Changes in soil pollen assemblages on footwear worn at different sites

J.B. Riding, B.G. Rawlins, and K.H. Coley: Changes in soil pollen assemblages on footwear worn at different sites 135CHANGES IN SOIL POLLEN ASSEMBLAGESON FOOTWEAR WORN AT DIFFERENT SITES

JAMES B. RIDINGBARRY G. RAWLINSBritish Geological SurveyKingsley Dunham CentreKeyworthNottingham NG12 5GGUnited Kingdome-mail: [email protected]; [email protected]

KIRSTIN H. COLEYDepartment of GeographyRoyal HollowayUniversity of LondonEghamSurrey TW20 0EXUnited 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 pollenfrom the locations where the boots/shoes have been worn most recently. This study investigated the changes in pollen assemblages onfootwear that had been worn at different sites. Six rural sites in the East Midlands of England, United Kingdom were visited wearing pristineboots (i.e. no mixing), and boots that were previously worn at other localities (i.e. potential mixing). Samples of adherent soil from theseitems of footwear, and control samples, were analysed palynologically in order to assess the degree and significance of mixing of the pollenassemblages. 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 widespreadpalynological 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 bootsetc. dominantly reflects that of the last site. This was evident from a visual examination of the raw data, and was confirmed using detrendedcorrespondence analysis applied to the eleven dominant taxa. These data showed clustering of samples based on the last site visited. Themore abundant the pollen/spores, the closer the samples were clustered. The clustering was less convincing at localities that yieldedrelatively sparse palynomorphs. However, sample material from footwear that was potentially contaminated with soil from previouslocalities typically exhibited some subtle differences. These were normally slight increases in diversity, and small variations in certainpollen types. The relative insignificance of these differences means that they would be difficult to discern consistently and quantify. It isthus 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.

Palynology, 31 (2007): 135–151© 2007 by AASP Foundation ISSN 0191-6122

INTRODUCTION

Forensic palynology has been used in many criminalcases to associate the pollen/spore assemblage from cloth-ing, fabrics, or footwear belonging to a suspect with a crimescene, or other locations associated with an investigationsuch as a body deposition site (Milne et al., 2005; Bryantand Jones, 2006). Palynology is the study of pollen, sporesand other organic remains (palynomorphs) that can be

either modern or fossil (Moore et al., 1991; Jansonius andMacGregor, 1996; Traverse, 2007). Palynomorphs areabundant, chemically/mechanically robust, and small; theyare therefore relatively ubiquitous. By far the most impor-tant palynomorphs in forensic studies are the two mainterrestrial groups, pollen and spores.

Forensic palynology generally assumes that due to con-tact between a suspect and the ground, other surfaces, orvegetation, an adherent pollen assemblage from a forensic

136 PALYNOLOGY, VOLUME 31 — 2007

sample will be distinctive for a particular location. This isbecause every locality apparently has a distinctive palyno-logical profile due to the huge variability in vegetationspectra and taphonomic factors, both of which affect thedistribution of pollen and spores (Wiltshire, 2006). Com-parison of control samples from the site of investigationand other locations with forensic sample(s) ought, there-fore, to establish the probability that a forensic samplecame from a specific crime scene.

Despite its use in criminal investigations, few studieshave been undertaken to test some of the basic assump-tions upon which the application of forensic palynologyare based. One study tested whether pollen/spores in soilsamples from a relatively small (localized) area, i.e.comparable to a crime scene, exhibited significant varia-tion (Horrocks et al., 1998). The palynological compara-bility of surface soil samples from other localized areas ofsimilar vegetation type was also tested (Horrocks et al.,1998). In this instance, surface soil samples from theprincipal control site were dominated by grass pollen andbracken spores, and overall these samples had similarpollen/spore contents, which was demonstrated statisti-cally (Horrocks et al., 1998). However, the pollen/sporeassociations from the control site differed considerablyfrom other sites with similar vegetation types. In anotherstudy, soil samples were collected from consecutive foot-prints made by clean shoes within a localized area andtheir pollen content analyzed (Horrocks et al., 1999). Theresultant data were compared with pollen and spore asso-ciations from the shoes that made the prints. In thisexperiment, soil samples from and between the prints, andfrom two soil samples from the shoes indicated a homog-enous pollen/spore assemblage. It was clearly demon-strated that ‘perfect matches’ do not occur, because minordifferences within this sample set were present (Horrockset al., 1999).

One of the potential limitations to the application offorensic palynology is the dilution or mixing of the pollenassemblage from a crime scene with that from sites visitedboth before and after the alleged crime. It is inevitable thatthe pollen association taken from a shoe or boot will neverperfectly match any specific locality because of the effec-tiveness of footwear at picking up pollen grains (Wiltshire,2006). Unless items of clothing or footwear are seized froma suspect immediately after a crime has been committed,the adherent pollen assemblage from the crime scene, andany pollen that was present before the crime scene wasvisited, will be mixed with pollen in material from any sitessubsequently attended. However it has been suggested thatpollen is efficiently retained on footwear over considerableperiods of time, even if the items are cleaned (Wiltshire,2006). This mixing of pollen will depend on numerous

characteristics of the clothing/footwear, and the ground orsurfaces at each site (e.g. moisture content and soil texture),and the nature of the contact between them. It is possiblethat pollen/spores from several localities will adhere tofootwear or clothing. An ideal, but unlikely, scenariowould be for a criminal to wear a new pair of boots, withdeep tread, at a damp site with clay-rich soil that adhereseasily. It has been assumed that the pollen on clothing orfootwear will predominantly reflect the assemblage at thesite that was visited last, but this has not been scientificallytested to date.

In this contribution, results are presented from an inves-tigation into the changes in the pollen assemblages ofmaterial adhering to footwear that has been worn at siteswith differing pollen assemblages. Pristine footwear wasworn at each of six rural sites, and the adherent materialsubsampled before the footwear was worn at subsequentsites, and further subsamples taken. Control soil sampleswere also collected from each site to determine its typicalpollen assemblage. Then each of these samples wereanalysed for their pollen assemblage. In doing so, the effectof mixing pollen/spores from soil at different sites overtime, with the pollen/spore assemblages from individualsites were investigated. The results were analysed statisti-cally in order to assess the degree of similarity between thepollen/spore assemblages at the six sites, and potentialmixtures between these sites. The implications of thesefindings for the application of forensic palynology arediscussed.

