THE ASSESSMENT OF A METHOD FOR THE DETERMINATION OF ABSOLUTE POLLEN FREQUENCIES BY J. MATTHEWS

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1 New PhytoL (1969) 68, i6i-i66. THE ASSESSMENT OF A METHOD FOR THE DETERMNATON OF ABSOLUTE POLLEN FREQUENCES BY J. MATTHEWS Department of Environmental Studies, University of Lancaster {Received 1 March 1968) SUMMARY One method for the estimation of absolute pollen frequencies involves the addition of a known amount of an exotic pollen to a known quantity of sediment. From the observed ratio of indigenous to exotic pollen, the absolute quantity of' indigenous per unit volume of sediment can be calculated. The pollen of Nyssa sylvatica was considered to be a suitable exotic for addition to northwestern European Post-glacial sediments to be measured by this method. A series of experiments was made to estimate the settling rate of fresh N. sylvatica pollen in pure glycerine, to determine the period of agitation required to produce an even suspension of A^.,S'toaitca pollen in pure glycerine, to observe the effects of KOH treatment and acetolysis on a mixture of fresh A^. sylvatica and Ailanthus eligantissima pollen and to estimate the experimental error of the method by using it to determine the quantity of pollen in a series of homogenized samples of gyttja. The value of the method for the estimation of absolute pollen frequencies is assessed. NTRODUCTON Benninghoff (196) has given a brief description of a method for the estimation of the absolute frequency of fossil pollen in sediments. A measured quantity of pollen of an exotic species is added to a measured quantity of sediment. The mixture is then prepared for analysis in the normal way. Both fossil and added exotic pollen is counted. The frequencies of the various pollen types can then be expressed either relatively as percentages of a total or absolutely as number of grains per unit of sediment. The method is briefly as follows. The quantity of fresh pollen suspended in pure glycerine is determined with a haemacytometer. A known volume of the suspension is added to a known quantity of sediment. The mixture is then treated with KOH and subjected to acetolysis. Samples of the mixture after treatment are counted and the ratios of the various types of fossil pollen and the added exotic observed. Relative frequencies can then be calculated directly. As a known quantity of exotic pollen is added to a known quantity of sediment, the amount of fossil pollen per unit of sediment will be a x ble, when a is the number of exotic grains added, b the number of fossil grains observed and c the number of exotic grains observed. EXPERMENTAL PROCEDURE Fresh defatted Nyssa sylvatica pollen (obtained from the Greer Drug and Chemical Corporation of Lenoir, North Carolina, U.S.A.) was suspended in pure glycerine, 161

2 i6 J. MATTHEWS specific gravity 1.. t was found that +50 mg pollen was a suitable working quantit)'. About 5 ml of 96% alcohol were added to such a quantity of pollen and the mixture made up to 0 ml with pure glycerine. A short period in a warm oven was then sufficient to evaporate the alcohol which was added merely to assist the even mixture of the pollen and glycerine. A magnetic stirrer was used to suspend the pollen in the glycerine. Samples of the suspension were transferred with a pipette to a haemacytometer. The haemacytometer used had a modified Fuchs-Rosenthal ruling and chamber depth of 0. mm. The concentration of the A'', sylvatica suspension was calculated from a series of haemacytometer counts, and a small amount, its volume determined by weight, was added to a weighed amount of sediment. The resulting mixture of fresh pollen in glycerine and fossil pollen in the sediment was treated with 10% KOH, subjected to acetolysis, stained with safranin and suspended in silicone fiuid (AK 000). A small quantity was transferred to a slide and the pollen then counted at a magnification of x 400, the traverses planned to sample either the whole or half of the area of the cover-slip. RESULTS Settling rate of Nyssa sylvatica pollen suspended in pure glycerine Twenty-five millilitres of pure glycerine were added to +50 mg A^^. sylvatica pollen in a 5 ml glass beaker and the mixture agitated with a magnetic stirrer for hours. The stirrer used (a Morgan model 50) had an adjustable control which was kept at the same position throughout all the trials to maintain a more or less constant rate of agita- Table i. Numbers of grains counted at ^-minute intervals after the end of agitation and moving totals for three counts Time (hours) after end of stirring i i i ij i i j i No. of grains counted/slide s SS Moving total for three counts U tion. Samples of the suspension were taken with a i.o ml pipette from +1 cm below the surface of the suspension, as were all similar samples during the succeeding experiments. The suspension was transferred from the pipette to the haemacytometer and the pollen counted at a magnification of x 100. Samples were similarly taken at intervals of 15 minutes during a period of ^ hours. The haemacytometer was carefully cleaned with 96% alcohol and the pipette washed with dichromate cleaning solution between sampling during this and subsequent experiments. The number of grains counted in each sample is given in Table i, which also gives the

