Bryant Jr, Vaughn M., and John H. Wrenn (editors), 1998.

Reviewed by Alwynne B. Beaudoin, Archaeological Survey,
Provincial Museum of Alberta, in CAP Newsletter 22(1):5-11 (1999)
and reprinted in PALYNOS 22(1):7-12.

New Developments in Palynomorph Sampling, Extraction and Analysis consists of 16 papers and an introduction by the editors. Most papers were presented in a symposium at the 27th AASP Annual Meeting held in College Station, Texas, in 1994. As Bryant and Wrenn observe in their overview, the papers span the range from "new ideas [to] refinements of older techniques and procedures". Techniques and data interpretation are the focus of most papers, which deal primarily with Quaternary palynology, including melissopalynology, entomopalynology, and forensic palynology, with only two papers (Wrenn, and Rich and Pirkle) focussing on pre-Quaternary palynology. However, the issues raised – among them, quality control, cost reduction, and statistical validity – are not the monopoly of the Quaternary community and indeed have implications for other areas of micropalaeontology beyond palynology.

An evocative, if light-hearted, subtitle for the volume might be "The Palynology of Weird Stuff". Certainly, several of the papers deal with the extraction of palynomorphs from difficult samples, many in archaeological context. Often, these samples are isolated and do not form part of a stratigraphic sequence. Indeed, the recovery of palynomorphs from objects, whether human-made artifacts such as amphorae or naturally-formed items such as mollusc shells and insect bodies, is a subsidiary theme. These objects and their pollen loads can often be transported far from their place of origin or initial assembly. Thus, as well as requiring creative solutions for extracting palynomorphs, the acquired data also require different interpretive strategies.

Four papers deal explicitly with archaeological or historical samples. Three of these concern the recovery of palynomorphs from artifacts, namely, amphorae and their contents and textiles. Jones, Bryant and Weinstein attempt the "Pollen Analysis of Ceramic Containers from a Late Iron Age II or Persian Period Shipwreck near Haifa, Israel". They concentrate on five sediment samples that they believe "represent decomposed materials originally stored in amphora" rather than later inwashed sediment (p. 62) and four samples of pitch and resin from ceramic containers. They were able to identify pollen from several plants of economic significance, such as grape, olive, and pistachio, and pollen from other plants, such as oak and hazel, that perhaps represent later contamination or the contemporary pollen rain. Pollen evidence for grape was supplemented by the recovery of seeds from two samples. Jones et al. note the coincidence of well-preserved pine pollen in some samples with olive or grape pollen or both. They suggest that this might result from amphorae sealed with pine pitch and used to transport olives, olive oil, grapes, or wine. In a related paper, Jacobsen, Bryant and Jones present "Preliminary Pollen Analysis of Terebinth Resin from a Bronze Age Mediterranean Shipwreck" found off the shore of southwest Turkey. The resin, traded and valued for its use as incense and presumed medicinal properties, was obtained from the sap of a species of pistachio tree. Based on the analysis of eight samples, the assemblage includes conifer (predominantly Pinus) and Pistacia pollen but is dominated by NAP, especially cereal pollen grains. Their data suggest an origin for the resin in the eastern Mediterranean, perhaps the area now encompassed by north Israel-south Syria-northwest Jordan.

In these studies, the extraction of palynomorphs from the pitch and terebinth resin required considerable experimentation. Jones et al. dissolved the pitch and resin by "soaking the samples for several days in acetone, then soaking them again in 95% ethanol", prior to acetolysis (p. 63). Jacobsen et al. were able to dissolve the terebinth resin in xylene. However, they next describe adding Lycopodium tracers and HCl acid. I assume a precursor step would have involved removal of the xylene and transfer of the palynomorphs to water for further processing. The description of how this was accomplished is missing. Thus it would be difficult for anyone to follow their procedure with similar samples.

Moving forward in time, Jarzen summarizes his "Pollen Analysis of the Gondar (Ethiopia) Hanging". Determining the item's origin, history, and authenticity were the objectives of the investigation. Jarzen analyzed debris from the packing and wash and rinse water from the conservation treatment of this historically-important two-hundred-year-old silk hanging. Most data were retrieved from the packing debris, and did yield two pollen types (Olea and Justicia) that suggested residence in Ethiopia for the artifact, although most pollen types were more cosmopolitan and probably related to its residence in Ontario since the late 19th century. The small sample size available, and the necessity to eliminate sources of contamination, involved processing techniques similar to those used in forensic palynology.

