PROTOCOL FOR THE PREPARATION OF SAMPLES FOR THE ANALYSIS OF POLLEN AND A SELECTION OF NON-POLLEN PALYNOMORPHS

Bas van Geel; 7 May 2003



Introduction

In pollen preparations other microfossils of various origin are also preserved. Among the 'extra' microfossils in raised bog peat are spores of fungi, remains of algae and invertebrates. In a series of papers the descriptions and illustrations of many non-pollen palynomorphs (NPP, or 'Types') were published and their indicator value was discussed (for references see van Geel, 2001). Morphological descriptions were always combined with stratigraphic information, often in the form of pollen and macrofossil diagrams. Several hundred Types have now been distinguished, each one with a Type-number. In most cases there was initially no, or hardly any taxonomic/ecological knowledge about the distinguished Types. The identification of the fossils was attempted with the aid of literature and by consulting colleagues in invertebrate zoology, phycology and mycology. Among the NPP there still are many taxa which are not properly identified, but some of them nevertheless can be used as palaeoenvironmental indicators. In such cases, the ecological information was inferred from the co-occurrence (curve matching) with identified taxa.
For Types in raised bog peat the publication by van Geel (1978) is relevant. For the ACCROTELM project only a selection of Types with a clear indicator value will be recorded (see below).

Preparation of microfossil samples:

  1. Boil the peat sample (ca 1 cc; volume should be measured first) in 10 % KOH.
  2. Sieve (meshes 215 µm); pour into centrifuge tubes in which the Lycopodium tablet(s) is/are already dissolved in 10% HCl. Add distilled water and centrifuge up to a speed of at least 4500 rpm. Do not decant the tubes, but take most of the water out of the tubes by using a water pump, so that no microfossils will be lost.
    NB: Sieve very carefully, so that no microfossils are left on the sieve. Burn the (metal) sieves before sieving, so that any microfossil from earlier samples will be destroyed.
  3. Wash the material with water and centrifuge until the supernatant is clear (washing two times, but sometimes more than twice is necessary).
    NB: the following steps (4-7) are undertaken in the fume cupboard.
  4. Dehydrate with 96% acetic acid and centrifuge.
  5. Prepare an acetolysis mixture by slowly adding 1 part H2SO4 to 9 parts acetic anhydride (stirring and cooling in a water bath is essential; no contact between acetolysis mixture and water!; safety glasses and gloves required).
  6. Acetolyse the material by heating the sample in the acetolysis mixture to 100 °C for ca 10 minutes in a water bath.
  7. Cool the sample tubes in a water bath and then centrifuge.
  8. Wash with distilled water and centrifuge until the supernatant is clear (wash at least twice).
  9. Wash with 96 % alcohol and centrifuge twice.
  10. Decant and wash the sample into a residue tube, using 96 % alcohol. Centrifuge (up to 4500 rpm) the residue tubes. Decant and add a drop/drops of glycerine to the residue (dependent upon the residue size).
  11. Put the residue tube for a night in the oven at 40 °C.
  12. Prepare microscope slides.

A selection of non-pollen palynomorphs in raised bog peat and their indicator potential

Many different fungal 'Types' occur in peat deposits, especially in peat layers which were formed under relatively dry conditions. In peat which was formed under very wet conditions fungal remains occur rarely.
From the various studies of fossil fungal spores, it became clear that the recorded spores in most cases are of strictly local occurrence. They were fossilised at, or near the place where they had been produced, or the spores were deposited only a short distance from the place where sporulation took place. A relevant selection of raised bog fungi is given here. ACCROTELM partners responsible for pollen analysis will receive an
illustration showing the following NPP:

- Gelasinospora spores (Types 1 and 2) indicate relatively dry conditions.
Spores are ellipsoidal, 22-30(-37) x 14-20(-24) µm. Spore surface almost black, evenly ornamented with ca 1 µm wide round, or ellipsoidal hyaline pits.

- Type 10 is an indicator for relatively dry conditions. It occurs on the roots of Calluna vulgaris.
Spores transversely (1-)2-3(-6) septate, (10-)20-30(-50) µm long and (7-)9-10(-12) µm wide, formed at the ends of septate, pigmented, (1-)1.5(-2) µm wide hyphae; basal cells paler (thinner-walled) than the others; not constricted at the septa or hardly so. In each septum a ca 0.3 µm wide pore.

