CHANGES IN ASSEMBLAGES J.B. Riding, B.G. Rawlins, and SOIL K.H. Coley:POLLEN Changes in soil pollen assemblages on footwear worn at different sites ON FOOTWEAR WORN AT DIFFERENT SITES
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JAMES B. RIDING BARRY G. RAWLINS British Geological Survey Kingsley Dunham Centre Keyworth Nottingham NG12 5GG United Kingdom e-mail:
[email protected];
[email protected] KIRSTIN H. COLEY Department of Geography Royal Holloway University of London Egham Surrey TW20 0EX United Kingdom
Abstract The application of palynology to forensic investigations relies on the similarity of pollen assemblages from forensic items, such as footwear, with control samples from a crime scene. The pollen from material adhering to footwear is likely to reflect some combination of pollen from the locations where the boots/shoes have been worn most recently. This study investigated the changes in pollen assemblages on footwear that had been worn at different sites. Six rural sites in the East Midlands of England, United Kingdom were visited wearing pristine boots (i.e. no mixing), and boots that were previously worn at other localities (i.e. potential mixing). Samples of adherent soil from these items of footwear, and control samples, were analysed palynologically in order to assess the degree and significance of mixing of the pollen assemblages. With the exception of one sample, the pollen adherent to footwear or in the soil samples from each of the six sites (no mixing) had a characteristic signature. This supports the general distinctiveness of pollen from individual sites, the concept of widespread palynological heterogeneity, and the utility of palynology in forensic geoscience. The data from this study show that when mixing occurs from wearing footwear at different sites, the pollen/spore content of the boots etc. dominantly reflects that of the last site. This was evident from a visual examination of the raw data, and was confirmed using detrended correspondence analysis applied to the eleven dominant taxa. These data showed clustering of samples based on the last site visited. The more abundant the pollen/spores, the closer the samples were clustered. The clustering was less convincing at localities that yielded relatively sparse palynomorphs. However, sample material from footwear that was potentially contaminated with soil from previous localities typically exhibited some subtle differences. These were normally slight increases in diversity, and small variations in certain pollen types. The relative insignificance of these differences means that they would be difficult to discern consistently and quantify. It is thus critical that, in relevant forensic investigations, footwear belonging to suspects is seized as soon as possible after a crime is committed.
Key words: forensic palynology; soil analysis; provenance determination; multivariate statistics.
INTRODUCTION Forensic palynology has been used in many criminal cases to associate the pollen/spore assemblage from clothing, fabrics, or footwear belonging to a suspect with a crime scene, or other locations associated with an investigation such as a body deposition site (Milne et al., 2005; Bryant and Jones, 2006). Palynology is the study of pollen, spores and other organic remains (palynomorphs) that can be
Palynology, 31 (2007): 135–151 © 2007 by AASP Foundation
ISSN 0191-6122
either modern or fossil (Moore et al., 1991; Jansonius and MacGregor, 1996; Traverse, 2007). Palynomorphs are abundant, chemically/mechanically robust, and small; they are therefore relatively ubiquitous. By far the most important palynomorphs in forensic studies are the two main terrestrial groups, pollen and spores. Forensic palynology generally assumes that due to contact between a suspect and the ground, other surfaces, or vegetation, an adherent pollen assemblage from a forensic
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sample will be distinctive for a particular location. This is because every locality apparently has a distinctive palynological profile due to the huge variability in vegetation spectra and taphonomic factors, both of which affect the distribution of pollen and spores (Wiltshire, 2006). Comparison of control samples from the site of investigation and other locations with forensic sample(s) ought, therefore, to establish the probability that a forensic sample came from a specific crime scene. Despite its use in criminal investigations, few studies have been undertaken to test some of the basic assumptions upon which the application of forensic palynology are based. One study tested whether pollen/spores in soil samples from a relatively small (localized) area, i.e. comparable to a crime scene, exhibited significant variation (Horrocks et al., 1998). The palynological comparability of surface soil samples from other localized areas of similar vegetation type was also tested (Horrocks et al., 1998). In this instance, surface soil samples from the principal control site were dominated by grass pollen and bracken spores, and overall these samples had similar pollen/spore contents, which was demonstrated statistically (Horrocks et al., 1998). However, the pollen/spore associations from the control site differed considerably from other sites with similar vegetation types. In another study, soil samples were collected from consecutive