are cellulose free. and no cellulose is evident in the acid treated grains. The sporopollenin Žstained pink. is apparent on both raw and acid treated pollen and is believed to show less staining on the raw pollen as a result of the presence of an external ‘protective’ polysaccaride layer ŽShaw and Yeadon, 1964.. When the same staining technique was applied to untreated sub-fossil Žca. 10 ka BP. pollen some cellulose was identifiable in a number of the pollen grains. This test may also prove useful when examining palaeosequence records to determine the extent of diagenesis downcore, prior to isotopic analysis. Examining the influence of processing time on pollen d13 C ŽFig. 1., there appears to be no d13 C dependence on times over 30 min ŽTable 2.. We adopt a processing time of 8 h because this yielded consistent d13 C ratios and, when examined under a microscope, samples treated for less time showed some partially extracted grains while those processed for longer showed signs of mechanical degradation. Similarly, it was found that grains that had been stirred extremely violently became so fragmented that the filter becomes blocked, trapping non-sporopollenin fragments in the sample. The raw and processed d13 C ratios are extremely strongly correlated Ž r ) 0.9. and have a 1:1 relationship Ž m s 1.02. ŽFig. 2.. It is noted that this relationship is valid for both the C3 and C4 species. There-
N.J. Loader, D.L. Hemmingr Chemical Geology 165 (2000) 339–344
343
Fig. 2. Comparison of the d13 C composition of raw and acid treated pollen. C3 and C4 species are shaded grey and black respectively. The acid digestion treated pollen is shown by the grey diamonds — C3 Ž Betula pendula, Pinus sylÕestris and Picea abies ., grey circle — C3 Ž Populus tricocarpa. and black circle — C4 Ž Zea mays .. Pollen treated with acetolysis is shown by the grey square — C3 Ž Populus tricocarpa. and black square — C4 Ž Zea mays ..
fore, the C3:C4 ratio, which is an important source of information for past changes in vegetation balance, is preserved ŽTeeris and Stowe, 1976; StreetPerrot et al., 1998.. The fact that the d13 C ratios of the two acetylated samples, also plotted on Fig. 2, clearly lie off the regression line of the other samples, is support for the acid treatment technique.
4. Conclusions Partial diagenesis of cellulose in the sub-fossil record requires the isolation of a single chemical component of pollen prior to its d13 C analysis. We have demonstrated a rapid and inexpensive technique for the preparation of pollen samples for d13 C analysis, which does not introduce contaminating carbonbearing compounds into the sample. By isolating the sporopollenin we are able to demonstrate a consistent d13 C signal between both raw and processed pollen. This means that pollen d13 C records may be utilised, together with conventional palynology, to assess plant responses to environmental changes. Great potential exists for such analyses, and while undoubtedly modifications and refinements may be
necessary in the future, this work represents an important first step towards quantitative isotopic palynology. [PD]
Acknowledgements We thank John Parker, Nick Shackleton, Roy Switsur, Tony Carter, Adam Gardner, Monni Kotilainen, Iain Robertson and Kathy Willis for their advice and support. Thanks also to Mike Hall for running the mass spectrometer, Mr. Sci Mato Jurkovic for supplying pollen for this study and to John Waterhouse, Rebecca Sharman and Abrar Jawaid ŽAnglia Polytechnic-University. for assistance with the cellulose staining technique. This work was partially funded by the British Ecological Society.
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