Cummings considers sample spatial variation in "Sampling Prehistoric Structures for Pollen and Starch Granules". Her samples were obtained in 1979 from an Anasazi Pueblo 1 pithouse in Colorado, a site over a thousand years old. By gridding the floor into 50 cm x 50 cm squares, and sampling within this grid, she was able to identify probable activity areas, based on characteristic pollen components. Not surprisingly, pollen from food plants was often found in squares near the hearth in areas where artifacts indicated food preparation had probably occurred. As a sidelight on contamination, she had to consider the problem associated with the interpretation of Nicotiana pollen as a consequence of tobacco use by the field crew. Cummings was able to identify certain areas of the pithouse that provided most information on probable plant use, then used this as a guide for sampling in a subsequent pithouse excavation. Cummings' analysis shows the importance of considering spatial variability in the assessment of archaeological pollen samples, and the importance of a research design that allows this variability to be revealed. Stratigraphic (and thus temporal) variability is often a consideration in pollen studies; it is less common to take areal variability into consideration. I was, however, mystified by the reference to starch granules in the title of this paper, since these are only mentioned briefly in one sentence and are not a focus of the research presented here.

Moving away from archaeology and stepping back in time, Rich and Pirkle look at "Steinkerns as Pollen Traps". Steinkerns, I found out, are not beer-glasses but are the sediment infillings of mollusc shells. Because these are likely to have formed soon after the mollusc shell became part of the deposit and because the shell has protected the contents from contamination or reworking, these may give a contemporary palynological signature. This is a concern for the authors working in the southeast US where the floras of the late Tertiary and Quaternary are very similar and so contamination from modern sources may be difficult to detect.

Two papers focus on entomopalynology and deal with the pollen loads of insect pests on economically-important crops. Jones and Coppedge explore "Pollen Analysis of the Boll Weevil Skeleton" by using SEM imagery to examine pollen adhering to the heads of these insects. Their results show that the insects forage on plants other than cotton, including oak, plum, and black willow. In the following paper, "Pollen Analysis of the Crop of Adult Corn Earworms", Jones and Lopez use light microscopy (LM) to look at pollen in the crop, an internal organ of the moth. Again, the results showed that the insects were visiting a variety of plants. Both papers are investigating where the insects are foraging and feeding, presumably so that better pest control strategies can be designed. These papers form an interesting contrast, since in the first the authors argue that SEM analysis of externally-adhering pollen gives a better impression of foraging strategy than the LM analysis of gut contents, whereas in the second paper, precisely the opposite argument is advanced! Whether this is related to the different morphologies or feeding and foraging strategies of the two insect taxa is not clear.

Another theme that is strongly evident in several papers in this compilation is the evaluation of costs of doing pollen analysis. Perhaps this is a reflection of the increasing importance of consulting and contract work in the palynological world. The opening paper in the volume by Wrenn on "The Importance of Palynological Sampling to the Oil Industry" introduces this theme. His central argument is that using external contractors to process samples, while initially perhaps attractive from a cost perspective, is not effective if there is no quality control over the product being produced. He illustrates this proposition by presenting the results produced by three consulting firms in southeast Asia who were asked to process mainly Eocene samples from the Chindwin Basin of Myanmar. Their preparations were generally poor and often debris-laden in comparison to the relatively clean slides produced by Amoco Production Company staff. These preparations would have involved significantly greater counting times. Chances are that debris may have also obscured some palynomorphs. The age assignments provided by the consultants were also broad and inaccurate. His results show quite cogently that there is no substitute for expertise, especially from in-house experts who are able to assess the validity of results being presented and maintain continuity in quality. This may be a powerful argument to present to managers who are simply looking at a "bottom line" assessment, rather than considering value and reliability. Certainly, when decisions are being made about spending millions of dollars in development, it would seem prudent to be confident of the validity of the data underlying those decisions. His paper takes tilt at the myth that somehow outside consultants can do things better, quicker, and cheaper than in-house staff.

