FORENSIC SCI SEM Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. 2157118X.6.1R3 Peer Reviewed Received 10 April 2016 Received in revised form 2 June 2016 Accepted 3 June 2016 Available online 18 June 2016

orensic Ecology, Botany, and Palynology: Some FAspects of Their Role in Criminal Investigation Patricia E.J. Wiltshire *, a a Department of Geography and Environment, University of Aberdeen, Elphinstone Road, Aberdeen AB24 3UF, UK; and Department of Natural and Social Sciences, University of Gloucestershire, Swindon Road, Cheltenham GI50 4AZ, UK. * Corresponding Author. E-mail : [email protected]

Abstract Ecology, botany, and palynology are now accepted as part of the armoury of forensic techniques. These disciplines have been tested in court and have provided evidence for contact of objects and places, location of clandestinely-disposed human remains and graves, estimating times of deposition of bodies, differentiating murder sites from deposition sites, and provenancing the origin of objects and materials. It is important that the forensic palynologist is a competent botanist and ecologist. Sadly, not all practitioners have this essential background and, therefore, produce work inadequate to withstand scrutiny in court. Palynology involves the identifi cation of many classes of microscopic entities, the most important being pollen, plant spores, and fungal spores. The practitioner needs to be able to identify palynomorphs in damaged and decayed states and this requires experience and skill. However, identifi cation is still the lowest level of palynological expertise, and interpretation of palynological assemblages requires knowledge of plant distribution, developmental responses, and phenology, as well as ecosystem structure and function. The forensic palynologist must also understand highly manipulated and artifi cial systems, and the complexities of taphonomic processes. There have been attempts to make forensic palynology ‘more scientifi c’ by the construction of test trials, the application of current statistical techniques, mathematical modelling, and reference to aerobiological data and pollen calendars. But these appear to be of limited use in the forensic context where outcomes are scrutinised in court. There is a high degree of heterogeneity and variability in palynological profi les, and every location is unique. It is impossible to achieve meaningful and forensicallyuseful databases of the palynological characteristics of places; predictive models will always be crude and unlikely to be of practical value. In spite of this, the experienced ecologist/palynologist has been able to identify places, demonstrate links between objects and places, estimate body deposition times, and differentiate pertinent from irrelevant places very successfully. Nevertheless, there has been no substitute for examination of every pertinent place, and every relevant exhibit in each criminal investigation. Keywords: Forensic ecology, Botany, Palynology, Criminal investigation, Forensic Science.

Introduction in the public domain, they are only science but, to be of use to a criminal accessible through court and police investigator, the essential requirements Ecology is the study of organisms records. are knowledge of soil, and of aquatic together with their environments The crime scenes which benefi t and terrestrial sediments. – the study of ecosystems. By its from palynological help are invariably Soils and sediments exhibit great nature, palynology is a subdiscipline ecosystems themselves but they may variability in the origin of their parent of botanical ecology and, to work in be highly modifi ed by human activities. materials, structure, and chemistry but a forensic context, the palynologist Therefore, as well as the understanding it is important for the forensic ecologist must have a sound botanical and of natural and semi-natural habitats, and palynologist to realise that soil is ecological training. In Britain, forensic the forensic ecologist/palynologist particularly complex because of its palynology is an acknowledged aid must also appreciate the complexities dynamic nature. It provides a habitat to criminal investigation, providing of highly manipulated systems, such where communities of organisms live valuable evidence in cases of murder, as gardens, parks, rubbish dumps, and complete their life-cycles, and manslaughter, rape, and abduction. The plantations, ponds, canals, roadsides these organisms profoundly affect the body of literature for the discipline in verges, hedgerows and wasteland. chemistry of the inorganic matrix as peer-reviewed journals is relatively Because of the breadth of the well as any organic object or material small and, although many reports and discipline, the forensic ecologist cannot present. interpretive material are technically be expert in every aspect of ecological Most plants rely on soil as a

