Forensic Science International 244 (2014) 186–195

Contents lists available at ScienceDirect

Forensic Science International

journal homepage: www.elsevier.com/locate/forsciint

Palynology and mycology provide separate classes of probative

evidence from the same forensic samples: A rape case from southern
England
Patricia E.J. Wiltshire a,b,*, David L. Hawksworth b,c,d, Judith A. Webb e, Kevin J. Edwards a,f
a
Department of Geography and Environment, School of Geosciences, University of Aberdeen, Elphinstone Road, Aberdeen AB24 3UF, UK
b
Mycology Section, Royal Botanic Gardens, Kew, Surrey TW9 3DS, UK
c
Departamento de Biologı´a Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal, Madrid 28040, Spain
d
Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, UK
e
2 Dorchester Court, Blenheim Road, Kidlington, Oxford OX5 2JT, UK
f
Department of Archaeology, School of Geosciences, University of Aberdeen, Elphinstone Road, Aberdeen AB24 3UF, UK

A R T I C L E I N F O A B S T R A C T

Article history: A young woman claimed to have been raped at night in a wooded strip of land 120 m from her home. The
Received 10 July 2014 suspect refuted her claim and said that they had had consensual sexual relations on an area of short turf
Received in revised form 18 August 2014 in a local park, 130 m distant from the alleged crime scene. Comparator samples from each place, and
Accepted 20 August 2014
clothing and footwear from each party, were obtained for assessment and analysis. All places that were
Available online 29 August 2014
considered to be relevant to the case were visited, and lists of plant species made. Results showed that
the palynological and mycological profiles yielded by the footwear and clothing of both parties closely
Keywords:
resembled that of the wooded area, but were dissimilar to that of the park. The profile of the wooded site
Spores
Pollen
reflected closely its own vegetation. Faced with the evidence, the suspect confessed. The study proves the
Fungi value of a multi-proxy approach to forensic investigation using exactly the same preparations.
Plants ß 2014 Elsevier Ireland Ltd. All rights reserved.
Profile
Soil

1. Introduction
Palynology is the study of palynomorphs. These are microscop-
ical, organic particulates derived from individual taxa1 of organ-
isms, and may be of botanical, mycological, or even zoological
origin. They are proxy indicators of the environments from which
they originate, and the total of palynomorph taxa observed during
analysis forms the palynological assemblage2 of any specific
sample. A characteristic profile of a sample is obtained by detailed
counting and calculation of the proportions of the palynomorph
taxa [1] in each sample.
Forensic palynology and mycology are sub-disciplines of
forensic botany and mycology. Botany is the study of every aspect
* Corresponding author at: Department of Geography and Environment, School of
Geosciences, University of Aberdeen, Elphinstone Road, Aberdeen AB24 3UF, UK.
Tel.: +44 1372 272087; fax: +44 1372 272087.
E-mail address: patricia.wiltshire1@btinternet.com (Patricia E.J. Wiltshire).
1
Taxa (plural of taxon): names of individuals or groups of organisms of any rank.
2 4
Assemblage: all the palynomorph taxa present in a sample.
of all plant groups, and mycology is the study of all fungi which
includes lichens, mushrooms, moulds, mildews, slime moulds, and
yeasts. Pollen grains are produced by conifers and flowering plants,
and plant spores are produced by mosses, ferns and their allies.
The various groups of fungi produce different kinds of spore in a
wide variety of microscopic and macroscopic structures; and these
structures can, themselves, form part of the palynological profile.
Many taphonomic3 factors are involved in determining the
representation of a plant in the air spora,4 a major one being
whether a plant is insect- or wind-pollinated. Put simply, wind-
pollinated plants produce copious amounts of well-dispersed
pollen, while insect-pollinated plants produce small amounts
which may never be found in the air spora. Any pollen residuum
left in the flower by animal vectors may only be released into the
immediate environs of the plant, or to the soil when the plant dies
3
Palynological taphonomy: the sum total of factors which influence the presence
of a palynomorph at a given place at a specific time.
Air spora: the total pollen and spore load of the air at any one time.