footprints made by clean shoes within a localized area and their pollen content analyzed (Horrocks et al., 1999). The resultant data were compared with pollen and spore associations from the shoes that made the prints. In this experiment, soil samples from and between the prints, and from two soil samples from the shoes indicated a homogenous pollen/spore assemblage. It was clearly demonstrated that ‘perfect matches’ do not occur, because minor differences within this sample set were present (Horrocks et al., 1999). One of the potential limitations to the application of forensic palynology is the dilution or mixing of the pollen assemblage from a crime scene with that from sites visited both before and after the alleged crime. It is inevitable that the pollen association taken from a shoe or boot will never perfectly match any specific locality because of the effectiveness of footwear at picking up pollen grains (Wiltshire, 2006). Unless items of clothing or footwear are seized from a suspect immediately after a crime has been committed, the adherent pollen assemblage from the crime scene, and any pollen that was present before the crime scene was visited, will be mixed with pollen in material from any sites subsequently attended. However it has been suggested that pollen is efficiently retained on footwear over considerable periods of time, even if the items are cleaned (Wiltshire, 2006). This mixing of pollen will depend on numerous
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characteristics of the clothing/footwear, and the ground or surfaces at each site (e.g. moisture content and soil texture), and the nature of the contact between them. It is possible that pollen/spores from several localities will adhere to footwear or clothing. An ideal, but unlikely, scenario would be for a criminal to wear a new pair of boots, with deep tread, at a damp site with clay-rich soil that adheres easily. It has been assumed that the pollen on clothing or footwear will predominantly reflect the assemblage at the site that was visited last, but this has not been scientifically tested to date. In this contribution, results are presented from an investigation into the changes in the pollen assemblages of material adhering to footwear that has been worn at sites with differing pollen assemblages. Pristine footwear was worn at each of six rural sites, and the adherent material subsampled before the footwear was worn at subsequent sites, and further subsamples taken. Control soil samples were also collected from each site to determine its typical pollen assemblage. Then each of these samples were analysed for their pollen assemblage. In doing so, the effect of mixing pollen/spores from soil at different sites over time, with the pollen/spore assemblages from individual sites were investigated. The results were analysed statistically in order to assess the degree of similarity between the pollen/spore assemblages at the six sites, and potential mixtures between these sites. The implications of these findings for the application of forensic palynology are discussed. MATERIAL AND METHODS General Strategy and Sampling Six rural sites in the East Midlands of England, United Kingdom were visited, where pristine boots were worn (Text-Figure 1; Appendix 1). With each new locality, boots were also used that had been worn at previous localities within two ‘pyramid’ structures (Text-Figure 2). Each ‘wearing’ comprised one of us (JBR) walking normally, and in random directions, for one minute at the specific locality within a small area (ca. 9 m3). The items of footwear were not worn between localities. Composite control soil/surface samples were also taken from each locality. Five subsamples were collected from a ca. 3 m transect at each locality, and thoroughly mixed. This subsampling strategy aims to provide a representative sample by avoiding the skewing of a single sample due to the proximity of a localized, particular highly polleniferous plant. The subsampling procedure followed the recommendations of Adam and Mehringer (1975). The control subsamples were collected by carefully scraping the up-
J.B. Riding, B.G. Rawlins, and K.H. Coley: Changes in soil pollen assemblages on footwear worn at different sites
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Text-Figure 1. Locality map of sample locations 1 to 6 within the East Midlands of England, U.K. a – a small scale map of England with the East Midlands indicated. b – a map of the counties of Nottinghamshire and Leicestershire illustrating the three areas where samples were taken. c – a detailed map illustrating location 1 near a wood, southwest of Nanpantan, Leicestershire. d – a detailed map illustrating location 6 at the margin of Wollaton Park Lake, Nottingham. e – a detailed map illustrating locations 2, 3, 4, and 5 at Ruddington Country Park, Nottingham.
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Text-Figure 2. Pollen dilution triangle for sites 1 to 4 illustrating the pure (bulk) soil samples in the left column, and the boot samples in the triangle to the right. The triangle indicates that the potential pollen dilution via contamination increases to the left and towards the base. Sample code 1234B (number 12) is hence potentially the most palynologically heterogeneous. The numbers refer to the sites visited; B denotes boot and S denotes soil.
