EPHE Department Paleoclimatology  and Marine Paleoenvironments
     Pollen sample preparation

Assessment of the microsieving technique for pollen analysis


Dept. EPHE Paleoclimatology and Marine Paleoenvironments, UMR EPOC, Bordeaux 1 Univ.


Our preparation technique for pollen (and spores) analysis includes a 10μm mesh

sieving to eliminate small nonpalynomorph particles. This technique is commonly used in

marine sediments (e.g. Heusser and Stock 1984; Leroy and Dupont 1994; Sánchez Goñi, et al.

1999; Dupont et al. 2008) to concentrate pollen grains and spores. However, microsieving

has been criticized in a number of works based on experiments comparing pollen counts of

sieved and unsieved residues (e.g. Tzedakis et al. 2004; Roucoux et al. 2005; Margari et al.

2010). Sieving has been reported to result in an unacceptable level of differential pollen loss,

particularly affecting Poaceae pollen grains because they can be small in size and often

crumpled (Roucoux et al. 2005). These experiments are presented in Roucoux PhD

dissertation (Roucoux 2000) but are still unpublished. Therefore, the protocol set up for

these experiments is not available.

To test whether microsieving affects substantially the proportion and composition of

the different morphotypes included in the pollen assemblages we have performed new

experiments in our laboratory. We have analyzed sample U1386A 15H CC (IODP expedition

n°339 “Mediterranean Outflow Expedition”) collected in the southwestern Iberian margin.

The sediment was divided in two aliquots. Our routine protocol, including the 10 μm mesh

sieving (http://ephepaleoclimat.com/ephe/Lab%20Facilities.htm), was applied to one

aliquot, and the same protocol without sieving to the other aliquot. A known concentration

of Lycopodium spores (one tablet of 20,428 spores) was added to each aliquot to calculate

the pollen concentration of sieved and unsieved residues. Slides were prepared using a

mobile mounting medium to allow rotation of pollen grains. Pollen quantification was done

under a Zeiss Primo Star light microscope at 400× magnifications with regular use of 1000×

magnification for the identification of pollen and spores. Pollen percentages for terrestrial

taxa were calculated against the main sum of terrestrial grains, while percentages for Pinus,

aquatics and spores were calculated against the total sum of all pollen and spores.




Counting one slide from the sieved residue was enough to reach a minimum of 100

pollen grains, excluding Pinus which is overrepresented in marine sediments from the

western European margin (Turon 1984a, 1984b), and 20 pollen morphotypes. These two

criteria are needed to obtain pollen percentage assemblages that accurately reconstruct the

vegetation cover and composition (McAndrew and King 1976; Rull 1987). In contrast, the

first slide of unsieved residue (1) showed low amount of pollen grains, most of the mounted

residue being composed of nonpalynomorph particles. We were obliged to count a second

slide to get a pollen sum higher than 100 pollen grains (slides 1+2). At that point, the total

pollen concentrations of the sieved and unsieved slides from the same sample disagreed.

However, the number of Lycopodium spores counted was still low and the sum of

Lycopodium spores and pollen grains did not reach 200, which is considered the minimum

number to obtain accurate concentration values (Finsinger and Tinner 2005). For this reason,

we mounted two additional slides, 3 and 4, with higher amount of residue than that of the

previous slides. Both slides contained a high amount of nonpalynomorph particles and were

difficult to analyse. Despite that, we reached more than 100 pollen grains and 20

morphotypes in each slide and summed up more than 200 items. Concentrations of slides 3

and 4 were considered reliable and more similar to the pollen concentration of the sieved

slide. The number of identified taxa was also similar between sieved and unsieved slides,

oscillating between 18 and 22 taxa.


Table 1 Amount of the two treated aliquots, sieved and unsieved, from the same sample

U1386A 15H CC and pollen concentration of the different slides counted.



We observed that the concentration of the sieved slide is 25% lower than that of the

unsieved slides. However, the losses affect all types of pollen grains in the same proportion,

excluding Pinus (Figure 1). There is no a differential loss of Poaceae pollen grains contrarily

to what has been suggested in previous unpublished works. In contrast, the concentration of

Pinus pollen grains was similar in both sieved and unsieved slides. The big size of Pinus

pollen, 5 times the mean size of the pollen grains of the studied area (20 μm), would

preclude any leak of this taxon through the 10 μm mesh.



Figure 1 Pollen concentrations of the main taxa (pollen grains/g) in the sieved and sum

unsieved slides.

We calculated the pollen percentages in the sieved and unsieved slides to evaluate if

the observed loss of pollen grains in the sieved residue produces a substantial difference in

the pollen percentages between the sieved and unsieved residues (Figures 2 and 3).



Figure 2 Pollen percentages of the main taxa in the sieved and sum unsieved slides.


Figure 2 shows that the difference of the pollen percentages between sieved and

unsieved slides of each morphotype, excluding Pinus and corroded grains, is less than 5%

that is within the counting error estimated for pollen sums of 100 grains (Fletcher and

Sanchez Goñi 2008). Therefore, our results showed that Poaceae pollen percentages were

not differentially affected by the application of the 10 μm microsieving technique. In

contrast, the comparison between the pollen assemblages from the sieved slide and the

unsieved slides 1+2 highlights the distortion produced by the counting of low amount of

palynomorphs in unsieved slides (Figure 3). The difference in the pollen percentages of each

taxon is of 50% in average between the sieved and unsieved slides.



Figure 3 Pollen percentages of the main taxa in the sieved and unsieved slides 1+2.


In conclusion, the microsieving technique at 10 μm mesh produces a loss of pollen

grains, which affects similarly all the morphotypes excluding Pinus. This lost does not

produce, however, any distortion in the percentages of the different morphotypes included

in the pollen assemblage. Therefore, we recommend the inclusion of microsieving in the

pollen preparation protocol applied to marine sediments. This technique is a valuable

technique to concentrate the pollen grains and produce clear residues that help the

identification and quantification of pollen grains. This test demonstrates, in turn, that

counting only 100 pollen grains in unsieved sediment does not give a fair representation of

the sample pollen assemblages.




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