http://dx.doi.org/10.1016/j.forsciint.2014.08.017
0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
 P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195
and decays. There is a high level of variability in the distribution of
vegetation, and this is reflected in the pattern of pollen deposition
on the ground. As yet, no two locations have been shown to yield
precisely the same palynological profiles, and this makes the
discipline a powerful technique for differentiating one place from
another. Taphonomy, and interpretation of dispersal patterning,
are complex and important factors in palynology [1–4].
The distribution of fungal spores is also variable as it depends
on an appropriate supply of food for the fungus. Many form
mutualistic (e.g. endo- or ectomycorrhizal)5 or antagonisic (e.g.
parasitic) relationships with plants, while others (saprotrophs) can
only use dead plant and animal material, including dung. Those
closely associated with living plants exhibit varying degrees of
specificity in host exploitation, but even saprotrophs may be
limited to the dead litter of a restricted range (and often a single
species) of plants [5,6]. Fungal spores alone can be indicative of
specific habitats as they are often secondary proxy indicators of
plant species. Their taphonomy is also complex, but there is
evidence that most fall very close to where they are produced and
fail to reach the air spora [7–9]. They can thus be highly
characteristic of a small area.
Plants and fungi may be undetected in a field survey, and their
presence (both past and present) may be indicated only by their
pollen and spores. By combining the pollen and plant spore profile
with that of the fungal spore profile, a higher degree of spatial
specificity is achieved than by one class of evidence alone.
To the best of our knowledge, this is the first instance where
both botanical and mycological trace evidence has been obtained
from the same samples, and an extensive knowledge of the ecology
and geographical distribution of the plants and fungi, involved in
the assemblage, was critical to data interpretation. The case
involves the rape of a young woman in the county of Wiltshire,
southern England. For legal reasons, details that may identify
locations and individuals have been omitted. The analytical results
of this case contributed to a confession, and we consider that the
procedures and the disciplines involved have relevance to a wide
range of criminal investigations.
2. Background
A woman claimed to have been raped by her boyfriend after
they had spent the evening together. He walked her home and she
maintained that instead of stopping outside her house, he forced
her to walk another 50 m to a wooded strip of land running
between two quiet roads (Figs. 1 and 2). The suspect said that they
had had consensual sexual relations on the turf in the middle of an
open park approximately 200 m to the southwest of the putative
crime scene. The couple had admitted to sexual activity on the day
of the alleged offence, so DNA analysis would not have been useful
in testing the veracity of the testimony of either party.
Experience in many forensic investigations has revealed that
contact with paving and metalled surfaces results in few
palynomorphs being transferred to footwear, unless there is a
layer of plant litter, or other palyniferous6 material such as soil, on
that surface. For clothing to accumulate large numbers of
palynomorphs, experience has shown, again, that it must have
been in contact with palyniferous surfaces or material. Thus, in this
putative rape case, if the clothing and footwear of the parties
yielded palynological profiles, it indicated that they had contacted
soil, vegetation, plant litter, or some other palyniferous surface.
5
Mycorrhizal fungus: a fungus which penetrates plant roots; it obtains
carbohydrate from the plant, and provides its plant partner with phosphate and
other nutrients. The endomycorrhizal fungus penetrates the root cells while the
ectomycorrhizal fungus grows only between them.
6
Palyniferous: any surface or material coated with, or containing, palynomorphs.
187
The defendant stated he had never been in the wooded area at
any time, while the woman claimed that he had forced her to lie on
the ground there. Therefore, if trace evidence obtained from both
parties were more similar to either the park or the wooded area,
this would provide support for one of the testimonies over the
other. Two alternative hypotheses can be constructed:
1. The parties had lain on the ground in the woodland.
2. The parties had lain on the ground in the park.
The results of palynological analysis of clothing and footwear,
from the male, female, and both sites, were used to test these two
hypotheses.
3. Methods
3.1. Rationale
As the woman claimed that she had been forced to the ground
and raped in the wooded area (Fig. 1), it was important to
confirm from her clothing and footwear that she could have been
on the ground in that place while wearing those garments. If
the accused had also lain in the same place, his clothing would be
expected to yield a similar profile of palynomorphs to that of the
woman. Thus, it was necessary to assess the palynological
profiles from the clothing and footwear of each party before any
microscopical examination of comparator (field) samples. A list
of the samples examined and/or analysed is given in
Table 1. Once the nature of each palynological profile was
established, the species lists from all survey places could be
compared, and locations (including that of the park where the
man asserted that the parties had had consensual sex) could
be differentiated by virtue of the vegetation alone. It was not
possible to carry out a comprehensive survey of the whole
catchment for each site, but predominant plant species noted at
each pertinent location are given in Table 2.
3.2. Site descriptions
A: Alleged crime scene: The gross pattern of roads, buildings, and
vegetation around the alleged crime is shown in Fig. 1. The scene
was a narrow strip of wooded land, dominated by conifer and
hardwood trees and shrubs, and bordered either side by roads
running parallel to it in an east/west direction. The road on the
southern side was bordered by a wide strip of partially mown,
weedy turf planted with occasional small trees and shrubs. The turf
contained a range of herbs, some of which were flowering, but
other areas had been too closely mown to allow them to flower.
The ground under the trees and shrubs consisted mostly of bare
soil, but it was also strewn with a layer of dead leaves and woody
litter, mostly derived from the large Quercus tree which dominated
the strip of ground. Mature Betula pendula, Pinus sylvestris, and
Chamaecyparis were also very prominent, along with Sambucus
nigra. The ground flora was dominated by Poaceae, Taraxacum,
Urtica dioica, Anthriscus sylvestris, and other sparsely growing
herbs. There were richly planted, well-tended gardens in
surrounding roads, and pollen from them could be expected to
have found its way into the alleged crime scene.
B: The path: The path was metalled and bordered by weedy,
mown grassland on either side and led into the wooded area of
the crime scene.
C: The park: This park consisted of open area (145 m  40 m) of
turf, with native and exotic trees and shrubs around the periphery.
The site where consensual sexual activity was claimed by the male
was 8.0 m from the nearest trees. This specific patch of ground was
covered by weedy turf that had been mown since the alleged
[(Fig._1)TD$IG]
188 P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195

Fig. 1. Map showing the locations mentioned in the text. A – Crime scene; B – path approaching crime scene; C – park; D – claimant’s back garden; E – claimant’s front and side
garden; F – open public space in the town; G – site where defendant discarded his over-trousers; and H – defendant’s lodgings.