32 Forensic Science Seminar Volume 6 number 1 Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. FORENSIC SCI SEM source of mineral nutrition, water, and it for cross examination in court on The study of palynomorphs gave physical support. Depending on their many occasions. Table 9.1 lists a range rise to the science of palynology, fi rst responses to climate, microclimate, and of objects and matrices from which I coined by Hyde and Williams in the their ecological tolerances and needs, have analysed thousands of samples 1940s (Hyde and Williams 1944). the geographical distribution of plant (see also Milne et al. 2005). There are Its derivation is from the Greek verb species can refl ect historical geography, many cases where ecology, botany, and palynein, meaning ‘to spread or and the patterning of soil types at local, palynology have successfully helped sprinkle around’. Hyde and Williams regional, and national levels. Plant in: (i) linking objects and places (e.g. were aeropalynologists, concerned with distribution is also profoundly affected Wiltshire 2006a; Mildenhall, 2006); airborne allergens, and had little interest by biotic factors – other plants, animals, (ii) locating hidden human remains and in soil palynomorphs. Their work micro-organisms and people. Again, provenancing of objects (Brown et al. involved trapping airborne particles the forensic ecologist and botanist 2002; Wiltshire 2005a); (iii) estimating and identifying temporal sequences must have an understanding of the temporal aspects of deposition of of anthesis (pollen release) for the factors underlying plant distribution, remains (Szibor et al. 1998; Wiltshire construction of ‘pollen calendars’. plant response to change, and to have 2002a; 2003b); and, (iv) differentiating The discipline of palynology is a grasp of the variability created by murder scenes from deposition sites now over 100 years old. The founder human intervention. This is achieved (Wiltshire 2002b). of modern pollen analysis was Swedish by strengthening and modifying Knowledge of the anatomy of geologist, Lennart von Post, but the theoretical knowledge with extensive plants, animals, and other organisms subject was developed and promoted fi eld experience. helps in the identifi cation of what by fellow Swedish botanists Rutger Over time, organic components victims have eaten or inhaled Sernander and Gustaf Lagerheim. The of soil will decompose to their before death, and whether or not an fi rst major work in the subject was constituent molecules. The speed of object is of biological rather than published by Erdtman (1921). decomposition will depend on the manufactured origin (Wiltshire 2003a, The fi rst recorded cases of communities of resident decomposer 2004a; 2006b). An understanding of palynology being used as a forensic organisms, and their function depends plant and fungal development, and tool were described by Erdtman (1969). largely on the physico-chemical nature the activity of scavenging animals, Although applied occasionally (e.g. Frei of the soil itself. Palynomorphs are has given valuable information on 1979; Nowicke and Meselson 1984), important organic particles in soils and the length of time a corpse has lain it has only been used more routinely sediments and, in recent years, these in situ or the length of time since in the last 15 years or so. Mildenhall have provided valuable trace evidence an offender walked on vegetation pioneered the techniques in New in criminal investigation. Originally, (Hawksworth 2008a; Wiltshire 2007a, Zealand, Bryant in the United States the term ‘palynomorph’ was used to b). Knowledge of soil stratigraphy, (Mildenhall 1982; Bryant et al. 1990; describe pollen grains and plant spores. coupled with plant development and Bryant and Mildenhall 1998), and I Over the years, however, the term has distribution, has resulted in establishing have developed forensic palynology, expanded to include: other microscopic the premeditated nature of a victim’s forensic ecology, and forensic botany plant remains such as trichomes grave (Wiltshire 2005b). Finally, in the British Isles. (plant hairs and glands); fungal spores exploitation of knowledge of plant and and other fungal bodies; diatoms; fungal taxonomy (Hawksworth 2008b; Palynomorphs and Their cyanobacteria; and microscopic animals Wiltshire 2005c, 2008) has been used Identifi cation such as testate amoebae, nematode in assessing the potential of plants eggs and mouth parts, mites, and other being involved in attempted murder The range of palynomorphs arthropod body parts. The palynologist by poisoning, or manslaughter through requiring identifi cation in criminal needs to be able to identify many of shamanism. investigation has grown, and every these kinds of palynomorph, or seek Therefore, it is clear that the attempt should be made to identify additional expertise. identity, structure, chemistry, life- anything of apparent signifi cance in a cycles, and growth responses of whole palynological preparation. That said, Applications of Ecology, Botany, organisms play an important role the most abundant and frequently and Palynology in Criminal in criminal investigation, and that encountered palynomorphs are pollen Investigation even fragments of organisms provide grains and plant spores. There are many valuable forensic evidence. web sites and publications providing I have contributed ecological, pictures, diagrams, and descriptions of botanical, and palynological evidence in Palynology: The Background pollen and spore structures to facilitate over 200 criminal cases, and presented their identifi cation (e.g. Moore et al.

June 2016 ISSN 2157-118X 33 FORENSIC SCI SEM Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42.

1991; Beug 2004). However, there is no genus (e.g. Quercus – oaks; Salix sometimes identifi ed as Sphagnum substitute for authenticated reference – willows; Polygala – milkworts; moss spores, or as modern fern spores. material. Any unknown pollen grain Aesculus – horse chestnuts), while Fossil fungal remains are also retrieved or spore must be compared with some can only be identifi ed to from exhibits and considered modern. actual, accurately identifi ed material family (Cupressaceae – cypress; Fungal spores have been misidentifi ed and every attempt made to obtain a Chenopodiaceae/Amaranthaceae – as pollen! prepared or fresh specimen. Serious goosefoot family; Poaceae – grasses). Many areas of palynological misidentifi cations have been made In some families such as the Rosaceae, investigation (e.g. palaeoecology, by those relying solely on pictures. taxa can be identifi ed to species melissopalynology) can achieve clean While this is regrettable in any area of (Rubus chamaemorus – cloudberry; samples with palynomorphs in good palynological study, it could have dire Agrimonia eupatorium – agrimony), condition (Figure 9.1a), but forensic consequences in forensic investigation. to genus (Geum – wood avens and palynology often involves examination Keys and pictures should only be water avens), to type (Potentilla – type of very poor samples (Figure 9.1b). used as guides; fi nal identifi cation which includes three genera), and to The samples may be laden with must involve critical comparative groups of genera and species whose cellulosic debris, fl y-ash, soot, and examination of actual palynomorphs morphologies merge one into another other materials which can obscure the under the microscope. and so are diffi cult to differentiate view of the palynomorph on the slide. Pollen and plant spores are reliably (e.g. many Prunus – cherry/ The palynomorphs themselves might identifi ed by their shape, size, plum/peach/almond species). It is also be crushed, crumpled, broken, outer wall (exine) structure, surface also diffi cult to differentiate between and partially decayed. In the case sculpturing, and the type, number, pollen taxa such as Rosa (roses), Rubus of forensic samples, there must be and arrangement of apertures. To (brambles and others), Sorbus (rowan no positive opinion given unless the achieve the highest resolution of and others), Crataegus (hawthorns), analyst can demonstrate criteria for identifi cation, it is essential to remove and some species of Prunus are in identifi cation which are robust enough the inner part of the grain so that only the rose-bramble-hawthorn group. As for legal challenge. the exine remains, involving use of introduced garden species and cultivars Scanning electron microscopy toxic and corrosive acids. It results in are important in the forensic context, (SEM) and other sophisticated the dissolution of background humic particular care must be taken in microscopical methods (e.g. confocal material, cellulose, and silica, and only identifi cation of these groups, but they electron microscopy) have little structures which are resistant to the can also provide surprisingly distinctive practical application for routine treatment will be retained (Moore et al. markers. forensic palynological work. Although, 1991). These include the outer walls Identifi cation will be relatively on occasion, a sample might consist of of pollen and plants spores which are crude in the absence of chemical a single taxon, identifi ed by SEM, this composed of sporopollenin, a very processing. If the inner part of the is rare. SEM presents a picture of only robust polymer. Fungal and arthropod pollen grain, and the soil matrix (or the outer surface of the pollen grain or remains, composed of chitin, and some other material), are not removed from spore. In the case of pollen, it is often testate amoebae, will also be left after the background, only identifi cation to the elements making up the outer wall treatment. Although not so important family may be possible. Another source (exine) seen in section, that are pivotal for the identifi cation of some non- of error is in the identifi cation of taxa for precise identifi cation. These details botanical palynomorphs, the chemical which are impossible to differentiate are more easily differentiated by high- processing of pollen and plant spores by standard techniques. Knowing powered, bright fi eld and phase contrast is critical for precise identifi cation. what is possible requires considerable microscopy. In forensic samples, it is Some sculpturing features of the experience of pollen and spores from usually necessary to identify and count palynomorph are small (e.g. 0.5 μm), of a wide range of species. Reference hundreds of palynomorphs found in needing observation under phase to text books will not always resolve a prepared slide and, although it is contrast microscopy at ×1000 or more. the problem of inter-generic and technically feasible using SEM (Jones The resolution in pollen and spore inter-species variation. Further, many and Bryant 2007), the experienced identifi cation is variable. In some fossil spores (from the Mesozoic to palynologist does not need SEM for plant families, taxa can be identifi ed Caenozoic eras), and other remains, identifi cation. If only SEM micrographs to species (e.g. Plantago lanceolata fi nd their way into palynological are available, identifi cation can be – ribwort plantain; Sanguisorba preparations; and these have been impossible on some occasions. offi cinalis – greater burnet; Centaurea known to be wrongly identifi ed as scabiosa – greater knapweed). Others modern taxa. This is particularly the Palynomorphs as Trace Evidence can only be identifi ed reliably to case for Pteridophyta (ferns and allies),