MATERIAL AND METHODS

General Strategy and Sampling

Six rural sites in the East Midlands of England, UnitedKingdom 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 previouslocalities within two ‘pyramid’ structures (Text-Figure2). Each ‘wearing’ comprised one of us (JBR) walkingnormally, and in random directions, for one minute at thespecific locality within a small area (ca. 9 m3). The itemsof footwear were not worn between localities. Compositecontrol soil/surface samples were also taken from eachlocality. Five subsamples were collected from a ca. 3 mtransect at each locality, and thoroughly mixed. Thissubsampling strategy aims to provide a representativesample by avoiding the skewing of a single sample due tothe proximity of a localized, particular highly polleniferousplant. The subsampling procedure followed the recom-mendations of Adam and Mehringer (1975). The controlsubsamples were collected by carefully scraping the up-

https://www.researchgate.net/publication/6868497_Consideration_of_some_taphonomic_variables_of_relevant_to_forensic_palyno-logical_investigation_in_the_United_Kingdom?el=1_x_8&enrichId=rgreq-00c52c41c32d667bf725df0a81860d90-XXX&enrichSource=Y292ZXJQYWdlOzIyNDk0NTgzOTtBUzoxMDM2ODY0NzkwMjQxMzNAMTQwMTczMjE5MTk0MA==





J.B. Riding, B.G. Rawlins, and K.H. Coley: Changes in soil pollen assemblages on footwear worn at different sites 137

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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 Englandwith the East Midlands indicated. b – a map of the counties of Nottinghamshire and Leicestershire illustrating the three areas wheresamples were taken. c – a detailed map illustrating location 1 near a wood, southwest of Nanpantan, Leicestershire. d – a detailedmap illustrating location 6 at the margin of Wollaton Park Lake, Nottingham. e – a detailed map illustrating locations 2, 3, 4, and5 at Ruddington Country Park, Nottingham.


permost 5 mm of surface materials using a clean knife (seealso Appendix 2). The samples of adherent soil from theitems of footwear and the control samples were analysedpalynologically to attempt to determine if the pollen/spore content becomes diluted by wearing the footwear atother sites. The control samples provide essential data forcomparison, even with adherent material taken from thepristine footwear, which should have a very similar com-position.

The collecting and sampling protocols are described indetail above, and also in Appendix 2. A cumulative strategywas adopted within two ‘pyramid’ structures. In the first,and larger, ‘pyramid’, only the first pair of boots was wornat locality one, the first and second pairs were worn atlocality two, the first, second and third pairs were worn atlocality three, and the first, second, third and fourth pairs offootwear were worn at locality four (Text-Figure 2). In thesecond, smaller, ‘pyramid’, the fifth pair of boots were

worn at both sites. Samples of the adherent soil on thefootwear were taken as appropriate, together with controlsamples (Text-Figure 2; Appendices 1, 2). The point of thisstrategy is to determine the magnitude of the dilution ormixing of the pollen/spore signals, assuming that palyno-logical dilution is a real phenomenon. The footwear usedcomprised sturdy ‘outdoor’ boots with a maximum treaddepth of between 7 and 9 mm deep. The six pairs used wereall new, and were thoroughly cleaned using a surfactantbefore the fieldwork. Similar footwear was used through-out this study. Representative examples of the footwearused are illustrated in Text-Figure 3.

In the sampling/subsampling from the footwear, theadherent mud/soil was carefully scraped away into a newsampling bag using a clean scalpel. No attempt was madeto differentiate a stratigraphy of the adherent materials, allthe mud/soil down to the sole was taken using the scalpelblade. In some cases, several subsamples were taken fromeach boot. The subsamples were taken from discrete areasof the tread that are described in Appendix 1. It wasassumed that the pollen content of the adherent materials isrelatively homogenous over the entire sole of the boot, andthat any bias introduced during subsampling was minimal.The times between the initial wearing, sampling/subsampling, and re-wearing where appropriate are also

Text-Figure 2. Pollen dilution triangle for sites 1 to 4illustrating the pure (bulk) soil samples in the left col-umn, and the boot samples in the triangle to the right. Thetriangle indicates that the potential pollen dilution viacontamination increases to the left and towards the base.Sample code 1234B (number 12) is hence potentially themost palynologically heterogeneous. The numbers referto the sites visited; B denotes boot and S denotes soil.

Text-Figure 3. Photographs of two pairs of clean boots used in this study, illustrating the treads. The left hand photograph is of pair3, ‘Dr Martens’ black 7-eye safety boots. The right hand photograph is of pair 4, ‘Century’ wellington type boots.


documented in Appendix 1. These data are importantbecause these parameters may determine how much dryingthere was, and whether any material became detached fromthe footwear.

It is acknowledged that, in forensic cases, several differ-ent techniques are used for obtaining pollen from footwearexhibits. These include washing or wiping the pollen fromthe upper part of the boot or shoe, and washing pollen fromthe laces separately. Pollen from the uppers and/or laces isoften a good indicator of the last place visited, and also if theowner has walked through crops, grass, herbs, other veg-etation, or in an open area. Furthermore, it is possible todissect specific layers of adherent material. This experi-ment simply used all the adherent materials from the solesof the footwear used, and washing was not undertaken. Thereason for this strategy is that the primary objectives wereconcerned with testing the transference of pollen in soil/surface materials taken from footwear and their dilutionpotential, 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 GeologicalSurvey palynology laboratory using standard palynologi-cal preparation procedures (Moore et al., 1991). They weredemineralized using hydrochloric acid (HCl) and hydrof-luoric acid (HF), the residual mineral grains were removedusing heavy liquid separation, and the residues subjected toacetolysis. The heavy liquid used was zinc bromide with aspecific gravity of 2.45–2.52. The microscope slides weremounted using the permanent mounting medium Elvacite.This is a permanent mounting medium and key grains caneasily be given a coordinate on the coverslip and re-examined. The disadvantage of a permanent mountant isthat problematic pollen grains cannot be moved in order tohelp identifications. If the mounting medium is viscous,e.g. glycerine or silicone oil, the grains can readily bemanipulated. However the grains can move in a viscousmounting medium and relocation can be a significantproblem. In a legal case, a key pollen grain crucial to theinvestigation must be readily relocatable so that it can beverified by both the legal teams. Sufficient slides wereproduced in order to allow statistically significant pollencounts to be made.

The palynomorphs were studied using an Olympus CH-2 transmitted light microscope. Pollen grains were identi-fied using standard European keys (e.g. Moore et al., 1991),and the pollen reference collection in the Department ofGeography, Royal Holloway, University of London, UnitedKingdom. The palynomorphs identified in this study arelisted in Appendix 3.

Statistical Analysis

From the full dataset, only those pollen types that ac-counted for more than 1% of the total pollen count wereselected and these eleven types are highlighted in Table 1,and listed in Table 2. The matrix of 18 samples with a subsetof 11 pollen types was analysed using Detrended Corre-spondence Analysis (DCA). This overcomes the arch/horseshoe effect. This phenomenon, and the tendency toconcentrate/compress the end of the ordination axis, are themajor problems associated with Correspondence Analysis(CA), which is often used for the analysis of ecological data(Hill and Gauch, 1980). Detrended Correspondence Analy-sis compensates for this by stretching and straightening thedata plots. The DCA was undertaken using the ‘vegan’package in an R-mode statistical environment (Oksanen etal., 2007).

The minor (<1%) components were not subjected toDCA because, if large numbers of trace taxa were included,the results would be skewed such that this technique wouldnot produce coherent plots and hence would be of limiteduse. It should be borne in mind however, distinctive pollentypes with low frequencies are potentially crucial in a legalsense. These may be of critical importance in providing keyevidence that a person visited a specific locality.

RESULTS

The 18 samples are listed in Appendix 1 and the resultantpollen data is presented in Table 1 and Text-Figure 4. Minorpollen types are those which account for less than 1% of thetotal pollen count in all samples; these are subsequentlyreferred to as trace components.