offence took place. However, as the grass clippings had been left in
situ and, and these would have contained palynomorphs likely to
have been picked up at the time of the offence, they were included
in the sampling. It was noted that there were considerable
amounts of Trifolium repens and Trifolium campestre in the cuttings
and in the intact turf.
D and E: Back, front, and side garden of complainant’s home: The
front and side gardens were open plan and consisted of narrow
strips of lawn with sparsely planted flowerbeds. The back garden
was of mown lawn, weedy edges, and unkempt flowerbeds.
F: Open public space: The public space in the centre of town
consisted of open, weedy turf and plantings of mature trees around
[(Fig._2)TD$IG] P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195
Fig. 2. Photographs of locations (see text and Fig. 1): top – the park (site C); middle –
alleged crime scene within the woodland (site A), with the entry path to the right
(site B); bottom – alleged crime scene (site A) within the woodland. The numbered
scene markers are visible in the top and bottom photographs [See online on article
for colour version.].
the periphery. Many of the trees were Tilia europaea. It was
known that both parties frequented this area. It was 1.0 km from
the putative crime scene (directional arrow in Fig. 1).
G: Site of discarded over-trousers: The garment was discarded
over a Cotoneaster bush on the boundary at the rear of two gardens
after the suspect left the complainant. These gardens were richly
planted with exotic shrubs and herbaceous plants. In addition,
there were non-native conifers planted along the street at the
back of the gardens.
H: Suspect’s lodgings: The suspect had been staying in a house on
an estate (450 m SSW of the putative crime scene). The estate backed
onto mature woodland about 80 m away. The woodland contained
189
Fagus sylvatica, Fraxinus excelsior, and Acer pseudoplatanus. The
estate itself was surrounded by typical suburban gardens and
weedy, grass verges with trimmed Cupressaceae trees, and an
occasional Prunus sp. A single B. pendula was seen on the estate.
I: Friend’s house: This was the address which the suspect was
visiting immediately before the alleged offence took place. It was
located 870 m NE of the crime scene and is not shown in Fig. 1. The
garden was well tended, most of the shrubs were clipped, and the
grass was closely mown. There was a large Magnolia tree, and other
trees and shrubs, in the back garden. The owner said that the
suspect had not entered the back garden at any time.
3.3. Field evaluation and sampling
For the evaluation of the vegetation of various locations
associated with the alleged offence, a field visit was made in the
month of July, within a week of the incident. Each of the parties
identified the exact place within each site where they claimed
intercourse had taken place, and comparator sampling was closely
targeted within those areas (Fig. 2).
In order to obtain a sample representative of the area over
which the prone bodies contacted the ground surface during
intercourse, a group of subsamples, 30 cm  30 cm (0.90 m2),
was obtained from each identified place (Fig. 2). In the case of the
grass in the park, all turf cuttings in the sample were taken as
well as growing grass shoots which were cut with scissors. The
wooded area was sampled in the same way although, here,
surface soil (no deeper than 0.5 cm), and all twiggy material from
the immediate surface, were collected. In both cases, the total
area sampled was 0.72 m2. Samples from the park were stored in
paper bags and dried quickly to prevent them from becoming
mouldy. The soil samples were placed into self-locking plastic
bags and refrigerated at 3 8C. Processing was carried out within
two days.
3.4. Laboratory sampling, preparation, and analysis
3.4.1. Sampling
Every precaution against contamination was given at all
stages of the work. Exhibits were examined by eye and under
10 magnification, and then photographed. Six comparator
samples were chosen for the wooded area, and five for the park.
This represented ground areas for each site of 0.54 m2 and
0.45 m2 respectively.
Some items obtained from the two parties were subdivided;
this enabled the determination of a greater resolution for
transference patterning of palynomorphs on the garments. Soles
of footwear were treated separately from uppers, while knees and
hems of jeans were treated separately, and independently of the
body area. This would make it possible to compare samples from
the exhibits with those from the comparator samples. It would also
enable us to establish whether or not those parts of the body had
contacted the ground.
3.4.2. Processing and sampling preparation
Palynomorphs were retrieved from exhibits by standard
techniques using a hot surfactant solution, sieving, and centrifu-
gation, followed by standard treatments [10]. Comparator samples
were prepared in the same way. Processing involved HCl, NaOH,
acetolysis, HF, safranine stain, embedding of palynomorphs in
glycerol jelly, and spreading of the palynomorph suspension on
microscope slides [3]. The fixing in jelly is important for forensic
samples in that each palynomorph must be able to be relocated by
any other palynologist from coordinates. Penile and vaginal swabs
were provided by the medical examiner. These were subjected to
the same protocols as other samples.
190 P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195
Table 1
List of exhibits obtained from each party for scanning and analysis.
Exhibits from female
Vulval and vaginal swabs 3 amalgamated into 1
Nylon tights Entirety treated
Shorts Buttock area treated
Top Back area treated
Shoes Two soles combined
Exhibits from male
Penile swabs 6 swabs amalgamated into 1
Trainers Two soles combined
Two sets of uppers combined
Jeans Fabric from two knees combined
Fabric from two hem areas combined
Over-trousers Two legs treated separately
Body area treated separately
3.4.3. Microscopical analysis
Two slides of each preparation were examined with a Zeiss
Phase Contrast Microscope at 400 and 1000 magnification. For
