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Forensic Science International 206 (2011) 1–11

Contents lists available at ScienceDirect

Forensic Science International


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

Review article

Forensic mycology: the use of fungi in criminal investigations


David L. Hawksworth a,b,*, Patricia E.J. Wiltshire c
a
Departamento de Biologı´a Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal, Madrid 28040, Spain
b
Department of Botany, The Natural History Museum, Cromwell Road, London SW7 5BD, UK
c
Department of Geography and Environment, 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: This is the first overview to be published of the whole field of forensic mycology. It is based on all
Received 27 April 2010 available information located in the literature, together with 13 examples from recent casework.
Received in revised form 7 June 2010 Background information on fungi is given, and this is followed by an outline of the value, and potentially
Accepted 10 June 2010
wide application, of mycology in criminal investigation. Applications include roles in: providing trace
Available online 14 July 2010
evidence; estimating time since death (post-mortem interval); ascertaining time of deposition;
investigating cause of death, hallucinations, or poisonings; locating buried corpses; and biological
Keywords:
warfare. Previous work has been critically evaluated, with particular attention to its evidential value, and
Clandestine burial
Deposition time
suitability for presentation in a court of law. The situations where mycology might assist an investigation
Hallucinogens are summarised, and issues relating to the further development of the subject are presented. A
Lichens comprehensive bibliography with 120 citations is provided.
Palynology ß 2010 Elsevier Ireland Ltd. All rights reserved.
Palynomorph
Poisons
Post-mortem interval
Trace evidence

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Trace evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
4. Time since death (post-mortem interval) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
5. Time of deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
6. Cause of death, hallucinations, or poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
7. Location of corpses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
8. Biological warfare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
9. When to consider forensic mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
10. Developing forensic mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1. Introduction use of mycological evidence in criminal investigations, and its


testing in the courts, i.e. forensic mycology, has until recent years
Mycology is the study of fungi of all kinds, including blights, largely been restricted to cases involving poisonous and psycho-
moulds, mildews, mushrooms, plant and human pathogens, tropic species. However, during the last 3 years we have found
lichens, rusts and smuts, slime-moulds, truffles, and yeasts. The various situations in which fungal data can provide critical
evidence. The objectives of this review are both critically to
consider the published information, and to draw attention to the
* Corresponding author at: Milford House, The Mead, Ashtead, Surrey KT21 2LZ,
range of situations where we now know mycological data can be
UK. Tel.: +44 1372 272087. informative – including our personal experience in criminal cases.
E-mail address: d.hawksworth@nhm.ac.uk (D.L. Hawksworth). Applications include roles in: providing trace evidence; estimating

0379-0738/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2010.06.012
2 D.L. Hawksworth, P.E.J. Wiltshire / Forensic Science International 206 (2011) 1–11

