# Pollen-methods

The fine fraction was treated with concentrated Hydrofluoric Acid (HF) overnight to remove silicates.  After completely neutralizing the acid with distilled water, the samples were treated with a solution of darvan, and sonicated in a Delta D-9 Sonicator for 30 seconds.  The Darvan solution was removed by repeated washing with distilled water and centrifuged (2,000 RPM) until the supernatant liquid was clear and neutral.  This procedure removed fine charcoal and other associated organic matter and effectively deflocculated the sample.

The samples were dehydrated in glacial acetic acid in preparation for acetolyis.  Acetolysis solution was added to each sample.  Centrifuge tubes containing the solution were heated in a boiling water bath for approximately eight minutes and then cooled for an additional eight minutes before centrifugation and removal of the acetolysis solution with glacial acetic acid followed by distilled water.  Centrifugation at 2,000 RPM for 90 seconds dramatically reduced the size of the sample, yet from periodic examination of the residue, did not remove fossil palynomorphs.

Heavy density separation ensued using zinc bromide, with a specific gravity of 2.00, to remove much of the remaining detritus from the pollen.  The light fraction was diluted with distilled water and concentrated by centrifugation.  The samples were washed repeatedly in distilled water until neutral.  The residues were rinsed in a one percent solution of potassium hydroxide for less than one minute, which was effective in removing the majority of the unwanted alkaline soluble humates.

The material was rinsed in Ethanol stained with safranin-O, rinsed twice with Ethanol, and transferred to 1-dram vials with Tertiary Butyl Alcohol.  The samples were mixed with a small quantity of glycerine and allowed to stand overnight for evaporation of the Tertiary Butyl Alcohol.

A drop of the polliniferous residue was mounted on a microscope slide for examination under an 18 by 18 mm cover slip sealed with fingernail polish.  The slide was examined using 200X or 100X magnification under an aus-Jena Laboval 4 compound microscope.  Occasionally, pollen grains were examined using either 400X or 1,000X oil immersion to obtain a positive identification to either the family or genus level.

Abbreviated microscopy was performed on each sample in which either 20 percent of the slide (approximately four transects at 200X magnification) or a minimum of 50 marker grains were counted.  Regardless of which method was used, the uncounted portion of each slide was completely scanned at a magnification of 100X for larger grains of cultivated plants such as Zea mays and Cucurbita, two types of Cactus (Platyopuntia and Cylindropuntia), and other large pollen types such as members of the Malvaceae, or Nyctaginaceae families.  Because corn pollen was very common in many of these samples, corn grains were tabulated during the scans only if an unequal distribution of this taxon on the microscope slide was observed.

For those samples warranting full microscopy, a minimum of 200 pollen grains per sample were counted, which allows the analyst to inventory the most common taxa present in the sample.  All transects were counted completely resulting in various numbers of grains counted beyond 200.  Pollen taxa encountered on the uncounted portion of the slide during the low magnification scan are tabulated separately.

Total pollen concentration values were computed for all taxa.  In addition, the percentage of "Indeterminate" pollen was also computed.  Statistically, pollen concentration values provide a more reliable estimate of species composition within the assemblage.  Traditionally, results have been presented by relative frequencies (percentages) where the abundance of each taxon is expressed in relation to the total pollen sum (200+ grains) per sample.  With this method, rare pollen types tend to constitute less than 1 percent of the total assemblage.   Pollen concentration values provide a more precise measurement of the abundance of even these rare types.  The pollen data are reported as pollen concentration values using the following formula:

$\mathrm{PC}=\frac{K*\mathrm{3p}\mathrm{3L}*S}{}$

Where:

• PC = Pollen Concentration
• K = Lycopodium spores added
• 3p = Fossil pollen counted
• 3L = Lycopodium spores counted
• S = Sediment weight

The following example should clarify this approach.  Taxon X may be represented by a total of 10 grains (1 percent) in a sample consisting of 1,000 grains, and by 100 grains (1 percent) in a second sample consisting of 10,000 grains.  Taxon X is 1 percent of each sample, but the difference in actual occurrence of the taxon is obscured when pollen frequencies are used.  The use of "pollen concentration values" is preferred because it increases the variability between samples in the occurrence of the taxon.  The variability, therefore, is more readily interpretable when comparing cultural activity to noncultural distribution of the pollen rain.

The pollen concentration values for pollen wash samples were calculated using a modification of the above formula.  This modification involved the substitution of the area washed (in cm2) for the sediment weight (S) variable in the denominator from the above equation because the sample was in liquid form.  The resulting concentration value is thus expressed as estimated grains per cm2.  The resulting pollen concentration values from pollen wash samples are treated independently of those from soil samples in the results and discussion sections, although the data are presented with the other samples in the tables.  The use of pollen concentration values from these particular samples is preferred, as explained above, in order to accentuate the variability between pollen wash samples.  The use of the area washed also provides a mechanism for the comparison of calculated pollen concentration values between artifacts.