intelligence purposes, and as a preamble to full analysis, scanning
for both plant and fungal palynomorphs was carried out on the
same slides. This involved identification and subjective scoring of
palynomorph abundances, and allowed determination of those
samples worthy of full analysis. For both scanning and full-count
studies, contiguous fields along equidistantly spaced transects
were examined. This allowed for any non-random distribution of
palynomorphs in the mountant. In full analysis of the comparators
and exhibits, the sum of palynomorphs ranged from 336 to 19,733.
After counting, the data were inevitably enhanced by further
examination aimed at identifying any rarer components that might
be present in the sample.
3.4.4. Production of pollen diagram
A partial diagram of palynomorph taxa (Fig. 3) was produced
within the program Tilia [11].
4. Results
4.1. Presentation of data
Table 2 lists the most obvious plant taxa observed at each site.
Supplementary Information Tables S1–S3, present the percent-
age dataset for each palynomorph taxon obtained from
comparators and exhibits. The count for each palynomorph
taxon was expressed as a percentage of the total count of
palynomorphs for all analysed samples. Fungal and non-plant
palynomorphs in each sample were expressed as a percentage of
the total count (including all pollen, plant spores, plus fungal
spores) in that sample. This practice ensures that the number of
fungal spores does not affect the proportions for plant spores and
pollen. Fungal spores are not formed in the same way as plant
spores and pollen, and constitute a data set independent of the
botanical profile; other non-plant and non-fungal remains found
are presented in a similar way. Taxa achieving less than 0.3%
are shown in Tables S2 and S3 as plus signs. Palynomorphs that
were identified by extra scanning, at the end of counting, were
shown as plus (+) signs in parentheses, and these were not
included in the calculations.
The values in Supplementary Information Tables S1–S3
represent the relative proportions of the various taxa in each
sample. In addition, for ease of inspection of the findings, each set
of samples (i.e. body swabs, clothing, footwear, and comparator
samples) were combined, and presented graphically as simplified
plots (Fig. 3). In this way, all profiles could be compared visually.
For convenience, Fig. 3 does not include any taxon which failed to
achieve at least 0.5% of the total count. Irrespective of their
percentage values, all taxa are given in Table 3 which provides a
summary of all taxa found in each set of samples (for the wooded
area, park, female, and male). The taxa are ordered for ease of
comparison.
4.2. Discussion
Table 2 presents the rich suite of trees, shrubs, and climbers
(34 taxa) and herbs (25 taxa) that were observed during field
evaluation. No ferns were recorded during the site visits, and no
attempt was made to identify the mosses. This is usual practice as
few moss taxa have spores with a sufficiently distinct morphology
to allow identification, though some identifiable moss spores were
found in the analysis (Supplementary Information Table S1). The
spore-producing bodies of most fungi are microscopic, and many
are not readily seen in the field, even by use of a hand-lens; it is
consequently impractical to include them in field surveys of
forensically pertinent sites.
Scanning of the preparations made from the garments and
shoes demonstrated a rich assemblage of taxa more similar to
vegetation at the putative crime scene (site A) and its environs (B)
than that in the park (C), or in any of the other locations examined
(Table 2). The analysis consequently concentrated on ground
samples from the wooded area, and from the park, for comparison
with those from the clothing and footwear.
The park and the alleged crime scene were only about 130 m
apart, but the environment offered by each was different. The
ground surface identified by the suspect at the park was mown,
weedy turf, with grass cuttings left in situ. It was not directly
influenced by trees or shrubs, whereas the alleged crime scene was
enclosed by trees and shrubs, and the ground was open soil strewn
with woody litter (dead twigs, small branches, dead leaves, and
fruits). The palynological profiles from the park and the wooded
area reflected the plants, litter, and conditions at each site.
4.2.1. Alleged crime scene and the park
4.2.1.1. Palynological profile – plants. A striking feature of the
woody plants represented in the alleged crime scene samples was
the number of taxa (28). It contained many native plants, but also
diverse aliens whose pollen would have originated from local
planted areas and gardens. The high proportion of pollen from
trees and shrubs reflected the wooded nature of the site and its
immediate vicinity. The profile was dominated by Betula, Quercus,
Pinus, and Sambucus nigra-type,7 while Rosaceae undiff.8 and
Cupressaceae were also well represented. Many of the taxa found
in the alleged crime scene were absent from the comparators from
the park. These included Alnus, Fagus, Hedera, Picea, Acer, Castanea,
Juglans, Lonicera periclymenum-type, Salix, Ceanothus, Rhus, and
Ulmus. The palynological profile indicated a mixture of artificial
habitats commonly found in suburban settings.
In contrast, the results from the park indicated a relatively
treeless place, even though there were trees and shrubs around its
perimeter. The actual area of ground, claimed by the suspect to be
the place where consensual sexual activity took place, was
sufficiently far from the peripheral trees and shrubs (8.0 m)
for their pollen to be relatively poorly represented (15 taxa). Only a
single grain of Betula was recorded while the Quercus record was
negligible. Fig. 3 shows a stark contrast between this location in the
park and that of the alleged crime scene. Of the 28 woody taxa
7
Type (-type): indicates cases where several species within the genus or species
complex are not differentiated by the pollen.
8
Undiff.: undifferentiated, indicates pollen identification only made to plant
family.
 P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195 191