time since death (post-mortem interval); ascertaining time of on or near the ground, their spores will be more readily caught up in
deposition; investigating cause of death, hallucinations, or wind currents and can then be distributed more widely, although
poisonings; locating buried corpses; and biological warfare. their concentration in air is normally low. In most cases, fungal
spores are rarely dispersed more than 100–200 m horizontally from
2. Background the source [5]. Nevertheless, spores of certain species that occur
abundantly on leaves and bark (e.g. Alternaria and Cladosporium), can
Fungi have traditionally been studied as a part of botany, but are be encountered in large numbers in air samples, particularly in late
now known to belong to the same major evolutionary group as summer and autumn. In contrast, fungi with more restricted
animals, and are not included in the plant kingdom. In common occurrences rarely contribute even 1% of the total air spora2 [6].
with animals, fungi do not manufacture their own food, but obtain The key reference work in mycology is the current edition of
nutrients directly from living or dead organisms, or other organic ‘‘Ainsworth & Bisby’s Dictionary of the Fungi’’ [7]. For more detailed
materials. information on biology, physiology, and ecology of fungi, several
The fungi were already diverse 600 million years ago, and have texts can be recommended [8–15]. For fungal identification, there
continued to diversify to the extent that they are classified into are 15 books to which we refer frequently [16–30]. These
numerous separate groups. About 100,000 species are known selections are necessarily eclectic, but key works are indicated
worldwide, and around 800 species are currently being named as genus by genus in the ‘‘Dictionary’’ [7]. Some additional specialist
new to science each year, even from relatively well-researched texts on particular groups are mentioned where they are
parts of the world. However, it is now generally accepted that there appropriate in the following sections.
may well be around 1.5 million fungal species on Earth [1].
As an example of species numbers in a single country, the UK 3. Trace evidence
Fungal Records Database (see Footnote 9) has around 14,000
species listed, and an additional 40–50 species are added each year. Like other palynomorphs,3 fungal spores and other remains
The newly found species include both ones previously recognised may be picked up by any object contacting them and are subject to
in other countries, and ones new to science; the latter even include similar taphonomic4 considerations [31]. Generally, any palynifer-
conspicuous mushrooms (e.g. Xerocomus chrysonemus from ous5 surface will yield palynomorphs, but the main sources in
southern England) as well as more easily overlooked microscopic criminal investigations are soils, sediments, vegetation, and plant
species (e.g. Psammina palmata on wood from East Anglia). The litter. Unlike plants, fungi (including lichen fungi) can also grow on,
14,000 figure compares with the about 2100 native flowering for example, stone, brick, tiles, paving stones, wooden objects,
plants and ferns in the UK, a total almost unchanged for a century. leather, plastics, rubber, and textiles [32]. Their spores may thus
That there are 6–7 times more native fungi than plants in the UK, provide trace evidence in situations where other palynomorphs are
means that, potentially, they provide a massive source of scarce or absent. Even fragments of lichens, or fragments of mouldy
additional information for forensic investigations. objects, can become detached and caught up in items that are
The number of fungi present in a single locality is enormous. involved in criminal investigation.
However, a comprehensive inventory of all the fungi in one place is Although there do not appear to be any cases reported in the
probably unachievable as many make spore-producing structures literature of fungi having been used as trace evidence in criminal
only rarely, sometimes decades apart, or require specialist methods cases, we have successfully used them in our own forensic
to determine their presence. The best documented sites in the UK casework6 and they have greatly augmented palynological7 data.
have yielded 1000–3500 species [2], but even after 30 years of study To find fungal species during their field surveys, mycologists
species continue to be found for the first time in those localities. generally rely on their eyesight, hand lens, and experience of which
Most fungi are associated with particular plants or animals; habitats might prove fruitful. We have, however, found that the
they may cause disease (pathogens), be beneficial to their hosts methods of sampling vegetation, soils, and forensic exhibits for
(mutualists), have no evident effects (commensals), or live on dead palynomorphs can yield evidence of species of which are
and decaying remains (saprobes). Consequently, distributions are extraordinarily rare. For example, we have encountered the
limited by the ranges of the organisms on which they depend. distinctive spores of Caryospora callicarpa in preparations made in
Fungal distribution patterns are not as well-studied as those of connection with forensic cases in the UK, although no specimen of
plant and many animal groups, but are generally similar in that the fungus itself has been collected in the country since 1865. This
they have distinct geographical distributions and habitat prefer- fungus must still be considered rare since it was present in only
ences. However, some mould fungi can be found almost every- seven samples out of 1100 examined over a 2-year period, but it is
where, especially ones that: (a) spoil foodstuffs; (b) cause the not extinct as might be supposed from field finds [33]. Occurrences
deterioration of manufactured materials; or (c) are involved in of such rare species make them especially valuable as trace evidence.
decomposition of organic matter in soil. In an investigation of the murder of a young woman, her body
Fungi are generally dispersed by spores that may be produced was dumped in a bed of stinging nettles. Nettle (Urtica dioica) can
either sexually, asexually, or both. These can be diagnostic to support at least 92 fungi, of which about 17 are known only from
species-level in many cases, but in others can only be differentiated this plant [23,34]. Spores of two fungi common on dead stinging
to, for example, family or genus. Some are forcibly ejected from the nettles (e.g. Periconia sp., Torula herbarum) were found in
sporophore1 for a distance of only a few millimeters but,
2
exceptionally, some can achieve 30 cm [3,4]. However, in many The air spora consists of organic particulates carried in the air – mostly pollen
and plant and fungal spores.
cases, dispersal is passive, and spores form slimy or dry masses and 3
The term ‘‘palynomorph’’ includes pollen of flowering plants and conifers,
are dispersed by outside agencies. Such fungi may never achieve spores of ferns and mosses, and spores of fungi. Hyphae of fungi and arthropod
aerial dispersal at all. Many are distributed: (a) on seeds; (b) on plant fragments are also included though not all palynologists consider these as
or wood fragments; (c) by insects; (d) in the faeces of herbivores; (e) palynomorphs in routine work.
4
Taphonomy in this context may be considered to be all factors which influence
by rain-splash; or (f) in water. Where fungi produce dry spores and
whether a palynomorph will be found at a certain location at a certain time.
grow on aerial parts of living plants or trees, rather than on materials 5
Containing, or having a covering of, palynomorphs.
6
Some of our cases have been anonymised for legal reasons but will be written-
1
Specialised part of the fungal body which produces spores. It may be up more fully in due course.
7
microscopic as, for example, in Penicillium species, or large as in mushrooms. Palynology is the study of palynomorphs.
D.L. Hawksworth, P.E.J. Wiltshire / Forensic Science International 206 (2011) 1–11 3