Site 1, Scrubland Near Wood, Southwest ofNanpantan, Leicestershire, U.K. (samples 1 and 2)

This rural locality (Text-Figures 1c, 5) is an area of roughscrubland and has a flora of small birch and oak trees, ferns,brambles, and herbs including grasses, nettles, thistles, andother weeds. Stands of mixed deciduous woodland sur-round the locality.

Pollen from samples 1 and 2 (i.e. pure locality 1) is ofrelatively low diversity (13 and 16 taxa respectively) and isdominated by Poaceae, with common Lactuceae, Pinus,and Cyperaceae (Text-Figure 4). Trace components in-clude Ambrosia, Aster type, Betula, Caryophyllaceae,Cedrus, cf. Centaurea type, Picea, Plantago type, Quercus,Tilia, undifferentiated broken saccate pollen, and undif-ferentiated trilete spores (Table 1). The pollen is rich inanemophilous (wind-pollinated) forms such as Pinus andPoaceae, and entomophilous (insect-pollinated) types such


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in sample 2 than sample 1; the remaining rare pollen typesoccur in similar numbers. The diversity of rare pollen typesis somewhat higher in sample 2; Ambrosia, Cedrus, cf.Centaurea type, and undifferentiated trilete spores wererecorded only in this sample. The only unique occurrencein sample 1 was a single grain of Tilia pollen. These results,unsurprisingly, support the expectation that material adher-ing to footwear that has only been worn at one locality willhave a virtually identical pollen signature as the localityitself. This is further supported by multivariate statisticalanalysis (Text-Figure 6).

The dominance of Poaceae at site 1 does not uniquelycharacterize the site. However of all the samples investi-gated as part of this study, the dominance of Poaceaetogether with common Lactuceae, Pinus, and Cyperaceae,in the complete absence of Alnus and the rarity of Plan-tago type is a characteristic signature. The rare compo-nents in both samples are also relatively similar.

Site 2, Elder/Hawthorn Woodland at RuddingtonCountry Park, Nottingham, U.K. (samples 3, 4 and 5)

This locality is adjacent to a footpath in a large countrypark (Text-Figure 1e). The flora comprises deciduoustrees and shrubs including elder and hawthorn withbrambles and stands of nettles (Text-Figure 7). The sur-rounding areas are fields.

Text-Figure 4. Percentages of pollen in each sample for the 11 dominant taxa (i.e. 1% and above of the total pollen in all samples).The samples are presented in sequence, i.e. 1-18. In the sample codes, the numbers refer to the sites visited in the sequence shown;B refers to boot, and S refers to soil. Therefore 123B means the sample was taken from a boot that had visited sites 1, 2, and 3.The control soil sample taken from site 6 is 6S.

Table 2. Pollen type (not species or sample) scores from thefirst two ordinates of the Detrended CorrespondenceAnalysis, and summary statisticsfrom the analysis.

as Lactuceae. This assemblage reflects the natural vegeta-tion at and around this site, which is a mixture of typicalscrubland trees and herbs. Sample 1 yielded more Lactuceaeand Cyperaceae, and less Pinus and Poaceae than sample 2(Text-Figure 4; Table 1). Quercus pollen is more common


This locality was the sparsest palynologically (Table 1).The reason for this is not clear; it may be due to the churningof the soil by animals such as rabbits and badgers, therebypromoting the mixing and rapid oxidation of the soil. Thepollen floras from samples 3, 4 and 5 are substantiallysimilar; the diversity is moderate, with 17, 17 and 21 taxarecorded respectively. These are overwhelmingly domi-nated by Pinus and Poaceae (Text-Figure 4; Table 1).Samples 4 and 5 respectively produced less and morePoaceae than sample 3 (Text-Figure 4). Moderate levels ofCyperaceae and Lactuceae are present, however otherforms are present in extremely low proportions (Text-

Figure 4; Table 1). Trace levels of Acer, Alnus, Artemisia,Aster type, Brassicaceae, Caryophyllaceae, Chenopod-iaceae/Amaranthaceae, Corylus, Fraxinus, Hedera helix,cf. Littorella, Picea, Plantago type, Polypodiaceae, Quercus,Ranunculus type, Salix, Sinapis cf. arvensis, Trifolium,undifferentiated broken saccate pollen, and Ulmus wererecorded (Table 1). The diversity of rare pollen types issomewhat higher in sample 5. Unique occurrences insample 5 are Artemisia, Corylus, cf. Littorella, and Sinapiscf. arvensis. Fraxinus and Salix are only present in sample4. Rare forms found in all three samples are Caryophyllaceae,Quercus, and Polypodiaceae. The pollen is dominated byanemophilous plant types such as Pinus and Poaceae;entomophilous types such as Aster type, Lactuceae, andRanunculus type are subordinate. This assemblage reflectsthe natural vegetation at this site and its environs, whichcomprise scrubland trees with some herbs.

The pollen/spore associations from pure samples fromsite 2 (i.e. samples 3 and 4) are significantly similar in bothtaxonomic spectrum and relative proportions. As at site 1,this observation supports the expectation that soil adheringto footwear only worn at one locality will have a virtuallyidentical pollen signature to the parent soil. This is sup-ported by multivariate statistical analysis (Text-Figure 6).This site is characterized by prominent Pinus and Poaceae,low numbers of Alnus, Cyperaceae, Lactuceae,Polypodiaceae, Quercus, and Ranunculus type, and thecomplete absence of Plantago type and cf. Primula. Boththe prominent and rare components are similar in thefootwear sample and the control.

Text-Figure 5. Site 1, scrubland near wood, southwest ofNanpantan, Leicestershire, U.K.

Text-Figure 6. Sample scores from the first two ordinates of the detrended correspondence analysis. In the sample codes, thenumbers refer to the sites visited in the sequence shown; B refers to boot, and S refers to soil. Therefore 1234B means the samplewas taken from a boot that had visited sites 1, 2, 3, and 4. The control soil sample taken from site 4 is 4S.


Sample 5 potentially includes material from both sites 2and 1. The gross similarity to samples 1 and 2 (Text-Figure4; Table 1) indicates that the majority of the material is fromsite 2, i.e. the last site visited. However there are somedifferences, which may be attributable to input (i.e. con-tamination) from site 1. For example, the diversity isslightly higher and there is more Acer, Brassicaceae,Lactuceae, Picea, Plantago type, Poaceae, Quercus, andundifferentiated broken saccate pollen in sample 5 than insamples 3 and 4 (Text-Figure 4; Table 1). Furthermore, atthis site, the occurrences of Artemisia, Corylus, Sinapis cf.arvensis, and cf. Littorella are unique to sample 5. Sample5 yielded significantly elevated levels of several pollentypes that are relatively common at site 1 and therefore maybe derived from there. These are Lactuceae, Picea, Plan-tago type, Poaceae, Quercus, and undifferentiated brokensaccate pollen. It is considered that these differences in bothdiversity and numbers recorded indicate input from bothsites 2 and 1 in sample 5.