34 Forensic Science Seminar Volume 6 number 1 Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. FORENSIC SCI SEM

Locard’s Principle (‘every contact have surfaces yielding distinctive aquifolium), oak (Quercus robur), leaves a trace’) is known to every police palynological profi les with areas of bare birch (Betula pendula), pine (Pinus), detective (White 2004). As outlined soil, lawns, weedy cracks in pavements honeysuckle (Lonicera periclymenum), above, palynomorphs, especially and corners, and some vegetation. and bramble (Rubus fruticosus), with pollen and spores, are excellent proxy But great care is required in planning a ground fl ora dominated by, amongst indicators of place. Offenders walk the sampling strategy, the refi nement others, bluebell (Hyacinthoides non- on soil, mud, or vegetation (short and of preparation, the resolution of scripta), wind-fl ower (Anemone tall); they have been known to hide in, palynomorph identifi cation, and the nemorosa), and bracken (Pteridium or walk through, hedges, lean against interpretation of the volume of data aquilinum). The palynomorph buildings, trees, and posts, or sit on gathered. assemblage in the comparator samples seats. Important evidence has been One of the most commonly-used was complex; many other taxa (some retrieved from very many objects and arguments against the expert witness relatively uncommon in palynological matrices and some of these are shown palynologist is that the observed assemblages) were recorded in addition in Table 9.1. If palynomorphs are profi le could have been picked up to the most obvious plants growing transferred from a place to an offender, from ‘anywhere’. Another common in the vicinity of the grave. The a victim, or any object, they can be challenge is that the observed profi le assemblages retrieved from footwear retrieved. The transferred assemblage had accumulated through repeated and a vehicle belonging to two can then be evaluated in terms of the contact with a variety of palyniferous suspects were very similar to that in likelihood of the offender, or victim, surfaces, each contributing to a the comparator samples from the crime or object having contacted the specifi c profi le that, collectively, happens to scene. Included in the assemblage were place (Wiltshire 2004c). be characteristic of the crime scene. abundant conidia (asexual spores) of Pollen grains have evolved for The reply would need to stress the Triposporium elegans. Although found sticking to the female part of the improbability of this happening. on a range of woody species, this plant and, unlike fi bres (which are Experience has shown that, while every fungus is very common on the cupules readily shed from clothing and other place will yield a unique profi le, some of beech fruits. objects), will embed into fabrics and places are similar to others in various An inevitable argument likely small interstices in footwear and other degrees. Considerable credibility as to have been presented by Defence objects; pollen and spores are not to the uniqueness of an assemblage is Counsel, and which needed addressing, easily removed. They are held fi rmly achieved when palynomorphs that are was that the assemblage could by their surface sculpturing and by generally rare (in palynological terms) have ‘come from any woodland in static charges, and are not easily shed, are found in the profi le. Hampshire and Sussex’. Investigation even from clothing and footwear Databases and Statistical revealed 14 woodlands that could, that have been subjected to washing Analyses conceivably have had the same species in a machine (Wiltshire 1997). This It might be considered that a composition as the crime scene. Each quality of tenacious adherence makes reference database could be compiled was extensively fi eld-walked with the them very valuable as trace evidence of palynological profi les of oak purpose of fi nding an environment and indicators of places or specifi c woodlands, grasslands, roadside verges, which might offer a similar surfaces. The value and advantages of or any other kind of recognisable palynological assemblage to that from palynology to forensic investigation are environment. This might be feasible the crime scene. Three were found, obvious and the discipline proven to in the broadest sense but only at a sampled, and analysed. None resembled be effective. However, it is not simple level where the resolution would never the crime scene closely, although all and the practitioner may need to apply be suffi ciently high to be of value the dominant species were growing in caveats to any conclusion. in criminal investigation. Databases the three places. Furthermore, only one for populations of palynomorph yielded Triposporium elegans, even Caveats and Limitations species can be constructed but not for though all three were thickly strewn assemblages from places. with beech cupules; this fungal spore It is important to be aware of This can be exemplifi ed by helped eliminate two of the sites. caveats and limitations in forensic Hampshire murder case (Wiltshire Pollen and spore reference palynology as the outcome of the work and Black 2006) where a man was material in a good collection will could result in someone losing their garrotted beneath a beech tree (Fagus contain examples taken from different liberty. It is not an academic exercise. sylvatica) and placed, face-down, in anthers/sporangia, different plants, All environments are highly variable, a shallow grave immediately adjacent and different places at different times particularly those outside buildings. to where he was killed. The area for each species; and to be competent, Even run-down inner-city estates can around the grave also had holly (Ilex a palynologist requires access to an