Table 2 pollen could be picked up from more than one place. Others, such
Plant taxa growing at pertinent sites: A – crime scene; B – path approaching crime
as Apiaceae undiff., Ranunculus-type, Rumex obtusifolius-type, and
scene; C – park; D – claimant’s back garden; E – claimant’s front and side garden;
F – open public space in the town; G – site where defendant discarded his over- Cyperaceae were either more abundant at the alleged crime scene
trousers; H – defendant’s lodgings; and I – house of defendant’s friend. than the park, or even absent from the park, whereas Artemisia,
Fabaceae undiff., and Amaranthaceae were more abundant in
Trees, shrubs, climbers A B C D E F G H I
the park.
Laburnum anagyroides + +
Malus sylvestris + +
4.2.1.2. Palynological profile – fungi. Supplementary Information
Betula pendula + + + + +
Cupressaceae + + + + Table S1 shows that there were abundant fungal spores, and a rich
Sorbus aucuparia + + + assemblage of fungal taxa, retrieved from the alleged crime scene;
Corylus avellana + + there were relatively few in the park samples. The spores of most
Cratageus monogyna + +
fungi making up the assemblage are passively discharged and
Pinus sylvestris + +
Quercus robur + +
poorly dispersed, and unlikely to be deposited more than a few cm
Rubus fruticosus + + from where they are produced. Their presence on any object
Hedera helix + + implies direct contact; this could be with the ground, with dead/
Populus sp. + decaying wood or leaves, or any other material on which the
Sambucus nigra +
fungus could grow.
Sorbus sp. +
Acer pseudoplatanus + + The bare soil at the alleged crime scene was littered with twiggy
Aesculus hippocastanum + + + material and fallen, decaying leaves. Fungi characteristic of dead
Fagus sylvatica + + + wood and litter of hardwoods (including Betula, Quercus, and Fagus)
Prunus (cultivar) + + +
were frequent (Brachysporium britannicum, Camposporium cam-
Tiliaeuropaea + +
Platanusacerifolia +
brense, and Diplocladiella scalaroides). In view of the abundance of
Robinia pseudoacacia + Betula litter, it is of particular interest that Bactrodesmium betulicola,
Ilex aquifolium + an indicator of this genus, was well represented. Clasterosporium
Rhododendron (cultivar) + flexum was the most abundant fungal spore from the alleged crime
Rosa (cultivar) +
scene, but this fungus is represented by only 11 records in the British
Carpinus betulus + +
Hebe (cultivar) + Mycological Society Fungal Records Database of Britain and Ireland
Cotoneaster sp. + (FRDBI).9 That species occurs on decaying leaves of Cupressaceae,
Forsythiaintermedia + and there was a tree belonging to this family growing immediately
Laurus nobilis +
adjacent to the scene, and abundant decaying leaves and litter from it
Lonicera periclymenum +
Fraxinus excelsior +
on the ground. Pestalotiopsis funerea, a parasite attacking living leaves
Magnolia sp. + of Cupressaceae, was also abundant in the comparator samples.
Buddleja davidii + Another marker of interest was Pseudovalsella (cf. 10), a taxon
Fuchsia (cultivar) + considered to be exceedingly rare. The discovery of Rhizophagus
Herbs A B C D E F G H I fasciculatus was of special note as it is an endomycorrhizal fungus
Anthriscus sylvestris + and its presence implies contact with exposed soil. There are only
Arum maculatum + four records of this species of Rhizophagus in the FRDBI, although it
Lapsana communis + has been found in woodlands in continental Europe, or associated
Poaceae + + + + with Platanus. Another rare fungus found at the alleged crime scene
Taraxacum officinale + + + + + + +
Poaceae + + + + + + + +
was a species of Didymosphaeria, and the species in question may
Urtica dioica + + even be new to science.
Trifolium repens + + + + + + Epicoccum nigrum was highly abundant in the samples from the
Bellis perennis + + + + + + + park but was represented by only two records from the alleged
Cirsium arvense + +
crime scene. It is a ‘spoilage’ fungus and would be expected to have
Plantago lanceolata + + + +
Ranunculus repens + + + + + been exploiting the grass cuttings on the surface of the turf in the
Anagallis arvensis + park. Cymadothea trifolii, which is only known to grow on leaves of
Geranium molle + Trifolium species, reflects the presence of this plant genus in the
Hieracium sp. + park samples. The Melanospora sp. was also relatively abundant,
Plantago major +
Rumex sanguineus +
and most species of that genus parasitise other fungi which are
Senecio jacobaea + saprotrophs11 of herbaceous plants.
Epilobium roseum + + Both the alleged crime scene and the park had, therefore,
Picris echioides + yielded spores of fungi characteristic of their respective plant
Stellaria media +
communities.
Senecio vulgaris +
Sonchus oleraceus +
Calystegia sepium + 4.3. Clothing and footwear