Site 3, Pine Plantation at Ruddington Country Park,Nottingham, U.K. (samples 6, 7, 8 and 9)

This locality is a pine plantation in a large country park(Text-Figure 1e). The flora is dominated by pine trees andgrasses, with some other herbs/weeds (Text-Figure 8). Allfour samples from site 3 yielded abundant pollen/sporeassociations (Text-Figure 4; Table 1). The floras from thissite are all substantially similar; in particular they are alloverwhelmingly dominated by Poaceae pollen (Text-Fig-ure 4). However, less Poaceae pollen was counted insamples 8 and 9 in comparison to numbers 6 and 7. Thediversity is generally relatively low; samples 6, 7, 8 and 9produced 10, 12, 14 and 16 taxa respectively. Moderate

levels of Aster type, Cyperaceae, Lactuceae, Pinus, andundifferentiated broken saccate pollen are present. Further-more Alnus, Apiaceae, Brassicaceae, Betula, Caryophyl-laceae, Chenopodiaceae/Amaranthaceae, Lycopodium,Malva, Myriophyllum, Picea, Plantago type, Quercus,Ranunculus type, Sinapis type, and Tilia were recorded inlow proportions (Text-Figure 4; Table 1). The diversity ofrare pollen types is low in sample 6 in comparison tosamples 7, 8, and 9. Unique occurrences are that of Tilia insample 7, Quercus, Lycopodium, and Myriophyllum insample 8, and Caryophyllaceae and Ranunculus type insample 9. The only rare form found in all three samples isAlnus. The dominance of Poaceae pollen reflects the pres-ence of abundant grasses in the plantation, and in thesurrounding park. Pinus is also unsurprisingly present,together with a variety of other tree and herb pollen. Thisassociation is more diverse than would have been predictedfrom the overall flora. This probably reflects significanttransportation of pollen into this site.

The pollen/spore associations from the two pure samplesfrom site 3 (i.e. samples 6 and 7) are remarkably similar indiversity, taxonomic spectrum, and relative proportions.As before, this phenomenon demonstrates that soil onpristine footwear normally yields a virtually identical pol-len assemblage to the parent soil; this is also indicated bystatistical analysis (Text-Figure 6). The site is character-ized by dominant Poaceae, with subordinate Alnus, Astertype, Betula, Cyperaceae, Brassicaceae, Lactuceae, Picea,Pinus, and undifferentiated broken saccate pollen. Bothprominent and rare components are similar, and Acer,Hedera helix, and cf. Primula are entirely absent.

Samples 8 and 9 are susceptible to contamination frommaterial from sites 2 and 1. The overall similarities

Text-Figure 8. Site 3, pine plantation at Ruddington Coun-try Park, Nottingham, U.K.

Text-Figure 7. Site 2, elder/hawthorn woodland atRuddington Country Park, Nottingham, U.K.


between the pollen/spores in samples 8 and 9 to thosefrom samples 6 and 7 (Text-Figure 4; Table 1) stronglysuggests that the majority of the material was derivedfrom the last locality visited, i.e. site 3. However somedifferences were noted. The diversity in samples 8 and 9is slightly higher than in samples 6 and 7; furthermorethere are more Alnus, Aster type, Betula, Brassicaceae,Cyperaceae, Lactuceae, and Pinus in samples 8 and 9 thanin samples 6 and 7 (Text-Figure 4; Table 1). Moreover, theoccurrences of Plantago type, Quercus, Lycopodium, andMyriophyllum were only recorded in sample 8, andCaryophyllaceae, Ranunculus type, and Sinapis type, areunique to sample 9. Because samples 8 and 9 producedhigher diversities and elevated levels of certain taxa thatare present at sites 1 and 2, these compositional differ-ences probably represent input from sites 1 and 2. Forexample, the relatively high levels of Cyperaceae andLactuceae in sample 8 probably are, at least in part,derived from site 1. Likewise the presence of Quercus insample 8 may be derived from sites 1 and 2, and theoccurrence of Caryophyllaceae and Ranunculus type insample 9 could have come from site 2. The Picea andPinus pollen in samples 8 and 9 is also likely to havederived from sites 1 and 2. By contrast, undifferentiatedbroken saccate pollen is, somewhat surprisingly, con-fined to samples 6 and 7. The bar diagram of the mostcommon pollen/spore types (Text-Figure 4) clearly illus-trates the difference between pure site 3 soil, and the bootsthat had visited sites 1 and 2.

Site 4, Mixed Deciduous Woodland atRuddington Country Park, Nottingham, U.K.

(samples 10, 11, 12, 13 and 14)

This site is a stand of dense mixed woodland borderinga large country park (Text-Figures 1e, 9). The five samplesfrom this site produced relatively abundant pollen/sporeassociations of moderate diversity (Text-Figure 4; Table1). The diversities in samples 10 to 14 are 17, 18, 17, 15and 15 respectively. The pollen/spore assemblages are allsubstantially similar and are dominated by Alnus, Astertype, Cyperaceae, Lactuceae, Pinus, and Poaceae. By farthe most prominent taxa are Alnus and Poaceae (Text-Figure 4). Minor levels of Betula, Brassicaceae,Caryophyllaceae, Chenopodiaceae/Amaranthaceae,Frankenia cf. laevis, cf. Littorella, Lycopodium, Malva,Picea, Plantago type, Quercus, Ranunculus type,Rhynchospora, Sinapis type, Tilia, and undifferentiatedbroken saccate pollen were also recorded (Table 1). Uniqueoccurrences are those of cf. Littorella and Tilia in sample10, Frankenia cf. laevis, Quercus, Ranunculus type andSinapis type in sample 11, and Malva and Rhynchospora

in sample 14 (Table 1). The only rare forms found in allfive samples are Brassicaceae, Chenopodiaceae/Amaranthaceae, Picea, and undifferentiated broken sac-cate pollen. The prominence of Alnus and Poaceae re-flects the common alder trees in this area of woodland,and the grass of the nearby parkland respectively. Othertrees include Betula and Quercus, and herbs are repre-sented also.

The pollen/spore associations from the two pure samplesof soil from site 4 (i.e. numbers 10 and 11) are similar indiversity, taxonomic make up, and relative proportions.This again demonstrates that soil from new footwear nor-mally yields virtually identical pollen assemblages to theparent soil (Text-Figures 4, 6). Pollen assemblages fromthe two samples are characterized by dominant Alnus andPoaceae, with lesser proportions of Aster type, Cyperaceae,Lactuceae, and Pinus. Both the common and rare compo-nents are similar, and Acer, Hedera helix, and cf. Primulaare entirely absent.

Samples 12, 13 and 14 are prone to contamination frommaterial from sites 3, 2 and 1. The similarities between thepollen/spores in samples 12, 13 and 14, and those fromsamples 10 and 11 indicate that the majority of the materialwas derived from the last locality visited, i.e. site 4. The bardiagram of the most common pollen/spore types (Text-Figure 4) clearly illustrates the similarities between all fivesamples from site 4. However some relatively minor differ-ences were observed. Unlike at sites 2 and 3, the diversityof the three samples from multiple sites is lower than thatof pure soil from site 4 (Table 1). Malva and Rhynchosporawere only recorded rarely in sample 14; no unique taxawere recovered from samples 12 and 13. More Poaceae andundifferentiated broken saccate pollen are present in samples12, 13, and 14 than the pure soil samples 10 and 11. It is not

Text-Figure 9. Site 4, mixed deciduous woodland atRuddington Country Park, Nottingham, U.K.


clear if these increases represent contamination, and if so,from which site(s) they were derived. By contrast, severaltaxa diminish in numbers in the multiple site samples 12,13, and 14; these are Aster type, Betula, Cyperaceae, andPicea (Text-Figure 4; Table 1). The numbers of Alnuspollen in sample 13 are significantly higher than the othersamples at site 4. This increased level apparently representsnatural variability at site 4 because Alnus proved extremelyrare at sites 1, 2 and 3. Similarly the numbers of Pinus insample 12 is relatively high; this may also be due to naturalvariations (Text-Figure 4).