palynological preparations from the crime scene and also in the setosa, Caryospora callicarpa (see above), and Melanospora zamiae.
suspect’s car. These supported ecological and palynological The latter species is mostly found in warmer regions of the world.
evidence demonstrating a link between the suspect and the place Like some of the pollen taxa, it is possible that at least some of the
where the corpse had been deposited [35]. fungal species were derived from the imported rabbit food. In any
In a drug-related shooting, a gunman hid himself up against the event, the complex, combined assemblages contributed to a
trunk of an oak tree that was growing in a cypress hedge along a specific trace signature from the farm that was also found on the
quiet lane in Romford, Essex and killed an associate who had footwear of the suspects [40]. With so many trace markers being
arranged to meet him in the lane. The cypress was growing sub- involved, and considering the rarity of some of these, the likelihood
optimally because of intense shading by the oak, and was infected of this specific assemblage being obtained from any other location
with a pathogenic fungus (Pestalotiopsis funerea). The gunman had was remote.
been standing in deep leaf and twiggy litter, and his body and feet While the precision of pollen data is enhanced by fungal spores,
could not avoid contacting the cypress branches, oak trunk, and the and they provide a heightened level of correlation between
litter. The pollen assemblage from the crime scene was very similar comparator samples and objects retrieved from suspects, they are
to that found associated with items seized from the suspect and his generally found in low concentration in palynological prepara-
associates. The spores of Pestalotiopsis funerea formed part of that tions. This means that large numbers of palynomorphs need to be
palynomorph assemblage; they were abundant in the leaf litter counted in order to even find the fungi. In our experience, it is often
and vegetation samples, and were found in a vehicle known to have the rare spores found in palynological preparations that enhance
been used by the gunmen and associates. In addition, spores of the value of this class of trace evidence. This is demonstrated by a
what most probably was an as yet undescribed Endophragmiella rape case investigated by Wiltshire Constabulary in 2009. The
species were also found in the leaf litter and the get-away car victim claimed she had been raped on the ground under some
[36,37]. This was a case where, although the palynological profiles trees, while the suspect insisted they had had consensual sex on
of the comparator samples and exhibits were similar, the fungal grass in a park about 200 m away. Nineteen fungi characteristic of
spores provided additional resolution that added powerful dead leaves and twigs (Fig. 1) of woody plants were found on the
pertinent information for the court. clothing and footwear of victim and/or suspect. Of these, 16 were
Another criminal case, where a relatively rare fungus provided represented in the comparator samples from the area under the
great resolution to the palynological profile, was in the first trees, and only four from the park. When combined with the other
successful prosecution for badger-digging for the Royal Society for palynological results, the fungal assemblage clearly showed that,
the Prevention of Cruelty to Animals (RSPCA) [38]. Again, the despite their close proximity, the two sites could be easily
palynological profile from soil samples obtained from the badger differentiated. The results indicated that the woman was telling
sett was very similar to that retrieved from a spade and shovel that the truth about where the incident took place [41,42], but such
had been seized from a suspect. Badgers had made their sett in a convincing findings needed a palynomorph count of over 38,000
pasture very close to a somewhat neglected hedge in the (Table 1). The outcome was rewarding for investigating officers
Staffordshire countryside. Two large oak trees were growing in since, when confronted with the palynological evidence, the
the boundary hedge, each about 100 m from the sett. It was very suspect confessed.
surprising to find spores of the white-veined truffle (Choiromyces In the present century, it might be thought that DNA profiles of
meandriformis) in the soil of the sett and in the soil adhering to the environmental samples that include fungi, also had potential as
suspect’s spade. The truffle is the sporophore of the fungus, and trace evidence. The reality is that the methods that have been
this forms beneath the soil surface in association with the roots of a attempted are not yet sufficiently robust and reproducible for
variety of deciduous trees, including oak. It is well known that pigs, acceptance in a court of law.
dogs, and humans enjoy truffles, but here was a case where
badgers must have been digging for them in the oak’s root system
and taking them back to their sett. This rare spore helped link that Table 1
Presence/absence of fungal spores on the clothing of victim and suspect in a rape
spade and the badger sett.
case with those in the two possible locations, investigated for Wiltshire
Although occasional rare fungal spores enhance the resolution Constabulary in 2009 [41,42]. ‘‘Palynomorphs counted’’ = pollen and plant spores
of the palynological data, when whole fungal and large pollen and as well as fungal spores. See text for further information.
plant spore assemblages are combined, evidence of contact
Fungi Suspect Victim Wood Park
becomes very powerful. Each fungal species has particular
nutritional and habitat requirements, so assemblages of fungi Clasterosporium flexum + + +
Pseudovalsella-like + + +
can indicate very specific situations. In Lincolnshire in 2008, four
Pestalotiopsis funerea + + + +
animal rights activists were arrested on suspicion of being involved Brachysporium britannicum + + +
in an attempted break-in at a farm that bred laboratory rabbits. Camposporium cambrense + +
Footwear from each of the suspects yielded palynological profiles Diplocladiella scalaroides + +
Glomus-type + +
that were similar to those in the comparator8 samples from the
Didymosphaeria sp. + +
farm. It was of interest that exotic plant species were represented, Melanospora sp. + + +
and it was interpreted that their pollen had been derived from the Bactrodesmium obovatum + +
imported rabbit food. In addition, the comparator samples and Dictyosporium toruloides + +
footwear yielded 21 fungal taxa which were identified to genus or Bactrodesmium betulicola + +
Periconia byssoides + + + +
species [39]. Of these, 19 were associated with the suspects’
Epicoccum nigrum + + + +
footwear; and three of these have few records in the FRDBI9; they Endophragmiella fagicola + +
must, therefore, be considered rare. These were Brachysporiella Asterosporium hoffmannii +
Cymadothea trifolii +
cf. Diporotheca sp. +
8
Samples which are collected a targeted way from areas of a crime scene or other Phaeotrichosphaeria brittanica +
pertinent place which are likely to have been contacted by an offender. Niesslia exosporioides +
9
Fungal Records Database of Britain and Ireland (FRDBI) maintained by the Ball of straw coloured cells + +
British Mycological Society; http://www.fieldmycology.net/GBCHKLST/gbchklst. Palynomorphs counted 3236 1680 3294 30,129
asp.
[(Fig._1)TD$IG]
4 D.L. Hawksworth, P.E.J. Wiltshire / Forensic Science International 206 (2011) 1–11

Fig. 1. A selection of fungal spores found in palynological preparations in a rape case investigated for Wiltshire Constabulary in 2009 [41,42]: (A) Clasterosporium flexum; (B)
Diplocladiella scalarioides; (C) Camposporium cambrense; and (D) Dictyosporium toruloides. Not to scale. Photographs by J.A. Webb.