3 Determination of absolute pollen frequencies 16 moving totals for grains in three counts. The latter indicate that A^. sylvatica pollen suspended in pure glycerine settles at an appreciable rate so that samples must be taken without delay after agitation on the magnetic stirrer has stopped. Period of agitation necessary for homogeneity A series of ten experiments was made to estimate the period of agitation with the magnetic stirrer necessary to produce a homogeneous suspension of A'^. sylvatica pollen in pure glycerine. For each trial approximately 5 ml of pure glycerine were added to an amount of pollen that was varied from +0 mg to ±60 mg. Samples were taken at intervals of 15 minutes during a period of hours agitation on the magnetic stirrer. Table. Number of grains counted per slide at intervals of 15- minutes dttring continuous agitation with magnetic stirrer Experiment 4 S S s Time (minutes) after beginning of agitation OS The numbers of grains counted at intervals of 15 minutes are given in Table. t will be seen that there is some variation in the counts for each experiment but that this variation tends to be more pronounced for the first two counts. The temperature of the suspension was found to rise from +0 C at the beginning of the agitation to + C at the end, the electric m^otor of the stirrer providing heat. The viscosity of the glycerine will be reduced as temperature rises and it is probable that smaller variation in the counts after half-an-hour's agitation is connected with this reduction in viscosity, although no direct relation between the temperature of the suspension and the variation in the number of grains counted could be observed. t therefore appears that continuous agitation on the magnetic stirrer produced a homogeneous suspension after threequarters-of-an-hour. Calculated and observed ratios in mixtures of fresh pollen To give an estimate of the reliability of the method, pollen of A'', sylvatica and Ailanthus elegantissima were separately suspended each in 5 ml pure glycerine. Each mixture was then agitated on the magnetic stirrer, seven samples being counted at 15 minute intervals after the first three-quarters-of-an-hour. The amount of pollen in each suspension was calculated from these counts. A weighed volume of one suspension was then added to a weighed volume of the other and the ratio of the two pollen types to be expected in the mixture calculated. The mixture was then treated with 10% KOH, subjected to acetolysis, stained with safranin and suspended in glycerine. The ratio of Nyssa sylvatica to Ailanthus elegantissima was then observed.