Variability in pollen concentration values can also be attributed to deterioration of the grains through natural processes.  A "1000 grains/g" rule was developed to assess the degree of pollen destruction.  This approach has been used by many palynologists working in other contexts as a guide to determine the degree of preservation of a pollen assemblage and, ultimately, to aid in the selection of samples to be examined in greater detail.  A pollen concentration value below 1000 grains/gm indicates that forces of degradation may have severely altered the original assemblage.  However, a pollen concentration value of fewer than 1000 grains/g can indicate the restriction of the natural pollen rain.  Samples from pit structures or floors within enclosed rooms, for example, often yield pollen concentration values below 1000 grains/g.

Pollen degradation also modifies the pollen assemblage because pollen grains of different taxa degrade at variable rates.  Some taxa are more resistant to deterioration than others and remain in assemblages after other types have deteriorated completely.  Many commonly occurring taxa degrade beyond recognition in only a short time.  For example, most (ca. 70 percent) Angiosperm pollen has either tricolpate (three furrows) or tricolporate (three furrows each with pores) morphology.  Because surfaces erode rather easily, once deteriorated, these grains tend to resemble each other and are not readily distinguishable.  Other pollen types (e.g. Cheno-am) are so distinctive that they remain identifiable even when almost completely degraded.

Pollen grains were identified to the lowest taxonomic level whenever possible and then interpretations of the vegetation made.  The majority of these identifications conformed to existing levels of taxonomy with a few exceptions.  For example, Cheno-am is an artificial, pollen morphological category which includes pollen of the family Chenopodiaceae (goosefoot) and the genus Amaranthus (pigweed), which are indistinguishable from each other.  All members are wind pollinated (anemophilous) and produce very large quantities of pollen.  In many sediment samples from the American Southwest, this taxon often dominates the assemblage.

Pollen of the Asteraceae (Sunflower) family was divided into four groups.  The high spine and low spine groups were identified on the basis of spine length.  High spine Asteraceae contains those grains that are divided into two spine lengths, short and long.  Artemisia pollen is identifiable to the genus level because of its unique morphology of a double tectum in the mesocopial (between furrows) region of the pollen grain.  Pollen grains of the Liguliflorae are also distinguished by their fenestrate morphology.  Grains of this type are restricted to the tribe Cichoreae which includes such genera as Taraxacum (dandelion) and Lactuca (lettuce).

Pollen of the Poaceae (Grass) family are generally indistinguishable below the family level, with the single exception of Zea mays, identifiable by its large size, relatively large pore annulus, and the internal morphology of the exine.  All members of the family contain a single pore, are spherical, and have simple wall architecture.  Identification of non-corn pollen is dependent on the presence of the single pore.  Only complete or fragmented grains containing this pore were tabulated as members of the Poaceae.

Clumps of four or more pollen grains (anther fragments) were tabulated as single grains to avoid skewing the counts.  Clumps of pollen grains (anther fragments) from archaeological contexts are interpreted as evidence for the presence of flowers at the sampling locale.  This enables the analyst to infer possible human behavior.

Finally, pollen grains in the final stages of disintegration but retaining identifiable features, such as furrows, pores, complex wall architecture, or a combination of these attributes, were assigned to the "Indeterminate" category.  The potential exists to miss counting pollen grains without identifiable characteristics.  For example, a grain that is so severely deteriorated that no distinguishing features exist closely resembles many spores.  Pollen grains and spores are similar both in size and are composed of the same material (Sporopollenin).  So that spores are not counted as deteriorated pollen, only those grains containing identifiable pollen characteristics are assigned to the "Indeterminate" category.  Thus, the "Indeterminate" category contains a minimum estimate of degradation for any assemblage.  If the percentage of "Indeterminate" pollen is between 10 and 20 percent, relatively poor preservation of the assemblage is indicated, whereas "Indeterminate" pollen in excess of 20 percent indicates severe deterioration to the assemblage.

In those samples where the total pollen concentration values are approximately at or below 1000 grains/g, and the percentage of "Indeterminate" pollen is 20 percent or greater, counting was terminated at the completion of the abbreviated microscopy phase.  In some cases, the assemblage was so deteriorated that only a small number of taxa remained.  Statistically, the concentration values may have exceeded 1000 grains/gm.  If the species diversity was low (generally these samples contained only pine, Cheno-am, members of the Asteraceae (Sunflower) family and Indeterminate category, counting was also terminated after abbreviated microscopy even if the pollen concentration values slightly exceeded 1000 grains/g.