Various molecular methods of characterising fungal communi- temperatures and human immune systems. They range from
ties in soil, in which species are not identified but ‘‘fingerprint’’-like dermatophytic fungi, that cause ringworm on the surface of the
patterns are produced, have been tried [43]. But, at the present time, skin, to invasive infections, such as candidiasis (thrush), and deep-
none is at a stage where it could be commended for either ecological seated infections in lungs (e.g. aspergillosis) and other tissues (e.g.
or forensic investigation. Fungi in soils are particularly diverse; 50– mycetomas, mycoses) [25,50,51]. In immunocompromised indi-
100 different species can be expected to be isolated into culture from viduals, however, a variety of less specialised fungi can occasion-
a single soil sample [44], and there can be as much as 3 km of fungal ally occur opportunistically within human tissues [52]. In our
hyphae in 1 g of dry soil [45]. This diversity makes the development experience, the fungi frequently found growing on and in corpses
of standard molecular, or other, approaches extremely difficult. are those that are not normally able to colonise living tissue.
Currently, precise molecular characterisation of any soil is Surprisingly, there is almost no information on the role of
impractical for a variety of reasons, including the following: (1) a particular fungi in the decomposition of human corpses [53,54].
single molecular approach is unlikely to be universally applicable However, remains interred directly in soil frequently have been
[46]. (2) The commonly used terminal restriction fragment length mentioned as showing signs of moist decomposition, with skin
polymorphism (TRFLP) approach is unreliable for due to several slippage and fungus development [55]. Janaway et al. [56]
problems, not least being peaks due to multiple fragments as a result commented that soil fungi might be involved on the body surface
of PCR enzyme inefficiency, and also whether conserved or more ‘‘after the major phase of decomposition’’, and mention that
variable genes are targeted [47]. (3) There can be huge differences in ‘‘moulds begin to appear on the surface of the body’’ in the first
profiles obtained from sample sites in close proximity; one study has week. No source for these statements was given although our own
shown that soils associated with the same tree species growing 20 m work confirms his observations. Janaway et al. [56] also note that
apart were very different [48]. (4) The amount of soil from which an unidentified Candida species had been isolated from an early
total DNA is extracted for analysis in molecular ecology studies is stage of decomposition (again with no source indicated), and that
generally 250–500 g – amounts hardly ever encountered on forensic fungi can be found growing in soil infused with decomposition
exhibits; sometimes mere traces are retrieved from exhibits. And (5) products – citing Sagara et al. [57], and also a paper mentioning
relatively few fungi are represented by correctly named species in two Penicillium species [58].
public DNA databases; this makes species-level identifications The only experimental study appears to be that of Parkinson
problematic (see below). There are, however, possibilities for the et al. [59] who endeavoured to compare the changes in fungal
detection of particular species of fungi in environmental samples communities in soil in response to human cadaver deposition on
using specially designed probes [49], but this approach requires the surface. This study was carried out at the Forensic Anthropol-
prior knowledge of what is present. ogy Center of the University of Tennessee, and used molecular
We have not traced work aimed at determining the extent of approaches; the control samples appeared ‘‘to cluster a little more
reproducibility in molecular profiles obtained from a single site, closely than the cadaver samples’’. No clear trend in succession was
but we suspect that this would be low due to the abundance and evident, and no attempt was made to identify the fungi detected.
wide variety of fungal propagules to be expected in a single sample. The fungi that have proved of most interest in estimating post-
mortem interval are neither specialised medically important fungi,
4. Time since death (post-mortem interval) nor ones restricted to dead human tissues, but rather are
decomposer or spoilage fungi that are able to directly colonise
Although healthy humans can have fungal infections, the fungi the surfaces of corpses and body parts after death (Fig. 2). It is not
involved are usually specialised species tolerant of high body surprising that fungi have occasionally been noted by pathologists
[(Fig._2)TD$IG] D.L. Hawksworth, P.E.J. Wiltshire / Forensic Science International 206 (2011) 1–11 5

subject, especially in relation to the pattern and rate of growth of


fungal species on cadavers.
When the body of a man was found by a tube line in Ruislip,
north-west London, the medical examiner considered it had been
there not more than 48 h. There was no evidence of any scavenger
or insect activity. However, botanical evidence, and a large circular
fungal colony under the chin, indicated that the body had lain in
situ for between 3 and 5 weeks [69]. It later transpired that the
actual time was 4 weeks 3 days. Decomposition had been delayed
because the weather had been exceptionally cold, and the low
temperatures had inhibited colonisation by flies. Security fencing
Fig. 2. Colonies of Penicillium griseofulvum growing on abdominal skin of a cadaver. had excluded scavenging animals and the body had remained
intact.
In the case of the serial killing of prostitutes around Ipswich,
on the surface of human cadavers. One key reference text for Suffolk at the end of 2006, two of the bodies were recovered from
forensic pathologists reports extensive growths on a boy after 6 water. These were ensheathed in fungal growth that had trapped
weeks [60], while another refers to development after several silt particles that had become graded as they accumulated in the
months on an embalmed cadaver [61]. However, the fungi involved hyphal10 weft. Several fungi were involved, including species of
have not generally been identified even to genus, nor considered as Fusarium, Geotrichum, Mucor, and Pythium. The first body that was
a forensic tool. retrieved was completely ensheathed. With the second body, the
The first researchers to appreciate that fungal growth on sheath was present though it was patchy and had collapsed due to
corpses had a role in the determination of time of death were van the much greater length of the hyphae than on the other body.
de Voorde and van Dijck [62]. These authors made isolations from Where there had been skin slippage and exposure of the
fungal growths on an eyelid and on inguinal skin of a murdered underlying dermis, there had been fresh fungal colonisation on
baroness found in a room in Belgium. They incubated the fungal some of the exposed areas. The fungi were intermixed and not
isolates at the same temperature as that in which the body had identified to species. Even though information on the growth of
been found (a constant 12 8C controlled by a thermostat in the these fungi in cold water was not available, it was obvious that the
room), and measured the colony sizes daily. They estimated that body of this victim had been in the water longer than that of the
the woman had died at least 18 days before her body was other. An estimate of at least 5 weeks submersion was suggested
discovered, and that agreed exactly with a subsequent admission by the condition of the fungal mycelium and other features of this
by the murderer. They considered that superficial fungi could help corpse. Using the same criteria, it was suggested that the woman
in determining the time of death, when this was 10–20 days who had been found first had been in the water for about 2 weeks.
earlier, provided those temperature data were available. The fungi These estimates agreed with the length of time the victims had
involved in this case were named as: Cladosporium sp., Fusarium been reported to be missing [70,71].
sp., Geotrichum candidum, Hormodendron sp., Mortierella sp., and Fungal colonies on, or associated with, human cadavers can give
Penicillium chysogenum (as P. notatum). indications of time since death as there is information on growth
Unaware of the Belgian case, Ishii et al. [63] reported on fungal rates of many moulds. But the reliability of any estimates will
growths found on the surface of a mummified body found in an depend on the accuracy of the identification of the fungus, the
abandoned house, and also on skeletal remains discovered in a storage methods for the body, and the availability of data on the
forest, that they suggested ‘‘may reveal local habitats’’. They temperature and humidity at the site. As yet there are few precise
identified these as Aspergillus chevalieri (as Eurotium chevalieri), A. data on actual rates of growth on dead human tissues, especially
repens (syn. E. repens) that was the most abundant, A. rubrum (as E. under different conditions of temperature and humidity. This
rubrum), and Gliocladium sp. (probably actually a species of means that it is necessary to undertake experiments mirroring, as
Clonostachys). These authors recognised that more cases were near as is possible, the environmental parameters with which the
needed to establish the approach as a forensic tool, but did not corpse was associated, using the actual strains found. We are
attempt to estimate PMI in these cases. They subsequently currently involved in such experimental work.
reported on white growths dotted on the face of a male corpse In a case undertaken for Tayside Police in 2009 [72], a man had
found in a kneeling position and partly in water at the bottom of an been repeatedly stabbed and died in a closed flat so that flies were
open well in Japan [64]. The humidity was nearly 100%, and the excluded. Fungal colonies had developed on parts of the carpet and
temperature constant at 12–13 8C; the fungi were isolated into on a sofa that had become soaked with body fluids (Fig. 3). After the
culture and identified as Aspergillus terreus and Penicillium sp. The discovery and removal of the body, data loggers were placed in the
deceased had been dead for 10 days, and although no growth room and consistently gave relative humidity readings of 30–34%.
measurements of the colonies were made, they suggested that the Colonies were measured in situ and subcultures made onto
fungal data were consistent with the time as the fungi ‘‘generally artificial media. Samples of the carpet were then removed and
colonise [in] 3–7 days’’ – a generalization based on the time-lag in kept dry for observation; no subsequent growth occurred within 5
attacking foodstuffs [65]. These workers independently suggested days. This result was expected since most mould fungi require at
that fungi could provide a useful means of estimating PMI where least 95% relative humidity for growth [73]. When the carpet
forensic entomological data were not available. samples were re-wetted with bovine blood and kept at a high
Menezes et al. [66] considered that Ishii et al.’s claim of fungal humidity, there was a spurt of new fungal growth. A comparison of
growths being of value in estimating PMI’s would have been more the sizes of the new colonies, both in culture and on the carpet,
convincing if they had investigated the growths empirically. Their with those at the crime scene, suggested that the death had
response reaffirmed their view of the potential, acknowledging occurred about 5 days prior to discovery of the body; this was
that there were unsolved problems regarding growth rates and the consistent with a subsequent admission of guilt. The three
actual fungi [67]. In a rejoinder, Menezes et al. [68] stressed the
need for more forensic cases to be documented from different 10
The vegetative threads of the fungus. A mass of hyphae (singular, hypha) is
climatic conditions, and for experimental work on a substitute termed ‘‘mycelium’’.
[(Fig._3)TD$IG]
6 D.L. Hawksworth, P.E.J. Wiltshire / Forensic Science International 206 (2011) 1–11