Dean in "Finding a Needle in a Palynological Haystack: A Comparison of Methods" attempts to devise a strategy for assessing the abundance of rare types in primarily archaeological samples. These rare types, which Dean defines as being those present as < 0.5% of the pollen spectrum, might include taxa of low abundance but high interpretive significance, such as corn (Zea mays). In these instances, even the occurrence of a single grain of the cultigen is significant. Thus the palynologist may be faced with the prospect of scanning a large amount of the preparation to be sure of counting (or not counting) a rare type. As Dean points out, if a rare type is not encountered within some target count, the palynologist may be in danger of concluding that the type is "not present" when in fact it is there but just has not been found. The objectives of her recommended procedures are three-fold: to maximize the probability of encountering rare types, to minimize counting time, and to provide an estimate of time needed to reach the target count for budgetary purposes. The methods that she describes, which she calls "Intensive Systematic Microscopy" or ISM, uses the spike palynomorph as an index to estimate the amount of counting needed to encounter a type present as a certain specified concentration, say an abundance of 1 grain g^-1. This approach does not eliminate the prospect of drawing a wrong conclusion of absence of a particular type, since, even if not encountered within the target count, it may still be present in the rest of the preparation. It does, however, provide the palynologist a way of quantifying what has been done in search of the rare type.

An embarrassment of samples was Gish's problem in "The Transwestern Pipeline Expansion Project Pollen Analysis". She describes her solution to the formidable challenge of dealing with more than a thousand samples from 90 archaeological sites and geomorphological study locales. Analysis had to follow a strict priority based on sample context. Sample counting was split between three analysts but with Gish, as the lead investigator, doing part of each count to minimize operator bias. Here both costs and project time constraints were limiting factors.

On the processing side, Jones and Ellin present "Improved Palynological Sample Preparation Using an Automated Focused Microwave Digestion System". They suggest that this system produces cleaner samples and reduces the needed amounts of processing chemicals, which are often both expensive and hazardous. They point out that there are now more stringent occupational health and safety regulations in the workplace and tighter controls over the use and disposal of hazardous chemicals. Hence processing methods that can be shown to be both safer and more environment-friendly are attractive. Their method is devised to deal primarily with rock samples. The advantages and results for the system certainly sound impressive, indicating success with samples that were not treatable by conventional means. But I was curious why the vital information about the cost of the system was left out of the article. Parenthetically, I note that it may be easier for labs to get money for chemicals ("supplies") than it is for new pieces of equipment ("capital"), so a new method that involves considerable outlay may not find wide acceptance.

Cost reduction is also a concern for Milne in the development of "Surface-embedding of Fossil Pollen for Time- and Cost-Effective Ultramicrotomy (TEM) and Multiple Microscopy (LM, SEM, TEM) of Single Grains". She describes a method that allows the same grain to be examined by different microscopy techniques, thus saving time and therefore reducing costs. Her especial concern was to reduce the costs involved in preparing a specimen for sectioning for TEM.

Several papers, besides that of Dean, focus on both processing techniques and aspects of the statistical validity of samples. Jones and Bryant explicitly examine whether a single-drop sample is representative in one of two papers dealing with melissopalynology ("Are All Counts Created Equal?"). Honey is characterized or classified according to the analysis of one drop of pollen residue. From this the honey will be judged as to its floral source, a judgement that may have financial implications for the producer. Hence, it is critically important to know whether the sample strategy used gives representative and reliable results. Not surprisingly, Jones and Bryant found that assemblage diversity increases with increasing pollen count. They counted 500 grains in each of five single drop samples, finding 130 taxa in total. They note that none of the samples contained more than 60% of the total number of taxa. The implication is that large counts, as a minimum 500 grains, are needed to adequately characterize a honey's floral sources. However, I was left questioning how these data relate to honey classification. Presumably, "clover honey", for example, would be expected to have an assemblage dominated by clover pollen. So how does the total number of taxa identified in a sample help in this assessment?

In their companion paper, "Pollen Recovery from Honey", Jones and Bryant explore two processing techniques (alcohol dilution and filtration), assessing their ability to maximize the chances of recovering a full spectrum of pollen types from a honey sample. They conclude that using ethyl alcohol to dilute honey and reduce its specific gravity as an initial processing step is likely to allow good recovery of pollen. The authors are not enthusiastic about the filtration technique, citing a number of disadvantages, especially in terms of the equipment required.