- Type 12 is also a dryness indicator. It grows on a variety of bog plants.
Spores curved, (7-)10-13(-14) µm long, with two transverse septa, constricted at the septa. Apical cell (4-)6-7(-8) µm wide, thick-walled, dark brown, with a sub-apical germ pore about 0.7 µm wide. Central cell brown; basal cell hyaline and thin-walled.

- Spores of Meliola niessleana (Type 14). The fungus is a parasite on Calluna vulgaris.
Spores 41-55 x 14-18 µm, three-septate, inequilateral (one side almost straight), slightly constricted at the septa. Septa thickened around the ca 1.5 µm wide pore.

- Spores of Neurospora (Type 55C) indicate local bog fires.
Spores ellipsoidal, non-septate, brown, 22-28 x 15-18 µm, with two protruding apical pores, about 1 µm wide; with about 16 fine, longitudinal grooves.

Some algal spores:
- The hyaline spores of Mougeotia, Spirogyra and Zygnema-type are produced by filamentous green algae, inhabiting shallow, stagnant, oxygen-rich freshwater pools.
- Spores of Mougeotia are square, laterally straight to concave; retuse angles often with depressions. Surface smooth or with small pits.
- Spores of Spirogyra are ellipsoidal. Walls are smooth or reticulate; spores show a longitudinal furrow which often encircles almost the whole spore.
- Spores of Zygnema-type are spheroidal and flattened, with pits all over the surface.

Testate Amoebae in pollen slides:
- The thecae of Amphitrema flavum and Assulina indicate local relatively wet conditions. Many species of Testate Amoebae do not 'survive' the pollen preparation method. See Charman et al. (2000) for illustrations and ecological information and a complete analysis of Testates.


Counting of microfossils should be continued until at least 400 pollen grains of taxa included in the pollen-sum are recorded. Pollen-sum taxa are: trees, shrubs and upland herbs. Aquatic plants, local bog plants (like Cyperaceae and Ericales) and spores are excluded from the pollen-sum. However, their percentages will be expressed on the pollen-sum.
Some spores (like Types 10 and 12) can be so frequent that counting them until the pollen-sum is reached is too time-consuming. Therefore counting of these highly frequently occurring spores can be stopped when 50 pollen-sum taxa (or a certain number of the added Lycopodium spores) are recorded. Before entering the data in the TILIA program (for drawing diagrams) the number of spores should be 'extrapolated' first, so that (rough) percentages can be calculated.

Please note that Bas van Geel (IBED-FNWI, Kruislaan 318, 1098 SM Amsterdam, Netherlands; e-mail: vangeel@science.uva.nl) is always willing to help in case of doubts about identification of non-pollen palynomorphs. If necessary samples can be photographed and sent as electronic attachments (.jpg files with compression so that the files are less than 1Mb).


References

Charman, D.J., Hendon, D. and Woodland, W.A. 2000. The identification of testate amoebae (Protozoa) in peats. QRA Technical Guide No. 9, Quaternary Research Association, London. 147 pp.
Transeau, E.N. 1951. The Zygnemataceae. Columbus Graduate School Monographs, Contributions in Botany, 1. Columbus, Ohio, 327 pp.
van Geel, B. 1978. A palaeoecological study of Holocene peat bog sections in Germany and the Netherlands. Rev. Palaeobot. Palynol. 25, 1-120.
van Geel, B. 1986. Application of fungal and algal remains and other microfossils in palynological analyses. In Berglund, B.E. (ed) Handbook of Holocene Palaeoecology and Palaeohydrology. Wiley, Chichester. p. 497-505.
van Geel, B., Pals J.P., van Reenen G.B.A. and van Huissteden J. 1995. The indicator value of fossil fungal remains, illustrated by a palaeoecological record of a Late Eemian/Early Weichselian deposit in the Netherlands. In Herngreen, G.F.W. & L. van der Valk (eds) Neogene and Quaternary geology of North-West Europe. Meded. Rijks Geol. Dienst 52: 297-315.
van Geel, B. and Grenfell H.R. 1996. Spores of Zygnemataceae. In Jansonius, J. & D.C. McGregor (eds) Palynology: principles and applications, Am. Ass. Strat. Palynol. Found., Vol. 1: 173- 179.
van Geel, B. 2001. Non-pollen palynomorphs. In: J.P. Smol, H.J.B. Birks and W.M. Last (eds.) Tracking environmental change using lake sediments.; Volume 3: Terrestrial, algal and silicaceous indicators. Kluwer, Dordrecht, p. 99-119.

 

For those who are interested to see more NPP's from various sediment types:
Ask Bas van Geel for a copy of a xerox-copy book showing hundreds of NPP.

 

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