growing on human skulls. In particular, the skull lichen (Parmelia


saxatilis or some related species) was supposed to be ‘‘worth its
weight in gold’’ as a cure for epilepsy. In 1640 Parkinson [75] stated
that it ‘‘groweth upon the bare scalps of men and women that have
lyne long’’, and refers to reports that for greatest efficacy ‘‘it should
be taken from the sculls of those who have been hanged or
executed for offences’’. There is a huge literature on the use of
lichens in dating surfaces by geomorphologists, glaciologists and,
to a lesser extent, archaeologists [76,77]. However, these mostly
use the ‘‘map lichens’’ of the Rhizocarpon geographicum complex
and these do not grow on calcareous materials.
In a recent case we found it possible to provide information on
the time that a plastic-wrapped body part had lain in situ from
botanical and lichen evidence. A broken twig that had the lichen
Xanthoria parietina growing on it was on the ground under the
body part. When well illuminated, this lichen is yellow-orange
with deeper orange sporophores (Fig. 5A). When growing naturally
in the shade, even on the underside of the same twig, the lichen is
grey but still has the orange sporophores (Fig. 5B). However, if kept
in the dark and in too-moist conditions, it will turn green (Fig. 5C).
A simple experiment was constructed whereby a sample of the
lichen from the same locality was covered by a plastic-covered
weight; compared with an adjacent but uncovered control, it
Fig. 3. Carpet saturated with body fluids and then dried, showing fungal colonies.
changed from yellow to green within 5 days. This indicated that the
The large, dark greyish colonies are of Mucor plumbeus. See text for details.
body part at the crime scene had probably not been in position for
more than a week [78]. Some other lichens can also change colour
principal fungi involved in this investigation were Mucor plumbeus, under adverse conditions (see below).
Penicillium brevi-compactum, and P. citrinum. The sporing cycle of fungi can also help in timing events. The
scavenged skeleton of a young female was found in a shallow grave
5. Time of deposition close to the A40 west of London in February 2004. It appeared that
she had lain there for a long time. However, the remains lay on
Exposed bones can be colonised by mould and other fungi some shooting bramble stems that indicated she could not have
under appropriate ambient conditions [24]. Although we could been there for more than 1 year. Also, there were some detached
find no modern published information on fungi growing on human and still-green leaves of bramble (Rubus fruticosus) in the grave fill.
bones, conspicuous fungal colonies were found on the scavenged The underside of the leaves had black pustules while the leaf tissue
skeletal remains of a woman whose remains had been found in a associated with them was red-purple. The pustules were caused by
Sussex woodland in 2008 (Fig. 4); 13 fungal isolates were obtained a common rust fungus, Phragmidium violaceum that produces
from the leg and pelvic bones [74]. It must be noted that there was orange spores in the spring and early summer (urediniospores),
still a very thin and patchy film of soft tissue on some of the bones and dark ones (teliospores) in the late summer and autumn.
and some were also rather greasy. These fungal isolates are the Combined with the evidence from the shoots, and the colour of the
subject of current research. pustules, an estimate was made that the body had been deposited
If bone is exposed for many years in well-lit situations, lichen between late September and early November. When the victim
colonies of, in particular, Caloplaca and Lecanora species can [(Fig._5)TD$IG]was identified, it transpired that she had been reported missing on
develop. Colony diameter can give a good indication of minimum
[(Fig._4)TD$IG]periods of exposure. There are also ancient reports of lichens