4.3.1. Plant palynomorphs

recorded at the alleged crime scene, just 13 were recorded at the
park, and the overall palynological profiles were completely
different.
A total of 29 herbs, ferns, and mosses was represented at the
alleged crime scene, and 26 in the park comparators. Fig. 3 shows
that at both places the dominant herbaceous taxa were Poaceae,
Urtica, Plantago lanceolata, Sinapis-type, and fenestrate Asteraceae.
These are all common plants in many open environments and their
Table 3 shows that of the 30 woody taxa represented in the
comparator samples, 29 were also found associated with the
defendant’s belongings. The most abundant of these associated
with the suspect were also those that were most frequent at the
alleged crime scene. These included Betula, Pinus, Quercus,
9
http://www.fieldmycology.net/FRDBI/FRDBIrecord.asp.
10
cf.: compare with, used to indicate the most similar taxon of pollen or spore.
11
Saprotroph: an organism whose food source consists of dead material.
192 P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195

Table 3 Table 3 (Continued)

Summary of pollen and spore taxa found in samples from the wooded area, park,
male, and female. Herbs Wood Park Male Female

Trees/shrubs/climbers Wood Park Male Female Poaceae + + + +

Urtica + + + +
Betula + + + + Brassicaceae (Sinapis-type) + + + +
Quercus + + + + Plantago lanceolata + + + +
Pinus + + + + Ranunculus-type + + + +
Sambucus nigra-type + + + + Asteraceae fenestrate + + + +
Rosaceae undiff. + + + + Rumex obtusifolius-type + + + +
Ligustrum/Syringa-type + + + + Cereal-type + + + +
Cupressaceae + + + + Fabaceae indet + + + +
Fraxinus + + + + Apiaceae (Heracleum cf.) + + + +
Tilia + + + + Bellis/Aster-type + + + +
Castanea sativa + + + + Plantago major + + + +
Aesculus + + + + Typha latifolia + + + +
Lonicera periclymenum-type + + + + Amaranthaceae + + + +
Buddleja + + + + Apiaceae indet + + +
Corylus avellana + + + Cyperaceae + + +
Calluna vulgaris + + + Rumex acetosa-type + + +
Fagus + + + Filipendula + + +
Salix + + + Artemisia + +
Rhus + + + Caryophyllaceae (Cerastium-type) + +
Juglans regia + + + Geum cf. + +
Hedera helix + + + Malva-type +
Ulmus + + Sanguisorba minor-type +
Ceanothus cf. + + + Papaver + + +
Picea + + + Achillea/Anthemis-type + + +
Acer + + + Mercurialis annua + + +
Carpinus + + 0 Potentilla-type +
Ericaceae undiff. + + + Oenothera +
Eucalyptus-type + + + Cirsium +
Magnoliaceae undiff. + + + Mercurialis perennis +
Taxus + + Allium + +
Platanus + Rumex crispus-type + +
Buxus + Thalictrum + +
Prunus-type + Fabaceae (Vicia-type) + +
Ilex + + Anemone + +
Cedrus + Juncaginaceae + +
Garrya + Liliaceae indet + +
Galium-type + +
Ferns/mosses Wood Park Male Female
Lamiaceae (Mentha-type) + +
Pteropsida monolete + + + + Silene dioica-type + +
Dryopteris dilatata + + + Lamiaceae (Prunella-type) +
Dryopteris filix-mas-type + + + Stachys sylvatica-type +
Sphagnum + + + Centaurea nigra-type +
Phyllitis cf. + Geranium +
Polypodium + Knautia +
Asplenium-type + Liliaceae (Hyacinthoides-type) +
Polygonum aviculare-type +
Fungi Wood Park Male Female Campanula-type +
Brassicaceae (Capsella-type) +
Dictyosporium toruloides + + + +
Symphytum +
Periconia byssoides + + + +
Impatiens (small) +
Bactrodesmium betulicola + + + +
Pestalotiopsis funerea + + + +
Clasterosporium flexum + + +
Pseudovalsella-like + + +
Brachysporium britannicum + + + Sambucus, Cupressaceae, and Rosaceae undiff. Other pollen taxa of
Camposporium cambrense + + woody plants were found on the clothing and footwear, but many
Diplocladiella scalaroides + + of these were absent from the park samples. The value for Rosaceae
Rhizophagus fasciculatum + + undiff. was high (55% of the whole assemblage) on the over-
Didymosphaeria sp. + + +
Melanospora sp. + +
trousers. This reflects their having being thrown over a large,
Bactrodesmium obovatum + + flowering Cotoneaster plant at site G as the defendant left the area.
Epicoccum nigrum + + The high values of Fraxinus pollen on the trainers also indicate
Endophragmiella fagicola + + that he had walked close to a Fraxinus tree, but the values on
Cymadothea trifolii +
his jeans resembled those in the profile of the alleged crime
Diporotheca cf. +
Asterosporium hoffmannii + scene even more closely.
Niesslia exposporioides + The herbaceous palynomorph profiles of both the alleged crime
Phaeotrichosphaeria britannica + scene and the park comparator samples were dominated by
Poaceae, Urtica, Plantago lanceolata, Sinapis-type, and fenestrate
Asteraceae. All these are common plants and could have been
transferred to clothing and footwear at either place. Others, such as
Apiaceae undiff., Ranunculus-type, Rumex obtusifolius-type, and
Cyperaceae, were either more abundant at the alleged crime scene
or absent from the park samples. Angelica sylvestris (Apiaceae)
plants were also growing in the wooded area but not in the park,
 P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195
and the suspect had accumulated pollen of this plant family on his
footwear and clothing. Conversely, pollen of Artemisia and
Fabaceae undiff. was more frequent (the latter overwhelmingly
so) in the park samples than in those from the alleged crime scene.
Although small amounts of Fabaceae pollen were found on the
belongings of both parties, the amounts were similar to those
found in the samples from the wooded area. Considering
the substantial amounts of Fabaceae pollen in the turf of the
park, this taxon could have been expected to contribute a much
larger proportion of the palynological profile from the defendant
and claimant if they had lain on the ground there.
The values for Poaceae and Urtica on the suspect’s belongings
were higher than in the comparator samples for the alleged crime
scene, but he may have picked up pollen directly from flowers and
foliage rather than the ground at that site. Flowering Poaceae and
Urtica plants were growing close to the place identified by the
complainant as the attack site. This contention is supported by
the presence of anther masses, immature Poaceae grains, and a
cluster of Pestalotiopsis funerea fungal spores. The complainant’s
belongings also yielded anther masses of Quercus, Betula,
Sambucus, and Poaceae. These are unlikely to have been dispersed
away from the plant or fungus, and their presence implies direct
contact with the catkins, flowers, or fungi.
The palynomorph profiles from both parties were similar; of the