Site 5, Edge of Lake at Ruddington Country Park,Nottingham, U.K. (samples 15 and 16)

This site is a low, grassy area next to a lake with nearbystands of mixed shrubs and trees, including birch trees(Text-Figures 1e, 10). The most prominent components arecommon grasses, low-level herbs/weeds, and the commonreed (Phragmites communis – Poaceae). Pollen fromsamples 15 and 16 (i.e. pure locality 5) are of moderatediversity (18 and 17 taxa respectively). Sample 15 isrelatively abundant, and is dominated by Alnus, Betula,Poaceae, and Sinapis cf. arvensis. Lower numbers of Astertype, Chenopodiaceae/Amaranthaceae, Lactuceae, Picea,Pinus, Quercus, and Sinapis type are also present (Text-Figure 4; Table 1). Trace components observed includeBrassicaceae, Cyperaceae, Isoëtes, cf. Lobelia, cf. Primula,and cf. Urtica. This assemblage reflects the occurrence ofalder and birch trees, grass and herbs.

By contrast, the assemblage from sample 16 is relativelysparse in pollen/spores; only 155 grains were counted fromthree microscope slides. This association is overwhelm-ingly dominated by Poaceae (Text-Figure 4). Lower pro-

portions of Aster type, Cyperaceae, Lactuceae, Picea, Pinus,Pediastrum, Plantago type, Quercus, and Sinapis type arealso present. Trace levels of Alnus, Betula, Cheno-podiaceae/Amaranthaceae, and Typha were observed.The area is grassy and this explains the preponderance ofPoaceae. The occurrence of Pediastrum in this sample isentirely consistent with a lakeside environment; this is awidespread genus of freshwater colonial algae (Batten,1996).

Unusually, the pollen floras of these two samples aresignificantly different. Of the four dominant taxa in sample15, sample 16 produced extremely low numbers of Alnus,Betula, and Sinapis cf. arvensis. Despite its abundance insample 15, no representatives of Sinapis cf. arvensis wereobserved in sample 16. By contrast, the proportion ofPoaceae in sample 16 is significantly higher than in sample15 (Text-Figure 4). The proportions of Aster type,Cyperaceae, Lactuceae, Picea, Pinus, Quercus, and Sina-pis type are relatively similar. Except for Pediastrum andPlantago type in sample 16, the only unique occurrences ofidentifiable pollen/spores are those that are relatively rare.These include Brassicaceae, Isoëtes, cf. Lobelia, cf. Primula,and cf. Urtica in sample 15, and Typha in sample 16 (Table1). The palynomorph associations from samples 15 and 16are hence substantially different, despite both being puresoil/lakeside mud from the same site. This is a highlyunusual situation, and demonstrates that substantial naturalvariability in pollen profiles does exist. This phenomenonmay also reflect the fact that this is a man-made lake whichis approximately 15 years old. It may be that the palynologi-cal heterogeneity observed reflects the abundant levels oftransported material during the landscaping process. Thesubstantial statistical distance between these samples isshown in Text-Figure 6. Normally, sites are characterized

Text-Figure 11. Site 6, margin of Wollaton Park Lake,Nottingham, U.K.

Text-Figure 10. Site 5, edge of lake at Ruddington CountryPark, Nottingham, U.K.


by a small number of abundant taxa. In this case, the twosamples from site 5 have similar proportions of Aster type,Cyperaceae, Lactuceae, Picea, Pinus, Quercus, and Sina-pis type, which are of low to moderate abundance (Text-Figure 4; Table 1).

Site 6, Margin of Wollaton Park Lake,Nottingham, U.K. (samples 17 and 18)

This locality is the immediate margin of a large lake closeto a path which is next to extensive grassed parkland withoccasional shrubs and trees (Text-Figures 1d, 11). Unlikesite 5, the pollen floras from samples 17 and 18 at site 6 aresubstantially similar in terms of both the spectrum of taxapresent, and their relative proportions (Text-Figure 4; Table1). This is confirmed by multivariate statistical analysis(Text-Figure 6). The diversity is moderate, with 19 and 18taxa recorded respectively. The two samples are dominatedby Poaceae, with significant proportions of Alnus, Lactuceae,Pinus, and Quercus (Text-Figure 4). Other forms are presentin relatively low proportions. These include Aster type,Betula, Brassicaceae, Caryophyllaceae, Cedrus,Chenopodiaceae/Amaranthaceae, Cyperaceae, Malva,Picea, Pediastrum, Plantago type, cf. Saxifraga, Sinapistype, Tilia, undifferentiated broken saccate pollen, and cf.Urtica (Table 1). Sample 18 yielded substantially lessPinus and Quercus than sample 17; otherwise the associa-tions from these two samples are broadly comparable(Text-Figure 4). This site is characterized by prominentPoaceae, with common/moderate numbers of Alnus,Lactuceae, Pinus, and Quercus. In particular, the relativelyhigh proportions of Quercus, together with the moderatelevels of Alnus, is highly characteristic. The dominantpollen reflects the grassed park with stands of alder and oaktrees, with some weeds/herbs. The presence of Pediastrumin sample 17 is consistent with a lakeside setting (Batten,1996).

Sample 18 potentially includes material from sites 5 and6, however the striking similarity of the two pollen/sporefloras indicates that the majority of the material is from site6, i.e. the last site visited (Text-Figure 4; Table 1). In anycase, contamination from site 5 would be difficult todiscern due to the markedly disparate nature of the florasfrom site 5 (see above). However, it is possible that theslightly raised levels of Alnus and Poaceae, and the occur-rences of rare Chenopodiaceae/Amaranthaceae and Plan-tago type, in sample 18 may be, at least in part, derived fromsite 5. It is also possible that this putative contaminationmay have diluted the levels of Cyperaceae, Pinus, andQuercus in sample 18 (Text-Figure 4). It must be stressedthat these differences are relatively minor, and well withinnormal levels of natural variability.

Detrended Correspondence Analysis

The first two ordinates explain most of the variation inthe data and the site scores for them are plotted in Text-Figure 6. The species scores are given in Table 2. Anotable feature of the site scores (Text-Figure 6) is theclustering of both ‘boot’ and ‘soil’ samples according tothe site visited last. For example, all the ‘boot’ sampleswith material derived from sites 1, 2, 3 and 4, and theirrespective soil samples, are enclosed by the dashed el-lipses drawn around them. The pollen composition for the‘soil’ from site 5 appears to be anomalous; the score on thefirst ordinate is far larger than those for the ‘boot’ samplefrom this site, and they are not closely associated. Theonly explanation for the large observed differences inpollen composition from this site is that it comprisestransported material (made ground) which has not beenthoroughly mixed. However, the pollen composition fromthe ‘boot’ which had been worn at sites 5 and 6 does occurin close proximity to both the ‘boot’ sample from site 5and the ‘soil’ sample at site 6.