Fig. 5. Xanthoria parietina growing on Sambucus nigra (elder) twigs: (A) natural
growth in full illumination; (b) natural growth in deep shade (underside of twig);
(C) sample grown in full illumination but then covered with a weighted object
Fig. 4. An example of fungal growth on defleshed human bone. wrapped in opaque plastic for 5 days. Not to scale.
D.L. Hawksworth, P.E.J. Wiltshire / Forensic Science International 206 (2011) 1–11 7

16th October in the previous year [79]. Similarly, as ergots hallucinogenic species has been compiled [86], and an on-line
(Claviceps purpurea) developing on cereals and other grasses in the identification program constructed [87,88]. Species-level identifi-
autumn have hooks at the tips that facilitate attachment to cation is necessary to ensure the correct diagnosis and also that any
clothing or animal coats, their discovery would also suggest an prescribed treatment is appropriate. In some continental European
incident in the autumn. countries where mushrooms are regularly collected and eaten,
Many mushrooms are also seasonal, and this is not only a poison control centres have accesses to mycological specialists or
matter of species that form sporophores in the autumn. Some are have local specialist controllers (e.g. Switzerland).
much more restricted, for example, the scarlet-cupped Sarcoscypha The use of fungi as hallucinogens, neurotropic or psychoactive
species appear in the early spring on fallen twigs. These fungi have drugs, has its roots in antiquity in both the Old and the New World
distinctive spores and might provide important trace evidence if [89,90]. In all countries that have signed the 1971 UN Convention
trodden on by suspects. Another potentially useful fungus is the on Pyschotropic Substances, their use is controlled by legislation.
mushroom Flammulina velutipes, that only appears after the first For example, under Section 21 of the UK Drugs Act 2005, psilocybin
frosts of winter. and its derivative psilocin are Class A drugs, and the knowing
Fungal colonies grow in a circular manner when on solid possession of mushrooms that produce these, ‘‘magic mush-
substrates, and the growth rates on artificial media are often cited rooms’’, is illegal. There are, however, at least 30 ‘‘magic mush-
in species descriptions. Those with non-septate hyphae belonging rooms’’ that grow in the UK, of which Psilocybe semilanceata is the
to the Mucorales (pin-moulds) are, in general, much more rapidly most commonly collected and used. The highest reported
growing than those belonging to other groups (e.g. ascomycetes, concentrations of psilocybin are from P. azurescens (‘‘blue angels’’)
basidiomycetes) that have repeatedly septate hyphae. In a recent which is to be found in the Pacific north-west of the USA [89].
case for Hertfordshire Constabulary, a mutilated murder victim However, concentrations may vary widely within a single species
was deposited on his ventral surface on a steep bank above a small, as a result of biological or ecological factors. Psilocybe cubensis, P.
sluggish stream. The absence of blood indicated that the murder mexicana, and P. semilanceata are the most-used species, but
and mutilation had occurred elsewhere. Small colonies of non- worldwide, there are at least 216 mushrooms known to have
sporing Mucor hiemalis had developed on the abdomen. Palyno- neurotropic effects; occurrences of these, listed by country, have
logical analysis showed that mud that covered the corpse had been been compiled [91]. Over 150 of these fungi are species of Psilocybe
derived from the stream and the victim had obviously been [90]; there are 230 or so species of this genus that are not easy to
dropped in or dragged through it. The colony size and immaturity identify without critical morphological and microscopical exam-
of the fungus indicated 1–2 days growth, and the pristine condition inations. The psilocybin-containing species generally turn bluish
of the mycelium showed that it had developed in situ after the body on handling or bruising, especially on the stalks (stipes), but this is
had been dragged through the stream. Experience has shown that not an unequivocal guide as various other fungi with different
fungi, such as Mucor species, are unable to colonise and grow on compounds can give a similar reaction. Particular neurotropic
freshly dead skin and it would appear, on average, that this is Psilocybe species can be encountered in forensic situations outside
unable to happen for at least a week after death. This reinforced the their normal geographical areas as they are exported from Central
hypothesis that the victim had been killed and stored elsewhere America into Europe, and some can be cultivated both indoors and
and had lain at the deposition site for a short time [80,81]. outside [92]. As some harmless species are superficially rather
similar to hallucinogenic ones, accurate identification is critical to
6. Cause of death, hallucinations, or poisoning law enforcement. Interestingly, as a result of recent molecular
phylogenetic studies, many of the non-hallucinogenic species
Mushroom poisoning can be accidental or deliberate, and may traditionally classified in Psilocybe are now being placed under the
be fatal. In most cases this arises from the consumption of wrongly generic name Deconica [93]. Some species that contain the
identified mushrooms by untrained collectors who often belong to prohibited compounds do not resemble Psilocybe species, for
the family of the victim(s). In most cases the results are not fatal example, the blue-green capped Stropharia aeruginosa. The
but vary according to the amount consumed and the tolerance of compounds psilocin and psilocybin can, however, be detected
the individuals. In some cases the onset of symptoms is rapid, by relatively inexpensive thin-layer chromatographic (TLC)
while in others, involving kidney damage, they may not be evident methods [94,95]. Procedures for the chemical determination of
for several days. Most toxic are the fungi producing amanitins (e.g. psilocybin and psilocin by gas chromatography and mass
Amanita virosa), gyromitrin (Gyromitra esculenta), muscarine (e.g. spectrometry (GC–MS), gas liquid chromatography (GLC), high
certain species of Conocybe and Inocybe), and orellanine (Cortinar- performance liquid chromatography (HPLC), and spectroscopy
ius orellanus), where only small amounts can prove fatal [82]. The have also been documented [95,96]. Chemical methods are
number of truly poisonous mushroom species is small, although especially valuable for testing magic mushroom preparations in
more can cause gastric problems in some individuals. Unfortu- the form of powders, tablets, or capsules.
nately some of the poisonous species are rather common and, to In view of the difficulty in differentiating the hallucinogenic
the non-specialist, can appear to be similar to certain edible Psilocybe species, several groups of researchers have endeavoured
species. In the case of any suspected mushroom poisonings, any to develop molecular tools for their discrimination. Lee et al. [97]
remaining mushrooms should be secured, and stomach contents compared the ITS-1 region sequences of five species and these
analysed. appeared useful at the generic level. However, a larger study
There are numerous books on mushroom identification, but involving 35 North American Psilocybe species, and six other
regional guides are seldom comprehensive as there are just so genera, demonstrated that the rDNA ITS-1 region was too variable
many species. Consequently, of particular value in the case of to provide satisfactory resolutions and that nLSU rRNA gave better
suspected poisonings are those dealing exclusively with the correlations [98]. Using ITS and LSU sequences of four Psilocybe
identification of poisonous species [83,84], and a comprehensive species, Maruyama et al. [99] provided an improved identification
list of species known to have particular adverse effects has been system. However, as there are over 200 species accepted in the
compiled [85]. When no intact mushrooms are available for genus, not all of which are hallucinogenic (see above), further work
examination, spores and other microscopic fungal remains in involving a more complete range of species is needed before the
stomach and gut contents can be used to determine the species molecular data will be robust enough to be used unequivocally in
consumed. A key for the identification of fragments of selected court.
8 D.L. Hawksworth, P.E.J. Wiltshire / Forensic Science International 206 (2011) 1–11