28 woody taxa found in the woodland samples, 24 were found on
[(Fig._3)TD$IG]
the claimant, and 27 on the suspect, and both yielded the rarer
Fig. 3. Simplified palynomorph plots of sample profiles. + indicates <0.5% of the relevant palynomorph sum (see text for details).
193
palynomorphs such as Picea, Lonicera, Castanea sativa, Rhus, Garrya,
Salix, Ceonothus, and Juglans (Table 3).
4.3.2. Fungal palynomorphs
Fig. 3 and Supplementary Information Table S1 show the
abundance and variety of fungi found on the clothing and footwear,
and include some which are rarely recorded and considered rare. It
is improbable that these taxa were transferred from the ground
surface in the park as they were not present in the comparator
samples from that place. High numbers of Epicoccum nigrum and
Melanospora sp. spores in the park comparator samples were
barely represented on the clothing and footwear, and higher values
would have been expected if the two parties had lain on the area of
ground identified by the defendant. Conversely, many of the fungal
palynomorphs found at the alleged crime scene were found on the
clothing and/or footwear of both parties. Clasterosporium flexum,
associated with decaying litter of Cupressaceae, and rarely found in
Britain (see above), as well as P. funerea, a parasite of Cupressaceae,
were found on the clothing and footwear of both claimant and
suspect. Of note is the presence of a mature, but shaded,
Chamaecyparis (Cupressaceae) tree immediately next to the spot
where the crime is alleged to have been committed. Pseudovalsella
(cf.) and Rhizophagus fasciculatus, both rarely recorded fungi, were
also found on the two parties; another rare fungus, Endophrag-
miella fagicola, which would have been growing on twigs, was
found only on the suspect. These rarely reported taxa were not
194 P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195
found in the comparator samples from the park which consisted of
grass cuttings. Phaeotrichosphaeria britannica and Asterosporium
hoffmannii were found on the suspect and, although they were not
found in the comparator samples from the alleged crime scene,
they are likely to derive from decaying twigs and leaf litter which
were present on the ground there. These fungi would not have
been able to grow on grass cuttings in the park.
The fungal spore assemblage found on the footwear and
clothing of the claimant was also characteristic of the alleged crime
scene. Again the fungi associated with Cupressaceae and Urtica
were particularly well represented along with the rare Pseudo-
valsella (cf.) and R. fasciculatus. A spore referred to Didymosphaeria
was also found on the claimant’s shorts; the species represented by
this spore has either not yet been observed in Britain and is
exceedingly rare, or has not yet been given a scientific name.
Another palynomorph, well represented at the alleged crime scene,
and found on the claimant’s shoes, shorts, and top, was described
as ‘a ball of straw-coloured cells’. It is not known whether this
palynomorph was fungal in origin, or belonged to some other
group of organisms; it was not found in the samples from the park.
4.4. Body samples
The suspect’s penile swabs yielded no palynomorphs, but
several pollen grains of Quercus and Poaceae were retrieved from
the claimant’s vaginal samples. No fungal spores were found. The
suspect’s penis or hands are likely to have contacted soil or
vegetation during the incident, and palynomorphs were probably
transferred into the girl during penetration. According to
circumstances, it might be prudent for vaginal swabs to be
routinely examined for palynomorphs in suspected rape cases
[12]. The place identified by the claimant was directly under the
canopy of a large Quercus tree. The value for Quercus pollen at the
alleged crime scene ranged between approximately 6.0 and 24.0%,
whereas only a trace was found in the park.
4.5. Summary discussion
Fig. 3 and Table 3 demonstrate the commonality of pollen and
spore taxa found at the alleged crime scene (site A) and on the
clothing and footwear of both parties. The high value for Rosaceae
undiff., retrieved from the male, reflects the Cotoneaster pollen
picked up by his over-trousers when he discarded the garment
over a bush at site G. Although Betula and Quercus were well
represented on the belongings of both parties, they failed to reach
the high values found in the alleged crime scene comparator
samples. Surface palynomorph distribution is often very patchy,
and it is possible that the samples from site A were not collected
from exactly the same area of ground supposedly lain on during the
offence. The significance of other taxa should also be emphasised.
The very high values for Fabaceae and Epicoccum nigrum would be
likely to have been much better represented on the footwear and
clothing if an offence had taken place in the park.
5. Conclusions
Analysis of comparator samples and exhibits showed that the
palynological profiles, obtained from the clothing and shoes of
both parties, resembled the profiles of the woodland more than
those from the park. Hence the palynological evidence strongly
supports Hypothesis 1 over Hypothesis 2. Thus, it was deemed
more likely that the girl’s testimony was true rather than that of
the defendant’s.
When presented with the evidence, the defendant confessed
that he had lain with the girl in the wooded area, as she had
claimed. Palynological analysis was thus instrumental in the police
obtaining a conviction without the need for lengthy and costly
court proceedings.
The following points of general applicability arise from this
investigation:
1. Plant and mycological palynomorphs can provide separate classes
of evidence from the same samples. The two independent sets of
evidential data can be used for challenging a hypothesis derived
from one sample set. Each set can corroborate the other in
supporting one hypothesis or another [13].
2. Finds of palynologically rare pollen, and spores from rarely
recorded fungi, can enhance the acceptance or refutation of
hypotheses.
3. Extensive experience of casework has indicated that, in the
course of day-to-day activity, pollen and spores in the air are not
deposited to a measurable extent on clothing. Some plants
produce small amounts of poorly dispersed pollen and, in the
case of most fungi, dispersal is particularly restricted. To yield