June 2016 ISSN 2157-118X 35 FORENSIC SCI SEM Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. authenticated and comprehensive will be found at a specifi c place at a moss, or fern will germinate and form reference collection of pollen, plant specifi c time’ (Wiltshire 2006a). The a new organism if conditions are spores, fungal spores, and other taphonomic processes infl uencing the amenable. Pollen produced by conifers microscopic entities. Although there pattern of palynomorph deposition and fl owering plants is carried to have been occasions where a single in any environment are numerous. female stigmas to effect fertilisation palynomorph taxon has been useful in Unlike populations of fi bres, glass and production of seeds. Both spores a criminal case (Mildenhall 2006), it and other man-made materials, those and pollen are carried by vectors, is rare for one palynomorph or single of palynological profi les are infi nite. mostly wind, insects, and rain-splash. palynomorph taxon to be of evidential The many variables that affect the Those pollen and spores, which achieve value. More usually, the evidence accumulation of palynomorphs in dispersal into turbulent air and get consists of palynological assemblages soils, on surfaces, and on items such carried up and away from the parent comprised of up to 200 or more as footwear, vehicles, and clothing, plant, form the ‘airspora’; this will different palynomorphs in varying preclude routine statistical techniques eventually fall as ‘pollen rain’ onto abundances. For many classes of trace from making useful contributions to surfaces. evidence (e.g. fi bres, glass, brick, paper, forensic palynological studies. In general, wind-pollinated paint, and ink), although each is huge, If two sampling points are spatially plants produce large amounts of their populations are fi nite; given time close, there will be a higher likelihood well-dispersed pollen, while insect- and resources, comprehensive reference of them being similar than if they pollinated ones produce relatively collections for these types of evidence were widely separated. Similarity small amounts of poorly-dispersed could be constructed for statistical diminishes with distance although, pollen. Pollen derived from wind- comparison. To date this has proved under some circumstances, samples in pollinated trees and shrubs, and tall, impossible for whole palynological close proximity to each other can be wind-pollinated herbs are common profi les. distinctly different. My interpretation components in the airspora, and are Some attempt has been made of many thousands of samples has often over-represented. But crime to invoke Bayes’ theorem to shown that every place will yield scenes are often dominated by insect- palynological data (Horrocks and a unique palynological profi le, and or self-pollinated plants. Invariably Walsh 1998). Unfortunately, there that even each sampling point within their pollen simply falls in a halo on were some false assumptions in their a site will have its own special the ground around the parent plant, or study, and the ‘cases’ appeared to be characteristics. To gain a palynological is released only when the plant dies and highly theoretical, simple, scenarios ‘picture of place’ requires the analysis falls to the ground. Pollen from these constructed to demonstrate their of many comparator samples to build plants, as well as many fungal spores hypotheses. If full datasets from real up a matrix of profi les, each one (including those of lichens), may never cases had been used, the conclusions contributing to the bigger picture. Such be found in the airspora, and contribute drawn may have been different. a bigger picture will be unique for that little to the pollen rain, but they may The Bayesian approach is important place. be the most abundant on an item of philosophically and is useful for The complexity of palynological footwear. The wearer may have walked framing the presentation of conclusions taphonomy makes the discipline an through organic debris or meadow but, at the present time, it is diffi cult exquisite tool in the hands of the vegetation, and abundant pollen from to see how it can be applied to the experienced forensic palynologist. the airspora at the time of the offence volume of data accrued from real However, it can be potentially may form only a small proportion of cases. Multivariate analysis has, on misleading and suspect when those the profi le relevant to the criminal occasion, proved useful in dealing with with inappropriate, or insuffi cient, investigation. data where there are high numbers experience are involved, and the Some palynomorph taxa are thus of exhibits and comparator samples caveats and constraints are not properly exceptionally useful markers of the (Wiltshire 2002c, 2004a; Riding addressed. place and, if there were many such et al. 2007), but only for guidance. Taphonomic Considerations plants at a specifi c location, that place Conclusions, and professional opinion Some of the major taphonomic would be endowed with a characteristic presented in court, must be based on factors affecting palynological palynological signature which would botanical and palynological criteria, profi les have been briefl y reviewed by be diffi cult to replicate artifi cially. skill, and experience. Mildenhall et al. (2006). They note that Analysis of many samples at numerous Taphonomy may be defi ned as ‘The of importance is the level of pollen and crime scenes has demonstrated that sum of all the factors that infl uence spore production, and the way these whatever the mode of pollination, the whether a palynomorph (pollen, entities are dispersed. major part of the pollen load of any spore, or other microscopic entity) A spore produced by a fungus, plant falls close to the parent.