DISCUSSION

The raw pollen/spore data from the 18 samples is dis-played in Table 1, and the relative proportions of the elevendominant taxa illustrated in Text-Figure 4. From bothTable 1 and Text-Figure 4, it is clear that each of the six siteshas an extremely distinctive pollen/spore association. How-ever, site 5 is unusual in that the two samples producedsubstantially different palynofloras. The distinctiveness ofthe palynofloras from these sites was also demonstratedstatistically using detrended correspondence analysis (Text-Figure 6). It is clear from Text-Figure 6 that the samplesfrom sites 1–4 and site 6 cluster closely together, demon-strating that the statistical distance between them is low.

There are two principal conclusions emanating from thisstudy. Firstly, that the six individual localities have distinc-tively different pollen/spore signatures. This is due to thenature of the virtually infinite variability of local vegetationspectra, and to the multiplicity of complex taphonomicprocesses which affect the pollen and spores deposited.Some examples of the many pertinent factors are altitude,nutrient availability, prevailing wind, soil type, and topog-raphy. This apparent palynological heterogeneity confirmsthe potential usefulness of palynomorph associations inforensic studies in order to establish provenance, and henceto link people or items to a specific location. The analysisherein shows that adherent materials on footwear domi-nantly reflect the last site that was visited, even if severalsites have been attended previously (Text-Figures 4, 6). Inother words, palynomorph associations derived from the


last site are not significantly diluted by the pollen fromprevious sites. Detrended correspondence analysis sup-ports this (Text-Figure 6). However, footwear samples thatwere potentially contaminated with materials from previ-ous localities normally exhibited some subtle differences.These are typically slight increases in diversity, and smallvariations in certain pollen types. The subtlety of thesedifferences means that they would be difficult to discernand/or quantify in a ‘real’ forensic investigation. Differ-ences in the pollen load on footwear which has been wornat other sites is to be expected because shoes/boots etc. areknown to pick up palynomorphs, especially if the sites areon soft/natural ground, as opposed to in the built environ-ment (Wiltshire, 2006).

The findings of this study show that in forensic investi-gations, it is imperative that footwear from suspects isseized as soon as possible, since the palynological signa-ture on the shoes/boots will probably reflect the last sitevisited. If several polleniferous sites are visited after thewearing of footwear at a crime scene, the crime scenepollen signature will normally be diluted, perhaps pro-foundly so. If there is a delay in seizing footwear, any mudetc. on the boots/shoes should be carefully studied anddissected prior to preparation. This is in order to attempt toestablish a pollen chronology or stratigraphy for the mate-rial entrained on the tread of the footwear. One possiblepertinent scenario is where a crime scene is characterizedby highly unusual, probably exotic, pollen type(s). If theunusual (?non-native) pollen was found in a background ofabundant, natural (?native) pollen picked up from otherlocalities, the former could link a suspect to a crime scenewhich was visited prior to other localities. In such a case,even small numbers of the unusual pollen that had persistedon the footwear could be critical to the case.

ACKNOWLEDGMENTS

This research was funded by projects managed by DrsMichael G. Petterson (BGS, Keyworth) and J. Russ Evans(BGS, Edinburgh). James B. Riding and Barry G. Rawlinspublish with the approval of the Executive Director,British Geological Survey (N.E.R.C.). This contributionreceived helpful and perceptive reviews from Drs. VaughnM. Bryant Jr. (Texas A&M University) and Stewart G.Molyneux (BGS, Keyworth).

References Cited

ADAM, D.P., and MEHRINGER, P.J. Jr.1975 Modern pollen surface samples – an analysis of

subsamples. U.S. Geological Survey Journal of Re-search, 3(6): 733–736.

BATTEN, D.J.1996 Chapter 7C. Colonial Chlorococcales. In: Jansonius,

J., and McGregor, D.C. (eds.). Palynology: prin-ciples and applications. American Association ofStratigraphic Palynologists Foundation, Dallas, 1:191–203.

BRYANT, V.M., and JONES, G.D.2006 Forensic palynology: Current status of a rarely used

technique in the United States of America. ForensicScience International, 163: 183–197.

HILL, M.O., and GAUCH, H.G. Jr.1980 Detrended Correspondence Analysis: an improved

ordination technique. Vegetatio, 42: 47–58.HORROCKS, M., COULSON, S.A., and WALSH, K.A.J.

1998 Forensic palynology: variation in the pollen contentof soil surface samples. Journal of Forensic Sci-ences, 43(2): 320–323.

1999 Forensic palynology: variation in the pollen contentof soil on shoes and in shoeprints in soil. Journal ofForensic Sciences, 44(1): 119–122.

JANSONIUS, J., and McGREGOR, D.C. (eds.)1996 Palynology: principles and applications. American

Association of Stratigraphic Palynologists Founda-tion, Dallas, 3 volume set, 1330 p.

MILNE, L.A., BRYANT, V.M. Jr., and MILDENHALL, D.C.2005 14. Forensic Palynology. In: Coyle, H.M. (ed.). Fo-

rensic Botany. Principles and applications to crimi-nal casework. CRC Press LLC, Boca Raton, Florida,U.S.A, 217–252.

MOORE, P.D., WEBB, J.A., and COLLINSON, M.E.1991 Pollen Analysis. Second Edition. Blackwell Scien-

tific Publications, Oxford, 216 p.OKSANEN, J., KINDT, R., LEGENDRE, P., and O’HARA, R.B.

2007 vegan: Community Ecology Package version 1.8-5.http://cran.r-project.org.

TRAVERSE, A.2007 Paleopalynology. Second edition. Springer,

Dordrecht, The Netherlands, 813 p.WILTSHIRE, P.E.J.

2006 Consideration of some taphonomic variables of rel-evance to forensic palynological investigation in theUnited Kingdom. Forensic Science International,163: 173–182.









https://www.researchgate.net/publication/257653484_Modern_pollen_surface_samples_-_An_analysis_of_subsamples?el=1_x_8&enrichId=rgreq-00c52c41c32d667bf725df0a81860d90-XXX&enrichSource=Y292ZXJQYWdlOzIyNDk0NTgzOTtBUzoxMDM2ODY0NzkwMjQxMzNAMTQwMTczMjE5MTk0MA==






Location 1: Scrubland near wood, southwest of Nanpantan,Leicestershire, U.K. (Text-Figures 1c, 5)

Rough scrubland dominated by shrubs.GPS coordinate: 449774 Easting, 316764 Northing; elevation

157 m.Wearing and subsampling: 15.20h; 30th November 2005.Boot type: new pair of ‘Dr Martens’ 6-eye black boots – ‘pair 1’.Soil type: surface soil, dark brown/black slightly sandy soil that is

relatively dry and well drained; much leaf/vegetable matter.Local geology: no outcrops visible; Charnian.Local flora: dominated by ferns/bracken. Some grasses and

herbs/weeds such as nettles and thistles. Some small treesespecially birch, hazel, and oak; mixed mature deciduouswoodland nearby (ca. 6 m).