While not normally fatal, irresponsible use of Psilocybe species but none is associated with human remains, and no such records
may lead to death either as a result of erratic behaviour while are known to specialists in the genus (H. Becker, pers. comm.). A
under the influence of the drugs they contain, or as a result of detailed review of the association between fungi and graves, or
imbibing extracts of the mushroom together with other drugs. excrement of different organisms, has been prepared [57] and this
At present it is only psilocin and psilocybin-containing mush- includes a list of 23 ammonia and post-putrefaction fungi, with
rooms that are prohibited from use by law in the UK. Other references to pertinent primary literature.
pyschotropic mushrooms include the fly agaric (Amanita muscaria) More practical in the location of corpses, are fungal indications
which has the familiar scarlet white-flecked cap. This has been of where tree branches or logs have been disturbed or moved.
used in religious ceremonies for centuries, has an inebriating Mushrooms are generally geotropic,11 with vertical stalks (stipes)
effect, and is not fatal itself unless consumed in large amounts. and horizontal caps (pilei), but if they are re-orientated, the stalks
With the prohibition of hallucinogenic Psilocybe species, dealers in may curve as they grow to regain a vertical position or their caps
north London have been found selling the fly agaric mushroom as skew to become horizontal once more [109]. In a case for South
an alternative. Wales Police, this phenomenon was used to refute the statement of
Some mould fungi also produce toxins directly (see below), or the finder of a murder victim’s grave where he claimed he had not
have products that can react with other compounds to produce touched or disturbed anything at the scene. Logs placed over the
toxic substances. Most notorious in this connection is Scopular- surface of the grave by the offenders supported numerous
iopsis brevicaulis. This fungus has been implicated in the sporophores of a Marasmius species. The orientation of the stipes
production of trimethyl arsine from arsenic-containing com- and pilei showed that several of the logs had been turned over
pounds. This gas can be produced by the fungus in damp conditions some (indeterminate) time before the find was reported [110] and
when growing on wallpaper containing ‘‘Paris Green’’, and its his testimony was refuted. If time were critical in such investiga-
inhalation can lead to death [100]. In the early 1990s, it was tions, simple experiments could be conducted to test the length of
suggested that this same fungus reacted with arsenic-containing time required for reorientation of the fungus.
fire-retardants in cot mattresses to produce the toxic gas and that Lichens, particularly foliose ones, normally occur on the better-
this was the cause of Sudden Infant Death Syndrome (SIDS). While illuminated sides of branches, so if they are found on an underside
that hypothesis did not stand up to critical examination [101], of a broken branch then it is likely to have been disturbed. Further,
other fungi occur in bedding materials, and some of these can some lichens undergo colour changes before they decompose if, for
produce other toxins. Where there is incidence of suspicious death example they become dislodged or are covered (see above). This
in beds, officers attending scenes should check for any substantial phenomenon is most easily seen in shrubby and foliose species
fungal growth. containing b-orcinol depsidones such as norstictic or salazinic
acids (e.g. certain species of Parmelia and Usnea). These assume a
7. Location of corpses pink–red colour if moved to densely shaded or wet situations and
they gradually die [111,112]. Investigators should be sensitive to
While we are not aware of any criminal case where fungi have such phenomena as they have the potential of indicating where
yet had a role in the location of buried corpses, there might be some branches have been disturbed or moved. This may help identify
potential. Some mushrooms are characteristic of disturbed ground, offender pathways at crime scenes, or recognition of clandestine
but will not produce sporophores until 1–2 years after the graves.
disturbance. Furthermore, some of the species with this ecology
are easy to recognise, notably the shaggy ink cap (Coprinus
comatus), and some morels (primarily Morchella species). 8. Biological warfare
Since Sagara [102] reported the association between shallowly
buried carcasses of a dog and a cat and the mushroom Hebeloma While the possibilities for the use of bacteria and viruses in
vinosophyllum in two different sites in Japan in 1976, there has biological warfare are well known [113], there is relatively little
been considerable interest in the potential of ‘‘corpse-finder’’ fungi. appreciation of the potential of fungi in this respect. Many moulds
That species is known to appear 3–8 months after urea or as well as mushrooms can produce toxins and, whilst the action of
ammonium application to soils and then to continue to produce most is long-term (for example they are carcinogens), some can be
mushroom sporophores for 1–2 years. The potential was empha- cultured in vats in large amounts and produce quicker-acting
sized by Carter and Tibbett [103,104], who distinguished between substances that have potential as biological weapons. They were
an assemblage of ‘‘ammonia fungi (AF)’’ making sporophores after wrongly implicated in ‘‘Yellow Rain’’ in Vietnam, but more
the addition of urea or ammonia, and ‘‘post-putrefaction fungi importantly, within the last 30 years, a certain middle-eastern
(PPF)’’ that form sporophores over animal cadavers without such country was found trying to purchase toxin-producing strains of
additions. Their paper was criticised by Bunyard [105] on the Fusarium from microbial culture collections in North America and
grounds that of the 35 species they listed, only Hebeloma radicosum Europe. A review of the potential of toxin-producing fungi as
and the North American Hebeloma syriense had been mentioned in biological weapons has been published [114]; most dangerous is
the literature as associated with corpses [106,107] rather than the Fusarium T2 toxin.
ammoniacal substrates, and there were ‘‘few first-hand claims’’. He Fungal parasites of plants can also be biological warfare agents
also stressed the problems of identifying these mushrooms, with as they can be developed to destroy crops in the same way that
over 200 species of the genus known in North America alone, and they are used for weed control [115]. The technology for spraying
because some of the listed species had different ecologies. In spore suspensions of fungi from aircraft is now developed to a
defending their position, Tibbett and Carter [108] accepted some of sophisticated level – a by-product of the use of Metarhizium
Bunyard’s comments and ‘‘emphasized that taphonomic mycota is anisopliae var. acridum as a mycoinsecticide (‘‘Green Muscle’’) for
little more than a concept at this stage and requires further the control of locusts and grasshoppers [116]. If suspected
research and development prior to practical application’’. That terrorists are found with what appear to be living cultures of
remains the situation today, and we have not traced any case fungi, the material should be confiscated and examined by a
where these fungi have assisted in finding, or been associated with, specialist.
a human grave. Hebeloma radicosum is widespread, but not very
common, in Western Europe; there are 187 records in the FRDBI 11
Affected by gravity.
D.L. Hawksworth, P.E.J. Wiltshire / Forensic Science International 206 (2011) 1–11 9