36 Forensic Science Seminar Volume 6 number 1 Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. FORENSIC SCI SEM

Many palynologists are engaged in of the sophistication of statistical There is some danger in adhering the reconstruction of past environments, modelling, the nature of the pollen to some of the conventional wisdoms vegetation change through time, trapping site is critical. There may in palynology where models are based and past land-use; this involves the be common elements in vegetation on the work of a limited number analysis of cores of mire or lake composition between the sampling of researchers in a limited range of sediments, or buried soils. Few carry site and a palaeoecological profi le, but scenarios. For example, it is often out independent and extensive analysis the chance of them having been truly assumed that some taxa such as Pinus of multiple surface samples at either representative of the ecology of another (pine) exhibit long distance transport. local or regional level. Most depend on is remote. Some grains are capable of being the researches of other palynologists The study of airborne transported many miles from source interested in the taphonomic problems palynomorphs is fraught with diffi culty but, if there are physical obstacles associated with dispersal and fall- and has been a focus of debate for between the source and accumulating out. The latter includes the Pollen many years. There is considerable surface, the pollen may be deposited Monitoring Programme, a research variation in the pollen rain at any one only very locally. In every plant, most initiative sponsored by the International place at any one time, and this variation of its pollen or spore production will Quaternary Association (INQUA) is refl ected in fall-out patterns onto fall near the parent. Enigmatically, (Hicks et al., 2001; Tinsley, 2001; surfaces. The variation will depend there have been cases where a Barkenow et al., 2007). Another on pollen production and dispersal prolifi c pollen producer such as pine initiative, POLLANDCAL (Pollen- characteristics, the presence of physical registered less than 2% of the total Landuse Calibrations), involves many and geographical barriers, the structure pollen sum even though mature trees palynologists collecting modern pollen and mass of the palynomorph affecting were within 10 m of the sampling site data and using sophisticated statistical sedimentation rates, and many other (Wiltshire unpubl.). In other instances, techniques to generate predictive factors. There are classic models insectpollinated plants such as Aesculus models (Sugita et al., 1999; Eklöf et constructed to explain the observed hippocastanum (horse chestnut), where al., 2004; Bunting et al., 2005; Bunting heterogeneity in the airspora (Tauber pollen production is thought to be and Middleton, 2005; Soepboer et al., 1965, 1967), and large-scale and small- low and dispersal poor, have achieved 2007). scale modern pollen studies which have the same values as Quercus (oak), a As with earlier studies on pollen led to the construction of predictive more prolifi c pollen producer, several productivity and dispersal, the work models, often quoted by palynologists hundred metres away from a mixed of the PMP and POLLANDCAL is, working on the reconstruction of past stand of horse chestnut and oak in a in the main, concerned with obtaining vegetation and land-use (Davies 1967; public park (Wiltshire unpubl.). information to enhance interpretation Jacobson and Bradshaw 1981; Prentice At a crime scene in Brierley Hill, of palaeoecological profi les. It 1985). near Birmingham, several samples aims to interpret past vegetation by A search on the internet provides from the vicinity of a young mature, extrapolation of results from patterns large numbers of papers, written over fruiting tree of Fagus sylvatica of modern pollen deposition to profi les the last 40 to 50 years or so, describing (beech) failed to yield any beech obtained from ancient deposits. and examining these taphonomic pollen (Wiltshire 2003c). A similar Airspora studies depend largely phenomena, as well as more recent situation was observed in a case in on the use of pollen traps (see Caulton research projects and papers. There is South Wales (Wiltshire 2004c) where et al. 1995; Levetin et al. 2000; little doubt that data collected through a murder victim was buried on a Wiltshire 2006a). Although there are the PMP and POLLANDCAL, and hillside, dominated by Picea sitchensis many trapping sites, the areas covered specifi c studies involving dispersal (sitka spruce). Analysis of the surface are, nevertheless, insignifi cant in from individual species, are not soils around the grave showed that comparison to the total land surface suffi ciently refi ned for use in forensic spruce pollen hardly registered in the available for deposition. Extrapolating studies. Inevitably, their investigations comparator samples, but that pine modern data to the past requires a leap concentrate either on palynomorphs, pollen was very well represented. of faith since there may be no past which become airborne and contribute In the lower fi ll of the grave-fi ll, the homologue or analogue of modern to the airspora, or are geared towards assemblage was dominated by pine. vegetation patterning. The situation plant/vector inter-relationships. The The spruce trees were very large and is less tenable when data from pollen former are mostly trees, shrubs, and it might be expected that their pollen traps are applied to more distant sites, wind-pollinated herbs which ave high would swamp the surface. However, and inappropriate when applied to pollen production and good dispersal, very few of the trees had reached ancient sites, hundreds of miles from and the latter related to crop plants sexual maturity. A single, small but the sampling locations. Irrespective which rely on insect pollination. mature, pine tree about 100 m from the