SAMPLE #1 – bulk soil sample. Collection number MPA 54840.SAMPLE #2 – pure locality 1 soil sample taken immediately

from the left-hand boot sole of pair 1. This was scrapings fromthe infilled treads all over the boot sole. Collection numberMPA 54841.

Location 2: Elder/Hawthorn woodland, Ruddington CountryPark, Nottingham, U.K. (Text-Figures 1e, 7)

South side of park, along a footpath/cycle track.GPS coordinate: 458090 Easting, 332140 Northing; elevation

46 m.Wearing and subsampling: 10.30h; 1st December 2005.Boot type: new ‘Caterpillar’ safety boots – ‘pair 2’.Soil type: surface soil, dark brown slightly sandy soil that is

relatively dry and well-drained. Much leaf/vegetable matter,largely hawthorn leaves with some red hawthorn berries.Abundant rabbit droppings were observed and a badger sett ispresent nearby. The samples were taken to the south side of thepath, among elder trees.

Local geology: no outcrops visible; till on Triassic mudstone.Local flora: trees are dense stands of elder and coppiced haw-

thorn; there are other (minor) mixed deciduous trees/shrubs.Some brambles and stands of nettles were observed. Therewere no grasses in the immediate area, but grassed fields arepresent nearby.


from the left-hand boot sole of pair 2. This was scrapings fromthe infilled treads all over the boot sole. Collection numberMPA 54845.

SAMPLE #5 – soil sample taken immediately from the right-hand boot sole of pair 1. This was scrapings from infilledtreads from the entire heel sector of the boot sole (mapped as‘sector no. 1’). This was scraped off with a clean scalpel, andthe scrapings are theoretically material from locations 1 and 2.There was 19 hours, 10 minutes between the visits to locations1 and 2. No unequivocal mixing was observed. Collectionnumber MPA 54848.

APPENDIX 1. List of samples.

Note that the geographic coordinates are in meters, and refer to the British National Grid. The data were captured using a standard globalpositioning system (GPS) unit.

Location 3: Pine plantation - Ruddington Country Park,Nottingham, U.K. (Text-Figures 1e, 8)

Pine woodland, close to children’s play area.GPS coordinate: 457043 Easting, 302226 Northing; elevation

41 m.Wearing and subsampling: 11.40h; 1st December 2005.Boot type: new pair of ‘Dr Martens’ black 7-eye safety boots –

‘pair 3’.Soil type: surface soil, mid/dark brown slightly sandy soil that is

relatively dry and well drained. Much leaf/vegetable matter,largely pine needles is present. The samples were takenseveral meters from the edge of the woodland.

Local geology: no outcrops visible; till on Triassic mudstone.Local flora: pine trees. Occasional immature birch trees; some

thistles. Grasses and low herbs/weeds on ground; close tograssed areas.


from the left-hand boot sole of pair 3. This was scrapings frominfilled treads all over the boot sole. Collection number MPA54850.

SAMPLE #8 – soil sample taken immediately from the right-hand boot sole of pair 1. This was scrapings from infilledtreads from the central sector of the boot sole (mapped as‘sector no. 2’). This was scraped off with a clean scalpel, andthe scrapings are theoretically material from locations 1, 2 and3. There was 19 hours, 10 minutes between the visits tolocations 1 and 2, and 1 hour, 10 minutes between the visits tolocations 2 and 3. No definite mixing was observed. Collec-tion number MPA 54853.

SAMPLE #9 – soil sample taken immediately from the right-hand boot sole of pair 2. This was scrapings from infilledtreads from the heel sector of the boot sole (mapped as‘sector no. 1’). This was scraped off with a clean scalpel, andthe scrapings are theoretically material from locations 2 and3. There was 1 hour, 10 minutes between the visits tolocations 2 and 3. No definite mixing was observed. Collec-tion number MPA 54854.

Location 4: Mixed deciduous woodland - Ruddington CountryPark, Nottingham, U.K. (Text-Figures 1e, 9)

Mixed deciduous woodland, close to the north part of the perim-eter road.

GPS coordinate: 457147 Easting, 332518 Northing; elevation26 m.

Wearing and subsampling: 13.20h; 1st December 2005.Boot type: new ‘Century’ wellington type boots - ‘pair 4’.Soil type: surface soil, black, sandy loam that is relatively dry and

well drained. Much leaf/vegetable matter, largely sycamoreand birch leaves. Samples were taken several meters from theedge of the woodland, close to the perimeter road.

Local geology: no outcrops visible; till on Triassic mudstone.


Local flora: coppiced mixed deciduous woodland dominated bysycamore with some birch and oak. Grass and herbs present atforest floor. Fungi (puffballs) also relatively common. Thesite is close to grassed areas and allotment gardens over theroad.

SAMPLE #10 – bulk soil sample. Collection number MPA54855.

SAMPLE #11 – pure locality 4 soil sample taken immediatelyfrom the left-hand boot sole of pair 4. This was scrapings fromthe infilled treads all over the boot sole. Collection numberMPA 54856.

SAMPLE #12 – soil sample taken immediately from the right-hand boot sole of pair 1. This was scrapings from infilledtreads from the entire upper part of the boot sole (mapped as‘sector no. 3’). This was scraped off with a clean scalpel, andthe scrapings are theoretically material from locations 1, 2, 3and 4. There was 19 hours, 10 minutes between the visits tolocations 1 and 2, 1 hour, 10 minutes between the visits tolocations 2 and 3, and 1 hour, 40 minutes between the visits tolocations 3 and 4. Some definite mixing was observed. Underthe darker soil was grey mud/soil, possibly from locality 1.Collection number MPA 54859.

SAMPLE #13 – soil sample taken immediately from the right-hand boot sole of pair 2. This was scrapings from infilledtreads from the entire upper part of the boot sole (mapped as‘sector no. 2’). This was scraped off with a clean scalpel, andthe scrapings are theoretically material from locations 2, 3 and4. There was 1 hour, 10 minutes between the visits to locations2 and 3, and 1 hour, 40 minutes between the visits to locations3 and 4. No definite mixing was observed. Collection numberMPA 54860.

SAMPLE #14 – soil sample taken immediately from the right-hand boot sole of pair 3. This was scrapings from infilledtreads from the whole of the boot sole. This was scraped offwith a clean scalpel, and the scrapings are theoretically mate-rial from locations 3 and 4. There was 1 hour, 40 minutesbetween the visits to locations 3 and 4. No definite mixing wasobserved, although some possible stratification was vaguelypresent. Collection number MPA 54861.

Location 5: Edge of lake at Ruddington Country Park, Nottingham,U.K. (Text-Figures 1e, 10)

Grassed area close to lake edge.GPS coordinate: 457464 Easting, 332010 Northing; elevation

34 m.Wearing and subsampling: 15.45h; 20th November 2005Boot type: new ‘Dr Martens’ 10-eye, black safety boots - ‘pair 5’.Mud type: wet, dark red-brown mud/soil at margin of lake.Local geology: no outcrops visible.Local flora: the most prominent components are the common

reed (Phragmites communis – Poaceae), common grass andlow-level herbs/weeds. Stands of mixed trees/shrubs close by.These include birch trees.