4 164 J. MATTHEWS Table shows that the calculated and observed ratios for ten such experiments agree as closely as can be expected when sampling error (Eaegri and versen, 1964) is taken into account. The method therefore gives an accurate estimate of the amount of pollen in each suspension of pure glycerine and the treatment with 10% KOH and acetolysis did not upset the ratio of the two types in the final preparation. Experiments with a mixture of Nyssa sylvatica and Liquidambar styraciflua pollen did not give such satisfactory results and it was observed that the L. styraciflua pollen did not settle in 10% KOH when in the centrifuge. Table. Calculated percentage ratios of Nyssa sylvatica and Ailanthus elegantissima and ratios observed after acetolysis Experiment Calculated percentage ratios Nyssa: Ailanthus 51:49 19:81 1:79 4:58 44:56 0:70 :68 9:71 44:56 48:5 Observed percentage ratios Nyssa: Ailanthus 5:47 18:8 1:79 9:61 41:59 1:69 0:70 0:70 45:55 51:49 No. of grains counted Estimation of the quantity of pollen in a series of homogenized gyttja samples A portion of a gyttja core taken from the bed of a shallow lake at Niederwil, near Frauenfeld in Switzerland, was digested with 10% KOH and the resulting mixture vigorously agitated and then divided into six fractions. The core had been taken by the Biologisch-Archaeologisch nstituut, Groningen, the Netherlands in connection with archaeological work. Previous pollen analysis had shown that the part of the core sampled in this context was rich in Corylus avellana pollen. Table 4. Estimated number of Corylus avellana grains/g of homogenized gyttja Tiple Estimated no. of 10* Nyssa grains/g sample 14,5 119, ,095 0,569 41,601 16,01 No. of Corylus grains counted No. of Nyssa grains counted Limits of estimated no. of 10* Corylus grains/g sample S The amount of Nyssa sylvatica pollen suspended in pure glycerine was estimated as before and a weighed volume of suspension was added to each of the six homogenized gyttja samples which had themselves been weighed previously. Each gyttja sample was then subjected to acetolysis, stained with safranin and the residue suspended in silicone fluid. The samples were then counted at a total magnification of x 400 and the absolute frequency of Corylus avellana pollen per gram of homogenized gyttja calculated. Table 4 sets out the number of Nyssa sylvatica grains estimated to have been added to one gram of gyttja, the number of Corylus avellana and Nyssa sylvatica grains counted

5 Determination of absolute pollen frequencies 165 and the range of the amount of Corylus avellana pollen estimated to be present in i g of homogenized gyttja. As has already been pointed out, there will be a sampling error when the ratios of two pollen types are observed (Faegri and versen, 1964). For example, when two types of pollen are found in the ratio of one grain of one type to one grain of the other, the sampling error can be taken to be +% when a total of 1000 has been counted. Thus when a mixture of two types of pollen is observed and a count of 1000 grains gives an observed percentage ratio of the two types as 50%: 50% the sampling error is such that the true percentage ratio must lie between the limits 5/0:47% on one hand and 47/0:5/o ori the other. n an example where 500 grains of an exotic pollen were added S Sample Fig.. Limits of estimated numbers of Corylus avellana grains per gram of homogenized gyttja. to one unit volume of sediment, a ratio of 5%:47% of exotic to indigenous grains would indicate a total of 44 indigenous grains per unit volume of sediment. A ratio of 47%: 5% would indicate a total of 564 indigenous grains per unit volume of sediment. Thus when a total of 1000 grains is counted and percentage ratios in the order of 50%: 50% observed, the exotic pollen method of the estimation of pollen quantities will have a sampling error of ±1%. The sampling error will vary according to the total number of pollen grains counted and the ratio observed. Fig. i demonstrates that, when the limitations of the sampling error are taken into account, none of the estimated concentrations of Corylus pollen in the samples of homogenized gyttja is significantly different from any other. t would appear therefore that the experimental error of the method examined here for the estimation of absolute pollen frequencies is negligible compared with the sampling error already discussed. ACKNOWLEDGMENTS am indebted to Dr W. van Zeist for his invaluable help during the initial stages of the investigation. Mrs J. Walsh gave technical assistance in the laboratory. Part of this work was carried out with the aid of a grant from the Wenner-Gren Foundation.

6 i66 J. MATTHEWS REFERENCES BENNNGHOFF, W. S. (196). Calculation of pollen and spore density in sediments by addition of exotic pollen in known quantities. Pollen Spores, 4(),. DAVS, M. B. (1965). A metbod for determination of absolute pollen frequency. n: Handbook of Palaeontological Techniques (Ed. by B. Kummel & D. Roup), pp Freeman, San Francisco. FAEGR, K. & VERSEN, J. (1964). Textbook of Pollen Analysis. Blackwell Scientific Publications, Oxford.

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