June 2016 ISSN 2157-118X 37 FORENSIC SCI SEM Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. site was the source of the surface pine be biological. The investigator must limited contact with soil, vegetation, pollen. That in the deeper profi le was be aware that footwear, the outside and other intensely palyniferous enigmatic, but could have represented of vehicles, and digging implements surfaces. As with footwear and the vegetation before the spruce might be irrelevant to a case and the vehicles, suffi cient comparator samples was planted about 40 years earlier. main source of evidence would be the are needed to be able to eliminate Without understanding such systems, clothing on the upper body, with no sources other than the crime scene but, or by not visiting the crime scene, the trace of soil. if the assemblage accumulated from the palynologist might well have assumed The palynological profi le from any crime scene is suffi ciently distinctive, pine rather than spruce woodland to be crime scene is built up from multiples multiple deposition need not be an associated with the suspects. of comparator samples; it is, therefore, insurmountable problem. These few examples demonstrate composed of a pattern of fragments. It A complication with fabric is that the danger of adhering strictly to also follows that, to gain a workable an offender may already have had soil simplistic scenarios. Further examples picture of the place, the larger the on clothing before committing the of the dangers of relying on airspora sampling area, and the greater the offence, or after visiting the burial site. data in forensic investigation are found number of samples obtained, the closer Palynomorphs from the crime scene in Wiltshire (2006a). the results will be to the actual profi le can then be superimposed on the pre- Source of Trace Evidence (even though that is unknowable in existing soil marks. In one case there Pollen and spores falling at any detail). An offender contacting a crime was an apparent confl ict of evidence one time will be mixed with pollen scene will pick up only a fragment where a soil scientist and palynologist previously accumulated on the of the crime scene’s palynological were not aware of each others’ roles surfaces. Plants (both insect and wind- profi le. If the trace evidence is then (Wiltshire 2001b). Soil on the suspect’s pollinated) colonising new ground secondarily transferred to, say, a sweatshirt was ‘innocent’, and was will also contribute to pre-existing vehicle, only a thirdorder fragment derived from deep sub-soil accumulated assemblages. This means that time will be retrieved. Palynological during the digging of arage foundations; is important in forensic sampling. A interpretation is, therefore, complex analysis of the soil from the excavation natural/semi-natural habitat such as and requires visualisation skills as well showed it to contain no palynomorphs. a woodland might yield very similar as an understanding of the complex While wearing the soiled clothing, profi les for many years, but there could taphonomy underlying assemblages. the offender buried the victim near a be drastic changes if the environment In spite of all the caveats that apply, hedge in a pasture. Before the grave were a manipulated one, such as a the assemblages distinctive enough was dug, there was little exposed soil plantation or garden, even within short to establish convincing links between in the meadow but, importantly, the periods. Further, any object contacting items, places, and vehicles have been offender picked up spores and pollen a palyniferous surface will receive repeatedly demonstrated. As previously from tall vegetation on the path to and only a fragment of the pollen rain that stated, palynological samples obtained from the area around the grave site on had accumulated on it over time, and a from exhibits are fragmentary in nature. his soiled sweatshirt. The palynomorph fragment of the biological signature of For links between them and crime assemblage on the garment was similar the habitat as a whole. This is why it is scenes to be acceptable to the Court, to that at and around the deposition site. essential for the palynologist to select there needs to be either: (a) a highly Thus, if only the soil evidence had been target locations within a crime scene. complex assemblage where there are taken, there would have been no link Although trace evidence is many points of similarity between between the garment and the burial site. transferred to the belongings of place and object, or (b) some unusual This case provided a salutary lesson offenders when they contact soil and or rare component or components. to investigators; the soil analyst and sediments, there have been cases Mixed Samples: Fabrics palynologist should work together to where soil has not played any role Garments worn repeatedly for gain the deepest level of understanding in investigations (Wiltshire 1997, considerable periods will pick up from the respective data. 2007b). Many surfaces are completely palynomorphs from various places, It is one of the strengths of vegetated, or covered in deep leaf litter, so any retrieved assemblage will be palynology that pollen grains and such that soil may not be contacted mixed. They are transferred easily from plant spores will embed themselves in by footwear, clothing, or tools, and palyniferous surfaces, but few seem to fabriucs such that they can be retrieved vehicles. Plant surfaces, plant litter, be picked up from air. Except where from exhibits even after being put humus, and compost can yield dust and, there is obvious soiling, it is impossible through the washing machine (Wiltshire perhaps, the fi ne fraction of the soil to separate various depositional events 1997). through rain splash and wind action, by sampling. But, unlike footwear, Mixed Samples: Footwear but the most important particulates will most items of clothing generally have Footwear presents another

38 Forensic Science Seminar Volume 6 number 1 Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. FORENSIC SCI SEM complex of problems and challenges. (sometimes thousands) of pollen and up from bare soil, mud, leaf litter, Usually, samples are taken from spores to achieve an assemblage large organic debris, and vegetation. Day- specifi c areas within the crime scene, enough to allow the differentiation to-day, most people do not usually known to have been walked upon by a of the crime scene from other places wear muddy or soiled shoes, and any suspect, so that they can be compared where palynomorphs may have been noticeable accumulations of soil or with palynological assemblages on transferred to the footwear. If footwear mud on footwear are obvious targets the footwear. An offender will have yielded sparse palynomorphs, low for sampling in criminal investigations. had to contact the edge (and inside) counts might still be useful, but only These have proved useful in linking of a grave during digging, and a rape if there were some very distinctive footwear with crime scenes and other victim might be able to locate the exact components present in the assemblage. sites but, more commonly, footwear places trodden by her attacker; samples My analysis of thousands of items from suspects in criminal cases is should be taken from such identifi ed of footwear, including wellington relatively clean or only slightly locations. Any footprints or depressions boots, baby’s bootees and Gucci soiled. If there were several obvious in soil and mud are obvious targets, but court shoes, has shown that variation depositions of soil/mud, then every these are usually seized by the police is so great that general models are attempt should be made to analyse each for casting and foot mark analysis. unlikely to be attainable goals. There one separately. However, even when It is now standard practice to scrape are many variables associated with sampling is meticulous, it is rare for away the deposit at the interface of the palyniferous material itself (soil, perfectly uncontaminated samples to be the underneath of the cast and the sediment, leaf litter, vegetation), obtained; the palynological preparations adhering soil layer to obtain the most but there are others which can will contain mixed assemblages. relevant comparator sample. Even affect the deposition and removal In some cases, the pertinent layer if the offender accumulated layers of palynomorphs. These include the of material on a shoe can be beneath of soil/mud from elsewhere prior, or materials making up the footwear, the subsequent, superfi cial layers of soil subsequent, to the offence, the mixed gait and wear patterns of the wearer, and mud, and it becomes impossible profi le on the footwear should contain the weight of the wearer, and even to differentiate them physically. Here, some of the trace evidence retrieved ambient weather conditions (and the situation is similar to that of the from the cast. The palynologist then hence the wetness of the surface). For relatively clean shoe where the whole has to differentiate the relevant profi le criminal investigation, in every case, it item must be sampled. There is no from the irrelevant one. is important that microscopic analysis substitute for counting many hundreds There has been some attempt of footwear and other items is carried of palynomorphs (or as many as to make forensic palynology more out so that they can be compared possible) and comparing them with the ‘scientifi c’ by setting up hypothetical directly with comparator samples from crime scene as well as other pertinent crime scenes and testing outcomes from the crime scene and other pertinent places for elimination purposes. various kinds of contact. Such studies places. In the forensic context, a model A complicated case was that are useful exercises and, for the objects is never likely to provide adequate of R vs Anthia (Wiltshire 2004d) used in the experiment, or places tested, information for prediction of events or concurrently investigated by two police the results might be valuable. However, outcomes. forces. Because of the modus operandi, some results presented, should be By its very nature, at any one time, the two forces suspected that the same considered to be preliminary in view of footwear will have a palynomorph load man was responsible for at least six the low pollen counts in each case, and accumulated from a range of different attacks in Hertfordshire and London. the limited number of treatments within places. Depending on the frequency I visited the six crime scenes, which the trials (e.g. Riding et al. 2007). For and pattern of wear, trace evidence varied from woodland, roadside verge different sets of footwear exposed to will continually be gained and lost. In hedges, golf courses, and wooded areas the same palyniferous surfaces, or other my experience relatively few pollen of parks. I was given the suspect’s footwear exposed to other palyniferous grains are picked up from paved or clothing, items from his vehicle, and surfaces, the outcomes might be very metalled surfaces, although spores can several items of footwear. There were different. It is dangerous to formulate be transferred from lichens growing convincing palynological similarities predictive models, or form fi rm on hard-standing. Palynomorphs can between one of the Hertfordshire conclusions, based on relatively few also be picked up from pavements and sites and the upper clothing, car test items, in a few test scenarios, with gravel paths on which decomposed seat, and one pair of footwear. The low pollen counts. Footwear invariably and decomposing plant litter palynology of another pair of boots accumulates multiple depositions of have accumulated. Invariably, the yielded very similar assemblages to palynomorphs. It is, therefore, often most signifi cant assemblages of another Hertfordshire crime scene as necessary to count many hundreds palynomorphs of all kinds are picked well as one of the London sites. These