9. When to consider forensic mycology unknown or unrecognised species are discovered every year. Semi-
popular guides rarely treat all species of a genus, and often do not
In view of the foregoing, it is evident that the situations in indicate that there are other similar species. There is not even a
which investigating officers should consider utilizing mycology pocket-sized identification manual on all hallucinogenic fungi,
can be summarised as follows: something that would make the psilocybin and psilocin possession
section of the Drugs Act 2005 more practical for police officers to
(1) As an integral part of the ecological assessment at crime scenes, enforce. Those undertaking aspects of forensic mycology that
especially in outdoor situations. involve numerous identifications, as in the provision of trace
(2) When the time of death or deposition is uncertain, and fungal evidence, consequently require access to a substantial library and
colonies are evident on human remains, clothing, or associated to reference collections of authoritatively named material. The
items (indoors or out of doors). This is particularly important if most reliable way to identify fungi is by direct comparison with
entomology is not appropriate but, if fungi are present as well correctly named reference material.
as flies, fungi can be considered as an independent line of Identifications using molecular sequence data can be made in
evidence. certain well-researched groups of fungi, especially those involved
(3) If trace evidence is being sought and fungal spores are found in in food spoilage, human diseases, or plant pathology [118]. The
palynological preparations. existing DNA databases, however, have only a small proportion of
(4) When mushrooms are found in the possessions of a suspect, in the known fungi represented in them, and many are represented
gut contents, or in food and drink associated with deaths or by only one or a few strains, so there is little representation of
neurotropic behaviour. within-species variability. Further, the gap between those known,
(5) If fungi are being grown in mass-culture (e.g. in liquid growth and those that are sequenced, may be growing; only 21% of the
media in large containers). species described in 2003–07 are represented in GenBank [119].
Also, and especially worrying, is that several independent studies
indicate that around 20% of the fungal sequences deposited in
10. Developing forensic mycology GenBank are based on wrongly identified material [120]. This
means that while in some cases DNA data may be able to provide
While mycology has been demonstrated to provide useful conclusive identifications, in many others either it will not, or it
forensic evidence in a variety of ways and, in some cases, has will yield erroneous results. Specialist interpretation is, therefore,
proved critical in securing convictions, it is currently rarely essential in using DNA (e.g. BLAST) approaches for fungal
employed. The principle reason for this is a lack of awareness identification; this is not something that is yet at a stage where
amongst crime scene investigators, and investigating officers, that it can be delegated to non-specialists.
fungi have the potential to make significant contributions. In
several of the cases in which we have been involved, it was the Acknowledgements
astuteness of officers in recognising there was fungal growth
present on human remains or artefacts that led to our being called We are grateful for the excellent assistance, support, and
in. expertise we have received from Dr. Judy Webb and Ms Julia
A secondary reason is a shortage of appropriately skilled Newberry in our forensic case work. We would also like to thank
mycologists. Some categories of mycological investigation, partic- Dr. Jane Nicklin for her enthusiastic help, Professor Henry Becker
ularly those involving the isolation and culture of fungi from for information on Hebeloma species, and two anonymous
remains and materials, can be undertaken by biologists with reviewers. Last, but not least, we gratefully acknowledge the
experience in microbiological methods, but those requiring cooperation and support of officers from many British Police forces.
identification need specialists of wide experience. There is a In particular in relation to cases discussed here, we thank
shortage of professional mycologists able reliably to identify fungi Hertfordshire Constabulary, Lincolnshire Police, Metropolitan
across different systematic groups. For example, in the UK, there Police Service, South Wales Police, Suffolk Constabulary, Sussex
has been a reduction in systematic mycology posts in the last 10– Police, Tayside Police, Thames Valley Police, Wiltshire Constabu-
15 years as a result of the restructuring of research institutes and lary, and the British Transport Police. This work was completed
retirements. The number of full-time fungal (including lichen) while D.L.H. was receiving support from the Ministerio de
systematists in the country (excluding curators of collections) is Educación y Ciencia of Spain (Proyectos I+D CGL 2008-01600).
currently a mere eight (of which two are lichenologists); and not
one is now employed anywhere in the university sector. This figure References
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