Smith's paper focusses on the comparison of samples prepared by different processing methods. The results that she obtained in her investigation of "Processing Pollen Samples from Archaeological Sites in the Southwest United States: An Example of Differential Recovery from Two Heavy Liquid Gravity Separation Procedures" are quite startling, especially for the concentration values. Her main conclusion is that "different procedures may not yield comparable data" (p. 29), a worrying issue. Besides statistical concerns, this paper raised questions about processing techniques. Analysts might perform HF acid treatment after heavy liquid separation to minimize the amount of HF acid needed to treat the sample by pre-removing silicates. This both reduces cost (HF acid is expensive) and reduces the amount of hazardous waste needing disposal. The implications of Smith's analysis are that this may not be the best procedure for pollen recovery. But the results raise other questions. For instance, I noted that some of the samples were described as containing clay. Yet no procedure for removal of clays and fine-grained material was apparently performed, as described for instance by Bates et al. (1978) and Cwynar et al. (1979). So are the differential results due to the influence of clay in the sample and, if so, would the effects be minimized with a precursor step for clay disaggregation and removal? A careful consideration of this paper will point the way to additional research questions.

In the last paper, Bryant and Mildenhall bring us a glimpse of the regrettable but necessary application of "Forensic Palynology: A New Way to Catch Crooks". They survey many cases in which pollen evidence was useful, especially to tie suspects or objects to a particular locale. All themes are well exemplified in this review: the necessity for meticulous and well-documented laboratory technique, concerns about statistical validity, and the need for cost-effective procedures. Besides having credentials as a scientist, the forensic palynologist must be prepared to deal with varied samples, from clothing to hair to drugs, maintain an impeccable chain of custody protocols, including locked storage, that will withstand legal scrutiny, and be ready to face, perhaps hostile, cross-examination in a courtroom. Given these strictures, I'm not surprised that most palynologists would opt for the calmer atmosphere of a research lab!

The editors have drawn together an interesting and thought-provoking set of papers. Many raise issues that deserve more consideration. Here, I am just going to discuss a couple of points that struck me as important as I read the book.

First, I was quite surprised that several of the papers (e.g., Dean, Smith, Gish) refer to a 200 grain count as though it were a standard. Several other papers (e.g., Jones et al., Rich and Pirkle) also mention this target. I was perplexed to find this thinking embedded in a volume devoted to new methods and approaches. Dean, for instance, states that a count of 200 grains has been "standard in palynology since the early years of this century" (p. 53). Although this may have been true at one time, I believe that palynological thinking has moved far from this view.

Several basic palynological textbooks provide guidance in this matter. As far back as 1980, Birks and Birks (pp. 165-166) were presenting data showing that a count of at least 300 – 500 grains is necessary to obtain stable pollen percentages for the main components of the assemblage. Moore et al. (1991: 168-169) suggest that a count of around 600 grains (in the pollen sum) may be adequate if the objective is "gross forest history". Much greater counts (over 1000 grains) will be necessary if minor components of the pollen assemblage are the focus of study. In another recent AASP volume, MacDonald (1996: 890) also summarizes these recommendations and indicates that "Quaternary palynologists generally count between 300 and 1000 grains of terrestrial plant pollen per sample". Berglund and Ralska-Jasiewiczowa (1986) suggest a minimum pollen sum 500 grains, and recommend that at least 1000 grains in the pollen sum be recorded where anthropogenic influence is suspected. Their experience suggests that "a pollen sum of 2000 will facilitate the identification of human impact" (p. 462). Because not all palynomorphs are included in the sum, the actual count may be much greater.

Larger counts may also be required if the pollen assemblage is dominated by one abundant type. For example, I recently undertook a study of pollen assemblages and variability from Lake O'Hara where the statistical validity of minor types was a particular concern. We set two counting targets for taxa included in the pollen sum: a minimum of 500 identifiable grains and a minimum of 100 grains over and above the abundant Pinus pollen. As a result, the mean number of grains counted was 1141, with a range from 534 to 5638 (Beaudoin and Reasoner 1992: 111).

A greater number of taxa (assemblage diversity) is also usually obtained with larger counts. This is illustrated quite neatly by Jones and Bryant's "Pollen Recovery from Honey" paper. Their data show that between about 7% and 17% new taxa were still being found when the count was increased from 400 to 500 grains (p. 110). In "Are All Counts Created Equal?", Jones and Bryant found 19 additional taxa in scanning one of their samples beyond the 500 grain count (p. 117). To assess adequately assemblage diversity, whether of a honey or a sediment sample, large counts are probably necessary.