June 2016 ISSN 2157-118X 39 FORENSIC SCI SEM Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. two crime scenes were suffi ciently yielded bluebell and this had probably found, key exhibits will include distinctive that both could be been carried in soil to the concrete on digging implements. Unless a spade recognised in the mixed assemblage on the grave-diggers’ feet. If both items of or shovel was bought for the criminal the boots. The defendant was convicted footwear had been amalgamated, the activity, it could have a palimpsest of and received 10 life sentences. case for his non-involvement would soil layers distributed heterogeneously Approaching 20,000 palynomorphs have been weaker. over the blade. Obtaining appropriate were counted in this case, and it was Evidence in a murder case in samples from such an item can be the exceedingly high resolution of the Greater Manchester was also enhanced fraught with diffi culty, and the best analysis which allowed the various by separate analysis of shoes (Wiltshire option might be to attempt a multiple crime scenes to be differentiated. 2003d). Palynologically, this was a sampling strategy. Again, a mixed It is now standard practice to very complex case and resulted in my sample will ensue and the skill of the analyse each item in a pair of footwear being cross-examined continuously for palynologist can be severely tested in separately. In many cases, both feet pick fi ve days, and the accused being given such cases. A soil-laden spade might up similar palynological assemblages a life sentence. The naked body of a also be laid on the ground where there and it may be thought unnecessary to woman, who had spurned her lover, is no bare soil but only a close cover do separate analysis. However, there was found lying on a path at a local of vegetation. Palynomorphs from the have been at least two cases where woodland beauty spot. She had been turf could dominate any number of each shoe differed, and the results were beaten and there were foot marks on profi les previously accumulated. Again, pivotal to interpretation of the cases. In her face. One aspect of the case was it is the rare or unusual assemblage a drugs-related case (Wiltshire 2001a), to confi rm a statement that events of palynomorphs, or even a very rare an informant claimed that, although he witnessed in the yard of the local public palynomorph, that might indicate a link had stood on an area of hard-standing at house might have been relevant. The with a specifi c place. It is unlikely to be the edge of a woodland where a grave victim had yellow stains on her jacket formed with widespread, common taxa. had been dug, he had not entered the sleeve; the stains were composed of In the case of the murder of Joanne scene and remained standing on an area Forsythia pollen and green algae. The Nelson, the ‘Valentine Girl’ (Wiltshire of muddy concrete about 30 m from the palynological assemblage from the 2005a), her lover killed her but forgot actual grave site. His statement needed suspect’s shoes was shown to have a where he had placed her body. His verifi cation. His shoes were analysed strong similarity to the actual crime statement to the police was incoherent and the palynological assemblages scene a few hundred metres away, and they were anxious to fi nd her on both of them showed that he but one had a large number of grains remains. From palynological analysis had, indeed, picked up woodland of fenestrate Lactuceae (dandelion- of his vehicle, footwear, and a garden palynomorphs, and the trees and shrubs like) pollen. The other shoe had only fork, I was able to eliminate his own, or were the same as those at the crime a couple of grains of dandelion-like his parents’ garden, as being the source scene. But, only one of his feet yielded pollen. A visit to the public house yard of the critical palynomorph assemblage. the assemblage characteristic of the showed a concrete fence covered in There were distinct similarities between gravesite itself; one foot had picked up green algae, with a fl owering Forsythia the profi les from the car, one pair of components of Alnus (alder), Quercus bush growing over its top. There shoes, and the garden fork and, from (oak), Pinus (pine), Corylus (hazel), was a very narrow verge along the them, I was able to envisage the kind of and other woody taxa and ferns. The fence, dominated by dense growths place her body had been deposited and other had the same assemblage and few of Taraxacum offi cinale (dandelion). the vegetation of the place in question. grains of Hyacinthoides (bluebell). The Extensive searches and sampling of The fork had not been used to bury her fl oor of the woodland was carpeted with the local area failed to fi nd anywhere body but to cover it with woody, forest- bluebells and Anemone (wind fl ower), which would offer such an assemblage fl oor litter. The signature of that litter and there were Rhododendron bushes of plants and palynomorphs in close was super-imposed on the pre-existing next to the grave. If he had walked to proximity to one another. It was mud. Again, the soil on the exhibit the grave site, he would not have been suggested that the suspect had started was irrelevant to the investigation, and able to avoid picking up larger amounts abusing the victim in the yard, pushed there was no soil from the crime scene of bluebell pollen, that of Anemone her against the fence, and whilst on the fork. If soil analysis alone had and, possibly, Rhododendron on both doing so, stepped on the verge and the been carried out, the girl would never feet. It would appear that he had picked dandelions with one foot. This was have been located. Police found her up the woodland palynomorphs from diffi cult for the accused to deny. body very quickly from the provision the muddy concrete but there was no Mixed Samples: Digging of an accurate description of the site. evidence that he had walked into the Implements and Vehicles The assemblage of palynomorphs woodland with both feet. Only one Whenever buried remains are was an unusual one because of the