sufficient palynomorphs for the construction of a profile, an
object needs to have direct physical contact with a palyniferous
surface (e.g. soil, vegetation, litter).
4. The analyst needs to recognise disparate palynological and/or
mycological assemblages in a mixed profile to enable elimina-
tion of locations irrelevant to the case. Clothing and footwear
can pick up palynomorphs from more than one place visited by
the wearer, but pollen and spores may also be lost in varying
amounts, so the profile can be modified after an event and the
initial transfer of palynomorphs. Different depositional events
may be represented on an item, and this will result in a mixed
assemblage, although this is more an issue for footwear than for
clothing. Further, when an item contacts a palyniferous surface,
it will inevitably pick up only part of its palynological profile.
This is the same for one or more comparator samples from a
precise location which will similarly contain a representation of
the actual palynological profile of the place. An item of clothing or
footwear might yield palynomorphs that were not seen in the
comparator samples even though they formed part of the
location’s profile. Conversely, a comparator sample might contain
palynomorphs that were not picked up by the clothing or
footwear. In spite of these factors, if sufficient proxy indicators of a
place are present on an item, contact between them will be
indicated [1,13,14]. More weight has to be accorded to presences
rather than absences because of this limitation.
5. At any crime scene, pollen and spores on the surface would
represent extant vegetation but, because of palynomorph
residuality, traces of previous vegetation could also be present.
Such residuality can be prolonged, depending on ambient
conditions, and the nature of the soil. Any presence of ‘historical’
palynomorphs increases the palynological specificity of the site
by enhancing the number of taxa available for transfer. Thus,
the palynological profile can be richer than that anticipated from
the vegetation even though palynomorphs from the extant
stand of vegetation would be likely to dominate. Once the
profiles of pertinent exhibits were established by scanning or
analysis, the forensic palynologist would probably be able to
eliminate any other location by taking note of the vegetation
currently growing there. In this case, it is unlikely that analysis
of surface samples other than those from the crime scene would
be necessary, except for corroborative purposes [1,13,14].
6. In most cases of rape, both parties would have contacted the
same palyniferous surfaces, with the same or different parts of
their bodies (and clothing). The botanical and mycological
profiles from the clothing and footwear of each party would be
expected to be similar, and there should be sufficient points of
similarity with that of the actual crime scene for a link to be
recognised.
 P.E.J. Wiltshire et al. / Forensic Science International 244 (2014) 186–195
7. The power of palynology is underlined by its ability to
differentiate between sites, even if they are in close proximity.
It is also possible to demonstrate the contact between specific
places and items, such as footwear, even though such items
invariably accumulate palynomorphs from other locations.
In making the above observations, we would point out that
they are supported by the results we have obtained in many
other cases which have been presented to, and tested, in court.
We have found no instance where the data sets obtained from
pollen and plant and fungal spores are in conflict, or where the
interpretations derived from them have proved to be at variance
with the facts established in court.
Acknowledgements
We thank Alison Sandison for artwork, and both Paul M. Kirk
and Christopher Walker for commenting on the identity of
particular fungal spores. This contribution was prepared while
D.L.H. was in receipt of funding from the Spanish Ministerio de
Ciencia e Innovación project CGL2011-25003. We are grateful to
Norman Fenton, Dallas Mildenhall, and an anonymous referee for
their comments on the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.forsciint.2014.08.017.
195
References
[1] P.E.J. Wiltshire, Forensic ecology, in: P. White (Ed.), Crime Scene to Court: The
ssentials of Forensic Science, 3rd ed., RSC Publishing, London, 2010, pp. 54–85.
[2] K. Faegri, L. van der Pijl, The Principles of Pollination Ecology, Pergamon Press,
Oxford, 1966.
[3] P.D. Moore, J.A. Webb, M.E. Collinson, Pollen Analysis, 2nd ed., Blackwell
Scientific Publications, Oxford, 1991.
[4] H. Tauber, Differential pollen dispersion and filtration, in: E.J. Cushing, H.E.
Wright, Jr. (Eds.), Quaternary Paleoecology, Yale University Press, New Haven/
London, 1967, pp. 131–141.
[5] M.B. Ellis, J.P. Ellis, Microfungi on Land Plants: An Identification Handbook, 2nd
ed., Richmond Publishing, Slough, 1997.
[6] D.F. Farr, G.F. Bills, G.P. Chamuris, A.Y. Rossman, Fungi on Plants and Plant
Products in the United States, American Phytopathological Society, St Paul,
MN, 1989.
[7] D. Malloch, M. Blackwell, Dispersal of fungal diasporas, in: G.C. Caroll, D.T.
Wicklow (Eds.), The Fungal Community: Its Organization and Role in the Ecosys-
tem, 2nd ed., Marcel Dekker, New York, 1992, pp. 147–171.
[8] J. Lacey, Air spora, in: D.L. Hawksworth, P.M. Kirk, B.C. Sutton, J.A. Stalpers (Eds.),
Ainsworth & Bisby’s Dictionary of the Fungi, 8th ed., CAB International, Wall-
ingford, 1995, pp. 10–11.
[9] T.E. Galante, T.R. Horton, D.P. Swaney, 95% of basidiospores fall within 1 m
of the cap: a field- and modelling-based study, Mycologia 103 (2001) 1175–
1183.
[10] P.E.J. Wiltshire, Hair as a source of forensic evidence in murder investigations,
Forensic Sci. Int. 163 (2006) 241–248.
[11] E.C. Grimm, Tilia v. 1.7.16, 2011, http://museum.state.il.us/pub/grimm/tilia/.
[12] D.C. Mildenhall, An unusual appearance of a common pollen type indicates the
scene of a crime, Forensic Sci. Int. 163 (2006) 236–240.
[13] D.L. Hawksworth, P.E.J. Wiltshire, Forensic mycology: the use of fungi in criminal
investigations, Forensic Sci. Int. 206 (2006) 1–11.
[14] P.E.J. Wiltshire, Forensic ecology, botany, and palynology: some aspects of their
role in criminal investigation, in: K. Ritz, L. Dawson, D. Miller (Eds.), Criminal and
Environmental Soil Forensics, Springer, Dordrecht, 2009, pp. 129–149.