permost 5 mm of surface materials using a clean knife (see also Appendix 2). The samples of adherent soil from the items of footwear and the control samples were analysed palynologically to attempt to determine if the pollen/ spore content becomes diluted by wearing the footwear at other sites. The control samples provide essential data for comparison, even with adherent material taken from the pristine footwear, which should have a very similar composition. The collecting and sampling protocols are described in detail above, and also in Appendix 2. A cumulative strategy was adopted within two ‘pyramid’ structures. In the first, and larger, ‘pyramid’, only the first pair of boots was worn at locality one, the first and second pairs were worn at locality two, the first, second and third pairs were worn at locality three, and the first, second, third and fourth pairs of footwear were worn at locality four (Text-Figure 2). In the second, smaller, ‘pyramid’, the fifth pair of boots were
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worn at both sites. Samples of the adherent soil on the footwear were taken as appropriate, together with control samples (Text-Figure 2; Appendices 1, 2). The point of this strategy is to determine the magnitude of the dilution or mixing of the pollen/spore signals, assuming that palynological dilution is a real phenomenon. The footwear used comprised sturdy ‘outdoor’ boots with a maximum tread depth of between 7 and 9 mm deep. The six pairs used were all new, and were thoroughly cleaned using a surfactant before the fieldwork. Similar footwear was used throughout this study. Representative examples of the footwear used are illustrated in Text-Figure 3. In the sampling/subsampling from the footwear, the adherent mud/soil was carefully scraped away into a new sampling bag using a clean scalpel. No attempt was made to differentiate a stratigraphy of the adherent materials, all the mud/soil down to the sole was taken using the scalpel blade. In some cases, several subsamples were taken from each boot. The subsamples were taken from discrete areas of the tread that are described in Appendix 1. It was assumed that the pollen content of the adherent materials is relatively homogenous over the entire sole of the boot, and that any bias introduced during subsampling was minimal. The times between the initial wearing, sampling/ subsampling, and re-wearing where appropriate are also
Text-Figure 3. Photographs of two pairs of clean boots used in this study, illustrating the treads. The left hand photograph is of pair 3, ‘Dr Martens’ black 7-eye safety boots. The right hand photograph is of pair 4, ‘Century’ wellington type boots.
J.B. Riding, B.G. Rawlins, and K.H. Coley: Changes in soil pollen assemblages on footwear worn at different sites
documented in Appendix 1. These data are important because these parameters may determine how much drying there was, and whether any material became detached from the footwear. It is acknowledged that, in forensic cases, several different techniques are used for obtaining pollen from footwear exhibits. These include washing or wiping the pollen from the upper part of the boot or shoe, and washing pollen from the laces separately. Pollen from the uppers and/or laces is often a good indicator of the last place visited, and also if the owner has walked through crops, grass, herbs, other vegetation, or in an open area. Furthermore, it is possible to dissect specific layers of adherent material. This experiment simply used all the adherent materials from the soles of the footwear used, and washing was not undertaken. The reason for this strategy is that the primary objectives were concerned with testing the transference of pollen in soil/ surface materials taken from footwear and their dilution potential, and that the resources available were limited. There is clearly much scope for future research in this area. Sample Preparation and Study The 18 samples were prepared in the British Geological Survey palynology laboratory using standard palynological preparation procedures (Moore et al., 1991). They were demineralized using hydrochloric acid (HCl) and hydrofluoric acid (HF), the residual mineral grains were removed using heavy liquid separation, and the residues subjected to acetolysis. The heavy liquid used was zinc bromide with a specific gravity of 2.45–2.52. The microscope slides were mounted using the permanent mounting medium Elvacite. This is a permanent mounting medium and key grains can easily be given a coordinate on the coverslip and reexamined. The disadvantage of a permanent mountant is that problematic pollen grains cannot be moved in order to help identifications. If the mounting medium is viscous, e.g. glycerine or silicone oil, the grains can readily be manipulated. However the grains can move in a viscous mounting medium and relocation can be a significant problem. In a legal case, a key pollen grain crucial to the investigation must be readily relocatable so that it can be verified by both the legal teams. Sufficient slides were produced in order to allow statistically significant pollen counts to be made. The palynomorphs were studied using an Olympus CH2 transmitted light microscope. Pollen grains were identified using standard European keys (e.g. Moore et al., 1991), and the pollen reference collection in the Department of Geography, Royal Holloway, University of London, United Kingdom. The palynomorphs identified in this study are listed in Appendix 3.
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Statistical Analysis From the full dataset, only those pollen types that accounted for more than 1% of the total pollen count were selected and these eleven types are highlighted in Table 1, and listed in Table 2. The matrix of 18 samples with a subset of 11 pollen types was analysed using Detrended Correspondence Analysis (DCA). This overcomes the arch/ horseshoe effect. This phenomenon, and the tendency to concentrate/compress the end of the ordination axis, are the major problems associated with Correspondence Analysis (CA), which is often used for the analysis of ecological data (Hill and Gauch, 1980). Detrended Correspondence Analysis compensates for this by stretching and straightening the data plots. The DCA was undertaken using the ‘vegan’ package in an R-mode statistical environment (Oksanen et al., 2007). The minor (