40 Forensic Science Seminar Volume 6 number 1 Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42. FORENSIC SCI SEM nature of the Forestry Commission conditions. However, they concluded origin of any organic particulate can plantings; it also included spores of that they could differentiate between only be crude, and there is no substitute Polypodium (polypody) fern which is winter/spring, spring/summer, and for detailed analysis of the crime very uncommon in the area. summer/autumn. To be suffi ciently scene, other places pertinent to the Seasonality and Temporal convincing to be useful in the forensic investigation, and the objects that are Interpretation context, a great deal more work would thought to have had contact with them. The time of an offence, or activities be necessary and the phenomenon of Worldwide, palynology is an surrounding criminal activity, are often residuality addressed. Depending on under-used resource for criminal important aspects of police intelligence. the environment, palynomorphs can investigation. This is due, in part, to Palynology has, on occasion, been used remain in situ for very long periods. the perennial dearth of competent to confi rm the temporal aspects of cases A soil sample might contain pollen palynologists who possess not only (Wiltshire, sub-judice cases ongoing). accumulated over decades, and it can comprehensive botanical knowledge, The timing of anthesis (pollen remain on foliage and bark for more ecological training, and appropriate release), especially of wind-pollinated than one year (Adam et al. 1967; and extensive fi eld experience, but plants, is critically important to those Groeneman-van Waateringe 1998). Any who can cope with the rigours of cross- involved in studying allergy. Pollen and pollen assemblage transferred during examination in the courts. fungal spores are important allergens, day-to-day activity would inevitably and considerable effort is focused on contain palynomorphs from a number Acknowledgements pollen calendars. Such calendars are of seasons. To rely on pollen calendar produced, and information exchanged, data for estimating seasonality in by various institutes, universities, and forensic work is imprudent. Grateful thanks are due to Judy Webb, Julia Newberry, and Peter hospitals in many countries (Hyde Murphy for the excellence of their 1969; Michel et al. 1976; O’Rourk Conclusions contribution to my case work over the years. I would also like to thank David 1990). The pollen calendars give start L. Hawksworth for assistance in the and fi nishing times, and the periods In natural ecosystems, organisms fi nalisation of this contribution. of peak release for pollen and spores occupy niches that may be narrow or References for selected species. However, such wide. Some have a wide geographical calendars have limited use for forensic distribution and others a narrow one. work because of the frequent, and This is useful for predicting the nature 1. Adam DP, Ferguson CW and LaMarch sometimes extreme, variation in pollen of places from which palynomorphs VC Jr. (1967). Enclosed bark as a pollen trap. Science 157:1067–1068. release times from region to region. were transferred to offenders. However, 2. Barnekow L, Loader NJ, Hicks S, In Britain there is a network of ecosystems are rarely natural in the true Froyd CA and Goslar T (2007). Strong correlation between summer temperature 10 pollen monitoring stations and 11 sense, with enormous environmental and pollen accumulation rates for Pinus stations which monitor only grass manipulation wherever people have sylvestris, Picea abies and Betula spp. in a high resolution record from northern pollen; the pollen stations are situated had infl uence. From place to place, Sweden. Journal if Quaternary Science in towns in lowland areas, so their and sample to sample, palynological 222:653–658. results may be unrepresentative of profi les are characterised by their 3. Beug H-J (2004). Leitfaden der Pollenbestimmung für Mitteleuropa much of the British airspora. Even variability and uniqueness. It follows und angrenzende Gebiete. Verlag Dr. at a local level, ‘pollen calendars’ that models for pollen dispersal and Friedrich Pfeil, München. 4. Brown AG, Smith A and Elmhurst O can never be precise – certainly, they pattern of fall-out, which might aid the (2002). The combined use of pollen and cannot offer the precision required for palaeoecologist in the interpretation soil analyses in a search and subsequent murder investigation. Journal of Forensic forensic investigation. If the pollen of past environments where human Sciences 47:614–618. calendar for a specifi c place were intervention was minimal, will be of 5. Bryant VM and Mildenhall DC (1998). known in detail, there might be some limited use in forensic case work. The Forensic palynology: a new way to catch crooks. In: New Developments application for interpretation of data amount of variation inherent in any in Palynomorph Sampling, Extraction, relating to that place; but, because of system makes it impossible to construct and Analysis (Eds. VM Bryant and JH Wrenn). American Association of specifi city and inherent variability, databases of palynological profi les Stratigraphic Palynologists Foundation. the uses of seasonal records are very that could be used with confi dence in Contributions Series Number 33:145– 155. limited. They can only be used in the criminal investigation and preparation 6. Bryant VM, Mildenhall DC and Jones crudest way. For example, Montali et of court statements. Every sample JG (1990). Forensic palynology in the al. (2006) showed that pollen retrieved from every crime scene, and every United States of America. Palynology 14:193–208. from corpses could not be related to assemblage retrieved from every 7. Bunting MJ, Armitage R, Binney H and the local pollen calendars because exhibit, will be unique and will need Waller M (2005). Estimates of ‘relative pollen productivity’ and ‘relevant source of the degree of variability in local independent evaluation. Predictions of area of pollen’ for major tree taxa in two

June 2016 ISSN 2157-118X 41 FORENSIC SCI SEM Patricia E.J. Wiltshire. Forensic ecology, botany, and palynology. FORENSIC SCI SEM, 2016, 6(1): 32-42.

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