SAMPLE #15 – bulk mud/soil sample. Collection number MPA54833.

SAMPLE #16 – pure locality 5 mud/soil sample taken immedi-ately from the left-hand boot sole. This was scrapings from theinfilled treads all over the boot. Collection number MPA54834.

Location 6: Edge of Wollaton Park Lake, Nottingham, U.K.(Text-Figures 1d, 11)

Lakeside margin, close to footpath.GPS coordinate: 452915 Easting, 338914 Northing; elevation

41 m.Wearing and subsampling: 13.20h; 23rd November 2005.Boot type: pair 5Mud type: wet, very dark red-brown mud/soil at margin of lake.Local geology: the lake bank comprises clay-rich till with com-

mon erratic pebbles.Local flora: grasses, herbs/weeds, and reeds. Stands of trees

(oak), catkin-bearing tree and shrubs are present nearby.SAMPLE #17 – bulk mud/soil sample. Collection number MPA

54835.SAMPLE #18 – mud/soil sample taken immediately from the

right-hand boot sole of pair 5. This was scrapings from infilledtreads from the bottom-left sector of the boot sole (mapped as‘sector no. 1’). This was scraped off with a clean scalpel, andthe scrapings are theoretically material from locations 5 and 6.There was 69 hours, 7 minutes between the visits to locations5 and 6. Collection number MPA 54837.


‘Pyramid 1’ – localities 1–4 (see Text-Figure 2).

1 At the first locality, a pair of new boots (‘Pair 1’) was worn.A sample of adherent material was collected from a mappedsector of the sole of the left boot of Pair 1; this sample waslabelled ‘Pair 1, Left - Locality 1’. The right boot of Pair 1was left intact. A surface/soil sample was taken as a control;theoretically this should be the same material as the adherentmaterial sample from the left boot of Pair 1.

2 At the second locality, another pair of new boots (‘Pair 2’)was worn. A sample of adherent material was taken from amapped sector of the sole of the left boot of Pair 2; thissample was labelled ‘Pair 2, Left - Locality 2’. The right bootof Pair 2 was left intact. A surface/soil sample was taken asa control; theoretically this should be the same material asthe adherent material sample from the left boot of Pair 2. Pair1 was worn again and a sample of adherent material wastaken from a mapped sector of the sole of the right boot; thissample was labelled ‘Pair 1, right - Localities 1+2’.

3 At the third locality, another pair of new boots (‘Pair 3’) wasworn. A sample of adherent material was taken from amapped sector of the sole of the left boot of Pair 3; thissample was labelled ‘Pair 3, left - Locality 3’. The right bootof Pair 3 was left intact. A surface/soil sample was taken asa control; theoretically this should be the same material asthe adherent material sample from the left boot of Pair 3.Pairs 1 and 2 were worn again and samples of adherentmaterial were taken from mapped sectors of the soles of theright boots; these samples were labelled ‘Pair 1, right -Localities 1+2+3’ and ‘Pair 2, right - Localities 2+3’ respec-tively.

4 At the fourth locality, another pair of new boots (‘Pair 4’)was worn. A sample of adherent material was taken from amapped sector of the sole of the left boot of Pair 4; thissample was labelled ‘Pair 4, left - Locality 4’. The right bootof Pair 4 was left intact. A surface/soil sample was taken asa control; theoretically this should be the same material asthe adherent material sample from the left boot of Pair 4.Pairs 1, 2 and 3 were worn again and samples of adherentmaterial were taken from mapped sectors of the soles of theright boots; these samples were labelled ‘Pair 1, right -Localities 1+2+3+4’, ‘Pair 2, right - Localities 2+3+4’ and‘Pair 3, right - Localities 3+4’ respectively.

‘Pyramid 2’ – localities 5 and 6.

5 At the fifth locality, a pair of new boots (‘Pair 5’) was worn.A sample of adherent material was collected from the sole ofthe left boot of Pair 5; this sample was labelled ‘Pair 5, Left- Locality 5’. The right boot of Pair 5 was left intact. Asurface/soil sample was taken as a control; theoretically thisshould be the same material as the adherent material samplefrom the left boot of Pair 5.

6 At the sixth locality, Pair 5 were worn. A sample of adherentmaterial was taken from a mapped sector of the sole of theright boot of Pair 5; this sample was labelled ‘Pair 5, Right- Locality 6’. A surface/soil sample was taken as a control.

APPENDIX 2. Detailed sampling protocol

Each visit to a site involved one of us (JBR) wearing the appropriate footwear and walking normally (i.e. not using exaggerated forcein order to deliberately entrain material into the boot tread), in random directions, for one minute. The wearing of the respective footwearwas confined to a small area of approximately 9 m3 at the specific locality. The items of footwear were not worn anywhere else, i.e. betweenlocalities. All the localities were on natural surfaces, i.e they were more or less vegetated. At each locality a composite sample of soil/surfacematerials was taken as a control. Five subsamples were collected from an approximately 3 m transect at each site, and thoroughlyhomogenized. This strategy of subsampling was undertaken in order to avoid the potential biasing of a single sample because of theproximity of specific plants. This should to lead to the collection of composite samples which are representative of the local pollen rain(Adam and Mehringer, 1975). The control samples were obtained by carefully scraping the uppermost 5 mm of surface material into a newsample bag using a clean knife. Furthermore, GPS readings were recorded, notes were made on the local vegetation, geology, and soilmoisture conditions, and some photographs taken.



Palynomorph taxa Common name

I POLLENAcer maple/sycamoreAlnus alderAmbrosia ragweedApiaceae (Umbelliferae) carrot family, e.g. celery, parsley, hogweedArtemisia mugwortAster type daisiesBetula birchBrassicaceae (Cruciferae) cabbage family, e.g. mustard, watercressCaryophyllaceae carnation family, e.g. chickweed, stichwortCedrus cedarcf. Centaurea type cornflower, knapweed, star thistle etc.Chenopodiaceae/Amaranthaceae goosefootCorylus hazelCyperaceae sedge familyFrankenia cf. laevis sea heathFraxinus ashHedera helix ivyLactuceae dandelion tribecf. Littorella shoreweedcf. Lobelia lobeliaMalva mallowMyriophyllum water milfoilPicea sprucePinus pinePlantago type plantainPoaceae grass familycf. Primula primrose/cowslipQuercus oakRanunculus type buttercups, crowfoots, and spearwortsRhynchospora star sedgeSalix willowcf. Saxifraga saxifrageSinapis type charlock/mustardSinapis cf. arvensis charlock/wild mustardTilia limeTrifolium cloverTypha bulrushUlmus elmcf. Urtica nettleUndiff. broken saccate pollen N/AUnidentified pollen N/A

II SPORESIsoëtes quillwortLycopodium clubmossPolypodiaceae polypod fern familyUndifferentiated trilete spores N/A

III MISCELLANEOUSPediastrum colonial freshwater alga

APPENDIX 3. Listing of the botanic and common names of the palynomorph taxa observed in this study.

Note that unidentified/undifferentiated forms could not be identified because they were either broken, or diagnostic features werenot visible.


Documents

Changes in soil pollen assemblages on footwear worn at different sites