There may be many situations in which the pollen count is limited by the sample itself. Here, the archaeological samples examined by Jones et al. and Jacobsen et al. are good examples, as are the forensic cases described by Mildenhall and Bryant. In other situations, where the sample or recovery does not impose limits, I would argue that, rather than adhering to a "standard", pollen counting strategies need to be flexible and devised according to the research questions. Indeed, this approach is illustrated by the analysis that Dean presents in the rest of her paper.

Second, given that several papers take a statistical approach and eight mention using tablets containing Lycopodium spores as a spike or tracer, I was surprised that more attention was not paid to the statistics of these. None of the papers give the batch number for the tracers, only four indicate whether one or more tablets were added, and only three acknowledge that the tablets have a range of contents, rather than an absolute number of spores. Jones et al. indicate that they added tablets containing 11,300±400 Lycopodium spores; Jones and Bryant (both papers) used tablets containing 11,300±300 spores. I was left wondering if these were actually from the same batch. The statistics associated with the use of a spike have been well explored by Maher (1981, 1997, see also Stockmarr 1971). Ideally, when assessing variability of spiked samples, the variability in the quantity of spike added needs to be taken into account. For instance, Dean's discussion of counting limits based on spike values would have been enhanced by a consideration of the confidence intervals on the amount of tracer added (see Maher 1997). I felt that a number of the papers, especially Smith's and Dean's, would have been much stronger with a more rigorous examination of the underlying statistical issues.

On the production side, while the book is nicely laid-out and designed, the volume would have benefitted from the attentions of a good copy-editor. There are inconsistencies in word use and there are noticeable typographical errors. The articles are generally well illustrated. With a few exceptions (the images in Wrenn's and Jones and Ellin's papers, and the laboratory view in Jarzen's paper), image reproduction is generally adequate, especially for the palynomorph photo-micrographs. The very modest price for the volume means that it should be in reach of a wide readership.

I greatly enjoyed reading this compilation. It would be a worthwhile addition to any palynologist's bookshelf. For me, the most abiding impression left by the volume is the sheer range of sample materials being investigated. As it extends from its traditional focus on peat, mud, or rock, palynology finds broader applications in other spheres, and makes a contribution to diverse fields in bio- and geosciences.

The publication can be ordered from Vaughn M. Bryant Jr, Secretary AASP Foundation, c/o Palynology Laboratory, Texas A&M University, College Station, Texas 77843-4352, USA. E-mail: vbryant@tamu.edu

References

Bates, C. D., P. Coxon, and P. L. Gibbard (1978) A New Method for the Preparation of Clay-Rich Sediment Samples for Palynological Investigation. The New Phytologist 81:459-463.

Beaudoin A. B., and M. A. Reasoner (1992) Evaluation of Differential Pollen Deposition and Pollen Focussing from Three Holocene Intervals in Sediments from Lake O'Hara, Yoho National Park, British Columbia, Canada: Intra-lake Variability in Pollen Percentages, Concentrations and Influx. Review of Palaeobotany and Palynology 75:103-131.

Berglund, B. E., and M. Ralska-Jasiewiczowa (1986) "Pollen Analysis and Pollen Diagrams". In Handbook of Holocene Palaeoecology and Palaeohydology, edited by B. E. Berglund, pp. 455-484. John Wiley and Sons, Chichester.

Birks, H. J. B., and H. H. Birks (1980) Quaternary Palaeoecology. Academic Press, New York.

Cwynar, L. C., E. Burden, and J. H. McAndrews (1979) An Inexpensive Method for Concentrating Pollen and Spores from Fine-Grained Sediments. Canadian Journal of Earth Sciences 16: 1115-1120.

MacDonald, G. M. (1996) "Non-Aquatic Quaternary". Chapter 22 in Palynology: Principles and Applications, Volume 2, edited by J. Jansonius and D. C. McGregor, pp. 879-910. AASP Foundation.

Maher Jr, L. J. (1997) Statistics for Lycopodium tablets. CAP Newsletter 20(2):26. See http://www.ualberta.ca/~abeaudoi/cap/articles/paper8.htm

Maher Jr, L. J. (1981) Statistics for Microfossil Concentration Measurements Employing Samples Spiked with Marker Grains. Review of Palaeobotany and Palynology 32:153-191.

Moore, P. D., J. A. Webb and M. E. Collinson (1991) Pollen Analysis, 2nd edition. Blackwell Scientific Publications, Oxford, U.K.

Stockmarr, J. (1971) Tablets with Spores Used in Absolute Pollen Analysis. Pollen et Spores 13:615-621.